Chemokine CXCL-2 Regulates Resistance to Platinum in Epithelial Ovarian Cancer by Mediating ATR/CHK1 Signaling Pathway

Backgrounds (cid:0) Platinum-resistance remains a challenge to recurrence and metastasis of epithelial ovarian cancer (EOC) and the mechanisms are unidentied. Tumor microenvironment (TME) and chemokine might play a key role in tumor chemoresistance. This study is designed to reveal the important role of CXCL-2 in causing EOC platinum-resistance. Materials and Methods: Differently expressed chemokines were selected based on the GSE114206 dataset of GEO database. Chemokines levels of platinum-sensitive and platinum-resistant EOC. Cell assays were performed and the cell stem characteristics were investigated for exploring the roles of CXCL-2 in EOC chemoresistance. We further explored the downstream signaling pathway regulated by CXCL-2 by detecting the expression of related molecules and rescue assay with the signaling inhibitor. Results: CXCL-2, CXCL-11 and CXCL-13 were found up-regulated in platinum-resistant EOC based on GSE114206 databases, and CXCL-2 was identied as key chemokine by validation. The cell assays showed overexpressing CXCL-2 and co-culturing with recombinant human CXCL-2 promoted cancer cell chemoresistance. Conversely, knocking down CXCL-2 and co-culturing with neutralizing antibody to CXCL-2 reduced cisplatin-resistance in cisplatin-resistant EOC cells. CXCL-2 levels regulated the stemness of cancer cells and activated ataxia telangiectasia and Rad3-related (ATR)/cell cycle checkpoint kinase1 (CHK1) signaling pathway. The cancer cell chemoresistance can be saved by CXCL-2 receptor inhibitor (SB225002) and CHK1 inhibitor (SAR-020106) in vitro. Conclusion: These results identied a CXCL-2 mediated platinum-resistance mechanism in EOC, and provided novel target for chemoresistance and


Background
Epithelial ovarian cancer (EOC) is the rst cause of gynecological malignancy-related death. According to statistics of American Cancer Society in 2020, there is estimated numbers of 21750 ovarian cancer cases and 13940 deaths in the United States in 2020 1 . The currently accepted treatment for EOC is the satis ed tumor reductive surgery and platinum-based chemotherapy. Although the tremendous progress has made in comprehensive therapy, the survival rate of patients with advanced tumor are poor even in highresource countries such as the United States and Canada, remained at only 47% 2 . EOC is most commonly treated with a combination of platinum and paclitaxel. Though initial response rates are 60-80%, majority of EOC patients acquire platinum-resistant during treatment with subsequent relapses and metastasis 3 . Platinum resistance remains an urgent challenge for patients with EOC, and the mechanism remains unidenti ed. There is lack of effective approach to prevent and against resistance for patients with EOC. Therefore, revealing the molecular mechanism contributing to platinum resistance and exploring therapeutic target to overcome the platinum resistance of EOC are clinical signi cances.
Chemokines contains a group of about 50 small (8-14kDa) secreted proteins, which regulate cell biological processes, including in malignancies progression 4,5 . These secreted proteins work by interacting with the corresponding receptors, a family of about 20 seven-transmembrane G-proteincoupled receptors 6 . Previous studies have suggested chemokines and their receptors are involved in malignancies progression mainly in the three mechanisms: attracting cancer cells for metastasis; mobilization of hematopoietic cell populations from the bone marrow to colonize at the tumor site and regulate tumor processes; acting as growth factors. which supporting tumor growth through an autocrine pathway 7,8 . Increasing evidences have supported that using immune checkpoint inhibitors that targeting chemokines and their receptors can be a novel approach in cancer therapy [9][10][11] . The anti-CCR4 monoclonal antibody and the CXCR4 receptor inhibitor are already in the clinical practice for hematological malignancies [12][13][14][15][16] . CXCL-10 was suggested as an immune checkpoint molecule in cancer, displaying a positive autocrine effect and directly suppressing tumor growth 17,18 20 . These studies suggested that chemokines and theri receptors might be potential novel therapeutic target in cancer chemoresistance.
Currently, gene expression pro les have been increasingly used in studies to identify candidate signi cant genes in various diseases, especially in malignancies 21 . Public genomics data repositories provided powerful systems biology approaches to detect the association between genes and cancers. In the present study, we devoted to explore the roles of chemokines in platinum-resistant EOC. We used bioinformatics methods to screened differentially expressed chemokines by comparing gene expression pro les based on platinum sensitivity status and PARP levels. Then, differentially expressed chemokines were validated the expression levels in EOC samples and cell lines to identify candidate chemokines eventually. Functional assays were further performed to investigate the molecule roles in EOC. In addition, we further explored the downstream signaling pathway which might in uence the process of DNA repairment.

Differentially expressed genes (DEGs) screening
The gene expression pro le was obtained from GSE114206 dataset of GEO database, which contained 6 EOC samples with low PARP and platinum-resistant and 6 EOC samples with high PARP and platinumsensitive We use the "limma" R package to screen the DEGs between the platinum-resistant and sensitive samples. The adjusted P-value < 0.05 and |log2fold change (FC)| > 1, were chosen as the cut-off threshold. The candidate genes were visualized by "circos" package (https://www.omicstudio.cn/tool).

