Abstract 937: Nef-M1 peptide inhibits oncogenic signaling and tumor angiogenesis in patient-derived xenografts of colorectal cancer

Abstract

Abstract Introduction: The Nef-M1peptide competes effectively with the natural ligand of CXCR4, SDF-1α, to inhibit tumor angiogenesis in colorectal and breast cancer cell models. However, these established cell models may not signify an adequate means for evaluating signaling mechanisms and therapeutic sensitivities. In this study, we evaluated the antiangiogenic effect of Nef-M1 and its inhibitory role in oncogenic signaling in patient-derived xenograft (PDX) of colorectal cancer (CRC). Experimental Design: Surgical specimens of human CRC were cut into 2-to 3-mm3 pieces in antibiotic-containing RPMI 1640 medium. Non-necrotic tumor pieces were selected and implanted subcutaneously in severe combined immunodeficient (SCID) mice. Tumors were harvested when they reached a size of 1500-mm3 (PDX1). The xenografts were repassaged multiple times with retention of genetically human characteristics. One week after implantation the mice started receiving intraperitoneal Nef-M1 injections at 2μg biweekly. Sections from PDX were evaluated for tumor angiogenesis, as measured by microvessel density (MVD) based on immunostaining of endothelial marker (CD31). MVD was determined by light microscopy in areas of invasive tumor containing the highest numbers of microvessels per high power field. Western blot analyses were performed on lysates of both PDX and CRC cell lines to assess the effect of Nef-M1 on the oncogenic signaling pathways. Results: Histological analysis revealed that the surgical specimens and the corresponding PDXs were morphologically similar. However, differences were seen in the untreated and treated PDXs. In mice bearing subcutaneous PDX tumors, Nef-M1 treatment inhibited tumor growth. Immunostaining analyses indicated that control PDXs had well established vascularity, but Nef-M1 treated PDXs had poor vascularization. The average MVD was reduced in Nef-M1 treated PDXs (n = 530) compared to control PDXs (n = 1132) (p<0.017).Western blot analyses of lysates of PDXs indicated that Nef-M1 effectively suppressed the expression of VEGF-A and the activation of p38 and extracellular signal-regulated kinase (ERK) MAP kinases. Suppression of Akt and Wnt/beta-catenin activation was significantly higher in Nef-M1 treated PDXs. Furthermore, Nef-M1 treated CRC cells displayed inhibition of Wnt/beta-catenin signaling as demonstrated by an increased expression of p21 and decreased expression of beta-catenin, cyclin D1, Axin 2 and survivin. The signaling appears linked to the CXCR4 receptor as cells expressing CXCR4 became susceptible to Nef-M1 induced inhibition of MAP kinase, Akt or Wnt/beta-catenin signaling pathways. Conclusions: Treatment with Nef-M1, a peptide antagonist of CXCR4, is a highly promising novel experimental therapeutic strategy against CRC. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251. Citation Format: Venkat R. Katkoori, Harvey L. Bumpers. Nef-M1 peptide inhibits oncogenic signaling and tumor angiogenesis in patient-derived xenografts of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 937.