Abstract B072: Claudin-4 modulates autophagy via cell-cell junctions as a cellular protective mechanism before genomic instability in ovarian cancer
Authors: Villagomez Fabian R. , Lang Julie , Webb Patricia , Neville Margaret , Woodruff Elizabeth R. , Bitler Benjamin G. 
Published: March 2024

Objectives: Claudin-4 is a protein upregulated in most high-grade serous carcinomas of the ovary, fallopian tube, and peritoneum (HGSC) and its overexpression is associated with cancer therapy resistance; nevertheless, disruption of the claudin-4 function is associated with increased genomic instability and sensibility of tumor cells to cancer therapeutics. The typical role of claudin-4 is on cell-cell junction regulation; however, this protein also participates in migration, mitosis, DNA repair. Furthermore, a key cellular process that regulates genomic instability is autophagy which has been linked to therapy resistance; however, the association of claudin-4, autophagy, and genomic instability is not known. Therefore, the goal of this work was to evaluate the claudin-4 participation in autophagy to regulate genomic instability. Methods: We used authenticated and routinely checked for mycoplasma HGSC cells (OVCA429, OVCAR3, OVCAR8) to evaluate the elimination (CRISPRi) and overexpression (lentivirus transduction) of claudin-4; complementary, we disrupted the function of claudin-4 using a claudin mimic peptide (CMP). We used immunofluorescence, live-cell imaging, and confocal microscopy to characterize markers of genome instability (micronuclei; Dapi) and its association with cell junctions (F-actin; LifeAct), and cell growth (cell cycle, propidium iodide and flow cytometry, FC). In addition, those cell lines were engineered to express (mCherry-GFP-LC3) to evaluate autophagy by FC under different conditions (chloroquine, autophagy inhibitor; rapamycin, autophagy activator, etc.); complementary assays included immunoblotting for LC3 A/B, and the up-stream regulators of autophagy, SLC1A5/Lat1. Finally, we evaluated the blocking of claudin-4 with CMP and compered such effect with a PARPi (niraparib) in a PDX-human system mice (unpaired t and One-way ANOVA with Tukey's multiple comparisons tests; p&lt; 0.05). Results: By modifying the claudin-4 function with CMP and CRISPRi, we determined this protein regulates cellular cytoskeletal connections and mitosis. Disruption of this axis leads to aberrant cell junctions, abnormal mitotic progression, and autophagic activity. Particularly, cell-cell connections become unstable which affects mitosis progression and drives genomic instability. In addition, genomic instability was associated with downregulation of the plasma membrane modulators of autophagy, SLC1A5/SLC7A5. Consequently, autophagy activity increased and associated with engulfment of cytoplasm-localized damaged DNA; thus, functioning to clear damaged genetic material. In addition, we found that targeting claudin-4 with CMP is as effective as niraparib treatment in disrupting the ovarian tumor progression, and a combinatorial therapy enhances the anti-tumor efficacy compared to the single agent. Conclusions: Claudin-4 promotes a protective cellular mechanism that links cell-cell junctions to genome integrity.

Citation Format: Fabian R. Villagomez, Julie Lang, Patricia Webb, Margaret Neville, Elizabeth R. Woodruff, Benjamin G. Bitler. Claudin-4 modulates autophagy via cell-cell junctions as a cellular protective mechanism before genomic instability in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B072.