Patients and clinical sample collection
All the ovarian cancer tissues and serum samples were obtained from patients who were diagnosed and undergone surgery in the First A liated Hospital with Nanjing Medical University between January 2010 to January 2019. Surgically resected specimens were immediately ash-frozen in RNA latter (AM7020, Invitrogen, USA) at −80°C for nucleic acid and protein extraction was performed. In the present study, 15 platinum-sensitive EOC samples and 9 platinum-resistant EOC samples were collected to investigate the molecular expression. Meanwhile, blood samples of 6 platinum-resistant and 9 platinum-sensitive EOC patients were collected to detect levels of chemokine in serum. All patients signed informed consent before using clinical specimens, and the use of specimens for this study has been proved by the ethics committee of the First A liated Hospital with Nanjing Medical University.

Plasmid and siRNA interference
The plasmid encoding transcript of CXCL-2 was designed and synthesized by Tsingke (Nanjing, China).
The siRNA targeted CXCL-2 and the negative control (NC) were purchased from GemmaPharma (Shanghai, China). Plasmid and siRNA transfected cells by Lipofectamine 3000 Transfection Reagent (L3000150, Invitrogen, USA) as manufacturer's instructions. Cells were treated with the indicated agents for analyses 24 or 48 hours after transfection. The CXCL-2 siRNA sequence was described in previous study 22 .

Cell viability assay
Preprocessed EOC cells were plated at a density of 6000-8000 cells per well in 96-well plates. were also used in cell viability assay.

RNA extraction and quantitative Real-time PCR (qRT-PCR)
The total RNA of cultured cells and EOC tissues was extracted with Trizol (15596026, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA HiScript Q RT SuperMix for qRT-PCR (R323, Vazyme, China) to prepare cDNA. qRT-PCR was performed with an SYBR Green PCR Kit (Q311, Vazyme, China). The sequences of gene primers used for qRT-PCR were synthesized by Tsingke (Nanjing, China) and showed in Supplementary Table 1. The ampli cation was performed as the manufacturer's instructions. The RNA expression level was normalized to GAPDH to calculate the relative gene expression.

Enzyme-linked immunosorbent assay
Platinum-resistant and platinum-sensitive patient's serum and cell culture supernatants were respectively extracted for cytokine analysis. Human CXCL-2 enzyme-linked immunosorbent assay (ELISA) kits, purchased from Hengyuan Biological Co. Ltd (H-10236, Shanghai, China) were used for analysis as the manufacturers' instructions. Each sample was duplicated.

Cell apoptosis assay
For the apoptosis assay, EOC cells were plated in 6-well plates and pretreated. After 48 hours cisplatin treating, 2× 10 4 cells and the cultural supernatant were collected. For cell staining, 5 μl of FITC Annexin V and 5 μl of propidium iodide (BD Biopharmingen, NJ, USA) were added to the collected cells and suspending in 300 μl of binding buffer for 15 min in dark. Cell apoptosis were eventually detected by a ow cytometer (FACScan; BD Biosciences, USA) using Cell Quest software (BD Biosciences, USA).

Statistical analysis
Each sample was analyzed based on results that were repeated at least three times and analyzed in Graphpad 8.0. The statistical signi cance of differences between two groups was determined by a standard Student's t-test.In all cases, differences at p < 0.05 were regarded as statistically signi cant (*), ones at p < 0.01(**) , p < 0.001(***) or p 0.0001(****) was considered higher statistical signi cances.

CXCL-2 regulated the cell apoptosis induced by cisplatin and the cell stemness in EOC
The results of cell apoptosis assay showed cell apoptosis induced by cisplatin were decreased after transfecting oeCXCL-2 plasmid or adding recombinant CXCL-2 in A2780 and HO8910 (Fig. 4A-F). On the contrary, transfecting CXCL-2 speci c siRNA or addition of CXCL-2 neutralizing antibody promoted cell apoptosis in cisplatin-resistant EOC cells (Fig. 4G-N). Meanwhile, we investigated the cell stem characteristics in EOC. The expression level of cell stem characteristics, including Nanog, SOX2 and OCT4, were also found be regulated by CXCL-2 (Fig. 5).
3.5 CXCL-2 promoted tumor resistance to cisplatin in EOC by mediating ATR/CHK1 signaling pathway Given that ataxia telangiectasia and Rad3-related (ATR)/cell cycle checkpoint kinase1 (CHK1) signaling pathway has recently be proved that play a signi cant role in DNA repair, as a PARP-independent approach. We detected the expression of ATR and CHK1 respectively in platinum-resistant and sensitive EOC samples and cell line. The results showed that ATR and downstream molecule CHK1 expressions were up-regulated in platinum-resistant EOC tissues and cisplatin-resistant EOC cells (Fig. 6A-F). The addition of CHK1 inhibitor reduced tumor resistance in vitro (Fig. 6G and 6H). Then, we further investigated the regulation of CXCL-2 and ATR/CHK1 signaling pathway. By detecting ATR and CHK1 expression, we found CXCL-2 expression could activating ATR/CHK1 signaling pathway, while knocking down CXCL-2 expression could suppress it (Fig. 6I-R). The cell function rescue assays suggested inhibiting ATR/CHK1 signaling pathway by using SAR-020106 reduced cell chemoresistance in cisplatinsensitive cells with oeCXCL-2 transfection (Fig. 6S and 6T).

