cancer-associated morc2-mutant m276i regulates an hnrnpm … · molecular cell biology...

Molecular Cell Biology

Cancer-Associated MORC2-Mutant M276IRegulates an hnRNPM-Mediated CD44 SplicingSwitch to Promote Invasion and Metastasis inTriple-Negative Breast CancerFang-Lin Zhang1,2,3, Jin-Ling Cao1, Hong-Yan Xie1,2,3, Rui Sun1, Li-Feng Yang1,Zhi-Ming Shao1,2,3,4,5, and Da-Qiang Li1,2,3,4,5,6

Abstract

Triple-negative breast cancer (TNBC) is the most lethalsubtype of breast cancer, with a high propensity for distantmetastasis and limited treatment options, yet its molecularunderpinnings remain largely unknown. Microrchidia familyCW-type zinc finger 2 (MORC2) is a newly identified chro-matin remodeling protein whose mutations have beencausally implicated in several neurologic disorders. Here, wereport that a cancer-associated substitution of methionine toisoleucine at residue 276 (M276I) of MORC2 confers gain-of-function properties in the metastatic progression of TNBC.Expression of mutant MORC2 in TNBC cells increased cellmigration, invasion, and lungmetastasis without affecting cellproliferation and primary tumor growth compared with itswild-type counterpart. TheM276Imutation enhancedbindingof MORC2 to heterogeneous nuclear ribonucleoprotein M(hnRNPM), a component of the spliceosome machinery. Thisinteraction promoted an hnRNPM-mediated splicing switch of

CD44 fromthe epithelial isoform(CD44v) to themesenchymalisoform (CD44s), ultimately driving epithelial–mesenchymaltransition (EMT). Knockdownof hnRNPM reduced the bindingofmutantMORC2 toCD44pre-mRNAand reversed themutantMORC2-induced CD44 splicing switch and EMT, consequentlyimpairing themigratory, invasive, and lungmetastatic potentialof mutant MORC2-expressing cells. Collectively, these findingsprovide the first functional evidence for the M276I mutation inpromoting TNBC progression. They also establish the firstmechanistic connection between MORC2 and RNA splicingand highlight the importance of deciphering unique patient-derived mutations for optimizing clinical outcomes of thishighly heterogeneous disease.

Significance:A gain-of-function effect of a singlemutation onMORC2 promotes metastasis of triple-negative breast cancer byregulating CD44 splicing. Cancer Res; 78(20); 5780–92.�2018 AACR.

IntroductionTriple-negative breast cancer (TNBC) is a distinct subtype of

breast cancer with low or no expression of estrogen receptor (ER),progesterone receptor (PR), and human epidermal growth factorreceptor 2 (HER2), which accounts for approximately 15% to20% of all breast cancers (1, 2). In contrast to other breast cancersubtypes, TNBC occurs most frequently in younger women,exhibits an extremely aggressive phenotype with higher rates of

early relapse and distant metastasis, and lacks the responsivenessto endocrine or HER2-targeted therapies, thus contributing to theworst clinical outcome (1, 2). Due to the lack of clinicallyavailable targeted therapies, cytotoxic chemotherapy remains themainstay of treatment for TNBC. However, treatment options arevery limited upon the development of chemoresistance anddistant metastasis (3). These clinical challenges are further rein-forced by its genetic heterogeneity. Recently, several genomic andtranscriptomic sequencing studies have revealed extensive muta-tional heterogeneity among TNBC tumors (4–6). In this context,in addition to a few common recurrent mutations that restrictprimarily to p53 and the phosphoinositide 3-kinase (PI3K)pathway, a large number of genes with lowmutational frequencywere found in TNBC tumors (4–6). Thus, deciphering the patternsof mutations in individual patient with TNBC is critical for per-sonalized cancer therapy and optimizing clinical outcomes (5).However, the functional consequences and related mechanisticunderpinnings for the majority of these identified unique patient-derived mutations in TNBC progression remain unknown.

Microrchidia family CW-type zinc finger 2 (MORC2) is amember of the evolutionarily conserved MORC nuclear proteinsuperfamily, which is characterized by the presence of a conservedGHKL (Gyrase, Hsp90, Histidine kinase, and MutL)-type ATPasedomain, a CW-type zinc finger domain, and several distinctcoiled-coil domains (7–9). Although MORC2 is ubiquitouslyexpressed in mammalian cells, its biological functions remain

1Shanghai Cancer Center and Institutes of Biomedical Sciences, ShanghaiMedical College, Fudan University, Shanghai, China. 2Cancer Institute, ShanghaiMedical College, Fudan University, Shanghai, China. 3Department of Oncology,Shanghai Medical College, Fudan University, Shanghai, China. 4Department ofBreast Surgery, Shanghai Medical College, Fudan University, Shanghai, China.5Key Laboratory of Breast Cancer in Shanghai, Shanghai Medical College,Fudan University, Shanghai, China. 6Key Laboratory of Medical Epigenetics andMetabolism, Shanghai Medical College, Fudan University, Shanghai, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Da-Qiang Li, Fudan University, 270 Dong-An Road,Building 2, Room 1312A, Shanghai 200032, China. Phone: 86-21-34777589; Fax:86-21-64172585; E-mail: [email protected]; and Zhi–Min Shao,E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-1394

�2018 American Association for Cancer Research.

CancerResearch

Cancer Res; 78(20) October 15, 20185780

on March 20, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst August 9, 2018; DOI: 10.1158/0008-5472.CAN-17-1394

http://crossmark.crossref.org/dialog/?doi=10.1158/0008-5472.CAN-17-1394&domain=pdf&date_stamp=2018-9-29

http://cancerres.aacrjournals.org/

largely unknown. Recently, we and others have defined MORC2as a chromatin remodeling protein with emerging roles in DNArepair (10) and gene transcription (11, 12). Interestingly, muta-tions inMORC2havebeen causally linkedwith several neurologicdisorders, such as Charcot–Marie–Tooth disease (11, 13, 14),cerebellar ataxia, axonal polyneuropathy, and nocturnal hypo-ventilation (15). In TNBC, a microarray-based gene-expressionprofiling study revealed that the expression levels of MORC2 areassociated with the recurrence risk of patients with TNBC (16). Inaddition, an exome sequencing analysis of primary TNBC tumorsrevealed that 1 of 65 patients with TNBC carried a conservedmutation of G to C at nucleotide 828 of the MORC2 gene,resulting in its encoding protein harboring a substitution ofmethionine to isoleucine at residue 276 (M276I; ref. 5). However,the functional andmechanistic roles forMORC2M276Imutationin TNBC development and progression remain unexplored.

The heterogeneous nuclear ribonucleoprotein M (hnRNPM) isa component of the spliceosomemachinery and plays key roles insuppression of precursor mRNA (pre-mRNA) splicing throughantagonizing the recognition of splice sites (17). Indeed,hnRNPM has been shown to regulate alternative splicing ofseveral cancer-associated genes, includingfibroblast growth factorreceptor 2 (FGFR2; ref. 18) and cell-surface molecule CD44 (19).FGFR2 pre-mRNA is alternatively spliced to form the epithelial-and mesenchymal-specific IIIb and IIIc isoforms, respectively,and the FGFR2 isoform switch potentially affects epithelial–mesenchymal transition (EMT) program and cancer progression(20). Similarly, human CD44 pre-mRNA contains nine variableexons between its constitutive exons. Inclusion of one or more ofthe variable exons generates CD44 variant isoforms (CD44v),whereas skipping all of the variable exons produces CD44 standardisoform (CD44s; ref. 19). Generally, expression of CD44v is com-mon in epithelial cells, while CD44s is expressed bymesenchymalcells (21). Emerging evidence shows that hnRNPM promotes thesplice isoform switch fromCD44v to CD44s, which is essential forEMT and breast cancer metastasis (19, 21). More importantly,upregulation of hnRNPM correlates with distant metastasis, poorprognosis, and increased CD44s in patients with breast cancer(19, 22). However, the upstream regulatory signals for hnRNPM-mediated splicing program in TNBC remain to be elucidated.

In this study, we report for the first time that the M276Imutation of MORC2 is a gain-of-function mutation that pro-motes metastatic progression of TNBC through regulatinghnRNPM-mediated CD44 splice isoform switch and EMT. Thesefindings provide novel mechanistic insights into MORC2 M276Imutation in promoting TNBC metastatic progression and high-light the importance of dissecting unique patient-derived muta-tions and their functional consequences for personalizedtreatments of this highly heterogeneous disease.

