miRNAs as prognostic and therapeutic tools in epithelial ovarian cancer.

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miRNAs as prognostic and therapeutic tools in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and is the fifth leading cause of cancer deaths in women. Developing adjuvant therapy to circumvent drug resistance represents an important aspect of current initiatives to improve survival in women with advanced EOC. A regulatory molecule that can act on multiple genes associated with a chemoresistant phenotype will be the ideal target for the development of therapeutics to overcome resistance and miRNAs constitute promising tools in this regard. In this review, we discuss the emerging role of miRNAs in regulating EOC phenotype with a focus on prognostic and therapeutic importance of miRNAs and the possibility of miRNA modulation as a tool to improve efficacy of chemotherapy in EOC.

Anil Belur Nagaraj1, Peronne Joseph1 & Analisa DiFeo*,1 Case Comprehensive Cancer Center, Case Western Reserve University, 2103 Cornell Road, Cleveland, OH 44106, USA *Author for correspondence: Tel.: +1 216 368 3117 Analisa.Difeo@ case.edu 1

Keywords: biomarkers • chemotherapy resistance • epithelial ovarian cancer • miRNA expression profiling • miRNAs • MRX34 • OncomiRs • pharmaco-miR • tumor heterogeneity • tumor suppressor miRs

Barriers in eradicating epithelial ovarian cancer Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and is the fifth leading cause of cancer deaths in women [1] . This year, it is estimated that approximately 22,000 women will be newly diagnosed and 14,200 will succumb to this lethal disease in the USA [1] . This poor overall survival is due to late-stage diagnosis and almost inevitable resistance to chemotherapy. Most cases of EOC are high-grade serous cancers (HGSOC), which are initially highly sensitive to standard treatment including cytoreduction surgery and platinum-based chemotherapy with response rate close to 85% [2] . In spite of such high initial response to platinum, the outcomes are poor with 5-year survival being less than 30% and the majority of women who initially respond to platinum therapy relapse due to development of chemoresistance [2] . Hence, there is immediate need of early diagnostic biomarkers and novel therapeutic tools that can enhance the efficiency of platinum-based therapy by overcoming chemoresistance in EOC.

10.2217/BMM.14.108 © 2015 Future Medicine Ltd

The world of miRNAs miRNAs are approximately 20–22 nucleotide length noncoding RNAs that regulate gene expression mainly by posttranslational mechanisms involving sequence-specific interaction with their targets [3,4] . miRNAs are transcribed as primary transcripts in the nucleus and undergo a series of tightly regulated cropping and cleavage events, which eventually result in functional mature miRNAs in the cytoplasm [3,4] . Being part of such a complex transcription and processing machinery [5] coupled with their nature of sequence-specific recognition of multiple targets, miRNAs are characterized by diverse expression profile and have emerged as important regulators of cell proliferation and differentiation processes [3] . miRNA deregulation is implicated in pathogenesis of a variety of tumors including ovarian cancer and miRNAs can be classified as tumor suppressor miRNAs or oncomiRs based on their functions in regulating tumor phenotype [6–8] . miRNAs have been identified as critical regulators of various aspects

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Review Nagaraj, Joseph & DiFeo of tumorigenesis including transformation events, metastatic events, cancer stem cell functions, chemoresistant mechanisms and hence miRNA modulation is emerging as an exciting prospect in cancer t herapeutics [9–11] . miRNAs & EOC PubMed search with ‘miRNAs and cancer’ lists around 12843 publications, whereas search with the term ‘miRNAs and epithelial ovarian cancer’ lists around 86 publications; thus, publications in EOC regarding miRNAs constitute a very tiny proportion of ~0.6% of overall publications describing miRNA roles in cancer indicating that there is quite some distance to be covered in this scenario. Around ~70% of these miRNA publications in EOC have been published in the last 2 years, indicating that miRNAs are currently an important focus in understanding mechanisms regulating EOC phenotype. Importance of novel & reliable prognostic markers in EOC

Failure to efficiently diagnose the disease in early stage and emergence of chemoresistance to platinum-based compounds are the major barriers to success in treating EOC. miRNA deregulation has been identified as an important feature in mechanisms underlying EOC [12,13] . A plethora of mechanisms including miRNA deregulation have been implicated in emergence of acquired resistance during the course of standard platinum-based treatment regimens in EOC [14] . The chemoresistance problem in EOC has many dimensions that need to be tackled. To begin with, there is lack of biomarkers that can identify the disease process in early stage in which treatment outcomes are most successful. Second, there is lack of reliable patient stratification strategies for platinum-based chemotherapy in EOC, which limits the application of targeted therapy strategies. Lastly, there is lack of reliable biomarkers that can be employed to efficiently monitor response during chemotherapy enabling early detection of chemoresistance. In spite of being greatly heterogeneous in terms of origin and composition, all EOC tumors are initially treated the same, i.e. with platinum compounds [15] . This is a major problem since platinum treatment is reported to increase the expression of stem-cell markers in tumors during the course of chemotherapy resulting in treatment failure and tumor relapse due to accumulation of stem-like cancer cells, which are drug resistant [16,17] . Hence, identification of potential responders (mostly with good survival outcome) and potential nonresponders (mostly with poor survival outcome) at the beginning of chemotherapy is critical

