Colorectal Cancer Overview - Signaling Pathway. Diagnostics Marker. Targeted Therapy and Clinical Trials.

Colorectal Cancer Signaling Pathway

Fig.1 Colorectal cancer signaling pathway. Targeted agents (listed in orange boxes) include those in clinical use (colored in green) and those in preclinical or early phase development (colored in red) for the treatment of advanced stage colorectal cancer.

An Introduction to Colorectal Cancer

Colorectal cancer (CRC) is the second largest cause of cancer-related deaths in Western countries. CRC arises from the colorectal epithelium as a result of the accumulation of genetic alterations in defined oncogenes and tumour suppressor genes (TSG). It appears that some individuals are more prone to colorectal cancer than others. It has been suggested that about 25% of colon cancer patients have some degree of familial background, and another 15% have a strong family history involving a first or second degree relative. Perhaps as many as 5% of colon cancers are caused by single-gene syndromes, most commonly familial adenomatous polyposis (FAP) or Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer, or HNPCC. The common affected pathways include Wnt signaling, receptor tyrosine kinase (RTK) signaling, TGFβ and TNF-α signaling, which inducing cell growth, proliferation and differentiation. Here, we show the main signaling pathway of altered gene regulation and therapy in gastric cancer.

1 Main Signaling Pathways in Colorectal Cancer Therapy

1.1 Wnt signaling cascade

Wnt signaling pathway is activated by Wnt ligands bind to the lipoprotein receptor-related protein and the frizzled receptor, the cytosolic disheveled protein is activated, and it inhibits CTNNB1 phosphorylation and its consequent degradation. Thus, the protein accumulates in the cytosol and eventually translocates to the nucleus, where it binds to T-cell specific transcription factor 7, and both participate in the activation of downstream target genes, such as c-Myc, promoting cell proliferation. Coloretal tumors showed the Wnt pathway was affected with inactivating mutations of APC and activating mutations of β-Catenin.

Therapy

1.2 RTK Signaling Cascade

An important group of signaling pathways involved in CRC is the group triggered by RTKs. These include the VEGFR, IGFR1, PI3K, EGFR, and MET pathways. RTKs are auto-phosphorylated upon ligand binding, which results in the activation of Ras and induction of serine/threonine kinase Raf. Raf phosphorylates MEK1/2 which in turn phosphorylate and activate Erk1/2, JNK1/2 and p38. PI3K is activated by RTK autophosphorylation and results in the activation of Akt which also induces mTOR within the mTORC1 complex. Akt is also regulated by mTORC2 complex. PLCγ activation leads to the activation of COX-2, promoting angiogenesis. These events play an essential role in proliferation, differentiation, survival and cell migration.

1.3 TGF-β Signaling Cascade

TGF-β signals through a heteromeric cell-surface complex of two types of transmembrane serine/threonine kinases, named ‘type I’ and ‘type II’ receptors. After ligand-induced activation of the receptor, Smad2 and/or Smad3 interact transiently with the TβRI receptor (RI), and then dissociate from the receptor to form a heterotrimeric complex comprising two receptor-activated Smads and Smad4. This complex then translocates into the nucleus, where it interacts at the promoter with transcription factors with sequence-specific DNA binding to regulate gene expression. Inactivation of TGF-β signaling in CRC also contributes to carcinogenesis. The increased proliferation results from the loss of the growth inhibition mediated by TGF-β.

1.4 TNF-α Signaling Cascade

Tumor necrosis factor alpha (TNF-α) activates both survival and proliferation pathways along with apoptotic pathways via TNFR. Upon binding of the homotrimer TNF-α, TNFR-1 trimerizes, and silencer of death domain protein is released. TNFR recruits the adaptor proteins receptor interacting protein (RIP), TNFR-associated factor (TRAF), and Fas-associated death domain (FADD). These adaptor proteins recruit key molecules that are responsible for further intracellular signaling. When TNFR signals apoptosis, FADD binds procaspase-8, which is subsequently activated. This activation initiates a protease cascade leading to apoptosis. Alternatively, the binding of TRAF initiates a pathway of phosphorylation steps resulting in the activation of NF-κB transcription factor via NIK and the IKK complex. The NF-κB induce transcription of antiapoptotic, proliferative, immunomodulatory, and inflammatory genes.

2  Colorectal Cancer Diagnosis

2.1 Molecular Markers for Colorectal Cancer

Rapidly growing insights into the molecular biology of colorectal cancer (CRC) and recent developments in gene sequencing and molecular diagnostics have led to high expectations for the identification of molecular markers to be used in optimized and tailored treatment regimens. However, However, many markers investigated suffer from technical shortcomings, resulting from lack of quantitative techniques to capture the impact of the molecular alteration. This understanding has recently led to the more comprehensive approaches of global gene expression profiling or genome-wide analysis to determine prognostic and predictive signatures in tumors. KRAS hopefully become biological prognostic and/or predictive markers.

MSI reflects the presence of a defective mismatch repair mechanism and is characterized by somatic alterations in the size of simple repeat microsatellite nucleotide sequences common throughout the genome. As a consequence, genes containing simple repeat sequences, such as TGFβRII, EGFR, or BAX, are often mutated in these tumors. The prognostic significance of both KRAS mutations (by PCR) and p21 staining (by IHC) has been assessed in a multitude of studies, with conflicting results. The majority of reported studies show KRAS mutation as an adverse prognostic indicator, indicating the need for adjuvant therapy. Some investigations have shown that KRAS mutation is prognostic only in some stages of the disease or only when associated with specific mutation types (transition or transversion, specific codons), or when related to specific types of recurrence or in combination with other molecular abnormalities (p53 mutation).

