The impact of ROR1, BMI-1 expression and PIK3CA mutation on the prognosis of luminal breast cancer

Breast cancer (BC) remains one of the leading causes of cancer mortality among women. Luminal BC subtypes, which are characterized by the expression of hormone receptors, account for about 70 % of all breast cancer cases. However, despite sensitivity to endocrine therapy, some patients demonstrate disease progression, which is associated with the molecular features of the tumor. The study of prognostic markers such as ROR1, BMI-1, and PIK3CA mutation is essential for understanding the mechanisms of resistance to therapy and metastasis. The aim of the study was to evaluate the prognostic significance of ROR1, BMI-1 protein expression and PIK3CA gene mutation in patients with luminal BC, who received hormone therapy with aromatase inhibitors, as well as their impact on clinical outcomes, including 5-year relapse-free survival. Material and Methods. The study included 80 patients with primary resectable luminal Her2-negative breast cancer (T1–2N0–1M0). All patients received adjuvant hormonal therapy with aromatase inhibitors. The expression of ROR1, BMI-1, cyclin D1 (immunohistochemistry), and PIK3CA mutation (real-time polymerase chain reaction) were assessed in tumor tissue. Results. Positive ROR1 expression was detected in 57.5 % of cases, BMI-1 in 82.5 %, and cyclin D1 overexpression in 37.5 %. The PIK3CA mutation was identified in 30 % of patients. ROR1 expression was observed in 100 % of cases with the luminal B subtype (14 out of 14) and in 48 % of cases with the luminal A subtype (32 out of 66), p=0.001. Cyclin D1 overexpression was observed in 58.7 % of patients with ROR1 expression and in 8.8 % of patients without ROR1 expression, p<0.0001. A high level of ROR1 expression (>50 %) was associated with cyclin D1 overexpression in 100 % of cases, p=0.044. Similarly, a high level of BMI-1 expression (>50 %) was associated with cyclin D1 overexpression in 64.7 % of patients compared to 31.6 % in those with low expression, p=0.03. Patients with PIK3CA mutations demonstrated significantly lower 5-year disease-free survival (p=0.03); disease progression was observed in 29 % of cases with the mutation versus 13 % in those without it (p=0.07). Conclusion. The study confirms the significance of ROR1, BMI-1 and PIK3CA mutation as potential prognostic markers in patients with luminal breast cancer. The identified relationships with cyclin D1 and molecular subtypes emphasize their role in tumor progression. The data obtained can be used for further personalization of treatment, including combination approaches with PI3K inhibitors and hormonal therapy.

1. Sancho-Garnier H., Colonna M. Epidemiologie des cancers du sein. Presse Med. 2019; 48(10): 1076–84. doi: 10.1016/j.lpm.2019.09.022.

2. Prat A., Perou C.M. Deconstructing the molecular portraits of breast cancer. Mol Oncol. 2011; 5(1): 5–23. doi: 10.1016/j.molonc.2010.11.003.

3. Onizuka Y., Nagai K., Ideno Y., Kitahara Y., Iwase A., Yasui T., Nakajima-Shimada J., Hayashi K. Association between FSH, E1, and E2 levels in urine and serum in premenopausal and postmenopausal women. Clin Biochem. 2019; 73:105–8. doi: 10.1016/j.clinbiochem.2019.08.009.

4. Zhao H., Zhou L., Shangguan A.J., Bulun S.E.Aromatase expression and regulation in breast and endometrial cancer. J Mol Endocrinol. 2016; 57(1): 19–33. doi: 10.1530/JME-15-0310.

5. Manna P.R., Ahmed A.U., Molehin D., Narasimhan M., Pruitt K., Reddy P.H. Hormonal and Genetic Regulatory Events in Breast Cancer and Its Therapeutics: Importance of the Steroidogenic Acute Regulatory Protein. Biomedicines. 2022; 10(6): 1313. doi: 10.3390/biomedicines10061313.

6. Agarwal V.R., Bulun S.E., Leitch M., Rohrich R., Simpson E.R. Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients. J Clin Endocrinol Metab. 1996; 81(11): 3843–49. doi: 10.1210/jcem.81.11.8923826.

