Mutations in the p53 tumor-suppressor gene are prevalent in human cancers. The majority of p53 mutations are missense, which can be classified into contact mutations (that directly disrupts the DNA-binding activity of p53) and structural mutations (that disrupts the conformation of p53). Both of the mutations can disable the normal wild-type (WT) p53 activities. Nevertheless, it has been amply documented that small molecules can rescue activity from mutant p53 by restoring WT tumor-suppressive functions. These compounds hold promise for cancer therapy and have now entered clinical trials. In this study, we show that cruciferous-vegetable-derived phenethyl isothiocyanate (PEITC) can reactivate p53 mutant under in vitro and in vivo conditions, revealing a new mechanism of action for a dietary-related compound. PEITC exhibits growth-inhibitory activity in cells expressing p53 mutants with preferential activity toward p53^R175, one of the most frequent ‘hotspot’ mutations within the p53 sequence. Mechanistic studies revealed that PEITC induces apoptosis in a p53^R175 mutant-dependent manner by restoring p53 WT conformation and transactivation functions. Accordingly, in PEITC-treated cells the reactivated p53^R175 mutant induces apoptosis by activating canonical WT p53 targets, inducing a delay in S and G2/M phase, and by phosphorylating ATM/CHK2. Interestingly, the growth-inhibitory effects of PEITC depend on the redox state of the cell. Further, PEITC treatments render the p53^R175 mutant sensitive to degradation by the proteasome and autophagy in a concentration-dependent manner. PEITC-induced reactivation of p53^R175 and its subsequent sensitivity to the degradation pathways likely contribute to its anticancer activities. We further show that dietary supplementation of PEITC is able to reactivate WT activity in vivo as well, inhibiting tumor growth in xenograft mouse model. These findings provide the first example of mutant p53 reactivation by a dietary compound and have important implications for cancer prevention and therapy.

We reported previously that phenethyl isothiocyanate (PEITC), a dietary compound, can reactivate p53R175H mutant in vitro and in SK-BR-3 (p53R175H) breast xenograft model resulting in tumor inhibition. Because of the diversity of human cancers with p53 mutations, these findings raise important questions whether this mechanism operates in different cancer types with same or different p53 mutations. In this study, we investigated whether PEITC recuses mutant p53 in prostate cancer cells harboring different types of p53 mutants, structural and contact, in vitro and in vivo. Cell proliferation, cell apoptosis and cell cycle arrest assays were performed to examine the effects of PEITC on prostate cancer cell lines with p53 mutation(s), wild-type p53, p53 null or normal prostate cells in vitro. Western blot analysis was used to monitor the expression levels of p53 protein, activation of ATM and upregulation of canonical p53 targets. Immunoprecipitation, subcellular protein fraction and qRT-PCR was performed to determine change in conformation and restoration of transactivation functions/ inhibition of gain-of-function (GOF) activities to p53 mutant(s). Mice xenograft models were established to evaluate the antitumor efficacy of PEITC and PEITC-induced reactivation of p53 mutant(s) in vivo. Immunohistochemistry of xenograft tumor tissues was performed to determine effects of PEITC on expression of Ki67 and mutant p53 in vivo. We demonstrated that PEITC inhibits the growth of prostate cancer cells with different “hotspot” p53 mutations (structural and contact), however, preferentially towards structural mutants. PEITC inhibits proliferation and induces apoptosis by rescuing mutant p53 in p53R248W contact (VCaP) and p53R175H structural (LAPC-4) mutant cells with differential potency. We further showed that PEITC inhibits the growth of DU145 cells that co-express p53P223L (structural) and p53V274F (contact) mutants by targeting p53P223L mutant selectively, but not p53V274F. The mutant p53 restored by PEITC induces apoptosis in DU145 cells by activating canonical p53 targets, delaying cells in G1 phase and phosphorylating ATM. Importantly, PEITC reactivated p53R175H and p53P223L/V274F mutants in LAPC-4 and DU145 prostate xenograft models, respectively, resulting in significant tumor inhibition. Our studies provide the first evidence that PEITC’s anti-cancer activity is cancer cell type-independent, but p53 mutant-type dependent.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer–related deaths worldwide, mainly because of its poor prognosis. A valid mechanism‐based prognostic biomarker is urgently needed. γ‐hydroxy‐1,N ²‐propanodeoxyguanosine (γ‐OHPdG) is an endogenously formed mutagenic DNA adduct derived from lipid peroxidation. We examined the relationship of γ‐OHPdG with hepatocarcinogenesis in two animal models and its potential role as a prognostic biomarker for recurrence in HCC patients. Bioassays were conducted in xeroderma pigmentosum group A knockout mice and diethylnitrosamine‐injected mice, both prone to HCC development. γ‐OHPdG levels in the livers of these animals were determined. The effects of antioxidant treatments on γ‐OHPdG and hepatocarcinogenesis were examined. Using two independent sets of HCC specimens from patients, we examined the relationship between γ‐OHPdG and survival or recurrence‐free survival. γ‐OHPdG levels in liver DNA showed an age‐dependent increase and consistently correlated with HCC development in all three animal models. Theaphenon E treatment significantly decreased γ‐OHPdG levels in the liver DNA of xeroderma pigmentosum group A knockout mice and remarkably reduced HCC incidence in these mice to 14% from 100% in the controls. It also effectively inhibited HCC development in the diethylnitrosamine‐injected mice. Using clinical samples from two groups of patients, our study revealed that higher levels of γ‐OHPdG are strongly associated with low survival (P < 0.0001) and low recurrence‐free survival (P = 0.007). Conclusion: These results support γ‐OHPdG as a mechanism‐based, biologically relevant biomarker for predicting the risk of HCC and its recurrence. (Hepatology 2018;67:159‐170).

We previously reported that phenethyl isothiocyanate (PEITC), a dietary-related compound, can rescue mutant p53. A structure–activity relationships study showed that the synthetic analog 2,2-diphenylethyl isothiocyanate (DPEITC) is a more potent inducer of apoptosis than natural or synthetic ITCs. Here, we showed that DPEITC inhibited the growth of triple-negative breast cancer cells (MDA-MB-231, MDA-MB-468, and Hs578T) expressing “hotspot” p53 mutants, structural (p53^R280K, p53^R273H) or contact (p53^V157F), at IC₅₀ values significantly lower than PEITC. DPEITC inhibited the growth of HER2+ (p53^R175H SK-BR-3, p53^R175H AU565) and Luminal A (p53^L194F T47D) breast cancer (BC) cells harboring a p53 structural mutant. DPEITC induced apoptosis, irrespective of BC subtypes, by rescuing p53 mutants. Accordingly, the rescued p53 mutants induced apoptosis by activating canonical WT p53 targets and delaying the cell cycle. DPEITC acted synergistically with doxorubicin and camptothecin to inhibit proliferation and induce apoptosis. Under these conditions, DPEITC delayed BC cells in the G1 phase, activated p53 canonical targets, and enhanced pS1981-ATM. DPEITC reduced the expression of MDR1 and ETS1. These findings are the first report of synergism between a synthetic ITC and a chemotherapy drug via mutant p53 rescue. Furthermore, our data demonstrate that ITCs suppress the expression of cellular proteins that play a role in chemoresistance.