https://openworks.mdanderson.org/sumexp21/1196/thumbnail.jp

Brown, Powel H

Han, Margaret Y

Ma, Yanxia

Mazumdar, Abhijit

Qian, Jing

Tahaney, William M

English

OpenWorks @ MD Anderson

v VCP expression is relatively higher in TNBC celllines than non-TNBC cell lines.v VCP binds with SOX9 in TNBC as shown by IPanalysis.v VCP regulates SOX9 downstream geneexpression (RGCC and PRTN3) in TNBC.v Knockdown of VCP suppresses TNBC cell growth.HypothesisVCP knockdown disrupts the function of SOX9 andreduces cancer cell growth and survival in TNBCIntroductionv Oncomine Analysis: VCP mRNA expression and5-year overall survival rates were compared for sixdifferent breast cancer datasets (6).v qRT-PCR and Western Blotting Assay: mRNAlevels and protein level of indicated factors wereexamined by qRT-PCR and Western blottingassays.v siRNA transfection for VCP knockdown:Transfection of cells was performed with a pool ofthree independent siRNA duplexes targeting VCPfor 48 hours.v Cell Growth Assay: After treatment with siRNA for48 hours, cells were harvested and reseeded into24-well plates. Cell number was recorded byCountess Automated Cell counter (Invitrogen, LifeTechnologies, Grand Island, NY) on Day 1, 3, 5, 7.v Immunoprecipitation: used to detect VCP as aSOX9 binding proteinv Bioinformatic Analysis: mRNA-seq data wereanalyzed by Drs. Ganiraju Manyam and WenyiWang from the Department of Bioinformatics andComputational Biology, Department ofBiostatistics, The University of Texas MDAnderson Cancer Center.Validation of Valosin-Containing Protein (VCP) as a Therapeutic Target for Triple Negative Breast CancerMargaret Y. Han1, 2, Yanxia Ma1, William M. Tahaney1, 3, Jing Qian1, Abhijit Mazumdar1, Powel H. Brown1, 3, 41Department of Clinical Cancer Prevention, The University of Texas M. D. Anderson Cancer Center, Houston, TX 770302Department of Biosciences, Rice University, Houston, TX 770053Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 770304Department of Breast Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030ResultsVCP Plays a Critical Role in TNBCMethodsReferencesConclusionsResultsVCP and SOX9 BindingVCP Knockdown and Cell GrowthSOX9 and VCP Expression There are few effective targeted therapies available fortriple-negative breast cancers (TNBCs), the mostaggressive form of breast cancer (1, 2). A betterunderstanding of critical molecular regulators of TNBCis necessary to develop effective targeted therapies.We previously determined that the transcription factorsSOX9 is highly expressed in TNBCs and is requiredfor TNBC cell survival and metastasis (3, 4). However,SOX9 is difficult to target directly for therapeuticsdevelopment, so it is of great interest to evaluate moredruggable SOX9 binding proteins as potential targets.Valosin-containing protein (VCP) is a member of theAAA-ATPase superfamily whose elevated expressionis correlated with increased metastatic potential andpoor prognosis in some cancers (5). VCP has beenidentified as a SOX9 binding protein throughimmunoprecipitation (IP)/mass spectrometry.CAFigure 2. Analysis of SOX9 and VCP expression in 3 Non-TNBC cell linesand 8 TNBC cell lines. A. Western blotting analysis shows that SOX9 has ahigher presence in TNBC cell lines (blue lanes) than Non-TNBC cell lines(black lanes). B. VCP also has a higher presence in TNBC cell lines thanNon-TNBC cell lines. Vinculin was included as a load control. C. HighermRNA expression of SOX9 (first row) and VCP (bottom two rows) in TNBCvs. Non-TNBC cell lines. Colors are z-score normalized to depict relativevalues within rows. The Oncomine™ Platform (Thermo Fisher, Ann Arbor, MI)was used for analysis and visualization.Figure 1. VCP plays critical role inTNBC. A. VCP presents relativelyhigher expression level in TNBCsthan non-TNBCs. B. Expression ofVCP correlated with SOX9expression C. VCP high expressionis associated with worse survivalrate in Curtis breast cancer dataset.