Distinct Functions of Epidermal and Myeloid-Derived VEGF-A in Skin Tumorigenesis Mediated by HPV8

Abstract

Beta human papillomaviruses (HPV) have been suspected to be carcinogenic in nonmelanoma skin cancers (NMSC), but the basis for potential viral contributions to these cancers is poorly understood. In particular, it is unresolved how HPV-infected keratinocytes escape cell-cycle control and whether their cross-talk with immune cells is critical for tumorigenesis. In nonviral preclinical models, the angiogenic cytokine VEGF-A has been identified as a critical regulator of NMSC. In this study, we dissected the contribution of epidermal versus myeloid cell-derived VEGF-A in HPVmediated skin cancer by interbreeding an HPV8 transgenic mouse model with a conditional disruption of VEGF-A restricted to either epidermal or myeloid cells. Although only epidermalderived VEGF-A was essential for initiation of skin tumor development, both spontaneously and UV-light triggered, both epidermal and myeloid cell-derived VEGF-A contributed to regeneration-induced tumorigenesis upon HPV8 overexpression, partly not only through a paracrine effect on endothelial cells, but also most probably through an additional autocrine effect on epidermal cells. Our findings offer new mechanistic insights into distinct functions of epidermal versus myeloid cell-derived VEGF-A during HPV-mediated tumorigenesis, with possible implications for preventing this disease. Cancer Res; 75(2); 330-43. Ó2014 AACR.

mTORC1 and mTORC2 regulate skin morphogenesis and epidermal barrier formation

“…Sections (10 μm) were stained with hematoxylin and eosin following a standard procedure60 and analysed using a light microscope (Leica DM4000B, Leica Microsystems, Wetzlar, Germany).…”

“…60). Bound primary antibody was detected by incubation with peroxidase-conjugated (EnVision System, Dako) secondary antibody, followed by incubation with peroxidase substrate (Sigma), or Alexa-Fluor 488- or Alexa Fluor 594-conjugated antibodies (Invitrogen).…”

“…Primary keratinocytes were isolated from newborns and cultured in low calcium medium60. For colony-forming assay 3,000 cells were plated in triplicates in collagen-coated 6-well plates (BD BioCoat) and cultured for 14 days in the presence of mitomycin-treated 3T3 feeders48.…”

“…For Real-time PCR epidermis was separated and total RNA was extracted from tissues using RNeasy Minikit (Qiagen), reverse transcription of isolated RNA was performed using the High Capacity cDNA RT Kit (Applied Biosystems)60. Amplification reactions were performed with PowerSYBR Green PCR Master Mix (Applied Biosystems) by using 7300 Real Time PCRsystem (Applied Biosystems).…”

Epidermal mammalian target of rapamycin complex 2 controls lipid synthesis and filaggrin processing in epidermal barrier formation

“…Hematoxylin and eosin staining was performed according to a standard procedure. 27 Images were analyzed with a light microscope (Leica DM4000B; Leica Microsystems, Wetzlar, Germany).…”