Distinctive clinical and histological characteristics of atrophic and hypertrophic facial photoageing.

BACKGROUND: Photoageing describes complex cutaneous changes which occur following chronic exposure to solar ultraviolet radiation (UVR). Amongst White Northern Europeans, facial photoageing appears as distinct clinical phenotypes: 'hypertrophic' photoageing (HP) and 'atrophic' photoageing (AP). Deep, coarse wrinkles predominate in individuals with HP, whereas those with AP have relatively smooth, unwrinkled skin with pronounced telangiectasia. AP individuals have an increased propensity for developing keratinocyte cancers. OBJECTIVES: To investigate whether histological differences underlie these distinct phenotypes of facial photoageing. METHODS: Facial skin biopsies were obtained from participants with AP (10 M, 10 F; mean age: 78.7 years) or HP (10 M, 10 F; mean age: 74.5 years) and were assessed histologically and by immunohistochemistry. RESULTS: Demographic characterization revealed 95% of AP subjects, as compared to 35% with HP, were Fitzpatrick skin type I/II; of these, 50% had a history of one or more keratinocyte cancers. There was no history of keratinocyte cancers in the HP cohort. Analysis of UVR-induced mitochondrial DNA damage confirmed that all volunteers had received similar lifetime cumulative doses of sun exposure. Histologically, male AP had a significantly thicker epidermis than did AP females or those of either sex with HP. HP facial skin exhibited severe solar elastosis, whereas in AP facial skin, solar elastosis was apparent only in females. Loss of papillary dermal fibrillin-rich microfibrils occurred in all HP and AP female subjects, but not in AP males. Furthermore, male AP had a significant reduction in collagen VII at the dermal-epidermal junction than did AP females or those of either sex with HP. CONCLUSIONS: This study provides further evidence that AP and HP represent distinct clinical and histological entities. Knowledge of these two phenotypes is clinically relevant due to the increased prevalence of keratinocyte cancers in those - particularly males - with the AP phenotype.

Loss of the laminin subunit alpha-3 induces cell invasion and macrophage infiltration in cutaneous squamous cell carcinoma.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is a common cancer that invades the dermis through the basement membrane. The role of the basement membrane in poorly differentiated cSCC is not well understood. OBJECTIVES: To study the effect that loss of the laminin subunit alpha-3 (α3) chain from the tumour microenvironment has on tumour invasion and inflammatory cell recruitment. METHODS: We examined the role of the basement membrane proteins laminin subunits α3, ß3 and γ2 in SCC invasion and inflammatory cell recruitment using immunohistochemistry, short hairpin RNA knockdown, RNA-Seq, mouse xenograft models and patient tumour samples. RESULTS: Analysis of SCC tumours and cell lines using antibodies specific to laminin chains α3, ß3 and γ2 identified a link between poorly differentiated SCC and reduced expression of laminin α3 but not the other laminin subunits investigated. Knockdown of laminin α3 increased tumour invasion both in vitro and in vivo. Western blot and immunohistochemical staining identified increased phosphorylated myosin light chain with loss of laminin α3. Inhibition of ROCK (rho-associated protein kinase) but not Rac1 significantly reduced the invasive potential of laminin α3 knockdown cells. Knockdown of laminin subunits α3 and γ2 increased monocyte recruitment to the tumour microenvironment. However, only the loss of laminin α3 correlated with increased tumour-associated macrophages both in xenografted tumours and in patient tumour samples. CONCLUSIONS: These data provide evidence that loss of the laminin α3 chain in cSCC has an effect on both the epithelial and immune components of cSCC, resulting in an aggressive tumour microenvironment.

Suppression of TGFß and Angiogenesis by Type VII Collagen in Cutaneous SCC.

