Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, associated with a high frequency of KRAS mutations (95%) and loss of negative growth constraints, due, in part, to frequent CDKN2A (85%), TP53 (75%) and SMAD4 (55%) mutations. PDACs also overexpress all three TGF-β isoforms, and high levels of TGF-βs correlates with decreased survival. Recently, we showed that in human PDAC and in murine PDAC (mPDAC) arising in biologically aggressive, oncogenic Kras-driven genetically engineered mouse models (GEMMs), proliferating pancreatic cancer cells (PCCs) often exhibit abundant phosphorylated Smad2 and RB indicating that in these PCCs, canonical TGF-β signaling pathways are active, whereas RB is inactive. Using PCCs derived from a GEMM that expresses oncogenic Kras, but lacks RB (KRC cells), we showed that TGF-β1 activates canonical and non-canonical (MEK, Src, PI3K) pathways, and enhances growth in 3-dimensional (3D) culture. This mitogenic effect is abrogated by type I TGF-β receptor (TβRI) kinase inhibition with SB505124, combined inhibition by MEK/Src or MEK/PI3K, or re-expression of wild-type, but not mutant RB. Thus, loss of RB function enables TGF-β to directly enhance PCC growth. Given that the EGF receptor (EGFR) family can activate these non-canonical pathways and that TGF-β cross-talks with EGFR, we investigated the role of EGFR family members in mediating the mitogenic effects of TGF-β on RB-deficient PCCs. In 3D culture, TGF-β1-enhanced proliferation was inhibited by targeting EGFR with erlotinib and completely suppressed by EGFR/HER2 inhibition with lapatinib. Moreover, lentiviral-mediated silencing of HER2 markedly attenuated TGF-β1-stimulated KRC growth, and this inhibitory effect was potentiated by erlotinib. Thus, TGF-β1's mitogenic effects are mediated, in part, through transactivation of EGFR/HER2, raising the possibility that concomitant use of TβRI inhibitors and lapatinib to inhibit EGFR/HER2 and may represent a novel therapeutic approach in PDAC. Citation Format: Jesse Gore, Samantha L. Deitz, Julie L. Wilson, Murray Korc. TGF-beta cross-talks with the EGF receptor family to promote proliferation of pancreatic cancer cells with dysfunctional RB. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 969. doi:10.1158/1538-7445.AM2014-969