Rapamycin-sensitive Cells Research Articles

Inhibition of MDM2 Re-Sensitizes Rapamycin Resistant Renal Cancer Cells via the Activation of p53

Background/Aims: Rapamycin is a potential anti-cancer agent, which modulates the activity of mTOR, a key regulator of cell growth and proliferation. However, several types of cancer cells are resistant to the anti-proliferative effects of rapamycin. In this study, we report a MDM2/p53-mediated rapamycin resistance in human renal cancer cells. Methods: Trypan blue exclusion tests were used to determine the cell viability. Changes in mRNA and protein expression were measured using real-time PCR and western blot, respectively. Xenograft models were established to evaluate the in vivo effects of rapamycin combined with a MDM2 inhibitor. Results: Rapamycin treatment suppresses the expression of MDM2 and exogenous overexpression of MDM2 in A498 cells contributes to rapamycin resistance. By establishing a rapamycin resistant cell line, we observed that MDM2 was significantly upregulated in rapamycin resistant cells than that in rapamycin sensitive cells. Importantly, the rapamycin resistant cells demonstrated attenuated accumulation of p53 in the nucleus in response to rapamycin treatment. Moreover, the inhibition of MDM2 by siMDM2 sensitizes A498 cells to rapamycin through the activation of p53. In both in vitro and in vivo models, the combination of rapamycin with the MDM2 inhibitor, MI-319, demonstrated a synergistic inhibitory effect on rapamycin resistant cells. Conclusion: Our study reports a novel mechanism for rapamycin resistance in human renal cancer and provides a new perspective for the development of anti-cancer drugs.

Abstract 1496: Epithelial to mesenchymal transition and rapamycin sensitivity in cancer cell lines.

Abstract Activation of the mTOR signaling pathway is common in multiple cancers. Rapamycin has inhibitory effects on solid tumors through inhibition of the mTOR kinase and alteration of downstream signaling. Though rapamycin and its analogs have yielded promising results from in vitro studies in sensitive cell lines, they have generated only a modest effect in clinical application. Epithelial to mesenchymal transition (EMT) in cancer cells involves not only the loss of cell-cell adhesion and acquisition of migratory and invasive properties but also resistance to cell death, senescence, and chemotherapy. We hypothesized that the EMT status of cancer cells regulates their sensitivity to rapamycin. We classified a panel of immortalized cancer cell lines as resistant or sensitive to rapamycin based on their IC50 as determined by Sulforhodamine B (SRB) assay. Using reverse phase proteomic array, we found that EMT markers were differentially expressed in sensitive compared to resistant immortalized cell lines. Rapamycin sensitive (RS) cell lines had increased expression of E-cadherin and N-cadherin while rapamycin resistant (RR) cell lines had increased expression of Smad3 (p<0.02). To validate these findings, we performed western blotting for EMT markers in selected RR and RS cell lines. E-cadherin expression was higher in RS cell lines than RR cell lines; in turn, RR cell lines expressed increased levels of the mesenchymal marker Vimentin. To determine if inducing EMT decreases rapamycin sensitivity, we assessed E-cadherin and Vimentin expression by western blotting in the epithelial, immortalized breast cancer cell line, MCF7, which was transfected with a constitutively-active mutant of Snail, a known EMT driver. Constitutively-active Snail mutant MCF7 demonstrated loss of E-cadherin and gain of Vimentin expression compared to cells transfected with nonfunctional mutant Snail or Wild-type Snail. Furthermore, we found decreased growth inhibition with rapamycin by SRB assay in constitutively-active Snail mutant MCF7 compared to controls (p<0.05). To determine if modulating EMT increases rapamycin sensitivity, we induced MET using ZEB1 siRNA knockdown and miR200b/c transfection in two mesenchymal cancer cell lines_ACHN and MDA-MB-231. Although miR200b/c transfection induced E-cadherin expression by immunoblotting in both cell lines, there was no significant increase in growth inhibition by SRB assay. In contrast, ZEB1 siRNA knockdown partially reversed EMT in these cells, which exhibited increased sensitivity to rapamycin by SRB assay. Most but not all mechanisms of EMT modulation altered rapamycin sensitivity in immortalized cancer cell lines. Future studies to determine the mechanism of EMT modulation that results in increased sensitivity to rapamycin could have important clinical applications for combination therapies utilizing rapamycin and its analogs. Citation Format: Ashley M. Holder, Farrell Adkins, Argun Akcakanat, Huiqin Chen, Kim-Anh Do, Mien-Chie Hung, Funda Meric-Bernstam. Epithelial to mesenchymal transition and rapamycin sensitivity in cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1496. doi:10.1158/1538-7445.AM2013-1496

