Stochastic phenotypes in RAS-dependent developmental diseases.

Germline mutations upregulating RAS signaling are associated with multiple developmental disorders. A hallmark of these conditions is that the same mutation may present vastly different phenotypes in different individuals, even in monozygotic twins. Here, we demonstrate how the origins of such largely unexplained phenotypic variations may be dissected using highly controlled studies in Drosophila that have been gene edited to carry activating variants of MEK, a core enzyme in the RAS pathway. This allowed us to measure the small but consistent increase in signaling output of such alleles in vivo. The fraction of mutation carriers reaching adulthood was strongly reduced, but most surviving animals had normal RAS-dependent structures. We rationalize these results using a stochastic signaling model and support it by quantifying cell fate specification errors in bilaterally symmetric larval trachea, a RAS-dependent structure that allows us to isolate the effects of mutations from potential contributions of genetic modifiers and environmental differences. We propose that the small increase in signaling output shifts the distribution of phenotypes into a regime, where stochastic variation causes defects in some individuals, but not in others. Our findings shed light on phenotypic heterogeneity of developmental disorders caused by deregulated RAS signaling and offer a framework for investigating causal effects of other pathogenic alleles and mild mutations in general.

Integrin-linked kinase tunes cell-cell and cell-matrix adhesions to regulate the switch between apoptosis and EMT downstream of TGFß1.

Epithelial-mesenchymal transition (EMT) is a morphogenetic process that endows epithelial cells with migratory and invasive potential. Mechanical and chemical signals from the tumor microenvironment can activate the EMT program, thereby permitting cancer cells to invade the surrounding stroma and disseminate to distant organs. Transforming growth factor ß1 (TGFß1) is a potent inducer of EMT that can also induce apoptosis depending on the microenvironmental context. In particular, stiff microenvironments promote EMT while softer ones promote apoptosis. Here, we investigated the molecular signaling downstream of matrix stiffness that regulates the phenotypic switch in response to TGFß1 and uncovered a critical role for integrin-linked kinase (ILK). Specifically, depleting ILK from mammary epithelial cells precludes their ability to sense the stiffness of their microenvironment. In response to treatment with TGFß1, ILK-depleted cells undergo apoptosis on both soft and stiff substrata. We found that knockdown of ILK decreases focal adhesions and increases cell-cell adhesions, thus shifting the balance from cell-matrix to cell-cell adhesion. High cell-matrix adhesion promotes EMT whereas high cell-cell adhesion promotes apoptosis downstream of TGFß1. These results highlight an important role for ILK in controlling cell phenotype by regulating adhesive connections to the local microenvironment.