Primary antibody diluted in 5% BSA in PBS-T or TBS-T (generally at 1:1000) was added to membrane overnight at 4o C

Primary antibody diluted in 5% BSA in PBS-T or TBS-T (generally at 1:1000) was added to membrane overnight at 4o C. its Ibudilast (KC-404) release into the cytosol. Consistent VAV2 with this notion, LapR cells possessed increased levels of 2 of the inhibitors of apoptosis (IAPs), survivin and c-IAP-2, which are reported to block Ibudilast (KC-404) caspase activation downstream of cytosolic cytochrome C release. Further, treatment with the mTOR kinase inhibitor AZD8055 or the Hsp90 inhibitor 17-AAG reversed expression of IAPs and overcame lapatinib resistance in LapR cells. Together, these data suggest that suppression of apoptosis downstream of cytosolic cytochrome C release, possibly through increased expression of IAPs or other caspase-suppressing proteins, may promote lapatinib resistance. Further, PI3K is thought to be the main driver of lapatinib resistance, but our findings indicate that PI3K inhibitors may be ineffective in some lapatinib-resistant HER2+ breast cancers with PI3K-independent activation of mTOR kinase, which may instead benefit from mTOR or Hsp90 inhibitors. cells rather than to vehicle-treated cell growth. Basal proliferation of AU565 LapR cells is lower than parental cells, and this shifts the AU565 LapR drug sensitivity curve upward; thus we normalized the LapR curve to parental to show that absolute cell number after 17-AAG treatment was similar between parental and LapR cells.) (B) AU565 LapR cells were treated with 1M birinapant for 2?days, followed by western blot. Because AU565 LapR cells appeared to possess PI3K/Akt-independent mTOR activation (Fig. 1B, C), we tested whether LapR cells might be sensitive to mTOR inhibitors but resistant to PI3K inhibitors. We treated parental and LapR cells with the mTOR kinase inhibitor AZD8055, which blocks the catalytic activity of mTOR,19 or the PI3K inhibitor GDC-0941.20 AU565 LapR cells were resistant to the PI3K inhibitor GDC-0941 compared to parental cells, indicating that LapR cells have decreased dependence on PI3K (Fig. 1D, top). On the other hand, AU565 LapR cells were sensitive to AZD8055 to a degree similar to AU565 parental cells, indicating that both cell lines are mTOR-dependent (Fig. 1D, bottom). These data reinforce the notion that AU565 LapR cells possess PI3K-independent mTOR activation that is required for their proliferation. We also found that catalytic inhibitors of mTOR which block both mTORC1 and mTORC2, such as AZD8055 and BEZ235, inhibited proliferation of AU565 LapR cells more effectively than rapalogs which block mTORC1 only, suggesting that both mTOR complexes may be important for the proliferation of AU565 LapR Ibudilast (KC-404) cells (data not shown). Therefore, we used the dual mTOR kinase inhibitor AZD8055 in subsequent experiments. PI3K-independent mTOR activation in AU565 LapR cells is independent of canonical mTOR activators To further explore the mechanism of PI3K/Akt-independent mTOR activation and S6 phosphorylation in AU565 LapR cells, we treated LapR cells (and parental cells as a control) with the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-220621 and analyzed S6 phosphorylation (Fig. 2A). GDC-0941 blocked S6 phosphorylation in AU565 parental cells but not in AU565 LapR cells, reaffirming that S6 phosphorylation in AU565 LapR cells is indeed PI3K-independent. Interestingly, Akt inhibition did not significantly inhibit S6 phosphorylation in parental cells suggesting that mTOR activation in parental cells is dependent on PI3K but not via Akt. Not surprisingly, Akt inhibition did not significantly inhibit the high level of S6 phosphorylation in AU565 LapR cells, which is independent of PI3K/Akt. Multiple known mTOR upstream activators can lead to mTOR activation.15 For example, Erk, Akt, IKK activate and AMPK and GSK-3 inhibit the TSC1/2 mTOR-suppressive complex, while Rheb and PRAS40 directly activate or inhibit mTORC1, respectively.15 We next tested whether these known mTOR upstream regulators were increased or Ibudilast (KC-404) decreased in LapR cells and confer mTOR activation and increased S6 phosphorylation compared with parental cells. We detected Erk, TSC2, PRAS40, AMPK, Rheb (Fig. 2B), GSK-3 and IKK (data not shown) and found that none of these signaling nodes were significantly different at total or phospho- protein levels in LapR cells compared to parental cells (Fig. 2B). To identify potential activating mutations in mTOR pathway genes and other mutational events that confer resistance in AU565 LapR cells, Ibudilast (KC-404) we performed whole-exome sequencing of AU565 parental and LapR cells. However, AU565 LapR cells did not gain apparent cancer-associated mutations or copy-number variations compared to parental cells (data not shown). Specifically, we carefully analyzed genes known to be involved in the mTOR pathway15 and genes from the Cancer Gene Census22 by looking for known point mutations present in LapR but not parental cells, and by comparing read depth as an indicator of copy number changes. We found that LapR cells did not possess mutations or copy number changes in mTOR pathway genes (or known oncogenes) compared to parental cells (data not shown). Together, these data indicate that a potentially novel, and as yet unclear, mechanism of mTOR activation may be involved in.