Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 

 
ORIGINAL ARTICLE
Ahead of print publication  

CXCR4 and RIF1 overexpression induces resistance of epithelial ovarian cancer to cisplatin-based chemotherapy


1 Department of Clinical Oncology and Nuclear Medicine, Tanta University, Egypt
2 Department of Pathology, Faculty of Medicine, Tanta University, Egypt

Date of Submission11-Jul-2019
Date of Acceptance21-Jan-2020
Date of Web Publication05-Nov-2020

Correspondence Address:
Lamiss Mohamed Abd Elaziz Sad,
Department of Clinical Oncology, Tanta University Hospital, Tanta
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_480_19

 > Abstract 


Introduction: The chemoresistance of epithelial ovarian cancer (EOC) is a major problem. Thus, the search for novel biomarkers associated with cisplatin sensitivity is overwhelming. Previous studies have shown that CXC chemokine receptor 4 (CXCR4) is associated with tumor growth, angiogenesis, and distant metastases, and replication timing regulatory factor 1 (RIF1) is responsible for the repair of double-strand DNA breaks. This study, thus, aimed to identify the correlation between CXCR4 and RIF1 overexpression and cisplatin sensitivity in EOC.
Materials and Methods: Fifty-five EOC patients were recruited to assess the chemosensitivity of EOC to cisplatin-based chemotherapy at the Oncology Department in Tanta University Hospitals, Egypt.
Results: The results showed that patients with a higher CXCR4 and RIF1 expression exhibited a significantly lower chemosensitivity, worse overall survival, and poorer progression-free survival. The only prognostic associated with overall survival was CXCR4.
Conclusion: Our study showed that CXCR4 and RIF1expression levels are not associated only with poor prognostic features of epithelial ovarian cancer but also with its chemoresistance to cisplatin, and consequently, with worse overall survival and progression free survival.

Keywords: Cisplatin, CXCR4, epithelial ovarian cancer, predictive, RIF1



How to cite this URL:
Sad LM, Mohamed DA, Elanwar NM, Elkady A. CXCR4 and RIF1 overexpression induces resistance of epithelial ovarian cancer to cisplatin-based chemotherapy. J Can Res Ther [Epub ahead of print] [cited 2020 Dec 2]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=300106




 > Introduction Top


Ovarian cancer is the leading cause of gynecologic malignancy-related deaths in women. It is the most common type (90%) of epithelial tumors.[1] Despite ovarian epithelial malignancy being among the most chemosensitive malignancies and the availability of modern treatment modalities incorporating radical tumor debulking followed by platinum plus paclitaxel-based chemotherapy, the prognosis of the patients is poor and the 5-year survival rate is still 25%.[2]

Age, tumor histology, performance status, and residual tumor volume have been reported as independent prognostic factors for the survival of epithelial ovarian cancer (EOC) patients.[3],[4] Even in patients with similar disease status and receiving the same treatment, the survival rates still vary, the reason of which is still unknown. Recognizing new biological prognostic variables could help anticipate patients' treatment outcome.

G-protein-coupled chemokine receptor, CXC chemokine receptor 4 (CXCR4), exerts its action through binding to its ligand stromal cell-derived factor 1 (also called CXCL12), changing the backbone of cell and tumor migration. It has been shown to play a role in tumor growth and metastasis.[5],[6],[7] However, there is still a debate on the correlation between CXCR4 and survival.[8],[9],[10]

Cisplatin cytotoxicity occurs mainly during the active replication of DNA by forming cisplatin-containing DNA adducts, leading to double-stranded DNA breaks.[11] Replication timing regulatory factor 1 (RIF1) is responsible for the repair of DNA strand breaks and the regulation of replication time.[12],[13] In breast cancer, RIF1 was found to be unregulated and silencing of RIF1 increased cisplatin sensitivity in cervical cancer.[14],[15] The role of RIF1 in EOC is still under investigation.

In the present study, we aimed to evaluate CXCR4 and RIF1 expressions and their impacts on cisplatin-based therapy in EOC patients.


 > Materials and Methods Top


Patients

Fifty-five individuals who diagnosed with EOC at the Oncology Department, Tanta University Hospital, Egypt, between January 2014 and January 2016 were included in the study. These patients were subjected to histopathological diagnoses at the Pathology Department of Tanta University Hospital, Egypt. These patients were staged according to the International Federation of Gynecology and Obstetrics.[16] Informed consent was taken from all patients. Chemotherapy was not allowed before surgery. Patients underwent surgical treatment followed by chemotherapy.

