Novel players in the development of chemoresistance in ovarian cancer: ovarian cancer stem cells, non-coding RNA and nuclear receptors

Ovarian cancer (OC) ranks as the fifth leading factor for female mortality globally, with a substantial burden of new cases and mortality recorded annually. Survival rates vary significantly based on the stage of diagnosis, with advanced stages posing significant challenges to treatment. OC is primarily categorized as epithelial, constituting approximately 90% of cases, and correct staging is essential for tailored treatment. The debulking followed by chemotherapy is the prevailing treatment, involving platinum-based drugs in combination with taxanes. However, the efficacy of chemotherapy is hindered by the development of chemoresistance, both acquired during treatment (acquired chemoresistance) and intrinsic to the patient (intrinsic chemoresistance). The emergence of chemoresistance leads to increased mortality rates, with many advanced patients experiencing disease relapse shortly after initial treatment. This review delves into the multifactorial nature of chemoresistance in OC, addressing mechanisms involving transport systems, apoptosis, DNA repair, and ovarian cancer stem cells (OCSCs). While previous research has identified genes associated with these mechanisms, the regulatory roles of non-coding RNA (ncRNA) and nuclear receptors in modulating gene expression to confer chemoresistance have remained poorly understood and underexplored. This comprehensive review aims to shed light on the genes linked to different chemoresistance mechanisms in OC and their intricate regulation by ncRNA and nuclear receptors. Specifically, we examine how these molecular players influence the chemoresistance mechanism. By exploring the interplay between these factors and gene expression regulation, this review seeks to provide a comprehensive mechanism driving chemoresistance in OC.


INTRODUCTION
Ovarian cancer (OC), ranking fifth in global women's mortality, recorded 313,959 incidences and 207,252 deaths [1] .Survival rates at 5 years for stages I-IV are 92.4%,72.9%, 72.9%, and 31.5%,respectively [2] .Earlystage diagnosis is challenging, with only 20% identified at stage I, while 70% are discovered at higher stages.The International Federation of Gynaecology and Obstetrics (FIGO) classifies OC based on spreading, with 5-year survival rates of 93%, 75%, and 31% for localized, regional, and distant cases, respectively [3] .Epithelial OC (EOC), causing 90% of cases, is a major OC death contributor.The correct staging of OC determines the specific treatment because it provides information about how many cancerous cells are present in body and their location, and cytoreductive surgery followed by chemotherapy is employed in most cases as a treatment strategy.Chemotherapy includes platinum-based drugs such as cisplatin or carboplatin in combination with taxane, generally paclitaxel, and they often develop resistance during chemotherapy or after a few months of the last chemotherapy.The cisplatin reacts N7 of deoxy-guanosine and deoxy-adenine (with low affinity) to form intrastrand, and interstrand crosslink leading to DNA replication blocks, and transcription to induce cell death, while paclitaxel induces cell death via preventing tubulin depolymerization through microtubules stabilization by interacting with β-subunit of tubulin leading to cell cycle arrest during anaphase which required separation of sister chromatids [4,5] .Each drug treatment course is termed a cycle, and six cycles of chemotherapy are provided, with each cycle being 3 weeks in length.The neoadjuvant chemotherapy, in which chemotherapy is given both before and after surgery, provides better responses and high 5-year relative survival rates [6] .The primary challenge in OC is early-stage detection, hindered by the absence of biomarkers and the asymptomatic nature in the initial stages.Another critical issue is chemotherapy-related mortality, wherein patients develop acquired chemoresistance or exhibit intrinsic chemoresistance.Initially, 70% respond to platinum and taxane-based therapies, but resistance develops via several mechanisms such as alteration of drug efflux/influx, increased antioxidant to neutralize reactive oxygen species (ROS) generated due to platinum-based drugs, decreased apoptosis, and hyperactive DNA repair contributing to increased mortality and relapse within 2 years for many patients [7][8][9] .Understanding chemoresistance involves complex mechanisms with poorly understood regulation of gene expression involved in chemoresistance mechanisms.Factors like ncRNA and nuclear receptors influencing chemoresistance lack comprehensive exploration, making them vital areas for further study.This review delves into chemoresistance related to transport systems, apoptosis, DNA repair, and OCSCs.It scrutinizes the modulation of key cellular processes that foster chemoresistance, encompassing efficient DNA repair, efflux transporter upregulation, OCSCs proliferation, apoptosis inhibition, and influx transporter downregulation.The discussion extends to the role of ncRNA and nuclear receptors for regulating genes associated with various chemoresistance mechanisms in OC.

