421 Over-expression of periostin is associated with clinical outcomes and poor prognosis in cutaneous squamous cell carcinoma

Abstract

421 Over-expression of periostin is associated with clinical outcomes and poor prognosis in cutaneous squamous cell carcinoma L Chao, V Gor, DF Murrell, M Kim, E O’Toole, A Ly, D Woodley, A Wysong and M Chen 1 University of Southern California, Los Angeles, CA, 2 University of New South Wales, Sydney, NSW, Australia and 3 Barts and the London School of Medicine and Dentistry, London, United Kingdom Cutaneous squamous cell carcinoma (cSCC), the second most common cancer in the US, is increasing in incidence globally. No specific molecular markers exist to evaluate the risk of cSCC progression and metastasis. Periostin (PN), an extracellular matrix macromolecule, is implicated in tumorigenesis and serves as a prognosis marker for many cancer types. However, there are no data on PN expression in cSCC. This study examined PN expression in patients with cSCC and explored its relationship with clinicopathologic factors and prognosis. We compared PN expression in 103 cSCCs representing a spectrum of cSCC aggressiveness: cSCC in situ (cSSCI) (n1⁄420), low-risk cSCC (LR-cSCC) (n1⁄446), high-risk cSCC (HR-cSCC) (n1⁄416), high-risk in immunocompromised hosts (HRIM-cSCC) (n1⁄413) and cSCC in recessive dystrophic epidermolysis bullosa (RDEB) patients (RDEB-SCC) (n1⁄48). Immunohistochemistry demonstrated PN expression within intra-tumoral stroma but not within tumor cells. PN levels significantly (p < 0.001) increased from cSSCI, LR-cSCC, HR-cSCC, HRIM-cSCC to RDEBSCC. RDEB SCCs represented the most aggressive SCC type. The high level of PN staining in RDEB cSCC was tumor specific with levels notably higher than the non-cancerous skin of RDEB patients. Moreover, the stroma of most SCCs contained cancer-associated fibroblasts with a myofibroblastic (a-SMA-positive) phenotype. Using Transwell assays and an in vivo xenograft tumor model, the addition of RDEB fibroblast-derived PN or recombinant PN significantly promoted in vitro RDEB cSCC invasion and in vivo tumor formation, suggesting that the over-expression of PN in RDEB fibroblasts may play a critical permissive role for SCC development. Our data show that PN expression is highly correlated with the aggressiveness of cSSC, thereby providing a molecular basis for development of novel diagnostic and therapeutic strategies targeting PN for high-risk cSCC. 422 Dsg2 increases exosome release and enhances EGFR/c-Src content: A mechanism for an intercellular mitogenic effect AM Overmiller, J Pierluissi, P Wermuth, U Martinez-Outschoorn, J Curry, A South and MG Mahoney Thomas Jefferson University, Philadelphia, PA Exosomes are nanoscale membrane-derived vesicles that serve as vehicles for the intercellular transfer of protein, mRNA, miRNA and DNA. Substantial evidence has shown that cancerderived exosomes, secreted by tumor cells into the blood and other bodily fluids, play a critical role in modulating the tumor microenvironment and the pathogenesis of cancer. Desmoglein 2 (Dsg2), a desmosomal cadherin often overexpressed in skin malignancies, can activate EGFR/c-Src signaling, enhance cell growth and migration, and promote oncogenesis. Here, we purified exosomes from SCC cells by ultracentrifugation or one-step polymer precipitation. Enrichment of exosomes was confirmed by Nanoparticle Tracking Analysis and by immunoblotting for exosome positive (CD9 and CD63) and negative (CoxIV and GM130) markers. We demonstrate that Dsg2 undergoes ectodomain shedding, and the residual 65 kDa membrane-associated C-terminal fragment (CTF) is enriched in exosomes. Cells were transfected with adenovirus expressing Dsg2 tagged with GFP (29 kDa) at the C-terminus, which was readily detected in exosomes via GFP fluorescence and immunoblotting detected a 95kDa CTF-GFP fusion product in exosomes. Confocal microscopy demonstrates uptake of the Dsg2-labeled exosomes by normal keratinocytes. While Dsg2 is expressed at low levels in the normal epidermis, it is highly upregulated in head and neck SCCs, and exosomes extracted from SCC patients’ sera are enriched in Dsg2 CTF. Overexpression of Dsg2 in SCC cells increased exosome release by 2 fold and enhanced not only total, but also activated, EGFR and c-Src in exosomes, providing a potent mechanism for paracrine mitogenic signal activation. In addition, Dsg2 down-regulates caveolin-1, loss of which has been shown to promote exosome endocytosis. This study defines a mechanism by which cancer cells modulate the tumor microenvironment through enhanced Dsg2 levels, leading to altered exosome dynamics with the potential to promote oncogenic signaling.