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to improve patient outcome and prevent tumor relapse in EOC [18] . Unfortunately, this is a great challenge in current EOC treatment scenario. Even though there are few ongoing-targeted therapeutic approaches in ovarian cancers employing antiangiogenic drugs and PARP inhibitors, the main challenges in these studies are lack of reliable biomarkers that enable efficient patient stratification and also reliably portray response to chemotherapy [19] . Over the last few decades, CA-125 has remained the most extensively employed prognostic factor in EOCs to predict survival, monitor disease progression and response to chemotherapy in ovarian cancer, but varying observations have been documented in different studies [20] . Analysis of The Cancer Genome Atlas (TCGA) gene expression dataset of high-grade serous EOC samples did not support a major effect of CA-125 in predicting overall survival and more importantly no correlation was observed between CA-125 expression levels and resistance to chemotherapy [21] highlighting the need for novel predictors of chemoresistance in EOC. miRNAs as prognostic markers in EOC miRNAs are attractive candidates as biomarkers in cancers including EOC because of their unique properties namely: miRNA deregulation is an established hallmark of cancers [6] ; miRNAs are characterized by tissue-specific expression and miRNA signatures can classify cancers [22] ; miRNAs are known to be very stable in formalin-fixed tissues, which are the common source of samples for biomarker analysis [23] ; and miRNAs are present in body fluids [24] , which makes their analysis possible by less invasive methods and hence more practical for biomarker analysis in clinical settings. miRNA expression profiling is being employed as a powerful tool to study miRNA deregulation in tumors and miRNAs are emerging as reliable prognostic tools in predicting survival outcome in EOC [25] . Various technologies are available to investigate the use of miRNAs as diagnostic entities in clinical settings (as summarized in Figure 1) and each method has its own advantages and disadvantages and the choice of these techniques is mostly dependent on the clinical settings encountered [26,27] . miRNAs & early diagnosis in EOC

miRNAs are known to play an important role in regulating ovarian physiology [28] and hence it is not surprising that miRNA deregulation is a prominent feature in EOCs [7] . Interestingly, miRNAs like let-7 tumor suppressor family, miR-125b and miR-29b that are known to be highly expressed in normal ovarian tissue [28] are often deregulated in EOCs, thus linking

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miRNAs as prognostic & therapeutic tools in epithelial ovarian cancer

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Samples Tissue microRNAs

Circulating microRNAs

[Fresh/fixed]

[Serum] RNA isolation ± enrichment

Real-time PCR

microRNA microarray

RNA sequencing

Bead-based flow cytometry

Fast

Slow

Slow

Fast

Input RNA

Moderate

Low-moderate

Moderate

Low-moderate

Sensitivity

High

Low

Moderate

High

Moderate

Moderate

High

Moderate

Low

High

High

Low

[Relative] Speed

Cost Infrastructure

Figure 1. Flowchart illustrating various techniques that are currently being employed in microRNA research in both laboratory- and clinic-based settings, and also summarizes the relative advantages associated with each methodology.

EOC pathogenesis to potential miRNA deregulation events during normal ovarian functioning. Functional delineation of miRNAs involved in early transformation events occurring in normal ovarian tissue is necessary to realize the possibility of employing miRNAs as tools for early diagnosis of the disease process leading to EOC. To date only one miRNA has been implicated in EOC in vitro transformation, namely miR-182 [29] . One of the major challenges in this context is the availability of reliable genetically engineered in vivo models that can faithfully recapitulate early events of EOC pathogenesis. EOC pathogenesis is highly heterogeneous both in terms of origin and composition of the tumor, which makes the generation of such oncogenic in vivo models a tough task, and this is further complicated by the fact that there is lack of understanding regarding specific genetic drivers of EOC phenotype. One potentially efficient model in this context was described recently wherein a de novo mouse model of HGSOC was generated through the deletion of p53, Brca1 or Brca2 and PTEN within the fallopian tube using the Pax8 promoter [30] . Combination of such genetically engineered EOC models with miRNA knockin or knockout mouse models should shed more light into understanding the role of miRNAs in regulating early transformation events in EOC pathogenesis and thus could pave the way for the development of reliable miRNA-based early diagnostic strategies in EOC.


The most common genetic alterations in EOC pathogenesis are reported to revolve around the two major tumor suppressor genes BRCA1 and TP53 [31] and interestingly both BRCA1 and TP53 are known to extensively cross talk with miRNA machinery, which is important for their functioning; thus, suggesting miRNA deregulation could be involved in the pathogenesis of EOC [32,33] . miR-29a and miR-29b expression was reported to be upregulated in high-grade serous EOC with BRCA1/2 abnormalities [33] . Loss of miR-31 is reported to be associated with defects in p53 pathway in EOC and overexpression of miR31 in p53-deficient background showed therapeutic effects [34] . Thus, miRNA deregulation events could potentially underlie the early transformation events in EOC pathogenesis and further studies are required in this context to develop a reliable miRNA diagnostic platform for early diagnosis of EOC. miRNAs predicting survival in EOC

One of the earliest reports describing miRNA deregulation in EOC was from the laboratory of Carlo Croce [7] in which miRNA signatures in EOC were analyzed and 39 miRNAs were found to be significantly deregulated in tumor tissues as compared with normal ovary [7] . The tumor suppressor miR-200 family was one of the earliest identified miRNA prognostic markers in EOC even though there are differences in observations from different groups about this family of miRNA,

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Review Nagaraj, Joseph & DiFeo which is probably due to the differences in methodology employed and cohorts investigated [7,35] . Since these initial findings in EOC, several other groups have reported differential miRNA expression in EOC, including miR-21 upregulation and miR-125b downregulation correlation with poor patient outcome in HGSOC [36] ; miR-34c was identified as sole independent predictor of recurrence-free survival with higher miR-34c levels favoring good prognosis in EOC [33] and Kim et al. reported that significant upregulation of miR-519a is associated with poor survival outcome in HGSOC [37] . In perhaps the most extensive analysis of miRNA signatures in EOC, TCGA provided a comprehensive analysis of HGSOC by integrated analyses of various parameters including miRNA expression profiles and identified four molecular subtypes of ovarian cancer [31] . miRNA expression clustering in this analysis revealed three subtypes with subtype 1 being associated with poor survival. Further refinement of these data identified a miRNA regulatory network that defined a mesenchymal subtype associated with poor survival [38] . Interestingly, only eight downregulated miRNAs identified in this analysis (miR-25, miR506, miR-29c, miR-182, miR-128, miR-101, miR-141 and miR-200a) were predicted to regulate 89% of the altered miRNA-associated genes, thus suggesting the importance of miRNA networks as predictors of survival in EOC. miRNAs predicting chemoresistance in EOC

