Abstract
Triple-negative (ER-PR-HER2-) breast cancers (TNBC) are highly aggressive and difficult to treat. TNBC exhibit high genomic instability, which enables them to adapt and become resistant to chemo/radiation therapy, leading to rapid disease relapse and mortality. The pro-survival factors that safeguard genome integrity in TNBC cells are poorly understood. LBH is an essential mammary stem cell-specific transcription regulator in the WNT pathway that is aberrantly overexpressed in TNBC, correlating with poor prognosis. Herein, we demonstrate a novel role for LBH in promoting TNBC cell survival. Depletion of LBH in multiple TNBC cell models triggered apoptotic cell death both in vitro and in vivo and led to S-G2M cell cycle delays. Mechanistically, LBH loss causes replication stress due to DNA replication fork stalling, leading to ssDNA breaks, ɣH2AX and 53BP1 nuclear foci formation, and activation of the ATR/CHK1 DNA damage response. Notably, ATR inhibition in combination with LBH downmodulation had a synergistic effect, boosting TNBC cell killing and blocking in vivo tumor growth. Our findings demonstrate, for the first time, that LBH protects the genome integrity of cancer cells by preventing replicative stress. Importantly, they uncover new synthetic lethal vulnerabilities in TNBC that could be exploited for future multi-modal precision medicine.
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References
Foulkes WD, Smith IE, Reis-Filho JS. Triple-negative breast cancer. N Engl J Med. 2010;363:1938–48.
Isakoff SJ. Triple-negative breast cancer: role of specific chemotherapy agents. Cancer J. 2010;16:53–61.
Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007;13:4429–34.
Nofech-Mozes S, Trudeau M, Kahn HK, Dent R, Rawlinson E, Sun P, et al. Patterns of recurrence in the basal and non-basal subtypes of triple-negative breast cancers. Breast Cancer Res Treat. 2009;118:131–7.
Cancer Genome Atlas N. Comprehensive molecular portraits of human breast tumours. Nature. 2012;490:61–70.
Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, et al. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature. 2012;486:346–52.
Staaf J, Glodzik D, Bosch A, Vallon-Christersson J, Reutersward C, Hakkinen J, et al. Whole-genome sequencing of triple-negative breast cancers in a population-based clinical study. Nat Med. 2019;25:1526–33.
Duijf PHG, Nanayakkara D, Nones K, Srihari S, Kalimutho M, Khanna KK. Mechanisms of genomic instability in breast cancer. Trends Mol Med. 2019;25:595–611.
Derakhshan F, Reis-Filho JS. Pathogenesis of triple-negative breast cancer. Annu Rev Pathol. 2022;17:181–204.
Briegel KJ, Joyner AL. Identification and characterization of Lbh, a novel conserved nuclear protein expressed during early limb and heart development. Dev Biol. 2001;233:291–304.
Al-Ali H, Rieger ME, Seldeen KL, Harris TK, Farooq A, Briegel KJ. Biophysical characterization reveals structural disorder in the developmental transcriptional regulator LBH. Biochem Biophys Res Commun. 2010;391:1104–9.
Rieger ME, Sims AH, Coats ER, Clarke RB, Briegel KJ. The embryonic transcription cofactor LBH is a direct target of the Wnt signaling pathway in epithelial development and in aggressive basal subtype breast cancers. Mol Cell Biol. 2010;30:4267–79.
Lindley LE, Curtis KM, Sanchez-Mejias A, Rieger ME, Robbins DJ, Briegel KJ. The WNT-controlled transcriptional regulator LBH is required for mammary stem cell expansion and maintenance of the basal lineage. Development. 2015;142:893–904.
Briegel KJ, Baldwin HS, Epstein JA, Joyner AL. Congenital heart disease reminiscent of partial trisomy 2p syndrome in mice transgenic for the transcription factor Lbh. Development. 2005;132:3305–16.
Conen KL, Nishimori S, Provot S, Kronenberg HM. The transcriptional cofactor Lbh regulates angiogenesis and endochondral bone formation during fetal bone development. Dev Biol. 2009;333:348–58.
Powder KE, Cousin H, McLinden GP, Craig Albertson R. A nonsynonymous mutation in the transcriptional regulator lbh is associated with cichlid craniofacial adaptation and neural crest cell development. Mol Biol Evol. 2014;31:3113–24.
Weir E, McLinden G, Alfandari D, Cousin H. Trim-Away mediated knock down uncovers a new function for Lbh during gastrulation of Xenopus laevis. Dev Biol. 2021;470:74–83.
