Androgen receptor functions as transcriptional repressor of cancer-associated fibroblast activation
Andrea Clocchiatti, Soumitra Ghosh, Maria-Giuseppina Procopio, Luigi Mazzeo, Pino Bordignon, Paola Ostano, Sandro Goruppi, Giulia Bottoni, Atul Katarkar, Mitchell Levesque, Peter Kölblinger, Reinhard Dummer, Victor Neel, Berna C. Özdemir, G. Paolo Dotto
Andrea Clocchiatti, Soumitra Ghosh, Maria-Giuseppina Procopio, Luigi Mazzeo, Pino Bordignon, Paola Ostano, Sandro Goruppi, Giulia Bottoni, Atul Katarkar, Mitchell Levesque, Peter Kölblinger, Reinhard Dummer, Victor Neel, Berna C. Özdemir, G. Paolo Dotto
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Research Article Dermatology Oncology

Androgen receptor functions as transcriptional repressor of cancer-associated fibroblast activation

Abstract

The aging-associated increase of cancer risk is linked with stromal fibroblast senescence and concomitant cancer-associated fibroblast (CAF) activation. Surprisingly little is known about the role of androgen receptor (AR) signaling in this context. We have found downmodulated AR expression in dermal fibroblasts underlying premalignant skin cancer lesions (actinic keratoses and dysplastic nevi) as well as in CAFs from the 3 major skin cancer types, squamous cell carcinomas (SCCs), basal cell carcinomas, and melanomas. Functionally, decreased AR expression in primary human dermal fibroblasts (HDFs) from multiple individuals induced early steps of CAF activation, and in an orthotopic skin cancer model, AR loss in HDFs enhanced tumorigenicity of SCC and melanoma cells. Forming a complex, AR converged with CSL/RBP-Jκ in transcriptional repression of key CAF effector genes. AR and CSL were positive determinants of each other’s expression, with BET inhibitors, which counteract the effects of decreased CSL, restoring AR expression and activity in CAFs. Increased AR expression in these cells overcame the consequences of CSL loss and was by itself sufficient to block the growth and tumor-enhancing effects of CAFs on neighboring cancer cells. As such, the findings establish AR as a target for stroma-focused cancer chemoprevention and treatment.

Authors

Andrea Clocchiatti, Soumitra Ghosh, Maria-Giuseppina Procopio, Luigi Mazzeo, Pino Bordignon, Paola Ostano, Sandro Goruppi, Giulia Bottoni, Atul Katarkar, Mitchell Levesque, Peter Kölblinger, Reinhard Dummer, Victor Neel, Berna C. Özdemir, G. Paolo Dotto

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Figure 13

Reversal of CAF activation by AR overexpression.

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Reversal of CAF activation by AR overexpression. (A) Multiple CAF strain...
(A) Multiple CAF strains infected with an AR-overexpressing lentivirus (pLenti AR) versus a β-galactosidase–expressing control (pLenti LacZ) were analyzed by RT-qPCR for expression of the indicated genes. Values for each strain are indicated as dots with mean ± SD. n(HDF strains) = 3; ***P < 0.005, 2-tailed unpaired t test. (B) Immunoblot analysis of AR versus CSL protein expression in 2 CAF strains infected with an AR-overexpressing lentivirus (AR) versus control (LacZ). Blots were sequentially probed with antibodies against AR and γ-tubulin and, on a separate membrane, CSL and β-actin. αSMA regulation by AR overexpression in CAF strains is shown in Supplemental Figure 12A. (C) SCC13 cells were cocultured at a 1:1 ratio with HDF PB1 or CAF16 infected with β-galactosidase–expressing (pLenti LacZ) or AR-expressing (pLenti AR) lentiviruses for 3 days, followed by immunofluorescence analysis with anti-keratin (red) and anti-vimentin (green) antibodies for cell identification. Shown are representative images together with quantification of large SCC13 cell clusters (>40 cells) per examined field with mean ± SD. Scale bar: 200 μm. n(number of fields) = 10; ***P < 0.005, 1-way ANOVA with Dunnett’s test. (D) SCC13 cells were mixed at a 1:1 ratio with HDF PB1 or β-galactosidase–expressing (pLenti LacZ) versus AR-expressing (pLenti AR) CAF16 and grown in Matrigel 3D cultures for 5 days. Shown are representative images of SCC13 spheroids together with quantification by ImageJ software of spheroid diameter with mean ± SD. Scale bar: 250 μm. n(number of spheroids) = 50; ***P < 0.005, 1-way ANOVA with Dunnett’s test.