Materials and MethodsCell culture and reagents

Human breast cancer cell lines (MDA-MB-231, MDA-MB-436,MDA-MB-468, Hs578T, BT20, BT549, HCC1937, and BT474),normal breast epithelial cell lineMCF10A, andhuman embryonickidney 293T (HEK293T) cell linewere obtained fromCell Bank ofType Culture Collection of Chinese Academy of Sciences. MDA-MB-231–derived LM2-4173 and LM2-4175 cells were kindlyprovided by Guohong Hu (University of Chinese Academy ofSciences, Shanghai, China). Both cell lines have enhanced lung

metastasis potential when compared with their parental cells(23). SUM149 and SUM159 cell lines were obtained from Aster-and. All cell lines were authenticated by short tandem repeatprofiling. Mycoplasma contamination was tested by a PCR-basedmethod as described previously (24). Cells were expanded andfrozen immediately into numerous aliquots after arrival in 2014.The cells revived from the frozen stock were used within 10 to 15passages and not exceeding a period of 6 months. MCF10A cellswere cultured in DMEM/F12 supplemented with 5% donor horseserum, 10 mg/mL insulin, 20 ng/mL epidermal growth factor,0.5 mg/mL hydrocortisone, and 100 ng/mL cholera toxin. Theculture medium for SUM159 and SUM149 is Ham's F12 contain-ing 10% fetal bovine serum (FBS), 5 mg/mL insulin, and 1 mg/mLhydrocortisone. Other cell lines were maintained in high-glucoseDMEM or RPMI1640 media supplemented with 10% FBS. Cul-ture media and supplements were obtained from BasalMedia.Horse serum and FBS were fromGibco. All chemicals and regentswere purchased from Sigma-Aldrich unless otherwise noted.

Expression vectorsMyc-DDK-MORC2 cDNA was purchased from Origene and

subcloned into the lentiviral vector pCDH-CMV-MCS-EF1-Puro(System Biosciences) to generate Flag-MORC2 construct.Myc-DDK-MORC2 cDNA was also subcloned into the lentiviralvector pLVX-IRES-Neo (Clontech) for reexpression of MORC2(G418 resistance) in MORC2 knockout (KO) cells (puromycinresistance). M276I mutation was generated by PCR-based muta-genesis and verified by DNA sequencing. Flag-His-hnRNPMcDNA was obtained from Vigene Biosciences and subcloned intopCDH-CMV-MCS-EF1-Puro vector to generate HA-hnRNPMexpression vector. GIPZ lentiviral short hairpin RNA (shRNA)vectors expressing hnRNPMshRNA (shhnRNPM) or nontargetingnegative control (shNC) were obtained from GE Healthcare.Detailed information concerning expression constructs and theprimers used for molecular cloning is provided in SupplementaryTables S1 and S2. Small interfering RNA (siRNA) targetinghnRNPL (sihnRNPL) and nontargeting negative control (siNC)were purchased form GenePharma (Supplementary Table S3).

Plasmid transfection and lentiviral infectionTransient plasmid transfection was performed using Neofect

DNA transfection reagent (TengyiBio) according to the manufac-turer's protocol. To generate stable cell lines expressing shRNAs orcDNAs, HEK293T cells were transfected with each lentivirusexpression vector and packaging plasmidmix using Neofect DNAtransfection reagents. The supernatant containing viruses wascollected 48 hours after transfection, filtered, and used for infect-ing target cells in the presence of 8 mg/mL of polybrene prior todrug selectionwith 2mg/mLof puromycin (CaymanChemical) or5 mg/mL of G418 (Sigma) for 1 to 2 weeks. MORC2 KO celllines were generated using the CRISPR/Cas9 system as describedpreviously (25) with LentiCas9-Blast and lentiGuide-Purovectors (Addgene). The short guide RNA sequence for MORC2(50-AGTAGACTCAGGTGTTCGCT-30) was chosen according tothe Web-based CRISPR design tool from the Zhang lab (http://www.genome-engineering.org/) and was cloned into lentiGuide-Puro vector following the standard protocol (25). SuccessfulMORC2 KO in the established cell lines was validated byimmunoblotting. SiRNA transfection was performed usingLipofectamine 2000 (Invitrogen). The efficiency of silencingwas assessed by immunoblotting 48 hours after transfection.

MORC2 M276I Mutation in TNBC Metastatic Progression

www.aacrjournals.org Cancer Res; 78(20) October 15, 2018 5781



http://www.genome-engineering.org/

http://www.genome-engineering.org/


Cell viability, colony-formation assay, and cell-cycle analysisCells were seeded in 96-well plates (2,000 cells/well) in trip-

licate, and cell viability was determined by Cell Counting Kit-8(CCK-8; Dojindo Laboratories). For colony-formation assay, cellswere seeded in 6-well plates (1,000 cells/well) in triplicate andcultured under normal growth conditions for 2 to 3 weeks.Colonies were stained with 1% crystal violet and counted. Forcell-cycle analysis, cells were harvested and fixed in 75% ethanolovernight. After PBS wash, cells were stained with cell-cyclestaining kit (MultiSciences Biotech), and analyzed on a BDFACSCanto II flow cytometer (BD Biosciences).

Cell migration and invasion assaysMigration and invasion assayswere conducted using 8-mmpore

noncoated polycarbonate transwell inserts (BD Biosciences) andBioCoat Matrigel Invasion Chambers (Corning), respectively.Briefly, 5 � 104 cells in 200 mL of serum-free growth mediumwere seeded in the top chamber.Growthmediumcontaining 10%FBS was used as a chemoattractant in the lower chamber. After 24hours, migrated and invaded cells were fixed and stained with 1%crystal violet. Cells were counted in 10 random fields undermicroscope.

Tumorigenicity and metastasis assaysAll animal studies were approved by the Institutional Animal

Care and Use Committee of Shanghai Cancer Center, FudanUniversity. For primary tumor and spontaneousmetastasis assays,2 � 106 LM2-4175 cells stably expressing empty vector pLVX,wild-type (WT), and M276I-mutant MORC2 were injected intothe fourth mammary fat pad of 6-week-old female nonobesediabetic/severe combined immunodeficient (NOD/SCID) mice(n ¼ 6; State Key Laboratory of Oncogenes and Related Genes,Shanghai Cancer Institute, Shanghai, China). The tumors weremeasured twice a week after appearance of tumors, and the tumorvolumewas calculated by the formula of (length�width2)/2. Themice were killed after 6 weeks of the inoculation, and primarytumor and the lung tissues were harvested for histologic analysis.As cancer cells formeddiffusemetastases in the lungs ofmice in anorthotropic xenograft model of spontaneous breast cancer metas-tasis, lung metastases were quantified by determining the percentmetastatic lung surface area relative to total lung surface area asdescribed previously (26). For experimental metastasis experi-ments, 2� 106MDA-MB-231 cells stably expressing empty vectorpCDH, WT, and M276I-mutant MORC2 in 200 mL of PBS wereinjected in the tail vein of 6-week-old BALB/c female nude mice(n ¼ 6; State Key Laboratory of Oncogenes and Related Genes).After 7 weeks of injection, the lungs were excised, fixed in Bouinsolution overnight, and lung colonies were counted under aNikon SMZ1500 stereomicroscope (Nikon). In addition,paraffin-embedded sections of lung tissues were stained byhematoxylin–eosin (H&E) staining to examine the presenceof micrometastases.

