Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

RB loss determines selective resistance and novel vulnerabilities in ER-positive breast cancer models

Abstract

The management of metastatic estrogen receptor (ER) positive HER2 negative breast cancer (ER+) has improved; however, therapeutic resistance and disease progression emerges in majority of cases. Using unbiased approaches, as expected PI3K and MTOR inhibitors emerge as potent inhibitors to delay proliferation of ER+ models harboring PIK3CA mutations. However, the cytostatic efficacy of these drugs is hindered due to marginal impact on the expression of cyclin D1. Different combination approaches involving the inhibition of ER pathway or cell cycle result in durable growth arrest via RB activation and subsequent inhibition of CDK2 activity. However, cell cycle alterations due to RB loss or ectopic CDK4/cyclin D1 activation yields resistance to these cytostatic combination treatments. To define means to counter resistance to targeted therapies imparted with RB loss; complementary drug screens were performed with RB-deleted isogenic cell lines. In this setting, RB loss renders ER+ breast cancer models more vulnerable to drugs that target DNA replication and mitosis. Pairwise combinations using these classes of drugs defines greater selectivity for RB deficiency. The combination of AURK and WEE1 inhibitors, yields synergistic cell death selectively in RB-deleted ER+ breast cancer cells via apoptosis and yields profound disease control in vivo. Through unbiased efforts the XIAP/CIAP inhibitor birinapant was identified as a novel RB-selective agent. Birinapant further enhances the cytotoxic effect of chemotherapies and targeted therapies used in the treatment of ER+ breast cancer models selectively in the RB-deficient setting. Using organoid culture and xenograft models, we demonstrate the highly selective use of birinapant based combinations for the treatment of RB-deficient tumors. Together, these data illustrate the critical role of RB-pathway in response to many agents used to treat ER+ breast cancer, whilst informing new therapeutic approaches that could be deployed against resistant disease.

Your institute does not have access to this article

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: RB-mediated cell cycle arrest by standard care of therapies.
Fig. 2: Vulnerability due to RB loss.
Fig. 3: Impact of RB loss on AURK and WEE1 inhibition.
Fig. 4: Regulatory function of RB on apoptosis.
Fig. 5: Combination treatment approach using birinapant.
Fig. 6: In vivo efficacy of Alisertib in combination with birinapant.
Fig. 7: Scheme illustrating the function of RB loss.

References

  1. Gong Y, Liu YR, Ji P, Hu X, Shao ZM. Impact of molecular subtypes on metastatic breast cancer patients: a SEER population-based study. Sci Rep. 2017;7:45411.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. Ahmad A. Breast cancer statistics: recent trends. Adv Exp Med Biol. 2019;1152:1–7.

    CAS  PubMed  Article  Google Scholar 

  3. Jensen EV, Jordan VC. The estrogen receptor: a model for molecular medicine. Clin Cancer Res. 2003;9:1980–9.

    CAS  PubMed  Google Scholar 

  4. Howell A. The endocrine prevention of breast cancer. Best Pract Res Clin Endocrinol Metab. 2008;22:615–23.

    CAS  PubMed  Article  Google Scholar 

  5. Mouridsen H, Gershanovich M, Sun Y, Perez-Carrion R, Boni C, Monnier A, et al. Superior efficacy of letrozole versus tamoxifen as first-line therapy for postmenopausal women with advanced breast cancer: results of a phase III study of the International Letrozole Breast Cancer Group. J Clin Oncol. 2001;19:2596–606.

    CAS  PubMed  Article  Google Scholar 

  6. McKeage K, Curran MP, Plosker GL. Fulvestrant: a review of its use in hormone receptor-positive metastatic breast cancer in postmenopausal women with disease progression following antiestrogen therapy. Drugs. 2004;64:633–48.

    CAS  PubMed  Article  Google Scholar 

  7. Manna S, Holz MK. Tamoxifen action in ER-negative breast cancer. Sign Transduct Insights. 2016;5:1–7.

    PubMed  PubMed Central  Google Scholar 

  8. Schiff R, Massarweh SA, Shou J, Bharwani L, Mohsin SK, Osborne CK. Cross-talk between estrogen receptor and growth factor pathways as a molecular target for overcoming endocrine resistance. Clin Cancer Res. 2004;10:331S–6S.

