NOTUM from Apc-mutant cells biases clonal competition to initiate cancer.

The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling1, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)2. Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.

Laminin matrix adhesion regulates basal mammary epithelial cell identity.

Mammary epithelium is a bilayered ductal network composed of luminal and basal epithelial cells, which together drive the growth and functional differentiation of the gland. Basal mammary epithelial cells (MECs) exhibit remarkable plasticity and progenitor activity that facilitate epithelial expansion. However, their activity must be tightly regulated to restrict excess basal cell activity. Here, we show that adhesion of basal cells to laminin α5-containing basement membrane matrix, which is produced by luminal cells, presents such a control mechanism. Adhesion to laminin α5 directs basal cells towards a luminal cell fate, and thereby results in a marked decrease of basal MEC progenitor activity in vitro and in vivo. Mechanistically, these effects are mediated through ß4-integrin and activation of p21 (encoded by CDKN1A). Thus, we demonstrate that laminin matrix adhesion is a key determinant of basal identity and essential to building and maintaining a functional multicellular epithelium.

Laminin alpha 5 regulates mammary gland remodeling through luminal cell differentiation and Wnt4-mediated epithelial crosstalk.

Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we show that Laminin α5 (Lama5) expression specifically in the luminal epithelial cells is necessary for normal mammary gland growth during puberty, and for alveologenesis during pregnancy. Lama5 loss in the keratin 8-expressing cells results in reduced frequency and differentiation of hormone receptor expressing (HR+) luminal cells. Consequently, Wnt4-mediated crosstalk between HR+ luminal cells and basal epithelial cells is compromised during gland remodeling, and results in defective epithelial growth. The effects of Lama5 deletion on gland growth and branching can be rescued by Wnt4 supplementation in the in vitro model of branching morphogenesis. Our results reveal a surprising role for BM-protein expression in the luminal mammary epithelial cells, and highlight the function of Lama5 in mammary gland remodeling and luminal differentiation.

Metabolic determination of cell fate through selective inheritance of mitochondria.

Metabolic characteristics of adult stem cells are distinct from their differentiated progeny, and cellular metabolism is emerging as a potential driver of cell fate conversions1-4. How these metabolic features are established remains unclear. Here we identified inherited metabolism imposed by functionally distinct mitochondrial age-classes as a fate determinant in asymmetric division of epithelial stem-like cells. While chronologically old mitochondria support oxidative respiration, the electron transport chain of new organelles is proteomically immature and they respire less. After cell division, selectively segregated mitochondrial age-classes elicit a metabolic bias in progeny cells, with oxidative energy metabolism promoting differentiation in cells that inherit old mitochondria. Cells that inherit newly synthesized mitochondria with low levels of Rieske iron-sulfur polypeptide 1 have a higher pentose phosphate pathway activity, which promotes de novo purine biosynthesis and redox balance, and is required to maintain stemness during early fate determination after division. Our results demonstrate that fate decisions are susceptible to intrinsic metabolic bias imposed by selectively inherited mitochondria.

Cellular shape reinforces niche to stem cell signaling in the small intestine.

Niche-derived factors regulate tissue stem cells, but apart from the mechanosensory pathways, the effect of niche geometry is not well understood. We used organoids and bioengineered tissue culture platforms to demonstrate that the conical shape of Lgr5+ small intestinal stem cells (ISCs) facilitate their self-renewal and function. Inhibition of non-muscle myosin II (NM II)-driven apical constriction altered ISC shape and reduced niche curvature and stem cell capacity. Niche curvature is decreased in aged mice, suggesting that suboptimal interactions between old ISCs and their niche develop with age. We show that activation of NM IIC or physical restriction to young topology improves in vitro regeneration by old epithelium. We propose that the increase in lateral surface area of ISCs induced by apical constriction promotes interactions between neighboring cells, and the curved topology of the intestinal niche has evolved to maximize signaling between ISCs and neighboring cells.

Oncogenic Ras Disrupts Epithelial Integrity by Activating the Transmembrane Serine Protease Hepsin.

Ras proteins play a causal role in human cancer by activating multiple pathways that promote cancer growth and invasion. However, little is known about how Ras induces the first diagnostic features of invasion in solid tumors, including loss of epithelial integrity and breaching of the basement membrane (BM). In this study, we found that oncogenic Ras strongly promotes the activation of hepsin, a member of the hepsin/TMPRSS type II transmembrane serine protease family. Mechanistically, the Ras-dependent hepsin activation was mediated via Raf-MEK-ERK signaling, which controlled hepsin protein stability through the heat shock transcription factor-1 stress pathway. In Ras-transformed three-dimensional mammary epithelial culture, ablation of hepsin restored desmosomal cell-cell junctions, hemidesmosomes, and BM integrity and epithelial cohesion. In tumor xenografts harboring mutant KRas, silencing of hepsin increased local invasion concomitantly with accumulation of collagen IV. These findings suggest that hepsin is a critical protease for Ras-dependent tumorigenesis, executing cell-cell and cell-matrix pathologies important for early tumor dissemination. SIGNIFICANCE: These findings identify the cell-surface serine protease hepsin as a potential therapeutic target for its role in oncogenic Ras-mediated deregulation of epithelial cell-cell and cell-matrix interactions and cohesion of epithelial structure.