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Medulloblastoma is the most common malignant brain tumor in children. Therapeutic approaches to medulloblastoma (combination of surgery, radiotherapy, and chemotherapy) have led to significant improvements, but these are achieved at a high cost to quality of life. Alternative therapeutic approaches are needed. Genetic mutations leading to the activation of the Hedgehog pathway drive tumorigenesis in ~30% of medulloblastoma. In a yeast two-hybrid proteomic screen, we discovered a novel interaction between GLI1, a key transcription factor for the mediation of Hedgehog signals, and PIN1, a peptidylprolyl cis/trans isomerase that regulates the postphosphorylation fate of its targets. The GLI1/PIN1 interaction was validated by reciprocal pulldowns using epitope-tagged proteins in HEK293T cells as well as by co-immunoprecipiations of the endogenous proteins in a medulloblastoma cell line. Our results support a molecular model in which PIN1 promotes GLI1 protein abundance, thus contributing to the positive regulation of Hedgehog signals. Most importantly, in vivo functional analyses of Pin1 in the GFAP-tTA;TRE-SmoA1 mouse model of Hedgehog-driven medulloblastoma demonstrate that the loss of Pin1 impairs tumor development and dramatically increases survival. In summary, the discovery of the GLI1/PIN1 interaction uncovers PIN1 as a novel therapeutic target in Hedgehog-driven medulloblastoma tumorigenesis.

In this study, we examined ErbB1 signaling in human basal and squamous cell carcinomas (BCC and SCC) of the skin in vivo. We used enzyme-linked immunosorbent assay, laser capture microdissection-coupled real-time reverse transcriptase-polymerase chain reaction, and immunohistochemistry to assess expression and activation levels of ErbB1 protein, ligands, and potential downstream effectors, in BCC and SCC tumors, stroma, and adjacent epidermis. Although total ErbB1 protein and mRNA were similar in cancerous and normal skin, we found that ErbB1 activation (phospho-Tyr(1068)) was greater in bulk SCC versus BCC or normal skin. In addition, three ErbB1 ligand transcripts (amphiregulin, heparin-binding epidermal growth factor-like growth factor, and transforming growth factor-alpha) were up-regulated in tumor cells of SCC but not BCC. Expression of these ligands was also increased in asymptomatic epidermis adjacent to both SCC and BCC, relative to normal skin. Interestingly, betacellulin transcript levels were inversely regulated compared with the other ligands. Consistently, downstream ErbB1 effectors (Erk1/2 and Akt) were activated in tumor cells of SCC but not of BCC and in adjacent epidermis of both BCC and SCC. These results demonstrate that ErbB1 signaling is hyperactive in tumor cells of SCC but not of BCC and in nearby asymptomatic epidermis of both tumor types. Our results suggest that targeting ErbB1 signaling might be of benefit in the treatment of SCC.

Medulloblastoma is the most common malignant brain tumor of childhood. Therapeutic approaches to medulloblastoma have led to significant improvements, but are achieved at a high cost to quality of life. Aberrant upregulation of the hedgehog (Hh) pathway drives cerebellar tumorigenesis in ∼30% of medulloblastoma patients. Hh pathway inhibitors such as the SMO antagonist Vismodegib are currently being tested in Hh-activated medulloblastoma and the preliminary results are encouraging. However, resistance to SMO inhibition can be acquired, leading to relapse. Alternative therapeutic approaches are needed. We aim to uncover novel Hh signal modulators that are essential in medulloblastoma to maintain tumorigenic potential. Using our proteomic platform for systematic protein interaction mapping, we discovered a novel interaction between GLI1, a key transcription factor for the mediation of Hh signals, and PIN1, a peptidylprolyl cis/trans isomerase that regulates the post-phosphorylation conformation of its substrates. Our results support a molecular model in which PIN1 protects GLI1 from proteasomal degradation, thus contributing to the positive regulation of Hh signals. Most importantly, our in vivo functional analyses of PIN1 in mouse models of Hh-driven medulloblastoma demonstrate that the loss-of-PIN1 impairs tumor development and increases survival by 3 fold, from 57 to 158 days (P < 0.0001), establishing PIN1 as a key factor in Hh-driven medulloblastoma tumorigenesis. Finally, in human medulloblastoma tumor samples, the GLI1 and PIN1 proteins are correlated in their expression, supporting the relevance of the GLI1/PIN1 interaction in this disease context. In summary, the discovery of the GLI1/PIN1 interaction uncovers PIN1 as a novel therapeutic target in medulloblastoma tumorigenesis. If our hypothesis is validated, i.e., PIN1 inhibitors can improve survival in mouse models of Hh-driven medulloblastoma, our project will strongly justify testing the clinical relevance of PIN1 blockade in medulloblastoma patients. Citation Format: Jean-Francois M. Rual, Tao Xu, Honglai Zhang, Sung-Soo Park, Sriram Venneti, Rork Kuick, Kimberly Ha, Lowell Michael, Mariarita Santi, Chiyoko Uchida, Takafumi Uchida, Ashok Srinivasan, Andrzej Dlugosz, Sandra Camelo-Piragua. PIN1 protects GLI1 from ubiquitination and promotes Hedgehog-driven medulloblastoma tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2447.

A plethora of growth factors regulate keratinocyte proliferation and differentiation that control hair morphogenesis and skin barrier formation. Wavy hair phenotypes in mice result from naturally occurring loss-of-function mutations in the genes for TGF-alpha and EGFR. Conversely, excessive activities of TGF-alpha/EGFR result in hairless phenotypes and skin cancers. Unexpectedly, we found that mice lacking the Trpv3 gene also exhibit wavy hair coat and curly whiskers. Here we show that keratinocyte TRPV3, a member of the transient receptor potential (TRP) family of Ca(2+)-permeant channels, forms a signaling complex with TGF-alpha/EGFR. Activation of EGFR leads to increased TRPV3 channel activity, which in turn stimulates TGF-alpha release. TRPV3 is also required for the formation of the skin barrier by regulating the activities of transglutaminases, a family of Ca(2+)-dependent crosslinking enzymes essential for keratinocyte cornification. Our results show that a TRP channel plays a role in regulating growth factor signaling by direct complex formation.