Cyclosporine a mediates pathogenesis of aggressive cutaneous squamous cell carcinoma by augmenting epithelial-mesenchymal transition: role of TGFβ signaling pathway

Abstract

Organ transplant recipients (OTRs) develop multiple aggressive and metastatic non-melanoma skin cancers (NMSCs). Yet, the underlying mechanism remains elusive. Employing a variety of immune-compromised murine models, immunoblotting, immunohistochemical and immunofluorescence techniques, we show that human squamous xenograft tumors in nude mice grow faster and become significantly larger in size following treatment with the immunosuppressive drug, cyclosporine A (CsA). Re-injected tumor cells isolated from CsA-treated xenografts continued to form larger tumors in nude mice than those from vehicle-controls and retained the CsA-signatures of calcineurin signaling inhibition. Similar results were obtained when these tumors were grown in SCID-beige mice or in immuno-competent mice inoculated with syngeinic tumor cells. Consistently, tumors in the CsA group manifested enhanced cellular proliferation and decreased apoptosis. Tumors in CsA-treated animals also showed an augmented epithelial-mesenchymal transition (EMT) characterized by an increased expression of fibronectin, α-SMA, vimentin, N-cadherin, MMP-9/-2, snail and twist with a concomitant decrease in E-cadherin. CsA-treated xenograft tumors manifested increased TGFβ1 expression and TGFβ-dependent signaling characterized by increased nuclear p-Smad 2/3. Our data demonstrate that CsA alters the phenotype of skin SCCs to an invasive and aggressive tumor-type by enhancing expression of proteins regulating EMT acting through the TGFβ1 signaling pathway providing at least one unique mechanism by which multiple aggressive and metastatic NMSCs develop in OTRs.

Fig 1. CsA augments xenograft tumor growth in immune deficient mice

CsA treatment induces proliferation and reduces apoptosis in xenograft tumors in nude mice. A,-Tumor volume (mean ± S.E.) of xenograft tumors grown in nude mice treated with CsA. B,-Growth curves of tumors in nude mice developed following inoculation with A431 cells treated in culture for 3 weeks with vehicle or CsA (5μM). C,-Growth-curves of tumors developed in nude mice following inoculation with tumor keratinocytes isolated from vehicle- or CsA-treated tumors. D,-Molecular phenotype of tumors excised from mice as shown in Figure-1c demonstrating downregulation of NFAT and calcineurin expression (immunofluorescence staining), (arrows show areas of intense staining) E,-Immunohistochemical staining for PCNA. F,-Immunofluorescence staining for Bcl-2 and TUNEL (arrows show areas of enhanced staining)

Fig 2

(A) Dose-dependent effects of CsA on xenograft tumor growth in nude mice. (B) Effects of CsA treatment on the growth of xenograft tumors in SCID-beige mice. (C) Histology of tumors harvested from CsA-treated and vehicle-treated mice. Inset shows mitotic figures in CsA-treated tumor and black arrow shows keratin pearl in vehicle-treated tumor. (D) Expression of cytokeratin 1 and 10 in vehicle and CsA treated tumors (E) Tumor volume of xenograft tumors developed in C57BL/6 immune competent mice following inoculation with LCC cells or LCC cells isolated from vehicle- or CsA-treated tumors developed in nude mice.

Fig 3

(A) Western blot analysis showing expression of proteins regulating proliferation, angiogenesis and apoptosis. (B) Immunofluorescence staining for VEGF.

Fig 4

Effects of CsA treatment on the (A) invasion and (B) wound healing capabilities of A431 cells. Cell invasion was measured with a blind well chemotaxis invasion chamber. 100μl of DMEM containing 20% fetal bovine serum was added into the lower chamber and covered with an 8μm pore size polycarbonate membrane filter. Cells (2.5 × 10⁴) in 100μL of serum free DMEM were seeded into the upper chamber. After incubating for 24 hours at 37°C, membranes were collected and non-invading cells were removed from the upper surface of the membrane using a cotton swab. The membranes were then fixed with methanol, stained with Harris hematolxylin, and photographed microscopically with a 10X objective. The number of invading cells on the lower surface of the membrane was counted in 10 fields per membrane. For wound healing assay, A431 cells were seeded in six-well plates and incubated overnight in starvation medium. Then, the cell monolayers were wounded with a sterile 100-μl pipette tip, washed with starvation medium to remove detached cells from the plates. Cells were left either untreated or treated with CsA, and kept for 24 hrs in CO2 incubator. After 24 hrs, medium was replaced with PBS and cells were photographed using an Olympus (Japan) IX-TVAD digital camera connected to an Olympus IX70 phase-contrast microscope (4X objective).

Fig 5. CsA increases TGFβ pathway-dependent signaling in xenograft human squamous tumors

(A) Immunofluorescence staining illustrates expression of TGFβ receptors and downstream signaling proteins Smads, Smad 2, 3, 4, and 7, in vehicle and CsA-treated tumors. (B) Western Blot analysis showing expression of TGFβ receptors and downstream signaling proteins Smads, Smad 2, 3, 4, and 7, in vehicle and CsA-treated tumors.

Fig 6. CsA increases epithelial-mesenchymal transition (EMT)

(A) Immunofluorescence staining showing expression of biomarkers of EMT in tumors excised from vehicle- and CsA-treated mice (B) Western Blot analysis of tumors showing the effects of CsA on the expression of EMT marker proteins

Fig. 7

Cartoon representing CsA-mediated activation of TGF β1-dependent signaling leading to epithelial-mesenchymal transition in epidermoid carcinoma cells. Green arrows represent levels of proteins in parental carcinoma cells and red arrows represent the alterations in the expression of these proteins following CsA treatment.