THE ROLE OF APOPTOSIS IN CHEMORESISTANCE IN OC
This segment of the review explores the intricate interconnection between drug resistance and apoptosis within the context of OC.In our thorough examination, we have delved into the pivotal modulators affected under both sensitive and resistant conditions.Under sensitive conditions, our review highlights key modulators, such as apoptotic protein mechanisms, that play essential roles in maintaining cellular homeostasis and preventing tumorigenesis.Conversely, in resistance conditions, these modulators undergo alterations, compromising their responsiveness to therapeutic interventions.Our comprehensive review has methodically summarized the modifications in these key modulators, elucidating their significance in mediating drug resistance in OC.
For convenience and improved understanding, we have compiled an extensive  1A] that visually represents the intricate pathways associated with

THE ROLE OF DNA REPAIR IN CHEMORESISTANCE IN OC
The role of DNA repair mechanisms in drug resistance within OC is a critical aspect of understanding the challenges and complexities associated with treatment.DNA repair processes have a significant role in the response of cancerous cells to various therapeutic agents [34] .Understanding the intricate relationship between DNA repair mechanisms and drug resistance in OC is crucial for formulating targeted therapies that can overcome or exploit these mechanisms, ultimately improving treatment outcomes for patients with this challenging disease.To address this complexity, we have chosen a strategic approach to streamline and simplify the information for our audience.We condensed the details concerning the role of drug resistance on DNA repair pathways into an extensive table [Table 2].This tabular format serves as a comprehensive reference, presenting the main insights and discoveries in an organized manner.It offers a concise yet informative overview of the subtle interactions within the DNA repair pathways influenced by drug resistance in OC.Additionally, alongside the table, we have created an illustrative figure [Figure 1B], that visually illustrates the impact of drug resistance on DNA repair pathways in OC.This visual representation aims to enhance comprehension of the intricate relationships between drug resistance mechanisms and the complex processes of DNA repair.

THE ROLE OF TRANSPORT SYSTEM IN CHEMORESISTANCE IN OC
The optimal effectiveness of a drug within cells depends on its optimal cytoplasmic concentration.Membrane transporters, particularly ABC transporter, P-type ATPase transporter, and solute carrier (SLC) transporter, are key factors influencing the bioavailability of drugs in ovarian cancer [Figure 2A and Table 3].

ABC superfamily transporters
ABC transporters, with seven families, use ATP to facilitate substrate efflux/influx.Comprising 49 members and 21 pseudo-members, they mainly efflux substrates.Structurally, ABC transporters have single polypeptides with two nucleotide binding domains (NBDs) and two transmembrane domains (TMDs) [65] .The SNAIL, ZEBs, and SLUG promote MDR, while VDR, ER, PXR, KLF, Gli, and Sp are also known to modulate ABC transporters [66] .The altered ABCB1 structure or drug binding, inhibition of its expression or knockout of the ABCB1 gene are the most potential strategies to overcome ABCB1-mediated drug resistance [67] .Similarly, Basic helix-loop-helix family member e40 (BHLHE40) is inversely related to ABCB1, suggesting that the upstream target of ABCB1 can be used to overcome ABCB1-mediated chemoresistance [68] .Recent research has explored the broad substrate specifity, and conversion of efflux to influx pump via engineering of ABC transporter, and the importance of membrane transporters is also highlighted in the development of precision medicine [69,70] .

