26.01.2015

Breast cancer journals impact factors

IMPACT OF LYMPHOEDEMA ON ARM FUNCTION AND HEALTH-RELATED QUALITY OF LIFE IN WOMEN FOLLOWING BREAST CANCER SURGERYDiana J.
Xiaoyan Li1,2,*, Qifeng Yang1,2,*, Haiyang Yu1, Lihua Wu1,3, Yuhan Zhao1, Cen Zhang1, Xuetian Yue1, Zhen Liu1, Hao Wu1, Bruce G. Breast cancer is one of the most common malignant tumors and the second most common cause of cancer related deaths in the United States. To directly investigate the effect of LIF on metastasis in breast cancer, two chamber trans-well assays were employed to determine the effects of LIF on the abilities of invasion and migration of MDA-MB-231, MCF7 and T47D cells. The effect of LIF on metastasis was further investigated in vivo by employing the in vivo lung metastasis assays.
In addition to promoting metastasis, LIF also promoted proliferation of breast cancer cells. Figure 2: LIF promotes proliferation and anchorage-independent growth of breast cancer cells and promotes the growth of xenograft breast tumors. Figure 4: Blocking the mTOR signaling largely abolishes the promoting effect of LIF on invasion and migration of breast cancer cells.
It has been reported that LIF activates the AKT pathway in several different cell types, including human embryonic kidney 293T, liver Hep3B, and oligodendrocytes [7, 27]. The AKT pathway crosstalks with and activates the mTOR pathway [28], which raises the possibility that LIF activates the mTOR pathway through AKT. To further determine whether the activation of AKT-mTOR pathway contributes to the effect of LIF on breast cancer tumorigenesis and metastasis, above-mentioned breast cancer cell lines were treated with LIF along with or without wortmannin to block AKT pathway. Figure 6: Blocking the AKT signaling inhibits the promoting effect of LIF on tumorigenesis and metastasis in breast cancer cells.
To evaluate the clinical importance of LIF in breast cancer, a cohort of 374 breast cancer patients was employed. Figure 7: High LIF expression levels are associated with a poor relapse free survival of breast cancer patients. Results from this study demonstrate that LIF promotes the growth and metastasis of breast cancer. LIF binds to its receptor complex composed of LIFR and gp130 to activate the LIF signaling pathway. It is currently unclear whether LIF activates the AKT-mTOR pathway to promote tumorigenesis and metastasis in other types of cancers.
In summary, results from this study demonstrate that LIF plays a vital role in promoting growth and metastasis of breast cancer. The trans-well system (24 wells, 8 µM pore size, BD Biosciences) was employed for cell migration and invasion assays as previously described [40]. Tissue microarray comprising duplicate cores of tumors from 374 breast cancer patients was used for this study. A computer program package SAS (Version 9.1, SAS Institute) was employed to manage the patient database and analyze the statistic difference.
All site content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 License. Alexandra Canonici1,*, Merel Gijsen2,*, Maeve Mullooly4, Ruth Bennett2, Noujoude Bouguern2, Kasper Pedersen1, Neil A O’Brien5, Ioannis Roxanis6, Ji-Liang Li3, Esther Bridge3, Richard Finn5, Dennis Slamon5, Patricia McGowan4, Michael J. HER2 is a member of the human epidermal growth factor receptor (HER) family which also includes EGFR (HER1), HER3 and HER4 [1]. Trastuzumab is a humanized monoclonal antibody which binds to extracellular domain IV of HER2. In an attempt to circumvent resistance to trastuzumab, anti-HER2 tyrosine kinase inhibitors (TKIs) were introduced. To further enhance HER2 inhibition, irreversible TKIs, such as neratinib, afatinib and dacomitinib have been developed. We determined sensitivity to neratinib in a panel of 36 breast cancer cell lines, including HER2 positive, luminal and basal-like cell lines (Figure 1A left panel, Supplementary Table 1).
As the HER2 positive cell lines were significantly more sensitive to neratinib, we investigated the effect of neratinib on HER2 signalling in two HER2 positive breast cell lines. Figure 1: Neratinib is most effective in HER2 positive breast cell lines and reduces HER2 phosphorylation. Figure 2: Combination of neratinib and trastuzumab has an additive effect and prevents re-activation of pHER3 and pAkt. We tested the effect of neratinib on acquired trastuzumab resistant BT474 (BT474R) and SKBR3 (SKBR3R) cells. Based on the additive interaction between neratinib and trastuzumab observed in the SKBR3 and BT474 cell lines, we extended the analysis of the combination treatment to 7 additional HER2 amplified breast cancer cell lines, including cell line models of innate trastuzumab resistance.
Figure 4: Combined neratinib and trastuzumab treatment in cell line models with varying trastuzumab sensitivity and resistance. In order to examine potential pharmacodynamic biomarkers of response to neratinib, we tested the effects of neratinib (200 nM) on HER2 and downstream signalling in a panel of HER2 amplified cell lines, with varying sensitivity to neratinib (Figure 5B).
Figure 6: Combination of trastuzumab and neratinib was additive in tumor growth inhibition in BT474 xenograft model.
Our results showed that the combination of trastuzumab and neratinib treatment was significantly more potent at reducing cell viability than trastuzumab alone in both sensitive and acquired resistant HER2 over-expressing SKBR3 and BT474 breast cancer cells.
Clinically, the withdrawal of trastuzumab treatment in patients who are no longer responding is controversial [33], partially due to the cost of continuing trastuzumab treatment [34]. Neratinib has shown promising activity in several clinical trials, particularly in HER2 positive breast cancer patients [29, 37].
It has been previously shown that EGFR and HER2 inhibitors failed to suppress HER3 phosphorylation due to ligand-dependent activation of HER3 via ADAM17 through an Akt feedback loop [19, 40, 41].
Although neratinib is a panHER inhibitor, we showed that it is selectively active in HER2 amplified compared to non-amplified breast cancer cell lines.
Despite the frequent overexpression of EGFR in triple negative breast tumors and cell lines, they were less sensitive to neratinib than HER2 amplified cells.
The levels of total HER2 protein and phosphorylated HER2 correlated with response to neratinib in the panel of HER2 overexpressing cell lines, similar to lapatinib [30].
Like lapatinib [30], neratinib can overcome trastuzumab resistance in cell line models of acquired trastuzumab resistance and can enhance response to trastuzumab in trastuzumab sensitive cell lines.
SKBR3 and BT474 were obtained from CRUK London Research Institute Cell Services, and were cultured in RPMI and DMEM respectively; both were supplemented with 10% FBS, penicillin-streptomycin.
Stock solutions of lapatinib (10 mM) (Sequoia Research Products) and neratinib (10 mM) (supplied by Pfizer) were prepared in dimethyl sulfoxide (DMSO).
For cell counting experiments, cells were seeded in duplicate at 2 – 3 x 104 cells per well, depending on the cell line, in 24 well plates. After neratinib treatment for 24h, cells were washed with PBS and lysed in RIPA buffer (Sigma) containing protease inhibitors, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM sodium orthovanadate. Formalin-fixed BT474 xenograft samples were embedded in paraffin and 4 µm thick sections were cut for immunohistochemical staining. Mann-Whitney U test was used to investigate the association between response to neratinib and breast cancer cell line subtype. We are grateful to all the members of Professor Adrian Harris’ lab for their help and advice.
Yun-Ru Chen1,2, Brittany Tsou1,2, Shuya Hu1, Huimin Ma1,2, Xiyong Liu1, Yun Yen1 and David K. Cell death is an important process in cancer therapy that occurs through three major pathways: apoptosis, autophagy, and necrosis - each of which can be characterized by various morphological criteria [1]. One therapeutic approach is to induce an imbalance of critical cellular components, leading to reduced proliferation and death of the cancer cells.
We have identified a novel small molecule RR inhibitor, COH29 that disrupts the interactions between the hRRM1 and RRM2 subunits of human RR [12].
Our previous study has shown that the regulation of the dNTP pool and RRM2 levels are inversely related to levels of autophagy [30]. Based on our previous work [30], we hypothesized that inducing autophagy would decrease RRM2 levels and sensitize cancer cells to COH29, a novel RR inhibitor [12, 31].
Next, we hypothesized that the TMX-induced, autophagy-mediated RRM2 downregulation sensitizes MDA-MB-231 cells to anti-RR agents, such as COH29. Figure 1: Tamoxifen enhances the cytotoxic effects of COH29 through autophagic degradation of RRM2. Next, we tested the hypothesis that reduced RRM2 levels would deplete intracellular dNTP pools. Lastly, we investigated the possibility that other cell death pathways were activated by the combined treatment. Next, we used an orthotopic tumor model and treated the tumor-bearing mice with vehicle, TMX, COH29 or the combination.
Next, the tumor samples were stained with anti-RRM2 and -Ki67 antibodies, respectively (Figure 3D and Supplementary Figure S4A).
In order to investigate the mechanism underlying breast cancer cell death by combined treatment with COH29 and TMX, fluorescence microscopy was used to examine the nuclear dynamics. In order to investigate the potential genomic instability induced by the combined of COH29 + TMX treatment, MDA-MB-231 cells undergoing various treatments were subjected to cytogenetic analysis (Figure 5). RR inhibitors, first introduced into clinic near 60 years ago, are widely used in cancer therapy. 60 years after the introduction of RR inhibitors into clinical oncology, their mechanism of action in human tumors is becoming increasingly clear.
The reciprocal regulation of autophagy and the dNTP pool has been observed and reported in human cancer cells [28]. Two different ubiquitin ligases are known to exert tight control over the steady-state levels of RRM2 in the G1- and G2-phases of cell cycle. Anti-mitotic agents have wide-ranging therapeutic potential for the treatment of various types of cancers [57, 58] and there has been a great deal of interest in identifying novel mitotic inhibitors that can overcome the various modes of resistance and promote improved pharmacological profiles [59]. We propose that autophagic degradation of a RRM2, the target of COH29, promotes cell death, whereas in other contexts autophagy can protect cancer cells from death [62, 63].
Many cytotoxicity assays are dependent on mitochondrial function and can generate artifacts if mitochondrial function is impaired without cell death. Cells treated with TMX, COH or combination treatment were harvested at the end of incubation period and lysed on ice for 30 min in RIPA buffer (Cell Signaling, #9806) containing a complete protease inhibitor cocktail (Roche, 11836145001) and PhosSTOP (Roche, 04906837001).
Interestingly, CSCs share many properties with normal stem cells, including immortality and resistance to stress, as well as asymmetric cell division [9, 10]. Clinically, there is an urgent need to identify new therapeutic strategies for selectively targeting CSCs. Here, we tested the efficacy of graphene oxide as a potential new anti-cancer agent, for the selective targeting of CSCs.
Initially, we tested the ability of GO to affect CSC proliferation, using MCF7 cells, a well-established ER(+) breast cancer cell line. Figure 2: Graphene oxide (GO) selectively targets cancer stem cells (CSCs) in breast cancer cells.
Since both the small and big GO flakes showed similar potency, we focused more on evaluating the efficacy of b-GO flakes. Figure 3 shows that b-GO flakes also effectively inhibited tumor-sphere formation in these 5 other cell lines.
Importantly, b-GO flakes also did not affect the viability of a normal skin fibroblast cell line (hTERT-BJ1), indicating that GO is relatively non-toxic for normal body cells (Figure 6). Figure 3: Graphene oxide (GO) selectively targets cancer stem cells (CSCs) of multiple cancer cell types. Figure 4: Graphene oxide (GO) selectively targets cancer stem cells (CSCs) of multiple cancer cell types. Figure 5: Graphene oxide (GO) does not affect cell viability of the total population of cancer cells.
For this purpose, we used a panel of MCF7 cell lines that were stably-transfected with different luciferase reporters, that allows one to quantitatively measure the activation-state of a given signal transduction pathway. Figure 7: Graphene oxide (GO) inhibits signaling pathways related to cancer stem cells, antioxidant responses and interferon. To further validate the idea GO was reducing stemness in MCF7-derived CSCs, we used a series of well-established breast cancer stem cell markers (CD44 and CD24), and quantitatively analyzed their expression by FACS analysis. Here, we show that treatment with GO is sufficient to inhibit tumor-sphere formation in six independent cancer cell lines, across multiple tumor types (breast, ovarian, prostate, lung, and pancreatic cancer, as well glioblastoma (brain cancer)). Importantly, our preliminary results indicate that GO treatment does not significantly affect oxidative mitochondrial metabolism (OXPHOS) in this context (data not shown), suggesting that GO does not target mitochondria. Also, since b-GO flakes are 5-to-20µm in size, they must be exerting their effects at the cell surface, as they are too large to be internalized within cells and are actually larger than a single cell. Interestingly, several studies have shown that GO is non-toxic for normal stem cells, and indeed GO promotes their differentiation.
Our new mechanistic studies suggest that GO could directly be used as a therapeutic for targeting CSCs, possibly as a differentiation agent.
A single cell suspension was prepared using enzymatic (1x Trypsin-EDTA, Sigma Aldrich, #T3924), and manual disaggregation (25 gauge needle) to create a single cell suspension. The Cignal Lenti reporter assay (luc) system (Qiagen) was chosen for monitoring the activity of several signal transduction pathways in MCF7 cells. The Luciferase Assay System (E1501, Promega Kit) was used on all luciferase reporter MCF7 cells treated with GO. Following GO treatment, the CSC population was enriched by seeding on low-attachment plates.
