Here we describe that CYLD acts as regulator of epidermal differentiation in humans. Our data demonstrate that alterations in CYLD expression in keratinocytes disrupt normal epidermal homeostasis: the forced expression of CYLDWT
in human HaCaT keratinocytes and the skin equivalents enhance keratinocyte differentiation. By contrast, the expression of a mutant CYLDC/S
that inhibits the endogenous CYLD-deubiquitination function impairs keratinocyte differentiation. We have found that JNK signaling pathway could be mediating the effects of CYLD in epidermal differentiation. In fact, it was previously reported that the inhibition of JNK activation in keratinocyte cultures in vitro
induces almost all aspects of epidermal differentiation in vivo
, including stratification and even production of cornified envelopes.2
By contrast, augmented JNK activity delays or inhibits keratinocyte differentiation.2
In line with these results, we have found diminished JNK activation in keratinocytes expressing CYLDWT
, concomitant with an enhanced epidermal differentiation of the CYLDWT
skin equivalents; additionally, we have found increased JNK activation in the CYLDC/S
keratinocytes associated to an impaired epidermal differentiation of the CYLDC/S
skin equivalents. This finding is relevant for our understanding of both the normal epidermal differentiation and its perturbations during pathological conditions.
Importantly, we have discovered that CYLDWT also promotes the differentiation of A431 tumoral keratinocytes through inhibition of JNK activation. It may be significant clinically, because attenuating JNK activity could become a differentiation therapy-based approach for skin cancer in general and for tumors developed by familial cylindromatosis patients in particular.
Another finding of this study is the increased rate of apoptosis induced by CYLDWT
both in tumoral and non-tumoral human keratinocytes. This effect could be the consequence of the lack of JNK-mediated cell survival signaling due to the diminished JNK activation in these cells.27
Our present results showing increased apoptosis in cells, and importantly, in skin tumors overexpressing CYLDWT
, constitute an important finding for the identification of the mechanisms through which CYLD exerts its role as a tumor suppressor.
In addition to the impairment of keratinocyte differentiation, we have found that CYLDC/S
expression provokes other alterations in skin equivalents such as the loss of cell polarity, likely due to the loss of β
-catenin membranous distribution and its cytoplasmic accumulation. Maintenance of cell polarity is an essential requirement for the functionality and homeostasis of epithelial tissues, being β
-catenin a key molecule in the maintenance of epithelial architecture as it regulates the polarity of cells and tissues.28
Other features of the H-CYLDC/S
skin equivalents are the formation of foci of basal invasion and resistance to apoptosis. Altogether, these changes could lead to epithelial dysplasia and reflect pre-malignant modifications, such as loss of epithelial differentiation and cell polarity, and the acquisition of invasive properties are frequently observed in malignant transformation. According to these pathological alterations found in H-CYLDC/S
skin equivalents, CYLDC/S
expression in A431 squamous carcinoma cells induces an enhancement of their malignancy in carcinogenesis assays.
Several mechanisms have been proposed through which loss of CYLD may exert a role in tumor promotion. One of them is the hyperactivation of antiapoptotic signals in absence of functional CYLD in tumoral cells;29
previously we described that CYLDC/S
induced resistance to apoptosis in mouse skin tumors;21
here we also extend this property to human skin cancer. We have also showed that CYLDC/S
expression enhances angiogenesis in mouse skin carcinomas,21
what constitutes a prominent feature of skin tumor progression.30
Here we have also confirmed that in human SCCs CYLDC/S
expression leads to a strong angiogenesis, increasing the size and permeability of blood vessels. Importantly, we have found as well that CYLDWT
overexpression in SCCs reduces the diameter of the blood vessels and augments its maturity, which possibly make them unable to supply the nutrients needed for tumor progression.
Therefore our study is relevant because it established the role that CYLD has in normal skin physiology. The use of our skin equivalent model has allowed us to determine the importance of CYLD for the maintenance of epidermal polarity, keratinocyte differentiation and apoptosis. In addition, our results show the reversion of the malignancy of the SCCs that express increased levels of CYLDWT. The increase in apoptotic cell death of the A-CYLDWT tumors, together with the reduced angiogenesis could be responsible of the A-CYLDWT tumor regression observed in the carcinogenesis assays. Accordingly, SCCs defective in CYLD function are very aggressive as revealed by their progression toward spindle cell carcinomas, resistance to apoptosis and increased angiogenesis. These findings of the in vivo influence of CYLD in cancer prognosis make our results relevant from a therapeutic point of view, and open new avenues for exploring novel cancer therapies.