概要

【Introduction】
　Cancer cells develop remarkable mechanisms to promote their uncontrolled proliferation, survival and motility. These characteristics contribute to their invasion of neighboring tissues and metastasis to distant organs. Treating cancers by various therapeutic modalities such as radiation and chemotherapeutic reagents is often unsatisfactory because the tumor cells gain therapeutic resistance leading to subsequent recurrence. The mechanisms of cancer therapeutic resistance have been the subject of intensive studies. Obviously, one of the most important causes of the therapeutic resistance is genetic and non-genetic heterogeneity and alterations of cancer cells, which becomes more prominent and elaborate by cancer cell evolution during the progression of the diseases and treatments. The actin- binding protein Girdin is a hub protein that interacts with multiple proteins to regulate motility and Akt and trimeric G protein signaling in cancer cells. Girdin expression correlates with poor outcomes in multiple human cancers. However, those findings are not universal, as they depend on study conditions. Those data suggest that multiple aspects of Girdin function and its role in tumor cell responses to anticancer therapeutics must be reconsidered. In the present study, we examined the effect of Girdin expression on cancer cells’ sensitivity to cytotoxic therapeutics.

【Methods】
　We selected ultraviolet C (UVC)-induced DNA damage as a model of radiation therapy. Two esophageal cancer cell lines KYSE 140 and KYSE 150, HeLa and HEK293T cell lines were used for in vitro study. Biopsy and surgically resected esophageal tissue samples from 28 esophageal squamous cell carcinoma patients were obtained at Nagoya University Hospital. Gene engineering was developed by the CRISPR/single-guide RNA (sgRNA)- directed synergistic activation mediator (SAM) system. Real time-qPCR, western blot analysis, flow cytometry, cell synchronization, immunohistochemistry (IHC), immunofluorescence, cell migration assay, colony formation assay, EdU proliferation assay, cell proliferation assay and microscopic imaging were performed in the present study.

【Results】
　Previous studies showed a significant correlation between Girdin (CCDC88A) mRNA expression levels and poor clinical outcomes in esophageal cancer. Our analysis of esophageal cancer cases in the TCGA dataset, however, resulted in variable Kaplan-Meier survival curves and log-rank P-values that fluctuated depending on cut-off values (Figure 1A-C). We used immunohistochemistry to examine Girdin protein expression in tissue sections of surgically resected tumors obtained from esophageal cancer patients, the results of which showed varying degrees of Girdin expression between cancer cells. We also examined Girdin expression in biopsy samples taken from the patients before treatment and stratified their outcome by a scoring system based on Girdin expression levels determined by IHC, but found no correlation between the patients’ survival and Girdin expression (Figure 1D). Given the heterogeneous expression of Girdin between tumor cells and its unclear prognostic significance in esophageal cancer, we speculated that Girdin may be involved in tumor cell sensitivity to anticancer therapeutics. We first found that high Girdin expression was associated with an increased sensitivity of cancer cells to UVC- mediated DNA damage (Figure 2). Interestingly, migratory cells, which are known to exhibit significant resistance to DNA damage-induced apoptosis, became prone to that by Girdin overexpression (Figure 3). This result suggested that high Girdin expression counteracts or eliminates the DNA damage protective effect of Girdin-mediated cell migration. We then attempted to address the mechanism of this observation by identifying a novel role of Girdin in cell cycle regulation. High Girdin expression was associated with dysregulated cell cycle progression and prolonged G1 and M phases (Figure 4A, B). These features were accompanied by p53 activation, which conceivably increases cancer cell vulnerability to UV exposure (Figure 4C). We also searched for other mechanisms that involve Girdin to regulate cell cycle progression, and found that Mad2, a key component of the spindle checkpoint machinery that is crucial for anaphase onset in M phase, was highly upregulated in Girdin-overexpressing cells compared to control cells at both the protein and mRNA levels (Figure 4D, E). An immunofluorescent staining for tubulin proteins revealed that the number of metaphase or early anaphase cells that undergo multipolar division was increased in Girdin-overexpressing cells compared to control cells. Thus, it may be possible that Girdin-overexpressing induces mitotic delay by interfering with Mad2-mediated regulation of the spindle checkpoint machinery. A correlation between Girdin and Mad2 expression was also observed in tissue samples of a human esophageal cancer cohort available from the TCGA database. Given the availability of both pre- and post-radiation tissue samples in a cohort of esophageal cancer patients (N = 28) in our institution, we used IHC to examine Girdin expression in the biopsies and surgical samples that were obtained both pre- and post-radiation, respectively. The data from both pre- or post-radiation samples, however, did not show a correlation between Girdin expression levels and histopathological evaluation of the response to radiation therapy. Although not conclusive (given the limited number of samples), it seems that elevated Girdin expression alone does not confer sensitivity to radiotherapy, at least in esophageal cancer patients. That suggests a far more complex mechanism of radiosensitivity or the need to stratify and select patients who will benefit from elevated Girdin expression.

【Discussion】
　The present study showed an unexpected link between Girdin and the sensitivity of cancer cells to DNA damage (Figure 5). In contrast to the general view that migratory cells are resistant to DNA damage, HeLa cells that expressed high levels of Girdin exhibited increased sensitivity to UVC-mediated DNA damage and subsequent apoptosis. Pathological analysis showed extensive intra-tumor heterogeneity of Girdin expression, supporting the view that the sensitivity to DNA damage is also variable among cancer cells. Together with previous studies that have shown a crucial role of Girdin in cancer cell invasion and metastasis, the data reveal complex effects of Girdin expression on cancer patients’ outcomes. Previous studies of Girdin function have shown that Girdin functions as a regulator of the migration-proliferation dichotomy. The migration-proliferation dichotomy is a hallmark of normal as well as cancer cells that cell migration and proliferation do not occur simultaneously. This mechanism helps to explain the resistance of migratory cancer cells to various cytotoxic therapeutics that target proliferating cells. Our present study added a new dimension to Girdin function, i.e., that high expression of Girdin enhances the vulnerability of cancer cells to DNA damage. These results seem to contradict the previous finding that Girdin promotes cell migration and cancer progression but may provide opportunities for therapeutic intervention if Girdin expression could be manipulated in human malignancies.
　An unexpected but intriguing finding of the present study was that overexpression of Girdin delayed G1 and M phases, both of which are sensitive to UVC. This might contribute to the increased sensitivity of Girdin-overexpressing cells to DNA damage. Our analysis identified that Mad2 expression was transcriptionally upregulated in Girdin OE cells, which may explain the dysregulation of the cell cycle in Girdin OE cells. We have not yet, however, delved into the detailed mechanisms underlying this observation. Given the role of Mad2 in prolonging checkpoint arrest caused by DNA damage, it is plausible that elevated Girdin and Mad2 are both involved in the dysregulation of the cell cycle and subsequent UVC-induced DNA damage and cell death.

【Conclusion】
　Our data suggested the presence of complex positive and negative roles of Girdin in cancer progression that depended on the cancer type and therapeutic context. These finding should be considered in the development of therapeutics that target pathways involving Girdin.

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