Synergistic cross-talk of hedgehog and interleukin-6 signaling drives growth of basal cell carcinoma.

Persistent activation of hedgehog (HH)/GLI signaling accounts for the development of basal cell carcinoma (BCC), a very frequent nonmelanoma skin cancer with rising incidence. Targeting HH/GLI signaling by approved pathway inhibitors can provide significant therapeutic benefit to BCC patients. However, limited response rates, development of drug resistance, and severe side effects of HH pathway inhibitors call for improved treatment strategies such as rational combination therapies simultaneously inhibiting HH/GLI and cooperative signals promoting the oncogenic activity of HH/GLI. In this study, we identified the interleukin-6 (IL6) pathway as a novel synergistic signal promoting oncogenic HH/GLI via STAT3 activation. Mechanistically, we provide evidence that signal integration of IL6 and HH/GLI occurs at the level of cis-regulatory sequences by co-binding of GLI and STAT3 to common HH-IL6 target gene promoters. Genetic inactivation of Il6 signaling in a mouse model of BCC significantly reduced in vivo tumor growth by interfering with HH/GLI-driven BCC proliferation. Our genetic and pharmacologic data suggest that combinatorial HH-IL6 pathway blockade is a promising approach to efficiently arrest cancer growth in BCC patients.

Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems.

UBE2W ubiquitinates N termini of proteins rather than internal lysine residues, showing a preference for substrates with intrinsically disordered N termini. The in vivo functions of this intriguing E2, however, remain unknown. We generated Ube2w germ line KO mice that proved to be susceptible to early postnatal lethality without obvious developmental abnormalities. Although the basis of early death is uncertain, several organ systems manifest changes in Ube2w KO mice. Newborn Ube2w KO mice often show altered epidermal maturation with reduced expression of differentiation markers. Mirroring higher UBE2W expression levels in testis and thymus, Ube2w KO mice showed a disproportionate decrease in weight of these two organs (~50%), suggesting a functional role for UBE2W in the immune and male reproductive systems. Indeed, Ube2w KO mice displayed sustained neutrophilia accompanied by increased G-CSF signaling and testicular vacuolation associated with decreased fertility. Proteomic analysis of a vulnerable organ, presymptomatic testis, showed a preferential accumulation of disordered proteins in the absence of UBE2W, consistent with the view that UBE2W preferentially targets disordered polypeptides. These mice further allowed us to establish that UBE2W is ubiquitously expressed as a single isoform localized to the cytoplasm and that the absence of UBE2W does not alter cell viability in response to various stressors. Our results establish that UBE2W is an important, albeit not essential, protein for early postnatal survival and normal functioning of multiple organ systems.

Viral Status Predicts the Patterns of Genome Methylation and Decitabine Response in Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine carcinoma that is classified as Merkel cell polyomavirus-positive (virus positive [VP]) or Merkel cell polyomavirus-negative (virus negative [VN]). Epigenetic changes, such as DNA methylation, can alter gene expression and influence cancer progression. However, patterns of DNA methylation and the therapeutic efficacy of hypomethylating agents have not been fully explored in MCC. We characterized genome-wide DNA methylation in 16 MCC cell lines from both molecular subclasses in comparison with other cancer types and found that the overall profile of MCC is similar to that of small-cell lung carcinoma. Comparison of VP MCC with VN MCC revealed 2,260 differentially methylated positions. The hypomethylating agent decitabine upregulated the expression of antigen-presenting machinery in MCC cell lines and stimulated membrane expression of HLA-A in VP and VN MCC xenograft tumors. Decitabine also induced prominent caspase- and large T antigen‒independent cell death in VP MCC, whereas VN MCC cell lines displayed decreased proliferation without increased cell death. In mouse xenografts, decitabine significantly decreased the size of VP tumors but not that of VN tumors. Our findings indicate that viral status predicts genomic methylation patterns in MCC and that decitabine may be therapeutically effective against MCC through antiproliferative effects, cell death, and increased immune recognition.

Characterizing the Therapeutic Potential of a Potent BET Degrader in Merkel Cell Carcinoma.

Studies on the efficacy of small molecule inhibitors in Merkel cell carcinoma (MCC) have been limited and largely inconclusive. In this study, we investigated the therapeutic potential of a potent BET degrader, BETd-246, in the treatment of MCC. We found that MCC cell lines were significantly more sensitive to BETd-246 than to BET inhibitor treatment. Therapeutic targeting of BET proteins resulted in a loss of "MCC signature" genes but not MYC expression as previously described irrespective of Merkel cell polyomavirus (MCPyV) status. In MCPyV+ MCC cells, BETd-246 alone suppressed downstream targets in the MCPyV-LT Ag axis. We also found enrichment of HOX and cell cycle genes in MCPyV- MCC cell lines that were intrinsically resistant to BETd-246. Our findings uncover a requirement for BET proteins in maintaining MCC lineage identity and point to the potential utility of BET degraders for treating MCC.