Predicting potential chemoresistant tumors both at the beginning and during the course of chemotherapy is critical to the planning and success of targeted therapy strategies and one of the critical constraints in this context is the requirement of reliable biomarkers that reliably reflect the response to chemotherapy [39] . miRNA deregulation is a characteristic feature of chemotherapy resistance in ovarian tumors [12] . In one of the earliest reports in this regard, Sorrentini et al. identified expression of five miRNAs (let-7e, miR-30c, miR125b, miR-130a and miR-335) to be altered in drug resistant ovarian tumor cell lines [40] . let-7i was the first miRNA reported to be significantly decreased in chemoresistant primary EOC samples and it was further observed in this study that lower let-7i levels correlated with shorter survival in EOC [41] . Eitan et al. reported miRNA signature predicting the response of EOC patients to platinum-based chemotherapy and this also correlated with survival outcome [39] . In addition, a cluster of eight miRNAs located at chrXq27.3 locus that were consistently downregulated in EOC patients who experienced early relapse and lower expression of this cluster correlated with shorter survival time

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in these patients [42] . Furthermore, Vecchione et al. identified a signature of 23 miRNAs associated with chemoresistance in EOC and from this set further validated that three miRNAs (miR-484, miR-642 and miR-217) that were able to predict chemoresistance in these tumors. Our lab recently identified miR-181a as a predictor of tumor recurrence and chemoresistance in HGSOC [43] . miR-181a levels were associated with poor outcome and shorter time to recurrence in advanced-stage EOC; higher expression predicting poor survival and early recurrence thus indicating that miR-181a could be an important prognostic tool in EOC. Circulating miRNAs as prognostic markers in EOC

One of the unique features of miRNAs is that they are present in body fluids and can be detected in serum samples, which is the one of the least invasive sample available for biomarker analysis. Hence, circulating miRNAs are increasingly gaining importance as biomarkers in various cancers even though there are some preprofiling factors that have to be taken into consideration that can significantly affect such diagnostic approaches [44,45] . miRNAs that are secreted from cells into circulation are known to be very stable because they exist in different forms like in complex with AGO2 protein and within vesicles called exosomes that confer resistance to degradation by RNAses [44] . One of the very first studies identifying circulating miRNAs as potential diagnostic entities in cancer was reported in 2008 by the laboratory of Muneesh Tewari [24] . In the same year, Resnick et al. identified miR-92 to be consistently overexpressed in serum of EOC patients as compared with healthy controls [46] . Following these studies, it was reported that miR-200 family miRNAs were greatly elevated in the serum of EOC patients as compared with age-matched healthy controls [47] . This observation was consistent with previous reports identifying miR-200 family miRNAs to be upregulated in EOC tissue samples. miRNAs in serum of EOC patients have been shown to correlate with survival outcome suggesting that circulating miRNAs could be of prognostic importance in EOC. High serum expression of miR-221 was identified as an independent unfavorable prognostic factor in EOC showing that serum miRNA expression can correlate with survival outcome in EOC [48] . miR21 was another miRNA to be identified in serum of EOC patients whose expression correlated with survival outcome; higher expression being associated with shortened overall survival [49] . A higher miR-92 level in serum EOC patients was observed to correlate with advanced disease stage [50] . In an interesting study that



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Table 1. Important studies describing the effect of miRNA modulation on platinum resistance in epithelial ovarian cancer. miRNA

Modulation

Platinum response

Targets

Study type

let-7e

Overexpression knockdown

Sensitivity upon overexpression

EZH2 CCND1

In vitro and in vivo

[95]

miR-29



COL1A1


[96]

miR-152

Overexpression

Sensitivity

DNMT1


[97]

miR-185

Overexpression

Sensitivity

DNMT1


[97]

miR-199b-5p

Overexpression

Sensitivity

JAG1


[98]

miR-449a

Overexpression

Sensitivity

NOTCH1


[99]

miR-484

Overexpression

Sensitivity

VEGFB VEGFR2


[100]

miR-9



BRCA1

In vitro

[101]

miR-21

Knockdown

Sensitivity

PDCD4

In vitro

[102]

miR-21


Sensitivity upon knockdown

PDCD4

In vitro

[103]

miR-30c-2–3p Overexpression knockdown


BCL9

In vitro

[104]

miR-30d

Overexpression

Sensitivity

ABCD2

In vitro

[105]

miR-93

Overexpression and knockdown

Resistance upon overexpression

PTEN

In vitro

[106]

miR-107

Overexpression

Sensitivity

RAD51 RAD51 D

In vitro

[107]

miR-103

Overexpression

Sensitivity

RAD51 RAD51 D

In vitro

[107]

miR-106a



Mcl-1

In vitro

[108]

miR-106a


Sensitivity upon knockdown

PDCD4

In vitro

[108]

miR-125b



Bak1

In vitro

[109]

miR-130a



PTEN

In vitro

[110]

miR-130a

Overexpression

Sensitivity

XIAP

In vitro

[111]

miR-130b



CSF-1

In vitro

[112]

miR-141

Overexpression

Resistance

KEAP1

In vitro

[113]

miR-193b-3p

Overexpression

Resistance

CRIM1

In vitro

[114]

miR-199a

Overexpression

Sensitivity

CD44

In vitro

[115]

miR-199a



IKKb

In vitro

[116]

miR-199a



mTOR

In vitro

[117]

miR-214



PTEN

In vitro

[118]

miR-302b

Overexpression

Sensitivity

HDAC4

In vitro

[119]

miR-376c

Overexpression

Resistance

ALK7

In vitro

[120]

miR-429

Overexpression

Sensitivity

Not tested

In vitro

[121]

miR-489



Akt3

In vitro

[122]

miR-519d



XIAP

In vitro

[123]

could potentially support the application of circulating miRNAs in early diagnosis of EOC events, Zheng et al. reported that combination of miR-205 and let-7f expression in plasma samples of EOC patients provided


Ref.

high diagnostic accuracy for stage I disease in EOC [51] . More interestingly, the accuracy of this miRNAbased prediction increased when combined with CA-125 levels suggesting that circulating miRNAs can


