Cutaneous SCC (cSCC) is the most frequent skin cancer with metastatic potential and can manifest rapidly as a common side effect in patients receiving systemic kinase inhibitors. Here we use massively parallel exome and targeted level sequencing 132 sporadic cSCC, 39 squamoproliferative lesions and cSCC arising in patients receiving the BRAF inhibitor vemurafenib, as well as 10 normal skin samples to identify significant NOTCH1 mutation as an early event in squamous cell carcinogenesis. Bisected vemurafenib induced lesions revealed surprising heterogeneity with different activating HRAS and NOTCH1 mutations identified in two halves of the same cSCC suggesting polyclonal origin.Immunohistochemical analysis using an antibody specific to nuclear NOTCH1 correlates with mutation status in sporadic cSCC and regions of NOTCH1 loss or down-regulation are frequently observed in normal looking skin. Our data indicate that NOTCH1 acts as a gatekeeper in human cSCC.

Ptd(4,5)P(2) is thought to promote and organize a wide range of cellular functions, including vesicular membrane traffic and cytoskeletal dynamics, by recruiting functional protein complexes to restricted locations in cellular membranes. However, little is known about the distribution of PtdIns(4,5)P(2) in the cell at high resolution. We have used the pleckstrin homology (PH) domain of phospholipase delta(1) (PLCdelta(1)), narrowly specific for PtdIns(4,5)P(2), to map the distribution of the lipid in astrocytoma and A431 cells. We applied the glutathione S-transferase-tagged PLCdelta(1) PH domain (PLCdelta(1)PH-GST) in an on-section labelling approach which avoids transfection procedures. Here we demonstrate PtdIns(4,5)P(2) labelling in the plasma membrane, and also in intracellular membranes, including Golgi (mainly stack), endosomes and endoplasmic reticulum, as well as in electron-dense structures within the nucleus. At the plasma membrane, labelling was more concentrated over lamellipodia, but not in caveolae, which contained less than 10% of the total cell-surface labelling. A dramatic decrease in signal over labelled compartments was observed on preincubation with the cognate headgroup [Ins(1,4,5)P(3)], and plasma-membrane labelling was substantially decreased after stimulation with thrombin-receptor-activating peptide (SFLLRN in the one-letter amino acid code), a treatment which markedly diminishes PtdIns(4,5)P(2) levels. Thus we have developed a highly selective method for mapping the PtdIns(4,5)P(2) distribution within cells at high resolution, and our data provide direct evidence for this lipid at key functional locations.

The acquisition of resistance to apoptosis, the cell's intrinsic suicide program, is essential for cancers to arise and progress and is a major reason behind treatment failures. We show in this article that small molecule antagonists of the -1 receptor inhibit tumor cell survival to reveal caspase-dependent apoptosis. antagonist-mediated caspase activation and cell death are substantially attenuated by the prototypic -1 agonists (؉)-SKF10,047 and (؉)-pentazocine. Although several normal cell types such as fibroblasts, epithelial cells, and even receptor-rich neurons are resistant to the apoptotic effects of antagonists, cells that can promote autocrine survival such as lens epithelial and microvascular endothelial cells are as susceptible as tumor cells. Cellular susceptibility appears to correlate with differences in receptor coupling rather than levels of expression. In susceptible cells only, antagonists evoke a rapid rise in cytosolic calcium that is inhibited by -1 agonists. In at least some tumor cells, antagonists cause calcium-dependent activation of phospholipase C and concomitant calcium-independent inhibition of phosphatidylinositol 3-kinase pathway signaling. Systemic administration of antagonists significantly inhibits the growth of evolving and established hormone-sensitive and hormone-insensitive mammary carcinoma xenografts, orthotopic prostate tumors, and p53-null lung carcinoma xenografts in immunocompromised mice in the absence of side effects. Release of a receptor-mediated brake on apoptosis may offer a new approach to cancer treatment.

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