We thank the University of Manchester for providing start-up funds that contributed to the success of this study (to Federica Sotgia and Michael P. Although the death rate of breast cancer has decreased with advances in prevention, surgical resection and adjuvant therapies, there are still approximately 232,340 new cases and 39,620 deaths of breast cancer in the United States in 2013 [1]. LIF promotes cell proliferation and anchorage-independent growth in soft agar of breast cancer cells in vitro, and the growth of xenograft breast tumors in vivo. Whereas the majority of these cells express LIF receptors (LIFR and gp130) at relatively similar levels (Supplementary Fig.
Tail vein injection of MCF7, T47D and MDA-MB-231 cells can all lead to the formation of lung metastatic tumors in mice.
Ectopic LIF expression promoted the proliferation of MCF-7, T47D and MDA-MB-231 cells, whereas knockdown of endogenous LIF significantly inhibited the growth of MDA-MB-231 cells (Fig.
Rapamycin treatment largely blocked the promoting effect of both exogenous LIF and ectopically expressed LIF in cells on invasion and migration in MCF-7, T47D and MDA-MB-231 cells (Fig.
The expression levels of LIF were determined in the tissue microarray containing the breast tumor tissues from these patients by using IHC staining. LIF plays an essential role in embryonic implantation and maintaining pluripotentiality of murine stem cells. LIF promotes proliferation, anchorage-independent growth in soft agar of breast cancer cells, and the growth rate of xenograft breast tumors.
During the preparation of this paper, a very recent study reported that exogenous LIF activates the mTOR pathway in nasopharyngeal carcinoma cell lines, which is consistent with our findings [37].
Our results show that higher LIF expression is associated with poorer relapse free survival of breast cancer patients, indicating that LIF could be an important prognostic marker for breast cancer patients.
Cells with stable LIF overexpression were established by transduction of a retroviral LIF expression vector (pLPCX-LIF) and selected by puromycin. LIF fragment was inserted into p3XFlag-CMV-14 vectors, then subcloned into pLPCX vectors along with the flag tag. In brief, cells in serum-free medium were seeded into upper chambers coated with or without matrigel (BD Biosciences) for invasion and migration assays, respectively.
For xenograft tumorigencity assays of T47D and MCF-7 cells, a 17β-estradiol pellet (Innovative Research of America) was implanted into each mouse 2 days before cell injection. The de-identified clinical information of these patients was obtained from the patient database that has been previously described [42, 43]. In brief, tissue sections were deparaffinized and antigen retrieval was achieved by Target Retrieval Solution (DAKO).
The relationship between LIF and clinicopathological variables was analyzed by standard Chi-square test. Slaets H, Dumont D, Vanderlocht J, Noben JP, Leprince P, Robben J, Hendriks J, Stinissen P and Hellings N. Wysoczynski M, Miekus K, Jankowski K, Wanzeck J, Bertolone S, Janowska-Wieczorek A, Ratajczak J and Ratajczak MZ. Freudenberg JM, Ghosh S, Lackford BL, Yellaboina S, Zheng X, Li R, Cuddapah S, Wade PA, Hu G and Jothi R.
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Zhou X, Tan M, Stone Hawthorne V, Klos KS, Lan KH, Yang Y, Yang W, Smith TL, Shi D and Yu D.
Ohbayashi N, Ikeda O, Taira N, Yamamoto Y, Muromoto R, Sekine Y, Sugiyama K, Honjoh T and Matsuda T. Feng Z, Zhang C, Kang HJ, Sun Y, Wang H, Naqvi A, Frank AK, Rosenwaks Z, Murphy ME, Levine AJ and Hu W.
Wander SA, Zhao D, Besser AH, Hong F, Wei J, Ince TA, Milikowski C, Bishopric NH, Minn AJ, Creighton CJ and Slingerland JM.
Gulhati P, Bowen KA, Liu J, Stevens PD, Rychahou PG, Chen M, Lee EY, Weiss HL, O’Connor KL, Gao T and Evers BM.
Zhang C, Liu J, Liang Y, Wu R, Zhao Y, Hong X, Lin M, Yu H, Liu L, Levine AJ, Hu W and Feng Z. Haffty BG, Yang Q, Reiss M, Kearney T, Higgins SA, Weidhaas J, Harris L, Hait W and Toppmeyer D.
The HER proteins are receptor tyrosine kinases which consist of an extracellular domain, an α-helical transmembrane region and an intracellular tyrosine kinase domain [2]. It has been shown to increase survival in HER2 positive metastatic and early breast cancer patients, especially when given in combination with chemotherapy [10, 11]. Other proposed mechanisms include the constitutive activation of the autophagy-related gene 12 (ATG12) [20], the presence of trastuzumab-refractory breast cancer stem cells (CSCs) [21], and the steric hindrance caused by association of HER2 with other cell surface signaling proteins, i.e. Neratinib, is an irreversible inhibitor of EGFR, HER2 and HER4 tyrosine kinase activity [26] shown to have promising preclinical activity against HER2-overexpressing cell lines [27].
The reactivation of pERK was not seen in BT474 cells, which are more sensitive than SKBR3 cells (Figure 1B). A) Trastuzumab resistant SKBR3 (SKBR3R, left) or BT474R (right) cells were treated for 1 hour or 1 day with increasing doses of neratinib.
In the MDA-MB-361 and EFM-192-A cell lines, which are moderately sensitive to trastuzumab, the combination of neratinib and trastuzumab showed greater inhibition of growth than either of the drugs alone (Figure 4A).
Sensitivity to trastuzumab and neratinib in A) two trastuzumab moderately sensitive and B) two innately trastuzumab resistant HER2 positive breast cancer cell lines. In all cell lines tested, neratinib significantly reduced phosphorylation of HER2, although the level of reduction did not correlate with sensitivity to neratinib (Figure 5C). A) Scatter plots showing the relationship between the levels of both HER2 protein and pHER2 (determined by western blotting) and neratinib sensitivity (IC50). In contrast to HER2, staining for pHER3 was weak but the lowest IRS scoring was seen in the combination arm (Supplementary Figure 3B). A) Left, Mice bearing BT474 xenograft tumors were treated with either control (vehicle), neratinib, trastuzumab or their combination for 16 days. In the trastuzumab-naïve SKBR3 and BT474 cells, acute neratinib treatment inhibited phosphorylation of EGFR, HER2, HER3 and HER4 as well as downstream pathways ERK and Akt, reflecting its immediate inhibitory effect on the tyrosine kinase activity of all the HER receptors. Our data revealed that the withdrawal of trastuzumab from the trastuzumab-resistant cell lines resulted in a significantly increased cell count compared to continuation of trastuzumab treatment. Neratinib and trastuzumab in combination with paclitaxel chemotherapy showed clinical benefit in patients who had been heavily pre-treated with anti-HER2 agents and chemotherapy, with a mean of 4 prior regimens [38]. However, some of the HER2 amplified cells listed in Supplementary Table1, including MDA-MB-361 and MDA-MB-453, have been classified as IHC 2+ or HER2 negative [43-46], as they express slightly lower levels of HER2 protein, at an expression level that is nearer to the original FDA definition of HER2 positivity [47]. Trastuzumab-resistant BT474 and SKBR3 cell lines were generated as previously described [19].
Trastuzumab was purchased from St Vincent’s University Hospital and Oxford University Hospitals NHS Trust.
After 20 min incubation on ice, lysate was passed through a 21-gauge needle and centrifuged at 10,000 g for 5 min at 4ºC.
Proteins were transferred to Hybond enhanced chemiluminescence nitrocellulose membrane (Amersham, Biosciences, Buckinghamshire, UK).
Anthony Kong and his lab members are supported by Breakthrough Breast Cancer through Holbeck Charitable trust. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A, et al.
Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, Gianni L, Baselga J, Bell R, Jackisch C, Cameron D, Dowsett M, Barrios CH, Steger G, Huang CS, Andersson M et al.
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Cufi S, Vazquez-Martin A, Oliveras-Ferraros C, Corominas-Faja B, Urruticoechea A, Martin-Castillo B, Menendez JA. Cufi S, Corominas-Faja B, Vazquez-Martin A, Oliveras-Ferraros C, Dorca J, Bosch-Barrera J, Martin-Castillo B, Menendez JA. Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T, Jagiello-Gruszfeld A, Crown J, Chan A, Kaufman B, Skarlos D, Campone M, Davidson N, Berger M, Oliva C, Rubin SD et al. Baselga J, Bradbury I, Eidtmann H, Di Cosimo S, de Azambuja E, Aura C, Gomez H, Dinh P, Fauria K, Van Dooren V, Aktan G, Goldhirsch A, Chang TW, Horvath Z, Coccia-Portugal M, Domont J et al. Untch M, Loibl S, Bischoff J, Eidtmann H, Kaufmann M, Blohmer JU, Hilfrich J, Strumberg D, Fasching PA, Kreienberg R, Tesch H, Hanusch C, Gerber B, Rezai M, Jackisch C, Huober J et al. Rabindran SK, Discafani CM, Rosfjord EC, Baxter M, Floyd MB, Golas J, Hallett WA, Johnson BD, Nilakantan R, Overbeek E, Reich MF, Shen R, Shi X, Tsou HR, Wang YF, Wissner A. Kramer-Marek G, Gijsen M, Kiesewetter DO, Bennett R, Roxanis I, Zielinski R, Kong A, Capala J. Montemurro F, Faggiuolo R, Redana S, Donadio M, Minischetti M, Durando A, Vietti-Ramus G, Buosi R, Aglietta M. Blackwell KL, Burstein HJ, Storniolo AM, Rugo H, Sledge G, Koehler M, Ellis C, Casey M, Vukelja S, Bischoff J, Baselga J, O’Shaughnessy J.
Blackwell KL, Burstein HJ, Storniolo AM, Rugo HS, Sledge G, Aktan G, Ellis C, Florance A, Vukelja S, Bischoff J, Baselga J, O’Shaughnessy J. Awada A, Dirix L, Manso Sanchez L, Xu B, Luu T, Dieras V, Hershman DL, Agrapart V, Ananthakrishnan R, Staroslawska E. Lin NU, Winer EP, Wheatley D, Carey LA, Houston S, Mendelson D, Munster P, Frakes L, Kelly S, Garcia AA, Cleator S, Uttenreuther-Fischer M, Jones H, Wind S, Vinisko R, Hickish T.
Juranic ZD, Borojevic N, Jovanovic D, Stanojevic-Bakic N, Zizak Z, Neskovic-Konstantinovic Z, Saric M, Stanojkovic T, Raonic T, Milosevic D. Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, Dowsett M, Fitzgibbons PL, Hanna WM, Langer A, McShane LM, Paik S, Pegram MD, Perez EA, Press MF, Rhodes A et al. Bose R, Kavuri SM, Searleman AC, Shen W, Shen D, Koboldt DC, Monsey J, Goel N, Aronson AB, Li S, Ma CX, Ding L, Mardis ER, Ellis MJ.
Kalous O, Conklin D, Desai AJ, O’Brien NA, Ginther C, Anderson L, Cohen DJ, Britten CD, Taylor I, Christensen JG, Slamon DJ, Finn RS. First, apoptosis is a form of programmed cell death that involves cell rounding, DNA fragmentation, and cell membrane blebbing. For instance, deoxyribonucleotide triphosphates (dNTP) are required building blocks for cells to carry out the basic functions of DNA synthesis and repair. COH29 is an aromatically substituted thiazole compound [N-(4-(3,4-dihyrophenyl)-5-phenylthiazol-2-yl) -3,4-dihydroxybenzamide], which we developed from a virtual screen of the National Cancer Institute (NCI) Diversity Set with 2000 compounds (NCI2000). In that report, reduced RRM2 abundance was associated with, increased autophagy and a decreased intracellular dNTP pool.
Consequently, we combined TMX, an autophagy inducer, with COH29 in ERα-negative (ER-) breast cancer cells and measured the resulting cytotoxicity [32, 33]. To test this possibility, MDA-MB-231 cells were maintained with the increasing concentrations of COH29 -in the presence or absence of TMX for 72 h.
Figure 2A reveals that MDA-MB-231 cells were more sensitive to treatment than their isogenic autophagy-deficient (ATG5-knockdown) counterparts, suggesting that the combinatorial effect of TMX and COH29 was autophagy-dependent (Figure 2A).
The percentage of anti-RRM2 positive cells per 500 cells in 2 tumor samples was enumerated.
As shown in Figure 4A, one striking feature was that COH29 treatment induced nuclear DNA leakage, as identified by DAPI-positive particles outside the nucleus, and the leaked DNA puncta were notably larger than micronuclei [36]. MDA-MB-231 cells were treated with TMX, COH or the combination for the indicated time periods and stained with DAPI A., anti-lamin B.
Serving a control, vehicle-treated MDA-MB-231 cells showed an abnormal female karyotype with hyperdiploid and near tetraploid populations.
MDA-MB-231 cells were treated with TMX, COH29 for 48 h or the combination for 24 and 48 h and subjected to cytogenetic analysis. The use of RR inhibitors as anti-cancer agents has been reviewed by Shao and colleagues [11, 41].
RR is a unique enzyme that utilizes free radicals to drive catalysis in a mechanism that involves components in both the RRM1 and RRM2 subunits [45, 46]. In this report, we have demonstrated that the ER-independent effect of TMX, autophagy induction, sensitizes ERα-negative MDA-MB-231 cells to COH29, via RRM2 reduction.
