Gene therapy for colon cancer pdf

Science, Technology and Medicine open access publisher.Publish, read and share novel research. Colon Cancer: Current Treatments and Preclinical Models for the Discovery and Development of New TherapiesSamuel Constant1, Song Huang1, Ludovic Wiszniewski1 and Christophe Mas1[1] OncoTheis, 14 Chemin des aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland1. The Intestinal Stem Cell Signature Identifies Colorectal Cancer Stem Cells and Predicts Disease Relapse. An anti-Wnt-2 monoclonal antibody induces apoptosis in malignant melanoma cells and inhibits tumor growth. Taniguchi H, Yamamoto H, Hirata T, Miyamoto N, Oki M, Nosho K, Adachi Y, Endo T, Imai K, Shinomura Y. The patch allows for nonsurgical treatment of colon cancer using three types of therapy at once. BOSTON, July 29 (UPI) -- A patch delivering one, two or three types of colon cancer treatment could make surgery for the disease unnecessary, according to researchers who developed it. Gene therapy, chemotherapy, thermal ablation, or a combination of the three, can be delivered by a hydrogel patch, a method shown to shrink tumors in mice, preventing recurrence and extending the rodents' survival, according to a study published in the journal Nature Materials.
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IntroductionMore than 10 years after the first sequencing of the human genome and despite major advances in scientific and technological expertise into drug research and development processes (R&D), the fact remains that we are facing a dearth of new drugs. 5 (UPI) -- Head Start early education programs were found in a recent study to have an especially good effect for children with disabilities, researchers report.
COLO-320 HSR cells were incubated with PBS (a) or pEGFP-N3-lipofectamine mixture (b) or gfp-VLP (c). The wild-type DPD gene contains a GT nucleotide sequence at exon 14 (wt), required for normal catabolism of 5-FU to dihydro-5-FU.
Overall success rate of clinical trials for phases I-III from 2003 to 2010 corresponding to 4275 drugs and 7300 indications (a), success rate for phase II and III divided according to therapeutic areas (b) and overall success rate within specific oncologic areas (c).
Estimated leading cancer sites mortality in US and in European Union (EU-27) for the year 2011 expressed as percent of total cancer deaths. This table gives an overview of the main colorectal cancer therapies being currently evaluated in clinical trials.
Sequential steps leading to the establishment of a CRC primary Patient-Derived Tumor Xenograft collection. Indeed, the number of drugs approved by the US Food and Drug Administration (FDA) has roughly fallen to 50% over the last ten years [1].
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The morphology of patient tumor tissues was compared with their corresponding xenografts using paraffin-embedded sections stained with hematoxylin and eosin (H&E).
Cells were fixed at 72 h post-transfection or infection and visualized by fluorescence confocal microscopy.
A G-to-A transition at the exon 14 splice site leads to deletion of the entire exon during transcription (mut), and synthesis of a nonfunctional protein. Column diagrams highlight the mortality rate within the population specifically affected by colon cancer.
Briefly, a CRC tumor fragment coming from surgical waste is directly xenografted in an immunodeficient mouse (Passage 0).
Unfortunately for pharmaceutical companies, at present this attrition in drug discovery combined with the expiration of major product patents logically lead to the development of generics. Patients with low hepatic DPD activity cannot efficiently metabolize 5-FU (which is used to treat colon and breast cancer) and its accumulation causes toxicity. After successful engraftment, new fragments are taken from the mouse hosted human tumor and xenografted again in multiple immunodeficient mice (Passage 1). Facing both a major medical need and an obvious economical challenge, there is an urgent need to make significant improvements in the research output.Analyses of the clinical trials landscape reveal that a large number of promising drug leads fail in late stages, mainly in phase II, with an overall failure rate of 67% (Fig.
A collection of fragments from the resulting tumors can then be cryopreserved in a tissue bank for subsequent experiments or directly re-engrafted in mice for expansion (P2, P3, etc…). In tumour tissue, the active metabolite of 5-FU, 5-fluorodeoxyuridine monophosphate (5-FdUMP), inhibits TS.
All studies agree on the reasons by pinpointing either insufficient efficacy (~55%) or safety issues (~20%) as major causes of human trials failure [2, 3].
Remarkably, the therapeutic area showing the largest number of failures is oncology, with only 29% of success rate in Phase II and 34% in Phase III (Fig.1b).
Within oncology indications, the status of colorectal cancer (CRC) is the most dramatic with an overall drug approval of only 3% (Fig.1c) over the last 10 years!
