Upregulation of SOX9 inhibits the growth of human and mouse melanomas and restores their sensitivity to retinoic acid
Thierry Passeron, … , Yoshinori Miyamura, Vincent J. Hearing
Thierry Passeron, … , Yoshinori Miyamura, Vincent J. Hearing
Published March 9, 2009
Citation Information: J Clin Invest. 2009;119(4):954-963. https://doi.org/10.1172/JCI34015.
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Research Article Oncology

Upregulation of SOX9 inhibits the growth of human and mouse melanomas and restores their sensitivity to retinoic acid

Abstract

Treatments for primary and metastatic melanomas are rarely effective. Even therapeutics such as retinoic acid (RA) that are successfully used to treat several other forms of cancer are ineffective. Recent evidence indicates that the antiproliferative effects of RA are mediated by the transcription factor SOX9 in human cancer cell lines. As we have previously shown that SOX9 is expressed in normal melanocytes, here we investigated SOX9 expression and function in human melanomas. Although SOX9 was expressed in normal human skin, it was increasingly downregulated as melanocytes progressed to the premalignant and then the malignant and metastatic states. Overexpression of SOX9 in both human and mouse melanoma cell lines induced cell cycle arrest by increasing p21 transcription and restored sensitivity to RA by downregulating expression of PRAME, a melanoma antigen. Furthermore, SOX9 overexpression in melanoma cell lines inhibited tumorigenicity both in mice and in a human ex vivo model of melanoma. Treatment of melanoma cell lines with PGD2 increased SOX9 expression and restored sensitivity to RA. Thus, combined treatment with PGD2 and RA substantially decreased tumor growth in human ex vivo and mouse in vivo models of melanoma. The results of our experiments targeting SOX9 provide insight into the pathophysiology of melanoma. Further, the effects of SOX9 on melanoma cell proliferation and RA sensitivity suggest the encouraging possibility of a noncytotoxic approach to the treatment of melanoma.

Authors

Thierry Passeron, Julio C. Valencia, Takeshi Namiki, Wilfred D. Vieira, Hélène Passeron, Yoshinori Miyamura, Vincent J. Hearing

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Figure 4

PGD2 restores RA sensitivity by increasing SOX9 expression, which in turn downregulates PRAME.

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PGD2 restores RA sensitivity by increasing SOX9 expression, which in tur...
(A) A375 melanoma cells were transduced with SOX9 lentivirus or with GFP lentivirus as a control. One week after transduction and selection with blasticidin, proteins were extracted and analyzed using immunoblotting with PRAME, SOX9, and GAPDH antibodies (upper panels). Expression levels of PRAME were determined using RT-PCR in A375 cells transfected for 48 hours with GFP or SOX9 cDNA; GAPDH expression served as the control (lower panel). (B) Proliferation assays with A375 and B16/F10 melanoma cells, which are resistant and sensitive to RA, respectively. Addition of RA at 10–7 M has no effect on A375 cells but decreases the proliferation of B16/F10 cells. Transfection of SOX9 induces a strong decrease in proliferation in both types of cells. The combination of SOX9 transfection and RA treatment (at 10–7 M) produces an additive decrease in proliferation in both B16/F10 and A375 cells, in which sensitivity to RA was restored. (C) Immunoblotting of A375 cells treated for 48 hours with PGD2 (0.5 μg/μl) with or without RA (10–7 M) compared with A375 cells treated in the same conditions but silenced for SOX9 by using an siRNA. Protein expression was analyzed using PRAME, p21, SOX9, and β-actin antibodies. (D) Metabolic 35S labeling of A375 cells treated with 0.5 μg/μl PGD2 for 4 and 24 hours. Labeled proteins were immunoprecipitated with PRAME or GAPDH antibodies. (E) Top panels: Proliferation assays of A375 cells treated with RA at 10–7 M and/or PGD2 at 0.5 μg/μl compared with untreated controls. The upper row shows macroscopic views of the proliferative clones and the lower row, the same clones at a magnification of ×10. Bottom panel: Graphic representation of the proliferation assays of A375 and B16/F10 cells treated with RA and/or PGD2. *P < 0.01, **P < 0.001.