    CAS  PubMed  Article  Google Scholar 

  9. Pu M, Messer K, Davies SR, Vickery TL, Pittman E, Parker BA, et al. Research-based PAM50 signature and long-term breast cancer survival. Breast Cancer Res Treat. 2020;179:197–206.

    CAS  PubMed  Article  Google Scholar 

  10. Dowsett M, Sestak I, Lopez-Knowles E, Sidhu K, Dunbier AK, Cowens JW, et al. Comparison of PAM50 risk of recurrence score with oncotype DX and IHC4 for predicting risk of distant recurrence after endocrine therapy. J Clin Oncol. 2013;31:2783–90.

    PubMed  Article  Google Scholar 

  11. Desmedt C, Sotiriou C. Proliferation: the most prominent predictor of clinical outcome in breast cancer. Cell Cycle. 2006;5:2198–202.

    CAS  PubMed  Article  Google Scholar 

  12. Mills JN, Rutkovsky AC, Giordano A. Mechanisms of resistance in estrogen receptor positive breast cancer: overcoming resistance to tamoxifen/aromatase inhibitors. Curr Opin Pharmacol. 2018;41:59–65.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Provenzano A, Kurian S, Abraham J. Overcoming endocrine resistance in breast cancer: role of the PI3K and the mTOR pathways. Exp Rev Anticancer Ther. 2013;13:143–7.

    CAS  Article  Google Scholar 

  14. Andre F, Ciruelos E, Rubovszky G, Campone M, Loibl S, Rugo HS, et al. Alpelisib for PIK3CA-mutated, hormone receptor-positive advanced breast cancer. N Engl J Med. 2019;380:1929–40.

    CAS  PubMed  Article  Google Scholar 

  15. Elkabets M, Vora S, Juric D, Morse N, Mino-Kenudson M, Muranen T, et al. mTORC1 inhibition is required for sensitivity to PI3K p110alpha inhibitors in PIK3CA-mutant breast cancer. Sci Transl Med. 2013;5:196ra99.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  16. Sherr CJ. Mammalian G1 cyclins. Cell. 1993;73:1059–65.

    CAS  PubMed  Article  Google Scholar 

  17. Rubin SM. Deciphering the retinoblastoma protein phosphorylation code. Trends Biochem Sci. 2013;38:12–9.

    CAS  PubMed  Article  Google Scholar 

  18. Thu KL, Soria-Bretones I, Mak TW, Cescon DW. Targeting the cell cycle in breast cancer: towards the next phase. Cell Cycle. 2018;17:1871–85.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Finn RS, Dering J, Conklin D, Kalous O, Cohen DJ, Desai AJ, et al. PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro. Breast Cancer Res. 2009;11:R77.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  20. Dickson MA. Molecular pathways: CDK4 inhibitors for cancer therapy. Clin Cancer Res. 2014;20:3379–83.

    CAS  PubMed  Article  Google Scholar 

  21. Turner NC, Slamon DJ, Ro J, Bondarenko I, Im SA, Masuda N, et al. Overall survival with palbociclib and fulvestrant in advanced breast cancer. N Engl J Med. 2018;379:1926–36.

    CAS  PubMed  Article  Google Scholar 

  22. Dickler MN, Tolaney SM, Rugo HS, Cortes J, Dieras V, Patt D, et al. MONARCH 1, A Phase II study of abemaciclib, a CDK4 and CDK6 inhibitor, as a single agent, in patients with refractory HR(+)/HER2(-) metastatic breast cancer. Clin Cancer Res. 2017;23:5218–24.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Dean JL, Thangavel C, McClendon AK, Reed CA, Knudsen ES. Therapeutic CDK4/6 inhibition in breast cancer: key mechanisms of response and failure. Oncogene. 2010;29:4018–32.

