*2.3.3. New anti-angiogenesis therapies*

shown to inhibit the expression of β-CATENIN [24]. Therefore, the modulation of KLF4 ex‐ pression may represent a novel therapeutic approach for β-CATENIN-driven malignancies. LOR-253 [25], a compound that stimulates KLF4 through the inhibition of the human metal-

It is noteworthy that despite the significance of this signaling axis for the treatment of spora‐ dic colorectal cancer, none of the therapies engaged to date in CRC clinical trials are directly targeting WNT/β-CATENIN pathway members. Nonetheless, considering the huge effort done at the research level to identify potential antagonists and the few candidate already en‐ gaged into preclinical studies, no doubt that innovative therapies will emerge from this

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 as‐ pects of anticancer therapy that is intrinsic or acquired drug resistance. Indeed, several stud‐ ies 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 mole‐ cules 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 thera‐ py 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 publica‐ tion reporting the apparition of resistance to PI3K and AKT inhibitors mediated by β-CATE‐ NIN overactivation, may temper this enthusiasm. Depending on the tumor status, from proapoptotic 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]. All to‐ gether, the complexity of these results supports the arrival of a personalized medicine, where a careful profiling of tumors will be useful to stratify patient population in order to test drugs sensitivity and combination with the ultimate goal to make treatments safer and

regulatory transcription 1 (MTF1), is currently in a Phase I clinical study (Table 2).

promising pathway in a near future.

440 Drug Discovery

more effective.

*2.3.2. Acquired tumor resistance and targeted therapies*

As 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 fac‐ tors, 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 treat‐ ment for metastatic CRC. Today, 4 additional therapeutic agents that target VEGF, Ramucir‐ umab [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 re‐ combinant peptide-antibody fusion protein (peptibody) which targets another signaling pathway involved in tumoral angiogenesis, the angiopoietin axis [41]. AMG386, which in‐ hibits 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 initiat‐ ed (May 2012) to evaluate TAS-102, a combination agent composed of the cytotoxic pyrimi‐ dine 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 phos‐ phorylase (TYMP) also known as the platelet-derived endothelial cell growth factor, a po‐ tent 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 pre‐ clinical models and clinical trials, when comparing antibodies with small molecules [35]. These discrepancies in clinical outcome underline the necessity to validate compounds on relevant models, preferentially based on human tissues, very early during drug develop‐ ment process.

#### *2.3.4. Other cellular mechanisms under target*

Modifications in the epigenetic landscape are commonly associated with cancer, but on the contrary to genetic mutations, these changes are potentially reversible and therefore drugga‐ ble. 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. An additional one, the histone deacetylase (HDAC) inhibitor Resmi‐ nostat [43] is currently being studied in patients with CRC, in a phase I/II trial (Table 2).


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 on‐ colytic virotherapies are currently evaluated in clinical trials for metastatic CRC indication, including ColoAd1, derived from an adenovirus [45], NV1020, derived from an Herpes sim‐ plex 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 modali‐ ty. However, it is noteworthy to mention that there are still some difficulties to viral infec‐ tion. 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]. These observations highlight how choosing the right experimental validation model, e.g. 3D cell cultures or spheroids *in vitro*, or patient primary-derived xenografts that retain tumoral

Colon Cancer: Current Treatments and Preclinical Models for the Discovery and Development of New Therapies

http://dx.doi.org/10.5772/53391

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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 com‐

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 prac‐ tical use in clinic [52] but large-scale genomics technology combined with advanced statisti‐ cal 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

It is worth mentioning that most of our understanding of the molecular mechanisms in‐ volved 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. Moreover, isolated cells grown on plastic dish flooded with growth factors appear retrospectively as a very poor model system to elucidate human CRC biolo‐

architecture complexity *in vivo*, will be critical for future clinical success.

pounds entering clinical trials.

to improve drug selection.

**3. Preclinical models**

**3.1. Colon cancer cell lines**

**Table 2.** Anti-cancer drugs in colorectal clinical trials. This table gives an overview of the main colorectal cancer thera‐ pies being currently evaluated in clinical trials. Drugs are presented sorted by type, i.e. small molecule or biologics. For each compound, the pathway target and clinical status is provided. Source: National Cancer Institute database, 2012 and the clinical database of the *Journal of Gene Medicine* (http://www.wiley.com/legacy/wileychi/genmed/clinical).

#### *2.3.5. Unconventional approaches*

Oncolytic viral therapy represents an appealing alternative therapeutic strategy for the treat‐ ment of CRC, both as single agent or in combination with existing clinical regimens. Onco‐ lytic 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 limit‐ ed 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 on‐ colytic virotherapies are currently evaluated in clinical trials for metastatic CRC indication, including ColoAd1, derived from an adenovirus [45], NV1020, derived from an Herpes sim‐ plex 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 modali‐ ty. However, it is noteworthy to mention that there are still some difficulties to viral infec‐ tion. 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]. These observations highlight how choosing the right experimental validation model, e.g. 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 com‐ pounds 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 prac‐ tical use in clinic [52] but large-scale genomics technology combined with advanced statisti‐ cal 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.
