Tailoring Chemotherapy for the African-Centric S47 Variant of TP53

Abstract

The tumor suppressor TP53 is the most frequently mutated gene in human cancer and serves to restrict tumor initiation and progression. Single-nucleotide polymorphisms (SNP) in TP53 and p53 pathway genes can have a marked impact on p53 tumor suppressor function, and some have been associated with increased cancer risk and impaired response to therapy. Approximately 6% of Africans and 1% of African Americans express a p53 allele with a serine instead of proline at position 47 (Pro47Ser). This SNP impairs p53-mediated apoptosis in response to radiation and genotoxic agents and is associated with increased cancer risk in humans and in a mouse model. In this study, we compared the ability of wild-type (WT) and S47 p53 to suppress tumor development and respond to therapy. Our goal was to find therapeutic compounds that are more, not less, efficacious in S47 tumors. We identified the superior efficacy of two agents, cisplatin and BET inhibitors, on S47 tumors compared with WT. Cisplatin caused dramatic decreases in the progression of S47 tumors by activating the p53/PIN1 axis to drive the mitochondrial cell death program. These findings serve as important proof of principle that chemotherapy can be tailored to p53 genotype.Significance: A rare African-derived radioresistant p53 SNP provides proof of principle that chemotherapy can be tailored to TP53 genotype. Cancer Res; 78(19); 5694-705. ©2018 AACR.

Figure 1.. S47 is an intrinsically poorer tumor suppressor.

(A, B) WT and S47 E1A/RAS MEFs were plated at a density of 20,000 cells per well in a 6-well plate under normal (A) or low serum (B) conditions and were counted daily for six days (n.s., not significant). * p<0.05. (C) WT and S47 E1A/RAS MEFs were plated in soft agar at a concentration of 8,000 cells per 60-mm dish and were incubated for 14 days (scale bar, 250 μm). (D) Quantification of (C) both by colony number and colony size, as measured by the area of colonies using the equation A=πr². Colony number quantification was performed by counting the number of colonies in five random fields of view, and colony size was performed by quantifying the area of ten random colonies per group. *** p<0.001. (E) 1×10⁶ WT or S47 E1A/RAS MEFs were injected subcutaneously into the right flanks of 8-week old NSG mice. Tumors were measured using a digital caliper and tumor volumes were derived from the equation v = (length x width²)/0.52, where the width was the shortest of the two sides (n=10 mice per group). Each experiment is representative of two independent clones of each genotype. (F) Measurements of the weights of WT and S47 E1A/RAS tumors taken at day 19. *** p<0.001. (G) IHC analysis of Ki-67 and p53 staining of WT and S47 E1A/RAS tumors (n=4 mice per group; scale bar, 100 μm). Rabbit IgG was used as a negative control.

Figure 2.. S47 transformed MEFs show increased sensitivity to cisplatin and BET inhibitors but not etoposide.

(A) WT or S47 E1A/RAS cells were plated at a density of 10,000 cells per well in 24-well plates. The next day, cells were treated with the indicated concentrations of cisplatin (CDDP) for 72 hours. Cells were then fixed with 10% formalin and stained with 0.5% crystal violet. * p<0.05 (B) WT and S47 E1A/RAS cells were pre-treated with sub-lethal concentrations of CDDP (0.5 μM) or Etoposide (0.02 μM) for 24 hours and were then plated in soft agar in the absence of drug for 14 days (scale bar, 1000 μm). (C) Quantification of (B). (D) WT and S47 E1A/RAS cells were plated at a density of 2,000 cells per well in a 96-well plate. The next day, cells were treated with the indicated doses of Nedaplatin or Carboplatin for 72 hours and subjected to Alamar Blue assays. (E) WT and S47 E1A/RAS cells were plated at a density of 1,000 cells per well in a 96-well plate. The next day, cells were treated with the indicated doses of the BET inhibitors JQ-1 or OTX-015 for 96 hours. Cells were then subjected to IC₅₀ Alamar Blue assays. (F) Colony formation assays of WT and S47 E1A/RAS cells that were treated with 10 μM OTX-015 for 24 hours prior to plating. Data are indicative of representative images of experiments done in triplicate and were performed on two independent clones of each genotype. (G) Quantification of (E). *** p<0.001

Figure 3.. Cisplatin and OTX-015 show superior efficacy in S47 tumors.

