Biomedicine & Pharmacotherapy 66 (2012) 397–402

Review

Review of MiR-200b and cancer chemosensitivity

Bing Feng 1, Rui Wang 1, Long-Bang Chen *

Department of Medical Oncology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, China

A R T I C L E I N F O

Article history:

Received 10 May 2012

Accepted 10 June 2012

Keywords:

MiR-200b

Cancer

Chemosensitivity

A B S T R A C T

Chemoresistance remains a major obstacle to successful cancer treatment and leads to poor prognosis of

the patients, yet the underlying mechanisms have not been fully understood. MicroRNAs (miRNAs) are

non-coding small RNAs of 19–22 nucleotides which could negatively regulate gene expressions mainly

through 30-untranslated region (30UTR) binding of target mRNAs. MiR-200 family (miR-200a, miR-200b,

miR-200c, miR-141, and miR-429) is a cluster of miRNAs highly correlated with epithelial-mesenchymal

transition (EMT), wherein miR-200b is identified as a critical regulator of tumor invasion, metastasis, and

chemosensitivity. Recent advances of miR-200b dysregulation in tumor chemoresistance were

summarized. Possible mechanisms and reversion strategies were also addressed.

� 2012 Elsevier Masson SAS. All rights reserved.

Available online at

www.sciencedirect.com

1. Introduction

Cancer is one of the most lethal diseases all over the world.Although significant advances have been made in the treatment ofvarious types of cancer, chemoresistance remains a major obstacleto successful clinical treatment and leads to poor prognosis of thepatients. Mechanisms include both genetic and epigeneticdysregulation of genes involved in drug transporters, drugmetabolism, repair of DNA damages, and pathway alterations ofapoptosis as well as cell cycle progress [1,2]. Emerging evidencehas demonstrated that microRNAs (miRNAs) are crucial regulatorsinvolved in these progresses by targeting different signalingpathways, yet there is still much to learn about the specificmechanisms by which they exert their effects.

Identified as new components of epigenetic gene-regulatorysystems, miRNAs are non-coding small RNAs of 19–22 nucleotidesthat could regulate gene expressions mainly through 30UTRbinding of target mRNAs, causing either degradation or inhibitionof gene translation [3]. Because of the short sequence requirementfor imperfect base pairing, a single miRNA could interplay with awide range of target transcripts, alter gene expression, andultimately dictate cell fate such as developmental transitions,organ morphology, cell proliferation and apoptosis. Recently,increasing studies have highlighted miRNAs related with che-moresistant phenotype of different types of tumors [4], mainlythrough dysregulation of cell cycle [5], apoptosis [6], and functionof drug transporters [7]. The miR-200 family consists of miR-200a,miR-200b, miR-200c, miR-429 and miR-141, which are regarded ashallmarks of epithelial cells. Recent reports indicate that miR-200b

* Corresponding author. Fax: +86 25 80860153.

E-mail address: Dr.Chenlb@163.com (L.-B. Chen).1 Co-first authors with equal contribution to this manuscript.

0753-3322/$ – see front matter � 2012 Elsevier Masson SAS. All rights reserved.

http://dx.doi.org/10.1016/j.biopha.2012.06.002

is a fundamental regulator of epithelial-mesenchymal transition(EMT) involved in cancer metastasis and chemoresistance [8,9].This review will focus on the association between miR-200bdysregulation and cancer chemoresistance.

2. MiR-200 family and MiR-200b

Located in two different loci in the genome [10], members ofmiR-200 family share seed sequences differed by one nucleotide.Although target prediction algorithms assume significant differ-ences in the genes targeted by miR-200b/200c/429 and miR-200a/141, evidence indicates a high degree of overlap in geneticregulation [8]. Recently, dysregulation of miR-200 family membershas been reported in various types of human cancer [11–14]. As amember of miR-200 family, miR-200b is generally recognized asone of the fundamental regulators of EMT [8,9]. Accumulatingevidence suggests that down-regulation of miR-200b and theaccompanied up-regulation of the EMT inducer ZEB1 and ZEB2 arehighly correlated with the mesenchymal and drug-resistantphenotypes of cancer cells [15]. Nevertheless, over-expression ofmiR-200b is also observed in several types of tumors [16–19].