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Review Nagaraj, Joseph & DiFeo complement CA-125 as biomarkers in EOC. This is very promising since the less invasive nature of circulating miRNA analysis could make them an important integral component of early diagnostic screening platforms in EOC and also could enhance the accuracy of CA-125-based prediction of the disease. Exosomes are an important source of circulating miRNAs and could constitute important tools in miRNA-based diagnostic modalities in cancers including EOC because exosomes are enriched source of miRNAs in circulation. In one of the first reports characterizing exosomal miRNAs in EOC, Taylor et al. conducted miRNA expression profiling of exosomes isolated from serum samples of highgrade serous ovarian cancer patients and compared them with expression profile of tumor cell-derived miRNAs from the same patients [52] . Some miRNAs were unique to the exosomes and tumor cells, but the global miRNA expression profile of exosomes was remarkably similar to the cell-derived miRNAs suggesting that exosomes faithfully recapitulate the disease process. Improvised protocols for isolation and enrichment of exosomes from serum samples are available now and hence this field is rapidly emerging as a diagnostic tool in cancers including EOC [53] . However, a very recent report by Chevillet et al. has shown that quantitative analysis of miRNAs in exosomes needs better models of evaluation and standard protocols and analysis methods currently in practice may not efficiently reflect miRNA biology associated with exosomes. Therefore, further studies are necessary to delineate efficient methods of exploring exosome-associated miRNAs [54] . miRNAs as therapeutic tools in EOC Since miRNA deregulation is a characteristic feature of cancers, this presents with a miRNA-based cancer therapeutics possibility to correct this deregulation either by replacing the downregulated tumor suppressor miRNAs using miRNA mimics (miRNA replacement therapy) or by inhibiting the upregulated oncomiRs using antisense miRs (miRNA inhibition therapy) [55,56] . Such miRNA-based therapeutics approach has the potential to have drastic effects on the tumor phenotype because of the fact that a single miRNA can regulate hundreds of targets and multiple targets can be regulated by a single miRNA, but at the same time this therapeutic approach would be pathway specific too. Since cells already express these miRNAs, exogenous delivery of miRNAs potentially would not result in adverse effects. Also, since miRNA deregulation drives tumor phenotype, correcting this deregulation would specifically affect cancer cells while potentially sparing normal cells. Hence,

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miRNA modulation is emerging as an exciting cancer therapeutics option. The first and the only ‘anticancer miRNA drug’ MRX34 that is a miR-34 miRNA mimic has entered Phase I clinical trials in patients with advanced hepatocellular carcinoma heralding the entry of miRNAs into cancer therapeutics in clinical settings. Interestingly, miR-34 family of miRNAs was found to be frequently downregulated in EOC and this downregulation was more evident in EOC patients with mutations in TP53 and was associated with metastatic clinical stage [57] . In the same study, it was further shown that miR-34 replacement therapy in the metastatic SKOV3 ovarian tumor cells significantly decreased invasion, migration and proliferation. Even though there is currently no miRNA drug in clinical settings in EOC, MRX34 could provide with an exciting possibility of clinical translation in EOC patients in future. miRNA modulation as a tool to enhance efficacy of chemotherapy in EOC

Numerous studies in preclinical models over the last few years have established that miRNA modulation is an exciting approach to target EOC mechanisms and improve chemosensitivity. Some of the important studies in this regard are summarized in Table 1. One of the major exciting observations in miRNA therapeutics is the ability of miRNAs to determine the efficacy of drugs [58] . Figure 2 illustrates some of the possible mechanisms by which miRNAs can improve the efficacy of chemotherapy and overcome c hemotherapy resistance. Combination of kinase inhibitors & miRNA replacement therapy

A classic example of this case would be the tumor suppressive miRNA, miR-34a, which is known to directly target EGFR [59] . The combination of miR-34a and the EGFR inhibitor Erlotinib exerted synergistic effect in hepatocellular carcinoma cells, which were resistant to Erlotinib alone [60] . miR-34a decreased the IC50 of Erlotinib by 46-fold; the reason behind this dramatic effect being miR-34a negatively targets multiple oncogenes known to confer resistance to Erlotinib. EGFR signaling pathway is known to be an important regulator of EOC phenotype; monoclonal antibodies targeting EGFR and small-molecule inhibitors of EGFR are in clinical trials in ovarian cancer but with limited success outcome [61] . This leads to an interesting speculation that miR-34 replacement therapy could greatly improve the efficacy of EGFR inhibitors in EOC, especially since the initial clinical trials of these drugs in EOC were met with limited success. EOC patients with low miR-34a expression along with altered EGFR



pathway components could be the subset that could greatly benefit from such combination studies. Apart from the exciting possibility of combining with drugs, miRNAs are also reported to enhance siRNA-based therapeutic approach. Tumor suppressor miRNAs can be combined with siRNAs targeting oncogenes and such combination therapies could have additional beneficial effects as compared with miRNAs or siRNAs employed alone. One such combination was described in EOC by Nishimura et al., wherein the tumor suppressor miRNA miR-520d-3p exhibited synergistic effect with siRNA against the oncogene EphA2 [62] . This advantage conferred by miR-520d-3p addition was found to be due to the transcriptional repression both EphA2 and EphB2 by miR-520d-3p. This further highlights the possibility that the unique nature of miRNAs, i.e ability to target multiple genes could have great application potential in designing miRNA-based combination therapeutic approaches either with drugs or siRNAs. Combination of platinum-based compounds with tumor suppressor miRNA mimics or antisense oligonucleotides against oncomiRs

A variety of mechanisms have been implicated in emergence of resistance to platinum-based compounds [63] . Some of the important constituents in this aspect are decreased drug accumulation due to upregulated ABC transporter family efflux pumps; upregulated DNA repair enzymes and upregulated antiapoptotic proteins that decrease cell death and potentially confer survival advantage to tumor cells in response to platinum-based compounds [14] . Supplementing platinum-based therapy with miRNA mimics targeting these drug resistance regulators or with antisense oligonucleotides that inhibit miRNAs promoting drug resistance could greatly improve the efficiency of cell death induced by platinum compounds. A couple of possibilities can be discussed in this context. The tumor suppressor miR-34 family that is frequently downregulated in EOC is known to promote apoptosis in cancer cells by directly targeting a variety of antiapoptotic genes [64] and hence it can be speculated that MRX34 addition could potentially improve the efficacy of platinum-based therapy in EOC. Similarly, miR-181a that is upregulated in chemoresistant EOC tumors [43] is reported to exert antiapoptotic effects by targeting the proapoptotic protein Bim [65] and Bim downregulation is known to contribute to cisplatin resistance in ovarian cancer [66] . Hence, antisense oligonucleotides inhibiting miR-181a could potentially improve the efficacy of platinum-based drugs by enhancing apoptotic cell death in response to cisplatin.


