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

Gastric Cancer Signaling Pathway

Fig.1 Gastric 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 gastric cancer.

An Introduction to Gastric cancer

Gastric cancer (GC) is one of the world's most common cancers. According to Lauren's histological classification gastric cancer is divided into two distinct histological groups-the intestinal and diffuse types. A more recent classification is based on mucin expression and distinguishes 4 types of gastric cancer: the gastric or foveolar type (G-type), the intestinal type (I-type), the gastric and intestinal mixed type (GI-type) and the neither gastric nor intestinal phenotypes (N-type). In I-type gastric cancer, p53 mutations and allelic deletions of the adenomatous polyposis coli (APC) gene are observed more frequently than in G-type gastric carcinoma. In contrast, microsatellite instability (MSI) is found more often in G-type than in I-type gastric cancer. In 1994, the World Health Organization has classified the Gram-negative spiral-shaped bacterium Helicobacter pylori as a class 1 carcinogen. Since then, it has become clear that one of the most prominent risk factors for spontaneous gastric carcinogenesis is infection with H. pylori. In tumor development, loss of control of cell growth and proliferation, loss of susceptibility to apoptosis, neovascularization through the induction of angiogenesis are important features of all tumors. Here, we show the main signaling pathway of altered gene regulation and therapy in gastric cancer.

1 Main Signaling Pathways in Gastric Cancer Therapy

1.1 RTK signaling cascade

This pathway is characterized by the action of four RTK receptors: c-Met, HER, EGFR, VEGFR. The MAPK/Erk signaling cascade is activated by the RTK receptor, the docking protein is Shc, which binds the receptor to a guanylate exchange factor. Then the signal is transduced to the small molecule Ras, which in turn activates the cascade core units, including Raf, MEK1/2, Erk. The activated Erk dimer can regulate the target protein in the cytoplasm, and can also be transported into the nucleus to phosphorylate various transcription factors that regulate gene expression in the nucleus. Activation of the PI3K/AKT/mTOR pathway can be triggered by the activation of RTKs, PI3K-activating mutations and amplifications, loss of PTEN function due to deletions or mutations, and overexpression or activating mutations of AKT1. AKT1 not only activates S6K, but also inhibits phosphorylation of p53, which affects apoptosis regulation BCL-2, promotes protein synthesis and thus cell proliferation.

1.2 Hippo signaling cascade

In mammals, the Hippo signaling pathway provides tumor-suppressor signaling involved in regulation of diverse cellular processes such as proliferation, apoptosis, survival, migration and differentiation. Once inactivated, the  downstream components in this pathway, such as YAP1, TAZ, will be activated, which lead to tumorigenesis at last. Core to the Hippo pathway is a kinase cascade, the upstream molecules of Hippo signaling pathway, such as Mst1, Mob and WW45, often form a conserved kinase cassette. These molecules can phosphorylate and activate LATS1/2, LATS1/2 in turn phosphorylate and inactivate the YAP/TAZ complex. When dephosphorylated, YAP/TAZ translocate into the nucleus and interact with TEAD family and other transcription factors such CDX2 to induce expression of genes that promote cell proliferation and inhibit apoptosis.

1.3 The Hh signaling cascade

The Hh ligand has been found to be deregulated during the progression in gastric cancer, Hh proteins initiate signaling through binding to the canonical receptor Patched (PTCH1), Hh binding to PTCH1 results in derepression of the proto-oncogene Smoothened (SMO) that results in SMO accumulation and phosphorylation. SMO mediates the large cytoplasmic complex dissociates from the microtubules, and the full-length Gli transcriptional activator is translocated to the nucleus, leading to transcriptional activation of Hh target genes such as cyclin E, cyclin D. And its target genes control the major hallmarks of cancer and cancer stem cells including proliferation, survival, metastasis, angiogenesis and self-renewal.

1.4 Wnt signaling cascade

Specific Wnt ligands bind to their target ‘frizzled’ membrane receptor and interfere with the multi‐protein destruction complex, resulting in downstream activation of gene transcription by β‐catenin. Simplistically, the multi‐protein destruction complex involves Axin and APC serving as scaffolds binding both β‐catenin and GSK3, to facilitate phosphorylation of β‐catenin by GSK‐3β. Phosphorylated β‐catenin is degraded in proteasomes by the ubiquination machinery. β-catenin abnormal activation occurs in gastric cancers and associates with nuclear transcription factors leading to the eventual transcription and expression of target genes such as c‐myc, c‐jun, and cyclin D1 that regulates cell proliferation, survival, morphology, migration, selfrenewal in stem cells, and specification of cell fate during embryonic development.

2 Gastric cancer diagnosis

2.1 Molecular Markers for Gastric cancer

As the significance of each genetic and epigenetic abnormality differs in gastric, and colorectal cancers, the roles of individual abnormalities should be sufficiently understood before these abnormalities are used in diagnosis. Over the past many years, integrated research in molecular pathology has clarified the details of genetic and epigenetic abnormalities of cancer-related genes in the course of the development and progression of gastric cancer. These abnormalities, which include telomerase activation, genetic instability, and abnormalities in oncogenes, tumor suppressor genes, cell-cycle regulators, cell adhesion molecules, and DNA repair genes, could be effective markers in the molecular diagnosis of gastric cancer.