P-type ATPase superfamily transporter
P-type ATPase superfamily transporter, with five subfamilies (P1-P5), transports ions across membranes using ATP hydrolysis energy.P-type ATPases have cytosolic domains (A, P, N) and transmembrane domains (M1-M6, with P1 having M7-M10) [88] .ATP7B knockdown enhances cisplatin sensitivity.ATP7A silencing lacks impact on resistance, but ATP7A polymorphism is linked to cisplatin resistance in ovarian cancer [89] .The transcription factor EB (TREB) binds the promoter's first intron region at coordinated lysosomal expression and regulation (CLEAR) sites of ATP7B, modulating its expression when exposed to platinum drugs in ovarian cancer [90] .

THE ROLE OF OCSC IN CHEMORESISTANCE IN OC
New research indicates that contrary to previous beliefs about a constant follicle in the ovary at birth, there are ovarian stem cells (OSCs) present.These include a dormant group of very small embryonic-like stem cells (VSELs) and a larger subset of dividing OSCs.OSCs are undifferentiated cells inherently capable of self-renewal, proliferation, multipotency, and differentiation.VSELs, which express embryonic markers such as octamer-binding transcription factor 4 (OCT-4), are located in ovary surface epithelium, and can divide asymmetrically to self-renew to form OSC [96] .OSCs are maintained by a niche microenvironment composed of ECM, immune cells, stromal cells, mesenchymal cells, and vascular network [97] .A recent study identified the tubal-peritoneal junction & hilum region as stem cell niche within the ovary [98] .Ovarian tumors exhibit heterogeneity with distinct cell types expressing stem cell markers cluster of differentiation 133 (CD133), leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), aldehyde dehydrogenase 1 (ALDH1), and cytokeratin 6B (CK6B).These cells, known as OCSCs, arise from genetic instability in OSCs, contributing to chemoresistance, cancer initiation, and treatment failure [98] .OCSC, characterized by various markers, may display diverse phenotypes, offering selective advantages [99] .OCSCs inherently resist chemotherapy.Targeting OCSC and pathways offers strategies against OC chemoresistance [99] [Figure 2B and Table 4].

The interplay between non-coding RNA and OCSCs
The non-coding RNAs are RNA molecules that are transcribed but not translated, and categorized based on length and shape, such as circular RNA (circRNA) with a circular structure, long non-coding RNA (lncRNA) exceeding 200 nucleotides, and microRNA (miRNA) with an average length of 22 nucleotides [129] .The lncRNA, miRNA, and circRNA are found to regulate the OCSCs, leading to tumor relapse and chemotherapy resistance [130] .The lncRNA SNORD89 is highly upregulated in OCSCs and promotes its OCSC: Ovarian cancer stem cell; SOX2: SRY-box transcription factor 2; MAPK: mitogen-activated protein kinase; OCT4: octamer-binding transcription factor 4; AKT: Ak strain transforming; ALDH1: aldehyde dehydrogenase 1; EMT: epithelial to mesenchymal transition; ABCG2: ATPbinding cassette subfamily G member 2; HLF: hepatic leukemia factor.

