Small Molecule Therapeutics Selective Disruption of Rb–Raf-1 Kinase Interaction Inhibits Pancreatic Adenocarcinoma Growth Irrespective of Gemcitabine Sensitivity

Inactivation of the retinoblastoma (Rb) tumor suppressor protein is widespread in human cancers. Inactivation of Rb is thought to be initiated by association with Raf-1 (C-Raf) kinase, and here we determined how RRD-251, a disruptor of the Rb–Raf-1 interaction, affects pancreatic tumor progression. Assessment of phospho-Rb levels in resected human pancreatic tumor specimens by immunohistochemistry (n1⁄4 95) showed that increased Rb phosphorylation correlated with increasing grade of resected human pancreatic adenocarcinomas (P 1⁄4 0.0272), which correlated with reduced overall patient survival (P 1⁄4 0.0186). To define the antitumor effects of RRD-251 (50 mmol/L), cell-cycle analyses, senescence, cell viability, cell migration, anchorage-independent growth, angiogenic tubule formation and invasion assays were conducted on gemcitabine-sensitive and -resistant pancreatic cancer cells. RRD-251 prevented S-phase entry, induced senescence and apoptosis, and inhibited anchorage-independent growth and invasion (P < 0.01). Drug efficacy on subcutaneous and orthotopic xenograftmodelswas tested by intraperitoneal injections of RRD-251 (50mg/ kg) alone or in combination with gemcitabine (250 mg/kg). RRD-251 significantly reduced tumor growth in vivo accompanied by reduced Rb phosphorylation and lymph node and liver metastasis (P < 0.01). Combination of RRD-251 with gemcitabine showed cooperative effect on tumor growth (P < 0.01). In conclusion, disruption of the Rb–Raf-1 interaction significantly reduces themalignant properties of pancreatic cancer cells irrespective of their gemcitabine sensitivity. Selective targeting of Rb–Raf-1 interaction might be a promising strategy targeting pancreatic cancer. Mol Cancer Ther; 12(12); 1–13. 2013 AACR. Introduction Pancreatic adenocarcinoma remains the fourth leading cause of cancer-related deaths (1). Even after potential curative surgery and adjuvant therapies, the overall 5year survival rate remains approximately 5% (2). In addition, chemotherapywith gemcitabine aloneprovides poor long-term outcome, with the possible acquisition of gemcitabine resistance by a variety of molecular aberrations that have not been fully defined (3, 4). Therefore, further understanding themolecularmechanisms involved in the genesis, progression, chemoresistance, and metastasis of pancreatic cancersmight lead to the development ofmore effective therapeutic strategies. The retinoblastoma (Rb) tumor suppressor protein is the major regulator of the mammalian cell-cycle progression (5–7). Rb prevents cell-cycle progression by physically interacting with E2F family of transcription factors repressing their transcriptional activity (8). Phosphorylation of Rb protein by cyclin-dependent kinases, mainly Cdk2, Cdk4, and Cdk6, leads to the dissociation of E2Fs from Rb, enabling them to induce downstream target genes that are necessary for cell-cycle progression (7). Thus, agents that can prevent Rb phosphorylation and maintain its functional state can be expected to have a cytostatic effect with subsequent antitumor activities (5). Although Rb phosphorylation is mediated mainly by cyclin-dependent kinases, Raf-1 could bind and phosphorylate Rb early in the cell cycle, facilitating subsequent phosphorylation and complete inactivation (5, 9). An 8 amino-acid peptide inhibiting Rb–Raf-1 interactions confirmed this hypothesis (10), and development of a smallmolecule inhibitor of the Rb–Raf-1 interaction, RRD-251, prevented Rb phosphorylation and subsequent growth of lung cancer and melanoma (7, 11). In invasive pancreatic adenocarcinoma, Rb expression correlates with Authors' Affiliations: Departments of Tumor Biology, Drug Discovery, and Anatomic Pathology, H. Lee Moffitt Cancer Center & Research Institute, Tampa; and Department of Surgery, University of Florida-Gainesville, Gainesville, Florida Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Current address forM. Verma: University ofMichigan, AnnArbor,Michigan; and current address for S. Singh: National Institute of Biomedical Genomics, Kalyani, West Bengal, India. Corresponding Author: S.P. Chellappan, H. Lee Moffitt Cancer Center andResearch Institute, Tampa, FL 33612. Phone: 813-745-6892; Fax: 813745-6748; E-mail: [email protected] doi: 10.1158/1535-7163.MCT-12-0719 2013 American Association for Cancer Research. Molecular Cancer Therapeutics www.aacrjournals.org OF1 on June 21, 2017. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Published OnlineFirst October 9, 2013; DOI: 10.1158/1535-7163.MCT-12-0719