Antibodies, immunoblotting, immunoprecipitation, andimmunofluorescence

The detailed information for primary antibodies used in thisstudy is provided in Supplementary Table S4. For immunoblot-ting analyses, cells were lysed in the modified RIPA buffer(50 mmol/L Tris–HCl, pH 7.4, 150 mmol/L NaCl, 1 % NP-40,0.25 % sodium deoxycholate, and 1 mmol/L EDTA) containing1 � protease inhibitor cocktail (Roche) and 1 � phosphatase

inhibitor cocktail (Bimake). Proteins were quantified using thebicinchoninic acid assay (Yeasen), resolved by SDS–PAGE, andtransferred onto PVDF membrane (Millipore). Antibody detec-tion was conducted using enhanced chemiluminescent substratekit (Yeasen). For immunoprecipitation (IP) analysis, cells werelysed in NP-40 lysis buffer (50 mmol/L Tris–HCl, pH8,150 mmol/L NaCl, 0.5% NP-40, 10% glycerol, 2 mmol/L MgCl2,and 1 mmol/L EDTA), and total 1 to 2 mg of exogenouslyexpressed proteins was incubated anti-Flag or anti-HA magneticbeads (Bimake) overnight at 4�C to pull down the protein–antibody complex. The resulting complexes were subjected toimmunoblotting analysis. For immunofluorescence staining,cells were fixed in 4% paraformaldehyde, permeabilized in0.1% Triton X-100, and blocked in 10% normal goat serum inPBS. Cells were incubated with primary antibodies, washed threetimes in PBS, and then incubated with the appropriate secondaryantibody conjugated with 555-Alexa (red) or 488-Alexa (green;Cell Signaling Technology), respectively. DNA staining wasperformed using Fluoroshield mounting medium with DAPI(Abcam). Microscopic analyses were performed using a Leica SP5confocal laser scanning microscopy (Leica Microsystems).

Proteomic analysisTo analyze MORC2-interacting proteins, total cellular lysates

from HEK293T cells stably expressing pCDH, Flag-MORC2 WT,andFlag-MORC2M276Iwere subjected to IP assayswith anti-Flagmagnetic beads (Bimake). After extensive washing, the boundproteins were eluted by boiling in SDS sample buffer, resolvedby SDS–PAGE, visualized by Coomassie Blue staining andsubjected to liquid chromatography–tandem mass spectrometry(LC-MS/MS) analysis as described previously (27). Data from LC-MS/MS analysis were searched against Swiss-Prot database bySEQUEST. Trans Proteomic Pipeline software (Institute of Sys-tems Biology, Seattle) was used to identify proteins based on thecorresponding peptide sequences with �95% confidence. A Pro-tein Prophet 3 probability of 0.95 was used for the proteinidentification results. The false positive ratewas less than 1%(27).

Quantitative PCRTotal RNA was isolated from cultured cells and xenograft

tumors from mice using TRIzol reagent (Invitrogen) and con-verted to cDNA using PrimeScript RT Master Mix (Takara). qPCRanalyses were performed in triplicate using SYBR Premix Ex Taq(Takara) on an Eppendorf Mastercycler ep realplex4 instrument(Eppendorf). All real-time data were normalized to GAPDH.Primer information is described in Supplementary Table S5.

Targeted exon sequencingGenomic DNA was extracted using Rapid Animal Genomic

DNA IsolationKit (SangonBiotech), and 100 ngofDNAwas usedto amplify MORC2 fragments surrounding the M276I mutation(c.828G >c) that span the exon10 region and adjacent two intronsby PCR using the indicated primers in Supplementary Table S6.Cycling conditionswere one cycle at 95�C for 3minutes, followedby 35 cycles of 94�C for 30 seconds, 58�C for 30 seconds, and72�C for 1 minute, with a final extension at 72�C for 10 minutes.PCR products were purified using the SanPrep Column DNA GelExtraction Kit (Sangon Biotech). DNA sequencing reaction wascarried out using 10 ng of purified PCR product with the BigDyeTerminator v3.1 Cycle Sequencing Kit (Applied Biosciences)under the following conditions: an initial denaturation at 96�C

Zhang et al.

Cancer Res; 78(20) October 15, 2018 Cancer Research5782




for 1 minute, followed by 25 cycles of 96�C for 10 seconds, 50 �Cfor 5 seconds, 60�C for 3 minutes. Sequencing was conductedwith a 3730XL DNA Analyzer (Applied Biosciences). Sequencechromatograms were aligned and analyzed by Sequencher soft-ware (Gene Codes).

RNA-binding protein immunoprecipitation assaysRNA-binding protein immunoprecipitation (RIP) assays were

performed using the EZ-Magna RIP Kit (Millipore) in accordancewith the manufacturer's instructions. Briefly, 6 � 107 cells werelysed in 300 mL of RIP lysis buffer containing RNase inhibitor andprotease inhibitors, and centrifuged at 14,000 rpm for 30 min-utes. About 10% of the total lysate was processed in parallel toobtain the input sample. The supernatants were incubated withanti-Flag magnetic beads (Sigma) at 4�C for 4 hours. Immuno-precipitates werewashed six timeswithwashing buffer containingRNase inhibitor and then incubated for 30 minutes at 55�C in150 mL of proteinase K buffer containing 117 mL of RIP washbuffer, 15 mL of 10% SDS, and 18 mL of 10mg/mL proteinase K torecover RNA. Immunoprecipitated RNA was purified using theconventional phenol-chloroform method and then subjected toreverse transcription and qPCR analysis as described above. qPCRprimers for amplifying the CD44 pre-mRNA region where alter-native splicing (v5) of CD44 occurs have been described previ-ously (Supplementary Table S5; ref. 28).

Statistical analysisAll data are presented as themean� standard deviation from at

least three independent experiments. The Student t test was usedfor assessing the difference between individual groups and P �0.05 was considered statistically significant.

ResultsCancer-associated mutations of MORC2 in breast cancer

The development andprogression of human cancer is driven bythe accumulation of pathogenic somatic mutations that conferoncogenic properties such as growth advantage, tissue invasionand metastasis, angiogenesis, and evasion of apoptosis (29). Toidentify cancer-associatedmutations ofMORC2 inhuman cancer,we analyzed publicly available cBioPortal for Cancer Genomics(http://www.cbioportal. org/) and Catalogue of Somatic Muta-tions in Cancer (COSMIC; https://cancer.sanger. ac.uk/cosmic)databases. In the cBioPortal database, total 379 mutations (330missense, 47 truncating, and 2 other mutations) in MORC2 havebeen reported in various types of human cancer. The positions ofthese identified mutations relative to the structural domains ofMORC2 are shown in Supplementary Fig. S1A. Unlike someoncoproteins (such as KRAS, BRAF, EGFR, and PIK3CA) withhotspotmutation sites or regions,MORC2mutations tend to spanalong the entire protein. In addition, the mutational frequency ofMORC2 varies significantly across different cancer types (Supple-mentary Fig. S1B). In the COSMIC database, total 225 mutationsin MORC2 were documented in various types of human cancer.An overview of the types of mutation and a breakdown of theobserved substitutionmutations are shown in Supplementary Fig.S2A and S2B, respectively. It was noticed that most MORC2mutations (69%) were missense mutation. These results suggestthat MORC2 mutations are relatively common in human cancer.

In breast cancer, three substitution mutations (K190N, M276I,and Y277C) of MORC2 protein were reported in both cBioPortal

and COSMIC databases and additional 4 missense mutations(R132H, P177A, I216T, andM253I)were only documented in thecBioPortal database (Supplementary Fig. S3A and S3B). Of spe-cific interest, we noticed that theM276I mutation had the highestmutational frequency, which was found in 1 of 65 (1.54%)patients with TNBC (5), and that this patient carrying theM276I mutation had angiolymphatic invasion at time of diag-nosis (Supplementary Fig. S4). To examine whether the M276Imutation is present in TNBC cell lines, we analyzed the mostcommonly used 11 TNBC cell lines in the literature and normalbreast epithelial MCF10A cell line by targeted exon sequencing(Supplementary Fig. S5). Unfortunately, we did not detect theM276Imutation ofMORC2 in those tested cell lines. This may bedue to the limited availability of TNBC cell lines and the relativelylow frequency of M276I mutation in TNBC tumors. Proteinstructural analysis showed that theM276Imutation falls betweenthe ATPase domain and the first coiled-coil domain of MORC2,and the M276 residue of MORC2 protein is highly conservedamong multiple species (Supplementary Fig. S6). Functionalanalysis through hiddenMarkovmodels (http://fathmm.biocompute.org.uk/) predicts that this mutation is pathogenic. As methi-onine (M) to isoleucine (I) substitution mutation in severalcancer relevant proteins, such as p53 (30), cyclin-dependentkinase inhibitor 2A (CDKN2A; ref. 31), and A-kinase anchoringprotein 9 (AKAP9; ref. 32), has been functionally linked withseveral types of human cancer, we set out to address the biologicalfunctions of MORC2 M276I mutation in TNBC in this study.

