Melanoma secretion of transforming growth factor-β2 leads to loss of epidermal AMBRA1 threatening epidermal integrity and facilitating tumour ulceration

Abstract

Background: For patients with early American Joint Committee on Cancer (AJCC)-stage melanoma the combined loss of the autophagy regulatory protein AMBRA1 and the terminal differentiation marker loricrin in the peritumoral epidermis is associated with a significantly increased risk of metastasis.

Objectives: The aim of the present study was to evaluate the potential contribution of melanoma paracrine transforming growth factor (TGF)-β signalling to the loss of AMBRA1 in the epidermis overlying the primary tumour and disruption of epidermal integrity.

Methods: Immunohistochemistry was used to analyse AMBRA1 and TGF-β2 in a cohort of 109 AJCC all-stage melanomas, and TGF-β2 and claudin-1 in a cohort of 30 or 42 AJCC stage I melanomas, respectively, with known AMBRA1 and loricrin (AMLo) expression. Evidence of pre-ulceration was analysed in a cohort of 42 melanomas, with TGF-β2 signalling evaluated in primary keratinocytes.

Results: Increased tumoral TGF-β2 was significantly associated with loss of peritumoral AMBRA1 (P < 0·05), ulceration (P < 0·001), AMLo high-risk status (P < 0·05) and metastasis (P < 0·01). TGF-β2 treatment of keratinocytes resulted in downregulation of AMBRA1, loricrin and claudin-1, while knockdown of AMBRA1 was associated with decreased expression of claudin-1 and increased proliferation of keratinocytes (P < 0·05). Importantly, we show loss of AMBRA1 in the peritumoral epidermis was associated with decreased claudin-1 expression (P < 0·05), parakeratosis (P < 0·01) and cleft formation in the dermoepidermal junction (P < 0·05).

Conclusions: Collectively, these data suggest a paracrine mechanism whereby TGF-β2 causes loss of AMBRA1 overlying high-risk AJCC early-stage melanomas and reduced epidermal integrity, thereby facilitating erosion of the epidermis and tumour ulceration.

Figure 1

Peritumoral AMBRA1 loss correlates with increased melanoma secretion of TGF‐β2. (a) Representative sections stained by immunohistochemistry for AMBRA1 or TGF‐β2 in AJCC all‐stage melanomas with decreasing levels of AMBRA1 in the peritumoral epidermis (percentage decrease of AMBRA1 in the peritumoral epidermis compared with nonperitumoral epidermis) or of an area of epidermis adjacent to an ulcerated melanoma (scale bar, 50 μm). (b) The percentage of tumour cells with detectable TGF‐β2 levels was determined in a cohort of 109 AJCC all‐stage melanomas and correlated to the percentage decrease of AMBRA1 in the peritumoral epidermis compared with nonperitumoral epidermis, with tumours grouped according to the degree of AMBRA1 loss or whether ulceration was present. The horizontal line represents median tumoral TGF‐β2 expression level (one‐way anova with Tukey’s post hoc correction; *P < 0·05, **P < 0·01, ****P < 0·0001). (c, d) The percentage of tumour cells with detectable TGF‐β2 levels in AJCC stage I melanomas (n = 30, derived from South Tees NHS Trust) grouped according to AMBRA1/loricrin levels (AMLo status: group 1 = high risk, group 2 = low risk) or whether tumours remained localized or metastasized after 7‐year follow‐up (t‐test; *P < 0·05, **P < 0·01). AJCC, American Joint Committee on Cancer; TGF, transforming growth factor.

Figure 2

TGF‐β2 induces AMBRA1 downregulation and deregulated epidermal differentiation. (a) Western blots of AMBRA1, loricrin (* lower band), CK14 and β‐actin protein from primary keratinocytes maintained in low calcium (0·06 mmol L^–1) or incubated in high calcium (1·3 mmol L^–1) for 5 days in the absence or presence of treatment with TGF‐β2 (1–15 ng mL^–1) for 5 days or the final 2 days. (b) qPCR mRNA expression analysis of AMBRA1, loricrin, CK14 and L34 in primary keratinocytes incubated in high calcium (1·3 mmol L^–1) for 0, 4 or 6 days in the presence or absence of treatment with TGF‐β2 (10 ng mL^–1) for the final 48 h. mRNA expression levels were normalized to L34 and presented relative to mRNA levels in control primary keratinocytes cultured in low calcium for 6 days (mean ± SD of two technical replicates, representative of n = 3). (c) Primary keratinocytes transfected with control (siCtrl) or AMBRA1 (siAMBRA1) siRNA were seeded onto a collagen 1 scaffold and cultured in high‐calcium medium for 7 days. Data are Western blots and qPCR mRNA expression analysis of AMBRA1 and GAPDH in primary keratinocytes after 48‐h siRNA transfection, and photomicrographs of haematoxylin and eosin‐stained epidermal equivalent sections (scale bar, 100 μm), or the number of nuclei per field of view (horizontal line represents the mean number of nuclei in five fields of view) in a representative skin equivalent (n = 3). CK, cytokeratin; L, loricrin; qPCR, quantitative polymerase chain reaction; TGF, transforming growth factor.

