Loss of Claudin-4 Reduces DNA Damage Repair and Increases Sensitivity to PARP Inhibitors

Abstract

High-grade serous ovarian cancer is the deadliest gynecologic malignancy due to progression to resistant disease. Claudin-4 is classically defined as a tight junction protein and is often associated with epithelial cancers. Claudin-4 is aberrantly expressed in nearly 70% of all ovarian cancer tumors and conveys a worse overall prognosis. Elevated claudin-4 expression correlates to increased DNA repair activity and resistance to DNA damaging agents. PARP inhibitors are emerging as an effective therapeutic option for patients with ovarian cancer and function by promoting DNA damage. The study examines the relationship between claudin-4 expression and the response to PARP inhibitors using both genetic and pharmacologic inhibition of claudin-4 in in vitro and ex vivo models of ovarian cancer to examine DNA repair markers and functional activity. Genetic inhibition of claudin-4 results in the downregulation of several DNA damage repair effectors, including 53BP1 and XRCC1. Claudin-4 knockdown did not change homology-directed repair but inhibited nonhomologous end-joining and reduced 53BP1 foci formation. In 15 primary ovarian cancer tumors, higher claudin-4 expression significantly correlated to a dampened PARP inhibitor-mediated antiproliferation response. Further, claudin-4 inhibition in high claudin-4 tumors sensitized tumor sections to PARP inhibition. These data highlight that claudin-4 expression in ovarian cancer tumors could serve as both a marker of PARP inhibitor response and a therapeutic target to improve PARP inhibitor response.

Figure 1:. Elevated claudin-4 expression correlates to DNA repair and response to DNA damaging agents.

A) Patient tumors within the TCGA (Firehose Legacy) were stratified based on the median expression of CLDN4 expression (Low, N=154 and High, N=153). B) Volcano plot of 1582 differentially regulated genes between low CLDN4 expressing tumors and high CLDN4 expressing tumor. C) Reactome pathway analysis of 1582 genes. D) Representative genes from Reactome DNA repair, replication and cycle pathways. E) Drug sensitivity of DNA damaging agents based on CLDN4 expression. Error bars, SEM. Statistical test, unpaired t-test with Benjamini-Hochberg multicomparison correction, ***p<0.001, ****p<0.0001.

Figure 2:. PARP inhibitor response in HGSOC cell lines with varying Claudin-4 expression.

A) Claudin-4 protein expression was measured via immunoblot in a panel of HGSOC cell lines. Loading control, β-actin. Olaparib and rucaparib dose response measured via colony formation in OVCAR8 (B), OVCAR10 (C), OVCAR4 (D). E) OVCAR3 cells transduced with shControl (shCtrl) or shCLDN4 (#1 and #2). Claudin-4 mRNA and protein levels were measured via qRT-PCR (Internal control, B2M) and immunoblot (Loading control, β-actin). F) Olaparib dose response in shCtrl and shCLDN4 cells. G) Rucaparib dose response in shCtrl and shCLDN4 cells. Experiments performed at least in triplicate. CI, confidence intervals. Error bars, SEM. Statistical test, one-way ANOVA with multicomparison correction, ***p<0.001, ****p<0.0001.

Figure 3:. Loss of claudin-4 downregulates DNA repair response effectors.

A) Protein isolated from OVCAR3 shCtrl (n=3) and shCLDN4#1 (n=3) cells and used for Reverse Phase Protein Array. Heatmap of the protein differentially regulated (p<0.05, FDR<15%). Arrows – DNA repair effectors. B) Protein expression of 53BP1 in OVCAR3 shCtrl, shCLDN4#1, and shCLDN4#2 cells. C) Protein expression of XRCC1 in OVCAR3 shCtrl, shCLDN4#1, and shCLDN4#2 cells. D) Claudin-4 mRNA expression (x-axis) and 53BP1 protein expression (y-axis) of 46 ovarian cancer cell lines. E) Claudin-4 mRNA expression (x-axis) and XRCC1 protein expression (y-axis) of 46 ovarian cancer cell lines. F) Cell viability screen of FDA-approved oncology drugs comparing OVCAR3 shCtrl (x-axis) and shCLDN4 (y-axis). Dotted lines represent – 2 standard deviations. Compound class is indicated by the color. G) Single cell sequencing from 5 HGSOC tumors, PAX8 (black, tumor cells), claudin-4 (red), 53BP1 (green), and XRCC1 (blue). Statistical test, one way ANOVA with multicomparison correction. *p<0.05, **p<0.01, ***p<0.001.

Figure 4.. Claudin-4 knockdown inhibits non-homologous end joining and reduces 53BP1 foci formation.

A) Homology-directed DNA repair activity measured in OVCAR3 shCtrl and shCLDN4 (#1 and #2) cells transfected without I-SceI and with I-SceI. B) Non-homologous end-joining DNA repair activity measured in OVCAR3 shCtrl and shCLDN4 (#1 and #2) cells transfected without I-SceI and with I-SceI. C) OVCAR3 shCtrl and shCLDN4 (#1 and #2) cells treated with 375 nM olaparib used for immunofluorescence against γH2ax (green) and XRCC1 (red). Nuclei stained using DAPI (blue). D) OVCAR3 shCtrl and shCLDN4 (#1 and #2) cells treated with vehicle control or 375 nM olaparib used for immunofluorescence against γH2ax (green) and 53BP1 (red). Nuclei stained using DAPI (blue). E) Same as D, quantification of 53BP1 foci per nuclei from five field of views. F) Low (2-69, n=95) and high (70-1899, n=107) mutation count from TCGA ovarian cancer tumors (x-axis) compared to claudin-4 mRNA expression (y-axis). Claudin-4 protein expression (CPTAC) versus Mutation count (y-axis) from 82 HGSOC TCGA tumors. Experiments performed at minimum in triplicate. Error bars, SEM. Statistical Test, one way ANOVA with multicomparison correction (A, B, E, F) or unpaired t-test (F) or Spearman Correlation (F). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5:. Olaparib and inhibition of claudin-4 inhibit proliferation of primary ovarian cancer tumors.

A) Workflow of the ex vivo experiments using primary tumor tissue. Primary tumors were classified as claudin-4 null/low or claudin-4 high based on immunofluorescence (IF, claudin-4, green and nuclei, DAPI). IHC against Ki67 was completed in all the tumors, tumors were de-identified, and a histology score (H-score) was calculated based on intensity of Ki67 signal and percentage of tumor cells from at least three fields of view. B) Representative H-score and claudin-4 IF for claudin-4 null/low expressing ovarian cancer tumors. C) Representative H-score and claudin-4 IF for claudin-4 high expressing ovarian cancer tumors. D) Representative Ki67 images of claudin-4 null/low and high expressing ovarian cancer tumors (Scale bar, 50 microns). E) Normalized Ki67 H-score based control (Ctrl) treated tumors for all the claudin-4 null/low (N=5) and high (N=7) expressing serous ovarian tumors. Three fields from each tumor were analyzed and each tumor is represented by three matched colored spots. Error bars, SEM. Statistical test, one way ANOVA with multicomparison correction. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.