The M276I mutation of MORC2 does not affect its expressionlevels, stability, and subcellular localization

To examine the functional impact of the M276I mutation onMORC2 protein, we first examined protein expression levels ofendogenous MORC2 in 6 commonly used TNBC cell lines byimmunoblotting. BT474 cell linewas used as a positive control fordetection of ER, PR, and HER2 expression. As shown in Fig. 1A,MDA-MB-436 and MDA-MB-468 cell lines expressed relativelyhigh levels of endogenous MORC2, while MDA-MB-231 andHs578T cells presented low levels of MORC2. As expected, all ofthose 6 TNBC cell lines were negative in ER, PR, and HER2expression. Then, MDA-MB-231 and Hs578T cell lines werechosen to generate stable cell lines expressing empty vectorpCDH, Flag-MORC2, and Flag-MORC2M276I by lentiviral infec-tion. The triple-negative breast epithelial cell line MCF10A wasalso included in this study as a normal control. Immunoblottinganalysis demonstrated that theM276Imutation did not affect theprotein expression levels of MORC2 as compared with its WTcounterpart (Fig. 1B). Consistently, pulse-chase experimentsusing protein synthesis inhibitor cycloheximide showed that theM276Imutation didnot significantly alter the half-live ofMORC2relative to its WT counterpart (Fig. 1C). Immunofluorescentstaining showed that both WT and M276I-mutant MORC2 local-ized primarily in the nuclear (Fig. 1D). These results indicate thatthe M276I mutation of MORC2 does not affect its proteinexpression levels, stability, and subcellular localization in normalbreast epithelial cells and TNBC cells.

MORC2 M276I mutation is dispensable for cell proliferationand cell-cycle progression but promotes cell migration andinvasion in vitro

To determine biological functions of the M276I mutation inTNBC development and progression, we carried out a series of





http://www.cbioportal

; https://cancer.sanger

http://fathmm.biocompute.org.uk/

http://fathmm.biocompute.org.uk/


functional assays, includingCCK-8, colony growth assay, and cell-cycle analysis. Results showed that stable expression of both WTand M276I-mutant MORC2 in MDA-MB-231, Hs578T, andMCF10A cells did not significantly affect cell proliferation (Sup-plementary Fig. S7A), colony formation (Supplementary Fig. S7Band S7C), and cell-cycle progression (Supplementary Fig. S7D) ascomparedwith its empty vector control. Asoneof thehallmarks ofTNBC is its highly invasive and metastatic behavior (2), we nextcarried out Transwell migration and Matrigel invasion assays toevaluate the migratory and invasive capability of MDA-MB-231,Hs578T, and MCF10A cells stably expressing pCDH, WT, andM276I-mutantMORC2. As shown in Supplementary Fig. S8A andS8B,M276I-mutant MORC2-expressing cells migrated faster thanempty vector- and WT MORC2-expressing cells. Moreover, stableexpression of M276I-mutant MORC2 significantly enhanced theinvasive potential ofMDA-MB-231,Hs578T, andMCF10A cells ascompared with its WT counterpart (Supplementary Fig. S8C andS8D). These results suggest that theM276Imutation promotes thepotential of TNBCcellmigration and invasion, two crucial steps inthe metastatic cascade.

To rule out the potential effects of endogenous MORC2 onthe biological functions of exogenous expression of MORC2,we knocked out endogenous MORC2 in MDA-MB-231 andHs578T cells using the CRISPR/Cas9 system (33) and thenreexpressed empty vector pLVX, WT, and M276I-mutantMORC2 in these MORC2 KO cells. Using MDA-MB-468 cells,which express relatively high levels of endogenous MORC2(Fig. 1A) as an internal control, we selected the stable cloneswhose exogenous MORC2 expression levels are comparableto endogenous MORC2 expression levels of MDA-MB-468 cellsfor subsequent functional studies (Fig. 2A). Consistently, aseries of functional assays demonstrated that stable expressionof both WT and M276I-mutant MORC2 in MORC2-depletedMDA-MB-231 and Hs578T cells did not affect cell proliferation(Fig. 2B), colony formation (Fig. 2C), and cell-cycle progression(Fig. 2D). In contrast, expression of M276I-mutant MORC2enhanced the migratory and invasive potential of MDA-MB-231 and Hs578T cells as compared with its WT counterpart(Fig. 2E and F, respectively). Together, these results suggest thatthe M276I mutation promotes migration and invasion of TNBC

Figure 1.

The M276I mutation does not affect the expression levels, stability, and subcellular localization of MORC2. A, Immunoblotting analysis of endogenous MORC2,ERa, PR, and HER2 expression in 6 TNBC cell lines and triple-positive BT474 cell line (positive control). B, MDA-MB-231, Hs578T, and MCF10A cells stablyexpressing empty vector pCDH, Flag-MORC2, and Flag-MORC2 M276I were generated by lentiviral infection and analyzed by immunoblotting. C, Cells stablyexpressing Flag-tagged WT and M276I-mutant MORC2 were treated with 100 mg/mL of cycloheximide (CHX) for the indicated times and then subjected toimmunoblotting with the indicated antibodies (top). Relative expression levels of exogenous MORC2 (Flag-MORC2/Vinculin) are shown at the bottom.D, Cells stably expressing empty vector pCDH, Flag-MORC2, and Flag-MORC2 M276I were subjected to immunofluorescence staining with an anti-Flag antibody.Cell nuclei were counterstained with DAPI.

Zhang et al.





cells without affecting cell proliferation and cell-cycle progres-sion in vitro.

MORC2 M276I mutation does not affect primary tumorgrowth but enhances spontaneous and experimental lungmetastasis in vivo

To examine whether expression of mutant MORC2 affectsprimary tumor growth and spontaneous lung metastasis ofTNBC cells in vivo, we chose LM2-4175 cells as a model systemas this cell line is highly metastatic to lung (23). First, weknocked out endogenous MORC2 in LM2-4175 cells by theCRISPR/Cas9 system (33) and then reexpressed empty vectorpLVX, WT, and M276I-mutant MORC2 in MORC2-depletedLM2-4175 cells (Fig. 3A and B). Second, we established anorthotopic xenograft model by incubation of these establishedcell lines into the mammary fat pads of NOD/SCID mice.Results showed that expression of M276I-mutant MORC2 hadno significant effects on the growth of primary LM2-4175tumors (Fig. 3C–E), but enhanced spontaneous lung metastasisof breast tumors as compared with its WT counterpart (Fig. 3Fand G).

To further assess the effects of expression of M276I-mutantMORC2 on TNBC metastatic colonization, we established anexperimental lung metastasis model using MDA-MB-231 cellsstably expressing pCDH, WT, and M276I-mutant MORC2 byinjection into tail vein of nude mice. After 7 weeks of injection,we observed that mutant MORC2-expressing cells significantlyincreased the number ofmetastatic nodules in the lungs ofmice ascompared with empty vector- and WT MORC2-expressing cells(Fig. 3H and I). These results are also supported by histologicexamination of lung sections of these mice via H&E staining(Fig. 3J). Together, these results suggest that MORC2 M276Imutation acts as a gain-of-functionmutation that promotes TNBCmetastatic progression.

TheM276Imutation enhances the binding ability ofMORC2 tohnRNPM

To address the molecular mechanisms by which the M276Imutation promotes TNBC invasion and metastasis, we nextexamined whether the M276I mutation could affect MORC2interactome by IP coupled with LC-MS/MS analysis (27). To dothis, we generated stable HEK293T cell lines expressing pCDH,

Figure 2.

The M276I mutation does not affect cell proliferation and cell-cycle progression but promotes cell migration and invasion in vivo. A, Endogenous MORC2 inMDA-MB-231 and Hs578T cells was knocked out by the CRISPR/Cas9 system. Reexpression of empty vector pLVX, WT, and M276I-mutant MORC2 in MORC2knockout cells was performed by lentiviral infection. Expression levels of exogenous MORC2were validated by immunoblotting. MDA-MB-468 cells were used as aninternal control. B–F, MDA-MB-231 and Hs578T cells stably expressing empty vector pLVX, WT, and M276I-mutant MORC2 were subjected to cell viabilityassays using CCK-8 kit (B), colony-formation assays (C), cell-cycle analysis by flow cytometry (D), Transwell migration (E), and Matrigel invasion (F) assays.