A previous report demonstrated that knockdown RRM2 and the addition of cisplatin induces inter- and intra-strand DNA crosslinks [60]. Why does autophagy promote tumor cell survival in some circumstances and death in other circumstances? The anti-Ki67 antibody (ab15580) was from Abcam, and the anti-p53R2 antibody (600-401-B67) was from Rockland. Therefor, ACP assays, which measures cellular acid phosphatase activity, were used to avoid the effects of mitochondria dysfunction. The Qproteome Mammalian Protein Prep Kit (Qiagen) was used to extract protein from harvested tumors.
Alternatively, 50 mg of tumor samples were added to 100 µl of 15% trichloroacetic acid. After washing with PBS, the cover slips were mounted over a microscope slide in Prolong anti-fade reagent that contained DAPI (Life Technologies, P-36931), and examined using a Olympus AX70 upright microscope (Olympus). Briefly, after de-paraffinization, endogenous peroxidase activity was blocked by pre-treatment with 3% H2O2.
Each cell biology experiment was performed in triplicate to obtain representative means and images. Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, Blagosklonny MV, El-Deiry WS, Golstein P, Green DR, Hengartner M, Knight RA, Kumar S, Lipton SA, Malorni W, Nunez G, et al. Qiu F, Chen YR, Liu X, Chu CY, Shen LJ, Xu J, Gaur S, Forman HJ, Zhang H, Zheng S, Yen Y, Huang J, Kung HJ and Ann DK. Nilsson R, Jain M, Madhusudhan N, Sheppard NG, Strittmatter L, Kampf C, Huang J, Asplund A and Mootha VK. Zhou B, Su L, Hu S, Hu W, Yip ML, Wu J, Gaur S, Smith DL, Yuan YC, Synold TW, Horne D and Yen Y.
Theriault RL, Carlson RW, Allred C, Anderson BO, Burstein HJ, Edge SB, Farrar WB, Forero A, Giordano SH, Goldstein LJ, Gradishar WJ, Hayes DF, Hudis CA, Isakoff SJ, Ljung BM, Mankoff DA, et al.
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Robert T, Vanoli F, Chiolo I, Shubassi G, Bernstein KA, Rothstein R, Botrugno OA, Parazzoli D, Oldani A, Minucci S and Foiani M. Chen MC, Zhou B, Zhang K, Yuan YC, Un F, Hu S, Chou CM, Chen CH, Wu J, Wang Y, Liu X, Smith L, Warden CD, Liu Z, Li H, Su L, et al. Gojo I, Tidwell ML, Greer J, Takebe N, Seiter K, Pochron MF, Johnson B, Sznol M and Karp JE. Lagadec C, Adriaenssens E, Toillon RA, Chopin V, Romon R, Van Coppenolle F, Hondermarck H and Le Bourhis X. D’Angiolella V, Donato V, Forrester FM, Jeong YT, Pellacani C, Kudo Y, Saraf A, Florens L, Washburn MP and Pagano M. Verre4, Maria Iliut4, Maria Peiris-Pagés1,2, Bela Ozsvari1,2, Ricardo Gandara1,2, Anna Rita Cappello3, Federica Sotgia1,2, Aravind Vijayaraghavan4 and Michael P.
As a consequence, they have been directly implicated in the disease pathogenesis of tumor recurrence and distant metastasis [4, 5]. A particular distinguishing characteristic of CSCs is their ability to initiate tumors and to undergo anchorage-independent growth, when cultured in suspension [11].
Graphene, described as two-dimensional sheets of carbon atoms without any additional functional groups, does not forms stable dispersions in water or other biologically relevant solvents [17].
Left and Right panels show atomic force microscopy images of graphene oxide on a silicon dioxide substrate indicating the flake size distribution and monolayer thickness of the flakes. We next evaluated whether GO also showed efficacy against CSCs from multiple cancer types, such as ovarian, prostate, pancreatic and lung cancers, as well as glioblastoma (brain) (the six cell lines tested are summarized in Table 1).
Thus, our results indicate that GO must be targeting a relatively specific and highly-conserved phenotypic property of CSCs, across multiple cancer types.
GO (big flakes) inhibits the anchorage-independent proliferation of SKOV3 ovarian cancer cells (A), U87 glioblastoma cells (B), PC3 prostate cancer cells (C), A549 lung cancer cells (D), as well as pancreatic cancer cells (E), in a concentration-independent manner.
Interestingly, Figure 7 shows that a number of signaling pathways were significantly inhibited by GO treatment. Then, cells were trypsinized and plated on low-attachment plates for 10 hours to induce anoikis and enrich for CSCs. This suggests that GO inhibits mammosphere formation by promoting the differentiation of breast cancer stem cells, supporting our results from the analysis of multiple signal transduction pathways.
These results suggest that GO specifically targets a global phenotypic property of CSCs that is highly conserved in multiple tumor types. This is in contrast to our previous studies where a number of mitochondrially-targeted FDA-approved antibiotics effectively eradiated CSCs [24]. This is consistent with our findings that GO-treatment dampens the activation of several stem cell associated signal transduction pathways, which are initiated at the cell surface.
More specifically, it was demonstrated that culturing normal pluripotent stem cells on GO as a substrate induces their terminal differentiation towards multiple cell lineages, including neurons, chondrocytes and adipocytes [29-33].
Our current mechanistic studies suggest that GO could directly be used as a therapeutic for targeting CSCs, because of its ability to induce differentiation.
The individual graphite oxide flakes contain carboxyl groups mainly at the edges, and epoxide, hydroxide and ketone groups mainly on the basal plane. The responsive luciferase constructs encode the firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of response elements for each pathway. Under these conditions, the non-CSCs undergo anoikis (a form of apoptosis induced by lack of proper attachment) and CSCs are believed to survive.
Ruiz ON, Fernando KA, Wang B, Brown NA, Luo PG, McNamara ND, Vangsness M, Sun YP and Bunker CE.
Lamb R, Ozsvari B, Lisanti CL, Tanowitz HB, Howell A, Martinez-Outschoorn UE, Sotgia F and Lisanti MP.
Bressan E, Ferroni L, Gardin C, Sbricoli L, Gobbato L, Ludovichetti F, Tocco I, Carraro A, Piattelli A and Zavan B. Marcano DC, Kosynkin DV, Berlin JM, Sinitskii A, Sun ZZ, Slesarev A, Alemany LB, Lu W and Tour JM. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractScreening mammograms is a repetitive task that causes fatigue and eye strain since for every thousand cases analyzed by a radiologist, only 3–4 are cancerous and thus an abnormality may be overlooked. Metastasis to vital organs such as lung, brain and bone is a major cause of death from breast cancer [2]. While the overexpression of LIF has been observed in several types of cancers including breast cancer [8, 11-15], the role of LIF in cancer is not well-understood. LIF also promotes invasion and migration of breast cancer cells in vitro and metastasis of breast cancer cells in vivo. 1a&b), the expression levels of LIF varied dramatically among these cell lines, which are correlated with the metastatic abilities of these cell lines (Fig.
The levels of total and phosphorylated AKT at Ser 473 (p-AKT-S473) were determined by Western-blot assays. 6a & b, wortmannin treatment largely blocked the promoting effect of both exogenous LIF and ectopically expressed LIF on invasion and migration in MCF-7, T47D and MDA-MB-231 cells.
Recently, we identified that LIF is a novel p53 target gene, and importantly, LIF mediates p53’s role in embryonic implantation [29-31].
LIF also promotes metastasis of breast cancer cells as determined by in vitro trans-well and in vivo lung and distant metastatic assays. It will be interesting to investigate whether LIF also activates the mTOR pathway through AKT in nasopharyngeal cancer, whether the activation of LIF-AKT-mTOR pathway exists in other types of tumors, and plays an important role in tumorigenesis in future studies.
Results from this study have the direct potential to develop LIF as an important biomarker for prognosis of breast cancer and a therapeutic target for breast cancer, especially for those with LIF overexpression. To knockdown endogenous LIF expression in cells, two validated shRNA vectors against LIF (SHCLNDNM_002309, Sigma) were transduced into cells and selected by puromycin.
The lower chamber was filled with 1:1 mix of medium supplemented with 10% FBS and NIH 3T3 cell-conditioned medium.
The numbers of lung and distant metastatic tumors were counted under a dissecting microscope and confirmed by histopathological analysis. In brief, none of the patients in this study received chemotherapy or irradiation therapy prior to the surgery. The Kaplan–Meier analysis was used to assess the relationship between LIF and the survival of patients. The leukemia inhibitory factor receptor (LIFR) gene is located within a cluster of cytokine receptor loci on mouse chromosome 15 and human chromosome 5p12-p13.
Leukemia inhibitory factor induces an antiapoptotic response in oligodendrocytes through Akt-phosphorylation and up-regulation of 14-3-3.
Leukemia inhibitory factor involvement in human ulcerative colitis and its potential role in malignant course. Cutaneous leukemia inhibitory factor and its potential role in the development of skin tumors. Leukemia inhibitory factor functions as a growth factor in pancreas carcinoma cells: Involvement of regulation of LIF and its receptor expression. OSM, LIF, its receptors, and its relationship with the malignance in human breast carcinoma (in situ and in infiltrative). Leukemia inhibitory factor triggers activation of signal transducer and activator of transcription 3, proliferation, invasiveness, and altered protease expression in choriocarcinoma cells.
Leukemia inhibitory factor increases the invasiveness of trophoblastic cells through integrated increase in the expression of adhesion molecules and pappalysin 1 with a concomitant decrease in the expression of tissue inhibitor of matrix metalloproteinases. Molecular cloning and expression of cDNA encoding a murine myeloid leukaemia inhibitory factor (LIF).
Leukemia inhibitory factor downregulates human papillomavirus-16 oncogene expression and inhibits the proliferation of cervical carcinoma cells. Recombinant leukemia inhibitory factor suppresses human medullary thyroid carcinoma cell line xenografts in mice.
Identification of gene expression profiles that predict the aggressive behavior of breast cancer cells. Role of mTOR in solid tumor systems: a therapeutical target against primary tumor growth, metastases, and angiogenesis. Leukemia inhibitory factor promotes nasopharyngeal carcinoma progression and radioresistance.
Spliced MDM2 isoforms promote mutant p53 accumulation and gain-of-function in tumorigenesis. Bcl-2 expression predicts local relapse for early-stage breast cancer receiving conserving surgery and radiotherapy. Locoregional relapse and distant metastasis in conservatively managed triple negative early-stage breast cancer.
Gene Amplifications in Well-Differentiated Pancreatic Neuroendocrine Tumors Inactivate the p53 Pathway.


Trastuzumab is thought to exert its anti-tumor activity partly by accelerating the internalization and degradation of HER2 and thus blocking downstream signaling. In patients who were resistant to trastuzumab, the combination of lapatinib with capecitabine increased median time to progression compared to capecitabine alone [23]. The results suggest that neratinib effectively inhibits HER2 activation and downstream signaling in HER2 positive breast cell lines. To understand the enhanced response to the combination, a time-course experiment was performed to assess the effect of the combination on HER receptor activation and downstream signaling. However in the trastuzumab-resistant cell lines (JIMT1, MDA-MB-453, HCC1419, HCC1954, UACC732) the combination treatment showed no enhancement compared to neratinib alone (Figure 4B and Supplementary Figure 2). Cells were treated with neratinib alone, trastuzumab alone or the combination at a fixed ratio for 5 days. Neratinib sensitivity was not significantly associated with p95-HER2, EGFR, HER3, Akt, ERK, PTEN, PI3K mutation status or ER status (Supplementary Table 2). B) The effects of neratinib on expression and phosphorylation of HER2, Akt and ERK in HER2 positive breast cancer cell lines. In contrast, trastuzumab did not decrease phosphorylation of EGFR, HER2, HER4 and ERK, reflecting the different mechanisms of action of the drugs. Furthermore, the combination of trastuzumab and neratinib was significantly more effective than neratinib alone even in the presence of trastuzumab resistance. A phase II multi-centre randomized study of neratinib in combination with weekly paclitaxel with or without trastuzumab followed by doxorubicin and cyclophosphamide (AC) as neoadjuvant therapy for women with HER2-positive locally advanced breast cancer (NSABP FB-7) is currently ongoing. We showed that although neratinib effectively inhibited phosphorylation of all HER receptors for up to 24 hours, reactivation of HER3 and Akt occurred within 3 days.
Since HER2 testing results may be variable according to the methods or assays of testing [48], it is not surprising that some of these cells have been classified differently.
In addition, neratinib but not lapatinib, has been shown to be active against HER2 somatic mutations present in about 1-3% of HER2 negative breast cancer patients [51]. The reduction in pAkt and pERK in response to neratinib treatment was also correlated with sensitivity to neratinib. The challenge will be to identify predictive markers to select those patients with HER2 positive breast cancer who may benefit from neratinib treatment but not from trastuzumab or lapatinib. In addition to SKBR3 and BT474, the following panel of breast cancer cell lines was used in this study: EFM192A, HCC1419, MDA-MB-361, MDA-MB-453, SUM190, SUM225, UACC732, UACC812, UACC893, CAMA1, EFM19, KPL1, MCF7, MDA-MB-134, MDA-MB-175, MDA-MB-415, T47D, ZR751, HCC1569, HCC1954, BT20, BT549, CAL51, HCC38, HCC70, HCC1143, HCC1187, HCC1395, HCC1806, HCC1937, JIMT1, MDA-MB157, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDA-MB-436. On the concluding day of the experiments, the cells were washed with phosphate buffered saline (PBS) before trypsinization and counted using ISOTON solution on the Coulter Z2 particle counter (Beckham Coulter, Inc). When tumors reached an average size of 125 mm3, the mice were divided into 4 groups, keeping average tumor size similar between groups. ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. A central role for HER3 in HER2-amplified breast cancer: implications for targeted therapy.
Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer.
PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer. Met receptor contributes to trastuzumab resistance of Her2-overexpressing breast cancer cells. HER2 phosphorylation is maintained by a PKB negative feedback loop in response to anti-HER2 herceptin in breast cancer. Autophagy-related gene 12 (ATG12) is a novel determinant of primary resistance to HER2-targeted therapies: utility of transcriptome analysis of the autophagy interactome to guide breast cancer treatment. Lapatinib with trastuzumab for HER2-positive early breast cancer (NeoALTTO): a randomised, open-label, multicentre, phase 3 trial. Lapatinib versus trastuzumab in combination with neoadjuvant anthracycline-taxane-based chemotherapy (GeparQuinto, GBG 44): a randomised phase 3 trial.
Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase. Association between patient and general practice characteristics and unplanned first-time admissions for cancer: observational study. Potential of PET to predict the response to trastuzumab treatment in an ErbB2-positive human xenograft tumor model. Randomized study of Lapatinib alone or in combination with trastuzumab in women with ErbB2-positive, trastuzumab-refractory metastatic breast cancer. Overall survival benefit with lapatinib in combination with trastuzumab for patients with human epidermal growth factor receptor 2-positive metastatic breast cancer: final results from the EGF104900 Study.
Safety and efficacy of neratinib (HKI-272) plus vinorelbine in the treatment of patients with ErbB2-positive metastatic breast cancer pretreated with anti-HER2 therapy. A phase I dose-escalation study evaluating weekly paclitaxel with neratinib and trastuzumab in women with metastatic HER2-positive breast cancer, NSABP FB-8.
A phase II study of afatinib (BIBW 2992), an irreversible ErbB family blocker, in patients with HER2-positive metastatic breast cancer progressing after trastuzumab.
HER2 oncogenic function escapes EGFR tyrosine kinase inhibitors via activation of alternative HER receptors in breast cancer cells. Dual blockade of HER2 in HER2-overexpressing tumor cells does not completely eliminate HER3 function. Update on the molecular profile of the MDA-MB-453 cell line as a model for apocrine breast carcinoma studies.
Quantitative assays for the measurement of HER1-HER2 heterodimerization and phosphorylation in cell lines and breast tumors: applications for diagnostics and targeted drug mechanism of action.
Gamma-heregulin: a novel heregulin isoform that is an autocrine growth factor for the human breast cancer cell line, MDA-MB-175.
Dacomitinib (PF-00299804), an Irreversible Pan-HER Inhibitor, Inhibits Proliferation of HER2-Amplified Breast Cancer Cell Lines Resistant to Trastuzumab and Lapatinib.
Protein-tyrosine phosphatase PTPN9 negatively regulates ErbB2 and epidermal growth factor receptor signaling in breast cancer cells. Subsequent engulfment of apoptotic cells by phagocytes prevents an inflammatory response to the dead cells.
Thus, imbalanced levels of dNTPs result in mutagenesis, genomic instability and cell death [8]. We screened for the ability to bind a pocket on the surface of the M2 subunit of RR, as predicted by protein structural analysis [12]. As a result, TMX is primarily used to reduce the recurrence of ERα-positive breast tumors [16]. On the other hand, overexpression of RRM2 or supplementation of exogenous deoxyribonucleotide monophosphates (dNMPs) to increase the intracellular dNTP levels attenuated autophagy [30]. Cell viability was determined using acid phosphatase (ACP) assays at 72 h post-treatment (Figure 1D). The viability data was further analyzed by Compusyn software to determine whether the drugs showed synergistic effects. Tumors were harvested at day 17 post-treatment and evaluated using hematoxylin and eosin (H&E) staining. Figure 3D reveals that the nuclear RRM2 abundance was slightly reduced in the TMX-only group, unchanged in the COH29-only group, and markedly decreased in the group treated with TMX+COH29.
The cells were characterized by structural abnormalities involving chromosomes X, 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 13, 15, 16, 18, and 21. Because rapidly dividing tumor cells have an increased need for dNTPs, they are far more sensitive to the cytotoxic effects of RR inhibition than normal cells.
The RRM1 subunit contains the active site and substrate specificity sites, whereas the RRM2 subunit contains the oxygen-linked diferric iron center and tyrosyl radical.
Studies on the combination of TMX with other agents, such as TRAIL [50] and rapamycin [51], have shown synergistic apoptotic activity. Also, a recent report from us showed that loss of RRM2b causes chromosomal instability and chromatid breakage in mice [61]. One possible explanation relates to what autophagy is degrading in each specific circumstance. The protein concentrations were determined using a Bio-Rad Protein Assay Kit (Bio-Rad, 500-0001). The solution was mixed and homogenized using a TissueLyser (Qiagen) for 1 min then kept on ice for 10 min. The slides were incubated with normal goat serum for 20 min at RT to block non-specific signal, then incubated with the primary antibody for 20 min at RT. Tina Patel of the Light Microscopy Digital Imaging Core at City of Hope for confocal and fluorescent microscopy analyses, Ms. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Arginine starvation impairs mitochondrial respiratory function in ASS1-deficient breast cancer cells. Arginine starvation-associated atypical cellular death involves mitochondrial dysfunction, nuclear DNA leakage, and chromatin autophagy. Metabolic enzyme expression highlights a key role for MTHFD2 and the mitochondrial folate pathway in cancer.
A small-molecule blocking ribonucleotide reductase holoenzyme formation inhibits cancer cell growth and overcomes drug resistance. Tamoxifen in early-stage estrogen receptor-positive breast cancer: overview of clinical use and molecular biomarkers for patient selection. Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials. Estrogen receptor-dependent and estrogen receptor-independent pathways for tamoxifen and 4-hydroxytamoxifen-induced programmed cell death.
Twenty-year follow-up of the Royal Marsden randomized, double-blinded tamoxifen breast cancer prevention trial.
Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy.
High-throughput ectopic expression screen for tamoxifen resistance identifies an atypical kinase that blocks autophagy. Autophagy is required for maintenance of amino acid levels and protein synthesis under nitrogen starvation. Radiation-induced autophagy is associated with LC3 and its inhibition sensitizes malignant glioma cells.
DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells. Prognostic and therapeutic significance of ribonucleotide reductase small subunit M2 in estrogen-negative breast cancers.
Induction of autophagy and cell death by tamoxifen in cultured retinal pigment epithelial and photoreceptor cells. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.
Standardization of counting micronuclei: definition of a protocol to measure genotoxic damage in human exfoliated cells.
Ribonucleotide reductase M2 subunit is a novel diagnostic marker and a potential therapeutic target in bladder cancer.
Expression of ribonucleotide reductase M2 subunit in gastric cancer and effects of RRM2 inhibition in vitro. Radical initiation in the class I ribonucleotide reductase: long-range proton-coupled electron transfer?
Structure and interactions of amino acid radicals in class I ribonucleotide reductase studied by ENDOR and high-field EPR spectroscopy. A phase I trial of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone in combination with gemcitabine for patients with advanced cancer. Phase I and pharmacokinetic study of Triapine, a potent ribonucleotide reductase inhibitor, in adults with advanced hematologic malignancies. Inhibition of mTOR activity restores tamoxifen response in breast cancer cells with aberrant Akt Activity. TNF-related apoptosis inducing ligand (TRAIL) and its receptors in tumor surveillance and cancer therapy.
Cyclin F-mediated degradation of ribonucleotide reductase M2 controls genome integrity and DNA repair. Lisanti1,2 1 The Manchester Centre for Cellular Metabolism (MCCM), Institute of Cancer Sciences, University of Manchester, UK 2 The Breakthrough Breast Cancer Research Unit, Institute of Cancer Sciences, University of Manchester, UK 3 The Department of Pharmacy, Health and Nutritional Sciences, The University of Calabria, Italy 4 School of Materials and National Graphene Institute, University of Manchester, UK Correspondence: Michael P. In addition, drug-resistant CSCs have been linked to unfavorable clinical outcomes, across different tumor types [6-8]. As such, our current study provides a new rationale for exploiting graphene oxide itself as an anti-cancer therapeutic, rather than simply as a drug-delivery agent [16]. On the other hand, graphene oxide [18] is the water-soluble derivative of graphene which can be produced with various sizes and bearing varied functional groups and which can be more easily manipulated experimentally, especially in biological systems. For this purpose, we assessed the effects of graphene oxide on the anchorage-independent clonal expansion of MCF7 CSCs, using the tumor-sphere assay. Note that GO (big and small flakes) inhibits the anchorage-independent proliferation of MCF7 CSCs, as evidenced by inhibition of mammosphere formation. Note that GO (big flakes) does not affect cell viability of the total cell population of SKOV3 ovarian cancer cells (A), U87 glioblastoma cells (B), PC3 prostate cancer cells (C), A540 lung cancer cells (D) as well as MIA-PaCa-2 pancreatic cancer cells. Note that GO (big flakes) does not affect cell viability of the total cell population of normal fibroblasts. MCF7-Luc reporter cells were treated with GO (big flakes) for 48 hours and luminescence was determined as a measure of pathway activation status. Then, cells were trypsinized and plated on low-attachment plates for 10 hours to induce anoikis and enrich for cancer stem cells. Thus, GO and mitochondrially-targeted antibiotics appear to work differently, via separate and distinct molecular mechanism(s). This could then mechanistically induce CSC differentiation, which we observed experimentally (summarized in Figure 9).
These properties are currently being actively exploited for tissue engineering and regenerative medicine, by using GO as a scaffold for bone reconstruction and neural regeneration. The C to O ratio is usually slightly lower or slightly higher than 1 as determined by X-ray photoemission spectroscopy. Multi-drug-resistant cells enriched from chronic myeloid leukemia cells by Doxorubicin possess tumor-initiating-cell properties. Cancer epigenetics: tumor heterogeneity, plasticity of stem-like states, and drug resistance.
Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy.
Measurement of Multicomponent Solubility Parameters for Graphene Facilitates Solvent Discovery.
Antibiotics that target mitochondria effectively eradicate cancer stem cells, across multiple tumor types: Treating cancer like an infectious disease. Energy metabolism analysis reveals the mechanism of inhibition of breast cancer cell metastasis by PEG-modified graphene oxide nanosheets.
The inhibition of migration and invasion of cancer cells by graphene via the impairment of mitochondrial respiration.
Graphene oxide as a chemosensitizer: diverted autophagic flux, enhanced nuclear import, elevated necrosis and improved antitumor effects. Graphene oxide promotes the differentiation of mouse embryonic stem cells to dopamine neurons. Dual Roles of Graphene Oxide in Chondrogenic Differentiation of Adult Stem Cells: Cell-Adhesion Substrate and Growth Factor-Delivery Carrier. Computer-aided detection (CAD) algorithms were developed to assist radiologists in detecting mammographic lesions. Patients and methods: A cross-sectional study, embedded within a pilot for an epidemiologic study, was undertaken involving women who had undergone surgery for unilateral stage I or II breast cancer. Therefore, there is an urgent need to further understand the molecular mechanisms underlying breast cancer tumorigenesis and metastasis.
Limited studies suggested the potential complex role of LIF in cancer depending upon the types of the cancer. To investigate whether autocrine secretion of LIF from cells has a similar promoting effect on invasion and migration of cells, MCF7, T47D and MDA-MB-231 cells with stable ectopic expression of LIF (MCF7-LIF, T47D-LIF and MDA-MB-231-LIF) were established by transduction of LIF-flag expression vectors (Fig. 1f, ectopic LIF expression in MCF7, T47D and MDA-MB-231 cells significantly increased the number of lung metastatic tumors. Furthermore, LIF promoted the anchorage-independent cell growth in soft agar; ectopic LIF expression increased the number and size of colonies formed by MCF7, T47D and MDA-MB-231 cells (Fig.
Taken together, these results demonstrate that LIF activates the mTOR pathway, which contributes to the promoting effect of LIF on breast cancer metastasis. 5a & b, both exogenous LIF treatment and ectopically expressed LIF in cells increased the phosphorylation of AKT at Ser-473 (p-AKT), which represents the activation of AKT, in MCF7, T47D and MDA-MB-231 cells.
Similarly, expressing DN-AKT in MCF7 and T47D cells to block AKT activity largely blocked the promoting effect of ectopically expressed LIF on invasion and migration in cells (Fig.
LIF is also involved in bone formation, neuronal survival, and acute immune response to inflammation [32, 33]. Both exogenous LIF and LIF expressed in cells show promoting effects on tumorigenesis and metastasis of breast cancer, suggesting that LIF functions in both autocrine and paracrine manners. The mTOR pathway is frequently activated in various cancers, including breast cancer, which plays a critical role in tumor growth and metastasis [34-36]. Therefore, using antibody against LIF to block LIF function or blocking LIF receptor complex are potential strategies for breast cancer therapy.
Antibodies against LIFR (sc-659), total AKT (sc-1618) and total STAT3 (sc-482) were purchased from Santa Cruz Biotechnology. Cells on the lower surface were fixed, stained and counted after culturing 24 h for MCF7 and MDA-MB-231 cells, and 48 h for T47D cells.
