Mutant p53 enhances the signal of hepatocyte growth factor (HGF) to endow cancer cells with drug resistance

Background

Approximately 50% of human cancers harbor p53 mutations, which often endow cancer cells with novel oncogenic functions, such as promoting resistance to various chemotherapeutic drugs. It was recently demonstrated that secreted molecules from the microenvironment can promote drug resistance as well. However, whether cancer cell determinants, such as mutant p53, can act cooperatively with stromal-derived factors, to promote tumorigenesis at large and drug resistance in particular, has not been comprehensively studied yet. In the current study, we were interested to investigate whether mutant p53 can act in a cooperative manner with various secreted molecules to promote resistance to pathway-targeted therapy.

Materials and Methods

We utilized the human lung adenocarcinoma cell-line PC9, which harbors an EGFR hyperactivating mutation and an endogenous “hotspot” mutation in p53 DNA binding domain, R248Q. To study mutant p53-dependent effects, we created PC9 sub-lines which stably express turbo-RFP (tRFP) and either an shRNA targeting mutant p53 (PC9 shp53) or control shRNA (PC9 Mut-p53). We then subjected both PC9 sub-lines to either gefitinib, an EGFR inhibitor, or DMSO as control, together with a library of 298 recombinant secreted molecules. We then followed the growth of each of the sub-lines in the different conditions by monitoring changes in their tRFP signal for another 6 days.

Results

We did not observe significant mutant p53-dependent resistance to gefitinib in the absence of secreted molecules. However, we identified several such molecules that endowed the Mut-p53 sub-line with increased survival compared to shp53 sub-line when treated by gefitinib. Interestingly, the secreted factor that exhibited the most significant mutant p53-dependent rescue from gefitinib was hepatocyte growth factor (HGF). We were able to demonstrate a similar rescuing effect of mutant p53 and HGF in colon cancer cells treated with a MEK inhibitor. We further observed that mutant p53 downregulation attenuated the activation of the HGF receptor MET and the reactivation of downstream pro-survival ERK pathway, which corroborates the results of our proliferation assay. Furthermore, we were able to demonstrate the synergistic effect of HGF and mutant p53 on gefitinib resistance, as well as on ERK and MET activation, by administering conditioned medium derived of HGF-secreting fibroblasts. Finally, in a mass-spectrometry analysis, we were able to identify potential binding partners of mutant p53 which could mediate this mutant p53 and HGF dependent effect on gefitinib resistance.

Conclusion

In all, we describe here a novel mechanism for mutant p53-mediated drug resistance, which is not solely dependent on cell-autonomous factors, but rather on a synergistic effect between mutant p53 and stromal-secreted HGF. In a broad sense, we demonstrate how a molecular determinant in the cancer cell modulates a stromal signal to promote a pro-oncogenic phenotype.