Flag-MORC2, and Flag-MORC2 M276I by lentiviral infection(Fig. 4A). Then, total cellular lysates from these established celllines were subjected to IP analysis using anti-Flag magnetic beads(Fig. 4B), and the IP complex were subjected to the LC-MS/MSanalysis (27). To optimize specificity, proteins detectable inpCDH-expressing sample (negative control) were classified asnonspecific contaminants and eliminated from the MORC2-interacting protein list. On the basis of these analyses, we foundthat total 38 and 23 proteins specially interacted with WT andM276I-mutantMORC2, respectively, while 48 proteins interactedwith bothWTandmutantM276IMORC2 (Fig. 4C). As expressionof both WT and M276I-mutant MORC2 promotes invasion andmetastasis of TNBC cells as compared with empty vector control(Figs. 2 and 3), we further analyzed the shared 48 proteins in bothgroups (Supplementary Table S7). Gene ontology analysisusing the Protein Analysis Through Evolutionary Relationships(PANTHER) program (http://www.pantherdb.org/) revealed themolecular functions of those 48 proteins are involved in poly(A)RNA-binding, RNA-binding, cadherin binding involved in cell–cell adhesion, protein binding involved in cell–cell adhesion and

cell adhesion (Fig. 4D). Among them, the molecular functions of22 proteins are enriched in ploy(A) RNA-binding and RNA-binding (Supplementary Table S8). Of specific interest, it wasnoticed that 5 of 22 proteins are involved in RNA alternativesplicing, including hnRNPM, splicing factor 3B subunit 1(SF3B1), serine/arginine-rich splicing factor 3 (SRSF3), splicingfactor proline and glutamine rich (SFPQ), and U2 small nuclearRNA auxiliary factor 1 (U2AF1).

To validate the above proteomic results, we next carried out aseries of sequential IP and immunoblotting analyses. As shownin Fig. 4E, Flag-MORC2 was associated with endogenoushnRNPM in HEK293T, MDA-MB-231, and Hs578T cells. More-over, this noted interaction between MORC2 and hnRNPM wasenhanced inM276I-mutantMORC2-expressing cells as comparedwith its WT counterpart, suggesting that the M276I mutationenhances the binding ability of MORC2 to hnRNPM. In contrast,there was no difference in the interaction of SRSF3 with both WTand M276I-mutant MORC2 (Fig. 4F). In addition, we didnot observe any interaction of both WT and mutant MORC2with SRSF1 (Fig. 4G), U2AF1 (Fig. 4H), and SFPQ (Fig. 4I).

Figure 3.

MORC2 M276I mutation does not affect primary tumor growth but enhances spontaneous and experimental lung metastasis of TNBC cells in vivo. A and B,Endogenous MORC2 was knocked out in LM2-4175 cells by the CRISPR/ Cas9 system (A) and then empty vector pLVX, WT, and M276I-mutant MORC2 werereexpressed in MORC2 knockout cells by lentiviral infection (B). The expression status of endogenous and exogenous MORC2 in these established cell lineswas validated by immunoblotting. C–G, LM2-4175 cells stably expressing empty vector pLVX, WT, and M276I-mutant MORC2 were transplanted into themammary fat pads of NOD/SCID mice. After 6 weeks of incubation, mice were killed and primary tumors and lung tissues were harvested. Images ofharvested xenograft tumors (C), tumor weight (D), growth curves of xenograft tumors (E), and representative images of H&E-stained lung sections of miceharboring orthotopic LM2-7175 tumors (F) are shown. G, Quantitative results of spontaneous lung metastasis determined by the percent metastatic lung surfacearea relative to total lung surface area. H–J, MDA-MB-231 cells stably expressing pCDH, WT, and M276I-mutant MORC2 were injected into nude mice through thetail vein, and the lungs were harvested after 7 weeks of injection. Representative images of lung metastasis (H), quantitative results of lung nodules (I), andrepresentative images of H&E-stained sections of lung tissues (J) are shown.

Zhang et al.




http://www.pantherdb.org/


Consistently, immunofluorescent staining showed that WT andmutant MORC2 colocalized with hnRNPM in MDA-MB-231 andHs578T cells (Fig. 4J). Further, the enhanced interaction betweenmutant MORC2 and hnRNPM was validated in cotransfectedHEK293T cells with exogenously expressed Flag-MORC2 andHA-hnRNPM plasmids (Fig. 4K).

In addition to hnRNPM (19), other abundant members of thehnRNP protein family, such as hnRNPA1 (34) and hnRNPL (35),are also involved in alternative splicing events driving tumori-genesis and cancer progression. To examine whether MORC2interacts with either hnRNPA1 or hnRNPL, HEK293T cellsstably expressing pCDH, Flag-MORC2, and Flag-MORC2M276I were subjected to IP analysis using anti-Flag magneticbeads. Immunoblotting analysis showed that there were nodetectable interactions of MORC2 with either hnRNPA1 orhnRNPL (Supplementary Fig. S9A). As a positive control, werepeatedly demonstrated an enhanced interaction between

mutant MORC2 and hnRNPM as compared with its WT coun-terpart. To further examine the specificity of the MORC2–hnRNPM interaction, we knocked down hnRNPL in HEK293Tcells stably expressing pCDH and Flag-MORC2 by specific siRNAstargeting hnRNPL. Results showed that knockdown of hnRNPLdid not affect the interaction of MORC2 with hnRNPM (Supple-mentary Fig. S9B). These results suggest that MORC2 interactionwith hnRNPM is specific.

hnRNPM is required for M276I-mutant MORC2-mediatedinvasion and metastasis of TNBC cells

hnRNPM has been shown to promote breast cancer invasionandmetastasis (19, 22). To examinewhether hnRNPM is involvedin mutant MORC2-mediated metastatic progression of TNBCcells, we knocked down endogenous hnRNPM in WT andM276I-mutant MORC2 expressing MDA-MB-231 and Hs578Tcells using two specific shhnRNPMs (Fig. 5A). Transwell

Figure 4.

The M276I mutation enhances the binding ability of MORC2 to hnRNPM. A and B, HEK293T cells stably expressing pCDH, Flag-MORC2, and Flag-MORC2M276I were validated by immunoblotting (A) and then subjected to IP analysis with anti-Flag magnetic beads. The bound proteins were isolated on 8% SDS–PAGEgel and stained using Coomassie brilliant blue (B). C, LC-MS/MS was used to identify MORC2-interacting proteins. The numbers of the identified proteins ineach group are shown.D, Bioinformatics analysis of themolecular functions of the identified proteins that interacted with both Flag-MORC2 and Flag-MORC2M276Iusing PANTHER program. E, Total cellular lysates from HEK293T, MDA-MB-231, and Hs578T cells stably expressing pCDH, Flag-MORC2, and Flag-MORC2M276Iwere subjected to IP analysiswith anti-Flagmagnetic beads, followedby immunoblottingwith the indicted antibodies. F–I, Total cellular lysates fromHEK293Tcells stably expressing pCDH, Flag-MORC2, and Flag-MORC2M276I were subjected to the sequential IP and immunoblotting analysis with the indicted antibodies. J,MDA-MB-231 and Hs578T cells stably expressing pCDH, Flag-MORC2, and Flag-MORC2 M276I were subjected to immunofluorescence staining with theindicated antibodies. Cell nuclei were counterstained with DAPI. K, HEK293T cells were transfected with the indicated expression vectors. After 48 hours oftransfection, total cellular lysates were subjected to the sequential IP and immunoblotting analysis with the indicated antibodies.






migration andMatrigel invasion assays demonstrated that knock-down of hnRNPM reduced cell migratory (Fig. 5B and C) andinvasive (Fig. 5D and E) potential of WT and mutant MORC2-expressing MDA-MB-231 and Hs578T cells when compared withcontrol shNC-infected cells. Consistently, in vivo experimentallung metastasis assays by injection of these established celllines into tail vein of immunodeficient mice also demonstratedthat knockdown of hnRNPM reduced WT- and M276I-mutantMORC2-induced lung metastatic potential (Fig. 5F–H). Collec-tively, these results suggest that MORC2 M276I mutation pro-motes invasion and metastasis of TNBC cells through, at least inpart, hnRNPM-mediated signaling.