Review

Combining platinum therapy with miRNAs targeting tumor-initiating cells in EOC

Emerging evidence supports the concept that platinum-based therapies are able to eliminate the bulk of differentiated cancer cells but are unable to eliminate tumor-initiating cells (TICs; also termed cancer stem cells) that are resistant to cytotoxic therapies [16,17,67] . These rare populations of TICs are characterized by their ability to self-renew indefinitely in an undifferentiated state and also result in differentiated progeny of cells with high proliferative and invasive capacity, thus driving the expansion of drug resistant tumors. Various stem cell markers have been reported to enrich for ovarian TICs and ALDH is the most convincing marker reported till date in this regard [68] . CD133 in combination with ALDH is reported to further increase the enrichment of TIC population in EOC [69] . The proven inability of platinum compounds to eradicate TICs in EOC provides an opportunity for miRNAs targeting TICs in EOC to be used in combination with cisplatin to eradicate TICs and overcome chemoresistance. Only very few miRNAs have been implicated to date in regulation of TICs in EOC [70] . One of the first miRNAs to be identified in this context was miR-214. miR-214 knockdown was shown to increase sensitivity to cisplatin and doxorubicin in ALDH-positive cells isolated from ovarian tumor cell lines [71] . Park et al. analyzed the miRNA expression profile of ALDH-positive chemoresistant SKOV3 ovarian tumor cells and found that miR-23b, miR-27a, miR-27b, miR-346, miR424 and miR-503 were overexpressed in ALDH-positive cells and these miRNAs were also upregulated in chemoresistant EOC tumors as compared with the chemosensitive group [72] . These reports suggest that modulation of miRNAs could be an exciting prospect in TIC-based therapeutic strategies in EOC in coming days. Predicting novel miRNAs that could improve drug efficacy in EOC

The observation that miRNAs can determine the efficacy of drugs has given rise to the field termed as ‘miRNA pharmacogenomics’ and these miRNAs are termed as ‘pharmaco-miRs’ [58] . As discussed earlier in Figure 2, the basis of this effect is that targeting miRNAs that regulate genes/pathways that are known to be involved in drug resistance/inactivation mechanisms could enhance drug efficacy by overcoming the ‘roadblocks’ affecting the drug action. Pharmaco-miRs provide with an exciting window of opportunity in overcoming chemoresistance in EOCs. Since platinum-based therapy is the mainstay of EOC treatment and most of the patients initially respond very well


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A Mutations

Kinase inhibitors or Phosphatase activators

Kinase

Mutations

Kinase

Kinase inhibitors or Phosphatase activators

Multiple targets in the pathway downregulated Chemo sensitivity

Down + regulation

+ Drug resistance Tumor suppressor microRNA mimics B

microRNA mimics Anti cancer drugs DNA-repair and anti-apoptotic proteins

+++ oncomiRs

Cell death Drug resistance

microRNA mimics

Efflux pumps

oncomiRs

Anti-sense miRs

++++ Cell death Chemo sensitivity

C Differentiated

EOC

TICs

p Tumor relapse TICs survive Cisplatin alone

Tum mor regression reg gressio Tumor Eradicates TICs Cisplatin + miRs t geting TICs tar targeting

Figure 2. Illustration of potential scenarios in which microRNAs could function as adjuvants to chemotherapy and enhance chemosensitivity. (A) Mutations in kinases could confer resistance to kinase inhibitors or phosphatase activators. Direct targeting of these kinases by microRNAs could overcome this resistance and enhance the efficacy in combination with these drugs. (B) Drug efflux pumps and mutations in DNA repair/anti-apoptotic proteins confer chemoresistance and hence microRNAs directly targeting these could confer chemosensitivity. (C) TICs may survive after platinum-based chemotherapy and constitute tumor recurrence and chemoresistance. Hence, microRNAs that directly target TICs could form an effective combination with cisplatin in eradicating TICs. TIC: Tumor-initiating cell.

to these drugs, employing pharmaco-miRs in combination with cisplatin could increase the efficiency of platinum-based therapy and more importantly could prevent the emergence of chemoresistance. A novel prediction database is available for identifying and characterizing potential pharmaco-miRs in the context of the drug of interest (http://www. pharmaco-mir.org/) [58] . Figure 3 lists one such prediction of potential pharmaco-miRs that can modulate cisplatin efficacy. Using this database, miRNAs that are known to be associated with targets that can affect cisplatin function can be identified and these miRNAs can be selected for further functional validation as tools to enhance cisplatin effectiveness. The miRNA association listed in this database includes both predicted miRNA binding sites in the 3’UTR of the targets and also experimentally verified targets (by 3’UTR reporter assays). As illustrated in Figure 3, several targets that are known to modulate cisplatin

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function and form important components of cisplatin resistance. For example, Pharmaco-miR associates the tumor suppressor miR-200c, which is commonly deregulated in EOCs with ABCB1 (MDR1) and very recently it has been shown that miR-200c antagonizes MDR1-dependent drug resistance in cancer cells [73] . These data show that pharmaco-miR prediction can reliably identify miRNAs that could modulate drug efficacy. miRNA modulation to target metastasis in EOC

