Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The tumor-suppressor gene ARHI (DIRAS3) suppresses ovarian cancer cell migration through inhibition of the Stat3 and FAK/Rho signaling pathways

Abstract

Ovarian cancers migrate and metastasize over the surface of the peritoneal cavity. Consequently, dysregulation of mechanisms that limit cell migration may be particularly important in the pathogenesis of the disease. ARHI is an imprinted tumor-suppressor gene that is downregulated in >60% of ovarian cancers, and its loss is associated with decreased progression-free survival. ARHI encodes a 26-kDa GTPase with homology to Ras. In contrast to Ras, ARHI inhibits cell growth, but whether it also regulates cell motility has not been studied previously. Here we report that re-expression of ARHI decreases the motility of IL-6- and epidermal growth factor (EGF)-stimulated SKOv3 and Hey ovarian cancer cells, inhibiting both chemotaxis and haptotaxis. ARHI binds to and sequesters Stat3 in the cytoplasm, preventing its translocation to the nucleus and localization in focal adhesion complexes. Stat3 siRNA or the JAK2 inhibitor AG490 produced similar inhibition of motility. However, the combination of ARHI expression with Stat3 knockdown or inhibition produced greatest inhibition in ovarian cancer cell migration, consistent with Stat3-dependent and Stat3-independent mechanisms. Consistent with two distinct signaling pathways, knockdown of Stat3 selectively inhibited IL-6-stimulated migration, whereas knockdown of focal adhesion kinase (FAK) preferentially inhibited EGF-stimulated migration. In EGF-stimulated ovarian cancer cells, re-expression of ARHI inhibited FAKY397 and SrcY416 phosphorylation, disrupted focal adhesions, and blocked FAK-mediated RhoA signaling, resulting in decreased levels of GTP-RhoA. Re-expression of ARHI also disrupted the formation of actin stress fibers in a FAK- and RhoA-dependent manner. Thus, ARHI has a critical and previously uncharacterized role in the regulation of ovarian cancer cell migration, exerting inhibitory effects on two distinct signaling pathways.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

References

  • Bast Jr RC, Hennessy B, Mills GB . (2009). The biology of ovarian cancer: new opportunities for translation. Nat Rev Cancer 9: 415–428.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bongard M . (2005). Visual Guide IV: measuring cells with ImageJ, http://naranja.umh.es/atg/tutorials/VGIV-MeasuringCellsImageJ.pdf.

  • Carragher NO, Frame MC . (2004). Focal adhesion and actin dynamics: a place where kinases and proteases meet to promote invasion. Trends Cell Biol 14: 241–249.

    Article  CAS  PubMed  Google Scholar 

  • Cheng GZ, Zhang WZ, Sun M, Wang Q, Coppola D, Mansour M et al. (2008). Twist is transcriptionally induced by activation of STAT3 and mediates STAT3 oncogenic function. J Biol Chem 283: 14665–14673.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chikumi H, Fukuhara S, Gutkind JS . (2002). Regulation of G protein-linked guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and LARG by tyrosine phosphorylation: evidence of a role for focal adhesion kinase. J Biol Chem 277: 12463–12473.

    Article  CAS  PubMed  Google Scholar 

  • Colomiere M, Findlay J, Ackland L, Ahmed N . (2009). Epidermal growth factor-induced ovarian carcinoma cell migration is associated with JAK2/STAT3 signals and changes in the abundance and localization of alpha6beta1 integrin. Int J Biochem Cell Biol 41: 1034–1045.

    Article  CAS  PubMed  Google Scholar 

  • Debidda M, Wang L, Zang H, Poli V, Zheng Y . (2005). A role of STAT3 in Rho GTPase-regulated cell migration and proliferation. J Biol Chem 280: 17275–17285.

    Article  CAS  PubMed  Google Scholar 

  • Etienne-Manneville S, Hall A . (2002). Rho GTPases in cell biology. Nature 420: 629–635.