Tumor Architecture and Notch Signaling Modulate Drug Response in Basal Cell Carcinoma.

Hedgehog (Hh) pathway inhibitors such as vismodegib are highly effective for treating basal cell carcinoma (BCC); however, residual tumor cells frequently persist and regenerate the primary tumor upon drug discontinuation. Here, we show that BCCs are organized into two molecularly and functionally distinct compartments. Whereas interior Hh+/Notch+ suprabasal cells undergo apoptosis in response to vismodegib, peripheral Hh+++/Notch- basal cells survive throughout treatment. Inhibiting Notch specifically promotes tumor persistence without causing drug resistance, while activating Notch is sufficient to regress already established lesions. Altogether, these findings suggest that the three-dimensional architecture of BCCs establishes a natural hierarchy of drug response in the tumor and that this hierarchy can be overcome, for better or worse, by modulating Notch.

Merkel Cell Polyomavirus Small T Antigen Initiates Merkel Cell Carcinoma-like Tumor Development in Mice.

Merkel cell carcinoma (MCC) tumor cells express several markers detected in normal Merkel cells, a nonproliferative population of neuroendocrine cells that arise from epidermis. MCCs frequently contain Merkel cell polyomavirus (MCPyV) DNA and express viral transforming antigens, sT and tLT, but the role of these putative oncogenes in MCC development, and this tumor's cell of origin, are unknown. Using a panel of preterm transgenic mice, we show that epidermis-targeted coexpression of sT and the cell fate-determinant atonal bHLH transcription factor 1 (ATOH1) leads to development of widespread cellular aggregates, with histology and marker expression mimicking that of human intraepidermal MCC. The MCC-like tumor phenotype was dependent on the FBXW7-binding domain of sT, but not the sT-PP2A binding domain. Coexpression of MCPyV tLT did not appreciably alter the phenotype driven by either sT or sT combined with ATOH1. MCPyV sT, when coexpressed with ATOH1, is thus sufficient to initiate development of epidermis-derived MCC-like tumors in mice. Cancer Res; 77(12); 3151-7. ©2017 AACR.

Merkel cell polyomavirus small T antigen is oncogenic in transgenic mice.

Merkel cell carcinoma (MCC) is a rare and deadly neuroendocrine skin tumor frequently associated with clonal integration of a polyomavirus, Merkel cell polyomavirus (MCPyV), and MCC tumor cells express putative polyomavirus oncoprotein small T antigen (sTAg) and truncated large T antigen. Here, we show robust transforming activity of sTAg in vivo in a panel of transgenic mouse models. Epithelia of preterm sTAg-expressing embryos exhibited hyperplasia, impaired differentiation, increased proliferation, and apoptosis, and activation of a DNA damage response. Epithelial transformation did not require sTAg interaction with the protein phosphatase 2A protein complex, a tumor suppressor in some other polyomavirus transformation models, but was strictly dependent on a recently described sTAg domain that binds Fbxw7, the substrate-binding component of the Skp1/Cullin1/F-box protein ubiquitin ligase complex. Postnatal induction of sTAg using a Cre-inducible transgene also led to epithelial transformation with development of lesions resembling squamous cell carcinoma in situ and elevated expression of Fbxw7 target proteins. Our data establish that expression of MCPyV sTAg alone is sufficient for rapid neoplastic transformation in vivo, implicating sTAg as an oncogenic driver in MCC and perhaps other human malignancies. Moreover, the loss of transforming activity following mutation of the sTAg Fbxw7 binding domain identifies this domain as crucial for in vivo transformation.

Merkel cell carcinoma dependence on bcl-2 family members for survival.

Merkel cell carcinoma (MCC), a rare but aggressive cutaneous neoplasm with high metastatic potential, has a poor prognosis at late stages of disease with no proven chemotherapeutic regimens. Using an enriched culture medium, we established and characterized 11 MCC cell lines for Bcl-2 family profiling and functional studies. Immunoblot analysis revealed collectively high protein levels of prosurvival Bcl-2 members in cell lines and a panel of MCC tumors. Downregulation of individual Bcl-2 proteins by RNAi promoted death in a subset of MCC cell lines, whereas simultaneous inhibition of multiple family members by using the small-molecule antagonist ABT-263 led to a marked induction of cell death in 10 of 11 lines. ABT-263 induced Bax-dependent apoptosis with rapid cleavage of caspase-3 and PARP, regardless of Bcl-2 family profile or the presence of Merkel cell polyomavirus. Furthermore, ABT-263 treatment led to rapid and sustained growth suppression of MCC xenografts from a representative cell line, accompanied by a striking increase in apoptosis. Our results establish that concurrent inhibition of multiple prosurvival Bcl-2 proteins leads to effective induction of apoptosis, and strongly support the concept that targeting MCC dependence on these molecules may be useful therapeutically by reversing an intrinsic resistance to cell death.

Cooperative Hedgehog-EGFR signaling.

