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Opposing roles for calcineurin and ATF3 in squamous skin cancer.

Wu X, Nguyen BC, Dziunycz P, Chang S, Brooks Y, Lefort K, Hofbauer GF, Dotto GP - Nature (2010)

Bottom Line: Squamous cell carcinoma (SCC) of the skin is a major complication of treatment with these drugs, with a 65 to 100-fold higher risk than in the normal population.By contrast, the incidence of basal cell carcinoma (BCC), the other major keratinocyte-derived tumour of the skin, of melanoma and of internal malignancies increases to a significantly lesser extent.ATF3, a member of the 'enlarged' AP-1 family, is selectively induced by calcineurin/NFAT inhibition, both under experimental conditions and in clinically occurring tumours, and increased ATF3 expression accounts for suppression of p53-dependent senescence and enhanced tumorigenic potential.

View Article: PubMed Central - PubMed

Affiliation: Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA.

ABSTRACT
Calcineurin inhibitors such as cyclosporin A (CsA) are the mainstay of immunosuppressive treatment for organ transplant recipients. Squamous cell carcinoma (SCC) of the skin is a major complication of treatment with these drugs, with a 65 to 100-fold higher risk than in the normal population. By contrast, the incidence of basal cell carcinoma (BCC), the other major keratinocyte-derived tumour of the skin, of melanoma and of internal malignancies increases to a significantly lesser extent. Here we report that genetic and pharmacological suppression of calcineurin/nuclear factor of activated T cells (NFAT) function promotes tumour formation in mouse skin and in xenografts, in immune compromised mice, of H-ras(V12) (also known as Hras1)-expressing primary human keratinocytes or keratinocyte-derived SCC cells. Calcineurin/NFAT inhibition counteracts p53 (also known as TRP53)-dependent cancer cell senescence, thereby increasing tumorigenic potential. ATF3, a member of the 'enlarged' AP-1 family, is selectively induced by calcineurin/NFAT inhibition, both under experimental conditions and in clinically occurring tumours, and increased ATF3 expression accounts for suppression of p53-dependent senescence and enhanced tumorigenic potential. Thus, intact calcineurin/NFAT signalling is critically required for p53 and senescence-associated mechanisms that protect against skin squamous cancer development.

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ATF3 up-regulation enhances keratinocyte tumour formation and suppresses cancer cell senescencea, HKCs infected with ATF3 expressing (ATF3) and control retroviruses were analyzed by real time RT-PCR for indicated genes. Error bars represent mean ± s.d (n = 3 replicates). b, HKCs plus/minus infection with ATF3 and H-rasV12 expressing retroviruses were analyzed for p53 expression by immunoblotting. c, HKCs plus/minus ATF3 knockdown and CsA/VIVIT treatment were analyzed by real time RT-PCR for p53 and DcR2 expression. Additional results are shown in Suppl. Fig. 10a. d, HKCs plus/minus H-rasV12 expression, ATF3 knockdown and CsA treatment as indicated were analyzed for ATF3 and p53 expression by immunoblotting. For densitometric quantification and experimental conditions see Suppl. Fig. 10b. e and f, H-rasV12 expressing HKCs (e) or SCC13 cells (f) infected with ATF3 expressing and control retroviruses were injected at the dermal-epidermal junction of Scid mice. Shown are histological images of lesions recovered 6 weeks later. For quantification see Suppl. Fig. 11a,b. g, H-rasV12 expressing HKCs plus/minus ATF3 knockdown were injected at the dermal-epidermal junction of Scid mice, followed by CsA treatment. Mice were sacrificed 10 days later. For histological images see Suppl. Fig. 11c. h Lesions from the previous experiment were analyzed for SA-β-galactosidase activity. Low magnification images and quantification are shown in Suppl. Fig. 11d.
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Figure 3: ATF3 up-regulation enhances keratinocyte tumour formation and suppresses cancer cell senescencea, HKCs infected with ATF3 expressing (ATF3) and control retroviruses were analyzed by real time RT-PCR for indicated genes. Error bars represent mean ± s.d (n = 3 replicates). b, HKCs plus/minus infection with ATF3 and H-rasV12 expressing retroviruses were analyzed for p53 expression by immunoblotting. c, HKCs plus/minus ATF3 knockdown and CsA/VIVIT treatment were analyzed by real time RT-PCR for p53 and DcR2 expression. Additional results are shown in Suppl. Fig. 10a. d, HKCs plus/minus H-rasV12 expression, ATF3 knockdown and CsA treatment as indicated were analyzed for ATF3 and p53 expression by immunoblotting. For densitometric quantification and experimental conditions see Suppl. Fig. 10b. e and f, H-rasV12 expressing HKCs (e) or SCC13 cells (f) infected with ATF3 expressing and control retroviruses were injected at the dermal-epidermal junction of Scid mice. Shown are histological images of lesions recovered 6 weeks later. For quantification see Suppl. Fig. 11a,b. g, H-rasV12 expressing HKCs plus/minus ATF3 knockdown were injected at the dermal-epidermal junction of Scid mice, followed by CsA treatment. Mice were sacrificed 10 days later. For histological images see Suppl. Fig. 11c. h Lesions from the previous experiment were analyzed for SA-β-galactosidase activity. Low magnification images and quantification are shown in Suppl. Fig. 11d.