Mouse experiments were approved by the University Institutional Animal Care and Use Committee. They were treated with breast conserving surgery with or without axillary lymph node dissection.
Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin.
Although HER2 has no known ligand, it is the preferred heterodimerization partner of the other HER receptors and is involved in the lateral transmission of signals between other HER family receptors [4]. As a result, lapatinib with capecitabine is currently licensed for use in patients who have previously failed trastuzumab. Since we have previously shown that trastuzumab-induced activation of all HER receptors may contribute to trastuzumab resistance [19], we assessed the effects of neratinib, to determine if it can improve response and overcome resistance to trastuzumab, in sensitive and resistant HER2 amplified breast cancer cells. The IC50 values for neratinib were lower than those previously published for lapatinib in the panel of HER2 positive cell lines [30].
A dose dependent inhibition of pHER2 was observed with increasing concentrations of neratinib in both cell lines (Figure 1B). Figure 2B shows the results after three days of treatment in the SKBR3 cells (data for BT474 cells are not shown as there were insufficient cells left after three days of the combination treatment). C) Scatter plots showing the relationship between the neratinib (200 nM) induced decrease in phosphorylation of HER2, Akt and ERK and neratinib sensitivity (IC50) in 9 HER2 positive breast cancer cell lines. B) At the end of the experiment from A, tumor samples were collected and embedded in paraffin.
This is supported by recent data which showed that lapatinib in combination with trastuzumab significantly improved overall survival and progression-free survival compared to lapatinib alone despite disease progression on prior trastuzumab-based therapy [35, 36].
As well as neratinib, other irreversible TKIs including dacomitinib (PF-00299804) and afatinib (BIBW2992) are also being tested in clinical trials. However, the combination of trastuzumab and neratinib significantly delayed reactivation of HER3 and Akt compared to neratinib alone. Nevertheless, some of the moderate HER2 expressing breast cancer cells including MDA-MB-453 and MDA-MB-361 show greater sensitivity to neratinib than trastuzumab.
This suggests that there may be both overlapping and non-overlapping mechanisms of resistance to different HER2 inhibitors.
Other potential biomarkers which may be predictive of response to neratinib, and other HER2 targeted therapies, include the dimerization states of the HER receptors [53], and levels of protein tyrosine phosphatases, such as PTPN9 which has been shown to regulate EGFR and HER2 activation [54, 55]. All cell lines used were obtained from the American Type Culture Collection (Rockville, MD, USA) unless otherwise stated. The reaction was stopped by adding 50 µl 1 M NaOH and absorbance was read at 405 nm with 620 nm as the reference wavelength.
The following day, sections were incubated with primary antibodies for 2 hours at room temperature, followed by 3 washes with PBS. We also thank the BREAST-PREDICT (CCRC13GAL) programme of the Irish Cancer Society for funding this work. Evaluation of lapatinib as a component of neoadjuvant therapy for HER2+ operable breast cancer: NSABP protocol B-41. We hypothesized that autophagy induction would synergize with a therapeutic agent targeting the autophagic cargo. Second, autophagy is a self-degradative pathway for disposal of obsolete parts of the cell, is accompanied by vacuolization in the cytoplasm and can lead to increased cell survival or cell death depending on the type and extent of self-degradation [2]. The intracellular supply of dNTPs is regulated by ribonucleotide reductase (RR), an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides, and plays a critical role in regulating the total rate of DNA synthesis [9].
Candidate compounds identified from this screening were further optimized by iterative virtual and structure activity relationship (SAR) analysis to yield COH29.
We hypothesized that adding an autophagy inducer may boost the effectiveness of COH29 by reducing the total levels of RRM2.
TMX treatment induced autophagy in MDA-MB-231 cells (Figure 1A, left panel), as evidenced by the accumulation of GFP-LC3 puncta over time (Figure 1A, right panel). As expected, the combination treatment was more effective at reducing cell viability than COH29 alone.
Likewise, Western analyses revealed that TMX-induced LC3-II accumulation and RRM2 reduction were retarded by knocking-down ATG5 (Figure 2E). The low percentage of cells undergoing early apoptosis suggested that apoptosis could not completely account for the observed death (Supplementary Figure S3A).
Lamins A and C, a structure that surrounds the nucleus, are the scaffolding components of the nuclear envelope in cells [37].
Clonal numerical gains of chromosomes 1, 2, 3, 10, 11, and 15 were detected, as well as clonal losses of chromosome 21 (Figure 5A). In addition, a growing body of evidence is showing that RRM2 is overexpressed in cancer and is associated with neoplasia, metastatic potential, and poor prognosis in human cancers [42-44]. Numerous small molecule inhibitors, such as hydroxyurea (HU), gemcitabine (both approved for human cancer clinical use) and triapine [47], interact with the RRM2 subunit and inhibit RR activity. Although the concept of combined TMX with TRAIL therapy is worthwhile, the therapeutic use of TRAIL is limited by concerns over its potential hepatotoxicity [52, 53].
In this report, we found that besides ubiquitin-mediated degradation of RRM2, the abundance of RRM2 was regulated by lysosomal degradation through autophagy induction. These findings showed that RRM2 plays a crucial role in maintaining chromosomal stability, suggesting that modulation of cellular RRM2 abundance may be important for the induction of mitotic cell death.
For example, starvation- or rapamycin-induced autophagy had been shown to decrease RRM2 level and accompanied by a decrease in RNR activity and dNTP pools in human cancer cells [30].
Rapamycin (R8781), tamoxifen (T5648), bafilomycin A1 (B1793) and Chloroquine diphosphate salt (C6628) were from Sigma. Cells were treated with TMX, COH or the combination at the indicated concentrations for 72 h.
Approximately 40 µg of protein was mixed with an equal volume of 2 x SDS loading buffer, boiled for 5 min, then separated by Tris-glycine SDS-PAGE and transferred to PVDF membranes. After reacting at room temperature (RT) for 20 min, 40 µl of the aliquots were applied to circular Whatman DE81 ion exchange paper (GE Healthcare Life Sciences, 3658-325). The slides were then incubated with polymer horseradish peroxidase-labeled secondary antibodies for 30 min at RT, then 3,3-Diaminobenzidine (DAB)-treated (0.05 g DAB and 100 ml 30% H2O2 in 100 ml PBS) for 5 and 10 min, respectively.
In striking contrast, the vast majority of non-CSCs undergo a specialized form of apoptosis in suspension cultures, called anoikis. Sterile GO dispersions were prepared in a 5% mixture of DMSO in double-distilled water for these studies. Interestingly, the viability of bulk non-CSCs from these five cancer cell lines (SKOV3, PC3, A549, Mia PaCa2, and U-87) was not affected by GO (Figure 5), further highlighting its specificity and selectivity for CSCs. Note that GO inhibits cancer stem cell signaling (WNT, STAT3 and Notch), NRF2-antioxidant responses, as well as INFΥ-STAT1 signaling. Finally, using a panel of specific well-established breast CSC markers (namely CD44 and CD24), we show that GO appears to reduce the number of CSCs by inducing their differentiation, as they now begin to express CD24. Alternatively, GO flakes could also be used as a lavage solution during surgery, to clear the tumor excision site or the peritoneal cavity (as in ovarian or other peritoneal cancers) of residual CSCs, with the aim of preventing tumor recurrence and distant metastasis, via differentiation-based nano-therapy (Figure 9). The graphene oxide flakes of different sizes were separated by centrifuging graphene oxide suspensions at various rpm and collecting different phases of the suspension.
In this paper, a computer-aided detection and diagnosis (CADD) system for breast cancer is developed. Two questionnaires (a lymphoedema screening questionnaire and the Disabilities of Arm, Shoulder and Hand questionnaire) were mailed and 72 of 204 responders reported having one or more symptoms of lymphoedema (prevalence 35%). LIF promotes cell proliferation and anchorage-independent growth of breast cancer cells in vitro, and the growth of xenograft breast tumors in vivo. LIF could inhibit the differentiation and promote proliferation in some cancers and cancer cell lines, and has been suggested to contribute to the progression of malignancies, including rhabdomyosarcoma, choriocarcinoma and melanoma [8, 16-18]. Importantly, the activation of AKT-mTOR signaling by LIF largely mediates the promoting effect of LIF on tumorigenesis and metastasis. The expression levels of LIF are much higher in MDA-MB-231 and HS578T cells that display higher metastatic abilities [22] compared to less metastatic breast cancer cells, including MCF7, MDA-MB-468, SK-Br-3, T47D and BT474 cells.
Furthermore, ectopic LIF expression in T47D and MDA-MB-231 cells promoted distant metastasis.
2b), whereas knock-down of endogenous LIF inhibited the anchorage-independent growth in soft agar of MDA-MB-231 cells (Fig. The role of LIF in cancer and its underlying mechanisms are not well-understood with limited studies suggesting that LIF may play a role in tumorigenesis. Furthermore, the promoting effect of LIF on tumorigenesis and metastasis of breast cancer is independent of ER status; similar effects of LIF were observed in both ER-positive breast cancer cells (MCF7 and T47D) and ER-negative breast cancer cells (MDA-MB-231).
Importantly, blocking mTOR activity largely abolished the promoting effect of LIF on breast cancer metastasis, suggesting that the activation of mTOR pathway mediates the promoting effect of LIF on breast cancer tumorigenesis and metastasis.
Furthermore, given the effect of LIF expression on the AKT-mTOR signaling, this pathway may serve as a novel therapeutic target to improve outcomes in breast tumors with LIF overexpression. Cells were treated with Rapamycin (Cell Signaling Technology), Wortmannin (Cell Signaling Technology) and Stattic (Sigma) for various time periods before being harvested for further analysis.
Following surgery, adjuvant systemic therapy was administered as clinically indicated in accordance with standard clinical practice.
Known positive controls were included in each experiment, and negative controls were obtained by omitting the primary antibody. The statistical differences in xenograft tumor growth among groups were analyzed by ANOVA, followed by Student’s t-test.
However, a significant proportion of HER2-positive patients are either inherently resistant or develop resistance to trastuzumab. However, many patients do not respond to trastuzumab and the majority who initially respond, progress within a year of initiating therapy [13]. Clinical trials have shown that the addition of trastuzumab and lapatinib to chemotherapy achieved a higher pathological complete response than either trastuzumab or lapatinib alone [24, 25]. The results were also confirmed by direct comparison of the two drugs in SKBR3 and BT474 cells, and both cell lines showed greater sensitivity to neratinib than lapatinib (data not shown).
Although total HER2 levels were not altered by treatment with neratinib for 1 hour, by 24 hours HER2 level was reduced in response to 50 nM neratinib in SKBR3 and was undetectable in BT474 cells. Neratinib monotherapy decreased cell numbers to a greater extent than trastuzumab replacement, in both resistant cell lines (Figure 3B). Although trastuzumab has been previously shown to downregulate HER2 [12, 19, 31], this effect was not seen with either trastuzumab, neratinib or the combination treatment in our xenograft study. Afatinib monotherapy was shown to have promising clinical activity in extensively pretreated HER2-positive breast cancer patients who had progressed following trastuzumab treatment [39]. In contrast a recent study showed that dual inhibition with trastuzumab and lapatinib or trastuzumab and pertuzumab does not completely eliminate HER3 function [42].
In NSABP B-41, lapatinib in combination with chemotherapy induced a greater pathological complete response than that of trastuzumab in combination with chemotherapy in breast cancer patients with IHC 2+ although the number of patients in each arm was small [49]. There is a need to further understand the biomarkers that can predict response to neratinib in different breast cancer subtypes. EFM192A, KPL1, EFM19 and CAL51 were supplied by the German Tissue Repository DSMZ (Braunschweig, Germany). The log of drug concentration was then plotted against the log growth inhibition and IC50 values were calculated using linear regression analysis as previously described [30].
The sections were then incubated with a horseradish peroxidise-coupled anti-rabbit secondary antibody (Vector laboratories) for 30 minutes at room temperature.
To test this hypothesis, we treated breast cancer MDA-MB-231 cells with tamoxifen (TMX), which induces autophagy through an estrogen receptor-independent pathway. During autophagy, organelles and parts of the cytoplasm are isolated in characteristic double-membrane organelles known as autophagosomes. RR is also one of the top 50 metabolic enzymes frequently overexpressed in human tumors [10]. Along the same lines, in vitro studies have suggested that TMX, besides inducing apoptosis in tumor cells, also promotes the accumulation of large-scale autophagic structures. Therefore, we evaluated the effects of combining TMX with COH29 in ER-negative MDA-MB-231 breast cancer cells both in vitro and in vivo. MDA-MB-231 cells with stable integration of GFP-LC3 were treated with TMX (5 or 10 µM) for the indicated time periods and analyzed with microscopy A. To further confirm the synergistic effect is autophagy-dependent, knockdown of BECLIN1 or ATG5, a key molecule for autophagy induction, also conferred a pro-survival effect, like in the context of combined treatment (Supplementary Figure S1C). Although, the expression of RRM1 was not affected, the abundance of RRM2 is more reduced by the combination of TMX and COH, which was consistent with the cytotoxicity data shown in Figure 2A. Western analyses further supported this finding by showing that the majority of caspase 3 remained inactivated (not cleaved) (Supplementary Figure S3B). Cell viability was determined using the acid phosphatase assay at 72 h post-combined treatment and significance was determined relative to the no treatment control. These results were validated by Western analyses using protein samples extracted from the indicated tumors (Supplementary Figure S4B). The near tetraploid sideline cells contained duplicate copies of most stemline abnormalities. However, these small molecules have problems that limit their use, including lack of specificity for the RRM2 protein, unwanted side effects, and susceptibility to resistance.
