Determinants of Rapamycin Sensitivity in Breast Cancer Cells

Abstract

Purpose: 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.Experimental Design: 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.Results: 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.Conclusions: 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.

Everolimus restores gefitinib sensitivity in resistant non-small cell lung cancer cell lines

“…This result is in agreement with that reported by Sun et al on H460 and H1299 cell lines treated with rapamycin [39]. The high basal level of p-AKT observed in H1650 and SKMES-1 may be associated with the higher sensitivity to everolimus as reported in ovarian cancer cells [40] and in breast cancer cells [41].…”

Phorbol 12-myristate 13-acetate and serum synergize to promote rapamycin-insensitive cell proliferation via protein kinase C-eta

“…As the mTOR pathway is activated in GBM tumor samples, rapamycin and its analogs have been tested as chemotherapeutic agents in the treatment of glioblastoma. Although rapamycin and its analogs have demonstrated potential as chemotherapeutic agents in the treatment of several cancers including GBMs, recent studies suggest certain populations of cancer cells are not susceptible to rapamycin inhibition of cell proliferation (Huang and Houghton, 2001;Dilling et al, 2002;Chen et al, 2003;Gera et al, 2004;Noh et al, 2004;Galanis et al, 2005).…”

“…Rapamycin also increases the effectiveness of radiation in U-87MG glioblastoma cells (Eshleman et al, 2002). However, certain breast cancer cell lines are resistant to rapamycin, whereas others are susceptible to rapamycin (Noh et al, 2004). This resistance has been correlated with the activity level of AKT and p70S6K.…”

“…This resistance has been correlated with the activity level of AKT and p70S6K. Expression of the cell cycle regulatory proteins c-myc and cyclin D1 has also been linked to rapamycin insensitivity in breast cancer and brain tumor cells (Gera et al, 2004;Noh et al, 2004).…”

Elevated phosphorylation and activation of PDK-1/AKT pathway in human breast cancer

“…Although increased phosphorylation of AKT and mTOR in breast cancer have been recently reported (Noh et al, 2004;Zhou et al, 2004;Zhang et al, 2005), limited number of studies has contrasted the phosphorylation profiles of large panel of protein kinases among normal and invasive breast cancer tissues. Further, although PDK-1 is one of the crucial downstream targets of PI3-K signalling and a key upstream kinase of AKT, it remains unclear whether PDK-1 is activated in primary breast cancer tissues.…”