175 The Ets-Transcription Factor Etv1 Regulates Stromal Expansion and Metastasis in Pancreatic Cancer

Background and Aims: Identification of new molecular targets is needed for better clinical care of colorectal cancer (CRC) patients. Na+/H+ exchanger regulatory factor 2 (NHERF2) was initially identified as a scaffold protein of NHE3, but NHERF2 interacts with a larger number of proteins and overexpression of NHERF2 in CRC has been reported. NHERF2 functions as a modulator of phospholipase Cβ and interacts with lysophosphatidic acid receptor type 2 (LPA2) to enhance LPA-dependent colon cancer cell growth in culture. We have previously shown that loss of LPA2 attenuates colon cancer progress in vivo. However, the importance of NHERF2 in cancer has not been studied and it is unclear whether the role of NHERF2 is limited to LPA2-dependent effects. The aim of the present work was to examine the role of NHERF2 in colonic tumor growth. Methods: HCT116 cells with stable knockdown of NHERF2 (shNHERF2), LPA2 (shLPA2) or control (shCont) were generated. The effects of NHERE2 knockdown on HCT116 cell growth was determined in vitro and in a xenograft model. The effects NHERF2 knockdown was compared with LPA2 knockdown. Apcmin/+ mice were crossed with Nherf2-/mice and the growth and number of intestinal tumors were determined in Apcmin/+, Apcmin/+;Nherf2+/-, and Apcmin/+;Nherf2-/mice. Results: Knockdown of NHERF2 significantly decreased the rates of cell proliferation in the presence of 1-10% FBS. Similarly, the rates of xenograft tumor growth of HCT116/ shNHERF2 cells were constantly slower compared with HCT116/shCont cells. Ki-67 staining of the xenografts confirmed the difference in tumor growth between shNHERF2 and shCont cells. Similar difference was obtained between shLPA2 and shCont cells. Intestinal tumor size was significantly smaller in Apcmin/+;Nherf2-/mice compared to Apcmin/+ mice. In contrast, there was no significant difference in the number of tumors between Apcmin/ +;Nherf2-/and Apcmin/+ mice. As a comparison, we reported earlier that both tumor numbers and sizes were decreased in Apcmin/+;Lpa2-/mice. To understand the molecular basis of NHERF2 depletion on colon cancer, we compared transcriptional profiling of control, shNHERF2 and shLPA2 xenograft cells by RNAseq. We found that expression of 549 and 778 genes in the shNHERF2 and shLPA2 cells, respectively, were altered (p< 0.05) compared with control. Interestingly, only 190 genes were altered in both shNHERF2 and shLPA2 cells, with 359 genes in the shNHERF2 and 588 genes in the shLPA2 cells uniquely regulated. Conclusion: Loss of NHERF2 attenuated tumor growth in both Apcmin/+ and xenograft models of colon cancer. Profile of dysregulated genes in NHERF2 deficient tumors indicates that the role of NHERF2 in tumor progress is potentially diverse and not limited to LPA signaling. This study highlights NHERF2 as a novel oncogenic factor and potential target for CRC treatment.