Supplementary Figure S1. Transgenic constructs and validation of transgene expression in vivo. Supplementary Figure S2. Paranuclear dot-like expression of K8 and K5 in mouse MCC-like tumors. Supplementary Table S1. Correlation between transgene expression level, based on immunostaining, and phenotype severity. Supplementary Table S2.Primary antibodies and dilutions.

Supplementary Figures 1-9, Tables 1-2 from Epidermal Growth Factor Receptor Signaling Synergizes with Hedgehog/GLI in Oncogenic Transformation via Activation of the MEK/ERK/JUN Pathway

Supplementary Figure Legends 1-9 from Epidermal Growth Factor Receptor Signaling Synergizes with Hedgehog/GLI in Oncogenic Transformation via Activation of the MEK/ERK/JUN Pathway

Basal cell carcinomas (BCCs) frequently possess immense mutational burdens; however, the functional significance of most of these mutations remains unclear. Here, we report that loss of Ptch1, the most common mutation that activates upstream Hedgehog (Hh) signaling, initiates the formation of nascent BCC-like tumors that eventually enter into a dormant state. However, rare tumors that overcome dormancy acquire the ability to hyperactivate downstream Hh signaling through a variety of mechanisms, including amplification of Gli1/2 and upregulation of Mycn. Furthermore, we demonstrate that MYCN overexpression promotes the progression of tumors induced by loss of Ptch1. These findings suggest that canonical mutations that activate upstream Hh signaling are necessary, but not sufficient, for BCC to fully progress. Rather, tumors likely acquire secondary mutations that further hyperactivate downstream Hh signaling in order to escape dormancy and enter a trajectory of uncontrolled expansion.

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.

Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11β2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.

Inhibitors of the Hedgehog (Hh) signaling pathway are effective at regressing basal cell carcinoma (BCC); however, tumor cells often persist and regenerate the original tumor upon drug stoppage. Using a mouse model of BCC, we show that these tumors are comprised of 2 molecularly and functionally distinct subcompartments. Whereas interior suprabasal cells display elevated Notch signaling and increased apoptosis in response to the Hh inhibitor vismodegib, peripheral basal cells possess increased Hh and persist throughout treatment. We further demonstrate that inhibiting Notch protects tumors against anti-Hh therapy, while activating Notch regresses already established lesions. Finally, we show that some human BCCs contain suprabasal tumor cells that activate Notch, whereas others do not. Altogether, our findings suggest that an interplay of Notch and Hh signaling may determine whether tumor cells persist or apoptose in response to therapeutic Hh pathway blockade. As Notch mutations are commonly observed in human BCCs, our findings suggest that eliminating Notch-low basal tumor cells is paramount for preventing recurrence.

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.

Abstract 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.

The role of STAT1 and STAT3 for colorectal carcinoma (CRC) development and progression is controversial. We evaluated 414 CRC patient samples on tissue microarrays for differential expression of STAT1 and STAT3 protein levels and correlated ratios with clinical parameters. Concomitant absence of nuclear STAT1 and STAT3 expression was associated with significantly reduced median survival by ≥33 months (p=0.003). To gain insight into underlying mechanisms, we generated four CRC cell lines with STAT3 knockdown. The cell lines harbor different known mutational drivers and were xenografted into SCID mice to analyze the influence of STAT3 on their tumor growth behavior. Experimental downregulation of STAT3 expression had differential, cell-line specific effects on STAT1 expression levels. STAT1 consistently showed nuclear localization irrespective of its tyrosine phosphorylation status. Two characteristic STAT1/3 expression patterns with opposite growth behavior could be distinguished: cell lines with a low STAT1/high STAT3 ratio showed faster tumor growth in xenografts. In contrast, xenografts of cell lines showing high STAT1 and low STAT3 levels grew slower. Importantly, these ratios reflected clinical outcome in CRC patients as well. We conclude that the ratio of STAT1 to STAT3 expression is a key determinant of CRC progression and that STAT1 counteracts pro-tumorigenic STAT3 signaling. Thus, we suggest that the STAT3/STAT1 ratios are better clinical predictors in CRC as compared to STAT3 or STAT1 levels alone.

Basal cell carcinoma (BCC) is characterized by frequent loss of PTCH1, leading to constitutive activation of the Hedgehog pathway. Although the requirement for Hedgehog in BCC is well established, the identity of disease-initiating cells and the compartments in which they reside remain controversial. By using several inducible Cre drivers to delete Ptch1 in different cell compartments in mice, we show here that multiple hair follicle stem cell populations readily develop BCC-like tumors. In contrast, stem cells within the interfollicular epidermis do not efficiently form tumors. Notably, we observed that innervated Gli1-expressing progenitors within mechanosensory touch dome epithelia are highly tumorigenic. Sensory nerves activate Hedgehog signaling in normal touch domes, while denervation attenuates touch dome-derived tumors. Together, our studies identify varying tumor susceptibilities among different stem cell populations in the skin, highlight touch dome epithelia as "hot spots" for tumor formation, and implicate cutaneous nerves as mediators of tumorigenesis.