For chemotherapy, the patients were subjected to intravenous infusion of 175 mg/m 2 paclitaxel over 3 h plus carboplatin area under the curve 5–6 IV over 1 h on day 1; and the cycle was repeated after every 21 days for six cycles.

Patients who responded to cisplatin-based chemotherapy or those who did not progressed for more than 6 months off treatment were considered platinum sensitive, whereas patients who progressed during treatment or within 6 months from the treatment were considered platinum resistant.

Immunohistochemistry

Surgically extracted tissue specimens were fixed in 10% formalin and embedded in paraffin.

Immunohistochemical staining of the CXCR4 and RIF1 antibodies was done (1:100, Sigma-Aldrich). Four micrometers thick tissue sections were cut from paraffin-embedded blocks. Then, the tissue sections were baked at 60°C for 30 min followed by the incubation in xylene for 3 × 10 min, and graded ethanol to distilled water was used for rehydration. Then, the samples were heated in 1 mmol/L ethylenediaminetetraacetic acid for 20 min for antigen retrieval. Then, the samples were stained with goat serum in phosphate-buffered saline (PBS) buffer for 20 min at room temperature for blocking nonspecific staining. Then, the samples were incubated in 3% H2O2 for 10 min for quenching the endogenous peroxidase activity.

The slides were incubated first with rabbit polyclonal nonspecific RIF1 antibody or PBS control at 4°C overnight followed by biotinylated goat anti-rabbit antibody and peroxidase-conjugated streptavidin. To visualize staining, 3,3'-diaminobenzidine tetrahydrochloride substrate kit (Zhongshan Golden bridge) was used, according to the manufacturer's instructions.

The intensity of CXCR4 staining was scored as 0, 1, 2, and 3 indicating no staining, weak, moderate, and marked, respectively.

The percentage scores were graded as follows: 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). Final score by multiplying the scores of each tumor sample was found to be between 0 and 12, In our study, CXCR4 expression was classified either low (-, +) or high (++, +++) based on the amount of protein expression [Figure 1],[Figure 3], [Figure 5], [Figure 7], [Figure 9] [Figure 12].
Figure 1: Clear cell carcinoma of the ovary showed high CXCR4 expression (×400)

Click here to view
Figure 3: Endometrioid carcinoma of the ovary showed high RIF1 expression (×400)

Click here to view
Figure 5: Serous carcinoma of the ovary showed high CXCR4 expression (×400)

Click here to view
Figure 7: Undifferentiated carcinoma of the ovary showed high CXCR4 expression (×400)

Click here to view
Figure 9: Endometrioid carcinoma of the ovary showed low CXCR4 expression (×400)

Click here to view
Figure 12: Serous carcinoma of the ovary showed low CXCR4 expression (×200)

Click here to view


Next, the RIF1 staining intensity was scored.[17] The RIF1 staining intensity was scored as 1, 2, 3, and 4 for <25%, ≥25% to 50%, ≥50% to 75%, and ≥75% to 100% positive cells, respectively. The staining intensity was scored as 0, 1, 2, and 3 for negative, weak, moderate, and strong, respectively. A final IRS score was then calculated by multiplying these two scores. The cutoff score was set to 4.0 according to receiver operating characteristic curves analysis. For the final score of <4 and ≥4, the tumor was considered to exhibit low RIF1 expression and high RIF1 expression, respectively [Figure 2], [Figure 6], [Figure 8], [Figure 10], [Figure 11], [Figure 13].
Figure 2: Endometrioid carcinoma of the ovary showed high CXCR4 expression (×400)

Click here to view
Figure 6: Serous carcinoma of the ovary showed high RIF1 expression (×100)

Click here to view
Figure 8: Clear cell carcinoma of the ovary showed high RIF1 expression (×400)

Click here to view
Figure 10: Endometrioid carcinoma of the ovary showed low RIF1 expression (×100)

Click here to view
Figure 11: Mucinous carcinoma of the ovary showed high RIF1 expression (×400)

Click here to view
Figure 13: Serous carcinoma of the ovary showed low RIF1 expression (×100)

