**2.3. Designing new therapies**

Moreover, in more advanced cases, when CRC has spread to distant organs in the form of metastasis and escape any surgical therapy, the 5-year survival dramatically drops to 12% [9]. 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 chemo‐ therapies 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, Panitumu‐ mab) to block mitogenic factors that promote cancer growth, have been introduced as possi‐ ble 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 recombi‐ nant protein active against angiogenesis, Aflibercept, has been approved by the FDA for the treatment of metastatic CRC in second-line therapy (Table 1). This new VEGF inhibitor has demonstrated a significant advance over currently available therapy in a Phase III study

Nonetheless, CRC remains a devastating disease since nearly 35-40% of all patients diag‐ nosed will die from the disease (Fig.2). Accordingly, the expansion and the development of new path of therapy, like drugs specifically targeting the self-renewal of intestinal can‐ cer stem cells - a tumor cell population from which CRC is supposed to relapse [12] –

**Table 1.** Anti-cancer colorectal drugs approved by the Food and Drug Administration. Drugs are presented sorted by type, i.e. small molecule or biologics (including recombinant protein and monoclonal antibody, noted mAb). Source:

(improvement in response rate and in overall survival; [11]).

remains relevant.

438 Drug Discovery

National Cancer Institute database, 2012.

A 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 meta‐ stasis. Because aberrant WNT signaling has been shown to drive the earliest step of colorec‐ tal tumorigenesis (see before), the WNT/β-CATENIN pathway appears critical for CRC and therefore represents a target of choice for the development of CRC therapeutics.

#### *2.3.1. Oncogenic WNT/β-CATENIN pathway as a therapeutic target*

Many experiments have demonstrated that disruption of the WNT signaling pathway lead to consistent growth inhibition and apoptosis of CRC cell lines and effective inhibition of tu‐ mor growth in CRC animal models. These results can be achieved by modulating the path‐ way at different levels, from the membrane receptor to the final nuclear transcription factors (Figure 3). A significant number of proof of principle studies have already been published, including targeted inhibition of WNT1-2, FZD or LRP5/6 receptors by antibodies or inhibito‐ ry fusion proteins [13-15], inactivation of the pathway by re-expression of WIF1 (WNT-in‐ hibitory factor-1) or through restoration of tumor suppressors APC and Axin expression [16], expression of a dominant-negative mutant to block the transcription factor TCF4 [17], and finally direct inhibition of β-CATENIN using RNA interfering technologies *in vitro* and *in vivo* [18, 19]. Taken together, these data provide a strong biological rationale for drugging the WNT/β-CATENIN signaling pathway.

In addition, recent evidence also points to a role for WNT/β-CATENIN signaling in the modulation of cancer stem cells. It is now well documented that a number of critical path‐ ways regulating stem cell maintenance and normal developmental processes (e.g. HEDGE‐ HOG-GLI, NOTCH, TGF) are also involved in the self-renewal and differentiation of cancer stem cells whose tumors are initiated [20]. Consequently, in a way similar to the HEDGE‐ HOG-GLI pathway [21], a large number of high-throughput cell-based screening strategies, mainly designed to disrupt TCF/β-CATENIN interaction, have led to the identification of promising molecules as inhibitors of WNT/β-CATENIN pathway (reviewed by [22]).

However, currently few of these compounds have progressed beyond the preclinical stages. To date, the only compound designed to specifically disrupt β-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 mu‐ cosa after ingestion by the patient [23]. CEQ508, which has shown efficiency in silencing β-CATENIN and preventing polyp formation in the APCmin FAP mouse model, is now in a Phase I clinical trial (Table 2).

Alternatively, a possible way of interfering with the WNT/β-CATENIN cascade, even if not direct, may reside in the manipulation of KLF4 levels. KLF4 (Kruppel-like factor 4) is a tu‐ mor suppressor factor which is typically deficient in a variety of cancers, including colorec‐ tal cancer. In addition to controlling the cell cycle regulator cyclin D1, KLF4 has also been 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 metalregulatory transcription 1 (MTF1), is currently in a Phase I clinical study (Table 2).

*2.3.3. New anti-angiogenesis therapies*

ment process.

*2.3.4. Other cellular mechanisms under target*

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‐

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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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).

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 promising pathway in a near future.

#### *2.3.2. 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 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 more effective.