More surprisingly, more than half of the drugs currently approved to treat CRC work through the general inhibition of DNA synthesis and cellular division, instead of targeting molecular processes specifically involved in CRC progression (Table 1).
This strategy may save years of efforts and millions of dollars, giving that the average usual time for developing a new drug is ten years and with a total cost amount to billions of dollars.But in contrast, because a new drug has to show a benefit compared to an already approved treatment, the number of patients involved in a pivotal trials is increasing more and more in order to reach significance, and a similar trend is noted for the duration of the trial, that is directly linked to safety. That was the time of the inevitable high-throughput screening, which combined with the “all-Omics” supposed to reduce costs and blew up success rates [4]. As we have seen, this approach, maybe too reductionist in the sense that it does not allow getting an idea of the full biological properties (ADME, toxicity,etc…) of a compound at an early stage, has favored the quantity instead of quality and has not kept its promises [1].Today, efforts have to be made to clearly address the early clinical discovery steps, with the goal to better qualify “leads” to increase the signal-to-noise ratio of drugs entering into clinical trials. This point of view is supported by the important failure rate subsisting in Phases III (Fig1b), suggesting an overestimation of the efficacy of candidate molecules during preclinical tests. One of the important reasons may be the use of irrelevant models or models not predictive enough.
Therefore, the development of relevant and predictive models is key to increase the quality of preclinical researches and to increase the success rate of new drugs. Consequently, the foundations of the drug discovery process have to be reconsidered by giving definitively more emphasis to the quality of preclinical validations and by encouraging the design of new pertinent models, including human 3D (three dimensional) in vitro cell models and tissue explants.
This article is intended to give an overview of the current knowledge about CRC and the different models commonly used to study CRC, in order to identify the most suitable bio-systems for optimal development of new CRC therapies. The first part will describe the pathology and its molecular basis, and the various drugs that are currently in clinical use or under development.
Then, in the second part we will review and discuss the use of cancer cell line collections, genetically engineered mouse models (GEM), primary human tumors xenografts (PDX) and ex vivo organotypic cultures (EVOC) to identify and validate anticancer colon therapeutics.

Colorectal CancerColorectal cancer is one of the major health concerns in the Western world. CRC is the second most frequently diagnosed cancer in men and women, right after lung cancer.
It represents the second leading cause of cancer-related deaths, both in the United States and in Europe, with a significant rate of 9% and 13% of total cancer deaths, respectively (Fig.2).
The vast majority (~75%) of colon cancers are sporadic adenocarcinomas, arising from mutations in the epithelial cells lining the wall of the intestine that is in continuous renewal. CRC often begins as an adenomatous polyp, a benign growth on the interior surface of the organ. Molecular mechanismsLoss of APC function is the initial molecular event that leads to adenoma formation. Indeed, germline mutations in the gene APC have been identified as the cause of familial adenomatous polyposis (FAP), an inheritable intestinal cancer syndrome [5], and APC is mutated in more than 80% of all sporadic cancers [6].
APC belongs to the WNT signaling pathway (Figure 3) where it interacts with other proteins like AXINS and GSK3? to make a complex that down-regulates the cellular levels of ?-CATENIN (see [7] for review). Activating mutations in ?-CATENIN gene have also been observed in more than 10% of CRC [8]. Through several cytoplasmic components, the signal is transduced to ?-CATENIN, which enters the nucleus and forms a complex with LEF and TCF4 to activate transcription of WNT target genes. Mutations in APC, Axin and ?-CATENIN genes lead to constitutive activation of WNT signaling and ultimately to cancer development. Clinical managementIt is commonly accepted that CRC results from complex interactions between inherited and environmental factors, with a large contribution of dietary and life style factors as suggested by wide geographical risk variations. However, the primary risk factor of CRC is age, as 90% of the cases are diagnosed over the age of 50 years [9].
Surgical removal remains the most efficient treatment for early stage colorectal cancer, and may be curative for cancers that have not spread. Patients whose cancer is detected at an early, localized stage present a 5-year survival around 90% [9]. For these reasons, US and European Union have implemented preventive screening programs that have contributed to slightly reduce morbidity and mortality [10].Unfortunately, as in many other forms of cancer, colon cancer does not display too many symptoms, develops slowly over a period of several years, and only manifests itself when the disease begins to extend. Adjuvant chemotherapy in combination with surgery or radiation is then the usual treatment. However, 5 of the 9 anti-CRC drugs approved by the FDA today are basic cytotoxic chemotherapeutics that attack cancer cells at a very fundamental level (i.e. These figures underline the urgent need to expand the standard therapy options by turning to more focused therapeutic strategies.