3. MiR-200b and cancer chemosensitivity

The association of miRNA expression and cancer chemosensi-tivity was firstly raised by Meng and colleagues in cholangiocar-cinoma [19]. It was found that inhibition of miR-21 and miR-200bcould enhance the sensitivity of cholangiocarcinoma cells togemcitabine, probably by modulation of CLOCK, PTEN, PTPN12 andthe downstream oncogene products such as c-Abl, Src, and Ras[19,20]. Furthermore, mounting evidence indicates the existenceof similarities between drug-resistant and metastatic cancer cellsin terms of resistance to apoptosis and enhanced invasiveness,

B. Feng et al. / Biomedicine & Pharmacotherapy 66 (2012) 397–402398

consistent with miR-200b dysregulation in chemoresistant cancercell lines. For instance, the decreased expression of miR-200b wasdetected in adriamycin- as well as cisplatin-resistant MCF-7human breast adenocarcinoma cells relative to the parental cells[21,22]. In ovarian cancer, a recent study with 55 advanced tumorsamples showed that a high expression of miR-200a, miR-200b,and miR-429 was associated with improved survival [23].According to miRNA microarray data in our previous study,miR-200b was identified as the most down-regulated miRNA indocetaxel-resistant human lung adenocarcinoma SPC-A1/DTXcells compared with the parental SPC-A1 cells [24]. DecreasedmiR-200b expression was also detected in tumor tissues sampledfrom lung adenocarcinoma patients treated with docetaxel-basedchemotherapy and was proved to be correlated with decreasedsensitivity to docetaxel and poor prognosis [25]. Nevertheless,adding to the complexity of miR-200b’s role in response toanticancer drugs and cancer survival is another study suggestingthat high expression of miR-200 family members is highlycorrelated with poor prognosis [26]. In this review, we willprovide potential mechanistic explanations for the associationbetween miR-200b dysregulation and cancer chemoresistancethrough aspects of EMT, cancer stem cell (CSC) maintenance,angiogenesis, apoptosis, and cell cycle distribution.

3.1. MiR-200b and EMT

EMT occurs during embryogenesis to support tissue remodelingand has been proposed as the fundamental step in the metastasis ofepithelial cancers, contributing to increased cell motility andinvasion [27]. This process in cancer pathogenesis involvescharacteristics such as elongated fibroblast morphology, down-regulation of epithelial marker E-cadherin, and up-regulation ofmesenchymal markers such as vimentin, ZEB1 and ZEB2 [28].Recent evidence indicates that EMT of cancer cells not only causesenhanced metastasis, but also contributes to chemoresistance.

The miR-200 family has been shown to be major determinantsin EMT by targeting ZEB transcription factors, subsequentlycontrolling cluster of gene expression including E-cadherin andvimentin [8,9,28]. Moreover, recent studies have discovered areciprocal feedback loop in which transcriptional repressors suchas ZEB1 and ZEB2 bind to E-boxes within the promoter regions ofthe miR-200 family to repress their transcription [10,29]. It wasalso proved that miR-200 family-mediated transcriptional up-regulation of E-cadherin in mesenchymal breast cancer cells wasdirectly associated with translational repression of ZEB1 andindirectly with increased acetylation of histone H3 at the E-cadherin promoter, which was probably due to the disruption ofrepressive complexes between ZEB1 and histone deacetylases andto the inhibition of SIRT1, a class III histone deacetylase. Theseevents not only inhibited EMT progression but also up-regulatedpro-apoptotic genes in the p53 apoptotic pathway and resensi-tized the cancer cells to doxorubicin [15], in accordance withthose reported in a very recent study [30]. Li et al. reported that theexpression of miR-200b was significantly down-regulated ingemcitabine-resistant pancreatic cancer cells with EMT char-acteristics. Re-expression of miR-200b resulted in the down-regulation of ZEB1, slug, and vimentin, accompanied by morpho-logical reversal of EMT phenotype leading to epithelial morphol-ogy [31].

In fact, EMT is generally recognized as the initiation of cancermetastasis, while at later stages, metastasizing cells settle down inthe target tissue with a differentiation process similar tomesenchymal-epithelial transition (MET) [32], resulting in up-regulation again of miR-200b. It is thereby not surprising that miR-200b has been reported to be down-regulated [15,24,33,34], up-regulated [16–19], or unchanged [35,36] in advanced cancers.

3.2. MiR-200b and CSCs

CSCs are cells within a tumor with the capacity of self-renewand maintenance of tumor-initiation through differentiation intothe heterogeneous lineages of cancer cells [37]. These tumor-initiating cells provide rich sources for cancer recurrence afteranticancer therapies. Emerging evidence suggests that theacquisition of EMT phenotype and induction of cancer stem cellphenotypes share biological similarities and synergisticallycontribute to cancer metastasis, recurrence, and chemoresistance.The emergence of CSCs occurs in part as a result of EMT [38,39].

