R1 서자희

Sonic hedgehog signaling pathway Cell proliferation and differentiation during embryonic development – Paracrine manner – Loss of signaling activity  congenital anomaly holoprosencephaly, polydactly, craniofacial defects, and skeletal malformations – Mutation, misregulation  cancer Medulloblastoma (MBL) Basal cell carcinoma (BCC) Rhabdomyosarcoma

Role of Hh signaling in cancer 1 st identified in Gorlin syndrome –Loss of function : PTCH1 –Numerous BCCs, MBL and rhabdomyosarcoma Most spordic BCCs & 1/3 of spordic MBLs –Inactivating mutation in PTCH1 –Activating mutation in SMO (more rarely)

Role of Hh signaling in cancer Tumor cell produce and respond to Hh ligand in an autocrine manner – In the absence of mutations in components of the Hh pathway, Hh signaling has been described to play a role in the growth of a variety of epithelial cancer types – Small cell lung cancer, prostate cancer, pancreatic and other GI tract malignacies

Inhibition of Hh pathway  therapeutic effect in tumors – Mutation level : PTCH1 or SMO – Hh ligand : overexpression in tumors  Natural compound cyclopamine  Small molecule drug optimized Hh-Antag Inhibition of SMO : interference of tumorogenesis GDC-0499 treatment : Tumor regression reported in advanced BCCs & MBLs Alternative model in three recent articles paracrine manner – Tumor-produced Hh ligand trigger signaling in the stromal environment in a paracrine manner

Hh Hh Hh GLI1, PTCH, cyclin D, E, Myc etc relocalization Positive & negative feedback loop Epithelial cell Mesenchymal cell De-repressing

Hh pathway in paracrine manner Hh ligand-expressing cancer cells of epithelial origin are refractory to ligand, whereas surrounding stroma cells are ligand responsive

Smo-antagonist-mediated growth inhibition  not correlate with Hh target gene expression – in vitro growth repression d/t off-target effects – compounds are used at high concentrations IC50* of Hh Antag to inhibit ptw in mesenchymal cell line >> IC 50 required to inhibit cell growth x 20 High drug concentration  not correlate with regulation of Hh target genes (GLI1, PTCH1) Studies using high concentrations of some of the SMO-inhibitors need to be interpreted with caution *half-maximal inhibitory concentration

Nature vol. 455, 2008 Hh-Antag-mediated growth inhibition

Colorectal, pancreatic, ovarian tumor – Hh ligand expression in particular SHH and/or IHH – levels of Hh ligand mRNA expression in tumor cells correlated with increased Gli1 and Ptch1 mRNA levels in the stroma and not the tumor compartment Hh ligands activate Hh signaling in the surrounding stroma and not the tumor epithelium

Therapeutic value of inhibition of paracrine signal Specific inhibition of Hh signaling in the stroma with either Hh-Antag or a neutralizing anti-Hh antibody  growth inhibition of Hh ligand-expressing xenografts Genetic ablation of Smo in the murine stroma  growth inhibition of human tumor xenografts  contribution of Hh-activated stroma in tumorigenesis Provide important clinical biomarker for selection of Hh-inhibitor responsive tumors – Hh ligand expression in tumor cells – Hh pathway activation in the stroma

Studies on mouse models of pancreatic ductal adenocarcinoma (PDAC) Genetic deletion of Smo from pancreatic epithelial cells – NOT affect the development and or progression of Kras G12D -driven PDAC – NOT affect Ptch1 and Gli1 expression levels – Smo expression was dispensable for the initiation and progression of PDAC

Epithelial expression of an oncogenic allele of Smoothened (SmoM2 *) – Not induce neoplastic transformation in murine pancreatic epithelium – Not affect tumor development and progression in Kras G12D -driven PDAC models * Smoothened (SmoM2) : triggers ligand-independent activation of Hh pathway constitutively SmoM2 failed to activate the Hh pathway in epithelial cells as measured – by measuring Gli1 mRNA levels using quantitative PCR following laser capture microdissection of the tumor and stromal compartments

GENES & DEVELOPMENT 23:24–

Epithelial expression of activated Gli2  cooperate with activated Kras G12D to induce U/D pancreatic tumors – expression of truncated form of Gli2 : resistant to key posttranscriptional regulatory mechanism – not reflect ability of cell to transduce signal – recent identification of mutations in the Gli transcription factors in human pancreatic cancer cell lines However, effect of these mutations on Gli activity needs to be determined before concluding – downstream Hh signaling components can contribute cell autonomously to adenocarcinoma formation

Gli activity in pancreatic tumor cells  stimulation of Hh independent manner by TGF-β and KRAS TGF- β treatment  upregulation of Gli1 & Gli3 – Smo expression status in PDAC cells – depletion of Kras expression  downregulation of Gli1 and Ptch1 mRNA Gli1  PDAC cancer cell survival  KRAS-dependent malignant cellular phenotype

Limitation In vitro cell culture Levels of Gli activity in the tumor cell compartment : much lower than in adjacent stromal cells – by quantitative mRNA analysis(RT-PCR) of microdissected cells from mouse or human PDAC and colorectal tumor cell culture It will be interesting to extend these results to in vivo models – i.e ; by specific deletion of Gli1 from epithelial compartment in the mouse PDAC model

Proc Natl Acad Sci USA 2009:106;4254-9

Hh pathway in paracrine manner Hh ligands secreted by the epithelial tumor cells activate Hh signaling in the surrounding stroma – Hh signaling  expression of soluble factors & ECM components acting upon tumor epithelium or other cell types promoting tumor growth

Sonic hedgehog pathway  contribute to fibrotic tissue formation in tumor microenvironment of orthotopic model of human pancreatic cancer  indirect angiogenic factor – induce expression of vascular endothelial growth factors and angiopoietins – inhibition of stromal Hh signaling  expression  of components of the Wnt and insulin-like growth factor signaling pathways insulin-like growth factor 2 : medulloblastoma Wnt/β-catenin signaling : skin hamartoma

Requirement for future studies  How these and other factors affect tumor growth  Which factors can be targeted for therapy – Hh signaling in the self-renewal and survival of various epithelial and nonepithelial cancer stem cells – Breast, multiple myeloma, chronic myelogenous leukemia stem cells Paracrine Hh signaling in stromal cells might also be involved in the establishment and maintenance of a cancer stem cell

In conclusion… Tumor-stromal interactions : critical to tumor establishment and growth, and the development of anti-angiogenic drugs – inhibition of signaling in the relatively normal stromal cell – less likely to acquire mechanisms of therapeutic resistance Challenging in a clinical setting – GDC-0449 Ovarian cancer : increase the time until tumor recurrence Metastatic colorectal cancer : improve response to therapy or increase the time until disease recurs

In BCCs… – Hh signaling in mutation-driven tumors – more difficult to characterize the role of paracrine signaling of Hh in cancer It may be possible that both paracrine and autocrine Hedgehog signaling cooperate in some tumors

Thank You ! Nikon F80 by Ja Hee