At time of EOC diagnosis, the majority of patients present with extensive metastatic dissemination and malignant ascites, which usually correlates with poor survival. Hence, targeting metastatic ovarian tumor cells is critical in improving patient response to chemotherapy and to prolong survival. Metastatic process is thought to be initiated by epithelial-mesenchymal transition (EMT) where in epithelial tumor cells acquire



mesenchymal features enabling them to migrate, invade and metastasize to distant organs [74,75] . miRNAs are important regulators of EMT in cancers [76] . The tumor suppressor miR-200 family that is one of the most commonly deregulated miRNAs in EOC and was one of the very first identified inhibitors of EMT process by directly targeting ZEB1, ZEB2 and vimentin, which are positive regulators of EMT [70,77,78] . miR-125a and miR-138 were later shown to inhibit EMT in ovarian cancer cells [79,80] . Furthermore, a comprehensive clustering analyses of 459 serous EOC samples identified a miRNA regulatory network, which consisted of miRNAs that were downregulated and inhibited EMT, correlated with the more aggressive mesenchymal ovarian cancer phenotype. Specifically, they found that miR-506 was an EMT inhibitor and positive prognostic predictor in ovarian cancer by targeting SNAI2 [38] . Enhanced expression of miR506 was found to upregulate E-cadherin, suppress EMT and reduced cancer cell proliferation, migration and invasion in EOC. Clearly, these reports identify a potential role for tumor suppressor miRNA mimics in targeting metastasis by inhibiting EMT processes. In the first report identifying miRNAs as promoters of EMT and metastasis in EOC, our lab showed that miR-181a is a critical regulator of metastasis in EOC by promoting EMT transition via activation of TGF-β signaling pathway [43] . We found that miR181a directly targets SMAD7, the critical inhibitor of TGF-β signaling pathway and promotes EMT transition in epithelial ovarian tumor cells and also miR181a inhibition decreased survival and migration in metastatic ovarian tumor cells. Our study suggests that antisense oligonucleotides targeting miR-181a could be efficient tools in targeting metastatic cells and improve survival in EOC. miRNA modulation targeting tumor microenvironment in EOC

Tumor microenvironment is an integral component of tumor heterogeneity, which greatly complicates diagnostic and therapeutic modalities [81] . This is one of the active areas of research in cancers including EOC, which will form one of the most important arms of EOC translational research aimed at overcoming chemoresistance [82] . miRNAs are known to play important functional roles in tumor microenvironment and hence miRNA modulation is emerging as an exciting tool to target tumor microenvironment in order to overcome chemoresistance [83] . Defining tumor microenvironment in EOC is more complicated as compared with other tumors because of the fact that the origin of EOC is greatly heterogeneous and identifying the site of origin of EOC is still a matter of intensive research.


Review

However, one of the components of tumor microenvironment, namely cancer-associated fibroblasts (CAFs) has been linked to EOC pathogenesis and miRNAs have been identified as regulators of these components of the microenvironment. In the first study reporting the regulation of CAFs in EOC by miRNAs, Mitra et al. identified three miRNAs as necessary and sufficient to reprogram normal fibroblasts into CAFs [84] . miR-214 and miR-31 were downregulated and miR-155 was upregulated in CAFs as compared with normal fibroblasts. Accordingly, miRNA modulation mimicking this deregulation-converted normal fibroblasts to CAFs and vice versa. This study highlights the importance of employing miRNAs as tools to target microenvironment; however, further studies are required to understand how miRNAs regulate other components of tumor stroma in EOC. Efficient miRNA delivery methods are important for success of miRNA therapeutics in EOC miRNA delivery methods in vivo are the most practical determinants or roadblocks of realizing miRNA transition into clinical settings because delivering small RNA molecules into the site of action in the human body bypassing all the tissue barriers is easier said than done. This concept is extensively gaining importance currently since this is the major obstacle in miRNA therapeutics. Systemic administration of miRNA mimics (miRNA replacement therapy) and antisense miRNA oligonucleotides (miRNA inhibition therapy) in vivo aimed at targeting tumor mechanisms could be a challenging task. A variety of miRNA modification methodologies are being tested in preclinical models for their stability and tissue-specific effects in vivo [55,85] . MRX34, which is the anticancer miRNA drug in clinical trial, is a liposome-based method of delivering miRNA in vivo [55] . Yang et al. reported nanoliposome-based method of delivering miR-506 intraperitoneally in EOC xenograft models and hence liposome-based miRNA delivery approaches could be important in EOC [38] . It is not surprising that the two miRNAs in clinical trials currently (miR-34 [MRX34] for hepatocellular carcinoma and miR-122 [miravirsen] for HCV infection) [55] are targeted against hepatic pathology. The first major barrier any therapeutic molecule has to successfully overcome after systemic administration is the hepatic circulation and this may not be a major problem in targeting hepatic pathology because accumulation of the ‘therapeutic miRNAs’ at the site of action is maximal in this scenario since most of the systemically administered miRNA molecules are known to be taken up by liver and kidney [55] .


249


Cisplatin resistance-associated targets ABC transporters DNA repair ABCB1

miR-222 miR-221 miR-200c miR-27a miR-331-5p miR-451

ABCC1

miR-217 miR-133b miR-133a miR-145 miR-873 miR-199a-5p miR-199b-5p miR-185 miR-7 miR-326 miR-330-5p miR-186 miR-224 miR-345

ERCC4

miR-544 let-7g miR-98 let-7c let-7a let-7i let-7b let-7e let-7d let-7f miR-202

miR-520h miR-520g miR-93 miR-519d miR-20b miR-20a miR-106b miR-106a miR-17 miR-205 miR-361-5p miR-200c miR-429 miR-200b miR-302e miR-302d miR-302b miR-302a miR-302c miR-372 miR-520a-3p

Angiogenesis

Apoptosis

VEGFA

miR-29b miR-520e miR-29a miR-373 miR-520c-3p miR-15b miR-520b miR-497 miR-424 miR-520d-3p miR-16 miR-378 miR-195 miR-203 miR-140-5p miR-15a miR-543 miR-613 miR-503 miR-1 miR-410 miR-206 miR-377 miR-199a-5p miR-199b-5p miR-299-3p miR-150 miR-185 miR-34b miR-186 miR-504 miR-125a-5p miR-134 miR-34a miR-126 miR-330-3p miR-383 miR-147 miR-107 miR-29c