As mentioned above, members of the miR-200 family couldinhibit EMT and suppress cancer invasion by directly repressing thetranslation factors ZEB1 and ZEB2. Additionally, these miRNAs havealso been shown to repress Suz12 and Bmi1 [40], two essentialcomponents of polycomb repressor complexes (PRCs) that areresponsible for the maintenance of embryonic and adult stem cells[41,42]. Iliopoulos et al. reported that in breast cancer, loss of miR-200b during CSC formation increased Suz12 expression, Suz12binding, H3-K27 trimethylation, and polycomb-mediated repres-sion of the E-cadherin gene. Re-expression of miR-200b or Suz12depletion blocked the formation and maintenance of mammo-spheres, and in combination with chemotherapy inhibited cancergrowth and prolonged remission in mouse xenografts. By contrast,ectopic expression of Suz12 in transformed cells promoted CSCformation, suggesting that the miR-200b-Suz12-E-cadherin path-way is involved in CSCs maintenance and invasive characteristics ofbreast cancer cells [43]. Sun et al. reported that miR-200b and miR-15b were significantly down-regulated in the chemoresistanttongue squamous cell carcinoma (TSCC) cells in comparison withthe parental cell lines. Forced re-expression of miR-200b and miR-15b effectively reversed the EMT and chemoresistant phenotypes ofthe resistant cells, while inhibition of both the miRNAs in thesensitive cell lines induced EMT and conferred chemoresistance[44]. Moreover, retrieving the expression of Bmi1, a direct target formiR-200b and miR-15b [45,46], recapitulated chemotherapy-induced EMT. In vivo, enforced miR-200b or miR-15b expressioninhibited metastasis of TSCC xenografts. Clinically, reduced miR-200b or miR-15b expression was associated with chemoresistancein TSCCs and poor survival.

Notch signaling pathway has been suggested to play a crucialrole in the regulation of both EMT and CSC formation during thedevelopment and progression of tumors [47,48]. As reported byKong et al., miR-200b could inhibit expression of ZEB1, ZEB2,Lin28B and Notch1 in prostate cancer. Reversal of EMT by forcedre-expression of miR-200b significantly suppressed clonogenicand prostasphere-forming ability and was associated with thedown-regulation of Notch1 and Lin28B [49]. Bao et al. demon-strated that ectopic expression of Notch1 could lead to acquisitionof EMT phenotype, potentially via down-regulation of miR-200b,and cause increased capacity of CSC-self-renewal consistent withup-regulation of CD44 and EpCAM expression [50].

Forkhead Box M1 (FoxM1) transcription factor is generallyinduced in multiple cell types within tumor lesions and involved inthe development and progression of the malignancies. Bao et al.established FoxM1 over-expressing pancreatic cancer cells withincreased cell growth, clonogenicity, and cell migration. Theyfound that over-expression of FoxM1 led to the acquisition of EMTphenotype by activation of mesenchymal cell markers ZEB1, ZEB2,Snail2, and vimentin, consistent with increased sphere-formingcapacity and expression of CSC surface markers CD44 and EpCAM.Ectopic FoxM1 expression also brought significant down-regula-tion of miR-200b, while re-expression of miR-200b inhibited theexpression of ZEB1, ZEB2, vimentin as well as FoxM1, induced theexpression of E-cadherin, and ultimately led to the reversal of EMTphenotype [51].

B. Feng et al. / Biomedicine & Pharmacotherapy 66 (2012) 397–402 399

3.3. MiR-200b and angiogenesis

Tumor angiogenesis is recognized to be a limiting factor intumor progression. Synergy occurs between anti-angiogenicagents and chemotherapy [52,53]. It has been broadly acceptedthat a partial decrease in vascular endothelial growth factor (VEGF)function, for example, by using an anti-VEGF receptor agent, couldreduce interstitial fluid pressure and ‘‘normalize’’ blood vesselmorphology [54]. Increased tumor blood flow may improve tumorresponse to chemotherapy with enhanced drug delivery [55].Furthermore, antibody-based anti-angiogenic agents will persistlonger in the tumor between cycles of chemotherapy, attenuatingtumor cell repopulation with more efficiency [52].

