Poly(ADP-ribose) polymerase inhibitors (PARPi) target tumours defective in homologous recombination (HR). Most BRCA-wild-type (WT) HR-proficient breast cancers are intrinsically resistant to PARP inhibitors, e.g., talazoparib. We evaluated the role of autophagy in this de novo resistance and determined the underlying mechanism to overcome this.
Autophagosome formation and autophagic flux were assessed by evaluating endogenous LC3-II levels and ectopic expression of EGFP-LC3 and mRFP-EGFP-LC3 in breast cancer cells. Autophagy-defective cells were generated by genetic depletion of BECN1, ATG5, p62/SQSTM1 and LAMP1 by using CRISPR-Cas9 double nickase system. The response of PARPi was evaluated in autophagy-proficient and -defective breast cancer cells and in xenograft SCID-mice model.
Pro-survival autophagy was significantly enhanced upon talazoparib treatment in BRCA-WT breast cancer cell lines. Autophagy-deficient cells were hypersensitive to talazoparib. Targeting autophagy synergistically enhanced the therapeutic efficacy of talazoparib in BRCA1-WT breast cancer cells in vitro and in vivo xenograft tumour mouse model. Mechanistically, autophagy inhibition by chloroquine promoted deleterious NHEJ mediated DSB-repair, leading to extensive genomic instability and mitotic catastrophe.
Autophagy confers de novo resistance to PARP inhibitor, talazoparib. Autophagy inhibition improves the therapeutic outcome of PARPi treatment in preclinical mice model, bearing HR-proficient breast tumours, warranting its usage in the clinical settings.
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Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A. & Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 68, 394–424 (2018).
Ray Chaudhuri, A. & Nussenzweig, A. The multifaceted roles of PARP1 in DNA repair and chromatin remodelling. Nat. Rev. Mol. Cell Biol. 18, 610–621 (2017).
Rojo, F., García-Parra, J., Zazo, S., Tusquets, I., Ferrer-Lozano, J., Menendez, S. et al. Nuclear PARP-1 protein overexpression is associated with poor overall survival in early breast cancer. Ann. Oncol. 23, 1156–1164 (2011).
Gilabert, M., Launay, S., Ginestier, C., Bertucci, F., Audebert, S., Pophillat, M. et al. Poly(ADP-ribose) polymerase 1 (PARP1) overexpression in human breast cancer stem cells and resistance to olaparib. PLoS ONE 9, e104302–e104302 (2014).
Naipal, K. A. & van Gent, D. C. PARP inhibitors: the journey from research hypothesis to clinical approval. Per. Med. 12, 139–154 (2015).
Ashworth, A. & Lord, C. J. Synthetic lethal therapies for cancer: what’s next after PARP inhibitors? Nat. Rev. Clin. Oncol. 15, 564–576 (2018).
Bryant, H. E., Schultz, N., Thomas, H. D., Parker, K. M., Flower, D., Lopez, E. et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434, 913–917 (2005).
Farmer, H., McCabe, N., Lord, C. J., Tutt, A. N. J., Johnson, D. A., Richardson, T. B. et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917–921 (2005).
Sonnenblick, A., de Azambuja, E., Azim, H. A. & Piccart, M. An update on PARP inhibitors—moving to the adjuvant setting. Nat. Rev. Clin. Oncol. 12, 27–41 (2015).
Malone, K. E., Daling, J. R., Doody, D. R., Hsu, L., Bernstein, L., Coates, R. J. et al. Prevalence and predictors of BRCA1 and BRCA2 mutations in a population-based study of breast cancer in white and black American women ages 35 to 64 years. Cancer Res. 66, 8297–8308 (2006).
Bouwman, P. & Jonkers, J. Molecular pathways: How can BRCA-mutated tumors become resistant to PARP Inhibitors? Clin. Cancer Res. 20, 540–547 (2014).
Edwards, S. L., Brough, R., Lord, C. J., Natrajan, R., Vatcheva, R., Levine, D. A. et al. Resistance to therapy caused by intragenic deletion in BRCA2. Nature 451, 1111–1115 (2008).
Gogola, E., Duarte, A. A., de Ruiter, J. R., Wiegant, W. W., Schmid, J. A., de Bruijn, R. et al. Selective loss of PARG restores PARylation and counteracts PARP inhibitor-mediated synthetic lethality. Cancer Cell 33, 1078–1093 (2018).
Chand, S. N., Zarei, M., Schiewer, M. J., Kamath, A. R., Romeo, C., Lal, S. et al. Posttranscriptional regulation of PARG mRNA by HuR facilitates DNA repair and resistance to PARP inhibitors. Cancer Res. 77, 5011–5025 (2017).