Discussions
TME signi cantly in uences therapeutic response and clinical outcomes. According to previous studies, chemokines in TME are associated with cancer cell proliferation, migration, invasion and stemness, which could partially explain the resistance of cancer cells to chemotherapeutic drugs [24][25][26] . In the present study, we explored the chemokines contributed to platinum resistance in EOC. As a result, we identi ed candidate chemokines by bioinformatics analysis. We then demonstrated that the CXCL-2 expression is signi cantly up-regulated in platinum-resistant EOC tissues and cisplatin-resistant EOC cell lines. Cell functional assays showed CXCL-2 could promote tumor resistance to cisplatin in vitro and reduce cell apoptosis induced by cisplatin. Mechanistically, we found CXCL-2 promotes tumor resistance to cisplatin in EOC by mediating ATR/CHK1 signaling pathway. CXCL-2, which was known as growth-related oncogene-2/-β or macrophage in ammatory protein-2α, is 90% identical in amino acid sequence 27 . CXCL-2 works in cells via the corresponding CXCR2, which is known as interleukin 8 receptor β, have been extensively studied with regard to their role in malignancies. CXCL-2/CXCR2 axis was reported to promotes chemotaxis of neutrophils and endothelial cells to contribute to angiogenesis, chemoresistance, growth, and transformation of tumors [28][29][30] . Natsume Makoto et al. suggested omental adipocytes trigger gastric cancer cells proliferation, migration and capacity to induce angiogenesis by CXCL2 secretion 31 . Monocytes-derived CXCL-2 and CXCL-8 as main factors in regulating the recruitment of neutrophils into tumor milieus of hepatocellular carcinoma, which could inhibit then cancer cell apoptosis 32 . Previous study has also suggested that concentrations of CXCL-2 were higher in serum of ovarian adenocarcinoma patients with chemoresistance. Overexpression of CXCR2 has been demonstrated promote ovarian cancer progression 33,34 . However, there is lack of research about the role of CXCL-2 and the regulation mechanism in platinum-resistant EOC. Herein, we systematically explored the molecule biological function in cisplatin-resistant EOC. As the results illustrated, high expression level of CXCL-2 in TME of EOC was related to the cisplatin resistance of tumor. We demonstrated that CXCL-2 overexpression in EOC cell could protected from cell apoptosis induced by cisplatin and maintain cancer cell stemness, which might result in cell chemoresistance phenotype.
As we known, cisplatin exerts anti-tumor effects via multiple mechanisms, yet its most prominent mode of action involves the generation of DNA lesions followed by the activation of the DNA damage response 35 . Interfering with cell cycle checkpoint signaling is a signi cant approach to modulate DNA repair activity in platinum resistant tumor 36 . In the present study, we found CXCL-2 might regulate ATR/CHK1 signaling pathway to promote platinum-resistance in EOC. ATR and its downstream kinase CHK1, can be activated by DNA damage and DNA replication stress 37,38 . Activating ATR/CHK1 signaling pathway results in arresting cell cycle, which allowing time for DNA replication and appropriate DNA repairment 39 . In addition, acticating ATR/CHK1 signaling pathway stabilizes replication forks and prevents collapse into DNA double strand breaks (DSBs) 40,41 . Those functions of ATR/CHK1 provided potential therapeutic targets. ATR/CHK1 inhibitors have been developed and are currently used either as single agents or paired with radiotherapy or a variety of genotoxic chemotherapies in preclinical and clinical studies, including in EOC 42 . Catherine J Huntoon et al suggested that inhibiting ATR/CHK1 broadly sensitizes ovarian cancer cells to chemotherapy independent of BRCA status 43 . Our nding suggested a novel regulation mechanism of CXCL-2 in tumor microenvironment and ATR/CHK1 signaling pathway. However, it is possible that CXCL-2 may also promote EOC chemoresistance in ATR/CHK1 -independent pathways Conclusions In summary, our ndings revealed the role of CXCL-2 in EOC cisplatin chemoresistance and demonstrated a new regulatory mechanism of CXCL-2 mediating ATR/CHK1 signaling pathway in platinum-resistant EOC. Our results suggest that inhibition the expression of CXCL-2 and the speci c receptor and regulatory pathways represents a potential strategy to overcome platinum resistance in EOC.

Data Availability
They could be achieved upon reasonable request to the authors.
Ethics approval and consent to participate All patients signed informed consent before using clinical specimens, and the use of specimens for this study has been proved by the ethics committee of the First A liated Hospital with Nanjing Medical University.