Currently, TMX is associated with depression, endometrial hyperplasia, and Coumadin drug interaction. In our study, we showed that COH29 along can induce cell death through inhibiting RR activity, and its cytotoxicity is dramatic enhanced when autophagy is induced by TMX, even though the cells are normally resistant to TMX.
The papers were dried, then washed 3 x 10 min with 5% Na2HPO4, and rinsed once with distilled water and once more with 95% ethanol. All animal experiments were approved by the institutional animal care and use committee at City of Hope. Lucy Brown of the Analytical Cytometry Core at City of Hope for flow cytometry analyses, Dr. Importantly, each 3D-spheroid originates from the clonal proliferation of a single CSC, is not to due to the self-aggregation of cancer cells. In contrast, GO (big and small flakes) does not affect cell viability of the total MCF7 cell population.
Thus, GO may reduce the number of bonafide CSCs that are capable of forming tumor-spheres, by inducing their differentiation and inhibiting their proliferation. The AFM characterization of graphene oxide flakes was performed on a Bruker Dimension FastScan AFM system by using taping mode. All experiments were performed in triplicate, three times independently, such that each data point represents the average of 9 replicates. Once cells were attached, the viral particles were diluted 1:10 in complete culture media containing polybrene (sc-134220, Santa Cruz), and added to the cells.
After 48 hours of treatment, luciferase assays were performed according to the manufacturer’s instructions. Then, the monolayer cells were trypsinized and seeded in low-attachment plates in mammosphere media.
The framework is based on combining principal component analysis (PCA), independent component analysis (ICA), and a fuzzy classifier to identify and label suspicious regions. LIF also promotes invasion and migration of breast cancer cells in vitro and metastasis of breast cancer in vivo. Meanwhile, LIF has also been reported to induce the differentiation of murine myeloid leukemia cells and inhibit proliferation and growth in some other cancer cell lines [19-21].
Furthermore, overexpression of LIF is significantly associated with a poorer relapse free survival in breast cancer patients.
The difference in LIF expression levels was confirmed at the protein level in MDA-MB-231, MCF7 and T47D cells (Fig 1b), which were chosen for the following experiments to study the role of LIF in breast tumorigenesis.
Compared with control cells transduced with control vectors, ectopic LIF expression greatly increased the abilities of invasion and migration of MCF7, T47D and MDA-MB-231 cells.
Two out of six mice injected with T47D-LIF cells developed metastatic breast tumors in the neck and muscle in addition to lung tumors, and two out of six mice injected with MDA-MB-231-LIF cells developed metastatic breast tumors in mediastinum, neck, back, underarm and muscle in addition to lung tumors. Furthermore, expression of DN-AKT greatly reduced the promoting effect of LIF on cell proliferation and anchorage-independent cell growth in soft agar in MCF7 and T47D cells (Fig.
The relationship between LIF expression levels and clinicopathological variables of breast cancer was analyzed. Further investigation on the mechanism by which the mTOR pathway is activated by LIF shows that the activation of mTOR is largely mediated by AKT. The tumor was considered positive for LIF staining when more than 10% of tumor cells were positive for LIF staining.
We assessed the effects of neratinib, an irreversible panHER inhibitor, in a panel of 36 breast cancer cell lines. HER2 signaling promotes cell proliferation via the RAS-ERK pathway and inhibits cell death via the PI3K-Akt-mTOR pathway [7]. Understanding the mechanisms responsible for resistance to trastuzumab is therefore crucial for the development of new therapeutic strategies and improving patient outcome. B) BT474 (right) and SKBR3 (left) cell were treated for 1 hour or 1 day with increasing concentrations of neratinib. Furthermore, the combination of trastuzumab and neratinib had a greater growth inhibitory effect than either drug alone, with or without trastuzumab withdrawal in SKBR3R (Figure 3B). The differences in means of the percentage of connective tissue between the groups were analyzed by Anova with Bonferroni’s multiple comparison test and statistically significant changes were indicated in the figure.
This may be because there was a significant heterogeneity in HER2 staining between xenograft tumor areas and the dose of trastuzumab used in this study was lower than in previously reported xenograft experiments [31], which may affect the amount of HER2 downregulation and assessment [32]. It would be interesting to determine if the reactivation of HER3 observed with neratinib is also induced by ADAM17 mediated heregulin release [19]. Thus it may be important to assess the effect of neratinib in patients whose tumors express lower levels of HER2 protein (IHC 2+).
The treatment continued for 17 days (5 trastuzumab treatments) and the mice were sacrificed 3 hours after the last neratinib treatment. Equal amounts of protein sample were prepared in 4X SDS with 10% beta-mercaptoethanol, boiled for 10 minutes at 95°C. Visualization of staining was performed using a diaminobenzidine (DAB) peroxidase substrate (Vector Laboratories).
Other funders supporting the co-authors and core facilities in Oxford include Cancer Research UK, Medical Research Council, Oxford Biomedical Research Centre, Oxford Experimental Cancer Medicine Centre and Cancer Research UK Oxford Cancer Research Centre.
Induction of autophagy reduced cellular levels of RRM2, a subunit of ribonucleotide reductase (RR), the rate limiting enzyme in the production of deoxyribonucleotide triphosphates (dNTPs). Fusion of the autophagosomes with lysosomes can allow for degradation and recycling as part of the quality control process that maintains bioenergetics and basic cell functions [3, 4]. We found that combined use of TMX and COH29 reduced intracellular dNTP levels, induced genomic instability, promoted tumor cell death, and reduced tumor volume in a xenograft model. These results suggested that TMX treatment induces autophagy in MDA-MB-231 cells in a concentration- and time-dependent manner. We further confirmed that the combination treatment also resulted in autophagy induction by analyzing GFP-LC3 puncta formation (Figure 1E, upper panel).


Next we determined if the combined inhibitory effect was autophagy-dependent by using an autophagy inhibitor, chloroquine (CQ). These results support our overall hypothesis that decreasing RR level decreases intracellular dNTP abundance.
Taken together, our results support the conclusion that the combined use of TMX and COH promoted cytotoxicity by autophagy induction.
The autophagy-competent, xenografted tumors showed significantly less growth in the presence of the combined TMX and COH29 treatment, relative to vehicle alone (Figure 3B) and also showed a reduced weight at the time of harvest (Figure 3C).
In autophagy-competent MDA-MB-231-derived tumors, RRM1 abundance was not affected by the treatments, whereas RRM2 levels were reduced by combined treatment. Using vehicle-treated MDA-MB-231 cells as a reference, we investigated the effects of drug treatment. For instance, HU blocks DNA synthesis by reducing the tyrosyl free radical of the RRM2 subunit to a normal tyrosine residue but is susceptible to resistance. 1C and 2C, the pretreatment of TMX (to induce autophagy) effectively reduced RRM2 level and sensitized cells to COH29. In summary, the present findings indicate that combination of TMX and COH29 effectively reduces cell viability in MDA-MB-231 cells via an autophagy-dependent pathway. The reaction was terminated by adding NaOH (1 N, 10 µl), and the absorbance was measured at 410 nm using a microplate reader.
The membranes were then washed with three times with PBST for 10 min, and incubated with HRP (horseradish peroxidase)-labeled secondary antibodies for 2 h at RT. After each paper was dried and deposited in a small test tube, and 5 ml of Ecoscint A was added to each tube. As a consequence, the survival of residual CSCs is thought to drive the onset of tumor recurrence, distant metastasis, and drug-resistance, which is a significant clinical problem for the effective treatment of cancer. Puromycin treatment (P9620, Sigma) was started 48 hours later in order to select stably infected cells. After 10h under low-attachment conditions, MCF7 cells were spun down and incubated with CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen cat.559942) antibodies for 15 minutes on ice. We found that LIF activates the AKT-mTOR signaling pathway to promote tumorigenesis and metastasis of breast cancer. Together, data from this study strongly suggest that LIF plays an important role in promoting the tumorigenesis and metastasis of breast cancer.
Furthermore, this promoting effect of LIF can be blocked by LIF neutralization antibody (Fig. In contrast, no distant metastatic tumor was observed in mice injected with T47D-Con and MDA-MB-231-Con cells within the same time period (Fig. Consistent with the results obtained from in vitro assays, ectopic LIF expression promoted the growth of xenograft tumors formed by MCF7, T47D and MDA-MB-231 cells (Fig.
Blocking AKT activity by wortmannin or the expression of DN-AKT largely abolished the activation of mTOR by LIF, and more importantly, largely abolished the promoting effect of LIF on tumorigenesis and metastasis of breast cancer. This study was reviewed and approved by University Human Investigations Committee Institutional Review Board.
We further assessed its effects with or without trastuzumab in several sensitive and resistant breast cancer cells as well as a BT474 xenograft model. Over-expression of HER2 occurs in approximately 20% of breast cancers and is associated with a poor prognosis [8, 9]. However, reactivation of HER3 and Akt phosphorylation was observed after 24 hours (Figure 2B and Supplementary Figure 1). This possibility might be investigated using an ADAM17 inhibitor or a monoclonal antibody to HER3. MDA-MB-134, MDA-MB-415, MDA-MB-436, MDA-MB-157, UACC893 and UACC812 were cultured in L15 medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 2 mM glutamine and 1 % penicillin G-streptomycin-fungizone solution (PSF) (Irvine Scientific, Santa Ana, CA, USA). Counterstaining was performed using Haemocytoxin prior to the mounting using aqueous-based mounting media (Aquatex). This autophagy inducer was combined with COH29, an inhibitor developed in our laboratory that targets RR through a novel mechanism.
However, we and others have also shown that prolonged starvation of arginine induces oxidative stress and triggers autophagic cancer cell death [5, 6]. RR consists of two protein subunits, RRM1 and RRM2, both of which are required for enzymatic activity. Unlike other RR inhibitors, COH29 does not appear to chelate iron, so side effects are likely to be reduced.
The role of autophagy in cancer cell death has been exploited by both pharmacological and genetic approaches that modulate autophagy [3, 24, 25]. The combined treatment with COH29 did not affect the ability of TMX to induce autophagy (Figure 1E, lower panel). The cells were treated with TMX and COH29, as indicated, in the presence and absence of CQ (20 µM). However, consistent with Supplementary Figure S1C, the combined treatment had no effect on tumors from ATG5-knockdown MDA-MB-231 cells (Figure 3B). Because dNTP depletion is known to induce genomic instability, we next wished to assess the presence of DNA strand breaks. The percentage of DNA leakage, γH2AX positive or containing both phenotype cells is calculated and plotted as a bar graph. Furthermore, 3-AP (triapine®), which was in human phase II clinical testing, relies on iron chelation to inactivate RR.
Theoretically, the side effects from the combination of TMX and COH29 should not exceed their respective adverse effect. Moreover, the addition of BafA1, an autophagy inhibitor, blocked the decrease of RRM2 after autophagy induction by TMX, rapamycin and arginine depletion (Figure 1C, Supplementary Figs. Furthermore, our data suggest that autophagy induction may serve as an adjuvant anticancer therapy, and that proper combined treatment with an autophagy inducer could synergistically enhance the therapeutic efficacy of a myriad of cancer treatments.
The interaction between drug combinations was analyzed using the Calcusyn software program (Biosoft, Cambridge, UK) to determine if the combination was antagonistic, additive or synergistic.
Immunoblots were visualized using the VersaDoc 5000 imaging system (Bio-Rad) and processed using quantity one (Bio-Rad). Victoria Bedell of the Cytogenetics Core of City of Hope for cytogenetic investigation, Ms. Thus, novel approaches to cancer therapy are needed urgently, to address this clinical need.
The CSC population is resistant to DNA-damage, and shows lower levels of ROS production, as well [14, 15].
Concentrations were obtained from UV-Vis spectra, which were recorded in 10 mm path length quartz cells using a PerkinElmer Lambda – 1050 UV-Vis-NIR spectrometer. Luminescence was normalized using SRB (to determine total cellular protein), as a measure of MCF7 cell viability. Inhibiting the AKT activity can largely block the activation of the mTOR pathway by LIF, suggesting that LIF activates the mTOR pathway through AKT.
2d), whereas knockdown of endogenous LIF reduced the growth of MDA-MB-231 xenograft tumors (Fig. The levels of p-AKT were much higher in T47D-LIF and MDA-MB-231-LIF xenograft tumors than T47D-Con and MDA-MB-231-Con tumors, respectively (Fig.
LIF can activate multiple signaling pathways, including STAT-3 pathway, to mediate some of LIF’s functions. Expression of DN-AKT also largely abolished the promoting effect of LIF on the growth of xenograft tumors formed by T47D cells (Fig. Notably, employing Kaplan-Meier survival analysis, we found that LIF expression levels were correlated with relapse free survival of breast cancer patients (p=0.0039). These findings demonstrate that LIF promotes tumorigenesis and metastasis of breast cancer through the AKT-mTOR signaling pathway (Fig. We confirmed that neratinib was significantly more active in HER2-amplified than HER2 non-amplified cell lines.
DMEM (Cellgro, Manassas, VA, USA) supplemented with 10 % heat-inactivated FBS and PSF (Irvine Scientific, Santa Ana, CA, USA) was used to culture CAL51 and KPL1. The combination therapy showed synergistic effects on cytotoxicity in vitro and in an in vivo xenograft model.