In recent years, combination of basic chemotherapies with targeted therapies, in the form of humanized monoclonal antibodies directed against the vascular endothelial growth factor VEGF (Bevacizumab) to prevent the growth of blood vessels to the tumor, or directed against the EGF receptor (Cetuximab, Panitumumab) to block mitogenic factors that promote cancer growth, have been introduced as possible therapeutic protocol and used routinely to treat standard CRCs, as well as metastatic CRCs (Table 1).
During the preparation of this manuscript (August 2012), another recombinant protein active against angiogenesis, Aflibercept, has been approved by the FDA for the treatment of metastatic CRC in second-line therapy (Table 1). Designing new therapiesA classical approach of drug design in oncology is to identify modulators of specific signal transduction pathways that are important for tumor growth, survival, invasion, and metastasis. These results can be achieved by modulating the pathway at different levels, from the membrane receptor to the final nuclear transcription factors (Figure 3).
It is now well documented that a number of critical pathways regulating stem cell maintenance and normal developmental processes (e.g. HEDGEHOG-GLI, NOTCH, TGF) are also involved in the self-renewal and differentiation of cancer stem cells whose tumors are initiated [20].
To date, the only compound designed to specifically disrupt ?-CATENIN is developed for the treatment of Familial Adenomatous Polyposis (FAP), an inherited form of colon cancer. This new RNAi-based therapeutic known as CEQ508 consists of a modified E.coli bacterium that is able to express and deliver a shRNA to the epithelial cells of the gastrointestinal mucosa after ingestion by the patient [23]. KLF4 (Kruppel-like factor 4) is a tumor suppressor factor which is typically deficient in a variety of cancers, including colorectal cancer. In addition to controlling the cell cycle regulator cyclin D1, KLF4 has also been shown to inhibit the expression of ?-CATENIN [24]. Therefore, the modulation of KLF4 expression may represent a novel therapeutic approach for ?-CATENIN-driven malignancies.
Acquired tumor resistance and targeted therapies In the recent years, a cohort of oncogenes, including BRAF, KRAS, NRAS, PI3K, PTEN and SMAD4, have been found mutated in CRC with significant frequencies ranging from 6% (NRAS) to 40% (KRAS) [26].
These observations pinpoint one of the most challenging aspects of anticancer therapy that is intrinsic or acquired drug resistance.
Indeed, several studies have shown that these mutations are associated with the lack of response to Cetuximab and Panitumumab (anti-EGFR therapies) observed in a subset of chemorefractory metastatic CRCs, suggesting that the corresponding deregulated signaling pathways are responsible for the occurrence of resistance of the tumor to the clinical treatment [27-28]. As a result, downstream key components (mostly protein kinases) of these constitutively activated growth-related signaling cascades have become targets for drug development. Small molecules inhibitors of BRAF (ARQ 736), MEK (Selumetinib, PD-0325901), PI3K (PX-866, BEZ235, BKM120), and MET (Tivantinib) that were able to reverse resistance to EGFR inhibitor therapy in pre-clinical studies [29-31] are currently in CRC Phase II clinical studies (Table 2).
This new class of drugs appears therefore as a promising third-line therapeutic strategy for colon cancer patients, especially after recurrence of tumor resistance. However, a recent publication reporting the apparition of resistance to PI3K and AKT inhibitors mediated by ?-CATENIN overactivation, may temper this enthusiasm. Depending on the tumor status, from pro-apoptotic tumor suppressor, PI3K or AKT inhibitors could become metastatic inducers [32].
Similar side effect induction mechanisms have also been reported in CRC for the BRAF(V600E) inhibitor Vemurafenib that triggers paradoxical EGFR activation [33]. New anti-angiogenesis therapiesAs previously mentioned, until recently the humanized monoclonal antibody Bevacizumab against VEGF was the only anti-angiogenesis agent approved by FDA. It is now completed by Aflibercept, a recombinant protein consisting of the key domains of VEGF receptors 1 and 2.
The compound captures and blocks all isoforms of VEGF-A and VEGF-B growth factors, as well as placental growth factors [34]. Due to improvement in the understanding of the critical role of angiogenesis in the maintenance of CRC tumors and the spread of their metastasis, anti-angiogenesis has become an area of active investigation [35]. However, the recent failure in Phase III first-line studies of two promising compounds (Sunitunib in 2009 and Cediranib in 2010) has cast serious doubt on that strategy. Therefore, the approval of Aflibercept provides timely support to the further development of anti-angiogenics as treatment for metastatic CRC. Today, 4 additional therapeutic agents that target VEGF, Ramucirumab [36], Icrucumab [37], Regorafenib [38] and Vatalanib [39-40] are under clinical evaluation (Table 2).