Liu Q, Guan X, Lv J, Li X, Wang Y, Li L. Limb-bud and heart (LBH) functions as a tumor suppressor of nasopharyngeal carcinoma by inducing G1/S cell cycle arrest. Sci Rep. 2015;5:7626.
Ekwall AK, Whitaker JW, Hammaker D, Bugbee WD, Wang W, Firestein GS. The rheumatoid arthritis risk gene LBH regulates growth in fibroblast-like synoviocytes. Arthritis Rheumatol. 2015;67:1193–202.
Matsuda S, Hammaker D, Topolewski K, Briegel KJ, Boyle DL, Dowdy S, et al. Regulation of the cell cycle and inflammatory arthritis by the transcription cofactor LBH gene. J Immunol. 2017;199:2316–22.
Jiang Y, Zhou J, Zou D, Hou D, Zhang H, Zhao J, et al. Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia. EBioMedicine. 2019;48:36–48.
Liu H, Giffen KP, Grati M, Morrill SW, Li Y, Liu X, et al. Transcription co-factor LBH is necessary for the survival of cochlear hair cells. J Cell Sci. 2021;134:jcs254458.
Chen J, Huang C, Chen K, Li S, Zhang X, Cheng J, et al. Overexpression of LBH is associated with poor prognosis in human hepatocellular carcinoma. Onco Targets Ther. 2018;11:441–8.
Deng M, Yu R, Wang S, Zhang Y, Li Z, Song H, et al. Limb-bud and heart attenuates growth and invasion of human lung adenocarcinoma cells and predicts survival outcome. Cell Physiol Biochem. 2018;47:223–34.
Yu R, Li Z, Zhang C, Song H, Deng M, Sun L, et al. Elevated limb-bud and heart development (LBH) expression indicates poor prognosis and promotes gastric cancer cell proliferation and invasion via upregulating Integrin/FAK/Akt pathway. PeerJ. 2019;7:e6885.
Young IC, Brabletz T, Lindley LE, Abreu M, Nagathihalli N, Zaika A, et al. Multi-cancer analysis reveals universal association of oncogenic LBH expression with DNA hypomethylation and WNT-Integrin signaling pathways. Cancer Gene Ther. 2023;30:1234–48.
Ashad-Bishop K, Garikapati K, Lindley LE, Jorda M, Briegel KJ. Loss of Limb-Bud-and-Heart (LBH) attenuates mammary hyperplasia and tumor development in MMTV-Wnt1 transgenic mice. Biochem Biophys Res Commun. 2019;508:536–42.
Liu L, Luo Q, Xu Q, Xiong Y, Deng H. Limb-bud and heart development (LBH) contributes to glioma progression in vitro and in vivo. FEBS Open Bio. 2022;12:211–20.
Lamb R, Ablett MP, Spence K, Landberg G, Sims AH, Clarke RB. Wnt pathway activity in breast cancer sub-types and stem-like cells. PLoS One. 2013;8:e67811.
Liu R, Wang X, Chen GY, Dalerba P, Gurney A, Hoey T, et al. The prognostic role of a gene signature from tumorigenic breast-cancer cells. N Engl J Med. 2007;356:217–26.
Honeth G, Bendahl PO, Ringner M, Saal LH, Gruvberger-Saal SK, Lovgren K, et al. The CD44+/CD24- phenotype is enriched in basal-like breast tumors. Breast Cancer Res. 2008;10:R53.
Khramtsov AI, Khramtsova GF, Tretiakova M, Huo D, Olopade OI, Goss KH. Wnt/{beta}-catenin pathway activation is enriched in basal-like breast cancers and predicts poor outcome. Am J Pathol. 2010;176:2911–20.
Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst. 2008;100:672–9.
Creighton CJ, Li X, Landis M, Dixon JM, Neumeister VM, Sjolund A, et al. Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl Acad Sci USA. 2009;106:13820–5.
Frontini M, Kukalev A, Leo E, Ng YM, Cervantes M, Cheng CW, et al. The CDK subunit CKS2 counteracts CKS1 to control cyclin A/CDK2 activity in maintaining replicative fidelity and neurodevelopment. Dev Cell. 2012;23:356–70.
Gu Y, Rosenblatt J, Morgan DO. Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. EMBO J. 1992;11:3995–4005.
Mass G, Nethanel T, Kaufmann G. The middle subunit of replication protein A contacts growing RNA-DNA primers in replicating simian virus 40 chromosomes. Mol Cell Biol. 1998;18:6399–407.
