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Background & Aims Lysophosphatidic acid (LPA) is a potent inducer of colon cancer and LPA receptor type 2 (LPA 2 ) is overexpressed in colon tumors. LPA 2 interacts with membrane-associated guanylate kinase with inverted orientation-3 (MAGI-3) and the Na+/H+ exchanger regulatory factor 2 (NHERF-2), but the biological effects of these interactions are unknown. We investigated the roles of MAGI-3 and NHERF-2 in LPA 2 -mediated signaling in human colon cancer cells. Methods We overexpressed or knocked down MAGI-3 in HCT116 and SW480 cells. The effects of MAGI-3 and NHERF-2 in LPA-induced cell migration, invasion, inositol phosphate generation, and nuclear factor-κB activation were determined. Expression of MAGI-3 and NHERF-2 in human colon tumor tissues was analyzed using tissue microarray analysis. Results NHERF-2 promoted migration and invasion of colon cancer cells, whereas MAGI-3 inhibited these processes. MAGI-3 competed with NHERF-2 for binding to LPA 2 and phospholipase C–β3. However, NHERF-2 and MAGI-3 reciprocally regulated LPA 2 -induced phospholipase C activity. MAGI-3 increased the interaction of LPA 2 with Gα 12 , whereas NHERF-2 preferentially promoted interaction between LPA 2 and Gα q . MAGI-3 decreased the tumorigenic capacity of LPA 2 by attenuating the activities of nuclear factor-κB and c-Jun N-terminal kinase. MAGI-3 and NHERF-2 were expressed differentially in colon adenocarcinomas, consistent with their opposing effects. Conclusions LPA 2 is dynamically regulated by 2 distinct PDZ proteins via modulation of G-protein coupling and receptor signaling. MAGI-3 is a negative regulator of LPA 2 signaling.

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.

G A A b st ra ct s the mechanisms by which polyamines regulate RBPs remain elusive. microRNAs (miRNAs) generally base-pair to sequences in the 3'-untranslated regions (UTR) of labile mRNAs and regulate gene expression posttranscriptionally. This study determined if polyamines regulate CUGBP1 expression through miRNA-503 (miR-503), thereby contributing to the control of intestinal mucosal growth. Methods: Studies were conducted in mice and cultured intestinal epithelial cells (IEC-6 line). CUGBP1 and miR-503 expression was examined by real-time PCR andWestern blotting analyses; CUGBP1 translation was measured by chimeric CUGBP1 3'-UTR luciferease reporter and newly synthesized CUGBP1 protein assays. Interaction of miR-503 with the CUGBP1 mRNA was examined by pmir-Glo reporter system and biotin labeled miR-503 pull-down assays. Polyamine levels were depleted by DFMO (a specific inhibitor of polyamine biosynthesis) but increased by ODC gene overexpression. The functions of miRNA-503 were examined by miR-503-directed siRNA and ectopic overexpression of its precursor. Results: Polyamine depletion by DFMO and fasting increased CUGBP1 protein expression (by ~2.6-fold) but decreased miR-503 in the small intestinal mucosa, which was associated with an inhibition of intestinal mucosal growth as indicated by a decrease in DNA synthesis and villous height. In cultured IECs, polyamine depletion also increased CUGBP1 protein (by ~3.5-fold) but decreased miR-503 (by ~70%), whereas elevated polyamines reduced CUGBP1 (by >85%) and increased miR-503 (by ~2-fold), neither intervention changed CUGBP1 mRNA levels. miR-503 directly bound the CUGBP1 mRNA via CUGBP1 coding region but not its 3'-UTR; this interaction was increased by increasing polyamines but it was repressed by polyamine depletion. miR-503 silencing reduced [miR-503/CUGBP1 mRNA] complex, stimulated CUGBP1 translation, increased its expression (by ~1.5-fold), and inhibited IEC proliferation. Conversely, miR-503 overexpression repressed CUGBP1 translation, decreased its protein levels (by >80%), and enhanced IEC growth. Ectopic CUGBP1 overexpression also repressed cell proliferation by ~65% when measured 6 days after the transfection. Conclusions: These results indicate that 1) polyamines repress CUGBP1 mRNA translation by increasing miR-503 and 2) induced CUGBP1 plays an important role in inhibition of intestinal mucosal growth after polyamine depletion.

mechanisms not fully understood. Since high level of plasma glucose is the main feature of diabetes mellitus, we hypothesized that hyperglycemia induces NPC1L1 expression in diabetic patients via modulation of gene transcription. Therefore, current studies were undertaken to examine the transcriptional regulation of NPC1L1 in human intestinal Caco2 cells in response to glucose. Cells were transiently transfected with different promoter constructs of NPC1L1 gene. Relative promoter activity was measured by luciferase activity normalized to β-galactosidase. The relative abundance of NPC1L1 mRNA was evaluated by real-time PCR. Incubation of Caco2 cells with D-glucose (25 mM, 24 h) significantly increased the NPC1L1 mRNA expression by ~6 fold. In contrast, the removal of D-glucose from the culture medium of Caco2 cells for 24 h significantly decreased the NPC1L1 promoter activity (45.1±2.5 % of control, P