TGFβ is well-known for its anti-proliferative activity and malignant progression is typically associated with the loss of responsiveness to TGFβ-induced cell growth inhibition. The cell-cycle machinery of proliferating cells is necessary for HPV replication. TGFβ profoundly suppresses epithelial cell proliferation. Studies in cervical cancer show that HPV-encoded proteins bind to TGFβ receptors and signal transducers, resulting in their degradation and promoting the proliferation of infected cells [
33]. Interestingly, we have previously shown [
18] that in both HPV-positive and negative HNC cell lines the TGFβ pathway is regularly activated and promotes transcription of mesenchymal genes. In particular, one of the main player of the TGFβ pathway SMAD4, whose expression is usually decreased in many tumours such as HNC, has been previously shown to be upregulated in HPV-positive HNC tumours compared to HPV-negative [
14]. Moreover, genetic alterations at the SMAD4 locus such as deletions or mutations, are much rare in HPV-positive compared to HPV-negative HNC [
14,
26]. In contrast, despite HPV-positive HNC and cervical carcinomas are both driven by HPV infection, cervical cancer samples showed a high frequency (41%) of LOH at SMAD4 gene locus [
34] and similar data were also reported in cervical cancer cell lines [
35]. In this study, although in a limited number of HNC samples, we confirmed the upregulation of SMAD4 mRNA in HPV-positive HNC tumours compared to HNC-negative and we show that also at protein level the expression of SMAD4 is higher in HPV-positive HNC. All these data were completely reproduced in a panel of HNC cell lines, showing how the use of these cell lines perfectly recapitulates the characteristic of the tumours. Moreover, we found that HPV16-encoded oncoproteins regulate SMAD4 expression at protein but not at mRNA level, without involving the action of p53. Nevertheless, another component of the p53 family, p63, is involved in the regulation of SMAD4 expression in HPV-positive HNC cell lines, explaining the lower SMAD4 level in one of the HPV-positive HNC cell lines used, the UMSCC47, which does not express the full-length ΔNp63α isoform, due to multiple integration of HPV16 at the TP63 locus [
27]. Interestingly, HPV16 affects the stability of SMAD4 protein by reducing its degradation. Many viruses and pathogens, and in particular the papillomaviruses, have been implicated in the regulation of posttranscriptional pathways such ubiquitination and SUMOylation, affecting the expression of the enzymes involved in these pathways [
36,
37]. In particular,
Listeria monocytogenes impairs the SUMO pathway, thus decreasing the stability of SMAD4 [
38], which is stabilized by SUMOylation [
39]. Here we show how HPV16 is responsible for the downregulation of the expression of one E3 ubiquitin ligase, the βTRCP1, which has been previously shown to induce SMAD4 ubiquitination and degradation. βTRCP1 leads to SMAD4 degradation upon sequential phosphorylation mediated by GSK3 and mitogen-activated protein kinase (MAPK) [
30]. Here we show how βTRCP1 preferentially degrades SMAD4 in HPV-negative and not in HPV-positive HNC cell lines, where SMAD4 is less ubiquitinated and has lower affinity to binding βTRCP1. One of the explanations of the different affinity of βTRCP1 to bind SMAD4, which differentiates HPV-negative and HPV-positive HNC cell lines, might be due to a different activation of the phosphorylation events that affects the binding of βTRCP1 to SMAD4 and its subsequent degradation. SMAD4 degradative defects in HPV-positive cell lines might be also explained by the fact that HPV downregulates βTRCP1 expression, leading to a lower expression of this ligase in HPV-positive HNC cell lines and tumours compared to HPV-negative. The ubiquitin pathway is not the only pathway influenced by viruses; the DNA repair pathway is a target of many viruses since DDR proteins play a role in the cellular response to viral infection, as well as in the lifecycle of many viruses. HPV both inhibits and activates different elements of the DDR pathway. This double behaviour has led to discrepancy in the interpretation of data studying the effect of HPV infection on the DDR machinery, which seems to be linked to a higher radiosensitivity of HPV-dependent tumours. In particular, the homologous recombination has been shown to be both inhibited [
40,
41] and activated by HPV16 oncoproteins [
42]. CHK1 is one of the key downstream molecules of DDR signalling. In response to DNA damage, CHK1 is phosphorylated primarily by ATR kinase [
43], leading to cell cycle arrest in S and at G2/M phases and promoting DNA repair before cell division. CHK1 phosphorylates many substrate proteins including the recombinase RAD51, the central molecule in HR pathway, that binds single-strand DNA at the sites of damage and forms filaments that are observed microscopically as nuclear foci. Here we show how HPV16, is able to upregulate the expression of both CHK1 and Rad51, together with the expression of other genes involved in the DDR signalling. This HPV-dependent upregulation leads to the increased expression of these proteins in both HNC tumours and cell lines and we also demonstrated that these two enzymes are still active in the presence of the virus. Indeed, upon cisplatin treatment in UDSCC2 cells, CHK1 is regularly phosphorylated and, in HKs transduced with HPV16 E6/E7, Rad51 foci increase compared to empty control cells and colocalize with pH2Ax, suggesting that Rad51 activity is maintained.
SMAD4 expression is deeply linked to the expression of genes involved in the DNA damage response [
6] and regulates the response to genotoxic [
6] and DNA repair inhibitors [
32]. In particular, here we highlight how both HNC cell lines and tumours recapitulate what shown upon interrogating the TCGA dataset [
32]. SMAD4 expression truly correlates with the expression of some genes involved in the ATR activation of the HR response, such as Rad51 and CHK1, and this is due to a transcriptional regulation mediated by SMAD4. Even more interestingly, we found that HPV-mediated upregulation of the DDR genes is dependent on the presence of SMAD4. Thus, as previously suggested in cervical cancer model [
44], HPV oncogenes promote initiation of the HR pathway but somehow impede the complete repair of the damage. This leads to an increased number of pH2Ax foci and might explain the greater sensitivity to radiotherapy of HPV-positive HNC tumours compared to the HPV-negative ones. Since HPV needs HR proteins to promote its replication, we hypothesize that the virus increases SMAD4 expression to upregulate HR and facilitate its replication. Finally, we show that in HPV-positive cell lines SMAD4-loss sensitizes cells to DNA-damaging agents, such as cisplatin, suggesting that SMAD4-deficient HPV tumours may be preferentially susceptible to similar treatments.