This battery of anti-angiogenics is supplemented by AMG386, a recombinant peptide-antibody fusion protein (peptibody) which targets another signaling pathway involved in tumoral angiogenesis, the angiopoietin axis [41]. AMG386, which inhibits the interaction between the ligands ANGIOPOIETIN-1 and ANGIOPOIETIN-2 with their TIE2 receptor, is currently in Phase II.
Finally, a phase III trial was also recently initiated (May 2012) to evaluate TAS-102, a combination agent composed of the cytotoxic pyrimidine analog TFT and a thymidine phosphorylase inhibitor (TPI) with antineoplastic activity (Table 2). TAS-102 mechanism of action is based on the inhibition of the thymidine phosphorylase (TYMP) also known as the platelet-derived endothelial cell growth factor, a potent angiogenic factor [42]. In this context, it is important to point out that differences in the efficiency to block angiogenesis and tumor progression have been observed between preclinical models and clinical trials, when comparing antibodies with small molecules [35]. Other cellular mechanisms under targetModifications in the epigenetic landscape are commonly associated with cancer, but on the contrary to genetic mutations, these changes are potentially reversible and therefore druggable.
Most of the epigenetic drugs discovered to date modulate DNA methylation or histone acetylation.

Four epigenetic drugs have already been approved by FDA for use in clinic against various cancers. Unconventional approachesOncolytic viral therapy represents an appealing alternative therapeutic strategy for the treatment of CRC, both as single agent or in combination with existing clinical regimens. Oncolytic viruses, like the vaccinia virus (a virus previously used for worldwide vaccination against smallpox), have the property to selectively infect and destroy tumor cells with limited or no toxicity to normal tissues.
These viruses efficiently replicate in tumor tissue, cause tumor lyses and stimulate antitumor immune response. During the last decade, numerous mutants have been engineered to improve their tumor specificity and antitumor efficacy, and to allow tracking of viral delivering by non-invasive imaging [44]. No less than five oncolytic virotherapies are currently evaluated in clinical trials for metastatic CRC indication, including ColoAd1, derived from an adenovirus [45], NV1020, derived from an Herpes simplex virus [46], Reolysin, a reovirus [47], and JX-594 [48] and GL-ONC1 [49] both derived from vaccinia viruses, reflecting the many hopes carried by this emerging treatment modality.
However, it is noteworthy to mention that there are still some difficulties to viral infection. Solid tumors have a complex microenvironment that includes disorganized surrounding stroma, poor vascular network as well as high interstitial fluid pressure.
All these parameters will limit viral delivery since viral penetration directly depends on cellular packing density and adhesion between cancer cells [50]. Moreover, hypoxia reduces viral replication, and therefore oncolytic efficiency, without affecting tumoral cells viability [51]. 3D cell cultures or spheroids in vitro, or patient primary-derived xenografts that retain tumoral architecture complexity in vivo, will be critical for future clinical success.This inventory of new drugs for the treatment of colorectal cancer highlights the diversity of approaches being considered to combat the disease. Whether based on small molecules, humanized antibodies or modified viruses, their success in further clinical assessment is largely related to the quality of their preclinical evaluation. This is why both the choice of appropriate existing model systems and the development of more clinically relevant and predictive pre-clinical models appear critical in overcoming the high attrition rates of compounds entering clinical trials.Current research is also focusing on the development of biomarkers that will be useful for the early detection of CRC, as well as for fine-tuning drug regimen and following efficacy during trials and treatments.
To date, only a few markers have been recommended for practical use in clinic [52] but large-scale genomics technology combined with advanced statistical analyses should generate soon new biomarker panels for CRC diagnosis [53]. Then, it will be interesting to see how these biomarkers could be implemented in preclinical stages to improve drug selection. 3.
Colon cancer cell linesIt is worth mentioning that most of our understanding of the molecular mechanisms involved in CRC come from studies done on mouse or human cell lines that represent only a highly selected fraction of the original tumor and that may have acquired in vitro additional genetic abnormalities. Clearly, the scientific community has taken into account these limitations, as shown by the growing interest for more complex models (e.g. However, although imperfect, colon cancer cell lines still represent a unique resource that can be extremely valuable in term of genetic manipulation and high-throughput screening, with cell viability, cell proliferation or promoter specific reporter activity being the usual endpoints followed. Several initiatives have been launched to maximize their utility in large scale drug discovery programs.
