Abstract 20: PHLDA1/2 contribute to tumor suppression in breast and lung cancer as downstream targets of oncogenic HER2 signaling

PHLDA1 (pleckstrin homology-like domain, family A, member 1) has been reported to act as a tumor suppressor gene in multiple cancers such as melanoma and breast cancer but appears to contribute to intestinal tumorigenesis. PHLDA2 is located in a cluster of imprinted genes on chromosome 11p15.5, an important tumor suppressor gene region. Alterations in this region may be associated with multiple cancers. Loss of imprinting of PHLDA2 was found to mediate placental growth retardation. The pleckstrin homology-like domain (PH domain) mediates protein recruitment to cellular membranes, which is of paramount importance for signal transduction. AKT and ERK are important protein kinases that play key roles in multiple cellular processes such as cell proliferation, apoptosis, transcription and cell migration. Activation and phosphorylation of AKT require binding to PIP through its PH domain. Another family member, PHLDA3 recently has been reported to be a major negative regulator of AKT signaling analogous to PTEN and it was shown that it functions as a negative AKT regulator by the quenching of membrane-bound phosphatidyl-inositol molecules. In our microarray studies of oncogenic EGFR and ErbB2 signaling using erlotinib-treated HCC827, CL-387,785-treated H1975 as well as lapatinib-treated Calu-3 and SKBR-3 cells, PHLDA1 and PHLDA2 were consistently identified as immediately downregulated genes upon oncogenic HER signaling inhibition and this was confirmed using quantitative RT-PCR as well as Western blotting studies. The regulation of PHLDA1/2 by HER signaling inhibition has not been investigated in detail previously. Our results show that the PI3K inhibitor, LY294002 significantly induces PHLDA1 expression but inhibits PHLDA2 expression in a time-dependent manner. Transient transfection mediated PHLDA1 over-expression in SKBR3 inhibited AKT activation and siRNA-mediated suppression of PHLDA2 in Calu3 cells dramatically induced ERK activation. Stable transfection mediated PHLDA1 over-expression in breast cancer cells inhibited both anchorage-dependent and independent cell growth in vitro. MTS cell proliferation studies showed that siRNA mediated suppression of PHLDA2 stimulated Calu3 cell growth. Studies are ongoing to test the potential role of PHLDA1/2 on inhibiting the direct activation of AKT by detailed PIP3 quantification studies. Our results provide strong evidence that PHLDA1/2 are downstream targets of HER2 signaling and suggest complex regulation of PHLDA1 and 2 by ERK and PI3K pathways. Our studies also suggest significant growth inhibitory effects for PHLDA1/2 and suggest that they might potentially act competitively with AKT and ERK to perform tumor suppression function. Our studies identify PHLDA1/2 as novel targets for therapeutic modulation in EGFR and ErbB2-driven cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 20. doi:1538-7445.AM2012-20