PIK3CA/PTEN Mutations and Akt Activation As Markers of Sensitivity to Allosteric mTOR Inhibitors

We sought to determine whether phosphoinositide 3-kinase (PI3K) pathway mutation or activation state and rapamycin-induced feedback loop activation of Akt is associated with rapamycin sensitivity or resistance. Cancer cell lines were tested for rapamycin sensitivity, Akt phosphorylation, and mTOR target inhibition. Mice injected with breast or neuroendocrine cancer cells and patients with neuroendocrine tumor (NET) were treated with rapalogs and Akt phosphorylation was assessed. Thirty-one cell lines were rapamycin sensitive (RS) and 12 were relatively rapamycin resistant (RR; IC(50) > 100 nmol/L). Cells with PIK3CA and/or PTEN mutations were more likely to be RS (P = 0.0123). Akt phosphorylation (S473 and T308) was significantly higher in RS cells (P < 0.0001). Rapamycin led to a significantly greater pathway inhibition and greater increase in p-Akt T308 (P < 0.0001) and p-Akt S473 (P = 0.0009) in RS cells. Rapamycin and everolimus significantly increased Akt phosphorylation but inhibited growth in an in vivo NET model (BON). In patients with NETs treated with everolimus and octreotide, progression-free survival correlated with p-Akt T308 in pretreatment (R = 0.4762, P = 0.0533) and on-treatment tumor biopsies (R = 0.6041, P = 0.0102). Patients who had a documented partial response were more likely to have an increase in p-Akt T308 with treatment compared with nonresponders (P = 0.0146). PIK3CA/PTEN genomic aberrations and high p-Akt levels are associated with rapamycin sensitivity in vitro. Rapamycin-mediated Akt activation is greater in RS cells, with a similar observation in patients with clinical responses on exploratory biomarker analysis; thus feedback loop activation of Akt is not a marker of resistance but rather may function as an indicator of rapamycin activity.

Determinants of rapamycin sensitivity in breast cancer cells.

Rapamycin inhibits the serine-threonine kinase mammalian target of rapamycin (mTOR), blocking phosphorylation of p70 S6 kinase (S6K1) and 4E-binding protein 1 (4E-BP1) and inhibiting protein translation and cell cycle progression. Rapamycin and its analogues are currently being tested in clinical trials as novel-targeted anticancer agents. Although rapamycin analogues show activity in clinical trials, only some of the treated patients respond. The purpose of this study is to identify determinants of rapamycin sensitivity that may assist the selection of appropriate patients for therapy. Breast cancer cell lines representing a spectrum of aberrations in the mTOR signaling pathway were tested for rapamycin sensitivity. The expression and phosphorylation state of multiple components of the pathway were tested by Western blot analysis, in the presence and absence of rapamycin. Cell proliferation was significantly inhibited in response to rapamycin in 12 of 15 breast cancer cell lines. The ratio of total protein levels of 4E-BP1 to its binding partner eukaryotic initiation factor 4E did not predict rapamycin sensitivity. In contrast, overexpression of S6K1, and phosphorylated Akt independent of phosphatase and tensin homologue deleted from chromosome 10 status, were associated with rapamycin sensitivity. Targeting S6K1 and Akt with small interfering RNA and dominant-negative constructs, respectively, decreased rapamycin sensitivity. Rapamycin inhibited the phosphorylation of S6K1, ribosomal S6 protein, and 4E-BP1 in rapamycin-resistant as well as -sensitive cells, indicating that its ability to inhibit the mTOR pathway is not sufficient to confer sensitivity to rapamycin. In contrast, rapamycin treatment was associated with decreased cyclin D1 levels in the rapamycin-sensitive cells but not in rapamycin-resistant cells. Overexpression of S6K1 and expression of phosphorylated Akt should be evaluated as predictors of rapamycin sensitivity in breast cancer patients. Furthermore, changes in cyclin D1 levels provide a potential pharmacodynamic marker of response to rapamycin.