This study showed that there is overexpression of CD44+/CD24− and RAD6 while there is lower expression of DDB2 in chemoresistance ovarian cancer patients. CD44+ (cluster of differentiation 44) and CD24− expression is associated with increased ovarian cancer oncogenesis and progression (5, 12). CD44+ overexpression has also been found in pancreatic cancer (13), breast cancer (14), gastric cancer (15), urothelial bladder cancer(16) and colorectal cancer (17). It is associated with metastasize, recurrence, chemoresistance, and poor survival rates in the ovarian cancer (18). CD24− is a cell surface adhesion molecule frequently detected in invasive ovarian carcinoma. High CD24− expression in invasive ovarian cancer predicts shorter overall survival than low CD24− markers (19).
CD44+/CD24− is a good predictor of ovarian cancer chemoresistance. We found that higher CD44+/CD24− has a significant result (p < 0,05) with moderate accuracy (AUC 0,7 − 0,8) with a sensitivity of 78% and specificity of 75%. Meng et al., (2012) found that ovarian cancer cells with high CD44+/CD24− expression have stem cells characteristics: higher aggressive, invasive, progressive, and multiplicative properties in each tumor histology type. This ovarian cancer cell also has higher chemoresistance properties, recurrence rate, and aggravating prognosis (5).
Li et al., (2017) found that high CD44+/CD24− protein levels indicated breast tumor malignancy higher rates of cell proliferation, tumorigenesis, and metastasize. Breast cancer with CSC has resistance to chemotherapy and radiotherapy (20). Yan et al., (2013) also found that cells with high CD44+/CD24− expression showed higher migration and invasion properties and were the cause of chemoresistance (21).
There was higher CD44+ expression in paclitaxel-resistant cell lines than paclitaxel-sensitive cell lines in a mouse model using ovarian cancer xenografts while patients with ovarian cancer showed that CD24− cells were more resistant to cisplatin and increased tumorigenesis ability (22). A meta-analysis study showed that CD44+ protein was associated with poorer cancer-specific survival rates in patients undergoing chemoradiotherapy (23). The study by Zhang et al., (2019) found that high expression of markers CD105, CD44+, and CD106 related to chemoresistance, poorly differentiated, and advanced-stage ovarian cancer (18).
RAD6 has a significant role in activating several DNA repair pathways and is substantial in chemoresistance in ovarian cancer (24). RAD6 overexpression is associated with mitotic abnormalities and tumor progression (25). We found that there was a significant increase in RAD6 levels (p < 0,05) in chemoresistance patients. However, ROC and AUC results were not significant (p > 0,05), and the accuracy was very weak (AUC 0,5 − 0,6), sensitivity 84%, specificity 46%.
Clark et al., (2018) investigated the role of RAD6 in chemoresistant ovarian cancer by inhibiting RAD6A and RAD6B in several ovarian cancer. These cells showed decreased expression of CSC markers, activation of DDR protein, and concomitant sensitivity to carboplatin responses suggesting that RAD6 expression increases after chemotherapy and causes chemoresistance in cancer cells through stimulating CSC protein expression and increasing DNA repair activity (25). The study by Somasagara et al., (2016) found association between chemoresistance and increased RAD6 in ovarian cancer cells through RAD6-mediated ubiquitin signaling, which led to increased DDR and CSC protein expression. In addition, a higher RAD6 (⩾5,1) was also associated with a disease recurrence rate of 70%. (26) Another study concluded that RAD6 related to the severity of ovarian cancer, breast cancer, and melanoma. Rad6 levels were significantly increased in severe ovarian cancer with platinum chemoresistance (27).
