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. 2014 Apr;144(2):287-298.
doi: 10.1007/s10549-014-2877-y. Epub 2014 Feb 22.

mTORC1/C2 and pan-HDAC inhibitors synergistically impair breast cancer growth by convergent AKT and polysome inhibiting mechanisms

Kathleen A Wilson-Edell #  1 , Mariya A Yevtushenko #  2 , Daniel E Rothschild  1 , Aric N Rogers  1 , Christopher C Benz  3
Affiliations

mTORC1/C2 and pan-HDAC inhibitors synergistically impair breast cancer growth by convergent AKT and polysome inhibiting mechanisms

Kathleen A Wilson-Edell et al. Breast Cancer Res Treat. 2014 Apr.

Abstract

Resistance of breast cancers to targeted hormone receptor (HR) or human epidermal growth factor receptor 2 (HER2) inhibitors often occurs through dysregulation of the phosphoinositide 3-kinase, protein kinase B/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Presently, no targeted therapies exist for breast cancers lacking HR and HER2 overexpression, many of which also exhibit PI3K/AKT/mTOR hyper-activation. Resistance of breast cancers to current therapeutics also results, in part, from aberrant epigenetic modifications including protein acetylation regulated by histone deacetylases (HDACs). We show that the investigational drug MLN0128, which inhibits both complexes of mTOR (mTORC1 and mTORC2), and the hydroxamic acid pan-HDAC inhibitor TSA synergistically inhibit the viability of a phenotypically diverse panel of five breast cancer cell lines (HR-/+, HER2-/+). The combination of MLN0128 and TSA induces apoptosis in most breast cancer cell lines tested, but not in the non-malignant MCF-10A mammary epithelial cells. In parallel, the MLN0128/TSA combination reduces phosphorylation of AKT at S473 more than single agents alone and more so in the 5 malignant breast cancer cell lines than in the non-malignant mammary epithelial cells. Examining polysome profiles from one of the most sensitive breast cancer cell lines (SKBR3), we demonstrate that this MLN0128/TSA treatment combination synergistically impairs polysome assembly in conjunction with enhanced inhibition of 4eBP1 phosphorylation at S65. Taken together, these data indicate that the synergistic growth inhibiting consequence of combining a mTORC1/C2 inhibitor like MLN0128 with a pan-HDAC inhibitor like TSA results from their mechanistic convergence onto the PI3K/AKT/mTOR pathway, profoundly inhibiting both AKT S473 and 4eBP1 S65 phosphorylation, reducing polysome formation and cancer cell viability.

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Figures

Fig. 1
Fig. 1
MLN0128 and TSA inhibit the proliferation of breast cancer cells more than non-transformed mammary epithelial cells. Viability assays were carried out after 72 h of treatment with the indicated doses of MLN0128 and TSA on the following cell lines: a SKBR3, b MDA-MB-231, c MCF7, d MCF7/HER2-18, e BT-474, and f MCF-10A. Error bars represent the standard deviation calculated from three triplicate wells. Each cell line was analyzed at least twice, and a representative experiment is shown
Fig. 2
Fig. 2
MLN0128 and TSA treatments enhance apoptosis in SKBR3 and MCF7 cells but not in MDA-MB-231 and MCF-10A cells. a SKBR3, b MCF7, c MDA-MB-231, and d MCF-10A cells were treated with MLN0128 (25 nM), TSA (100 nM), or Adriamycin (0.5 μg/mL) for 48 h. Whole cell lysates were collected and immunoblots were performed to detect cleaved PARP and a β-tubulin loading control. e MCF7 cells were treated with MLN0128 (25 nM) and/or TSA (100 nM) for 24 h. Annexin V was stained for and visualized by microscopy. The percentage of Annexin V-positive cells relative to total cells is expressed for four biological replicates. Error bars represent standard deviation. f MCF7 cells were treated with MLN0128 (25 nM) and/or TSA (100 nM) for 48 h. Cell cycle analysis was performed as described in materials and methods. Error bars represent the standard deviation from three biological replicates
Fig. 3
Fig. 3
MLN0128 reduces AKT, S6, and 4eBP1 phosphorylation, while the combination with TSA further reduces AKT phosphorylation. Cells were treated with MLN0128 and TSA for 8 h. Whole cell lysates were collected and immunoblots were performed using the indicated antibodies. The following cell lines were analyzed: a SKBR3, b MDA-MB-231, c MCF7, d MCF7/HER2-18, e BT-474, and f MCF-10A
Fig. 4
Fig. 4
The reduction of AKT phosphorylation by MLN0128 and TSA is accompanied by reduced 4eBP1 phosphorylation and impaired polysome formation: a SKBR3 cells were treated with 50 nM of MLN0128 and/or 500 nM of TSA for 24 h in single agent and in combination treatment. Whole cell lysates were collected and western blots were performed with the indicated antibodies. Ratios of phosphorylated to total protein were quantified using ImageJ. b Polysome profiling was performed from the same set of samples that were treated in (a). c SKBR3 cells were treated with 500 nM of MLN0128 or GDC0941 for 24 h. Whole cell lysates were collected and western blots and quantification were performed as in (a). d Polysome profiles were performed from samples treated in part (c)
Fig. 5
Fig. 5
Schematic of signaling pathways targeted by MLN0128 and TSA. AKT is activated in many breast cancers through dysregulation of receptor tyrosine kinases (RTKs) and/or PI3K. MLN0128 inhibits AKT activation by inhibiting TORC2, which activates AKT. MLN0128 also inhibits signaling downstream of AKT by inhibiting TORC1. Furthermore, TSA inhibits AKT through mechanisms involving increased dephosphorylation of AKT via activation of the PP1 phosphatase or increased AKT destabilization through HSP90 inhibition. Down-regulation of AKT by MLN0128 and TSA results in decreased polyribosome function and decreased translation of mRNAs involved in carcinogenesis. TSA also induces the acetylation of histones, thus increasing the transcription of various tumor suppressors
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