    Article  CAS  PubMed  Google Scholar 

  • Feng W, Marquez RT, Lu Z, Liu J, Lu KH, Issa JP et al. (2008). Imprinted tumor suppressor genes ARHI and PEG3 are the most frequently downregulated in human ovarian cancers by loss of heterozygosity and promoter methylation. Cancer 112: 1489–1502.

    Article  CAS  PubMed  Google Scholar 

  • Ferreira T, Rasband W . (2011). The ImageJ User Guide; p 144, http://rsb.info.nih.gov/ij/docs/user-guide.pdf.

  • Forcet C, Etienne-Manneville S, Gaude H, Fournier L, Debilly S, Salmi M et al. (2005). Functional analysis of Peutz–Jeghers mutations reveals that the LKB1 C-terminal region exerts a crucial role in regulating both the AMPK pathway and the cell polarity. Hum Mol Genet 14: 1283–1292.

    Article  CAS  PubMed  Google Scholar 

  • Geiger B, Bershadsky A . (2001). Assembly and mechanosensory function of focal contacts. Curr Opin Cell Biol 13: 584–592.

    Article  CAS  PubMed  Google Scholar 

  • Gough DJ, Corlett A, Schlessinger K, Wegrzyn J, Larner AC, Levy DE . (2009). Mitochondrial STAT3 supports Ras-dependent oncogenic transformation. Science 324: 1713–1716.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang M, Page C, Reynolds RK, Lin J . (2000). Constitutive activation of stat 3 oncogene product in human ovarian carcinoma cells. Gynecol Oncol 79: 67–73.

    Article  CAS  PubMed  Google Scholar 

  • Jang AC, Starz-Gaiano M, Montell DJ . (2007). Modeling migration and metastasis in Drosophila. J Mammary Gland Biol Neoplasia 12: 103–114.

    Article  PubMed  Google Scholar 

  • Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D . (2011). Global cancer statistics. CA Cancer J Clin 61: 69–90.

    Article  PubMed  Google Scholar 

  • Kaibuchi K, Kuroda S, Fukata M, Nakagawa M . (1999). Regulation of cadherin-mediated cell–cell adhesion by the Rho family GTPases. Curr Opin Cell Biol 11: 591–596.

    Article  CAS  PubMed  Google Scholar 

  • Kakinuma N, Roy BC, Zhu Y, Wang Y, Kiyama R . (2008). Kank regulates RhoA-dependent formation of actin stress fibers and cell migration via 14-3-3 in PI3K–Akt signaling. J Cell Biol 181: 537–549.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Katabuchi H, Okamura H . (2003). Cell biology of human ovarian surface epithelial cells and ovarian carcinogenesis. Med Electron Microsc 36: 74–86.

    Article  PubMed  Google Scholar 

  • Kurokawa K, Nakamura T, Aoki K, Matsuda M . (2005). Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells. Biochem Soc Trans 33: 631–634.

    Article  CAS  PubMed  Google Scholar 

  • Lidor YJ, Xu FJ, Martinez-Maza O, Olt GJ, Marks JR, Berchuck A et al. (1993). Constitutive production of macrophage colony-stimulating factor and interleukin-6 by human ovarian surface epithelial cells. Exp Cell Res 207: 332–339.

    Article  CAS  PubMed  Google Scholar 

  • Lim Y, Lim ST, Tomar A, Gardel M, Bernard-Trifilo JA, Chen XL et al. (2008). PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility. J Cell Biol 180: 187–203.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liotta LA, Wewer U, Rao NC, Schiffmann E, Stracke M, Guirguis R et al. (1987). Biochemical mechanisms of tumor invasion and metastasis. Anticancer Drug Des 2: 195–202.