THE ROLE OF NUCLEAR RECEPTORS IN CHEMORESISTANCE IN OC
Nuclear receptors, activated by lipid-soluble signals like steroid hormones, regulate gene expression via hormone response elements (HRE), impacting proliferation, apoptosis, and metabolism [146] .There are 48 nuclear receptors in humans, and when their normal functioning is disrupted, it is frequently associated with various diseases.Nuclear receptors are classified into seven families, i.e., NR0-6, based on sequence homology [147] .Nuclear receptors are structured into four segments: the unstructured N-terminal domain (NTD) housing activation function 1 (AF-1), DNA binding domain (DBD), Hinge region, and ligand binding domain (LBD).The LBD binds to ligands and engages with co-regulator proteins via activation function 2 (AF-2) [Figure 3A] [147] .The nuclear receptor co-regulators are divided into two categories, i.e., coactivators and corepressors, which directly interact with AF-1 and AF-2 regions of nuclear receptors.Coactivators bind via LXXLL motifs, and corepressors via CoRNR box motifs [147] .Nuclear receptors are also classified into classes I, II, III, and IV based on ligand binding and DNA binding [Figure 3B].Class I nuclear receptors are sequestered in cytoplasm with chaperon proteins, but upon ligand (cholesterol-derived steroidal hormones) activation, they enter inside the nucleus to bind DNA response elements (RE) composed of two inverted repeats as homodimers [147] .Class II nuclear receptors are present in nucleus with corepressor, but upon ligand activation, corepressor is swapped with coactivators and binds to DNA RE consisting of direct repeat sequence as heterodimers [147] .Class III nuclear receptors are similar in working mechanism to class II, Table 5 except they bind to DNA RE comprising direct repeat sequence as homodimers [147] .Class IV are also similar to the working mechanism of class II, except they bind to extended half-sites within DNA RE as monomers [147] .Chemotherapy is among the top three commonly used treatments for OC.Unfortunately, its effectiveness is restricted by OC cells that have become resistant to the drugs [148] .The importance of various nuclear receptor families in controlling drug metabolism and distribution is gaining recognition, and therapies aimed at these receptors offer new possibilities to mitigate or potentially prevent drug resistance [149] .In this context, we will explore the latest findings concerning the roles and control of different nuclear receptors in the emergence of drug resistance in OC.We will also shed light on how nuclear receptors are linked to drug resistance during chemotherapy and nuclear receptors associated with chemoresistance in OC [Table 5 and Figure 3C].
expression reverses EMT and stem cell characteristics in OC [189] .AR from family 3 with class I features facilitates growth in CSPC-rich OVTC PA1 cells, governing the self-renewal of stem cells.AR is more abundant in CD133+ cells, and its enrichment downregulates p53 and p16 [190] .AR is upregulated in OCSC and androgen 5α-dihydrotestosterone (DHT) promotes OC stemness by enhancing NANOG expression [191] .

THE ROLE OF NCRNAS IN CHEMORESISTANCE IN OC
ncRNAs serve as gene expression regulators across multiple biological processes, such as cell division, programmed cell death, cellular transport, EMT, OCSCs, and DNA mending [192] .Recent studies highlight ncRNAs as crucial regulators of chemoresistance in ovarian, breast, and lung cancers [193][194][195] .Understanding the identification and mechanisms of ncRNAs in gene expression regulation can aid in biomarker development for early detection.Additionally, targeting ncRNAs in chemotherapy can enhance cell death, ultimately leading to a higher 5-year survival rate.This review summarizes the roles of ncRNAs, particularly lncRNA, circRNA, and miRNA, in chemoresistance in OC [Table 6 and Figure 4].

Figure 2 .
Figure 2. (A) Schematic diagram of drug transport in chemoresistance in OC.ABC transporters such as ABCB1, ABCC1, ABCC2, ABCC4, ABCC10, and ABCG2, P-type ATPase ATP7A, and SLC transporter CTR2 responsible for drug efflux are upregulated, while SLC transporter CTR1 that functions for drug influx is downregulated, and P-type ATPase transporter ATP7B does not directly facilitate drug efflux, but its genetic polymorphism can influence drug efflux; (B) OCSCs confer chemoresistance in OC.OSCs are transformed into OCSCs due to genetic alteration, which then proliferate into progenitor cells and subsequently differentiate into cells that contribute to the relapse of ovarian tumors after some time instead of undergoing cell death upon chemotherapy.Created with BioRender.com.OC: Ovarian cancer; ABCB1: ATP-binding cassette subfamily B member 1; ABCC: ATP-binding cassette subfamily C; ABCG2: ATP-binding cassette subfamily G member 2; SLC: solute carrier; CTR1/2: copper transporter 1/2; OCSCs: ovarian cancer stem cells.

Figure 3 .
Figure 3. (A) Nuclear receptors domain architecture; (B) Diagram illustrating the categorization of nuclear receptors associated with chemoresistance in OC, with a focus on their interaction with ligands; (C) Demonstrating various mechanisms associated with the involvement of nuclear receptors in OC drug # resistance, primarily encompassing the inhibition of apoptosis, drug efflux, and the presence of OCSCs.Created with BioRender.com.OC: Ovarian cancer; OCSCs: ovarian cancer stem cells.

table [ Table 1 ]
detailing the observed alterations in key modulators under both sensitive and resistant conditions.Additionally, we have created a comprehensive figure [Figure