Jaspers, J. E., Kersbergen, A., Boon, U., Sol, W., van Deemter, L., Zander, S. A. et al. Loss of 53BP1 causes PARP inhibitor resistance in Brca1-mutated mouse mammary tumors. Cancer Discov. 3, 68–81 (2013).
Fojo, T. & Bates, S. Mechanisms of resistance to PARP inhibitors—three and counting. Cancer Discov. 3, 20–23 (2013).
Johnson, S. F., Cruz, C., Greifenberg, A. K., Dust, S., Stover, D. G., Chi, D. et al. CDK12 inhibition reverses de novo and acquired PARP inhibitor resistance in BRCA wild-type and mutated models of triple-negative breast cancer. Cell Rep. 17, 2367–2381 (2016).
Karakashev, S., Zhu, H., Yokoyama, Y., Zhao, B., Fatkhutdinov, N., Kossenkov, A. V. et al. BET bromodomain inhibition synergizes with PARP inhibitor in epithelial ovarian cancer. Cell Rep. 21, 3398–3405 (2017).
Ji, Y., Wang, Q., Zhao, Q., Zhao, S., Li, L., Sun, G. et al. Autophagy suppression enhances DNA damage and cell death upon treatment with PARP inhibitor niraparib in laryngeal squamous cell carcinoma. Appl. Microbiol. Biotechnol. 103, 9557–9568 (2019).
Zai, W., Chen, W., Han, Y., Wu, Z., Fan, J., Zhang, X. et al. Targeting PARP and autophagy evoked synergistic lethality in hepatocellular carcinoma. Carcinogenesis 41, 345–357 (2019).
Patel, A. G., Sarkaria, J. N. & Kaufmann, S. H. Nonhomologous end joining drives poly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells. Proc. Natl Acad. Sci. USA 108, 3406–3411 (2011).
Liu, E. Y., Xu, N., O’Prey, J., Lao, L. Y., Joshi, S., Long, J. S. et al. Loss of autophagy causes a synthetic lethal deficiency in DNA repair. Proc. Natl Acad. Sci. USA 112, 773–778 (2015).
Saha, B., Patro, B. S., Koli, M., Pai, G., Ray, J., Bandyopadhyay, S. K. et al. Trans -4,4’-dihydroxystilbene (DHS) inhibits human neuroblastoma tumor growth and induces mitochondrial and lysosomal damages in neuroblastoma cell lines. Oncotarget 8, 73905–73924 (2017).
Chou, T.-C. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 70, 440–446 (2010).
Wu, W., Bi, C., Credille, K. M., Manro, J. R., Peek, V. L., Donoho, G. P. et al. Inhibition of tumor growth and metastasis in non–small cell lung cancer by LY2801653, an inhibitor of several oncokinases, including MET. Clin. Cancer Res. 19, 5699–5710 (2013).
Hidau, M. K., Kolluru, S. & Palakurthi, S. Development and validation of a high-performance liquid chromatography method for the quantification of talazoparib in rat plasma: application to plasma protein binding studies. Biomed. Chromatogr. 32, e4046 (2018).
Keung, M. Y., Wu, Y., Badar, F. & Vadgama, J. V. Response of breast cancer cells to PARP inhibitors is independent of BRCA status. J. Clin. Med. 9, 940 (2020).
de Bono, J., Ramanathan, R. K., Mina, L., Chugh, R., Glaspy, J., Rafii, S. et al. Phase I, dose-escalation, two-part trial of the PARP inhibitor talazoparib in patients with advanced germline BRCA1/2 mutations and selected sporadic cancers. Cancer Discov. 7, 620–629 (2017).
Sui, X., Chen, R., Wang, Z., Huang, Z., Kong, N., Zhang, M. et al. Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment. Cell Death Dis. 4, e838 (2013).
Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010).
Wang, X., Shen, C., Liu, Z., Peng, F., Chen, X., Yang, G. et al. Nitazoxanide, an antiprotozoal drug, inhibits late-stage autophagy and promotes ING1-induced cell cycle arrest in glioblastoma. Cell Death Dis. 9, 1032 (2018).
Steinman, R. M., Mellman, I. S., Muller, W. A. & Cohn, Z. A. Endocytosis and the recycling of plasma membrane. J. Cell Biol. 96, 1–27 (1983).
Mauthe, M., Orhon, I., Rocchi, C., Zhou, X., Luhr, M., Hijlkema, K.-J. et al. Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Autophagy 14, 1435–1455 (2018).
Kimura, S., Noda, T. & Yoshimori, T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3, 452–460 (2007).