MORC2 M276I mutation regulates hnRNPM-mediated CD44splicing switch

hnRNPM has been shown to regulate alternative splicing ofFGFR2 and CD44, which are implicated in EMT and cancerprogression (18, 19). We next examined whether the M276I

mutation promotes TNBC progression through regulatinghnRNPM-mediated alternative RNA splicing and EMT. To addressthis question, we chose MCF10A cell line as a model system,which can undergo EMT in response to various extracellular andintracellular signals. MCF10A cells stably expressing pCDH, WT,andM276I-mutantMORC2were generated by lentiviral infectionand overexpression ofMORC2was validated by immunoblotting(Fig. 6A). qPCR analysis showed that mutant MORC2-expressingcells had a significant upregulation of CD44s with a concomitantdownregulation of CD44v5/6 (Fig. 6B). In contrast, expression ofboth WT and mutant MORC2 did not significantly affect thealternative splicing of FGFR2 (Fig. 6C). To verify these results, wenext examined the expression levels of CD44s and CD44v5/6 byqPCR inprimary tumors from the LM2-4175orthotopic xenograftmodels (Fig. 3A–G). Results showed that the expression levels ofCD44s were increased, whereas the levels of CD44v5/6 weredecreased, in M276I-mutant MORC2-expressing tumors as com-pared with its WT controls (Fig. 6D). Furthermore, knockdown of

Figure 5.

hnRNPM is required for M276I-mutant MORC2-mediated migration, invasion, and lung metastasis of TNBC cells. A, MDA-MB-231 and Hs578T cells stablyexpressing Flag-MORC2 and Flag-MORC2 M276I were infected with shNC and two different shRNAs targeting hnRNPM. After antibiotic selection, knockdowneffects of hnRNPM by shhnRNPMs were validated by immunoblotting. B–E, The resultant stable cell lines were subjected to Transwell migration (B–C) andMatrigel invasion (D–E) assays. Representative images of cell migration (B) and invasion (D) and quantitative results (C and E for cell migration and invasion,respectively) are shown. F–G, MDA-MB-231 cells stably expressing WT and M276I-mutant MORC2 were infected with lentiviral vectors expressing shNC andshhnRNPM #C. The resultant stable cell lines were injected into nude mice through the tail vein, and the lungs were harvested after 7 weeks of injection.Representative images of lung metastasis (F), representative images of H&E-stained sections of lung tissues (G), and quantitative results of lung nodules(H) are shown.

Zhang et al.





hnRNPM by shRNAs in mutant MORC2-expressing MCF10Acells showed decreased CD44s mRNA with a concomitantincrease in CD44v5/6 mRNA (Fig. 6E), indicating that hnRNPMis involved in mutant MORC2-mediated CD44 splicing switch.Following these observations, we carried out RIP assays (36) toexamine the binding of MORC2 to CD44 pre-mRNA. RNA thatwas bound to immunoprecipitated MORC2 was analyzed byqPCR. Results showed that the M276I mutation enhanced thebinding ability of MORC2 to CD44 pre-mRNA as comparedwith its WT counterpart (Fig. 6F). Moreover, knockdown ofhnRNPM by a specific shRNA targeting hnRNPM (shhnRNPM#C) in MCF10A cells stably expressing Flag-MORC2 and Flag-MORC2 M276I (Fig. 6G) attenuated the noted binding ofMORC2 to CD44 pre-mRNA (Fig. 6H). These results suggest

that hnRNPM mediates MORC2 recruitment to the variableexon region of CD44 pre-mRNA.

The CD44 isoform switching is required for breast cancer cellsto undergo EMT (19, 21). EMT is characterized by the loss ofepithelial characteristics and the acquisition of a mesenchymalphenotypewith enhancedmigratory and invasive properties (37).This phenotypic conversion is tightly controlled by a network oftranscription factors, including Snail, Slug, Twist, andZeb1,whichtranscriptionally suppress E-cadherin expression (38). To deter-mine whether MORC2 M276I mutation regulates hnRNPM-mediated EMT, we examined the expression levels of 6 EMT-associated marker molecules, including E-cadherin, N-cadherin,Snail, Slug, Twist, and Zeb1, in MCF10A cells stably expressingpCDH, WT, and M276I-mutant MORC2. Immunoblotting

Figure 6.

The M276I mutation promotes hnRNPM-mediated CD44 splicing switch. A, MCF10A cells stably expressing pCDH, Flag-MORC2, and Flag-MORC2 M276I werevalidated by immunoblotting. B and C, qPCR analysis of CD44s and CD44 v5/6 (B) and FGFR2 IIIb and FGFR2 IIIc (C) expression in MCF10A cells stablyexpressing pCDH, Flag-MORC2, and Flag-MORC2 M276I. D, qPCR analysis of CD44s and CD44 v5/6 expression in primary tumors from the LM2-4175 orthotopicxenograft models (Fig. 3A–G). E, MCF10A cells stably expressing Flag-MORC2 M276I were infected with lentiviral vectors expressing shNC and two differentshRNAs targeting hnRNPM. After antibiotic selection, CD44s and CD44 v5/6 expression levels were determined by qPCR. F, MCF10A cells stably expressingpCDH, Flag-MORC2, andFlag-MORC2M276Iwere subjected toRIP assays. The immunoprecipitatedRNAwas analyzedbyqPCR. The resultswere normalized to inputof each sample. G and H, MCF10A cells stably expressing pCDH, Flag-MORC2, and Flag-MORC2 M276I were infected with lentiviral vectors expressing shNCand shhnRNPM (#C). Knockdown effects of hnRNPM were verified by immunoblotting (G). The resultant cell lines were subjected to RIP assays as describedabove (H). I, Lysates from MCF10A cells stably expressing pCDH, WT, and M276I-mutant MORC2 were subjected to immunoblotting analysis with the indicatedantibodies. J, qPCR analysis of E-cadherin, N-cadherin, Snail, Slug, Twist, and Zeb1 mRNA levels in MCF10A cells stably expressing pCDH, WT, and M276I-mutant MORC2. K and L, MCF10A cells stably expressing Flag-MORC2 M276I were infected with lentiviral vectors expressing shNC and two different shRNAstargeting hnRNPM. After antibiotic selection, cells were subjected to immunoblotting (K) and qPCR (L) analysis.






(Fig. 6I) and qPCR (Fig. 6J) analyses showed that the mesenchy-mal markers N-cadherin and Slug were upregulated, while theepithelial marker E-cadherin was downregulated, in M276I-mutant MORC2-expressing cells as compared with pCDH andWT MORC2 expressing cells. Interestingly, expression of M276I-mutant MORC2 in MCF10A cells did not significantly affect theprotein and mRNA levels of Snail, Twist, and Zeb1 as comparedwith its WT counterpart (Fig. 6I and J). These results suggest thatSlug may be involved in the MORC2–hnRNPM–CD44 pathway-mediated EMT event. Furthermore, knockdown of hnRNPM inMCF10A cells expressing mutant MORC2 led to an increase in E-cadherin and a decrease in N-cadherin at both protein andmRNAlevels (Fig. 6K and L). Together, these results indicate that theM276I-mutant MORC2 promotes TNBC invasion and metastasisthrough, at least in part, regulating hnRNPM-mediated CD44splicing program and EMT.

DiscussionIn this study, we present several important findings concerning

the functional and mechanistic role of MORC2 M276I mutationin TNBC progression (Fig. 7).

First, the M276I mutation is a gain-of-function mutation thatpromotes TNBC invasion and metastasis. MORC2 is a newlyidentified chromatin remodeling protein with emerging roles inthemaintenance of genome integrity in response to DNA damage(10) and epigenetic regulation of gene transcription (11, 12). Notsurprisingly, genetic alternations of this gene may be linked tohuman diseases. Indeed, MORC2 has been shown to be upregu-lated in human cancers (39) and promotes the development andprogression of breast (16, 40), gastric (41), and liver cancers (42).More interestingly, mutations of MORC2 have been causallyimplicated in several neurologic diseases (11, 13–15). Althougha total of 379 mutations of MORC2 have been documented invarious types of human cancer (Supplementary Fig. S1A), thefunctional consequences of these identified mutations incancer development and progression have not yet been explored.In the present study, we provide the first evidence that one of suchmutations (M276I) was functionally important for TNBCprogression. These findings could advance our understandingof the functional role for MORC2 mutations in human cancer,in addition to the documented functional connection betweenMORC2mutations and several neurologic disorders (11, 13–15).In addition, these results provide a clue to further investigate the

Figure 7.

The proposed working model.hnRNPM directly binds to CD44pre-mRNA and promotes skipping ofCD44 variable exons, resulting ingenerating the mesenchymal-specificCD44s splice isoform. MORC2,especially M276I-mutant MORC2,forms a complex with hnRNPM tofacilitate the expression of themesenchymal isoformCD44s over theepithelial isoform CD44v. This CD44isoform switch is necessary for EMTand breast cancer progressionthrough multiple downstreampathways.

