Claudins (CLDNs) are tetraspan transmembrane proteins playing important function for tight junction formation and cell polarity (Krause et al., 2008). Structurally, claudins contain four transmembrane helices, two extracellular loops that form trans or cis protein-protein interactions, and intracellularly located N-terminal and C-terminal tails (Suzuki et al., 2017). The C-terminal tail can interact with other scaffolding proteins to regulate paracellular tightness and ion permeability (Heinemann and Schuetz, 2019). CLDNs are frequently overexpressed in various types of cancer. In tumor cells, CLDNs often fail to assemble into functional tight junctions, suggesting alternative roles beyond barrier formation. To date, 27 distinct CLDN family members have been identified in mammals, each exhibiting tissue-specific expression patterns and playing distinct roles in tumorigenesis across different cancer types (Mineta et al., 2011). CLDN1 is overexpressed in colorectal cancer and gastric cancer (Eftang et al., 2013, Hahn-Stromberg et al., 2017). CLDN3 and CLDN4 are overexpressed in ovarian cancer and promote cancer cell survival and metastasis, but not cell proliferation (Agarwal et al., 2005). CLDN3 overexpression increases cellular motility in breast cancer (Todd et al., 2015) and malignant potential in colorectal cancer (de Souza et al., 2013). Nuclear expression of claudin-3 could be used as a biomarker for colorectal adenocarcinoma (Tokuhara et al., 2018). Claudin-4 was identified as a potential marker and therapeutic target for prostate cancer (Landers et al., 2008). CLDN6 overexpression was associated with enhanced proliferation and invasion of gastric cancer (Yu et al., 2019) and leads to a worse overall survival in endometrial cancer (Zhang et al., 2021). Splicing isoform 2 of CLDN18 (CLDN18.2) is characteristically overexpressed in gastric cancer and pancreatic cancer (Hong et al., 2020).

Claudins were targeted for cancer treatment in various cancer cells. An anti-claudin-6 antibody drug conjugate (IMAB027-vcMMAE) induced potent antitumor activity in claudin-6 positive ovarian and testicular cancer cells in a preclinical study (Özlem et al., 2018). In nonclinical models, Zolbetuximab, an antibody targeting claudin-18.2, had strong antitumor activity against claudin-18.2 positive pancreatic cancer cells, and improved the median progression-free survival (mPFS) for patients with advanced or metastatic claudin-18.2 positive gastric adenocarcinoma (Tureci et al., 2019, Zhang et al., 2020). Interestingly, Clostridium perfringens enterotoxin (CPE), found in food poisoning Clostridium species, can specifically interact with claudin-3/4 with high affinity (Shinoda et al., 2016). A CPE-toxin fusion proteins demonstrated efficacy in CLDN3/4 overexpressing cancer models (Pahle et al., 2017).

The expression of claudins and the functional integrity of tight junction barriers are dynamically regulated by diverse microenvironmental cues, depending on the physiological or pathological context. TNFα can induce upregulation of CLDN3 in kidney tubular epithelial cells through NF-κB and CREB1 (Anwer et al., 2021). In colon cancer cells, it was found that the increased CLDN1 expression is mediated through transcription factors Cdx1, Cdx2 and GATA4 in cooperation with Wnt signaling (Bhat et al., 2012). The PKC/MAPK/AP-1 dependent pathway played an important role in regulation of CLDN18.2 expression in PMA stimulated MKN45 gastric cells (Yano et al., 2008). An increased expression of CLDN1 and decreased expression of CLDN3 was observed in invasive breast cancer patients following chemotherapy (Skalova et al., 2019).

Previous reports showed that the expression of CLDN3 and CLDN4 in ovarian cancer cells is regulated by epigenetic mechanisms, i.e. DNA methylation and histone H3 acetylation (Honda et al., 2007, Honda et al., 2006). It remains unclear whether chemotherapy drugs regulate CLDN3 and CLDN4 expression in ovarian cancer cells. We hypothesize that CLDN3 and CLDN4 expression is modulated in response to chemotherapy treatment and may be associated with the development of chemoresistance. The goal of this study is to investigate the regulation mechanism of CLDN3 and CLDN4 by gemcitabine in ovarian cancer cells. We primarily used A2780 cancer line to study the regulation of CLDN3 and CLDN4 by gemcitabine and probed the downstream signaling pathways using specific pathway inhibitors. We overexpressed CLDN3 or CLDN4 in A2780 cancer cell line to study their effects on chemotherapy protection. Finally, we investigated the potential of enhanced targeting of ovarian cancer cells using a claudin-3 and claudin-4 dual-specific monoclonal antibody-drug conjugate (ADC) in combination with gemcitabine.