Zhou, C., Zhong, W., Zhou, J., Sheng, F., Fang, Z., Wei, Y. et al. Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells. Autophagy 8, 1215–1226 (2012).
Liang, X. H., Jackson, S., Seaman, M., Brown, K., Kempkes, B., Hibshoosh, H. et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402, 672–676 (1999).
Scarlatti, F., Maffei, R., Beau, I., Codogno, P. & Ghidoni, R. Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells. Cell Death Differ. 15, 1318–1329 (2008).
He, C. & Klionsky, D. J. Regulation mechanisms and signaling pathways of autophagy. Annu. Rev. Genet. 43, 67–93 (2009).
Colicchia, V., Petroni, M., Guarguaglini, G., Sardina, F., Sahún-Roncero, M., Carbonari, M. et al. PARP inhibitors enhance replication stress and cause mitotic catastrophe in MYCN-dependent neuroblastoma. Oncogene 36, 4682–4691 (2017).
Maya-Mendoza, A., Moudry, P., Merchut-Maya, J. M., Lee, M., Strauss, R. & Bartek, J. High speed of fork progression induces DNA replication stress and genomic instability. Nature 559, 279–284 (2018).
Schoonen, P. M., Talens, F., Stok, C., Gogola, E., Heijink, A. M., Bouwman, P. et al. Progression through mitosis promotes PARP inhibitor-induced cytotoxicity in homologous recombination-deficient cancer cells. Nat. Commun. 8, 15981 (2017).
Karantza-Wadsworth, V., Patel, S., Kravchuk, O., Chen, G., Mathew, R., Jin, S. et al. Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis. Genes Dev. 21, 1621–1635 (2007).
Vessoni, A. T., Filippi-Chiela, E. C., Menck, C. F. & Lenz, G. Autophagy and genomic integrity. Cell Death Differ. 20, 1444–1454 (2013).
Mizushima, N. & Levine, B. Autophagy in mammalian development and differentiation. Nat. Cell Biol. 12, 823–830 (2010).
Levine, B. & Kroemer, G. Biological functions of autophagy genes: a disease perspective. Cell 176, 11–42 (2019).
Lord, C. J. & Ashworth, A. BRCAness revisited. Nat. Rev. Cancer 16, 110–120 (2016).
Chapman, J. R., Sossick, A. J., Boulton, S. J. & Jackson, S. P. BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair. J. Cell Sci. 125, 3529–3534 (2012).
Baldwin, P., Likhotvorik, R., Baig, N., Cropper, J., Carlson, R., Kurmasheva, R. et al. Nanoformulation of talazoparib increases maximum tolerated doses in combination with temozolomide for treatment of Ewing sarcoma. Front. Oncol. 9, 1416 (2019).
Gottipati, P., Vischioni, B., Schultz, N., Solomons, J., Bryant, H. E., Djureinovic, T. et al. Poly(ADP-Ribose) polymerase is hyperactivated in homologous recombination–defective cells. Cancer Res. 70, 5389–5398 (2010).
Hewitt, G., Carroll, B., Sarallah, R., Correia-Melo, C., Ogrodnik, M., Nelson, G. et al. SQSTM1/p62 mediates crosstalk between autophagy and the UPS in DNA repair. Autophagy 12, 1917–1930 (2016).
Somyajit, K., Mishra, A., Jameei, A. & Nagaraju, G. Enhanced non-homologous end joining contributes toward synthetic lethality of pathological RAD51C mutants with poly (ADP-ribose) polymerase. Carcinogenesis 36, 13–24 (2014).
We would like to acknowledge the central animal facility at Bhabha Atomic Research Centre, Mumbai. We would also like to thank R. Krishna Mohan, Jasraj Vaishnav and Unique Biodiagnostics Enterprises, Mumbai for their help in the conduct of animal experiments, HPLC and histopathology and biochemical analysis, respectively.
Ethics approval and consent to participate
No human-derived samples were used in this study and hence there is no human-specific ethical approval to report. All the animal experiments were performed upon receipt of approval from the institutional animal ethics committee (IAEC). Mice were maintained in BARC SCID mouse facility, and experiments were performed as per the institutional guidelines and regulations laid down by the BARC animal ethics committee.
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This work was supported financially by the internal funding of Bhabha Atomic Research Centre, Department of Atomic Energy, India.
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Pai Bellare, G., Saha, B. & Patro, B.S. Targeting autophagy reverses de novo resistance in homologous recombination repair proficient breast cancers to PARP inhibition. Br J Cancer (2021). https://doi.org/10.1038/s41416-020-01238-0