    CAS  PubMed  Article  Google Scholar 

  24. O’Leary B, Cutts RJ, Liu Y, Hrebien S, Huang X, Fenwick K, et al. The genetic landscape and clonal evolution of breast cancer resistance to palbociclib plus fulvestrant in the PALOMA-3 trial. Cancer Discov. 2018;8:1390–403.

    PubMed  PubMed Central  Article  Google Scholar 

  25. O’Leary B, Hrebien S, Morden JP, Beaney M, Fribbens C, Huang X, et al. Early circulating tumor DNA dynamics and clonal selection with palbociclib and fulvestrant for breast cancer. Nat Commun. 2018;9:896.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  26. Arnedos M, Bayar MA, Cheaib B, Scott V, Bouakka I, Valent A, et al. Modulation of Rb phosphorylation and antiproliferative response to palbociclib: the preoperative-palbociclib (POP) randomized clinical trial. Ann Oncol. 2018;29:1755–62.

    CAS  PubMed  Article  Google Scholar 

  27. Condorelli R, Spring L, O’Shaughnessy J, Lacroix L, Bailleux C, Scott V, et al. Polyclonal RB1 mutations and acquired resistance to CDK 4/6 inhibitors in patients with metastatic breast cancer. Ann Oncol. 2018;29:640–5.

    CAS  PubMed  Article  Google Scholar 

  28. Wander SA, Cohen O, Gong X, Johnson GN, Buendia-Buendia JE, Lloyd MR, et al. The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors in patients with hormone receptor positive metastatic breast cancer. Cancer Discov. 2020;10:174–93.

    Article  Google Scholar 

  29. Li Z, Razavi P, Li Q, Toy W, Liu B, Ping C, et al. Loss of the FAT1 tumor suppressor promotes resistance to CDK4/6 inhibitors via the hippo pathway. Cancer Cell. 2018;34:893–905e8.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  30. Presti D, Quaquarini E. The PI3K/AKT/mTOR and CDK4/6 pathways in endocrine resistant HR+/HER2-metastatic breast cancer: biological mechanisms and new treatments. Cancers. 2019;11:1242.

    CAS  PubMed Central  Article  Google Scholar 

  31. Vora SR, Juric D, Kim N, Mino-Kenudson M, Huynh T, Costa C, et al. CDK 4/6 inhibitors sensitize PIK3CA mutant breast cancer to PI3K inhibitors. Cancer Cell. 2014;26:136–49.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. Michaloglou C, Crafter C, Siersbaek R, Delpuech O, Curwen JO, Carnevalli LS, et al. Combined inhibition of mTOR and CDK4/6 is required for optimal blockade of E2F function and long-term growth inhibition in estrogen receptor-positive breast cancer. Mol Cancer Ther. 2018;17:908–20.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Guarducci C, Bonechi M, Benelli M, Biagioni C, Boccalini G, Romagnoli D, et al. Cyclin E1 and Rb modulation as common events at time of resistance to palbociclib in hormone receptor-positive breast cancer. NPJ Breast Cancer. 2018;4:38.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  34. Asghar US, Kanani R, Roylance R, Mittnacht S. Systematic review of molecular biomarkers predictive of resistance to CDK4/6 inhibition in metastatic breast cancer. JCO Precis Oncol 2022;6:e2100002.

    PubMed  PubMed Central  Article  Google Scholar 

  35. Dean JL, McClendon AK, Knudsen ES. Modification of the DNA damage response by therapeutic CDK4/6 inhibition. J Biol Chem. 2012;287:29075–87.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Oser MG, Fonseca R, Chakraborty AA, Brough R, Spektor A, Jennings RB, et al. Cells Lacking the RB1 tumor suppressor gene are hyperdependent on Aurora B kinase for survival. Cancer Discov. 2018;9:230–47.

    PubMed  PubMed Central  Article  Google Scholar 

  37. Gong X, Du J, Parsons SH, Merzoug FF, Webster Y, Iversen PW, et al. Aurora-A kinase inhibition is synthetic lethal with loss of the RB1 tumor suppressor gene. Cancer Discov. 2019;9:48–63.