**: p< 0.01AABVCP Regulates SOX9 Downstream Genes Figure 3. IP: VCP binds to SOX9A. Western blotting analysis of IPin MDA MB-231.B. Immunoprecipitation todetermine VCP as a SOX9binding protein. Graphic createdusing BioRender software.Figure 4. Knockdown of VCP in MDA MB-231 suppressed cell growth invitro. Cells were treated with siLuc, siVCP ORF, and siVCP 3’UTR for 48hours before harvest and reseeding.A. Western blotting analysis showing successful knockdown of VCP insiVCP ORF and siVCP 3’UTR. VCP was only present in the control.B. Cell growth curve. Cells were harvested and reseeded into 24-wellplates after treatment with siRNA. Cell number was recorded on day 1,3, 5, and 7 using the Countess Automated Cell. **: p< 0.01; ***: p< 0.001A BBFigure 5. Knockdown of VCP reduces SOX9 downstream geneexpression in MDA MB-468. A. Western blotting assay showingsuccessful knockdown of VCP with no protein present in siRNA treatedsamples. B. Knockdown of VCP reduces relative mRNA expression ofSOX9 downstream genesAcknowledgementsSpecial thanks to Dr. Yanxia Ma and Dr. PowelBrownfor their guidance and mentorship and the restof the Brown Lab for their technical assistance. I amalso grateful to the CPRIT-CURE program for thisopportunity.VCP Cell Line Western Blot v Perform qRT-PCR to evaluate mRNA levels ofVCP in TNBC and non-TNBC cell lines.v Further confirm the effect of VCP expression onTNBC cell growth and invasion throughoverexpression and knockout in TNBC cell linesv Determine the effect of VCP inhibition on tumorgrowth in vivo.v Investigate other SOX9 binding proteins such asCAV1 and GSK-3β as potential therapeutic targetsin TNBC.Future Plans1. Anders CK, et al. Biology, metastatic patterns, andtreatment of patients with triple-negative breast cancer. ClinBreast Cancer 2009;9 Suppl 2:S73-812. Lehmann BD, et al. Identification of human triple-negativebreast cancer subtypes and preclinical models for selection oftargeted therapies. J Clin Invest 2011;121:2750-673. Ma Y, et al. SOX9 is a critical regulator of triple-negativebreast cancer cell growth and invasion. Cancer Res. 2017;774. Ma Y, et al. SOX9 is essential for triple-negative breastcancer cell survival and metastasis. Mol Cancer Res.2020;18(12):1825–385. Cui Y, et al. High expression of valosin-containing proteinpredicts poor prognosis in patients with breast carcinoma.Tumour Biol. 2015;36(12):9919-276. Curtis C, et al. The genomic and transcriptomic architectureof 2,000 breast tumours reveals novel subgroups. Nature.2012;486(7403):346-352.VCP Knockdown Western BlotC BA0 1 2 3 4 50246Correlation of VCP andSOX9 ExpressionVCPSOX9Y = 0.2138*X + 2.2790.01116R squared =0 20 40 60607080901001105 Year Overall Survival in Curtis Breast (n=2136)Follow Up (Months)Probability of SurvivalVCP LowVCP HighLog-rank (Mantel-Cox) testp = 0.0360TNBC Non-TNBC02468VCP Expression in Curtis BreastLog2 Median Centered IntensityTNBCNon-TNBC**n = 250 n = 17250 20 40 6060708090100110Non-TNBC 5 Year Survivalin Curtis BreastFollow Up (Months)Probability of SurvivalVCP LowVCP HighLog-rank (Mantel-Cox) testp = 0.20070 2 4 6 801×1062×1063×106MDA MB-231DayCell NumbersiLucsiVCP ORFsiVCP 3'UTR*****LAMB2P1MIOXRGCCFLJ46906PRTN3LYNC00173CEACAM10.00.51.01.52.0MDA MB-468Relative mRNA Expression(2-DD Ct )siLucsiVCP* **** * * *** ** *B0 20 40 606080100TNBC 5 Year Overall Survival in Curtis BreastFollow Up (Months)Probability of SurvivalVCP LowVCP HighLog-rank (Mantel-Cox) testp = 0.0650SOX9VCPVCPn = 250n = 1725

Validation of Valosin-Containing Protein (VCP) as a Therapeutic Target for Triple Negative Breast Cancer

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1196&context=sumexp21

Validation of Valosin-Containing Protein (VCP) as a Therapeutic Target for Triple Negative Breast Cancer

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