Click here to view


Statistical analysis

The correlation between patient characteristics and expression levels of both CXCR4 and RIF1 expression was evaluated using the Chi-square test. The survival was analyzed using the Kaplan–Meier method version 21. For univariate analysis of prognostic factors, the log-rank test was used. Statistical significance was defined at P < 0.05. For multivariate analysis, hazard ratios and their 95% confidence intervals were calculated using Cox's multivariate analysis for overall survival (OS) and progression-free survival (PFS).[18],[19]


 > Results Top


CXCR4 and RIF1 expression was evaluated in 55 ovarian cancer patients. High and low expression of CXCR4 was observed in 30 (54.5%) and 25 (45.5%) patients, respectively. High and low expression of RIF1 was found in 47 (85.5%) and 8 (14.5%) patients, respectively [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13].
Figure 4: Mucinous carcinoma of the ovary showed high CXCR4 expression (×400)

Click here to view


[Table 1] summarizes the clinical characteristics of the patients with respect to CXCR4. High CXCR4 expression was significantly associated with bad performance, advanced stage (Stage III and IV), high grade, and chemoresistant cancer.
Table 1: Patients' characteristics according to CXC chemokine receptor 4

Click here to view


In addition, we found that a high RIF1 level was associated with advanced stage (P = 0.028) and chemoresistant (P = 0.033) cancer, as shown in [Table 2].
Table 2: Patients' characteristics according to replication timing regulatory factor 1

Click here to view


The OS of the patients was 29.4% and 91.7% under high and low CXCR4 expression, respectively (P < 0.0001), whereas it was 36.7% and 85.7% under high and low RIF1, respectively (P = 0.017) [Figure 14] and [Figure 15].
Figure 14: Overall survival in relation to CXCR4

Click here to view
Figure 15: Overall survival in relation to RIF 1

Click here to view


In univariate analysis, the statistically significant factors were performance status (0.004), FIGO (P = 0.000), early versus late stage (P = 0.001), optimal debulking versus suboptimal debulking (P = 0.000), grade (P = 0.000), chemosensitive versus chemoresistant (P = 0.000), and expression levels of both CXCR4 and RIF1 (P < 0.000 and 0.017, respectively).

In multivariate analysis, only chemotherapy sensitivity and CXCR4 expression were statistically significant with OS, as shown in [Table 3].
Table 3: Univariate and multivariate analysis of overall survival

Click here to view


The 3-year PFS was 36.5% and 84.4% for patients with high and low CXCR4 expression, respectively (P = 0.001), whereas it was 46.7% and 875% for patients with high and low RIF1 expression, respectively (P = 0.035) [Figure 16] and [Figure 17]. Univariate analysis revealed that the following clinical parameters correlated with improved PFS: performance status, FIGO staging (early), optimal debulking, well-differentiated tumor, chemotherapy sensitivity, and low expressions of CXCR4 and RIF1 [Table 4]. The multivariate analysis revealed that no factor was significantly associated with PFS [Table 4].
Figure 16: Progression-free survival in relation to CXCR4

Click here to view
Figure 17: Progression-free survival in relation to RIF 1

Click here to view
Table 4: Univariate and multivariate analysis of progression-free survival

Click here to view



 > Discussion Top


In order to eradicate ovarian cancer, cisplatin is used which leads to improved OS through the formation of crosslinks.[11],[20] Nevertheless, EOC chemoresistance against cisplatin has been reported.[20],[21] Hence, identifying novel biomarkers that predict response to cisplatin-based chemotherapy is essential to tailor the treatment strategies for EOC patients.

Several human tumors, such as pancreatic, breast, ovarian, and non-small cell lung cancer, overexpress CXCR4.[9],[10],[22],[23] Angiogenesis, metastases, and poor prognosis have been shown to be associated with the overexpression of CXCR4;[9],[10],[11] however, its correlation with chemosensitivity to cisplatin has not yet been studied.

In addition, RIF1 is responsible for the regulation of telomere length and repair double-strand DNA breaks which is the cisplatin mode of action.[24],[25] RIF1 has been found to be upregulated in breast cancer, and knockdown of RIF1 in cervical cancer is associated with cisplatin sensitivity. Thus, it could be a potential therapeutic target in EOC.[9]

Therefore, we assessed the prognosis of EOC with these biomarkers (CXCR4 and RIF1 expressions) and their association with chemosensitivity to cisplatin.

