Cholesterol regulates insulin-induced mTORC1 signaling.

The rapid activation of the crucial kinase mechanistic target of rapamycin complex-1 (mTORC1) by insulin is key to cell growth in mammals, but the regulatory factors remain unclear. Here, we demonstrate that cholesterol plays a crucial role in the regulation of insulin-stimulated mTORC1 signaling. The rapid progression of insulin-induced mTORC1 signaling declines in sterol-depleted cells and restores in cholesterol-repleted cells. In insulin-stimulated cells, cholesterol promotes recruitment of mTORC1 onto lysosomes without affecting insulin-induced dissociation of the TSC complex from lysosomes, thereby enabling complete activation of mTORC1. We also show that under prolonged starvation conditions, cholesterol coordinates with autophagy to support mTORC1 reactivation on lysosomes thereby restoring insulin-responsive mTORC1 signaling. Furthermore, we identify that fibroblasts from individuals with Smith-Lemli-Opitz Syndrome (SLOS) and model HeLa-SLOS cells, which are deficient in cholesterol biosynthesis, exhibit defects in the insulin-mTORC1 growth axis. These defects are rescued by supplementation of exogenous cholesterol or by expression of constitutively active Rag GTPase, a downstream activator of mTORC1. Overall, our findings propose novel signal integration mechanisms to achieve spatial and temporal control of mTORC1-dependent growth signaling and their aberrations in disease.

BMP2K phosphorylates AP-2 and regulates clathrin-mediated endocytosis.

Phosphorylation of the central adaptor protein complex, AP-2 is pivotal for clathrin-mediated endocytosis (CME). Here, we uncover the role of an uncharacterized kinase (BMP-2 inducible kinase-BMP2K) in AP-2 phosphorylation. We demonstrate that BMP2K can phosphorylate AP-2 in vitro and in vivo. Functional impairment of BMP2K impedes AP-2 phosphorylation leading to defects in clathrin-coated pit (CCP) morphology and cargo internalization. BMP2K engages AP-2 via its extended C-terminus and this interaction is important for its CCP localization and function. Notably, endogenous BMP2K levels decline upon functional impairment of AP-2 indicating AP-2 dependent BMP2K stabilization in cells. Further, functional inactivation of BMP2K in zebrafish embryos yields gastrulation phenotypes which mirror AP-2 loss-of-function suggesting physiological relevance of BMP2K in vertebrates. Together, our findings propose involvement of a novel kinase in AP-2 phosphorylation and in the operation of CME.

Phenotypic heterogeneity drives differential disease outcome in a mouse model of triple negative breast cancer.

The triple negative breast cancer (TNBC) subtype is one of the most aggressive forms of breast cancer that has poor clinical outcome and is an unmet clinical challenge. Accumulating evidence suggests that intratumoral heterogeneity or the presence of phenotypically distinct cell populations within a tumor play a crucial role in chemoresistance, tumor progression and metastasis. An increased understanding of the molecular regulators of intratumoral heterogeneity is crucial to the development of effective therapeutic strategies in TNBC. To this end, we used an unbiased approach to identify a molecular mediator of intratumoral heterogeneity in breast cancer by isolating two tumor cell populations (T1 and T2) from the 4T1 TNBC model. Phenotypic characterization revealed that the cells are different in terms of their morphology, proliferation and self-renewal ability in vitro as well as primary tumor formation and metastatic potential in vivo. Bioinformatic analysis followed by Kaplan Meier survival analysis in TNBC patients identified Metastasis associated colon cancer 1 (Macc1) as one of the top candidate genes mediating the aggressive phenotype in the T1 tumor cells. The role of Macc1 in regulating the proliferative phenotype was validated and taken forward in a therapeutic context with Lovastatin, a small molecule transcriptional inhibitor of Macc1 to target the T1 cell population. This study increases our understanding of the molecular underpinnings of intratumoral heterogeneity in breast cancer that is critical to improve the treatment of women currently living with the highly aggressive TNBC subtype.