Our group has recently identified ARHI (ras homolog I), a novel tumor suppressor gene that can inhibit growth, motility and invasiveness of breast and ovarian cancers. ARHI is expressed in normal breast and ovarian epithelial cells, but its expression is lost or markedly downregulated in a majority of breast and ovarian cancers. The mechanisms by which ARHI inhibit growth, motility and invasion of cancer cells are still poorly understood. To identify proteins that interact with ARHI, a yeast two-hybrid was performed with a normal breast cDNA library. ARHI was found to interact with a MAP-1 (microtubule-associated protein) like protein, suggesting that ARHI may interfere the function of microtubules in the cytoskeleton. Using immunoprecipitation with anti-ARHI antibody, we carried out proteomic analysis to identify potential ARHI associated proteins. Mass spectrographic analysis showed that ARHI interacts with tubulin β. Immunopreciptation of the adenovirus-ARHI-infected SKBr3 cell lysate using either anti-ARHI or anti-tubulin β further confirmed that ARHI binds tubulin β, directly or indirectly. Re-expression of ARHI showed that ARHI could reduce tubulin expression and significantly inhibit mitosis in cancer cells by about 10-fold. Immunofluorescent staining of breast cancer cell lines (SKBr3 and MDA-MD-231) demonstrated intense perinuclear localization of microtubules with well-ordered cytoplasmic patterns. After re-expressing ARHI in these cells, however, the pattern of the microtubuler organization was altered with markedly decreased perinuclear localization and weak staining of cytoplasmic microtubules. Taken together, our results indicate that ARHI interacts directly or indirectly with tubulin and inhibits tumor cell mitosis. Immunostaining results also suggest that the inhibition of tumor cell growth by ARHI is related to the disorganization of microtubular structure.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]