Mechanistic target of rapamycin (mTOR) regulates self-sustained quiescence, tumor indolence, and late clinical metastasis in a Beclin-1-dependent manner.

Self-sustained quiescence (SSQ) has been characterized as a stable but reversible non-proliferative cellular state that limits the cloning of cultured cancer cells. By developing refined clonogenic assays, we showed here that cancer cells in SSQ can be selected with anticancer agents and that culture at low cell density induced SSQ in pancreas and prostate adenocarcinoma cells. Pre-culture of cells in 3D or their pretreatment with pharmacological inhibitors of mechanistic target of rapamycin (mTOR) synergize with low cell density for induction of SSQ in a Beclin-1-dependent manner. Dissociated pancreatic adenocarcinoma (PAAD) cells rendered defective for SSQ by down-regulating Beclin-1 expression exhibit higher tumor growth rate when injected subcutaneously into mice. Conversely, dissociated PAAD cells in SSQ promote the formation of small indolent tumors that eventually transitioned to a rapid growth phase. Ex vivo clonogenic assays showed that up to 40% of clonogenic cancer cells enzymatically dissociated from resected fast-growing tumors could enter SSQ, suggesting that SSQ could significantly impact the proliferation of cancer cells that are naturally dispersed from tumors. Remarkably, the kinetics of clinical metastatic recurrence in 124 patients with pancreatic adenocarcinoma included in the TGCA-PAAD project could be predicted from Beclin-1 and Cyclin-A2 mRNA levels in their primary tumor, Cyclin A2 mRNA being a marker of both cell proliferation and mTOR complex 1 activity. Overall, our data show that SSQ is likely to promote the late development of clinical metastases and suggest that identifying new agents targeting cancer cells in SSQ could help improve patient survival.

Transient exposure to androgens induces a remarkable self-sustained quiescent state in dispersed prostate cancer cells.

Cellular quiescence is a reversible cell growth arrest that is often assumed to require a persistence of non-permissive external growth conditions for its maintenance. In this work, we showed that androgen could induce a quiescent state that is self-sustained in a cell-autonomous manner through a "hit and run" mechanism in androgen receptor-expressing prostate cancer cells. This phenomenon required the set-up of a sustained redox imbalance and TGFß/BMP signaling that were dependent on culturing cells at low density. At medium cell density, androgens failed to induce such a self-sustained quiescent state, which correlated with a lesser induction of cell redox imbalance and oxidative stress markers like CDKN1A. These effects of androgens could be mimicked by transient overexpression of CDKN1A that triggered its own expression and a sustained SMAD phosphorylation in cells cultured at low cell density. Overall, our data suggest that self-sustained but fully reversible quiescent states might constitute a general response of dispersed cancer cells to stress conditions.

SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy.

Metastasis involves the dissemination of single or small clumps of cancer cells through blood or lymphatic vessels and their extravasation into distant organs. Despite the strong regulation of metastases development by a cell dormancy phenomenon, the dormant state of cancer cells remains poorly characterized due to the difficulty of in vivo studies. We have recently shown in vitro that clonogenicity of prostate cancer cells is regulated by a dormancy phenomenon that is strongly induced when cells are cultured both at low cell density and in a slightly hypertonic medium. Here, we characterized by RT-qPCR a genetic expression signature of this dormant state which combines the presence of both stemness and differentiation markers. We showed that both TFGß/BMP signaling and redox imbalance are required for the full induction of this dormancy signature and cell quiescence. Moreover, reconstruction experiments showed that TFGß/BMP signaling and redox imbalance are sufficient to generate a pattern of genetic expression displaying all characteristic features of the dormancy signature. Finally, we observed that low cell density was sufficient to activate TGFß/BMP signaling and to generate a slight redox imbalance thus priming cells for dormancy that can be attained with a co-stimulus like hypertonicity, most likely through an increased redox imbalance. The identification of a dual regulation of dormancy provides a framework for the interpretation of previous reports showing a restricted ability of BMP signaling to regulate cancer cell dormancy in vivo and draws attention on the role of oxidative stress in the metastatic process.

A dormant state modulated by osmotic pressure controls clonogenicity of prostate cancer cells.

Cell dormancy constitutes a limiting step of the metastatic process by preventing the proliferation of isolated cancer cells disseminated at distant sites from the primary tumor. The study of cancer cell dormancy is severely hampered by the lack of biological samples so that the mechanisms that regulate cell dormancy have not been extensively explored. In this work, we describe the rapid induction in vitro of a dormant state in prostate cancer cells by exposure to a slightly hypertonic growth medium. This quiescence is observed only when cells are seeded at low density and, once established, requires additional stimuli besides osmotic pressure to be reversed. Media conditioned by cells grown at high density can partially prevent or reverse dormancy, a phenomenon which can be reproduced with citric acid. In addition to this role of small metabolites, inactivation of the p53 and smad pathways also counters the entry into dormancy, whereas exposure to activin A induces it to some extent. Thus, this easily inducible dormancy reproduces several features associated with the dormancy of stem cells and cancer cells in vivo.