Third, necrosis is defined as a type of cell death that lacks features of the two previous pathways and is accompanied by swelling of the cell, loss of membrane integrity, and inflammatory response, as the cellular contents are released into the extracellular environment [1].
Two RRM1 subunits and two RRM2 subunits join together to form the active tetrameric enzyme. Through interference with DNA repair and replication pathways, COH29 upsets the balance of dNTP in cells, leading to cell death.
Modulating autophagy requires extra caution because autophagy can protect cancer cells in the early stages of chemotherapy, but promote tumor cell death afterwards [3, 24, 25]. The effects of the drug combination were far greater than the effects of either drug alone.
Moreover, TMX induced RRM2 reduction was also observed in the ERα-positive breast cancer T47D and MCF7 cells (Supplementary Figure S1A), suggesting that it is not breast cancer subtype is specific. The addition of CQ resulted in a significant rescue of cell viability in cells treated with 10 or 20 µM each of TMX and COH29 (Figure 2C).
Furthermore, TMX and COH29 treatment, when delivered individually, did not reduce tumor growth (Figure 3C). Data from a phase I trial of 3-AP showed that patients developed side effects such as hypoxia, respiratory distress, and methemoglobulin, due to chelation of iron in the patients’ red blood cells [48]. This program is based on the Chou-Talalay method to calculate a CI, and CI values below 1 indicate synergistic effect. Towards this end, here we have investigated the therapeutic potential of graphene oxide to target cancer stem cells. 3D-tumor-spheres derived from breast cancer cells are also known as mammo-spheres [12, 13]. Importantly, both small and big GO flakes did not affect the viability of the bulk non-CSC population of MCF7 cells, indicating selectivity towards CSCs (Figure 2). Inhibiting the AKT activity as well as inhibiting the mTOR activity largely block the promoting effect of LIF on tumorigenesis and metastasis.
Knock-down of endogenous LIF by two shRNA targeting LIF in MDA-MB-231 cells, which have high endogenous LIF levels, clearly decreased the abilities of invasion and migration of cells (Fig. Consistently, mice injected with MDA-MB-231 cells with stable knock-down of LIF (MDA-MB-231-LIFshRNA) formed much less metastatic lung tumors compared to mice injected with MDA-MB-231-ConshRNA cells (Fig. The prognosis analysis demonstrates that higher expression of LIF had a poorer relapse free survival in breast cancer patients (Fig. Neratinib decreased the activation of the 4 HER receptors and inhibited downstream pathways.
The scoring criteria is composite score based on staining intensity (SI) and percentage of positive cells (PP) using the formula IRS = SI × PP. This cytotoxicity was blocked by knockdown of the autophagy protein ATG5 or addition of chloroquine, an autophagy inhibitor. Cell death pathways play important roles in tumorigenesis, cancer progression, and resistance to therapy and thus are important therapeutic targets in cancer [7]. RRM1 and RRM2 are encoded by different genes on separate chromosomes and, most importantly, their messenger RNAs (mRNAs) are differentially expressed throughout the cell cycle. Because COH29 has limited effects as a single therapeutic agent, we used various biochemical, cell biological, and xenograft approaches to find a combination therapy that reduced the toxicity and increased the efficacy of COH29.
Furthermore, autophagy can be induced by various stimuli, including nutrient deprivation, serum starvation, metabolic stress, radiation, and anticancer drugs in multiple cancer cells [24, 26-29]. This study proposes an effective anti-tumor regimen and, more broadly, establishes aggressive autophagy induction as a plausible method for increasing the sensitivity of cells to chemotherapy. Along the same line, a dose-dependent cytotoxic effect by the combination of COH29 and TMX was also noted in T47D and MCF7 cells (Supplementary Figure S1B). The percentage of cells containing GFP-LC3 puncta was enumerated by counting 300 cells and plotted (right panel). Moreover, pre-treatment with TMX for 24 h to induce autophagy notably enhanced the cytotoxicity induced by TMX and COH29 (20 µM each). Immunohistochemistry staining (IHC) of Ki67, a cellular marker for proliferation, was performed to assess tumor cell growth or not. The percentage of cells that showed DNA leakage, γH2AX positivity or both was enumerated. In the cells co-treated TMX and COH29 for 24 h, the overall chromosomal morphology was significantly worse than that in vehicle-treated cells and chromosomal breakage was apparent in approximately 35% of the cells (Figure 5D).
Therefore, approach to improve efficacy of existing RRM2 inhibitor or development of new RRM2 inhibitors are needed [49]. All these suggest that autophagy-dependent lysosomal degradation plays a critical role in controlling RRM2 level in breast cancer cells.
The CIs were determined from cell viability ACP assays as the fraction of cells killed by individual drugs, or combination of drugs, compared to vehicle-treated cells.
Graphene and its derivatives are well-known, relatively inert and potentially non-toxic nano-materials that form stable dispersions in a variety of solvents. This suggests that GO inhibits mammosphere formation by promoting the differentiation of breast cancer stem cells. Together, these results demonstrate that LIF promotes proliferation, anchorage-independent growth of breast cancer cells and the growth of xenograft breast tumors. The levels of p-AKT were much lower in MDA-MB-231 LIFshRNA xenograft tumors than MDA-MB-231-con tumors (Fig.
Taken together, these results demonstrate that LIF activates the AKT-mTOR pathway, which in turn promotes tumorigenesis and metastasis of breast cancer. 7b), suggesting that LIF could be a prognostic marker for poor prognosis of breast cancer patients.
However, HER3 and Akt were reactivated at 24 hours, which was prevented by the combination of trastuzumab and neratinib. All other cell lines were maintained in RPMI 1640 (Cellgro) supplemented with 10 % FBS (heat inactivated), 2 mM glutamine and 1 % PSF (Irvine Scientific, Santa Ana, CA, USA). The membrane was then incubated with primary antibody in 3% BSA in PBS-Tween overnight at 4°C.
The combined therapy also induced dNTP depletion and massive genomic instability, leading us to hypothesize that combining autophagy induction with RR inhibition can lead to mitotic catastrophe in rapidly dividing cells.
The cellular RRM1 protein level remains relatively stable through the entire cell cycle and has a relatively long half-life of 18 to 24 h.
We propose to exploit autophagy-dependent cell death to promote the efficacy of COH29-based cancer therapies. However, the non-tumorigenic mammary MCF-10A cells were less sensitive to the combined treatment (Supplementary Figure S1B). Consistently, the enhanced cytotoxic effect of sequential treatment was also retarded by CQ addition (Figure 2C). The line shown in the plot indicates CI = 1 and values below 1 are defined as synergistic effects. The staining indicated that less Ki67 signals were detected in xenografted tumor cells from mice receiving the combined treatment of TMX and COH29 (Supplementary Figure S4A), consist with the significant reduction of tumor weights among four treatment groups (Figure 3C).
Western blot analysis showing the change in γH2AX abundance after TMX, COH and combination treatment.
At 48 h after combined treatment, chromosome and chromatid breaks and gaps were observed in almost all cells. However, the underlying mechanism of how autophagy recognized RRM2 for degradation is still unclear and additional study is warranted.
For the apoptosis assay, cells were treated with COH29 and TMX for the indicated time periods. Hsiu-Ming Shih and the RNAi Consortium at Academia Sinica for providing the lentiviral constructs against human ATG5, members of Ann’s laboratory for helpful discussions, and Dr.
Here, we show that graphene oxide (of both big and small flake sizes) can be used to selectively inhibit the proliferative expansion of cancer stem cells, across multiple tumor types. Taken together, our data strongly suggest that LIF plays an important role in the tumorigenesis and metastasis of breast cancer, and could be an important prognostic marker for breast cancer.
These results demonstrate that LIF promotes the abilities of invasion and migration of breast cancer cells in both autocrine and paracrine manners.
MCF7-LIF, T47D-LIF, MDA-MB-231-LIF and their control cells (Con) were injected into nude mice via tail vein, and the number of metastatic tumors in lung and distant metastatic tumors was determined after 10 weeks. 3, exogenous LIF treatment dramatically increased the phosphorylation of STAT-3 at Thy705, which represents the activation of the STAT-3 pathway. We have previously shown that rapamycin decreased RRM2 level in an autophagy-dependent manner in KB cells [30].
Altogether, our results clearly show that TMX and COH29 have anti-tumor effects both in vitro and in vivo and suggest that autophagy is required for the combinatorial effect.
In addition, all cells undergoing combined treatment for 48 h were arrested in metaphase, accompanied by shorter chromosome length and loss of chromosomes (Figure 5E). Although, we cannot rule out the possibility that increase in COH29-mediated cytotoxicity is due to increased drug intake via autophagy, we postulated that TMX-induced aggressive autophagy promotes the death of COH29-treated cells.
After trypsinization, cells were washed twice with cold PBS and collected by centrifugation at 1000 rpm.
For this purpose, we employed the tumor-sphere assay, which functionally measures the clonal expansion of single cancer stem cells under anchorage-independent conditions. Virtually identical results were also obtained using 7-AAD (7-Aminoactinomycin D; Life Technologies) to distinguish between the live and dead populations of cells (cell viability), during anoikis. Approximately one in 100 women have had a diagnosis of breast cancer some time in the past 15 years, thus there are an estimated 162,600 breast-cancer survivors in Canada (1).Unfortunately some survivors experience long-term sequelae that include physical impairments, psychological distress and sub-optimal health-related quality of life (HRQL) (2, 3).
Representative H&E images of lung sections (left panels) and distant metastatic tumors (right top panels) are shown.
However, blocking STAT-3 activity by employing Stattic, a specific STAT-3 inhibitor, did not have a significant effect on the activation of the mTOR pathway by LIF, suggesting that STAT-3 does not play a major role in the activation of the mTOR pathway by LIF. Neratinib in combination with trastuzumab had a greater growth inhibitory effect than either drug alone in 4 HER2 positive cell lines. Therefore, RR enzyme activity is largely determined by the RRM2 protein levels in normal cells. Taken together, these results suggested that COH29 treatment induced DNA damage, resulting in DNA leakage, which was markedly aggravated by combination with TMX. This possibility is supported by the observation that some of the leaked DNAs were within lamin-labeled structures (Figure 4B), consistent with the report by Gonzalez-Suarez et al. More specifically, we show that graphene oxide effectively inhibits tumor-sphere formation in multiple cell lines, across 6 different cancer types, including breast, ovarian, prostate, lung and pancreatic cancers, as well as glioblastoma (brain).
Lymphoedema is one of the predominant physical sequelae and has an impact on physical function, psychological distress and HRQL (4). Taken together, our results demonstrate that LIF activates the mTOR pathway mainly through AKT.
Furthermore, trastuzumab in combination with neratinib was growth inhibitory in SKBR3 and BT474 cells which had acquired resistance to trastuzumab as well as in a BT474 xenograft model. Bands were then visualized and the blot developed using an enhanced chemiluminescent system (ECL, GE Heathcare).
In contrast, a more abundant γH2AX signal with extended duration was detected in combination-treated cells. Lymphoedema following breast cancer surgery is caused by mechanical lesion of the lymphatic system; protein accumulates in the tissues causing fibrosclerosis. MDA-MB-231-LIFshRNA and MDA-MB-231-ConshRNA cells were injected into nude mice via tail vein, and the number of lung metastatic tumors was determined after 12 weeks. Innately trastuzumab resistant cell lines showed sensitivity to neratinib, but the combination did not enhance response compared to neratinib alone. Together, these results support a model where autophagy induction, through TMX, sensitizes cancer cells to COH29-induced cell death.
MDA-MB-231 cells were pre-treated with vehicle or TMX for 24 h prior to exposure to TMX and COH29, as indicated, for 72 h.
It has been shown that autophagy plays a role in DNA damage response by cleaning up damaged DNAs [38-40]. The mixture was gently vortexed and incubated for 15 min at RT and 1 x Binding Buffer (400 µl) was added to each tube before analyzing by flow cytometry. Mechanistically, we present evidence that GO exerts its striking effects on CSCs by inhibiting several key signal transduction pathways (WNT, Notch and STAT-signaling) and thereby inducing CSC differentiation. Metabolic processes in the interstitium are disturbed by the oedema, and inflammatory processes are facilitated. Actin abundance was used as an internal control to ensure that equal amounts of proteins were loaded in each lane. Chloroquine (CQ, 20 µM) addition significantly reversed the cytotoxic effects of COH29 and TMX. Thus, graphene oxide may be an effective non-toxic therapeutic strategy for the eradication of cancer stem cells, via differentiation-based nano-therapy.
Our data indicate that neratinib is an effective anti-HER2 therapy and counteracted both innate and acquired trastuzumab resistance in HER2 positive breast cancer.
The prevalence of chronic lymphoedema is difficult to assess due to the varying definitions, populations and methods of measurement. Our results suggest that combined treatment with trastuzumab and neratinib is likely to be more effective than either treatment alone for both trastuzumab-sensitive breast cancer as well as HER2-positive tumors with acquired resistance to trastuzumab. RRM2-positive cells per 500 cells were enumerated from each sample (n = 2) and the percentage was calculated by comparing to the control (designated as 100%).
A variety of physical therapeutic interventions are used to reduce oedema, including elevation, massage, exercise and the application of external pressure, and some of these therapies are used in combination (a€?complex physiotherapya€?). MDA-MB-231 cells were cultured with increasing concentrations of COH29 with and without 20 µM TMX for indicated time periods prior to cytotoxicity assays.