Zhang et al.





biological functionsof all identifiedmutationsofMORC2 in cancerprogression and therapeutic response using well-establishedrecombination-based mutation barcoding library (43, 44) andgenetically engineered mouse models in the near future.

Second, the M276I mutation of MORC2 enhances its bindingability to hnRNPM, an abundant component of human hnRNPcomplexes. The splicing of pre-mRNAs is an essential step ofeukaryotic gene expression, which is tightly controlled by theactivities of splicing regulators, such as hnRNPs or serine/ argi-nine-rich (SR) proteins (17). By means of a combination ofbiochemical approaches, we demonstrated that the M276I muta-tion does not affect MORC2 protein expression levels, stability,and nuclear localization (Fig. 1), but enhances the binding abilityof MORC2 to hnRNPM (Fig. 4). Previous studies have demon-strated that methionine and isoleucine residues are important forforming the protein–protein interaction interface (45, 46). Forinstance, it hasbeen reported that theM531Imutation inCDKN2Aloses its ability to interact with cyclin-dependent kinase 4 (CDK4;ref. 31). In support of our findings, a recent study on crystalstructure of MORC2 suggests that the M276I mutation wouldcause a conformational change that may affect its interaction withotherproteins. In addition, thismutationwouldnot be expected tosubstantially alter the stability or half-life of MORC2 (47).

Third, we establish a mechanistic link between the oncogenicactivity of theM276Imutation and the hnRNPM-mediated CD44splicing program. Previous studies have shown that hnRNPM haskey roles in regulating pre-mRNA splicing of several cancerrelevant genes such as CD44 (19). CD44 splice isoform switchingfrom the splicing variants (CD44v) to the standard form (CD44s)is essential for EMT and breast cancer metastasis (19, 21). Con-sequently, depletion of CD44s inhibits breast cancermetastasis inmice (48), and expression of CD44s rescues the impaired met-astatic phenotype by hnRNPM depletion (19). qPCR analysisdemonstrated that expression of M276I-mutant MORC2 pro-motes a switch of CD44 splice isoform from CD44v to CD44s(Fig. 6). These findings are consistent with a recent report thathnRNPM promotes breast cancer metastasis through activatingCD44 splicing program and EMT (19). Moreover, knockdown ofhnRNPM attenuates the M276I mutation-mediated CD44 splic-ing switch, EMT, cell migration, invasion, and metastasis (Figs. 5and 6). These results indicate that the M276I mutation promotesbreast cancer invasion and metastasis through, at least in part,activating hnRNPM-mediated CD44 alternative splicing switchand EMT. Given that alternative splicing events of CD44 havebeen widely documented in various types of human cancerincluding melanoma (49), we cannot rule out the possibility thatother MORC2 mutations may regulate the development andprogression of human cancer through regulating CD44 splicingprogram. Interestingly, a recent report showed that CD44s acti-vates the expression of EMT-inducing transcription factor Zeb1,which in turn controls CD44s splicing by repression of epithelial

splicing regulatory protein 1, thus forming a CD44s–Zeb1 feed-back loop tomaintain EMTand stemness properties in cancer cells(50). In our study, we noticed that expression of M276I-mutantMORC2 in MCF10A cells upregulates the protein and mRNAlevels of Slug, but not Snail, Twist, and Zeb1, as compared with itsWT counterpart (Fig. 6I and J). Therefore, it remains to bedetermined whether the hnRNPM–CD44s pathway promotesEMT and breast cancer metastasis through regulating the expres-sion of Slug, in addition to Zeb1.

In conclusion, findings presented here demonstrate a gain-of-function mutation of MORC2 in TNBC progression and providemechanistic insights into the oncogenic mutation of MORC2involving hnRNPM-mediated alternative splicing and EMT. Thesefindings broaden our understanding of the genetic heterogeneityof TNBC and highlight the importance of analyzing uniquepatient-derived mutations for the development of personalizedtherapies in the era of precision oncology.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Z.-M. Shao, D.-Q. LiDevelopment of methodology: F.-L. ZhangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F.-L. Zhang, J.-L. Cao, H.-Y. Xie, R. Sun, L.-F. YangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F.-L. Zhang, J.-L. Cao, H.-Y. Xie, L.-F. YangWriting, review, and/or revision of the manuscript: F.-L. Zhang, D.-Q. LiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F.-L. Zhang, J.-L. Cao,H.-Y. Xie, R. Sun, L.-F. YangStudy supervision: D.-Q. Li

AcknowledgmentsWe would like to acknowledge the staff members of the pathology core

facility (Shanghai Cancer Center, Fudan University), the proteomic center(Institute of Biomedical Sciences, Fudan University), and the animal resourcecenter (State Key Laboratory of Oncogene and Related Gene, Shanghai JiaotongUniversity) for technical assistance. We are grateful to members of the Lilaboratory for technical assistance.

The work in the Li laboratory is supported, in whole or in part, by theNational Natural Science Foundation of China (No. 81372847, 81572584, and81772805), the Program for Professor of Special Appointment (Eastern Schol-ar) at Shanghai Institutions of Higher Learning (No. 2013-06), the Science andTechnology Innovation Action Plan of Shanghai Municipal Science and Tech-nology Commission (No. 16JC1405400), and start-up fund for new investi-gators from Fudan University (to D.Q. Li).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 17, 2017; revised April 14, 2018; accepted July 31, 2018;published first August 9, 2018.

References1. Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L. Triple-negative

breast cancer: challenges and opportunities of a heterogeneous disease.Nat Rev Clin Oncol 2016;13:674–90.

2. Foulkes WD, Smith IE, Reis-Filho JS. Triple-negative breast cancer. N Engl JMed 2010;363:1938–48.

3. Cleator S,HellerW,CoombesRC. Triple-negative breast cancer: therapeuticoptions. Lancet Oncol 2007;8:235–44.

4. Comprehensive molecular portraits of human breast tumours. Nature2012;490:61–70.

5. Shah SP, Roth A, Goya R, Oloumi A, Ha G, Zhao Y, et al. The clonal andmutational evolution spectrum of primary triple-negative breast cancers.Nature 2012;486:395–9.

6. Craig DW, O'Shaughnessy JA, Kiefer JA, Aldrich J, Sinari S, Moses TM, et al.Genome and transcriptome sequencing in prospective metastatic triple-negative breast cancer uncovers therapeutic vulnerabilities. Mol CancerTher 2013;12:104–16.

7. Li DQ, Nair SS, Kumar R. The MORC family: new epigenetic regulators oftranscription and DNA damage response. Epigenetics 2013;8:685–93.






8. Iyer LM, Abhiman S, Aravind L.MutL homologs in restriction-modificationsystems and the origin of eukaryotic MORC ATPases. Biol Direct 2008;3:8.

9. Perry J, Zhao Y. The CW domain, a structural module shared amongstvertebrates, vertebrate-infecting parasites and higher plants. Trends Bio-chem Sci 2003;28:576–80.

10. Li DQ, Nair SS, Ohshiro K, Kumar A, Nair VS, Pakala SB, et al. MORC2signaling integrates phosphorylation-dependent,ATPase-coupled chromatinremodeling during the DNA damage response. Cell Rep 2012;2:1657–69.

11. Tchasovnikarova IA, Timms RT, Douse CH, Roberts RC, Dougan G, King-ston RE, et al. Hyperactivation of HUSH complex function by Charcot-Marie-Tooth disease mutation in MORC2. Nat Genet 2017;49:1035–44.

12. Liu N, Lee CH, Swigut T, Grow E, Gu B, Bassik MC, et al. Selective silencingof euchromatic L1s revealed by genome-wide screens for L1 regulators.Nature 2018;553:228–32.

13. Albulym OM, Kennerson ML, Harms MB, Drew AP, Siddell AH, Auer-GrumbachM, et al. MORC2mutations cause axonal Charcot-Marie-Toothdisease with pyramidal signs. Ann Neurol 2016;79:419–27.

14. Sevilla T, Lupo V, Martinez-Rubio D, Sancho P, Sivera R, Chumillas MJ,et al. Mutations in the MORC2 gene cause axonal Charcot-Marie-Toothdisease. Brain 2016;139:62–72.