Mentions: Functionally, ectopic ATF3 expression, at levels comparable to those occurring in SCCs from CsA-treated patients (Fig. 2f), blocked expression of p53 and senescence-associated genes (Fig. 3a,b). Conversely, ATF3 knockdown induced these genes, counteracting the CsA and VIVIT effects (Fig. 3c,d, Suppl. Fig. 10). In vivo, ectopic ATF3 promoted tumourigenicity of H-rasV12-expressing HKCs and SCC cells, produced aggressive tumours with reduced senescence as those caused by calcineurin/NFAT inhibitors (Fig. 3e,f; Suppl. Fig. 11a,b,e). Conversely, ATF3 knockdown overcame the tumour promoting effects of these compounds and restored senescence (Fig. 3g,h; Suppl. Fig. 11c,d).


Opposing roles for calcineurin and ATF3 in squamous skin cancer.

Wu X, Nguyen BC, Dziunycz P, Chang S, Brooks Y, Lefort K, Hofbauer GF, Dotto GP - Nature (2010)

ATF3 up-regulation enhances keratinocyte tumour formation and suppresses cancer cell senescencea, HKCs infected with ATF3 expressing (ATF3) and control retroviruses were analyzed by real time RT-PCR for indicated genes. Error bars represent mean ± s.d (n = 3 replicates). b, HKCs plus/minus infection with ATF3 and H-rasV12 expressing retroviruses were analyzed for p53 expression by immunoblotting. c, HKCs plus/minus ATF3 knockdown and CsA/VIVIT treatment were analyzed by real time RT-PCR for p53 and DcR2 expression. Additional results are shown in Suppl. Fig. 10a. d, HKCs plus/minus H-rasV12 expression, ATF3 knockdown and CsA treatment as indicated were analyzed for ATF3 and p53 expression by immunoblotting. For densitometric quantification and experimental conditions see Suppl. Fig. 10b. e and f, H-rasV12 expressing HKCs (e) or SCC13 cells (f) infected with ATF3 expressing and control retroviruses were injected at the dermal-epidermal junction of Scid mice. Shown are histological images of lesions recovered 6 weeks later. For quantification see Suppl. Fig. 11a,b. g, H-rasV12 expressing HKCs plus/minus ATF3 knockdown were injected at the dermal-epidermal junction of Scid mice, followed by CsA treatment. Mice were sacrificed 10 days later. For histological images see Suppl. Fig. 11c. h Lesions from the previous experiment were analyzed for SA-β-galactosidase activity. Low magnification images and quantification are shown in Suppl. Fig. 11d.
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Figure 3: ATF3 up-regulation enhances keratinocyte tumour formation and suppresses cancer cell senescencea, HKCs infected with ATF3 expressing (ATF3) and control retroviruses were analyzed by real time RT-PCR for indicated genes. Error bars represent mean ± s.d (n = 3 replicates). b, HKCs plus/minus infection with ATF3 and H-rasV12 expressing retroviruses were analyzed for p53 expression by immunoblotting. c, HKCs plus/minus ATF3 knockdown and CsA/VIVIT treatment were analyzed by real time RT-PCR for p53 and DcR2 expression. Additional results are shown in Suppl. Fig. 10a. d, HKCs plus/minus H-rasV12 expression, ATF3 knockdown and CsA treatment as indicated were analyzed for ATF3 and p53 expression by immunoblotting. For densitometric quantification and experimental conditions see Suppl. Fig. 10b. e and f, H-rasV12 expressing HKCs (e) or SCC13 cells (f) infected with ATF3 expressing and control retroviruses were injected at the dermal-epidermal junction of Scid mice. Shown are histological images of lesions recovered 6 weeks later. For quantification see Suppl. Fig. 11a,b. g, H-rasV12 expressing HKCs plus/minus ATF3 knockdown were injected at the dermal-epidermal junction of Scid mice, followed by CsA treatment. Mice were sacrificed 10 days later. For histological images see Suppl. Fig. 11c. h Lesions from the previous experiment were analyzed for SA-β-galactosidase activity. Low magnification images and quantification are shown in Suppl. Fig. 11d.
