The effect of endogenous parathyroid hormone-related protein (PTHrP) on gene expression in breast cancer cells was studied. We suppressed PTHrP expression in MDA-MB-231 cells by RNA interference and analyzed changes in gene expression by microarray analysis. More than 200 genes showed altered expression in response to a PTHrP-specific small interfering (si) RNA (siPTHrP). Cell cycle-regulating gene CDC2 and genes (CDC25B and Tome-1) that control CDC2 activity showed increased expression in the presence of siPTHrP. CDC2 activity was also found to be higher in siPTHrP-treated cells. Studies with PTHrP peptides 1-34 and 67-86, forskolin, and a PTH1 receptor (PTH1R)-specific siRNA showed that PTHrP regulates CDC2 and CDC25B, at least in part, via PTH1R in a cAMP-independent manner. Other siPTHrP-responsive genes included integrin ␣6 (ITGA6), KISS-1, and PAI-1. When combined, siRNAs against ITGA6, PAI-1, and KISS-1 could mimic the negative effect of siPTHrP on migration, whereas siKISS-1 and siPTHrP similarly reduced the proliferative activity of the cells. Comparative expression analyses with 50 primary breast carcinomas revealed that the RNA level of ITGA6 correlates with that of PTHrP, and higher CDC2 and CDC25B values are found at low PTHrP expression. Our data suggest that PTHrP has a profound effect on gene expression in breast cancer cells and, as a consequence, contributes to the regulation of important cellular activities, such as migration and proliferation.

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Rho GDP dissociation inhibitor B (Rho-GDIB), an inhibitor of Rho GTPases, is primarily expressed by hematopoietic cells but is also found in epithelial cancer cells. Recently, we have identified Rho-GDIB as a target of the transcription factor Ets1. Here, we show that, in breast cancer cells, Ets1 regulates Rho-GDIB expression and binds to the upstream region of the Rho-GDIb gene. Furthermore, in primary breast cancer, Rho-GDIB is coexpressed with Ets1. Studying the function of Rho-GDIB in breast cancer, we found that a Rho-GDIB-specific small interfering RNA increased cellular migration but also decreased the expression of cyclooxygenase-2 (Cox-2) oncogene as shown by microarray, quantitative reverse transcription-PCR, and Western blot analyses. Further studies revealed that Rho-GDIB regulates Cox-2 gene at least partly on the transcriptional level, most likely by activating nuclear factor of activated T cells 1 (NFAT-1). Vav-1, an interaction partner of Rho-GDIB, was also found to interfere with Cox-2 expression and NFAT-1 cellular distribution, suggesting a cooperative action of Rho-GDIB and Vav-1 on Cox-2 expression. To explore the importance of Rho-GDIB for the survival of breast cancer patients, two cohorts, including 263 and 117 patients, were analyzed for clinical outcome in relation to Rho-GDIB RNA and protein levels, respectively. Expression of Rho-GDIB was not associated with either disease-free or overall survival in the two patient population. Our data suggest that the expression of Rho-GDIB in breast cancer is neither beneficial nor disadvantageous to the patient. This may be the net effect of two opposing activities of Rho-GDIB, one that suppresses tumor progression by inhibiting migration and the other that stimulates it by enhancing Cox-2 expression. [Cancer Res 2007;67(22):10694-702]

Supplementary Figure 2 from <i>Cyclooxygenase-2</i> Is a Target Gene of Rho GDP Dissociation Inhibitor β in Breast Cancer Cells