Liaw H, Lee D, Myung K. DNA-PK-dependent RPA2 hyperphosphorylation facilitates DNA repair and suppresses sister chromatid exchange. PLoS One. 2011;6:e21424.
Ashley AK, Shrivastav M, Nie J, Amerin C, Troksa K, Glanzer JG, et al. DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation, fork restart, homologous recombination and mitotic catastrophe. DNA Repair (Amst). 2014;21:131–9.
Toledo L, Neelsen KJ, Lukas J. Replication catastrophe: when a checkpoint fails because of exhaustion. Mol Cell. 2017;66:735–49.
Zeman MK, Cimprich KA. Causes and consequences of replication stress. Nat Cell Biol. 2014;16:2–9.
Marechal A, Zou L. DNA damage sensing by the ATM and ATR kinases. Cold Spring Harb Perspect Biol. 2013;5:a012716.
Smith J, Tho LM, Xu N, Gillespie DA. The ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer. Adv Cancer Res. 2010;108:73–112.
Zou L, Elledge SJ. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science. 2003;300:1542–8.
Lecona E, Fernandez-Capetillo O. Targeting ATR in cancer. Nat Rev Cancer. 2018;18:586–95.
Kwon M, Kim G, Kim R, Kim KT, Kim ST, Smith S, et al. Phase II study of ceralasertib (AZD6738) in combination with durvalumab in patients with advanced gastric cancer. J Immunother Cancer. 2022;10:e005041.
McMullen M, Karakasis K, Loembe B, Dean E, Parr G, Oza AM. DUETTE: a phase II randomized, multicenter study to investigate the efficacy and tolerability of a second maintenance treatment in patients with platinum-sensitive relapsed epithelial ovarian cancer, who have previously received poly(ADP-ribose) polymerase (PARP) inhibitor maintenance treatment. Int J Gynecol Cancer. 2020;30:1824–8.
Lord CJ, Ashworth A. PARP inhibitors: synthetic lethality in the clinic. Science. 2017;355:1152–8.
Garrido-Castro AC, Lin NU, Polyak K. Insights into molecular classifications of triple-negative breast cancer: improving patient selection for treatment. Cancer Discov. 2019;9:176–98.
Marine JC, Dawson SJ, Dawson MA. Non-genetic mechanisms of therapeutic resistance in cancer. Nat Rev Cancer. 2020;20:743–56.
Lehmann BD, Jovanovic B, Chen X, Estrada MV, Johnson KN, Shyr Y, et al. Refinement of triple-negative breast cancer molecular subtypes: implications for neoadjuvant chemotherapy selection. PLoS One. 2016;11:e0157368.
Minn AJ, Kang Y, Serganova I, Gupta GP, Giri DD, Doubrovin M, et al. Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. J Clin Invest. 2005;115:44–55.
Acknowledgements
We thank the Analytic Imaging Core of the Miami Project to Cure Paralysis for confocal imaging. This work was supported by NIH/NIGMS Grant R01GM113256 (K.J.B.), the Department of Defense (DoD)/Breast Cancer Research Program (BCRP) Breakthrough Award W81XWH-19-1-0255 (K.J.B.); a Tumor Biology Program Trainee Award (K.G.), and research funds from the Sylvester Comprehensive Cancer Center (K.J.B.). Research reported in this publication was performed in part at the Flow Cytometry Shared Resource (FCSR; RRID: SCR_022501) and the Cancer Modeling Shared Resource (CMSR; RRID: SCR_022889) of the Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine, which is supported by the National Cancer Institute Cancer Center Support Grant (CCSG) P30-CA240139.
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K.G. and K.J.B. conceived the study and designed the experiments. K.G. performed most experiments, analyzed the data, prepared the figures, and wrote the first draft of the manuscript. I.-C.Y. and S.H. generated stable LBH knockdown cell lines. I.-C.Y. performed in vivo Xenograft experiments and data analysis. P.R. provided essential reagents and feedback on the study. C.J. reviewed and edited the manuscript. K.J.B. directed the study, reviewed the data, wrote the manuscript, and secured funding.
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Garikapati, K., Young, IC., Hong, S. et al. Blocking LBH expression causes replication stress and sensitizes triple-negative breast cancer cells to ATR inhibitor treatment. Oncogene 43, 851–865 (2024). https://doi.org/10.1038/s41388-024-02951-3
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DOI: https://doi.org/10.1038/s41388-024-02951-3