It has been known for many years that cooperative interactions between oncogenes (e.g. RAS, MYC, BCL2) can fuel cancer growth (1-5), but the restricted druggability of many of those interacting cancer genes has hampered translation of combined targeting to medical cancer therapy. The identification and characterization of cooperative cancer signaling pathways amenable to medical therapy is therefore a crucial step towards the establishment of efficient targeted combination treatments urgently needed to improve cancer therapy. Here we review recent findings of our group and colleagues on the molecular mechanisms of cooperative Hedgehog/GLI and Epidermal Growth Factor Receptor (EGFR) signaling, two clinically relevant oncogenic pathways involved in the development of many human malignancies. We also discuss the possible implications of these findings for the design of a therapeutic regimen relying on combined targeting of key effectors of both pathways.

Hedgehog-EGFR cooperation response genes determine the oncogenic phenotype of basal cell carcinoma and tumour-initiating pancreatic cancer cells.

Inhibition of Hedgehog (HH)/GLI signalling in cancer is a promising therapeutic approach. Interactions between HH/GLI and other oncogenic pathways affect the strength and tumourigenicity of HH/GLI. Cooperation of HH/GLI with epidermal growth factor receptor (EGFR) signalling promotes transformation and cancer cell proliferation in vitro. However, the in vivo relevance of HH-EGFR signal integration and the critical downstream mediators are largely undefined. In this report we show that genetic and pharmacologic inhibition of EGFR signalling reduces tumour growth in mouse models of HH/GLI driven basal cell carcinoma (BCC). We describe HH-EGFR cooperation response genes including SOX2, SOX9, JUN, CXCR4 and FGF19 that are synergistically activated by HH-EGFR signal integration and required for in vivo growth of BCC cells and tumour-initiating pancreatic cancer cells. The data validate EGFR signalling as drug target in HH/GLI driven cancers and shed light on the molecular processes controlled by HH-EGFR signal cooperation, providing new therapeutic strategies based on combined targeting of HH-EGFR signalling and selected downstream target genes.

IL-37: a new anti-inflammatory cytokine of the IL-1 family.

The IL-1 family of cytokines encompasses eleven proteins that each share a similar ß-barrel structure and bind to Ig-like receptors. Some of the IL-1-like cytokines have been well characterised, and play key roles in the development and regulation of inflammation. Indeed, IL-1α (IL-1F1), IL-1ß (IL-1F2), and IL-18 (IL-1F4) are well-known inflammatory cytokines active in the initiation of the inflammatory reaction and in driving Th1 and Th17 inflammatory responses. In contrast, IL-1 receptor antagonist (IL-1Ra, IL-1F3) and the receptor antagonist binding to IL-1Rrp2 (IL-36Ra, IL-1F5) reduce inflammation by blocking the binding of the agonist receptor ligands. In the case of IL-37 (IL-1F7), of which five different splice variants have been described, less is known of its function, and identification of the components of a heterodimeric receptor complex remains unclear. Some studies suggest that IL-37 binds to the α chain of the IL-18 receptor in a non-competitive fashion, and this may explain some of the disparate biological effects that have been reported for mice deficient in the IL-18R. The biological properties of IL-37 are mainly those of down-regulating inflammation, as assessed in models where human IL-37 is expressed in mice. In this review, an overview of the role of IL-37 in the regulation of inflammation is presented. The finding that IL-37 also locates to the nucleus, as do IL-1α and IL-33, for receptor-independent organ/tissue-specific regulation of inflammation is also reviewed.

Epidermal growth factor receptor signaling synergizes with Hedgehog/GLI in oncogenic transformation via activation of the MEK/ERK/JUN pathway.

Persistent activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of a number of human cancers. The GLI zinc finger transcription factors act at the end of the HH signaling cascade to control gene expression, and recent studies have shown that the activity of GLI proteins can be additionally modified by integration of distinct signals, such as the MEK/extracellular signal-regulated kinase (ERK) and phosphinositide-3 kinase (PI3K)/AKT pathway. However, little is known about the identity of the upstream activators of these HH/GLI interacting signaling pathways in cancer. Here, we provide evidence that integration of the HH/GLI and epidermal growth factor receptor (EGFR) pathway synergistically induces oncogenic transformation, which depends on EGFR-mediated activation of the RAS/RAF/MEK/ERK but not of the PI3K/AKT pathway. EGFR/MEK/ERK signaling induces JUN/activator protein 1 activation, which is essential for oncogenic transformation, in combination with the GLI activator forms GLI1 and GLI2. Furthermore, pharmacologic inhibition of EGFR and HH/GLI efficiently reduces growth of basal cell carcinoma (BCC) cell lines derived from mice with activated HH/GLI signaling. The results identify the synergistic integration of GLI activator function and EGFR signaling as a critical step in oncogenic transformation and provide a molecular basis for therapeutic opportunities relying on combined inhibition of the HH/GLI and EGFR/MEK/ERK/JUN pathway in BCC.