S oF LECTuRES (in alphabetical order of speakers) BLANpAIN, Cèdric .................................................................................................................................. 9 CLARKE, Michael F. .............................................................................................................................10 DICK, John E. ........................................................................................................................................11 FRENETTE, Paul ...................................................................................................................................12 GIANCoTTI, Filippo G. ..........................................................................................................................13 GLIMM, Hanno .....................................................................................................................................14 HANAHAN, Douglas .............................................................................................................................15 JAMIESoN, Catriona ............................................................................................................................16 HuELSKEN, Joerg ................................................................................................................................. 17 KIM, Carla .............................................................................................................................................18 LEvINE, Ross L. ....................................................................................................................................19 LIu, Hai-Kun ..........................................................................................................................................20 MCKAy, Ronald ....................................................................................................................................21 MILSoM, Michael .................................................................................................................................22 NIEHRS, Christof ..................................................................................................................................23 pARADA, Luis F. ...................................................................................................................................24 pLASS, Christoph .................................................................................................................................25 RICH, Jeremy N. ...................................................................................................................................26 RuDoLpH, K. Lenhard .........................................................................................................................27 DE SAuvAGE, Frederic .........................................................................................................................28 TRuMpp, Andreas ................................................................................................................................29 vALIENTE, Manuel ...............................................................................................................................30 4 ABSTRACTS oF poSTERS (in alphabetical order of authors) ABERGER, Fritz ....................................................................................................................................35 ARNDT, Kathrin ....................................................................................................................................36 AuGuSTIN, Iris ...................................................................................................................................... 37 BASER, Avni ..........................................................................................................................................38 BELLvIS, Pablo .....................................................................................................................................39 BEyELER, Sarah ...................................................................................................................................40 BHAvASAR, Shefalee .......................................................................................................................... 41 BRECKwoLDT, Michael O. ..................................................................................................................42 CABEZAS-wALLSCHEID, Nina ............................................................................................................43 DHAwAN, Abhishek .............................................................................................................................44 EDGAR, Bruce A. ..................................................................................................................................45 EIBL, Robert H. .....................................................................................................................................46 ESpINET HERNáNDEZ, Elisa .............................................................................................................. 47 GIACHINo, Claudio ..............................................................................................................................48 GLAuCHE, Ingmar ................................................................................................................................49 GoIDTS, Violaine ..................................................................................................................................50 GoLESTANEH, Azadeh Fahim ............................................................................................................. 51 GRALLA, Robert ...................................................................................................................................52 HAAS, Simon ........................................................................................................................................53 HEILER, Sarah ......................................................................................................................................54 KAEBISCH, Constanze .........................................................................................................................55 KIJoNKA, Marek ..................................................................................................................................56 KIM, Ella L. ............................................................................................................................................57 KLIMMECK, Daniel ...............................................................................................................................58 KNApp, Silvana ....................................................................................................................................59 KRITSCH, Daniel ..................................................................................................................................60 LAuRENTI, Elisa ...................................................................................................................................61 MAuRER, Jochen .................................................................................................................................62 MELE, Valentina ...................................................................................................................................63 MENDE, Nicole .....................................................................................................................................64 MuLLINS, Christina Susanne ..............................................................................................................65 5 NoLL, Elisa M. ......................................................................................................................................66 NuNuKovA, Alena ................................................................................................................................67 oBoRoTovA, Marina ...........................................................................................................................68 pApADIMITRopouLoS, Adam ............................................................................................................69 pETERSSoN, Monika ...........................................................................................................................70 pHILLIpS, Emma .................................................................................................................................. 71 pRENDERGAST, Áine M. ....................................................................................................................72 RAHMIG, Susann .................................................................................................................................73 SCHIppER, Dorothee ........................................................................................................................... 74 SCHMIDT, Christin ...............................................................................................................................75 SCHüLE, Roland ................................................................................................................................... 76 SCoGNAMIGLIo, Roberta ...................................................................................................................77 SKoDA, Jan ..........................................................................................................................................78 uCKELMANN, Hannah .........................................................................................................................79 voN

Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor in many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling depend profoundly on interactions with other pathways, such as epidermal growth factor receptor-mediated signaling, which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. Our experimental data demonstrated that the Daoy human medulloblastoma cell line possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic HH or Smoothened agonist induced expression of GLI1 protein and simultaneously prevented the processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed at the transcriptomic and proteomic levels. The Daoy cells responded to the HH/EGF co-treatment by downregulating GLI1, PTCH, and HHIP at the transcript level; this was also observed when Amphiregulin (AREG) was used instead of EGF. We identified a novel crosstalk mechanism whereby EGFR signaling silences proteins acting as negative regulators of HH signaling, as AKT- and ERK-signaling independent process. EGFR/HH signaling maintained high GLI1 protein levels which contrasted the GLI1 downregulation on the transcript level. Conversely, a high-level synergism was also observed, due to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile. This suggests that there are more wide-spread, yet context-dependent interactions, between HH/GLI and growth factor receptor signaling in human malignancies.

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.

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.

Abstract 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. [Cancer Res 2009;69(4):1284–92]