Higher levels of STAT3 have been demonstrated in CSCs isolated from liver, bone, cervical and brain thereby cancers, and furthermore treatment of putative glioblastoma stem cells with Stattic results in a dramatic reduction in their formation. Although the Stat3 gene itself was not methylated in any of our studies, qRT PCR analysis demonstrated that compared to non invasive cells, the invasive cells had a significant increase in e pression of Stat3 and ICC detected an increase in active protein as well. However, as seen in Figure S3B, there was a significant reduction in cell proliferation with Stattic treatment. To determine if this was the reason why we observed such a significant reduction in invasion, we took the remaining cells which survived treatment and further placed them through an invasion assay.

The cells were unable to invade toward SCM, indicating that the cells resistant to Stattic induced apoptosis were still sen sitive at inhibiting invasion by lowering STAT3. A similar result was observed in the GBM SCs, since different isolates of the cells responded differ ently to treatment with Stattic. The authors concluded that GBM SCs derived in serum respond to Stattic by undergoing apoptosis, however in those derived using stem cell media they do not. They state that this could be a result of certain GBM SC lines being more differentiated, and are thus more sensitive to STAT3 inhibition. Since inhibition of SO 1 with shRNA and BM ulti mately with LFM A13 decreased invasion toward SCM, we sought to determine if an interaction might be occurring between these differentially methylated genes and STAT3.

To test this, an IP was performed to see if either BM or SO 1 directly interact with STAT3. We found that only SO 1 could directly interact with STAT3 and not BM , and this interaction occurs in both the cytoplasm and the nucleus. In these sub cellular frac tions, we still see an association between SO 1 and STAT3 in shSO 1 cells since e pression of the protein was not fully ablated. Interestingly, decreased e pression of either BM or SO 1 does result in significantly less active STAT3 and a decrease in its DNA binding activity. This observation is not too surprising since BM has been shown to regulate such cellular processes as differentia tion, motility, invasion, apoptosis, and more recently, when inhibited, a delay in tumor growth.

Specifically, within the prostate, BM is up regulated in tumors from both mouse and human specimens com pared to benign tissues, and when over e pressed in cell lines, led to an increase in proliferation and elevated levels of AKT and STAT3. Anacetrapib Albeit having a role in the formation of leukemia, our research is the first to demonstrate that BM may play a significant role in the regulation of prostate CSCs. Both STAT3 and SO 1 are transcription factors that regulate cell fate and differentiation. however a direct interaction between these proteins has never been identi fied.