Figure 3

Identification of ALK5 signalling as a potential mechanism of AMBRA1 regulation in keratinocytes. (a) Quantitative polymerase chain reaction mRNA expression analysis of ALK1, ALK5 and L34 in HUVEC or primary keratinocytes (Kerat.) ± differentiation (Diff) in high calcium (1·3 mmol L^–1) for 5 days. mRNA expression levels were normalized to L34 and presented relative to mRNA levels in HUVEC cells (mean ± SD, n = 3) (one‐way anova with Dunnet’s post hoc correction; **P < 0·01). (b–d) Western blots of p‐Smad 2, Smad 2, p‐Smad 3, Smad 3, p‐Smad 1/5/9, Smad 1, loricrin and GAPDH protein in primary keratinocytes differentiated in high calcium (1·3 mmol L^–1) for 5 days in the presence or absence of treatment with TGF‐β2 (10 ng mL^–1) and/or ALX‐270‐455 (ALK5i; 50 nmol L^–1) for the final 2 h (b, c) or 72 h (d). Protein levels were quantified by densitometry, normalized to GAPDH and presented relative to the mean protein/GAPDH value for each experiment (mean ± SD, n = 3) (unpaired t‐test; *P < 0·05, **P < 0·01, ***P < 0·001). HUVEC, human umbilical vein endothelial cells; L, loricrin; TGF, transforming growth factor.

Figure 4

ATG7 is not required for keratinocyte differentiation. Western blot and qPCR mRNA expression analysis of ATG7, loricrin, LC3 I/II and GAPDH or RPL13A from primary keratinocytes transfected with control (siCtrl) or ATG7 siRNA and incubated in high calcium (1·3 mmol L^–1) for 5 days. Protein levels were quantified by densitometry, normalized to GAPDH, and presented relative to siCtrl (mean ± SD, n = 3). mRNA expression levels were normalized to RPL13A and presented relative to siCtrl (mean ± SD, n = 4) (unpaired t‐test; ***P < 0·001, **P < 0·01, *P < 0·05). qPCR, quantitative polymerase chain reaction.

Figure 5

TGF‐β2‐induced downregulation of claudin‐1 is dependent on AMBRA1. (a) Western blots of claudin‐1 and GAPDH protein from primary keratinocytes differentiated in high calcium (1·3 mmol L^–1) for 5 days in the presence or absence of treatment with TGF‐β2 (10 ng mL^–1) and/or ALX‐270‐455 (ALK5i; 50 nmol L^–1) for the final 72 h. Protein levels were quantified by densitometry, normalized to GAPDH and presented relative to the mean protein/GAPDH value for each experiment (mean ± SD, n = 3). (b) Western blots of AMBRA1, claudin‐1 and GAPDH protein from primary keratinocytes transfected with control (siCtrl) or AMBRA1 siRNA prior to treatment with TGF‐β2 (10 ng mL^–1) for 72 h. Protein levels were quantified by densitometry, normalized to GAPDH and presented relative to the mean protein/GAPDH value for each experiment (mean ± SD, n = 5) (one‐way anova with Tukey’s multiple comparison test to compare treatment with control; *P < 0·05, **P < 0·01). TGF, transforming growth factor.

Figure 6

AMBRA1 loss correlates with decreased claudin‐1 in the peritumoral epidermis of stage I melanomas. (a) Representative sections stained by immunohistochemistry for AMBRA1 or claudin‐1 and (b) claudin‐1 quantitation (H‐score), in a subcohort of 11 nonulcerated AJCC stage I melanomas, and (c) features of consumption of the epidermis (defined as epidermal thinning and loss of rete ridges in areas of direct contact with melanoma cells) and cleft formation identified in a cohort of 42 nonulcerated AJCC stage I melanomas. Tumours were grouped according to whether AMBRA1 expression was maintained or lost in the peritumoral epidermis compared with an area of epidermis adjacent to the tumour (scale bar, 100 μm) (paired t‐test; *P < 0·05). AJCC, American Joint Committee on Cancer.