PIK3R3-STAT3 Feedback Loop Mediates Trastuzumab Resistance

Object:To determine the role of PIK3R3 in mediating Trastuzumab resistance in HER2+ breast cancer and gastric cancer.Method:First, we analyzed the relationship of PIK3R3 with clinicalpathology breast cancer using online database, including The Cancer Genome Atlas (TCGA). Then, we obtained the Trastuzumab-resistant cell lines (BT474-R, SKBR3-R, NCI-N87-R) after a long time (more than six months) Trastuzumab treatment in Trastuzumab-sensitive breast cancer cell lines (BT474, SKBR3) and gastric cancer cell line (NCI-N87). An analysis of the expression of PIK3R3 in Trastuzumab sensitive and resistant cell lines was following. Again, we overexpressed PIK3R3 in Trastuzumab sensitive cell lines, then observed the effect of PIK3R3 in Trastuzumab resistance in vitro and in vivo. Finally, we observed the effect of Trastuzumab treatment after low express PIK3R3 in Trastuzumab-resistant cell lines.Results:First, PIK3R3 was relateved to HER2+breast cancer patients poor Prognosis and survival without tumours. Second, Trastuzumab-resistant cell lines had higher PIK3R3 level than the patnenal cell lines. Third, overexpress of PIK3R3 in Trastuzumab sensitive cell lines induced Trastuzumab resistance in vitro and in vivo. Fourth, low expression of PIK3R3 in Trastuzumab resistant lines sensitive to cell Trastuzumab treatment in vitro.Conclusion:PIK3R3 was relateved to HER2+ breast cancer patients poor Prognosis. PIK3R3 was highly expressed in Trastuzumab-resistant cell lines. PIK3R3 expression could induce Trastuzumab resistance. Low expression of PIK3R3 restored Trastuzumab sensitivity in Trastuzumab resistance in cell lines.Object:To study the mechanism of PIK3R3 facilitating Trastuzumab resistance in HER2+ breast cancer and gastric cancer.Method:Firstly, we overexpressed PIK3R3 in Trastuzumab sensitive cell lines or low expressed PIK3R3 in Trastuzumab resistant cell lines, followed by detecting of changes of resistance-associated downstream signaling pathways (AKT and STAT3). Then, after over-expression of PIK3R3, we inhibited downstream signaling pathways with specific inhibitors or specific siRNA repressor in vitro, and detected the effects of Trastuzumab treatment in cell lines. Finally, after over-expression of PIK3R3, we inhibited downstream signaling pathways with specific inhibitors in vivo, observed the effect of Trastuzumab treatment.Result:Firstly, After high or low expression PIK3R3 expression, no significant changes were detected in AKT phosphorylation levels. And we found that PIK3R3 had significantly affect on the level of phosphorylation of STAT3. Secondly, PIK3R3 can active IL-6/STAT3 inflammatory loop. Thirdly, after high expression PIK3R3 followed by STAT3 inhibitor, we found STAT3 inhibitor can significantly eliminate the Trastuzumab resistant effect of PIK3R3 in vitro. And similar effect was detected in vivo.Conclusion:PIK3R3 causes Trastuzumab resistance through STAT3 pathway instead of AKT pathway. And inhibiting STAT3 pathway eliminates the Trastuzumab resistant effect of PIK3R3.Object:To study the exact mechanisms of PIK3R3 activating STAT3 and the relationship of PIK3R3 and STAT3.Method:Firstly, we explored the relationship between PIK3R3 and STAT3 through co-immunoprecipitation (Co-immunoprecipitation, IP), immunofluorescence co-localization (Immunofluorescence colocalization, IFC), pull-down metheds. Then, PIK3R3 expression was detected after inhibiting STAT3 activity by STAT3 specific small or low expression STAT3 with STAT3 siRNA. Moreover, after constructing a PIK3R3 reporter gene containing a promoter region, we detected the effect of STAT3 activity on PIK3R3 promoter activity. Finally, we confirmed that STAT3 can directly bind to PIK3R3 promoter region and regulate the expression of PIK3R3 through chromatin immunoprecipitation (Chromatin Immunoprecipitation, ChIP).Results:PIK3R3 directly bound to STAT3, and induced STAT3 phosphorylation and activated STAT3. In addition, PIK3R3 mRNA levels and protein levels were decreased when STAT3 activity was inhibited. And inhibition of STAT3 aslo inhibited the promoter activity of PIK3R3. Finally, Activated STAT3 directly bound to the promoter region of PIK3R3 and directly induced the PIK3R3 expression.Conclusion:PIK3R3 activates STAT3 through direct binding. On the contrary, activated STAT3 can directly regulate the expression of PIK3R3, thereby forming a positive feedback loop between PIK3R3 and STAT3.