    CAS  PubMed  Google Scholar 

  • Long W, Yi P, Amazit L, LaMarca HL, Ashcroft F, Kumar R et al. (2010). SRC-3Delta4 mediates the interaction of EGFR with FAK to promote cell migration. Mol Cell 37: 321–332.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lu Z, Luo RZ, Lu Y, Zhang X, Yu Q, Khare S et al. (2008). The tumor suppressor gene ARHI regulates autophagy and tumor dormancy in human ovarian cancer cells. J Clin Invest 118: 3917–3929.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Luo RZ, Fang X, Marquez R, Liu SY, Mills GB, Liao WS et al. (2003). ARHI is a Ras-related small G-protein with a novel N-terminal extension that inhibits growth of ovarian and breast cancers. Oncogene 22: 2897–2909.

    Article  CAS  PubMed  Google Scholar 

  • Marcoux N, Vuori K . (2005). EGF receptor activity is essential for adhesion-induced stress fiber formation and cofilin phosphorylation. Cell Signal 17: 1449–1455.

    Article  CAS  PubMed  Google Scholar 

  • Martinez-Maza O, Berek JS . (1991). Interleukin 6 and cancer treatment. In vivo 5: 583–588.

    CAS  PubMed  Google Scholar 

  • Naora H, Montell DJ . (2005). Ovarian cancer metastasis: integrating insights from disparate model organisms. Nat Rev Cancer 5: 355–366.

    Article  CAS  PubMed  Google Scholar 

  • Nishimoto A, Yu Y, Lu Z, Mao X, Ren Z, Watowich SS et al. (2005). A Ras homologue member I directly inhibits signal transducers and activators of transcription 3 translocation and activity in human breast and ovarian cancer cells. Cancer Res 65: 6701–6710.

    Article  CAS  PubMed  Google Scholar 

  • Okamura H, Katabuchi H . (2005). Pathophysiological dynamics of human ovarian surface epithelial cells in epithelial ovarian carcinogenesis. Int Rev Cytol 242: 1–54.

    CAS  PubMed  Google Scholar 

  • Orr FW, Wang HH . (2001). Tumor cell interactions with the microvasculature: a rate-limiting step in metastasis. Surg Oncol Clin N Am 10: 357–381, ix-x.

    Article  CAS  PubMed  Google Scholar 

  • Orr FW, Wang HH, Lafrenie RM, Scherbarth S, Nance DM . (2000). Interactions between cancer cells and the endothelium in metastasis. J Pathol 190: 310–329.

    Article  CAS  PubMed  Google Scholar 

  • Parsons JT . (2003). Focal adhesion kinase: the first ten years. J Cell Sci 116: 1409–1416.

    Article  CAS  PubMed  Google Scholar 

  • Raftopoulou M, Etienne-Manneville S, Self A, Nicholls S, Hall A . (2004). Regulation of cell migration by the C2 domain of the tumor suppressor PTEN. Science 303: 1179–1181.

    Article  CAS  PubMed  Google Scholar 

  • Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G et al. (2003). Cell migration: integrating signals from front to back. Science 302: 1704–1709.

    Article  CAS  PubMed  Google Scholar 

  • Roger L, Gadea G, Roux P . (2006). Control of cell migration: a tumour suppressor function for p53? Biol Cell 98: 141–152.

    Article  CAS  PubMed  Google Scholar 

  • Romer LH, Birukov KG, Garcia JG . (2006). Focal adhesions: paradigm for a signaling nexus. Circ Res 98: 606–616.

    Article  CAS  PubMed  Google Scholar 

  • Rosen DG, Wang L, Jain AN, Lu KH, Luo RZ, Yu Y et al. (2004). Expression of the tumor suppressor gene ARHI in epithelial ovarian cancer is associated with increased expression of p21WAF1/CIP1 and prolonged progression-free survival. Clin Cancer Res 10: 6559–6566.

    Article  CAS  PubMed  Google Scholar 

  • Rustin GJ, van der Burg ME, Berek JS . (1993). Advanced ovarian cancer. Tumour markers. Ann Oncol 4 (Suppl 4): 71–77.

    Article  PubMed  Google Scholar 

  • Schaller MD . (2001). Biochemical signals and biological responses elicited by the focal adhesion kinase. Biochim Biophys Acta 1540: 1–21.

