Although tissue microenvironments play critical roles in epithelial development and tumorigenesis, the factors mediating these effects are poorly understood. In this work, we used a genomic approach to identify factors produced by cells in the microenvironment of basal cell carcinoma (BCC) of the skin, one of the most common human cancers. The global gene expression programs of stromal cell cultures derived from human BCCs showed consistent, systematic differences from those derived from nontumor skin. The gene most consistently expressed at a higher level in BCC tumor stromal cells compared with those from nontumor skin was GREMLIN 1, which encodes a secreted antagonist of the bone morphogenetic protein (BMP) pathway. BMPs and their antagonists are known to play a crucial role in stem and progenitor cell biology as regulators of the balance between expansion and differentiation. Consistent with the hypothesis that BMP antagonists might have a similar role in cancer, we found GREMLIN 1 expression in the stroma of human BCC tumors but not in normal skin in vivo. Furthermore, BMP 2 and 4 are expressed by BCC cells. Ex vivo, BMP inhibits, and Gremlin 1 promotes, proliferation of cultured BCC cells. We further found that GREMLIN 1 is expressed by stromal cells in many carcinomas but not in the corresponding normal tissue counterparts that we examined. Our data suggest that BMP antagonists may be important constituents of tumor stroma, providing a favorable microenvironment for cancer cell survival and expansion in many cancers.cancer biology ͉ stem cell regulation ͉ tissue microenvironment ͉ tumor stroma

Regulated protein destruction controls many key cellular processes with aberrant regulation increasingly found during carcinogenesis. Gli proteins mediate the transcriptional effects of the Sonic hedgehog pathway, which is implicated in up to 25% of human tumors. Here we show that Gli is rapidly destroyed by the proteasome and that mouse basal cell carcinoma induction correlates with Gli protein accumulation. We identify two independent destruction signals in Gli1, D N and D C , and show that removal of these signals stabilizes Gli1 protein and rapidly accelerates tumor formation in transgenic animals. These data argue that control of Gli protein accumulation underlies tumorigenesis and suggest a new avenue for antitumor therapy. Factors controlling protein destruction are critical for the timing of key processes such as the cell cycle, apoptosis, and cell fate decisions, with aberrant regulation increasingly found during carcinogenesis (Pickart 2004;Yamasaki and Pagano 2004). Inappropriate Sonic hedgehog (Shh) signaling results in a panoply of birth defects and is implicated in up to 25% of human tumors (Callahan and Oro 2001;Lum and Beachy 2004). While the Gli family of proteins mediates the transcriptional effects of Shh (Methot and Basler 2001;Ruiz i Altaba et al. 2002), the mechanism by which Gli proteins are regulated to achieve changes in pathway output remains poorly understood. Studies in mice and humans show that Shh target gene induction is sufficient to induce a variety of tumors including basal cell carcinomas (BCCs) (Oro et al. 1997;Nilsson et al. 2000;Hutchin et al. 2005). However, there is a wide variability in the onset and severity of phenotypes among patients with mutations in the Shh pathway (Wicking et al. 1997), and a noticeably wide variability of tumor onset in animal models (Oro and Higgins 2003;Hutchin et al. 2005). This suggests the possibility that additional, previously uncharacterized, cellular processes regulate pathway output. Here we show that Gli protein accumulation correlates with tumor formation and stabilizing mutations in Gli protein dramatically accelerate tumor induction. Results and DiscussionWhile expression of either Gli1 or Gli2 in the epidermis of transgenic mice induces BCCs (Fig. 1a), we have observed a considerable delay in the appearance of Gli-dependent tumors. Analysis of transgenic mice expressing Gli2 revealed an average latency of 7 mo before tumor appearance (Fig. 1b). We ruled out changes in transcription of the transgene with age as a cause of the tumors, as similar levels of RNA are seen in both age groups as measured by quantitative PCR (Fig. 1c). This suggested the existence in keratinocytes of additional processes, whose loss or dysregulation is required to permit Gli activity and direct tumor formation. Our previous studies indicated that differential accumulation of Gli protein plays an important role in restricting Shh target gene induction in interfollicular epithelium (Oro and Higgins 2003). Indeed, we detected no transgenic Gli protein in normal...

Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen owing to mutations in the gene COL7A1 and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We generated patient-derived COL7A1-corrected epithelial keratinocyte sheets for autologous grafting. We demonstrate the utility of sequential reprogramming and adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity, and these cells secreted wild-type type VII collagen, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice. Sequencing of corrected cell lines before tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1-corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a clinical platform to use iPSCs in the treatment of debilitating genodermatoses, such as RDEB.

Sonic hedgehog (Shh) signaling plays a critical role during development and carcinogenesis. While Gli family members govern the transcriptional output of Shh signaling, little is known how Gli-mediated transcriptional activity is regulated. Here we identify the actin-binding protein Missing in Metastasis (MIM) as a new Shhresponsive gene. Together, Gli1 and MIM recapitulate Shh-mediated epidermal proliferation and invasion in regenerated human skin. MIM is part of a Gli/Suppressor of Fused complex and potentiates Gli-dependent transcription using domains distinct from those used for monomeric actin binding. These data define MIM as both a Shh-responsive gene and a new member of the pathway that modulates Gli responses during growth and tumorigenesis.Supplemental material is available at http://www.genesdev.org.

During hair follicle morphogenesis, dermal papillae (DPs) function as mesenchymal signaling centers that crosstalk with overlying epithelium to regulate morphogenesis. While the DP regulates hair follicle formation, relatively little is known about the molecular basis of DP formation. The morphogen Sonic hedgehog (Shh) is known for regulating hair follicle epithelial growth, with excessive signaling resulting in basal cell carcinomas. Here, we investigate how dermal-specific Shh signaling contributes to DP formation and hair growth. Using a Crelox genetic model and RNAi in hair follicle reconstitution assays, we demonstrate that dermal Smoothened (Smo) loss of function results in the loss of the DP precursor, the dermal condensate, and a stage 2 hair follicle arrest phenotype reminiscent of Shh -/-skin. Surprisingly, dermal Smo does not regulate cell survival or epithelial proliferation. Rather, molecular screening and immunostaining studies reveal that dermal Shh signaling controls the expression of a subset of DP-specific signature genes. Using a hairpin/cDNA lentiviral system, we show that overexpression of the Shh-dependent gene Noggin, but not Sox2 or Sox18, can partially rescue the dermal Smo knockdown hair follicle phenotype by increasing the expression of epithelial Shh. Our findings suggest that dermal Shh signaling regulates specific DP signatures to maintain DP maturation while maintaining a reciprocal ShhNoggin signaling loop to drive hair follicle morphogenesis.