Evidence for G-quadruplex in the promoter of vegfr-2 and its targeting to inhibit tumor angiogenesis

Tumor angiogenesis is mainly mediated by vascular endothelial growth factor (VEGF), a pro-angiogenic factor produced by cancer cells and active on the endothelium through the VEGF receptor 2 (VEGFR-2). Here we identify a G-rich sequence within the proximal promoter region of vegfr-2, able to form an antiparallel G-quadruplex (G4) structure. This G4 structure can be efficiently stabilized by small molecules with the consequent inhibition of vegfr-2 expression. Functionally, the G4-mediated reduction of VEGFR-2 protein causes a switching off of signaling components that, converging on actin cytoskeleton, regulate the cellular events leading to endothelial cell proliferation, migration and differentiation. As a result of endothelial cell function impairment, angiogenic process is strongly inhibited by G4 ligands both in vitro and in vivo. Interestingly, the G4-mediated antiangiogenic effect seems to recapitulate that observed by using a specific interference RNA against vegfr-2, and it is strongly antagonized by overexpressing the vegfr-2 gene. In conclusion, we describe the evidence for the existence of G4 in the promoter of vegfr-2, whose expression and function can be markedly inhibited by G4 ligands, thereby revealing a new, and so far undescribed, way to block VEGFR-2 as target for anticancer therapy.     

The Akt Inhibitor ISC-4 Synergizes with Cetuximab in 5-FU-Resistant Colon Cancer

Phenylbutyl isoselenocyanate (ISC-4) is an Akt inhibitor with demonstrated preclinical efficacy against melanoma and colon cancer. In this study, we sought to improve the clinical utility of ISC-4 by identifying a synergistic combination with FDA-approved anti-cancer therapies, a relevant and appropriate disease setting for testing, and biomarkers of response. We tested the activity of ISC-4 and 19 FDA-approved anticancer agents, alone or in combination, against the SW480 and RKO human colon cancer cell lines. A synergistic interaction with cetuximab was identified and validated in a panel of additional colon cancer cell lines, as well as the kinetics of synergy. ISC-4 in combination with cetuximab synergistically reduced the viability of human colon cancer cells with wild-type but not mutant KRAS genes. Further analysis revealed that the combination therapy cooperatively decreased cell cycle progression, increased caspase-dependent apoptosis, and decreased phospho-Akt in responsive tumor cells. The synergism between ISC-4 and cetuximab was retained independently of acquired resistance to 5-FU in human colon cancer cells. The combination demonstrated synergistic anti-tumor effects in vivo without toxicity and in the face of resistance to 5-FU. These results suggest that combining ISC-4 and cetuximab should be explored in patients with 5-FU-resistant colon cancer harboring wild-type KRAS.     

The prevalence and prognostic significance of KRAS mutation in bladder cancer, chronic myeloid leukemia and colorectal cancer

Mutations in the KRAS gene have been shown to play a key role in the pathogenesis of a variety of human tumours. However the mutational spectrum of KRAS gene differs by organ site. In this study, we have analysed the mutational spectrum of KRAS exon 1 in bladder tumours, colorectal cancer (CRC) and chronic myeloid leukemia (CML). A total of 366 patients were included in the present study (234 bladder tumours, 48 CRC and 84 CML). The KRAS mutations are absent in BCR/ABL1 positive CML. This result suggests that BCR/ABL1 fusion gene and KRAS mutations were mutually exclusive. The frequency of KRAS mutations in bladder cancer was estimated at 4.27 %. All of mutations were found in codon 12 and 90 % of them were detected in advanced bladder tumours. However the correlation between KRAS mutations and tumour stage and grade does not report a statistical significant association. The KRAS mutations occur in 35.41 % of patients with CRC. The most frequent mutations were G12C, G12D and G13D. These mutations were significantly correlated with histological differentiation of CRC (p = 0.024). Although the high frequency of KRAS in CRC in comparison to bladder cancer, these two cancers appear to have the same mutational spectrum (p > 0.05).     

Hypoxia Activates the K-Ras Proto-Oncogene to Stimulate Angiogenesis and Inhibit Apoptosis in Colon Cancer Cells

The KRAS proto-oncogene plays a key role in the development of many human tumors and is commonly activated by somatic mutation or signaling through specific growth factor receptors. However, the interaction between the micro-environment and K-ras activity has not been defined. Hypoxia invariably develops as tumors outgrow their supply of oxygen. A series of well-orchestrated cellular adaptations occur that stimulate angiogenesis and enhance survival of the tumor in hypoxic conditions. Our previous studies demonstrated that mutant KRAS alleles can interact with hypoxia to induce vascular endothelial growth factor (VEGF) in colon cancer. We sought to determine whether similar hypoxic responses are also present in tumors without a KRAS mutation. Hypoxia consistently increased the levels of activated, GTP-bound K-ras in colon cancer cell lines with a wild-type KRAS gene, and this depended upon the activation of c-Src. Inhibition of c-Src by PP2 treatment or siRNA knockdown blocked the hypoxic activation of K-ras. This activation of K-ras did not depend upon EGFR and resulted in the phosphorylation of Akt and induction of VEGF expression. In addition, activation of K-ras significantly blocked apoptosis in hypoxic conditions. These studies reveal a unique adaptive mechanism in hypoxia that activates K-ras signaling in the absence of a mutant KRAS oncogene.     

Reflections on drug resistance to KRASG12C inhibitors and gene silencing/editing tools for targeting mutant KRAS in cancer treatment

KRAS is the most commonly mutated oncogene in human tumors, especially in lung, pancreatic, and colorectal cancers. Small-molecule inhibitors targeting mutant KRAS^G12C demonstrated promising anti-tumor effect in patients with non-small cell lung cancer harboring KRAS^G12C mutation, while the intrinsic and acquired drug resistance occurred frequently and might be inevitable. Unlike the protein-level inhibition approach, gene silencing/editing tools for DNA-level knockout and RNA-level knockdown of mutant KRAS may be advantageous since these approaches directly eliminate the production of mutant KRAS-encoded protein. An in-depth understanding of KRAS biology, drug resistance to KRAS^G12C inhibitors and gene silencing/editing methods applied for anti-KRAS therapy may give new insight into the therapeutic strategy for cancer treatment.