2.2 Protein Markers for Colorectal Cancer

The existing diagnostic methods for colorectal cancer include fecal occult blood test, colonoscopy and carcinoembryonic antigen, but their sensitivity and accuracy is inadequate. Therefore, it is essential to identify novel serum biomarkers for CRC with high sensitivity and specificity.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) are high throughput techniques for the analysis of complex biological specimens with high sensitivity and specificity. SELDI-TOF-MS combined with ProteinChip technology has been used to identify novel biomarkers for CRC, breast cancer, thyroid cancer, endometriosis and other diseases. This technology can detect biomarkers in serum samples effectively, but it can only obtain the molecular weight data of biomarkers without protein sequences. In the present study, several technologies were combined to purify and identify a CRC-related protein.

3 Targeted therapy for colorectal cancer

Over the past years, the number of targeted agents used in various malignancies has increased dramatically. Currently there are seven FDA approved targeted agents in colorectal cancer with many more in development and in clinical trials (Table 1-11）. These targeted agents fall under the broad classification of mAbs, fusion proteins and small molecule inhibitors.

3.1 Colorectal cancer therapy for Wnt pathway

A proposed strategy has been the development of inhibitors against molecules that do not constitute the central core of the pathway. Inhibition of the interaction between CTNNB1 and Creb-binding protein by the small molecule ICG 001, the Creb-binding

protein/CTNNB1 antagonist PRI-724 has been proposed for testing with chemotherapy and bevacizumab in patients with newly diagnosed metastatic colorectal cancer (mCRC).

3.2 Colorectal cancer therapy for RKT pathway

Among these molecules, VEGF is the most important regulator of the angiogenic process identified to date and has shown markedly increased expression in advanced colorectal tumors. Bevacizumab, Ramucirumab and Aflibercept are the anti-angiogenic agents available for therapy for mCRC that binds directly to all major isoforms of VEGF-A, forming a protein complex that prevents further binding to VEGF receptors. Cetuximab and Panitumumab block downstream signaling by binding to the EGFR’s extracellular domain, which prevents further ligand binding, sterically hinders dimerization with other RTKs and induces EGFR degradation. Onartuzumab and Rilotumumab are monoclonal antibodies developed against the extracellular domain of cMET and the hepatocyte growth factor, respectively. Buparlisib, BYL719, is an oral compound that specifically inhibits PI3K in the PI3K/AKT signaling pathway. MK-2206 binds to and inhibits the activity of AKT isoforms. Several clinical trials have been completed of mTOR inhibitors such as Everolimus or Temsirolimus, which are analogs of rapamycin. Trametinib binds to and inhibits the activity of MEK1/2.

Table 1 Clinical trials of VEGF mAB Bevacizumab

According to statistics, a total of 70 Bevacizumab projects targeting colorectal cancer VEGF are currently in clinical stage, of which 30 are recruiting and 40 are not recruiting.

Table 2 Clinical trials of VEGF mAB Ramucirumab

Table 3 Clinical trials of VEGF mAB Aflibercept

Table 4 Clinical trials of EGFR mAB Cetuximab

According to statistics, a total of 59 Cetuximab projects targeting colorectal cancer EGFR are currently in clinical stage, of which 31 are recruiting and 28 are not recruiting.

Table 5 Clinical trials of EGFR mAB Panitumumab

According to statistics, a total of 31 Panitumumab projects targeting colorectal cancer EGFR are currently in clinical stage, of which 22 are recruiting and 9 are not recruiting.

Table 6 Clinical trials of PI3K inhibitor Buparlisib

According to statistics, a total of 31 Buparlisib projects targeting colorectal cancer PI3K are currently in clinical stage, of which 22 are recruiting and 9 are not recruiting.

Table 7 Clinical trials of PI3K inhibitor BYL719

Table 8 Clinical trials of AKT inhibitor MK-2206

Table 9 Clinical trials of mTOR inhibitor Temsirolimus, Everolimus, Sirolimus

Table 10 Clinical trials of MEK1/2 inhibitor Trametinib

3.3 Colorectal cancer therapy for TGF-β pathway

Inactivation of TGF-β signaling in CRC also contributes to carcinogenesis. The genes encoding the downstream proteins of TGF-β signaling are also affected in CRC, such as the loss of the genes SMAD2 and SMAD4 caused by the deletion of chromosome 18q. More clinical trials regarding this topic are needed.

3.3 Colorectal cancer therapy for TNF-α pathway

TNF-α stimulates the acute phase response, mediates the inflammatory response, activates other cytokines, and increases vascular permeability. The compound Lenalidomide has been used in clinical trials for the treatment of patients with CRC. This drug inhibits TNF-a production, stimulates T cells, reduces the serum levels of VEGF and basic fibroblast growth factor, and inhibits angiogenesis. Dulanermin binds to and activates TRAIL receptors 1 and 2, which might activate caspases and induce p53-independent apoptosis in TRAIL receptor 1/2-expressing tumor cells.

Table 11 Clinical trials of TNF-α inhibitor Lenalidomide