Lifelong self-renewal of the adult intestinal epithelium requires the activity of stem cells located in mucosal crypts. Lgr5 and Bmi1 are two molecular markers of crypt-cell populations that replenish all lineages over time and hence function as stem cells. Intestinal stem cells require Wnt signaling, but the understanding of their cellular niche is incomplete. Lgr5-expressing crypt base columnar cells (CBCs) reside deep in the crypt, mingled among mature Paneth cells that are well positioned for short-range signaling. Partial lineage ablation previously had implied that Paneth cells are nonessential constituents of the stem-cell niche, but recently their absence was reported to interfere with Lgr5 + CBCs, resurrecting an appealing idea. However, previous mouse models failed to remove Paneth cells completely or permanently; defining the intestinal stem-cell niche requires clarity with respect to the Paneth cell role. We find that Lgr5 + cells with stem-cell activity cluster in future crypts early in life, before Paneth cells develop. We also crossed conditional Atoh1 −/− mice, which lack Paneth cells entirely, with Lgr5 GFP mice to visualize Lgr5 + CBCs and to track their stemcell function. In the sustained absence of Paneth cells, Lgr5 + CBCs occupied the full crypt base, proliferated briskly, and generated differentiated progeny over many months. Gene expression in fluorescence-sorted Lgr5 + CBCs reflected intact Wnt signaling despite the loss of Paneth cells. Thus, Paneth cells are dispensable for survival, proliferation, and stem-cell activity of CBCs, and direct contact with Lgr5-nonexpressing cells is not essential for CBC function. S tem cells in selected adult tissues, such as the bone marrow, skin, and digestive tract, play a vital role in replenishing multiple cell types throughout life, and their unique and potent capacity for self-renewal is replicated in cancer (1). These stem cells occupy specialized niches and respond to the local environment (2). The functions of such niches range from delivering trophic signals that control cell proliferation and prevent stemcell depletion to preventing unrestrained cell replication (3). Defining the cellular and molecular constituents of adult stem-cell niches therefore is an important challenge in biology and medicine.Intestinal stem cells reside in mucosal crypts and generate four distinct cell types. Enterocytes, goblet cells, and enteroendocrine cells line deep crypts in the colon and villi that project into the small bowel lumen; Paneth cells lie at the crypt base in the small intestine, increasing in number from duodenum to ileum, but are absent from the colon (4). Two small intestine crypt-cell populations are able to generate all four cell types over extended periods: Lgr5-expressing crypt base columnar cells (CBCs), which lie deep in the crypt, interspersed among Paneth cells (5), and Bmi1-expressing cells that occupy several crypt tiers, most notably the +4 position (6). Although recent evidence suggests that each of these cell populations can engende...
Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequencespecific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85D V12 activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.R AS signaling is a critical control point for a host of cellular functions ranging from cellular survival and proliferation to cellular endocytosis and motility (1). The on or off state of RAS is dictated by nucleotide exchange. GTP-bound RAS is the activated form that engages its downstream effectors with high avidity. The endogenous GTPase activity of RAS hydrolyzes GTP to GDP and inactivates signaling. This biochemical process is further regulated by GTPase-activating proteins (GAPs) that impair RAS signaling through increasing endogenous GTPase activity and guanine-nucleotide exchange factors (GEFs) that enhance RAS signaling by facilitating GDP release and, thus, GTP association. Given the central roles of RAS in cellular growth and metabolism, it is not surprising that cancer cells usurp its prosurvival activities to achieve immortality.Activating mutations in KRAS represent the most frequent oncogenic driving force among the RAS homologs K-, N-, and H-RAS, and are associated with poor prognosis and chemoresistance (2). KRAS mutations are present in ∼30% of human tumors and at even higher frequencies in cancers of the pancreas, lung, thyroid gland, colon, and liver. For example, in pancreatic ductal adenocarcinomas (PDAC) that carry a 5-y survival rate of less than 5%, activating KRAS mutations are present in more than 90% of tumors (3). Thus, therapeutic inhibition of RAS is among the highest priority goals of the cancer field. Because oncogenic forms of KRAS typically harbor single-point mutants that stabilize its active GTP-bound form, a host of recent small molecule and peptide development efforts have been aimed at disarming this pathologic biochemical state. The extremely high affinity of KRAS for its GTP substrate has hampered the development of competitive GTP inhibitors. However, a GDP mimetic that covalently modifies the mutant cysteine of KRAS G12C represents a promising approach to plugging the nucleotid...
Understanding the molecular alterations that confer cancer cells with motile, metastatic properties is needed to improve patient survival. Here we report that p38γ MAPK regulates breast cancer cell motility and metastasis, in part by controlling expression of the metastasis-associated small GTPase RhoC. This p38γ-RhoC regulatory connection was mediated by a novel mechanism of modulating RhoC ubiquitination. This relationship persisted across multiple cell lines and in clinical breast cancer specimens. Using a computational mechanical model based on the finite element method, we demonstrated that p38γ-mediated cytoskeletal changes are sufficient to control cell motility. This model predicted novel dynamics of leading edge actin protrusions, which were experimentally verified and established to be closely related to cell shape and cytoskeletal morphology. Clinical relevance was supported by evidence that elevated expression of p38γ associated with lower overall survival of breast cancer patients. Taken together, our results offer a detailed characterization of how p38γ contributes to breast cancer progression, presents a new mechanics-based analysis of cell motility, and discovers a leading edge behavior in motile cells to accommodate modified cytoskeletal architecture. In summary, these findings not only identify a novel mechanism for regulating RhoC expression but also advance p38γ as a candidate therapeutic target.
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