The major conclusion from this study is that patients on long term ARD therapy have increased P-Smad2 signaling within benign KA, CIS, malignant SCC, as well as in adjacent non-lesional skin. Although a tumor suppressive role for this enhanced signaling pathway in OTRs cannot be excluded, this observation, together with a wealth of pre-clinical data on TGFβ activities in skin tumorigenesis, would support a role for activation of the TGF-β/Smad2 pathway in outgrowth and progression of SCC in OTRs (16
). In mouse skin, over-expression of TGF-β1 acts as a suppressor of the early outgrowth of benign tumors (28
). As carcinomas progress, oncogene activation blunts this negative growth response and tumor cells acquire enhanced cellular plasticity in response to increased TGF-β1, resulting in an elevated incidence of NMSC and a more invasive phenotype (28
). TGF-β also acts on the tumor microenvironment to stimulate tumor progression (30
). The hypothesis that ARDs might enhance tumorigenesis by increasing TGF-β1 levels is therefore plausible.
In an immunodeficient SCID–beige mouse model, injection of anti-TGF-β blocking antibodies inhibited a CsA–stimulated increase in the number of metastases resulting from injection of A549 adenocarcinoma cells (16
). Since the host mice lacked both T and NK cells, it was concluded that this was a direct effect of TGF-β acting on the tumor cell per se
rather than on the immune system. This interpretation was supported by the fact that A549 cells treated with CsA in vitro
undergo morphological changes characteristic of invasive cells, and that these changes are completely prevented by the use of TGF-β blocking antibodies (16
). Similar findings have been made in a rat model of metastatic colon cancer using a small molecule inhibitor of TβRI (31
In contrast to earlier reports on SCC from non-OTRs (32
), we found that the majority of SCC from both non-OTRs and OTRs showed active P-Smad2 signaling. The reason for this discrepancy between previous studies and ours could be multifold. Guasch et al. (34
) restricted their studies to genital SCC which might have a very different etiology from that of cutaneous SCC. Other studies utilized antibodies against total Smads rather than P-Smads (33
), or used smaller samples sizes (32
). Nonetheless, consistent with findings by Hoot et al. (33
), P-Smad2 staining was slightly but significantly (P
=0.005) decreased in SCC compared to normal adjacent skins of non-OTRs ( and 3S
), an effect that was overridden in OTRs.
In contrast to P-Smad2, staining intensities of TGF-β1 and TβRII were relatively unaffected by OTR status, if anything they showed a tendency for decreased levels in SCC from OTRs. This observation, regarding ligand level, is surprising in light of several reports of increased systemic TGFβ-1 levels in response to ARDs in humans (25
) as well as in mice (16
). It might be that examination of steady-state levels of the ligand by immunohistochemistry is misleading. In vivo, once activated, TGF-β1 is very rapidly cleared (36
), as such, TGF-β1 protein levels might appear depleted at the sites of activation. Similarly, with respect to decreased TβRII staining in OTRs, the activation of TGF-β receptors results in their degradation via the endosomal pathway (37
). It is therefore conceivable that rapid receptor turnover during chronic activation might lead to lower immunohistochemically detectable levels of TβRII in OTRs, despite enhanced signaling.
It is possible that other TGF-β superfamily ligands that activate P-Smad2 may be elevated in OTRs. TGF-β2 and TGF-β3, which act through the same TβRI/TβRII complex, were ruled out by immunostaining. However, activins, nodal and myostatin can also activate P-Smad2 via their own distinct serine threonine receptor kinase complexes (38
). Another possibility is that increased nuclear localization of P-Smad2 may be caused by changes in nuclear shuttling and turnover of this signaling molecule. P-Smad2 shuttling and stability is regulated by interaction with other transcription factors and by ubiquitylation (18
), as well as by phosphorylation of the centrally located Smad “linker region”, for example by the ERK/MAPK pathway (39
). Activation by TGF-β1/TβRII is therefore not required in order to explain elevation of P-Smad2 levels. Nevertheless, reports of elevated systemic TGF-β1 levels in response to ARD treatment (25
) would suggest that this ligand contributes some component to enhanced P-Smad2 activation.
The observation of reduced levels of the activated BMP pathway components in KA compared to adjacent skin of non-OTRs, and the increased activation of BMP-Smads in KA in response to long term ARD therapy, is particularly intriguing. KA are benign lesions that are thought to arise from the hair follicle (42
). It is well established that the BMP signaling pathway plays a major role in follicular stem cell maintenance and the adult hair follicle cycle (43
). BMP ligands, BMP2, BMP4 and BMP6, in particular, are known to be elevated in the stem/progenitor niche of the hair follicle, where they signal through the BMPR1A receptor to limit stem cell expansion and to induce hair cell differentiation (43
). Horsley et al. (44
) recently demonstrated that BMPs maintain bulge stem cells through the transcriptional activation of NFATc1, which acts to limit stem cell proliferation. NFATc1 activation and nuclear localization is regulated by the phosphatase, Calcineurin, which in turn, is inhibited by the most commonly used ARD, CsA. Thus ARDs would release stem cells from the BMP-NFAT1c mediated proliferative constraint. The activation of BMP signaling seen within KA from OTRs compared to non-OTRs may therefore result from preferential expansion of a BMP-responsive follicular stem/progenitor compartment that occurs when the consequent down-stream negative growth response is short-circuited by CsA inhibition of NFATc1 activity.
In conclusion, this study together with a wealth of published preclinical data suggests that ARD-enhanced TGF-β signaling might contribute to enhanced skin malignancy in the OTR patient population. Several specific TGF-β inhibitory drugs are now in clinical trials for various applications (47
), and the established anti-hypertensive drug, losartan, has been shown to reduce circulating TGF-β1 levels (48
). Such drugs should be used with caution due to possible inhibition of the tumor suppressive arm of TGFβ signaling that could potentially result in more benign lesions, such as KA. However, moderate down-regulation of excessive P-Smad2 levels in OTRs by the use of topical TGF-β inhibitors (50
) might be useful to reduce cutaneous malignancy and further tumor progression. It might even be considered worthwhile to administer losartan for difficult to manage OTRs with many SCCs.