In gastric cancer, p53, APC, and CD44 have been used as markers for differential diagnosis, and TGFα, EGFR, c-Met, HER2, cyclin E, p27Kip1, and CDC25B for degree of malignancy. For the screening of genetic instability, hMLH1 expression is examined. With PCR-SSCP and PCR-RFLP, deletions or mutations of the APC gene and the p53 gene are examined. Mutations of the p53 gene are analyzed in exons 5 to 8, and LOH is analyzed using polymorphism at the BamH1 site of the 3-untranslated region of the gene. Genetic instability is monitored by microsatellite assay. Four loci of two CA repeats and two poly A tracts are examined, and if two or more loci with replication errors are detected, MSI-H is considered to be present. For MSI-H, the presence or absence of mutation of hMLH1 and hMSH2 is determined by PCR-SSCP methods.

2.2 Protein Markers for Gastric cancer

An early diagnosis coupled with good treatment strategy for gastric cancer can significantly improve the survival rates. Hence, serum biomarkers that identify patients with high risk for gastric adenocarcinoma would increase the effectiveness of endoscopy and improve the early diagnosis rate. In past decades, studies have revealed several serum biomarkers for gastric cancer which comprise carcinoembryonic antigen, cancer antigen (CA) 19-9, and CA 72-4, among others.

Traditionally, the detection of serum markers was done using the enzyme-linked immunosorbent assay (ELISA). However, the disadvantage of this method is that it requires larger volumes of sample and incurs high costs compared to the recent method of multiplex detection. The Luminex® xMAP® technology enables the large numbers of biological tests to be conducted and analyzed quickly, cost-effectively, and accurately as compared to the ELISA tests.

3 Targeted therapy for gastric cancer

By identifying the molecular characteristics of GC, a classification of GC subtypes has been proposed, intracellular pathways that contribute to carcinogenesis have been elucidated, and driver genes have been recognized as potential therapeutic targets. Alterations in specific genes that play important roles in diverse cellular functions, such as cell adhesion, signal transduction, cell differentiation, development, metastasis, DNA repair, and glycosylation changes, have been identified. In Table 1-10, selected clinical trials of novel therapeutic targets for the treatment of GC are presented.

3.1 Gastric cancer therapy for RTK pathway

Trastuzumab, TS-1 are designed to target and block HER2 by inhibiting dimerization, by inducing antibody dependent cellular cytotoxicity, and by increasing receptor endocytosis. Cetuximab is an IgG1 monoclonal antibody that inhibits ligand binding to the EGFR and stimulates cell-mediated cytotoxicity. Panitumumab is a recombinant, fully human, IgG2-monoclonal antibody that is highly selective for EGFR. Anti-VEGFR monoclonal antibody Ramucirumab is directed against the VEGFR2 that mediated the majority of downstream effects of VEGF in angiogenesis by binding to VEGFR as a receptor antagonist blocking VEGF/VEGFR. Apatinib highly selectively binds to and strongly inhibits VEGFR2 and decreases the VEGF-mediated endothelial cell migration, proliferation, and tumor microvascular density. Onartuzumab, ABT-700, AMG 337 blocks the binding of HGF to its receptor and inhibits HGF/c-MET-mediated response. The PI3K/AKT/mTOR pathway is frequently activated in GC, two classes of PI3K inhibitors have been evaluated for the treatment of GC: BKM120, PX-886, XL147, which target all PI3K family members; BYL719, which specifically target the p110 catalytic subunit of PI3K. MK-2206, GDC0068 as AKT inhibitors been evaluated in clinical trials. Everolimus is an oral mTOR inhibitor and was established as standard therapy in several types of cancer.

Table 1 Clinical trials of HER2 mAB Trastuzumab

According to statistics, a total of 22 Bevacizumab projects targeting gastric cancer HER2 are currently in clinical stage, of which 14 are recruiting and 8 are not recruiting.

Table 2 Clinical trials of HER2 inhibitor TS-1

Table 3 Clinical trials of EGFR mAB Cetuximab

Table 4 Clinical trials of EGFR mAB Panitumumab

Table 5 Clinical trials of VEGFR mAB Ramucirumab

Table 6 Clinical trials of VEGFR inhibitor Apatinib

According to statistics, a total of 20 Apatinib projects targeting gastric cancer VEGFR are currently in clinical stage, of which 16 are recruiting and 4 are not recruiting.

Table 7 Clinical trials of c-Met inhibitor AMG 337

According to statistics, a total of 23 AMG 337 projects targeting gastric cancer c-Met are currently in clinical stage, of which 15 are recruiting and 7 are not recruiting.

Table 8 Clinical trials of AKT inhibitor GDC0068

Table 9 Clinical trials of mTOR inhibitor Everolimus

3.2 Gastric cancer therapy for Hippo pathway

Verteporfin was identified as YAP1 inhibitors. When livers were treated with VP, overgrowth induced by YAP overexpression, or by inactivation of NF2, was inhibited, thereby demonstrating the therapeutic potential of disrupting YAP1/TAZ-TEAD interactions.

3.3 Gastric cancer therapy for Hh pathway

Sonidegib, Buparlisib, Vismodegib, inhibit Hedgehog signaling through binding to SMO. However, in a phase II clinical trial of patients with metastatic colorectal cancer, treatment with vismodegib exhibited no incremental benefit in combination with FOLFOX.

Table 10 Clinical trials of SMO inhibitor Vismodegib

3.4 Gastric cancer therapy for Wnt pathway

Despite the fact that the Wnt signaling pathway is more difficult to target compared with the Notch and Hh pathways, receptor/ligand interactions, cytosolic signaling components, and nuclear signaling components of the Wnt signaling pathway have been inhibited(e.g., the β-catenin antagonist, ICG-001 and PRI-724).