Claudin 4 Is Differentially Expressed between Ovarian Cancer Subtypes and Plays a Role in Spheroid Formation

Kristin L M Boylan,Stefan E Pambuccian,Katherine M Harrington,Benjamin Misemer,John D Andersen,Steve E Kalloger,Amy P N Skubitz,C Blake Gilks,Melissa S Derycke

doi:10.3390/ijms12021334

Kristin L M Boylan, Stefan E Pambuccian + Show 7 more

Open Access

https://doi.org/10.3390/ijms12021334

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Abstract

Claudin 4 is a cellular adhesion molecule that is frequently overexpressed in ovarian cancer and other epithelial cancers. In this study, we sought to determine whether the expression of claudin 4 is associated with outcome in ovarian cancer patients and may be involved in tumor progression. We examined claudin 4 expression in ovarian cancer tissues and cell lines, as well as by immunohistochemical staining of tissue microarrays (TMAs; n = 500), spheroids present in patients’ ascites, and spheroids formed in vitro. Claudin 4 was expressed in nearly 70% of the ovarian cancer tissues examined and was differentially expressed across ovarian cancer subtypes, with the lowest expression in clear cell subtype. No association was found between claudin 4 expression and disease-specific survival in any subtype. Claudin 4 expression was also observed in multicellular spheroids obtained from patients’ ascites. Using an in vitro spheroid formation assay, we found that NIH:OVCAR5 cells treated with shRNA against claudin 4 required a longer time to form compact spheroids compared to control NIH:OVCAR5 cells that expressed high levels of claudin 4. The inability of the NIH:OVCAR5 cells treated with claudin 4 shRNA to form compact spheroids was verified by FITC-dextran exclusion. These results demonstrate a role for claudin 4 and tight junctions in spheroid formation and integrity.

Highlights

Ovarian cancer is the most lethal gynecological malignancy, resulting in approximately 125,000 deaths yearly, worldwide [1]
To further validate our gene expression studies, we examined the expression of claudin 4 RNA in ovary tissues and cell lines
We demonstrated that claudin 4 is differentially expressed across histological subtypes of ovarian cancer; no difference in survival was observed between claudin 4 positive vs. negative tumors

Summary

Introduction

Ovarian cancer is the most lethal gynecological malignancy, resulting in approximately 125,000 deaths yearly, worldwide [1]. Due to the paucity of specific symptoms and the lack of an effective screening method, the majority of ovarian cancers are diagnosed at late stages of malignancy, after the tumor has spread beyond the ovary [2]. In contrast to other solid tumors, the most common method for ovarian cancer metastasis is direct peritoneal spread. Late stage cancers are frequently associated with ascites, and tumor cells can be shed into the ascites fluid either as single cells or multicellular aggregates called spheroids. We and others have shown that ovarian cancer spheroids are a source of tumor invasion and metastasis [4,5,6,7,8]. Spheroids are chemoresistant [9,10], implicating spheroids as a factor in disease persistence or recurrence

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