Hence, it is important to detect lymphoedema because there are effective treatments, but it is also important to go beyond limb size and evaluate all the impairments, activity limitations and participation restrictions that women with breast cancer experience in order that the intervention will have the intended impact on function and quality of life.
The functional problems faced by women with breast cancer have been classified by Brach et al. Specifically, women report reduced strength in elbow flexors, shoulder abductors and grip, increased pain and oedema (13, 14).
Women post-ALND report significantly more limitations in activities of daily living because of shoulder impairments than women post-sentinel lymph node biopsy (14). Lymphoedema has been found to be independently associated with decreased quality of life scores (15).
The impairments associated with lymphoedema lead to functional limitations that can be targeted successfully by specific interventions (16). To improve the efficacy of strategies or interventions to reduce the occurrence of lymphoedema and minimize its impact on function and HRQL, it is important to understand how lymphoedema relates to function and HRQL.
The objective of this study was to estimate the extent to which the impairments associated with lymphoedema are linked to activity limitations, participation restrictions and sub-optimal HRQL. METHODSThis study was a sub-study of a pilot for an epidemiologic study to test the feasibility of developing a clinically-based prevalence study of lymphoedema and arm dysfunction among women operated on for stage I or II breast cancer. Through this pilot it was possible to add the measures needed to test relationships among variables related to lymphoedema and arm disability. The epidemiologic pilot study used a postal survey which included: (i) a questionnaire on arm function, the Disabilities of Arm Shoulder and Hand (DASH), and (ii) 5 questions, informed by the work of Kwan et al. For this study, we had the opportunity to invite women who responded positively to any one of the signs and symptoms to participate in a comprehensive physical assessment in our clinic. ParticipantsThe study population for the epidemiologic pilot comprised a subset of women treated surgically for stage I or II breast cancer at the McGill University Health Centre, Montreal, Canada from January 1992 to January 2002. The focus of the pilot study was to identify feasible sampling strategies, particularly for persons who were difficult to trace. For this study, the available sample was women who responded to the first wave of questionnaires. The study was approved by the Research Ethics Board (Surgery) of the McGill University Health Centre. Subjects provided written informed consent to participate in this study.Measures used for the comprehensive assessment (Phase II) The WHO ICF framework was used to inform the measurement strategy for this study (18). The requirement is to move the maximum number of small blocks from one compartment of a box across to another identical compartment within 1 min.
The Jamar dynamometer (Simmonds Preston Inc., Bolingbrook, USA) was used to measure grip strength (kg), 3 attempts were made with each hand, and the average of the 3 scores used.
Scores are transformed to a 0a€“100 scale, where 0 reflects good function and 100 reflects considerable disability. The scores on all subscales range from 0 to 100 (higher scores indicate better health status).
It includes 8 multi-item scales measuring physical functioning, role functioning, emotional functioning, cognitive functioning, social functioning, fatigue, nausea, pain, as well as dyspnoea, insomnia, appetite loss, constipation, diarrhoea, and financial difficulties.
The core questionnaire has high internal consistency, good inter-scale correlation, and discriminative validity (26). The supplementary breast module shows high internal consistency of most scales and good known-group discriminative ability (27).To place in context the extent of disability experienced by women with lymphoedema, we provide normative data as well as data for representative cancer survivors. The Box and Block, Nine-hole Peg and grip strength normative data is from a sample of 310 male and 328 female adults, ages 20a€“94 years, from the 7-county Milwaukee area (21, 22, 28). SF-36 norms are from a cohort of 9423 randomly selected Canadian men and women aged 25 years or more living in the community (29). The values for the EORTC breast cancer module (BR23) come from data collected from 158 American women 3-months post breast cancer surgery (27). Statistical methodsCharacteristics of those not recruited into the study and participants were compared using logistic regression. For continuous variables, age, time since diagnosis and number of nodes examined, between-group differences and 95% CI in the means were estimated. The extent of arm disability for women with or without symptoms of lymphoedema was compared by difference in means and 95% CI.
The relationships between screening items and volume and disability were estimated using linear regression.
Differences in scores on various tests between women include in this study sample and normative or reference values were tested using t-tests. To delineate the relationships between impairments, activity limitations, participation restrictions and sub-optimal health-related quality of life, a path analysis was completed using Mplus software.
Path analysis is an extension of a multiple regression model and is used to test the fit of the correlation matrix against 2 or more causal models that are being compared. The theoretical model used to inform this path analysis was that proposed by Wilson & Cleary (30), a model of HRQL that integrates biological and psychological aspects of health. There are 5 different levels in this model, namely, physiological factors, symptom status, functional health, general health perceptions, and overall quality of life. The regression weights predicted by the model are compared with the observed correlation matrix for the variables, and a goodness-of-fit statistic is calculated. Single indicator variables are depicted as squares (circles are for multiple indicator latent variables); single arrows indicate potentially a€?causala€? relationships and double-headed arrows indicate correlation with no directionality assumed. RESULTSIn response to the 596 questionnaires mailed, 204 women completed and returned questionnaires, giving an unprompted response rate of 34%. In all, 72 women (35%) indicated that they had one or more of the signs and symptoms of lymphoedema and 50 women (69%) attended for the comprehensive assessment (Fig.
Flow chart of patients invited to participate in study.The characteristics of the women who were invited to participate in the study are shown in Table I. Non-participants were, on average, 4 years older than participants, but there was little difference by a€?time since diagnosisa€?. Participants were more likely to have characteristics predisposing them to lymphoedema (extensive breast surgery, numerous nodes examined).
Also given in Table I is the OR for participation according to different levels of variables under study.
Odds ratio of participating in the study for each level of variable compared to the referent category. These women were more likely than those without symptoms to have had axillary lymph node dissection (87% vs 62%), but there was no significant difference between these 2 groups in age, number of nodes examined, type of breast surgery or use of adjuvant radiotherapy or chemotherapy.
Table II shows how women without symptoms of lymphoedema (n = 132), women with symptoms (n = 72) and those attending for the physical assessment (n = 50) differed by age, by number of symptoms, and by arm disability as measured by the DASH. Table III presents the results of measurements of arm volume for affected and unaffected arms.
Tape measurement indicated a slight difference between the 2 limbs not exceeding the clinically meaningful difference of 2 cm. Measurements by water displacement indicated that the affected arm was greater in size than the unaffected arm by an average difference of 154 ml. The differences in arm volume ranged from a€“160 ml to +740 ml with 16 of the 50 women having a difference of more than 200 ml and 5 women having a difference of more than 400 ml.Table III. For disability, there was a large difference between those without and those with symptoms (see Table II), but for symptomatic women, having more symptoms did not affect the DASH score. There was no association between volume of oedema and the DASH scores (correlation a€“0.05).
Women with a volume difference between arms of < 200 ml (n = 34) and a‰? 200 ml (n = 16) are contrasted because differences of greater than 200 ml between the affected and non-affected arms are indicative of lymphoedema (32).
Also presented are the values for all women combined and normative values, where available.
The 2 volume groups were similar on age, body mass index (BMI), co-morbidity and DASH scores; they were also similar on grip strength, but the total sample of women differed significantly from normative values. The most common co-morbidities found were osteoarthritis (48%) and back pain (54%).Table V. These values did not differ significantly by volume group but did differ significantly and substantially from norms. The values on the subscales of the SF-36 were all significantly lower than norms for Canadian women (29) with the exception of Mental Health Index and, consequently, the Mental Component Scale, but not significantly different between volume groups. Of note is that women with volume differences < 200 ml scored, on average, 10 points higher than women with volume differences of a‰? 200 ml, a value which is clinically meaningful, but this study did not have sufficient power to detect this difference. For pain, measured by the McGill Pain Questionnaire, there were no differences between groups. Pain was also measured using the SF-36 and the study sample of women had significantly more limitation due to pain than did the normative sample. Women from the different volume groups did not differ significantly on subscales of the EORTC QLQ-C30 or on the Arm Module. The dark lines indicate statistically significant paths; the light lines indicate variables that were required for explanation but were themselves not statistically significant. The significant paths were between co-morbidity and disability, pain and disability, volume and physical function aspect of HRQL, and disability and HRQL. There is a strong correlation between the number of symptoms of lymphoedema and the volume difference between arms (double-headed arrow). Path model of the relationships between impairments, disability and health-related quality of life. 3 shows the proportion of women responding positively to each of the symptom questions according to the size of the volume difference between arms.
As shown in Table IV, the sensitivity of 3 or more symptoms was 87% and the specificity was 61%. 4a shows the strong relationship between co-morbidity and disability, such that for every additional co-morbidity, the DASH score was greater by 4.76 units. This sub-study should provide valid estimates of associations between variables because 69% of symptomatic women agreed to attend for assessment and those who declined were very similar to those agreeing.
Arm swelling was not the principal contributor to arm disability post breast cancer surgery, but pain and co-morbidity were predominant factors. The numbers of symptoms reported as present were associated with volume but not disability. This suggests that it is the perceived presence of swelling that is associated with disability rather than the degree of swelling and, within this range of oedema, pain is a stronger determinant of disability than swelling.
A limitation of this study was that the screening questionnaire we used had not previously been tested for validity, although the content of the items was drawn from questions used in other studies (17). The data generated from this study contributes preliminary evidence for the validity of this screening questionnaire (see Fig.
There was also support for concurrent validity because of the strong relationship between symptoms and arm disability as measured by the DASH (see Table IV).
There was a significant relationship between pain and activity limitation and participation restriction. No positive relationship was shown between any of the anthropometric measurements and activity limitation or participation restriction. This may be due to insufficient sample size, or it may be that the perception of having oedema or not having oedema causes the limitation not the observed severity of the lymphoedema.
Women with self-reported lymphoedema had a sub-optimal HRQL; they did not achieve normative values on the SF-36 or the EORTC. A strong relationship was found between pain (McGill pain questionnaire) and decreased HRQL (SF-36, EORTC-QLQ30 and BR23). One of the SF-36 and EORTC subscales is pain, so that subscale would be expected to correlate with the McGill pain questionnaire, but other subscales also had high correlations.
A limitation of this study was that women were not asked to discriminate between pain arising from the breast surgery and pain or discomfort arising from carrying a heavy, lymphoedematous limb. It also added to the information by demonstrating a relationship between volume and one aspect of HRQL, physical function.Treatment is usually aimed at reducing the size and volume of oedema, and oedema reduction is measured to assess the outcome of treatment.
The findings from this study suggest that, for the majority of women, treatment should be aimed at decreasing pain, activity limitations and participation restrictions, and that the success of treatment should be measured by the womena€™s ability to participate. Treatment aimed at reducing arm volume is likely to be more relevant to those with very large arm volumes. The importance of co-morbidity indicates that treatment should focus not only on the sequelae of surgery, but also consider concurrent health conditions and exacerbation of existing musculoskeletal co-morbidities. Further research needs to be undertaken with a sufficiently large sample size to fully understand the role of pain and lymphoedema. As treatment of lymphoedema is aimed at reducing volume it should be ascertained whether a certain volume decrease correlates with a decrease in activity limitation, participation restriction, or HRQL, and if there is a clinically significant level of volume reduction.
Women with self-reported lymphoedema do report activity limitations, participation restrictions and a sub-optimal HRQL. The greater the pain an individual is experiencing the greater are their activity limitation and participation restrictions and the lower is their quality of life.
We were unable to demonstrate a positive relationship between any other individual measurement of impairment and activity limitation, participation restriction or health-related quality of life. It may be that psychological factors play a large part in the reporting of lymphoedema, not impairment, that thinking one has lymphoedema and experiencing pain reduces quality of life and increases activity limitation and participation restriction.With continuing research into treatments for lymphoedema, the use of measurements of pain, activity limitation, participation restriction, and health-related quality of life would add meaningful information to supplement that obtained from traditional volume measurements. Decreasing arm volume alone may not decrease a persona€™s activity limitations, participation restrictions or improve their quality of life.REFERENCES1. Morbidity following sentinel lymph node biopsy versus axillary lymph node dissection for patients with breast carcinoma. Improving surgical outcomes: standardizing the reporting of incidence and severity of acute lymphedema after sentinel lymph node biopsy and axillary lymph node dissection. A systematic review of common conservative therapies for arm lymphoedema secondary to breast cancer treatment. A randomized, controlled, parallel-group clinical trial comparing multilayer bandaging followed by hosiery versus hosiery alone in the treatment of patients with lymphedema of the limb. Treatment-related upper limb morbidity 1 year after sentinel lymph node biopsy or axillary lymph node dissection for stage I or II breast cancer. The effect of physiotherapy on shoulder function in patients surgically treated for breast cancer: a randomized study. Chronic arm morbidity after curative breast cancer treatment: prevalence and impact on quality of life. Upper-extremity volume measurements in women with lymphedema: a comparison of measurements obtained via water displacement with geometrically determined volume.
The European Organization for Research and Treatment of Cancer QLQ-C30: a quality-of-life instrument for use in international clinical trials in oncology. The European Organization for Research and Treatment of Cancer breast cancer-specific quality-of-life questionnaire module: first results from a three-country field study.
Have you noticed that your arms (hand, lower arm, or upper arm) are different sizes from each other?Yesi€SNoi€S2. Do you find that clothing feels tighter around your arm on the operated side than around your arm on the non-operated side?



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Comments to «Breast cancer journals impact factors»

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