RAD6 overexpression can increase stem cell characteristics, making them more aggressive, metastasize and relapse. The epigenetic influence of RAD6 causes the ubiquitination of some histone variants which then regulates genes related to DNA repair, cell resistance, and chemoresistance (27). RAD6 is also closely related to RAD18, a protein E3 ubiquitin ligase that regulates the DNA repair pathway in Fanconi anemia and the BRCA gene in breast cancer (26) R.AD6 was involved in breast cancer chemoresistance in which researchers inhibited RAD6 with a small molecule inhibitor and found an increased sensitivity to cisplatin (28). In bladder cancer, it was also found that overexpression of enzymes from the UBE2 group, one of which was RAD6, could affect the growth of bladder cancer cells. An experiment was carried out by stopping the expression of UBE2, then the cells would stop growing in the G2/M phase and increase the apoptosis of these cancer cells (29).
DDB2 is a protein localized in the cell nucleus that contribute to gene transcription, cell cycle progression, and protein degradation (30). The decrease in DDB2 also affects ovarian cancer’s aggression, metastasize, and severity (8). This study found that DDB2 protein expression was found significantly lower in chemotherapy-resistant patients (p < 0,05). ROC and AUC analysis results are p > 0,05, weak accuracy (AUC 0.6–0.7), sensitivity 37,5%, specificity 37,5%.
DDB2 levels are high in the chemo-sensitive cancer patient group because DDB2 participates in the tumor suppression process in at least three ways, namely: promoting the nucleotide excision repair (NER) process, supporting apoptosis, and inducing cell aging after DNA damage has occurred.(31) The loss of DDB2 function in normal cells can lead to susceptibility to tumor growth. DDB2 gene mutation causes loss of function and gives rise to the phenotypic characteristics of Xeroderma Pigmentosum group E, which is characterized by malignant skin tumors. Mice with low DDB2 levels are not only hypersensitive to UV-related carcinogenesis processes but also have a high incidence of broad-spectrum spontaneous malignant tumors from internal organs. Thus, DDB2 acts as a mediator in the suppression of the p53 and BRCA1 pathways, thereby being a tumor cell suppressor, protecting against cancer by regulating the cell cycle and increasing the occurrence of apoptosis as opposed to being directly involved in the repair of DNA damage.(32)
Barakat et al. investigated the expression of DDB2 in several cisplatin-resistant ovarian cancer cell lines, and the results obtained were lower DDB2 expression than cisplatin-sensitive cells (33). The study also further explained that low DDB2 expression is associated with chemoresistance poor patient prognosis (9). DDB2 can reduce excess CSC protein (CD44+/CD24−) in large ovarian cancer. Overexpression of DDB2 in human ovarian cancer cells decreased the ability of these tumor cells to replicate (9). Han et al., (2014) stated that low DDB2 protein expression was associated with poor prognosis ovarian cancer patients. DDB2 deficiency causes ovarian tumors to relapse due to the expansion of the CSCs population (34).
Yang et al., (2018) found that DDB2 protein expression was associated with the onset, progression, and prognosis of colorectal cancer. Increased DDB2 expression was significantly associated with a better colorectal cancer prognosis (35). DDB2 was found to be able to inhibit colon cancer metastasizing through the mechanism of decreasing gene expression, which is an activator of Epithelial-to-Mesenchymal Transition (EMT) in colon cancer, and NF-κB found in breast cancer (9). Decreased DDB2 also affects EMT activation, triggering squamous cell carcinomas in the head and neck region (10). A Study on non-small cell lung cancer (NSCLC) found that DDB2 can facilitate the process of breaking the cancer cell growth. Conversely, if DDB2 decreases, the G2 cycle will continue, tumor growth and therapy resistance will occur (36).
This study applies multivariate analysis. Multivariate analysis with logistic regression found that two variables with almost the same effect and can be used as logistic regression models are the CD44+/CD24− and Ca-125 variables. There have been no internationally published studies describing the CD44+/CD24− and Ca-125 regression models. Furthermore, it may be necessary to carry out further research to confirm this regression model.
In this study, some shortcomings made us unable to prove that RAD6 and DDB2 can predict ovarian cancer chemoresistance. Although the results showed a significant difference (p < 0,05), the accuracy was still weak. It cannot yet be used as a good predictor of ovarian cancer chemoresistance and need further research.