In our study, we found that the high expression of CXC4 was associated with bad performance, advanced stage (stage III and IV), high grade, and chemoresistance which was similar to that reported by Li et al., 2014.[8]

Consistent with previous studies, the high expression of CXCR4 was associated with worse OS and PFS in the univariate analysis. Furthermore, the high expression of CXCR4 was the independent prognostic factor of OS but not PFS in the multivariate analysis.[8],[9]

Our study showed that high RIF1 expression is associated with advanced stage and chemoresistant EOC similar to that reported by Mei et al., 2017.[15],[26]

Corroborating previous studies, high RIF1 was associated with worse OS and PFS.[15],[26] However, its statistical significance for both OS and PFS was not revealed by multivariate analysis, which might be due to a small sample size.


 > Conclusions Top


Our study showed that CXCR4 and RIF1 expression levels are not associated only with poor prognostic features of EOC but also with its chemoresistance to cisplatin, and consequently, with worse overall survival and PFS. Therefore, these factors might be used as novel biomarkers for tailoring the treatment.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published, and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

1.
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69:7-34.  Back to cited text no. 1
    
2.
Peres LC, Cushing-Haugen KL, Köbel M, Harris HR, Berchuck A, Rossing MA, et al. Invasive epithelial ovarian cancer survival by histotype and disease stage. J Natl Cancer Inst 2019;111:60-8.  Back to cited text no. 2
    
3.
Cuylan ZF, Meydanli MM, Sari ME, Akbayir O, Celik H, Dede M, et al. Prognostic factors for maximally or optimally cytoreduced stage III nonserous epithelial ovarian carcinoma treated with carboplatin/paclitaxel chemotherapy. J Obstet Gynaecol Res 2018;44:1284-93.  Back to cited text no. 3
    
4.
Maheshwari A, Kumar N, Gupta S, Rekhi B, Shylasree TS, Dusane R, et al. Outcomes of advanced epithelial ovarian cancer treated with neoadjuvant chemotherapy. Indian J Cancer 2018;55:50-4.  Back to cited text no. 4
[PUBMED]  [Full text]  
5.
Lacalle RA, Blanco R, Carmona-Rodríguez L, Martín-Leal A, Mira E, Mañes S. Chemokine receptor signaling and the hallmarks of cancer. Int Rev Cell Mol Biol 2017;331:181-244.  Back to cited text no. 5
    
6.
Hassan S, Buchanan M, Jahan K, Aguilar-Mahecha A, Gaboury L, Muller WJ, et al. CXCR4 peptide antagonist inhibits primary breast tumor growth, metastasis and enhances the efficacy of anti-VEGF treatment or docetaxel in a transgenic mouse model. Int J Cancer 2011;129:225-32.  Back to cited text no. 6
    
7.
Fujita T, Chiwaki F, Takahashi RU, Aoyagi K, Yanagihara K, Nishimura T, et al. Identification and characterization of CXCR4-positive gastric cancer stem cells. PLoS One 2015;10:e0130808.  Back to cited text no. 7
    
8.
Li J, Jiang K, Qiu X, Li M, Hao Q, Wei L, et al. Overexpression of CXCR4 is significantly associated with cisplatin-based chemotherapy resistance and can be a prognostic factor in epithelial ovarian cancer. BMB Rep 2014;47:33-8.  Back to cited text no. 8
    
9.
Sekiya R, Kajiyama H, Sakai K, Umezu T, Mizuno M, Shibata K, et al. Expression of CXCR4 indicates poor prognosis in patients with clear cell carcinoma of the ovary. Hum Pathol 2012;43:904-10.  Back to cited text no. 9
    
10.
Pils D, Pinter A, Reibenwein J, Alfanz A, Horak P, Schmid BC, et al. In ovarian cancer the prognostic influence of HER2/neu is not dependent on the CXCR4/SDF-1 signalling pathway. Br J Cancer 2007;96:485-91.  Back to cited text no. 10
    
11.
Xiong Y, Huang BY, Yin JY. Pharmacogenomics of platinum-based chemotherapy in non-small cell lung cancer: Focusing on DNA repair systems. Med Oncol 2017;34:48.  Back to cited text no. 11
    