XIAP

MCL1

miR-193a-3p miR-193b miR-582-5p miR-181a miR-181c miR-181d miR-181b miR-133b miR-133a miR-93 miR-519d miR-20b miR-20a miR-106b miR-106a miR-17 miR-153 miR-661 miR-892b miR-302e miR-302d

miR-302b miR-302a miR-302c miR-372 miR-520a-3p miR-520e miR-373 miR-520c-3p miR-520b miR-520d-3p miR-367 miR-363 miR-32 miR-92a miR-92b miR-25 miR-135a miR-135b miR-101 miR-125b miR-125a-5p

miR-302a miR-518a-5p miR-29c miR-29b miR-29a miR-16 miR-15a miR-876-3p miR-512-5p miR-886-3p miR-148b* miR-148* miR-193a miR-518 miR-582

miR-96 miR-181a miR-340 miR-181c miR-181d miR-23a miR-181b miR-23b miR-93 miR-7 miR-186 miR-519d miR-320b miR-20b miR-320c miR-20a miR-106b miR-320d miR-106a miR-320a miR-17 miR-219-5p miR-495 miR-136 miR-421 miR-200c miR-216a miR-429 miR-410 miR-200b miR-377 miR-27b miR-27a miR-494 miR-374a miR-371-5p miR-374b miR-1271

microRNAs associated with cisplatin resistance-associated targets

Figure 3. Pharmaco-miR database based prediction of microRNAs that could modulate the efficacy of cisplatin-based chemotherapy. ABC transporters (ABCB1, ABCC1), DNA repair components (ERCC4), angiogenesis regulators (VEGFA) and apoptosis regulators (MCL1, XIAP) constitute some of the important components of cisplatin resistance in EOC and hence microRNAs directly targeting these drug resistance regulators could be employed as effective tools in overcoming cisplatin resistance in EOC. Important microRNAs deregulated in EOCs are highlighted.

Delivering miRNA therapeutic molecules targeting EOC cells is a great challenge since the site of action is very distant from systemic administration as compared with hepatic tissue and also the miRNA molecules have to survive in the circulation long enough to reach the destination in sufficient concentration. Hence, intraperitoneal administration of miRNA therapeutic molecules could be an improvised way of targeted delivery especially in EOC since intraperitoneal administration of platinum-based compounds is often employed in EOC chemotherapy [86] . Importantly, this method could be of specific importance in targeting metastatic cells in EOC since EOC rarely disseminates outside of the abdominal cavity. Thus, it would be interesting to speculate that intraperitoneal administration of tumor suppressor miR-200 mimics and antisense oligonucleotides against miR-181a could emerge as important tools in miRNA-based EOC therapy by targeting metastatic ovarian tumor cells. Since the tumor suppressor family miR-34 is frequently downregulated

250


in EOC, MRX34 could have potential implications too in miRNA-based EOC chemotherapy in future. Nanoparticle-based intraperitoneal delivery of therapeutic molecules specifically targeted at ovarian tumor cells have been reported recently [87] . Combination of miRNAs with such nanoparticle-based ovarian tumor cell-targeted therapy administered intraperitoneally could greatly improve the application potential of miRNAs in EOC therapeutics. Current challenges & future directions of miRNA applications in EOC clinical settings The first miRNA anticancer drug MRX34 entered clinical trial approximately a decade after the discovery that miRNAs are deregulated in cancers. This is an impressive time period for a basic science discovery to translate into therapeutics in clinical settings considering that these are small RNA molecules that offer tremendous hope for cancer therapeutics in coming times. However, the dream of miRNA therapeutics becoming



realistic in cancer treatment still has to overcome various obstacles and this is more so in EOC considering that EOC is greatly heterogeneous both in terms of origin and composition of tumors and still is the most lethal gynecological malignancy. Some important perspectives in this regard can be summarized as below: miRNAs & early diagnosis of EOC pathogenesis

The role of miRNAs in early transformation events in EOC pathogenesis remains largely unexplored even though it is known the miRNAs regulate ovarian physiology. Reliable biomarkers for diagnosis of EOC pathogenesis could greatly improve patient outcome in EOC because chemotherapy intervention at early stage is highly successful with low recurrence. This is one important scenario where miRNAs could dramatically improve patient outcome in EOC. To date, there is lack of knockin or knockout miRNA mouse models specific for ovarian or fallopian tube tissue. These kinds of models are crucial in understanding the role of miRNAs in early transformation events in EOC. The utility of these models can be greatly enhanced in combination with the recently reported BRCA-TP53-PTEN targeting EOC mouse model that can be used to study the effects of miRNA modulation on early EOC events. Candidate miRNA knockin or knockout in the background of this BRCA-TP53-PTEN model can be tested to identify miRNAs that aggravate or reduce the occurrence of tumors in these mice and thus could be selected for diagnostic and therapeutic considerations. miRNAs & intratumoral heterogeneity in EOC

Expression analysis of different EOC cohorts has reported different miRNA signatures in EOCs, thus complicating the identification of common miRNA signatures across EOC patients to enable translation into clinical settings. Even though these kinds of variations are generally attributed to the differences in the methodologies employed and nature of cohorts studied, tumor geneity appears to play an important role in these conditions. Intratumoral heterogeneity presents one of the greatest challenges in clinical translation of novel diagnostic and therapeutic modalities that are successful in preclinical models [88,89] . This could further complicate the identification of common miRNA prognostic and therapeutic signatures across EOC patients since intratumoral heterogeneity is beginning to be identified in cancer patients [90] . This also leads to the interesting possibility of developing patientspecific miRNA signatures that will enable patient stratification and individualized miRNA therapeutics approach (Figure 4) . This kind of targeted miRNA prognostic and therapeutic approach is more likely to be successful in clinical settings and could offer


Review

maximal utility toward improving patient outcome in EOC. One of the greatest challenges in understanding intratumoral heterogeneity in tumors is the selection of right kind of samples that need to be used for analysis. Classical approach has been acquiring biopsies from multiple tumor sites in patients but this technique relies more on rules of probability and chance than specificity and hence, the accuracy of these samples in reflecting true heterogeneity can sometimes be questionable. Circulating tumor cells (CTCs) are increasingly gaining importance as potential reflectors of intratumoral heterogeneity [91] . Since CTCs shed by multiple tumor lesions in a patient enter a common streamline in the circulation, they are more likely to offer a dynamic view of disease pathogenesis in real time as compared with tumor biopsies that provide a more static outlook of the disease. CTC identification in ovarian cancers has not been very successful and the results have been disappointing due to lot of technical limitations that make sample range insufficient for analysis [92] . This provides a great opportunity for circulating miRNAs in EOCs to be employed as biomarkers to develop individualized therapeutic approaches. Since miRNAs that are secreted from multiple tumor lesions in the same patient are channeled into a common pool in circulation, these miRNAs can provide a reliable outlook of EOC pathogenesis events in real-time specific for each patient. Since isolation and analysis of circulating miRNAs in EOC patients are very well established, these circulating miRNAs could greatly revolutionize individualized prognostic and therapeutic approaches in EOCs in future. miRNAs as translational activators

