Abstract B4: Everolimus inhibits invasion and angiogenesis in a KRAS-mutated mesenchymal-differentiated colorectal cancer xenografts
Authors: Tijeras-Raballand Annemilai , Santos Celia Dos , Serova Maria , Riveiro Maria Eugenia , de Gramont Armand , Faivre Sandrine , Raymond Eric 
Published: March 2013

Background: KRAS mutations accounting for 32% to 40% of colorectal cancers and epithelial-to-mesenchymal transition (EMT) have been associated with resistance to targeted therapies in colorectal carcinomas. The growth factor receptor- and/or oncogenic KRAS-dependent AKT-activations may lead mTOR to promote the expression of transcription factors involved in EMT activation in cancer cells. mTOR also plays an important role in survival of cancer cells in response to hypoxia during tumor angiogenesis. In this study, we evaluated the effects of everolimus on KRAS-mutated mesenchymal differentiated colon cancer cells in culture and in xenografts.

Methods: The cellular and signaling effects of everolimus was investigated in parental COLO205-S cells (WT KRAS) that display an epithelial phenotype and their derived KRAS mutated (G13D/exon 2) mesenchymal counterparts, COLO205-R cells (Serova, 2010) using MTT assay and Western Blot. COLO205-S and COLO205-R were engrafted in nude mice treated with placebo or 10mg/kg everolimus for 3 weeks. Tumor invasion in mice was evaluated by counting tumor cells invading the skin. Angiogenesis and necrosis were evaluated using CD31 and HE staining, respectively.

Results: Acquisition of KRAS mutation and EMT in COLO205-R cells was associated with constitutive AKT activation and resistance to several targeted therapies including lapatinib, and enzastaurin. Inhibition of mTOR with everolimus decreased the proliferation in COLO205-S and COLO205-R cells at high concentrations. KRAS mutation and EMT in COLO205-R tumors were associated with an increased tumor growth rate compared to COLO205-S tumors (mean tumor volume after 17 days of treatment: 1650mm3 vs. 953mm3, respectively, p&lt;0.05). Pathological examinations also showed that COLO205-R cells were more invasive (number of invading cells in surrounding tumor tissues: 321 vs. 202) and more angiogenic than COLO205-S cells in tumors. In COLO205-S and COLO205-R xenografts everolimus significantly reduced tumor growth in nude mice compared to placebo (mean tumor growth inhibition: 35% and 34%, respectively). Everolimus also decreases the invasive potential of cancer cells in tissues surrounding the tumors in COLO205-R xenografts (number of invading cells per field: 63 vs. 321). The number and the size of vessels were significantly reduced (&gt;2-fold) in everolimus treated tumors, suggesting antiangiogenic properties of this mTOR inhibitor. In addition, everolimus also induced larger necrosis area in tumors as compared with placebo (in COLO205-R xenografts: 59% vs. 28%, respectively, p&lt;0.05; in COLO205-S xenografts: 31% vs 15%, respectively, p&lt;0.05).

Conclusions: Everolimus displayed effects on cancer cell proliferation and invasion and may also inhibit tumor angiogenesis in KRAS-mutated mesenchymal-differentiated colorectal cancer models. This suggested that odds antiangiogenic effects might be expected using everolimus despite adverse genetic and differentiation profiles of colon cancer cells.