BACKGROUND: Individuals with severe generalized recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering disorder caused by mutations in the COL7A1 gene, develop unexplained aggressive squamous cell carcinomas (SCC). Here we report that loss of type VII collagen (Col7) in SCC results in increased TGFß signaling and angiogenesis in vitro and in vivo. METHODS: Stable knockdown (KD) of Col7 was established using shRNA, and cells were used in a mouse xenograft model. Angiogenesis was assessed by immunohistochemistry, endothelial tube-forming assays, and proteome arrays. Mouse and zebrafish models were used to examine the effect of recombinant Col7 on angiogenesis. Findings were confirmed in anonymized, archival human tissue: RDEB SCC tumors, non-EB SCC tumors, RDEB skin, normal skin; and two human RDEB SCC cell lines. The TGFß pathway was examined using immunoblotting, immunohistochemistry, biochemical inhibition, and siRNA. All statistical tests were two-sided. RESULTS: Increased numbers of cross-cut blood vessels were observed in Col7 KD compared with control xenografts (n = 4 to 7 per group) and in RDEB tumors (n = 21) compared with sporadic SCC (n = 24, P < .001). Recombinant human Col7 reversed the increased SCC angiogenesis in Col7 KD xenografts in vivo (n = 7 per group, P = .04). Blocking the interaction between α2ß1 integrin and Col7 increased TGFB1 mRNA expression 1.8-fold and p-Smad2 levels two-fold. Increased TGFß signaling and VEGF expression were observed in Col7 KD xenografts (n = 4) compared with control (n = 4) and RDEB tumors (TGFß markers, n = 6; VEGF, n = 17) compared with sporadic SCC (TGFß markers, n = 6; VEGF, n = 21). Inhibition of TGFß receptor signaling using siRNA resulted in decreased endothelial cell tube formation (n = 9 per group, mean tubes per well siC = 63.6, SD = 17.1; mean tubes per well siTßRII = 29.7, SD = 6.1, P = .02). CONCLUSIONS: Type VII collagen suppresses TGFß signaling and angiogenesis in cutaneous SCC. Patients with RDEB SCC may benefit from anti-angiogenic therapy.

The receptor tyrosine kinase Axl regulates cell-cell adhesion and stemness in cutaneous squamous cell carcinoma.

Axl is a receptor tyrosine kinase (RTK) upregulated in various tumors including cutaneous squamous cell carcinoma (SCC). Axl expression correlates with poor prognosis and induction of epithelial-mesenchymal transition (EMT), hence we hypothesized that Axl is involved in the disruption of cell-cell adhesion to allow invasion and chemotherapy resistance of the cancer stem cell population. Cutaneous SCC cell lines with stable knockdown of Axl were generated using retroviral vectors. Axl depletion altered expression of intercellular junction molecules increasing cell-cell adhesion with downregulation of Wnt and TGFßR signaling. Furthermore, Axl expression correlated with the expression of putative cancer stem cell markers, CD44 and ALDH1, increased resistance to chemotherapy drugs, enhanced sphere formation ability and expression of EMT features by cancer stem cells. Axl depletion resulted in loss of tumor formation in an in vivo zebrafish xenograft model. In conclusion, these data suggest that abrogation of Axl results in loss of cancer stem cell properties indicating a role for Axl as a therapeutic target in chemotherapy-resistant cancer.

Endo180 modulation by bisphosphonates and diagnostic accuracy in metastatic breast cancer.

BACKGROUND: Endo180 (CD280; MRC2; uPARAP)-dependent collagen remodelling is dysregulated in primary tumours and bone metastasis. Here, we confirm the release and diagnostic accuracy of soluble Endo180 for diagnosing metastasis in breast cancer (BCa). METHODS: Endo180 was quantified in BCa cell conditioned medium and plasma from BCa patients stratified according to disease status and bisphosphonate treatment (n=88). All P-values are from two-sided tests. RESULTS: Endo180 is released by ectodomain shedding from the surface of MCF-7 and MDA-MB-231 BCa cell lines. Plasma Endo180 was significantly higher in recurrent/metastatic (1.71±0.87; n=59) vs early/localised (0.92±0.37; n=29) BCa (P<0.0001). True/false-positive rates for metastasis classification were: 85%/50% for the reference standard, CA 15-3 antigen (28 U ml(-1)); ≤97%/≥36% for Endo180; and ≤97%/≥32% for CA 15-3 antigen+Endo180. Bisphosphonate treatment was associated with reduced Endo180 levels in BCa patients with bone metastasis (P=0.011; n=42). True/false-positive rates in bisphosphonate-naive patients (n=57) were: 68%/45% for CA 15-3 antigen; ≤95%/≥20% for Endo180; and ≤92%/≥21% for CA 15-3 antigen+Endo180. CONCLUSION: Endo180 is a potential marker modulated by bisphosphonates in metastatic BCa.