Links between alpha-catenin, NF-kappaB, and squamous cell carcinoma in skin

Abstract

Cancers display a diverse set of cellular defects, which are thought to be elicited by multiple genetic mutations. In this study, we show that when a single adherens junction protein, alpha-catenin, is removed by conditional targeting, the entire skin epidermis systematically transforms to a hyperproliferative, invasive tissue replete with inflammation. Transcriptional profiling and biochemical analyses reveal that alpha-catenin ablation is accompanied by activation of NF-kappaB and its proinflammatory target genes, along with genes involved in proliferation, wound healing, angiogenesis, and metastasis. Many of these alterations occur in vitro and in the embryo, and thus seem at least partly to be intrinsic to the loss of alpha-catenin. We show that reductions in alpha-catenin, activation of NF-kappaB, and inflammation are common features of human squamous cell carcinomas of the skin.

Fig. 1.

Progression to SCC in the absence of α-catenin. E18.5 WT and cKO skins were grafted onto Nude mice for the days indicated. Seventy-day WT grafts were analogous to 40 days and are not shown. (A, C, and D) Far left images in A and C are of grafts (note the absence of follicles in cKO grafts). Hematoxylin and eosin (H&E)-stained sections are from grafts, with the exception of D Lower Right, which is of a grade III hSCC (arrows denote atypical cells, classical morphological signs of SCC, and prevalence in the dermis of the 70-day cKO graft). Color coding of immunostained sections is according to secondary Abs; some sections are counterstained with DAPI (blue). Note that in C most sections are of invasive keratinocytes within the dermis as indicated in upper left of each frame. Arrows in A and C denote perturbations at dermo-epidermal borders (hemidesmosome/basement membrane), more extensive at 70 days than 40 days. Arrowheads denote epidermal cells displaying weak or no E-cadherin, which in C are also weak for K14-GFP+. epid, epidermis; der, dermis; hf, hair follicles; Lam5, laminin-5; Ecad, E-cadherin; αcat, α-catenin; K14GFP, transgenic epifluorescence expression of GFP; Ki67, a proliferating nuclear antigen; K, keratin. Asterisks denote keratinized pearls. (B) EM of ultrathin sections that correspond to boxed areas in Fig. 6 A′ and B′. Shown are the dermo-epidermal boundaries. Note that the basal lamina (bl) and hemidesmosomes (hd) are intact in WT but are perturbed in cKO (region flanked by arrowheads).

Fig. 2.

Changes in gene expression elicited by loss of α-catenin. (A) mRNAs in five relevant categories that scored by microarray analyses as up-regulated or down-regulated in E18.5 cKO vs. WT epidermis. Numbers in parentheses correspond to fold change. Asterisks refer to mRNAs not expressed by keratinocytes. EMT, epithelial-mesenchymal transition. (B) Real-time PCR of mRNAs from E18.5 epidermis (epid), FACS-purified keratinocytes from epidermis (GFPepid), and primary keratinocytes (cult) cultured in low calcium medium to prevent intercellular adhesion. Fold change is relative to WT-epid (=1).

Fig. 3.

Early activation of the NF-κB pathway in α-catenin null epidermis. (A) Putative NF-κB target genes whose mRNAs were scored by microarray as up-regulated in E18.5 cKO vs. WT epidermis. Numbers in parentheses correspond to fold change. (B) NF-κB activation and inflammation in E18.5 cKO skin. (Left) Immunoblot with panel and phospho-specific Abs: P-NF-κB (activated p65 subunit), P-IκBα (inhibited state), and P-STAT3 (activated). (Right) Immunostainings with Abs against P-NF-κB, Mac1 (macrophage-specific), and P-STAT3. Asterisks denote positive dermal nuclei; additional identification of inflammatory cells is in Fig. 9. (C) Real-time PCR of NF-κB target genes. mRNAs were from FACS-purified GFP-positive epidermal cells and cultured keratinocytes (low calcium). Fold change was relative to WT-GFPepid (=1). (D) Anti-NF-κB and P-NF-κB immunofluorescence of KO and WT keratinocytes that are also GFPactin transgenic. Cells were cultured in low-calcium medium (LCa) and high-calcium medium (HCa). (E) NF-κB-luciferase and Cxcl1-luciferase reporter gene assays on cultured keratinocytes (LCa) ± TNF-α. Values shown are relative to Renilla luciferase levels. ∗, t test statistical P ≤ 0.005. (F) Nude mice harboring K14-GFP-expressing E18.5 WT and cKO skin grafts were treated ± Dex to suppress NF-κB activation. Grafts were processed at 30 days for hematoxylin and eosin (H&E) staining, and immunomicroscopy was performed with Ab against α-catenin (αcat). Pigmented melanocytes from grafted skin (C57/Bl6) are black.

Fig. 4.

Status of α-catenin (αcat) expression, β-catenin (βcat) expression, E-cadherin (Ecad) expression, and NF-κB/STAT activity in hSCCs. Immunostainings of hSCCs ranged from well to poorly differentiated (dif). Normal human skin is from unaffected areas of hSCC sections. Arrows point to cell–cell border staining. Chart summarizes immunodata from 40 hSCCs (+, present; +/−, weak; −, not detected). Note that some background staining was detected in the anti-β-catenin immunohistochemistry, which was comparable to that seen in the poorly differentiated hSCC sample shown. epid, epidermis; der, dermis.