VEGF acts as a fundamental factor and modulator of angiogen-esis. In the VEGF signaling pathway, the key components are VEGFand its receptors, Flt-1 and KDR. In a recent study, Choi et al.demonstrated that transfection of synthetic miR-200b reducedprotein levels of VEGF, Flt-1, and KDR. In human lung adenocarci-noma cells, transfection of synthetic miR-200b targeted thepredicted binding sites in the 30UTR of VEGF, Flt-1 and KDR, andreduced their protein levels. In human umbilical vein endothelialcells (HUVECs), ectopic miR-200b expression conferred decreasedability to form a capillary tube on matrigel and VEGF-inducedphosphorylation of ERK1/2. These results suggest that miR-200bnegatively regulates VEGF signaling by targeting VEGF and itsreceptors and that miR-200b may have therapeutic potential as anangiogenesis inhibitor [56]. Hypoxia is recognized as a general cuethat drives angiogenesis and is associated with advanced stage andpoor outcome in a range of tumor types. In a recent study, Chanet al. reported that similar to epithelial cells, miR-200b suppressedendothelial cell migration and the tube formation ability ofendothelial cells, establishing that miR-200b serves as an anti-angiogenic miRNA under physiological conditions [57]. Accordingto their results, miR-200b down-regulation was required to relieveEts-1 repression under hypoxic conditions, resulting in successfulangiogenesis.

Interestingly, it was also observed that ZEB1 could negativelyregulate angiogenesis in macrovascular endothelial cells. Knock-down of ZEB1 exaggerated tube formation and matrigel invasion inhuman umbilical vascular endothelial cells [58,59], suggesting themultiplicity and complexity of the involvement of miR-200 familymembers in tumoral angiogenesis.

3.4. MiR-200b and apoptosis

Resistance to apoptosis signaling is a common feature of tumorcells and leads to multi-drug resistance, mediated by Bcl-2 familyproteins [60]. Bcl-2 and Bcl-XL are pro-survival members of theBcl-2 family which have been shown to antagonize the pro-apoptotic activity of Bax and promote cell survival throughblocking Bax translocation from the cytosol to mitochondria andby preventing the release of cytochrome c.

It was recently found that Bcl-2 was a predicted target of miR-200b. Bcl-2 protein level was negatively regulated by miR-200bin gastric cancer cells, causing increased apoptosis and sup-pressed cell proliferation [61]. In our previous study, Bcl-2 wasalso predicted as a candidate target of miR-200b by bioinformat-ics cross-analysis. However, the negative correlation betweenmiR-200b and Bcl-2 was not detected at neither mRNA norprotein level, suggesting that the predicted binding sites for miR-200b in Bcl-2 30UTR were nonfunctional or alternative mecha-nisms were crucial for the regulation of the eventual proteinexpression. Mutations on the miRNA-binding sites and alterna-tive cleavage or polyadenylation of the 30UTRs are very prevalentin cancer and might be the mechanisms underlying thisobservation [62,63].

3.5. MiR-200b and cell cycle distribution

Cell cycle-mediated drug resistance is described as a relativeinsensitivity to chemotherapeutic agents due to the position of thecells in the cell cycle. This is most frequent in combinationchemotherapy, where one chemotherapeutic agent impacts thecell cycle so that the other chemotherapeutic agent givenimmediately in sequence becomes less effective. The best exampleis the combination chemotherapy involving taxanes, the typicalclass of cell cycle-specific chemotherapeutic agents [64].

Taxanes act by stabilizing microtubules, which could markedlyenhance microtubule assembly, disrupt the transition of a cellthrough mitosis, and ultimately cause a G2/M arrest followed byapoptosis [65,66]. In humans, there are at least eight differentisotypes of b-tubulin with variant expression pattern in tumoraltissue [67], wherein expression of b-tubulin III has been closelyrelated with response to taxanes and patient survival in a variety ofcancers [68,69]. Leskela et al. demonstrated that miR-200b/200c/429-binding sites were predicted in the 30UTR of the b-tubulinisotypes I, IIa, and III [70]. In a well-characterized series of ovariancarcinomas, low tumoral expression of miR-200 family wassignificantly associated with high b-tubulin III protein contentand had a trend toward poor progress-free survival (PFS),suggesting the potential significance of miR-200 family membersas new biomarkers predictive for response to the standardpaclitaxel–carboplatin treatment, as in agreement with ourprevious experimental results [25]. According to our miRNAmicroarray data, miR-200b was identified as the most down-regulated miRNA in docetaxel-resistant human lung adenocarci-noma SPC-A1/DTX cells compared with parental SPC-A1 cells.Ectopic miR-200b expression reversed docetaxel chemoresistanceof lung adenocarcinoma cells through proliferative suppression,apoptosis enhancement, and G2/M cell cycle arrest, mediated atleast partially by targeting E2F3, which was critical for themaintenance of normal cell cycle progress. Furthermore, it wasreported that miR-200b directly reduced the expression of RND3, amembers of the Rho GTPase family, thereby promoting expressionof the downstream protein cyclin D1 and S-phase entry of humancervical cancer Hela cells [71].