15. ZanniG,NardellaM,Barresi S,BellacchioE,NicetaM,CiolfiA, et al.Denovop.T362R mutation in MORC2 causes early onset cerebellar ataxia, axonalpolyneuropathy and nocturnal hypoventilation. Brain 2017;140:e34.

16. Chen LH, Kuo WH, Tsai MH, Chen PC, Hsiao CK, Chuang EY, et al.Identification of prognostic genes for recurrent risk prediction in triplenegative breast cancer patients in Taiwan. PLoS One 2011;6:e28222.

17. Wahl MC, Will CL, Luhrmann R. The spliceosome: design principles of adynamic RNP machine. Cell 2009;136:701–18.

18. Hovhannisyan RH, Carstens RP. Heterogeneous ribonucleoprotein m is asplicing regulatory protein that can enhance or silence splicing of alter-natively spliced exons. J Biol Chem 2007;282:36265–74.

19. Xu Y, Gao XD, Lee JH, Huang H, Tan H, Ahn J, et al. Cell type-restrictedactivity of hnRNPM promotes breast cancer metastasis via regulatingalternative splicing. Genes Dev 2014;28:1191–203.

20. Warzecha CC, Sato TK, Nabet B, Hogenesch JB, Carstens RP. ESRP1 andESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing. MolCell 2009;33:591–601.

21. Brown RL, Reinke LM, Damerow MS, Perez D, Chodosh LA, Yang J, et al.CD44 splice isoform switching in human and mouse epithelium isessential for epithelial-mesenchymal transition and breast cancer progres-sion. J Clin Invest 2011;121:1064–74.

22. Sun H, Liu T, Zhu D, Dong X, Liu F, Liang X, et al. HnRNPM and CD44sexpression affects tumor aggressiveness and predicts poor prognosis inbreast cancer with axillary lymph node metastases. Genes ChromosomesCancer 2017;56:598–607.

23. Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, et al. Genes thatmediate breast cancer metastasis to lung. Nature 2005;436:518–24.

24. Young L, Sung J, Stacey G, Masters JR. Detection of Mycoplasma in cellcultures. Nat Protoc 2010;5:929–34.

25. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genomeengineering using the CRISPR-Cas9 system. Nat Protoc 2013;8:2281–308.

26. Wagenblast E, Soto M, Gutierrez-Angel S, Hartl CA, Gable AL, Maceli AR,et al. A model of breast cancer heterogeneity reveals vascular mimicry as adriver of metastasis. Nature 2015;520:358–62.

27. Zhao J, Yu H, Lin L, Tu J, Cai L, Chen Y, et al. Interactome study suggestsmultiple cellular functions of hepatoma-derived growth factor (HDGF).J Proteomics 2011;75:588–602.

28. Tripathi V, Sixt KM, Gao S, Xu X, Huang J,Weigert R, et al. Direct regulationof alternative splicing by SMAD3 through PCBP1 is essential to the tumor-promoting role of TGF-beta. Mol Cell 2016;64:549–64.

29. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell2011;144:646–74.

30. Chen WG, Chen YY, Kamel OW, Koo CH, Weiss LM. p53 mutations inHodgkin's disease. Lab Invest 1996;75:519–27.

31. Sun S, Pollock PM, Liu L, Karimi S, Jothy S, Milner BJ, et al. CDKN2Amutation in a non-FAMMMkindredwith cancers atmultiple sites results ina functionally abnormal protein. Int J Cancer 1997;73:531–6.

32. Frank B, Wiestler M, Kropp S, Hemminki K, Spurdle AB, Sutter C, et al.Association of a common AKAP9 variant with breast cancer risk: a collab-orative analysis. J Natl Cancer Inst 2008;100:437–42.

33. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al.Genome-scale CRISPR-Cas9 knockout screening in human cells. Science2014;343:84–7.

34. Loh TJ, Moon H, Cho S, Jang H, Liu YC, Tai H, et al. CD44 alternativesplicing and hnRNP A1 expression are associated with the metastasis ofbreast cancer. Oncol Rep 2015;34:1231–8.

35. Loh TJ, Cho S, Moon H, Jang HN, Williams DR, Jung DW, et al. hnRNP Linhibits CD44 V10 exon splicing through interacting with its upstreamintron. Biochim Biophys Acta 2015;1849:743–50.

36. Selth LA, Close P, Svejstrup JQ. Studying RNA-protein interactions in vivoby RNA immunoprecipitation. Methods Mol Biol 2011;791:253–64.

37. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymaltransitions in development and disease. Cell 2009;139:871–90.

38. Lamouille S, Xu J, Derynck R. Molecular mechanisms of epithelial- mes-enchymal transition. Nat Rev Mol Cell Biol 2014;15:178–96.

39. Qian-Shan D, Li Z, Bi-Cheng W, Zhi Z, Xian-Qiong Z, Peng-Bo C, et al.Aberrant high expression level of MORC2 is a common character inmultiple cancers. Hum Pathol 2018;76:58–67.

40. Liao XH, Zhang Y, Dong WJ, Shao ZM, Li DQ. Chromatin remodelingproteinMORC2 promotes breast cancer invasion andmetastasis through aPRD domain-mediated interaction with CTNND1. Oncotarget 2017;8:97941–54.

41. Wang G, Song Y, Liu T, Wang C, Zhang Q, Liu F, et al. PAK1-mediatedMORC2 phosphorylation promotes gastric tumorigenesis. Oncotarget2015;6:9877–86.

42. Wang T,Qin ZY,Wen LZ,Guo Y, LiuQ, Lei ZJ, et al. Epigenetic restriction ofHippo signaling by MORC2 underlies stemness of hepatocellular carci-noma cells. Cell Death Differ 2018;[PMID: 29555977].

43. Chen L, Yang L, Yao L, Kuang XY, Zuo WJ, Li S, et al. Characterization ofPIK3CA and PIK3R1 somatic mutations in Chinese breast cancer patients.Nat Commun 2018;9:1357.

44. Ng PK, Li J, Jeong KJ, Shao S, Chen H, Tsang YH, et al. Systematicfunctional annotation of somatic mutations in cancer. Cancer Cell2018;33:450–62.

45. Kysilka J, Vondrasek J. Towards a better understanding of the specificity ofprotein-protein interaction. J Mol Recognit 2012;25:604–15.

46. Ma B, Nussinov R. Trp/Met/Phe hot spots in protein-protein interactions:potential targets in drug design. Curr Top Med Chem 2007;7:999–1005.

47. Douse CH, Bloor S, Liu Y, ShaminM, Tchasovnikarova IA, Timms RT, et al.Neuropathic MORC2 mutations perturb GHKL ATPase dimerizationdynamics and epigenetic silencing by multiple structural mechanisms.Nat Commun 2018;9:651. doi: 10.1038/s41467-018-03045-x.

48. Zhao P, Xu Y, Wei Y, Qiu Q, Chew TL, Kang Y, et al. The CD44s spliceisoform is a central mediator for invadopodia activity. J Cell Sci2016;129:1355–65.

49. Prochazka L, Tesarik R, Turanek J. Regulation of alternative splicing ofCD44 in cancer. Cell Signal 2014;26:2234–9.

50. Preca BT, Bajdak K, Mock K, Sundararajan V, Pfannstiel J, Maurer J, et al. Aself-enforcing CD44s/ZEB1 feedback loop maintains EMT and stemnessproperties in cancer cells. Int J Cancer 2015;137:2566–77.


Zhang et al.




2018;78:5780-5792. Published OnlineFirst August 9, 2018.Cancer Res Fang-Lin Zhang, Jin-Ling Cao, Hong-Yan Xie, et al. Metastasis in Triple-Negative Breast CancerhnRNPM-Mediated CD44 Splicing Switch to Promote Invasion and Cancer-Associated MORC2-Mutant M276I Regulates an

Updated version

10.1158/0008-5472.CAN-17-1394doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2018/08/09/0008-5472.CAN-17-1394.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/78/20/5780.full#ref-list-1

This article cites 49 articles, 5 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/78/20/5780To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-17-1394

http://cancerres.aacrjournals.org/content/suppl/2018/08/09/0008-5472.CAN-17-1394.DC1

http://cancerres.aacrjournals.org/content/78/20/5780.full#ref-list-1

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/78/20/5780


cancer-associated morc2-mutant m276i regulates an hnrnpm … · molecular cell biology...

Documents