(A) 5×10⁵ WT and S47 E1A/RAS MEFs were injected subcutaneously into the right flanks of 7–8-week-old NSG mice. Once tumors reached an approximate size of 100 mm³, mice were randomized into three groups (n=8 mice per group): (1) vehicle, (2) CDDP (4 mg/kg, every four days), and (3) OTX-015 (50 mg/kg, daily). Tumor volumes were measured every other day as previously described. Note the different scales for tumor volume between WT and S47. * = p<0.05, ** = p<0.01, and *** p < 0.001. (B) Tumor weights of WT and S47 tumors treated with either CDDP or OTX-015. (C) WT and S47 E1A/RAS MEFs were injected subcutaneously as in (A) to compare treatment efficacy between CDDP (4 mg/kg, every four days) and Etoposide (10 mg/kg, every four days). (D) Tumor weights of WT and S47 tumors treated with either CDDP or Etoposide. Etoposide did not cause a statistically significant decrease in tumor growth compared to control. (E-G) A mixed model with spline linear regression analysis showing that cisplatin and OTX-015, but not etoposide, caused significantly greater reduction in tumor volume per treatment in S47 tumors compared to WT. (H) IHC analysis of Ki-67, Cleaved Caspase-3, and p53 staining of WT and S47 E1A/RAS tumors +/− CDDP (scale bar, 100 μm). Rabbit IgG was used as a negative control.

Figure 4.. S47 tumor cells show increased utilization of the mitochondrial cell death pathway.

(A) WT and S47 E1A/RAS cells were treated with 10 μM CDDP for 24 hours in the presence or absence of the protein synthesis inhibitor cycloheximide (CHX, 2.5 μg/mL). Cell lysates were subjected to Western blot analysis, and immunoblotted for cleaved caspase-3, p53, p21, and HSP90 (loading control). CDDP: cisplatin (B) WT and S47 E1A/RAS cells were treated with 10 μM cisplatin (CDDP) for 24 hours. Cells were then fractionated into three fractions: whole cell lysate (W), mitochondria (M), and cytosol (C). Equal μg of lysates were probed for the mitochondrial protein BAK and cytochrome c (cyto c) and the nuclear/cytosolic protein PCNA to assess purity. Arrows depict p53 protein levels in mitochondrial fractions of WT vs. S47 cells treated with CDDP. Increased localization of S47 protein to mitochondria was evident in multiple independent experiments, including those where the levels of total WT p53 and S47 were closely matched. (C) An in-situ proximity ligation assay (PLA) was performed in WT and S47 E1A/RAS cells treated with 10 μM CDDP for 12 hours. Each red dot represents interaction between endogenous p53 and PIN1 proteins (scale bar, 25 μm). Cells stained with p53 antibody alone were used as a negative control. DAPI nuclear staining is shown in blue. The white boxed image is magnified in the panel to the right. Shown on the right is the quantification of (C), measured as the average number of PLA signals per nuclei. Data was quantitated by counting the number of cells in five random fields of view per experimental group. CDDP: cisplatin. * p<0.05 and *** p<0.001 (D) S47 E1A/RAS cells were transfected with si-PIN1 for 48 hours. Cells were then treated with 10 μM CDDP for 12 hours and were subjected to an in-situ PLA assay. Each red dot represents interaction between endogenous p53 and TOMM20 proteins (scale bar, 25 μm). Cells stained with p53 antibody alone were used as a negative control. Shown on the right are Western blot analyses showing efficient knockdown of PIN1, as well as similar expression of p53 and TOMM20 in si-ctr and si-PIN1 S47 E1A/RAS cells. ** p<0.01

Figure 5.. The S47 variant of p53 shows increased ability to oligomerize BAK.

Equal amounts of whole cell (WCE), cytosolic (Cyto) and mitochondrial (Mito) extracts prepared from p53-null human H1299 cells were immunoblotted for mitochondrial protein BAK and the nuclear-cytoplasmic protein PCNA (left). Purified mitochondria (25 μg) were incubated with 100 pmol of full-length (FL) GST-p53 (middle panel) or His-tagged p53 (right panel) recombinant proteins, as indicated. The asterisk in the His-tagged panel reflects a likely degradation product. BAK oligomers were crosslinked with BMH, resolved by SDS-PAGE and detected by Western blotting with a BAK-specific antibody (anti BAK NT). The BAK oligomerization blots were reprobed with an antibody against p53 to verify equal quantity and purity of added p53 recombinant proteins used in the BAK activation assays, as indicated.

Figure 6.. Increased mitochondrial trafficking of S47 protein in tumors.

(A) Detection of p53 and TOMM20 interactions in Formalin Fixed Paraffin Embedded (FFPE) WT and S47 E1A/RAS tumors using the Duolink PLA Brightfield kit. Staining without primary antibodies was used as a negative control. PLA signals are shown as reddish-brown dots, indicated by black arrowheads (scale bar, 20 μm). (B) Quantification of p53-TOMM20 PLA of WT and S47 tumors +/− CDDP. S47 tumors treated with CDDP show a significantly greater number of PLA-positive dots compared to WT treated tumors. Quantification was performed by counting the number of PLA positive signals in five random fields of view for each experimental group, each done in triplicate. (C) Proposed model highlighting the increased usage of the direct mitochondrial pathway of p53-mediated cell death in S47 tumors treated with cisplatin.