7. Utsumi T., Harada N., Maruta M., Takagi Y. Presence of alternatively spliced transcripts of aromatase gene in human breast cancer. J Clin Endocrinol Metab. 1996; 81(6): 2344–49. doi: 10.1210/jcem.81.6.8964875.

8. Zhou C., Zhou D., Esteban J., Murai J., Siiteri P.K., Wilczynski S., Chen S. Aromatase gene expression and its exon I usage in human breast tumors. Detection of aromatase messenger RNA by reverse transcriptionpolymerase chain reaction. J Steroid Biochem Mol Biol. 1996; 59(2): 163–71. doi: 10.1016/s0960-0760(96)00100-8.

9. Valla M., Klæstad E., Ytterhus B., Bofin A.M. CCND1 Amplification in Breast Cancer -associations With Proliferation, Histopathological Grade, Molecular Subtype and Prognosis. J Mammary Gland Biol Neoplasia. 2022; 27(1): 67–77. doi: 10.1007/s10911-022-09516-8.

10. Jeffreys S.A., Becker T.M., Khan S., Soon P., Neubauer H., de Souza P., Powter B. Prognostic and Predictive Value of CCND1/Cyclin D1 Amplification in Breast Cancer With a Focus on Postmenopausal Patients: A Systematic Review and Meta-Analysis. Front Endocrinol (Lausanne). 2022; 13: 895729. doi: 10.3389/fendo.2022.895729.

11. Montalto F.I., de Amicis F. Cyclin D1 in Cancer: A Molecular Connection for Cell Cycle Control, Adhesion and Invasion in Tumor and Stroma. Cells. 2020; 9(12): 2648. doi: 10.3390/cells9122648.

12. Aleksakhina S.N., Kramchaninov M.M., Mikushina A.D., Kubrina S.E., Petkau V.V., Ivantsov A.O., Moiseyenko V.M., Imyanitov E.N., Iyevleva A.G. CCND1 and FGFR1 gene amplifications are associated with reduced benefit from aromatase inhibitors in metastatic breast cancer. Clin Transl Oncol. 2021; 23(4): 874–81. doi: 10.1007/s12094-020-02481-w.

13. Azarnezhad A., Tabrizi M., Javan F., Mehdipour P. Detection of CCND1, C-MYC, and FGFR1 amplification using modified SYBR Green qPCR and FISH in breast cancer. Turk J Med Sci. 2018; 48(4): 759–67. doi: 10.3906/sag-1710-93.

14. Marin A., Morales F., Walbaum B. Fibroblast growth factor receptor signaling in estrogen receptor-positive breast cancer: mechanisms and role in endocrine resistance. Front Oncol. 2024; 14: 1406951. doi: 10.3389/fonc.2024.1406951.

15. Giltnane J.M., Hutchinson K.E., Stricker T.P., Formisano L., Young C.D., Estrada M.V., Nixon M.J., Du L., Sanchez V., Ericsson P.G., Kuba M.G., Sanders M.E., Mu X.J., Van Allen E.M., Wagle N., Mayer I.A., Abramson V., Gόmez H., Rizzo M., Toy W., Chandarlapaty S., Mayer E.L., Christiansen J., Murphy D., Fitzgerald K., Wang K., Ross J.S., Miller V.A., Stephens P.J., Yelensky R., Garraway L., Shyr Y., Meszoely I., Balko J.M., Arteaga C.L. Genomic profiling of ER+ breast cancers after short-term estrogen suppression reveals alterations associated with endocrine resistance. Sci Transl Med. 2017; 9(402). doi: 10.1126/scitranslmed.aai7993. Erratum in: Sci Transl Med. 2019; 11(479). doi: 10.1126/scitranslmed.aaw7620.

16. Lim W., Mayer B., Pawson T. Cell Signaling: Principles and Mechanisms. 1st Edition. New York, USA. c2014. 412р. ISBN: 9780429258893.

