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. 2012 Sep;103(9):1665-71.
doi: 10.1111/j.1349-7006.2012.02359.x. Epub 2012 Aug 1.

Efficacy of everolimus, a novel mTOR inhibitor, against basal-like triple-negative breast cancer cells

Mayu Yunokawa  1 , Fumiaki Koizumi, Yuka Kitamura, Yasufumi Katanasaka, Naoko Okamoto, Makoto Kodaira, Kan Yonemori, Chikako Shimizu, Masashi Ando, Kenkichi Masutomi, Teruhiko Yoshida, Yasuhiro Fujiwara, Kenji Tamura
Affiliations

Efficacy of everolimus, a novel mTOR inhibitor, against basal-like triple-negative breast cancer cells

Mayu Yunokawa et al. Cancer Sci. 2012 Sep.

Abstract

Patients with triple-negative breast cancers (TNBCs) typically have a poor prognosis because such cancers have no effective therapeutic _targets, such as estrogen receptors for endocrine therapy or human epidermal growth factor receptor 2 (HER2) receptors for anti-HER2 therapy. As the phosphatidylinositol 3' kinase (PI3K)/Akt/mammalian _target of rapamycin (mTOR) cascade is activated in TNBCs, mTOR is a potential molecular _target for anticancer therapy. In this study, we investigated the antitumor activities of everolimus, an oral mTOR inhibitor, in nine TNBC cell lines. Everolimus effectively inhibited cell growth at concentrations under 100 nM (IC(50)) in five cell lines and even in the 1-nM range in three of the five cell lines. To identify specific characteristics that could be used as predictive markers of efficacy, we evaluated the expressions of proteins in the mTOR cascade, basal markers, and cancer stem cell markers using western blotting, fluorescent in situ hybridization (FISH), or immunohistochemistry. All five of the sensitive cell lines were categorized as a basal-like subtype positive for either epidermal growth factor receptor (EGFR) or CK5/6, although resistant cell lines were not of this subtype and tended to exhibit the characteristics of cancer stem cells, with decreased E-cadherin and the increased expression of Snail or Twist. In vivo assays demonstrated antitumor activity in a mouse xenograft model of basal-like breast cancer, rather than non-basal breast cancer. These results suggest that everolimus has favorable activity against basal-like subtypes of TNBCs. Epidermal growth factor receptor and CK5/6 are positive predictive markers of the TNBC response to everolimus, while cancer stem cell markers are negative predictive markers.

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Figures

Figure 1
Figure 1
IC 50 values for everolimus of nine triple‐negative breast cancer (TNBC) cell lines. Each cell line was treated with the indicated concentrations of everolimus for 96 h. Viable cell numbers were relatively quantified using the CCK‐8 assay and were expressed as a percent of the untreated control.
Figure 2
Figure 2
Baseline expressions of mammalian _target of rapamycin (mTOR) cascade proteins and mTOR cascade modulation by everolimus in nine triple‐negative breast cancer (TNBC) cell lines. (a) Baseline protein expressions of PTEN, p‐Akt, Akt, p‐mTOR, mTOR, p‐S6, S6, p‐4EBP1, 4EBP1, and β‐actin in nine TNBC cell lines. Ten micrograms of protein were prepared from the indicated cell lines at 60–70% confluence. The cell lines are arranged in decreasing order of sensitivity to everolimus, from left to right. β‐Actin was used as a loading control. (b) Cells were untreated or treated with 0.5, 5, or 50 nM of everolimus for 1 h. Five micrograms of protein were prepared from the indicated cell lines. Equivalent reductions of p‐S6 and S6 were observed with only 0.5 nM in the two sensitive cell lines (MDA‐MB‐468 and BT549) and the two resistant cell lines (MDA‐MB‐231 and MDA‐MB‐157).
Figure 3
Figure 3
Determination of breast cancer subtypes using basal markers and stem cell‐like characteristics. (a) Protein expressions of p‐EGFR, EGFR, HER2, E‐cadherin, Snail, Twist, and β‐actin in nine triple‐negative breast cancer (TNBC) cell lines. Ten micrograms of protein were prepared from the indicated cell lines at 60–70% confluence. The cell lines are arranged in order of decreasing sensitivity to everolimus, from left to right. β‐Actin was used as a loading control. (b) Epidermal growth factor receptor (EGFR) gene fluorescence in situ hybridization (FISH) analysis. MDA‐MB‐468 and BT20 showed the gene amplification of EGFR. The other seven TNBC cell lines did not exhibit the gene amplification of EGFR. Two positive cell lines and two negative cell lines are shown. (c) Immunohistochemical analysis of CK5/6. MDA‐MB‐468, BT20, and HCC38 were positive for CK5/6, and the other six cell lines were negative. Three positive cell lines and two negative cell lines are shown. Cell membranes were stained by the CK 5/6 antibody in all MDA‐MB‐468 cells and in some BT20 and HCC38 cells (indicated by arrows).
Figure 4
Figure 4
Effect of PTEN or epidermal growth factor receptor (EGFR) modulation on everolimus sensitivity. (a) MDA‐MB‐231 and BT20 cells were transfected with siRNA specific to human PTEN or nonspecific control siRNA. After 24 h, the cells were treated with the indicated concentrations of everolimus for 72 h. The results for MDA‐MB‐231 are shown. (b) MDA‐MB‐231 and MDA‐MB‐436 cells were transfected with retrovirus containing either an empty vector or an EGFRwt vector and then were treated with the indicated concentrations of everolimus for 96 h. The results for MDA‐MB‐231 are shown.
Figure 5
Figure 5
Effect of RAD001 on the growth of breast cancer cell lines in vivo. Athymic nude mice were inoculated with MDA‐MB‐468 cells (a) or MDA‐MB‐231 cells (b). When the tumors reached an average size of 120 mm3, mice were treated with placebo or 10 mg/kg per day RAD001, three times per week for 3 weeks. The tumors were measured twice weekly and tumor size was averaged for each treatment group. Points, mean; bars, standard deviation (SD); *P < 0.05, significantly different from placebo‐treated mice.
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