Melanoma is one of the most aggressive cancers, and its incidence is increasing. These tumors derive from the melanocyte lineage and remain incurable after metastasis. Here we report that SONIC HEDGEHOG (SHH)-GLI signaling is active in the matrix of human hair follicles, and that it is required for the normal proliferation of human melanocytes in culture. SHH-GLI signaling also regulates the proliferation and survival of human melanomas: the growth, recurrence, and metastasis of melanoma xenografts in mice are prevented by local or systemic interference of HH-GLI function. Moreover, we show that oncogenic RAS-induced melanomas in transgenic mice express Gli1 and require Hh-Gli signaling in vitro and in vivo. Finally, we provide evidence that endogenous RAS-MEK and AKT signaling regulate the nuclear localization and transcriptional activity of GLI1 in melanoma and other cancer cells. Our data uncover an unsuspected role of HH-GLI signaling in melanocytes and melanomas, demonstrate a role for this pathway in RAS-induced tumors, suggest a general integration of the RAS/AKT and HH-GLI pathways, and open a therapeutic approach for human melanomas.cancer ͉ melanocyte ͉ hair follicle ͉ metastasis ͉ skin

Human colon cancers often start as benign adenomas through loss of APC, leading to enhanced βCATENIN (βCAT)/TCF function. These early lesions are efficiently managed but often progress to invasive carcinomas and incurable metastases through additional changes, the nature of which is unclear. We find that epithelial cells of human colon carcinomas (CCs) and their stem cells of all stages harbour an active HH-GLI pathway. Unexpectedly, they acquire a high HEDGEHOG-GLI (HH-GLI) signature coincident with the development of metastases. We show that the growth of CC xenografts, their recurrence and metastases require HH-GLI function, which induces a robust epithelial-to-mesenchymal transition (EMT). Moreover, using a novel tumour cell competition assay we show that the self-renewal of CC stem cells in vivo relies on HH-GLI activity. Our results indicate a key and essential role of the HH-GLI1 pathway in promoting CC growth, stem cell self-renewal and metastatic behavior in advanced cancers. Targeting HH-GLI1, directly or indirectly, is thus predicted to decrease tumour bulk and eradicate CC stem cells and metastases.

The Gli code hypothesis postulates that the three vertebrate Gli transcription factors act together in responding cells to integrate intercellular Hedgehog (Hh) and other signaling inputs, resulting in the regulation of tissue pattern, size and shape. Hh and other inputs are then just ways to modify the Gli code. Recent data confirm this idea and suggest that the Gli code regulates stemness and also tumor progression and metastatic growth, opening exciting possibilities for both regenerative medicine and novel anticancer therapies.

1Cx36 is the predominant connexin isoform expressed by pancreatic ␤-cells. However, little is known about the role of this protein in the functioning of insulin-secreting cells. To address this question, we searched for a cell line expressing Cx36 and having glucose-induced insulin secretion comparable to that of primary ␤-cells. T he well coordinated functioning of cells within tissues is ensured by multiple mechanisms, including the direct exchange of ions, second messengers, and other metabolites through connexin channels, clustered at gap junctions (1,2). These channels connect almost all types of vertebrate cells, including those of pancreatic islets (3). Previous studies suggest that connexin-mediated communication between ␤-cells is required for the control of insulin secretion. Thus, glucose stimulation of single ␤-cells is reduced compared with that of cell clusters (4), pharmacological blockade of gap junction channels markedly decreases insulin release (5), and transgenic mice whose ␤-cells express Cx32 show altered insulin secretion in response to glucose (6). While these findings provide evidence that connexin-dependent communication contributes to the control of insulin release, they do not specifically address the function of native connexins expressed by pancreatic islet cells.We recently found that Cx36 is the predominant isoform expressed by ␤-cells (7). As yet, however, the specific role of this somewhat unusual connexin isoform (8,9) and, in particular, its possible contribution to insulin secretion have not been investigated. Moreover, no data are available to show how connexin-mediated signaling may influence ␤-cell function even though gap junctions have been implicated in the synchronization of [Ca 2ϩ ] i oscillations in ␤-cells (10 -13).To address these questions, we screened MIN6 cells, which, unlike some other cell lines, retain glucose-induced insulin secretion and express Cx36 like primary ␤-cells (14 -19). We decreased Cx36 expression via the stable transfection of an antisense construct. Here, we show that the loss of Cx36 resulted in impaired electrical coupling, desynchronization of [Ca 2ϩ ] i oscillations, and altered insulin release of MIN6 cells. These results support the hypothesis that Cx36 plays a critical role in glucoseinduced insulin secretion. RESEARCH DESIGN AND METHODSCell lines and tissues. RIN2A (14), INS1 (15), and HIT cells (16) were cultured in RPMI-1640 medium, whereas ␤TC3 (17) and MIN6 cells (18) were cultured in Dulbecco's modified Eagle's medium as indicated in the original reports. Control tissues were obtained from OFA rats (olfactory bulb) or C57BL/6 mice (liver and heart), which were frozen in liquid nitrogen and stored at Ϫ80°C until use. RNA analysis. Samples of total RNA were extracted, reverse-transcribed, and amplified as previously described (7). To detect native Cx36, we amplified either a 980-bp or a 559-bp fragment, using the oligonucleotide pairs 5Ј-CACAGCGATGGGGGAATGGA-3Ј/5Ј-TGCCCTTTCACACATAGGCA-3Ј and 5Ј-GAGCCCAGGCCAAGAGGAAGTC-3Ј/5Ј-GG...

Constitutive activation of canonical WNT-TCF signaling is implicated in multiple diseases, including intestine and lung cancers, but there are no WNT-TCF antagonists in clinical use. We have performed a repositioning screen for WNT-TCF response blockers aiming to recapitulate the genetic blockade afforded by dominant-negative TCF. We report that Ivermectin inhibits the expression of WNT-TCF targets, mimicking dnTCF, and that its low concentration effects are rescued by direct activation by TCFVP16. Ivermectin inhibits the proliferation and increases apoptosis of various human cancer types. It represses the levels of C-terminal β-CATENIN phosphoforms and of CYCLIN D1 in an okadaic acid-sensitive manner, indicating its action involves protein phosphatases.In vivo, Ivermectin selectively inhibits TCF-dependent, but not TCF-independent, xenograft growth without obvious side effects. Analysis of single semi-synthetic derivatives highlights Selamectin, urging its clinical testing and the exploration of the macrocyclic lactone chemical space. Given that Ivermectin is a safe anti-parasitic agent used by > 200 million people against river blindness, our results suggest its additional use as a therapeutic WNT-TCF pathway response blocker to treat WNT-TCF-dependent diseases including multiple cancers.