In an attempt to identify new active molecules, over 100,000 chemical compounds were pharmacologically tested in this cell line set.
But disappointingly, most of the selected positive candidates were typical cytotoxics, affecting cancer cells via general fundamental cellular processes, like cell cycle regulation. These cell lines are under further characterization by sequencing for mutations in known human oncogenes.
Interestingly, this resource can be screened on demand for any chemical or biological agent.
The Cancer Genome ProjectThe emergence of tumor acquired resistance to pharmacological inhibitors linked to mutations in driver oncogenes has recently revived the interest for cancer cell lines.
Indeed, an extensive characterization of cell lines at the genomic and genetic levels will allow determining a genetic profile predictive of drug sensitivity. Such a signature will help to stratify patient population and identify efficient therapeutics combination, as long as cell lines reflect real tumor biology. Using current high throughput techniques this program intends to provide information on mutations, copy number variations, single nucleotide polymorphisms (SNPs) and microsatellite instability of usual cancer cell lines. Biomimetic cell culture modelsThe derivation of a cancer cell line from the primary tumor is not an obvious process, and for many cancers, few if any cell line can be obtained. A success rate of less than 10% has been reported for the establishment of human colon cancer cell lines grew immediately in vitro from fresh tumors [56].
Elasticity of the surrounding microenvironment has been pointed out as a critical parameter of in vitro cell growth. Indeed, culture plastic dishes are much more rigid than the epithelial wall of the intestine (10000 kPa vs 40 kPa). More importantly, depending on the stiffness of the substrate, cells can be differentially sensitive to drugs in term of spreading and apoptosis-induction, notably because of the expression and presentation of surface receptors [57].
Therefore, the choice of an appropriate biomimetic substrate that will preserve the in vivo phenotype appears decisive not only for cell survival but also for clinical relevance. Colon cancer stem cell modelsCancer stem cells (CSCs) are a discrete self-renewing tumor cell subpopulation that can differentiate into multiple lineages, drive tumor growth and metastasis. Moreover, CSCs are thought to be responsible for tumor recurrence after chemotherapy and radiotherapy. One of the characteristic of the CSCs is their ability to form spherical cell colonies when they are cultured in chemically defined serum-free medium at a relative low density [58]. This model, also called colonospheres, constitutes a unique in vitro system to elaborate therapeutic strategies that specifically target colon CSCs, like oncolytic adenoviruses developed to target specific CSCs antigens (e.g. Multicellular Spheroid modelsEarly stage development of novel anti cancer treatment requires in vitro methods able to deliver fast, reliable and predictive results. To select the most active molecule lead in a library, pharmaceutical industry has turned its attention to High Throughput Screening (HTS) tests which mimic human tissues. Furthermore, 3-Dimensional (3D) test system has been widely accepted as being more informative and relevant than classical 2D cell systems. Combination of HTS and 3D models such as the multicellular tumor spheroid model has been pointed out having the potential to increase predictability of clinical efficacy from in vitro validation therefore contributing savings in both development cost and time [59].
Advantages of spheroids compared to classical 2D cell line culture have been reported [60]. Indeed, proteomic analysis of multicellular spheroids versus monolayers cultures identifies differential protein expression relevant to tumor cell proliferation, survival, and chemoresistance. Consequently, spheroids strategy has been used for the screening of new anticancer agents, like compounds that modulate apoptosis pathways [61].Standardized spherical microtissue production in a 96 or 384-well hanging-drop multiwell plate format on robotic platform has been successfully achieved by 3D Biomatrix and Insphero AG. Formation of standardized spheroids rely on the use of A431.H9, a human epithelial carcinoma cells, [62] or the colon cancer cell line HCT116 [63]. Interestingly, loss of cancer drug activity in HCT-116 cells during spheroid formation in a 3D spheroid cell culture system has been reported [64]. Spheroid cell models also enable the study of colon cancer chemoresistance and metastasis [65]. Chemically induced animal modelsColon cancer can be induced in mouse by specific carcinogens like 1,2-dimethylhydrazine (DMH) and azoxymethane (AOM). Exposure of the mouse intestine to these chemicals triggers rapid and reproducible tumor induction which recapitulates the adenoma-carcinoma sequence that occurs in human sporadic CRCs, with the notable exception however of the invasive and metastatic stage. Interestingly, differences in genetic mutations that arise in chemically induced colon tumor models are largely carcinogen specific.
K-Ras mutations are predominant in the DMH model, while AOM treated mice exhibit tumors with activating mutations in the ?-catenin gene [66].

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