Gastroenterology

Gastroenterology

Volume 140, Issue 3, March 2011, Pages 924-934
Gastroenterology

Basic—Alimentary Tract
MAGI-3 Competes With NHERF-2 to Negatively Regulate LPA2 Receptor Signaling in Colon Cancer Cells

https://doi.org/10.1053/j.gastro.2010.11.054Get rights and content

Background & Aims

Lysophosphatidic acid (LPA) is a potent inducer of colon cancer and LPA receptor type 2 (LPA2) is overexpressed in colon tumors. LPA2 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 LPA2-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 LPA2 and phospholipase C–β3. However, NHERF-2 and MAGI-3 reciprocally regulated LPA2-induced phospholipase C activity. MAGI-3 increased the interaction of LPA2 with Gα12, whereas NHERF-2 preferentially promoted interaction between LPA2 and Gαq. MAGI-3 decreased the tumorigenic capacity of LPA2 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

LPA2 is dynamically regulated by 2 distinct PDZ proteins via modulation of G-protein coupling and receptor signaling. MAGI-3 is a negative regulator of LPA2 signaling.

Section snippets

Cells

HCT116 and SW480 human colon cancer cells were grown and transfected as previously described.12 pcDNA3.1 harboring MAGI-3 or NHERF-2 was described previously.6, 12 Knockdown of MAGI-3, NHERF-2, or LPA2 was performed as previously described.11 Stable expression of LPA2 was achieved by using retroviral pLPCX harboring vesicular stomatitis virus glycoprotein-tagged LPA2, pLPCX/VSVG-LPA2, or pLPCX (Roche, Indianapolis, IN). Unless otherwise stated, cells were serum-starved for 24 hours followed by

NHERF-2 and MAGI-3 Reciprocally Regulate LPA2-Mediated Cellular Functions

To determine the role of MAGI-3 and NHERF-2, we used human colon cancer HCT116 cells, which express NHERF-2 and MAGI-3. We have shown previously that LPA2 is the major LPA receptor in Caco-2 and other colon cancer cells.6 Consequently, silencing of LPA2 expression abrogated LPA-induced migration of HCT116 cells, whereas overexpression of LPA2 enhanced cell migration (Figure 1A and B; Supplementary Figure 1A and B). Consistent with previous reports that NHERF-2 enhances LPA2-evoked cell

Discussion

The role of LPA signaling in the progression of cancer is an active area of study. Since the initial demonstration of the effect of LPA on cell proliferation, the identification of LPA as the ovarian-cancer activating factor in malignant ascites together with the finding of increased levels of LPA in ovarian and other gynecologic cancers have heightened the relevance of LPA to cancer.22, 23, 24 The recent report that free fatty acid generation in cancer cells produces oncogenic lipids, such as

Acknowledgments

The authors thank Dr Pann-Ghill Suh for phospholipase C–β clones; and the authors acknowledge the Emory Digestive Disease Research Development Center (supported by grant DK064399) for providing HCT116 and SW480 cells.

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    Conflicts of interest The authors disclose no conflicts.

    Funding This work was supported by grants from the National Institutes of Health (R01DK071597 and R01DK071597-03S1 to C.C.Y. and R01NS055179 to R.A.H.).

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