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

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.

MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation.

The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can disrupt tissues and contribute to age-related pathologies, including cancer. MTOR inhibition suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin reduced IL6 and other cytokine mRNA levels, but selectively suppressed translation of the membrane-bound cytokine IL1A. Reduced IL1A diminished NF-κB transcriptional activity, which controls much of the SASP; exogenous IL1A restored IL6 secretion to rapamycin-treated cells. Importantly, rapamycin suppressed the ability of senescent fibroblasts to stimulate prostate tumour growth in mice. Thus, rapamycin might ameliorate age-related pathologies, including late-life cancer, by suppressing senescence-associated inflammation.

RPL24: a potential therapeutic target whose depletion or acetylation inhibits polysome assembly and cancer cell growth.

Partial loss of large ribosomal subunit protein 24 (RPL24) function is known to protect mice against Akt or Myc-driven cancers, in part via translational inhibition of a subset of cap(eIF4E)-dependently translated mRNAs. The role of RPL24 in human malignancies is unknown. By analyzing a public dataset of matched human breast cancers and normal mammary tissue, we found that breast cancers express significantly more RPL24 than matched normal breast samples. Depletion of RPL24 in breast cancer cells by >70% reduced cell viability by 80% and decreased protein expression of the eIF4E-dependently translated proteins cyclin D1 (75%), survivin (46%) and NBS1 (30%) without altering GAPDH or beta-tubulin levels. RPL24 knockdown also reduced 80S subunit levels relative to 40S and 60S levels. These effects on expression of eIF4E-dependent proteins and ribosome assembly were mimicked by 2-24 h treatment with the pan-HDACi, trichostatin A (TSA), which induced acetylation of 15 different polysome-associated proteins including RPL24. Furthermore, HDAC6-selective inhibition or HDAC6 knockdown induced ribosomal protein acetylation. Via mass spectrometry, we found that 60S-associated, but not, polysome-associated, RPL24 undergoes HDACi-induced acetylation on K27. Thus, RPL24 K27 acetylation may play a role in ribosome assembly. These findings point toward a novel acetylation-dependent polysome assembly mechanism regulating tumorigenesis.