OTRs are highly susceptible to early cancer development in multiple organs. In this population the risk of NMSCs is particularly enhanced by many fold as compared to normal cohorts [1
]. Interestingly, the early molecular changes underlying the development of NMSCs in OTRs are identical to those which occur in immune competent populations [3
]. However, we and others have demonstrated that immune suppressive drugs manifest direct effects on tumor cells. Some of these molecular alterations appear to be important in mediating a highly aggressive and invasive tumor phenotype [7
]. In this study, we further defined the molecular mechanism by which skin cancer cells achieve an invasive phenotype in OTRs.
It has been shown that CsA enhances the expression of TGFβ in multiple human carcinoma cells [13
]. We confirmed these earlier reports, and also found a significant induction of TGFβ1 in human epidermoid carcinoma A431 cells [7
]. In addition, in these studies we showed that TGFβ-dependent signaling proteins including its receptors TGFβRI and TGFβRII and their target proteins, Smads, are increased in CsA-treated tumors [7
]. Similar results were reported for the skin SCCs excised from OTRs [14
]. Further strength for the notion that TGFβ1 is involved in the pathogenesis of CsA-mediated invasive neoplasm is provided by studies in which SCID-beige mice carrying lung and bladder xenograft tumors manifested increased pulmonary metastatic lesions following treatment with CsA, whereas metastatic tumor growth was significantly reduced in mice receiving treatments with anti-TGFβ antibodies [13
]. We also demonstrated that A431 xenograft tumors from mice treated with CsA overexpress EMT markers and manifest enhanced migration and invasion [7
TAK1 is an upstream member of the MAP kinase super-family which functions as a pivotal integrator of membrane-bound signals elicited by cytokines, particularly TGFβ1 [15
]. The mechanism by which TAK1 functions involves formation of its complexes with TAB1 and TAB2. The observations in this study that CsA enhances phosphorylation of TAK1 and promotes binding of TAK1 with TAB1 and TAB2 suggest the involvement of this pathway in CsA tumorigenesis. Furthermore, Smad7 has been shown to bind with TAB2 and TAB3 to block recruitment of TAK1 [16
]. In this regard, in earlier studies, we observed a reduced expression of Smad7 following CsA treatment [17
]. Downstream signaling of TGFβ that involves TAK1 and TABs has been shown to activate NFκB transcriptional functions [10
]. Our observations that CsA enhances nuclear localization of transcriptionally active NFκB protein p65, with a concomitant enhancement in the expression of its transcriptional targets, cyclin D1, Bcl2, and COX2, suggest that TAK1 mediates enhanced proliferation and reduced apoptosis through CsA-dependent NFκB. CsA in non-tumor cells inhibits COX-2 expression and –dependent angiogenesis. However, in OTRs chronic use of calcineurin inhibitors enhances tumor angiogenesis (microvessel density) and COX-2 expression in NMSCs [20
]. Our results confirm these observations in OTRs and provide an explanation for increased VEGF and COX-2 in NMSCs.
In addition to its role in NFκB activation, TAK1-TAB1/TAB2-dependent pathways may enhance MAPK-p38 expression and –dependent signaling pathway [10
]. CsA in mesangial cells increases p38 signaling by augmenting the binding of TGFβ to TGFβRII [21
]. However, its effects on p38 pathway in tumor cells in OTRs remain so far unknown. Our studies showing an increased expression of p38 in CsA-treated tumors and an enhancement in its downstream signaling proteins, MAPKAPK-2 and p-Hsp27, suggest that CsA augments p38-dependent pathway during the progression of skin carcinogenesis. Furthermore, enhancement in ERK signaling by CsA in this xenograft model suggests that CsA acts at multiple targets in tumor cells to augment tumor growth and invasion.
This study also provides insight into the upstream signaling pathway that activates TAK1/TAB1/TAB2 pathway. In this regard, our observations that CsA enhances the accumulation of ZMP with a concomitant activation of AMPK and IRAK suggest novel molecular targets involved in the pathogenesis of CsA-mediated tumor growth in OTRs. In conclusion, CsA directly affects SCC pathogenesis under the predisposed conditions of immune suppression by promoting tumor growth and invasion invoking TAK1/TAB1/TAB2 pathway that blocks apoptosis and enhances cell survival. These effects are mediated through aberrantly activated p38, MAPK and NFκB pathways.