Mentions: Functionally, ectopic ATF3 expression, at levels comparable to those occurring in SCCs from CsA-treated patients (Fig. 2f), blocked expression of p53 and senescence-associated genes (Fig. 3a,b). Conversely, ATF3 knockdown induced these genes, counteracting the CsA and VIVIT effects (Fig. 3c,d, Suppl. Fig. 10). In vivo, ectopic ATF3 promoted tumourigenicity of H-rasV12-expressing HKCs and SCC cells, produced aggressive tumours with reduced senescence as those caused by calcineurin/NFAT inhibitors (Fig. 3e,f; Suppl. Fig. 11a,b,e). Conversely, ATF3 knockdown overcame the tumour promoting effects of these compounds and restored senescence (Fig. 3g,h; Suppl. Fig. 11c,d).

Bottom Line: Squamous cell carcinoma (SCC) of the skin is a major complication of treatment with these drugs, with a 65 to 100-fold higher risk than in the normal population.By contrast, the incidence of basal cell carcinoma (BCC), the other major keratinocyte-derived tumour of the skin, of melanoma and of internal malignancies increases to a significantly lesser extent.ATF3, a member of the 'enlarged' AP-1 family, is selectively induced by calcineurin/NFAT inhibition, both under experimental conditions and in clinically occurring tumours, and increased ATF3 expression accounts for suppression of p53-dependent senescence and enhanced tumorigenic potential.

View Article: PubMed Central - PubMed

Affiliation: Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA.

ABSTRACT
Calcineurin inhibitors such as cyclosporin A (CsA) are the mainstay of immunosuppressive treatment for organ transplant recipients. Squamous cell carcinoma (SCC) of the skin is a major complication of treatment with these drugs, with a 65 to 100-fold higher risk than in the normal population. By contrast, the incidence of basal cell carcinoma (BCC), the other major keratinocyte-derived tumour of the skin, of melanoma and of internal malignancies increases to a significantly lesser extent. Here we report that genetic and pharmacological suppression of calcineurin/nuclear factor of activated T cells (NFAT) function promotes tumour formation in mouse skin and in xenografts, in immune compromised mice, of H-ras(V12) (also known as Hras1)-expressing primary human keratinocytes or keratinocyte-derived SCC cells. Calcineurin/NFAT inhibition counteracts p53 (also known as TRP53)-dependent cancer cell senescence, thereby increasing tumorigenic potential. ATF3, a member of the 'enlarged' AP-1 family, is selectively induced by calcineurin/NFAT inhibition, both under experimental conditions and in clinically occurring tumours, and increased ATF3 expression accounts for suppression of p53-dependent senescence and enhanced tumorigenic potential. Thus, intact calcineurin/NFAT signalling is critically required for p53 and senescence-associated mechanisms that protect against skin squamous cancer development.

Show MeSH
Related in: MedlinePlus