4. Mechanisms for MiR-200b dysregulation

Previous work on miR-200b has broadly focused on mecha-nisms of repression, including transcriptional inhibition andepigenetic modifications [10,29,72,73]. The recently character-ized promoter of miR-200b has a CpG island (length 2,541, % GC65.0, O/E ratio 0.69, 183 CpGs) [73]. Interestingly, Volodymyr et al.demonstrated that rat hepatocarcinogenesis induced by a methyl-deficient diet was characterized by a prominent inhibition ofexpression of miRNA genes responsible for the maintenance ofbalance between cell proliferation and apoptosis such as E2F3,Notch1, ZFHX1B, and Bcl-2. In the livers of rats fed with themethyl-deficient diet, miR-34a, miR-127, and miR-200b becamesignificantly down-regulated after 9 and 18 weeks on the diet[74].

A recent report suggested that p53/p63/p73 family bindingsites could modulate promoter activity of the miR-200 familymembers. Both p73 and p63 were proved to be directly associatedwith the miR-200b/a/429 promoter by chromatin immunoprecip-itation analysis [75]. Given that p63 also serves as a transcriptionalregulator of Dicer to modulate miRNA maturation, includingprocessing of the miR-200 family [76], these studies indicate thatp63 could act both directly and indirectly to alter miRNAexpression, although the mechanisms may be cellular context-dependent. In another study, Ahn et al. found that miR-200 familymembers were over-expressed in Smad3-over-expressed gastric

Fig. 1. Selected regulators and targets of miR-200b involved in cancer chemoresistance according to the current literatures. Upper: DNMT1 and HDAC1 could suppress the

transcription of pri-miR-200b, while Smad3, p63, and p73 could promote pri-miR-200b transcription. Lower: the validated targets of miR-200b associated with cancer

chemosensitivity via EMT, CSC maintenance, angiogenesis, apoptosis, and cell cycle progress.

B. Feng et al. / Biomedicine & Pharmacotherapy 66 (2012) 397–402400

cancer cell lines. Luciferase reporter assay revealed that Smad3could directly bind to a Smad-binding element located in thepromoter region of miR-200b/a and function as an activator at thetranscriptional level [77].

5. Discussion

In conclusion, evidence suggests that miR-200b is a criticalregulator of cancer chemosensitivity, mainly through signalingpathways of EMT, CSC maintenance, angiogenesis, apoptosis, andcell cycle distribution (Fig. 1). Aberrant expression of miR-200bhas been detected in various types of human cancers with changesof response to anticancer agents such as gemcitabine [19],adriamycin [21], cisplatin [22], and docetaxel [24]. The diagnosticand prognostic miRNA profiles including miR-200b have alreadybeen validated [25,78,79]. We propose that the therapeuticdelivery of miR-200b or its inhibitors may represent an additionalstrategy to potentially resensitize cancer cells to chemothera-peutic agents.

Recent advances in the miRNA field have lead to theunderstanding of new mechanisms of cancer chemoresistance.These studies provide a strong rationale for the development ofmiRNA-based diagnostic, prognostic, and ultimately therapeuticstrategies aiming to overcome cancer chemoresistance. Theadvantage of miRNAs is their ability of affecting multiple targetswith a single hit, involved in a whole network of interactingmolecules. When translating these fundamental research advancesinto medical practice, the efficiency and safety of miRNAs/anti-miRNAs delivery in vivo are the major obstacles to overcome. Tothis aim, modified miRNA molecules with longer half-lives andefficiency have been developed, such as anti-miRNA oligonucleo-tides [80], locked nucleic acid (LNA)-modified oligonucleotides[81], cholesterol-conjugated antagomiRNAs [82], and ‘‘miRNAsponges’’ [83]. A better understanding of the specific mechanismsof miRNA-mediated cancer chemoresistance, coupled withimprovements of drug delivery systems, will enable miRNA-based

therapy a promising new strategy to overcome cancer chemore-sistance in the future.

Disclosure of interest

The authors declare that they have no conflicts of interestconcerning this article.

Financial disclosure statements: there are no financial disclosuresfrom any authors.

Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (Grant Nos. 81071806 and 81172106).

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