We therefore examined whether activation of STAT3 via IL-6 stimulation led to repression of Necdin expression in the prostate cancer cell lines DU145 and PC3. These cell lines harbor low levels of constitutively active STAT3 , which can be further induced by stimulation with IL-6. Cells were serum starved for 3 h prior to treatment with IL-6 for 12 or 24 h. Total protein was prepared and analyzed by Western blot. Figure 4A shows that IL-6 stimulation resulted in increased STAT3 activity within the cells and demonstrated corresponding down-regulation of Necdin expression upon IL-6 stimulation, in both cell lines. This confirms that IL-6 is capable of repressing Necdin expression via STAT3 in prostate cancer cells. Since EGFR and Src signaling pathways contribute to STAT3 activation in breast cancers , we evaluated Necdin expression levels in human breast cancer cell lines with different levels of endogenous STAT3 activity. Figure 4B shows that p- STAT3 protein levels were high in MDA-MB-468 cells, slightly lower in MDA-MB-231 and very low in MCF-7 cells. Necdin protein expression inversely correlated with p-STAT3 levels, being expressed at a low level in MDA-MB-468 and MDA-MB-231 cells, but exhibited much CP-358774 higher expression in MCF-7 cells. To test the hypothesis that constitutively activated STAT3 has a causal role in suppressing Necdin expression in tumor cells, we examined whether transient activation of STAT3 signaling could down-regulate Necdin expression. MCF7 cells express high levels of Necdin , however when transiently transfected with v-Src or STAT3-C for 48 h, Necdin protein expression is inhibited. This demonstrates that even a transient 2- fold increase in STAT3 activation in these cells is sufficient to effectively repress the expression of Necdin . The transcriptional profile of a cell expressing constitutivelyactive STAT3 is predicted to be very different compared to a cell where STAT3 is under tight regulation. Our initial hypothesis was that STAT3 promotes widespread changes in global gene expression patterns, including both direct and indirect targets. We took a broad approach by studying global gene expression changes using microarray analysis in cells expressing constitutively- activated STAT3.