The classic outlook of miRNA functioning has been that miRNAs negatively regulate gene expression mainly by posttranslational mechanisms. In a breakthrough finding few years ago, Vasudevan et al. reported that miRNAs could also function as translational activators [93] and since then concept of regulation of gene expression by miRNAs has taken a new look. Recently, one such scenario was identified in cancer cells suggesting translational activation by miRNAs could be important regulators of tumor phenotype [94] . It is very likely that all these years, important miRNAs could have been missed out being identified as regulators of tumorigenesis since miRNA-cancer studies are most commonly based on the outlook of negative regulation of gene expression by miRNAs. Even though this phenomenon is yet to be identified in EOCs, miRNAs that function as translation activators could play an important role in translation of miRNA application in EOC clinical settings in future.


251


EOC tumor samples (biopsies, serum etc.)

Predicted good survival microRNA expression profiling

(potential chemo sensitive)

Standard chemotherapy

Predicted poor survival (potential chemoresistant) Patient stratification

Targeted therapy personalized

microRNA inhibition therapy

microRNA replacement therapy

Tumors with upregulated oncomiRs

Tumors with downregulated tumor suppressor miRs

miR-A miR-B Anti-sense miRs + standard chemotherapy

miR-C miR-D miR-E microRNA mimics + standard chemotherapy

Figure 4. A futuristic outlook of microRNA application in EOC diagnostics and therapeutics. microRNA signatures could be employed to predict patient survival and plan relevant treatment strategies in EOC clinical settings. microRNA signatures could form an integral component of targeted therapeutic approaches in EOC by enabling reliable patient stratification. microRNA inhibition therapy aimed at downregulating onco-miRs and microRNA replacement therapy aimed at upregulating tumor suppressor microRNAs could constitute future EOC therapeutics aimed at eradicating EOC.

To summarize, the discovery of miRNAs and the subsequent finding that they are deregulated in cancers a decade ago ushered in astronomical hopes that these small RNA molecules could be the magic bullets that mankind was in search of to eradicate cancer and the review of the progress in this regard in the last decade has been promising. If MRX34 shows favorable results in the ongoing clinical trial it could start a new era of miRNA cancer therapeutics and these tiny RNAs could greatly strengthen our fight in eradicating cancer.

in way of realistic application of miRNAs in oncology clinics on a daily basis. The hurdles to overcome are tough if not impossible. These tiny RNA molecules could go a long way in revolutionizing cancer diagnostics and therapeutics in coming years. Personalized therapeutic strategies could well form the basis of cancer diagnostics and therapeutics in the future and miRNAs will have a significant role to play in realizing this possibility. Financial & competing interests disclosure

Future perspective miRNAs have made a significant leap into translational research settings within a decade of being discovered to be deregulated in cancers. A lot of enthusiasm revolves around the success of MRX34, which is the miRNA currently in clinical trial for the treatment of cancer. miRNA and cancer research field has exploded considerably over the last few years and the main focus of research is now to overcome the challenges that stand

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The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.



Review

Executive summary Unique nature of gene expression regulation by miRNAs • miRNAs are small RNAs that regulate cell proliferation and differentiation by associating with their targets in a sequence-specific manner and have been identified as critical regulators of tumorigenesis mechanisms.

miRNA expression deregulation in cancers • miRNA expression profiling and miRNA modulation strategies are emerging as promising entities in cancer diagnostics and therapeutics. • The Cancer Genome Atlas (TCGA) provided a comprehensive analysis of high-grade serous EOCs by integrated analyses of various parameters including miRNA expression profiles and identified four molecular subtypes of ovarian cancer and miRNA expression clustering in this analysis revealed three subtypes with subtype 1 being associated with poor survival.

miRNA functioning in ovarian physiology & tumorigenesis processes • miRNAs are known to play an important role in regulating ovarian physiology and hence it is not surprising that miRNAs have been implicated in regulating EOC mechanisms. • miRNA deregulation is a characteristic feature of chemotherapy resistance and metastasis in ovarian tumors.

Circulating miRNAs as biomarkers • miRNAs are present in body fluids and can be detected in serum samples, which are one of the least invasive samples available for biomarker analysis and hence, circulating miRNAs are increasingly gaining importance as biomarkers in various cancers including EOCs. • Circulating miRNAs can provide a reliable outlook of EOC pathogenesis events in real-time specific for each patient and hence could revolutionize individualized therapy approach in EOC in future.

Application of miRNAs as therapeutic tools in EOC • The two main components of miRNA-based cancer therapeutics are replacing the downregulated tumor suppressor miRNAs using miRNA mimics (miRNA replacement therapy) or by inhibiting the upregulated oncomiRs using antisense miRs (miRNA inhibition therapy). • miRNA modulation can enhance efficacy of chemotherapy and also target metastatic cells in EOC. • miRNA delivery methods in vivo are the most practical determinants or roadblocks of realizing miRNA transition into clinical settings. • MRX34, which is the anticancer miRNA drug in clinical trial, is a liposome-based method of delivering miRNA in vivo. • Nanoliposome-based method of delivering miR-506 intraperitoneally in EOC xenograft models is reported to inhibit metastasis.

miRNAs & early diagnosis of EOC pathogenesis • The role of miRNAs in early transformation events in EOC pathogenesis is an important area to be investigated and miRNA knockin and knockout mouse models are critical requisites in this context.

miRNA in clinical trial • The progress of miRNA cancer research over the last decade is promising and miRNAs could be well set to enter clinical settings and there are great hopes about the success of MRX34 in clinical trial.

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