**2.2. Clinical management**

**Figure 2.** Cancer deaths anticipated in 2011. Estimated leading cancer sites mortality in US and in European Union (EU-27) for the year 2011 expressed as percent of total cancer deaths. Column diagrams highlight the mortality rate within the population specifically affected by colon cancer. Rates are standardized to the World Standard Population.

Loss 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 adenoma‐ tous 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 (Fig‐ ure 3) where it interacts with other proteins like AXINS and GSK3β to make a complex that down-regulates the cellular levels of β-CATENIN (see [7] for review). Activating mutations in β-CATENIN gene have also been observed in more than 10% of CRC [8]. When activated, β-CATENIN interacts in the nucleus with the transcriptional complex LEF/TCF to induce the expression of growth promoting genes, like MYC and CYCLIN D1. Additional waves of genetic and epigenetic alterations (KRAS, P53, etc…) will follow this early set of molecular changes to sustain the progression of the transformation process until carcinoma and meta‐

Source: American Cancer Society and Malvezzi et al, Annals of oncology, 2011, 22(4):947-56.

**2.1. Molecular mechanisms**

436 Drug Discovery

stasis stages.

It 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 suggest‐ ed 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 can‐ cers 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 mor‐ bidity 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 radia‐ tion 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. the cell division machinery) without specific targets, resulting in poor effectiveness and strong side-effects (e.g., oxaliplatin; Table 1).

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

**2.3. Designing new therapies**

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

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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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

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

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

therefore represents a target of choice for the development of CRC therapeutics.

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

the WNT/β-CATENIN signaling pathway.

Phase I clinical trial (Table 2).

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] – remains relevant.


**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: National Cancer Institute database, 2012.