12.
Zaaijer S, Shaikh N, Nageshan RK, Cooper JP. Rif1 Regulates the Fate of DNA Entanglements during Mitosis. Cell Rep 2016;16:148-60.  Back to cited text no. 12
    
13.
Alver RC, Chadha GS, Gillespie PJ, Blow JJ. Reversal of DDK-Mediated MCM phosphorylation by RIF1-PP1 regulates replication initiation and replisome stability independently of ATR/Chk1. Cell Rep 2017;18:2508-20.  Back to cited text no. 13
    
14.
Mohapatra P, Satapathy SR, Das D, Siddharth S, Choudhuri T, Kundu CN. Resveratrol mediated cell death in cigarette smoke transformed breast epithelial cells is through induction of p21Waf1/Cip1 and inhibition of long patch base excision repair pathway. Toxicol Appl Pharmacol 2014;275:221-31.  Back to cited text no. 14
    
15.
Mei Y, Peng C, Liu YB, Wang J, Zhou HH. Silencing RIF1 decreases cell growth, migration and increases cisplatin sensitivity of human cervical cancer cells. Oncotarget 2017;8:107044-51.  Back to cited text no. 15
    
16.
Šišovská I, Minář L, Felsinger M, Anton M, Bednaříková M, Hausnerová J, et al. Current FIGO staging classification for cancer of ovary, fallopian tube and peritoneum. Ceska Gynekol Summ; 82:230-6.  Back to cited text no. 16
    
17.
Zeng ZL, Luo HY, Yang J, Wu WJ, Chen DL, Huang P, et al. Overexpression of the circadian clock gene Bmal1 increases sensitivity to oxaliplatin in colorectal cancer. Clin Cancer Res 2014;20:1042-52.  Back to cited text no. 17
    
18.
Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc 1985;53:457-81.  Back to cited text no. 18
    
19.
Bland JM, Altman DG. The log rank test. BMJ 2008;328:1073.  Back to cited text no. 19
    
20.
Xie X, Huang N, Zhang Y, Wei X, Gao M, Li M, et al. MiR-192-5p reverses cisplatin resistance by targeting ERCC3 and ERCC4 in SGC7901/DDP cells. J Cancer 2019;10:1039-51.  Back to cited text no. 20
    
21.
Kim S, Han Y, Kim SI, Kim HS, Kim SJ, Song YS. Tumor evolution and chemoresistance in ovarian cancer. NPJ Precis Oncol 2018;2:20.  Back to cited text no. 21
    
22.
Shakir M, Tang D, Zeh HJ, Tang SW, Anderson CJ, Bahary N, et al. The chemokine receptors CXCR4/CXCR7 and their primary heterodimeric ligands CXCL12 and CXCL12/high mobility group box 1 in pancreatic cancer growth and development: Finding flow. Pancreas 2015;44:528-34.  Back to cited text no. 22
    
23.
Zheng CH, Chen XM, Zhang FB, Zhao C, Tu SS. Inhibition of CXCR4 regulates epithelial mesenchymal transition of NSCLC via the Hippo-YAP signaling pathway. Cell Biol Int 2018;42:1386-94.  Back to cited text no. 23
    
24.
Wang J, Zhang H, Al Shibar M, Willard B, Ray A, Runge KW. Rif1 phosphorylation site analysis in telomere length regulation and the response to damaged telomeres. DNA Repair (Amst) 2018;65:26-33.  Back to cited text no. 24
    
25.
Foti R, Gnan S, Cornacchia D, Dileep V, Bulut-Karslioglu A, Diehl S, et al. Nuclear architecture organized by Rif1 underpins the replication-timing program. Mol Cell 2016;61:260-73.  Back to cited text no. 25
    
26.
Liu YB, Mei Y, Long J, Zhang Y, Hu DL, Zhou HH. RIF1 promotes human epithelial ovarian cancer growth and progression via activating human telomerase reverse transcriptase expression. J Exp Clin Cancer Res 2018;37:182.  Back to cited text no. 26
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

 
Top
 
 
  Search
 
     Search Pubmed for
 
    -  Sad LM
    -  Mohamed DA
    -  Elanwar NM
    -  Elkady A
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

  >Abstract>Introduction>Materials and Me...>Results>Discussion>Conclusions>Article Figures>Article Tables
  In this article
>References

 Article Access Statistics
    Viewed94    
    PDF Downloaded4    

Recommend this journal