    Article  Google Scholar 

  38. Witkiewicz AK, Chung S, Brough R, Vail P, Franco J, Lord CJ, et al. Targeting the vulnerability of RB tumor suppressor loss in triple-negative breast cancer. Cell Rep. 2018;22:1185–99.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Weigelt B, Warne PH, Downward J. PIK3CA mutation, but not PTEN loss of function, determines the sensitivity of breast cancer cells to mTOR inhibitory drugs. Oncogene. 2011;30:3222–33.

    CAS  PubMed  Article  Google Scholar 

  40. Gomez Tejeda Zanudo J, Mao P, Alcon C, Kowalski K, Johnson GN, Xu G, et al. Cell line-specific network models of ER(+) breast cancer identify potential PI3Kalpha Inhibitor Resistance Mechanisms and Drug Combinations. Cancer Res. 2021;81:4603–17.

    PubMed  Article  Google Scholar 

  41. Diehl JA. Cycling to cancer with cyclin D1. Cancer Biol Ther. 2002;1:226–31.

    CAS  PubMed  Article  Google Scholar 

  42. Knudsen ES, Nambiar R, Rosario SR, Smiraglia DJ, Goodrich DW, Witkiewicz AK. Pan-cancer molecular analysis of the RB tumor suppressor pathway. Commun Biol. 2020;3:158.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Bertucci F, Ng CKY, Patsouris A, Droin N, Piscuoglio S, Carbuccia N, et al. Genomic characterization of metastatic breast cancers. Nature 2019;569:560–4.

    CAS  PubMed  Article  Google Scholar 

  44. Kumarasamy V, Vail P, Nambiar R, Witkiewicz AK, Knudsen ES. FunctionaL determinants of cell cycle plasticity and sensitivity to CDK4/6 inhibition. Cancer Res. 2021;81:1347–60.

    CAS  PubMed  Article  Google Scholar 

  45. Kumarasamy V, Ruiz A, Nambiar R, Witkiewicz AK, Knudsen ES. Chemotherapy impacts on the cellular response to CDK4/6 inhibition: distinct mechanisms of interaction and efficacy in models of pancreatic cancer. Oncogene. 2020;39:1831–45.

    CAS  PubMed  Article  Google Scholar 

  46. Rawlinson R, Massey AJ. gammaH2AX and Chk1 phosphorylation as predictive pharmacodynamic biomarkers of Chk1 inhibitor-chemotherapy combination treatments. BMC Cancer. 2014;14:483.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  47. Webster JD, Vucic D. The balance of TNF mediated pathways regulates inflammatory cell death signaling in healthy and diseased tissues. Front Cell Dev Biol. 2020;8:365.

    PubMed  PubMed Central  Article  Google Scholar 

  48. Lalaoui N, Merino D, Giner G, Vaillant F, Chau D, Liu L, et al. Targeting triple-negative breast cancers with the Smac-mimetic birinapant. Cell Death Differ. 2020;27:2768–80.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. Petersen SL, Wang L, Yalcin-Chin A, Li L, Peyton M, Minna J, et al. Autocrine TNFalpha signaling renders human cancer cells susceptible to Smac-mimetic-induced apoptosis. Cancer Cell. 2007;12:445–56.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. Yardley DA, Noguchi S, Pritchard KI, Burris HA 3rd, Baselga J, Gnant M, et al. Everolimus plus exemestane in postmenopausal patients with HR(+) breast cancer: BOLERO-2 final progression-free survival analysis. Adv Ther. 2013;30:870–84.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. Franco J, Balaji U, Freinkman E, Witkiewicz AK, Knudsen ES. Metabolic reprogramming of pancreatic cancer mediated by CDK4/6 inhibition elicits unique vulnerabilities. Cell Rep. 2016;14:979–90.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. Knudsen ES, Shapiro GI, Keyomarsi K. Selective CDK4/6 inhibitors: biologic outcomes, determinants of sensitivity, mechanisms of resistance, combinatorial approaches, and pharmacodynamic biomarkers. Am Soc Clin Oncol Educ Book. 2020;40:115–26.