17. Miller T.W., Rexer B.N., Garrett J.T., Arteaga C.L. Mutations in the phosphatidylinositol 3-kinase pathway: role in tumor progression and therapeutic implications in breast cancer. Breast Cancer Res. 2011; 13(6): 224. doi: 10.1186/bcr3039.

18. Chalhoub N., Baker S.J. PTEN and the PI3-kinase pathway in cancer. Annu Rev Pathol. 2009; 4: 127–50. doi: 10.1146/annurev.pathol.4.110807.092311.

19. Martínez-Sáez O., Chic N., Pascual T., Adamo B., Vidal M., González-Farré B., Sanfeliu E., Schettini F., Conte B., Brasó-Maristany F., Rodríguez A., Martínez D., Galván P., Rodríguez A.B., Martinez A., Muñoz M., Prat A. Frequency and spectrum of PIK3CA somatic mutations in breast cancer. Breast Cancer Res. 2020; 22(1): 45. doi: 10.1186/s13058-020-01284-9.

20. Karvonen H., Barker H., Kaleva L., Niininen W., Ungureanu D. Molecular Mechanisms Associated with ROR1-Mediated Drug Resistance: Crosstalk with Hippo-YAP/TAZ and BMI-1 Pathways. Cells. 2019; 8(8): 812. doi: 10.3390/cells8080812.

21. Zhang S., Chen L., Cui B., Chuang H.Y., Yu J., Wang-Rodriguez J., Tang L., Chen G., Basak G.W., Kipps T.J. ROR1 is expressed in human breast cancer and associated with enhanced tumor-cell growth. PLoS One. 2012; 7(3). doi: 10.1371/journal.pone.0031127.

22. Cui B., Zhang S., Chen L., Yu J., Widhopf G.F. 2nd, Fecteau J.F., Rassenti L.Z., Kipps T.J. Targeting ROR1 inhibits epithelial-mesenchymal transition and metastasis. Cancer Res. 2013; 73(12): 3649–60. doi: 10.1158/0008-5472.CAN-12-3832.

23. Henry C.E., Llamosas E., Djordjevic A., Hacker N.F., Ford C.E. Migration and invasion is inhibited by silencing ROR1 and ROR2 in chemoresistant ovarian cancer. Oncogenesis. 2016; 5(5). doi: 10.1038/oncsis.2016.32.

24. Zhang S., Chen L., Wang-Rodriguez J., Zhang L., Cui B., Frankel W., Wu R., Kipps T.J. The onco-embryonic antigen ROR1 is expressed by a variety of human cancers. Am J Pathol. 2012; 181(6): 1903–10. doi: 10.1016/j.ajpath.2012.08.024.

25. Sauvageau M., Sauvageau G. Polycomb group proteins: multifaceted regulators of somatic stem cells and cancer. Cell Stem Cell. 2010; 7(3): 299–313. doi: 10.1016/j.stem.2010.08.002.

26. Kim J.H., Yoon S.Y., Jeong S.H., Kim S.Y., Moon S.K., Joo J.H., Lee Y., Choe I.S., Kim J.W. Overexpression of Bmi-1 oncoprotein correlates with axillary lymph node metastases in invasive ductal breast cancer. Breast. 2004; 13(5): 383–88. doi: 10.1016/j.breast.2004.02.010.

27. Chen D., Wu M., Li Y., Chang I., Yuan Q., Ekimyan-Salvo M., Deng P., Yu B., Yu Y., Dong J., Szymanski J.M., Ramadoss S., Li J., Wang C.Y. Targeting BMI1+ Cancer Stem Cells Overcomes Chemoresistance and Inhibits Metastases in Squamous Cell Carcinoma. Cell Stem Cell. 2017; 20(5): 621–34. doi: 10.1016/j.stem.2017.02.003.

28. Dong H., Claffey K.P., Brocke S., Epstein P.M. Inhibition of breast cancer cell migration by activation of cAMP signaling. Breast Cancer Res Treat. 2015; 152(1): 17–28. doi: 10.1007/s10549-015-3445-9.