    Article  CAS  PubMed  Google Scholar 

  • Schlaepfer DD, Mitra SK . (2004). Multiple connections link FAK to cell motility and invasion. Curr Opin Genet Dev 14: 92–101.

    Article  CAS  PubMed  Google Scholar 

  • Silver DL, Naora H, Liu J, Cheng W, Montell DJ . (2004). Activated signal transducer and activator of transcription (STAT)3: localization in focal adhesions and function in ovarian cancer cell motility. Cancer Res 64: 3550–3558.

    Article  CAS  PubMed  Google Scholar 

  • Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N et al. (1997). Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc Natl Acad Sci USA 94: 3801–3804.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tomar A, Schlaepfer DD . (2009). Focal adhesion kinase: switching between GAPs and GEFs in the regulation of cell motility. Curr Opin Cell Biol 21: 676–683.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Van Aelst L, D'Souza-Schorey C . (1997). Rho GTPases and signaling networks. Genes Dev 11: 2295–2322.

    Article  CAS  PubMed  Google Scholar 

  • Vij N, Sharma A, Thakkar M, Sinha S, Mohan RR . (2008). PDGF-driven proliferation, migration, and IL8 chemokine secretion in human corneal fibroblasts involve JAK2–STAT3 signaling pathway. Mol Vis 14: 1020–1027.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yamashita S, Miyagi C, Carmany-Rampey A, Shimizu T, Fujii R, Schier AF et al. (2002). Stat3 controls cell movements during zebrafish gastrulation. Dev Cell 2: 363–375.

    Article  CAS  PubMed  Google Scholar 

  • Yu Y, Luo R, Lu Z, Wei Feng W, Badgwell D, Issa JP et al. (2006). Biochemistry and biology of ARHI (DIRAS3), an imprinted tumor suppressor gene whose expression is lost in ovarian and breast cancers. Methods Enzymol 407: 455–468.

    Article  CAS  PubMed  Google Scholar 

  • Yu Y, Xu F, Peng H, Fang X, Zhao S, Li Y et al. (1999). NOEY2 (ARHI), an imprinted putative tumor suppressor gene in ovarian and breast carcinomas. Proc Natl Acad Sci USA 96: 214–219.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhai J, Lin H, Nie Z, Wu J, Canete-Soler R, Schlaepfer WW et al. (2003). Direct interaction of focal adhesion kinase with p190RhoGEF. J Biol Chem 278: 24865–24873.

    Article  CAS  PubMed  Google Scholar 

  • Zhong Z, Wen Z, Darnell Jr JE . (1994). Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 264: 95–98.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by Grants NCI P01 CA064602 and 1RO1 CA135354-01 from the National Institutes of Health. We also acknowledge the support of the MD Anderson SPORE in Ovarian Cancer 5P50 CA83639 and the CCSG shared resources funded, in part, by CA 5 P30 CA016672. Support was also provided by the Ovarian Cancer Research Fund through a Program Project Award. DBB was supported by an Excellence Award from the Ovarian Cancer Research Fund. AAA was supported by a Cancer Research UK Clinician Scientist fellowship. We thank Jodie Polan for excellent technical support with confocal microscopy and Jared Burks from the Flow Cytometry and Cell Imaging core laboratory for the live-cell time-lapse microscopy partially supported by the MD Anderson Cancer Center CCSG NCI P30 CA16672.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to R C Bast Jr.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Badgwell, D., Lu, Z., Le, K. et al. The tumor-suppressor gene ARHI (DIRAS3) suppresses ovarian cancer cell migration through inhibition of the Stat3 and FAK/Rho signaling pathways. Oncogene 31, 68–79 (2012). https://doi.org/10.1038/onc.2011.213

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2011.213

Keywords

  • tumor-suppressor gene ARHI
  • migration suppression
  • Stat3
  • RhoA GTPase
  • cytoskeleton

This article is cited by

Search

Quick links