    PubMed  PubMed Central  Article  Google Scholar 

  53. Lee JW, Parameswaran J, Sandoval-Schaefer T, Eoh KJ, Yang DH, Zhu F, et al. Combined Aurora Kinase A (AURKA) and WEE1 inhibition demonstrates synergistic antitumor effect in squamous cell carcinoma of the head and neck. Clin Cancer Res. 2019;25:3430–42.

    PubMed  PubMed Central  Article  Google Scholar 

  54. Knudsen ES, Pruitt SC, Hershberger PA, Witkiewicz AK, Goodrich DW. Cell cycle and beyond: exploiting new RB1 controlled mechanisms for cancer therapy. Trends Cancer. 2019;5:308–24.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. Condon SM, Mitsuuchi Y, Deng Y, LaPorte MG, Rippin SR, Haimowitz T, et al. Birinapant, a smac-mimetic with improved tolerability for the treatment of solid tumors and hematological malignancies. J Med Chem. 2014;57:3666–77.

    CAS  PubMed  Article  Google Scholar 

  56. Feoktistova M, Geserick P, Kellert B, Dimitrova DP, Langlais C, Hupe M, et al. cIAPs block Ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death complex differentially regulated by cFLIP isoforms. Mol Cell. 2011;43:449–63.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. Chau BN, Chen TT, Wan YY, DeGregori J, Wang JY. Tumor necrosis factor alpha-induced apoptosis requires p73 and c-ABL activation downstream of RB degradation. Mol Cell Biol. 2004;24:4438–47.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. Boutillier AL, Trinh E, Loeffler JP. Caspase-dependent cleavage of the retinoblastoma protein is an early step in neuronal apoptosis. Oncogene. 2000;19:2171–8.

    CAS  PubMed  Article  Google Scholar 

  59. Knudsen ES, Balaji U, Mannakee B, Vail P, Eslinger C, Moxom C, et al. Pancreatic cancer cell lines as patient-derived avatars: genetic characterisation and functional utility. Gut. 2018;67:508–20.

    CAS  PubMed  Article  Google Scholar 

  60. Knudsen ES, Kumarasamy V, Ruiz A, Sivinski J, Chung S, Grant A, et al. Cell cycle plasticity driven by MTOR signaling: integral resistance to CDK4/6 inhibition in patient-derived models of pancreatic cancer. Oncogene. 2019;38:3355–70.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

The authors thank all members of the laboratory group and colleagues in the discussion and preparation of the manuscript. We thank Emily Schultz for acquiring the patient information and the genetic characteristics of the tumor tissues. Dr. Steven Pruitt provided the lentiviral vector for CDK2 sensor. Drug screening was performed through Small Molecule Screening facility at RoswellPark Cancer Center. We would like to thank Dr. Sandra Sexton, Facility director of Laboratory Animal Resources at Roswell Park for assisting us with xenografts. The research was supported by a grant to AKW and ESK from National Cancer Institute (NCI).

Author information

Authors and Affiliations

Authors

Contributions

Study concept and design: VK, ESK, and AKW. Acquisition of data: VK, JW, HR, and RN. Analysis and interpretation of data: VK, JW, RN, ESK, and AKW. Study supervision: ESK and AKW.

Corresponding authors

Correspondence to Agnieszka K. Witkiewicz or Erik S. Knudsen.

Ethics declarations

Competing interests

ESK and AKW have received research funding from Eli Lilly, Novartis and Pfizer over the last 5 years. There is no current research support from these entities and the study was written in the absence of input from any pharmaceutical company.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kumarasamy, V., Nambiar, R., Wang, J. et al. RB loss determines selective resistance and novel vulnerabilities in ER-positive breast cancer models. Oncogene 41, 3524–3538 (2022). https://doi.org/10.1038/s41388-022-02362-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41388-022-02362-2

Search

Quick links