Human tumors often contain slowly proliferating cancer cells that resist treatment, but we do not know precisely how these cells arise. We show that rapidly proliferating cancer cells can divide asymmetrically to produce slowly proliferating "G0-like" progeny that are enriched following chemotherapy in breast cancer patients. Asymmetric cancer cell division results from asymmetric suppression of AKT/PKB kinase signaling in one daughter cell during telophase of mitosis. Moreover, inhibition of AKT signaling with smallmolecule drugs can induce asymmetric cancer cell division and the production of slow proliferators. Cancer cells therefore appear to continuously flux between symmetric and asymmetric division depending on the precise state of their AKT signaling network. This model may have significant implications for understanding how tumors grow, evade treatment, and recur.quiescence | epigenetics | cell signaling | drug resistance T umors generally evolve through years of mutation and clonal selection (1). This favors the outgrowth of rapidly proliferating cancer cells over time. However, even advanced tumors contain many cancer cells that appear to be proliferating slowly (2). This proliferative heterogeneity correlates closely with time to clinical detection, growth, metastasis, and treatment response across all tumor types, but we still do not understand clearly how it arises. The rate of mammalian cell proliferation is generally determined by the time spent in G1 of the cell cycle. Critical genetic and epigenetic changes within cancer cells accelerate G1 transit, whereas a suboptimal microenvironment with imbalance of growth factors, nutrients, or oxygen can slow G1 progression (3). Therefore, individual cancer cells within a tumor are thought to vary significantly in their proliferative rate depending on the precise balance of these intrinsic and extrinsic factors. Interestingly, however, many tumor-derived cancer cell lines also produce slowly proliferating cells. These established lines have many acquired mutations that drive cell proliferation. They have also been grown ex vivo for years in a stable microenvironment to promote unbridled proliferation. These factors ought to favor a strong purifying selection against slow proliferators. We worked to understand how slowly proliferating cells seem to arise paradoxically in cancer cell lines.Results G0-Like Cancer Cells in Vitro. We began by studying MCF7, a highly proliferative, aneuploid, ER + /HER2 − human breast cancer cell line. This line displays significant proliferative heterogeneity despite mutations in CDKN2A and PIK3CA that cooperatively drive cell-cycle progression (4). We first hypothesized that slowly proliferating MCF7 cells might produce low levels of reactive oxygen species (ROS). This hypothesis was based on previous observations that slowly cycling hematopoietic, neural, and breast adult stem cells and cancer stem cells produce low levels of ROS (5-7). We stained MCF7 cells with 5-(and-6)chloromethyl 1-2′,7′-dichlorohydrofluorescein diacet...

All cancers contain an admixture of rapidly and slowly proliferating cancer cells. This proliferative heterogeneity complicates the diagnosis and treatment of cancer patients because slow proliferators are hard to eradicate, can be difficult to detect, and may cause disease relapse sometimes years after apparently curative treatment. While clonal selection theory explains the presence and evolution of rapid proliferators within cancer cell populations, the circumstances and molecular details of how slow proliferators are produced is not well understood. Here, a β1-integrin/FAK/mTORC2/AKT1-associated signaling pathway is discovered that can be triggered for rapidly proliferating cancer cells to undergo asymmetric cell division and produce slowly proliferating AKT1low daughter cells. In addition, evidence indicates that the proliferative output of this signaling cascade involves a proteasome-dependent degradation process mediated by the E3 ubiquitin ligase TTC3. These findings reveal that proliferative heterogeneity within cancer cell populations, in part, is produced through a targetable signaling mechanism, with potential implications for understanding cancer progression, dormancy, and therapeutic resistance.

Melanomas and other solid tumors commonly have increased ploidy, with near-tetraploid karyotypes being most frequently observed. Such karyotypes have been shown to arise through whole-genome doubling events that occur during early stages of tumor progression. The generation of tetraploid cells via whole-genome doubling is proposed to allow nascent tumor cells the ability to sample various pro-tumorigenic genomic configurations while avoiding the negative consequences that chromosomal gains or losses have in diploid cells. Whereas a high prevalence of whole-genome doubling events has been established, the means by which whole-genome doubling arises is unclear. Here, we find that BRAFV600E, the most common mutation in melanomas, can induce whole-genome doubling via cytokinesis failure in vitro and in a zebrafish melanoma model. Mechanistically, BRAFV600E causes decreased activation and localization of RhoA, a critical cytokinesis regulator. BRAFV600E activity during G1/S phases of the cell cycle is required to suppress cytokinesis. During G1/S, BRAFV600E activity causes inappropriate centriole amplification, which is linked in part to inhibition of RhoA and suppression of cytokinesis. Together these data suggest that common abnormalities of melanomas linked to tumorigenesis – amplified centrosomes and whole-genome doubling events – can be induced by oncogenic BRAF and other mutations that increase RAS/MAPK pathway activity.

Melanoma is commonly driven by activating mutations in the MAP kinase BRAF; however, oncogenic BRAF alone is insufficient to promote melanomagenesis. Instead, its expression induces a transient proliferative burst that ultimately ceases with the development of benign nevi comprised of growth-arrested melanocytes. The tumor suppressive mechanisms that restrain nevus melanocyte proliferation remain poorly understood. Here we utilize cell and murine models to demonstrate that oncogenic BRAF leads to activation of the Hippo tumor suppressor pathway, both in melanocytes in vitro and nevus melanocytes in vivo. Mechanistically, we show that oncogenic BRAF promotes both ERK-dependent alterations in the actin cytoskeleton and whole-genome doubling events, which independently reduce RhoA activity to promote Hippo activation. We also demonstrate that functional impairment of the Hippo pathway enables oncogenic BRAF-expressing melanocytes to bypass nevus formation and rapidly form melanomas. Our data reveal that the Hippo pathway enforces the stable arrest of nevus melanocytes and represents a critical barrier to melanoma development.