The p53 transcription factor family members are highly homologous and play critical roles in development, DNA damage responses, and tumorigenesis. Consequently, potential mechanisms of functional collaboration and antagonism among family members are much discussed, but with few exceptions are poorly understood. Through our characterization of p63-regulated miRs, we have uncovered what we believe is a new mechanism of p63/p73 functional crosstalk that mediates induced chemoresistance in SCC. This mechanism involves direct transcriptional regulation of miR-193a by both p63 and p73 as well as miR-mediated feedback regulation to p73 itself. Our finding that p53 does not participate in direct transcriptional regulation of this miR (Figure ) is in keeping with the somewhat weaker homology observed within its DNA-binding domain compared with p63 and p73 (14
). Importantly, our data together with other published work suggest that this regulatory circuit may have a particularly prominent role in tumors such as SCC that express both p63 and p73 but exhibit mutational inactivation of p53 (18
). Identifying a p53-independent mechanism linked specifically to chemosensitivity is particularly relevant in SCC, as inactivation of p53 in this tumor subtype is associated with both chemoresistance and a poorer overall prognosis (39
Our study demonstrates that p63 functions as a transcriptional repressor of miR-193a, while p73 functions as a direct transcriptional activator of this miR (Figures and ). These findings are in keeping with the established ability of ΔNp63α, the major isoform expressed in the epithelium, to function as a transcriptional repressor of genes, including CDKN1A
, which are regulated by other p53 family members (28
). The ability of TAp73 to regulate this miR in response to cisplatin is also in agreement with a large body of data demonstrating a critical role for p73-dependent transactivation in the DNA damage response and chemosensitivity (reviewed in ref. 41
). It is of note that the feedback loop from miR-193a to p73 potentially involves UTR-dependent regulation of multiple p73 isoforms, including ΔNp73 isoforms. Nevertheless, our data suggest that feedback to TAp73 is most relevant in SCC, as shRNA directed specifically against TAp73 isoforms reverses effects of miR-193a inhibition in both human and murine SCC cells (Figure ).
Transcriptional repression of miR-193a by p63 may begin to explain the somewhat paradoxical association of elevated p63 expression in tumor cells with chemosensitivity (25
). Expression of p63 is linked to proliferation, cellular survival, and regenerative potential, which likely explains selection for its overexpression and genomic amplification in SCC. Our findings suggest that p63 expression contributes to chemosensitivity through direct repression of miR-193a and consequent upregulation of p73. This regulatory circuit may also explain the unfortunately limited success of chemotherapy in SCC. Thus, treatment with cisplatin leads to p63 degradation and p73 activation (11
), which together induce miR-193a and inhibit p73, thereby limiting the therapeutic response. We show directly that this induced chemoresistance mechanism can be overcome by inhibiting miR expression in vivo, which converts a completely ineffective chemotherapy dose to a potent suppressor of tumor progression (Figure ).
Given that our findings point to miR-193a as a key regulator of p63/p73-dependent chemosensitivity, it is reasonable to ask whether expression of this miR itself might be useful in predicting the response of SCCs to chemotherapy. Indeed, we found that primary HNSCC tumors exhibiting high levels of miR-193a are likely to have lower p73 levels, potentially limiting chemosensitivity (Figure ). In the series of 23 HNSCC cases we studied, each patient was treated preoperatively with the same cisplatin-containing chemotherapy regimen. While most patients exhibited a partial response to therapy, 2 patients showed de novo resistance and disease progression with treatment, and their tumors were among the 3 highest in miR-193a expression (not shown). While the small number of patients and the uniform responses in this series preclude definitive conclusions, this observation is consistent with our central hypothesis. Future studies in additional uniformly treated cohorts, incorporating expression of p63/p73, miR-193a and other markers, are likely to refine our ability to predict which of these patients will benefit most from chemotherapy.
While several factors may contribute to p73 upregulation in tumor cells (42
), the importance of the miR-dependent mechanism we have identified for controlling p73 levels is clear. We observe an inverse correlation between miR-193a and p73 levels in human primary HNSCC tumors, and endogenous miR inhibition alone impedes tumor growth through a p73-dependent effect. In addition to the established role for p73 as a major effector of DNA damage–induced cell death, recent data have begun to suggest that p73-dependent cell death is a key mechanism in the response to targeted inhibition of growth-factor–mediated survival pathways in tumor cells (44
). Together, these findings position p73 as a central mediator in the response to varied environmental insults including DNA damage and growth factor withdrawal. As a result of these and other data, an intensive search is now underway to identify a means to activate p73 specifically in tumors as a therapeutic strategy (45
). Our results uncover a new means of eliciting specific p73 upregulation through miR inhibition. This approach provides the possibility of enhancing the therapeutic effect of both chemotherapy and the future generation of targeted anticancer agents.