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. 2023 Nov 15;27(1):19.
doi: 10.3892/ol.2023.14152. eCollection 2024 Jan.

The inhibitory effect of trastuzumab on BT474 triple‑positive breast cancer cell viability is reversed by the combination of progesterone and estradiol

José A López-Méndez  1   2   3 , José L Ventura-Gallegos  2   3   4 , Ignacio Camacho-Arroyo  5 , Marcela Lizano  2   6 , Alberto J Cabrera-Quintero  2   3 , Sandra L Romero-Córdoba  2   3   4 , Mariano Martínez-Vázquez  7 , Nadia J Jacobo-Herrera  3 , Alfonso León-Del-Río  2   4 , Adrian A Paredes-Villa  2 , Alejandro Zentella-Dehesa  2   3   4   8   9
Affiliations

The inhibitory effect of trastuzumab on BT474 triple‑positive breast cancer cell viability is reversed by the combination of progesterone and estradiol

José A López-Méndez et al. Oncol Lett. .

Abstract

Breast cancer expressing the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) is known as triple-positive (TPBC). TPBC represents 9-11% of breast cancer cases worldwide and is a heterogeneous subtype. Notably, TPBC presents a therapeutic challenge due to the crosstalk between the hormonal (ER and PR) and HER2 pathways. Patients with TPBC are treated with trastuzumab (TTZ); however, several patients treated with TTZ tend to relapse. The present study aimed to investigate the effect of the PR on inhibitory effect of TTZ on cell viability. BT474 cells (a model of TPBC) and BT474 PR-silenced cells were treated with either TTZ, progesterone (Pg), the PR antagonist mifepristone (RU486) or estradiol (E2) alone or in combination for 144 h (6 days). Cell viability assays and western blotting were subsequently performed. The results showed that Pg and E2 interfered with the inhibitory effect of TTZ on cell viability and this effect was potentiated when both hormones were combined. Pg was revealed to act through the PR, mainly activating the PR isoform B (PR-B) and inducing the protein expression levels of CDK4 and cyclin D1; however, it did not reactivate the HER2/Akt pathway. By contrast, E2 was able to increase PR isoform A (PR-A) expression, which was inhibited by Pg. Notably, in most of the experiments, RU486 did not antagonize the effects of Pg. In conclusion, Pg and E2 may interfere with the inhibitory effect of TTZ on cell viability through PR-B activation and PR-A inactivation.

Keywords: estradiol; progesterone; trastuzumab; triple-positive breast cancer.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
Inhibitory effect of TTZ on the viability of BT474 cells is interfered with by Pg, RU486 and their combination. Data are presented as the mean ± SD of three independent experiments performed in triplicate (n=9). *P<0.05, **P<0.01 and ***P<0.001 vs. T144 or as indicated. T144, control cells; V, vehicle (DMSO); Pg, progesterone; TTZ, trastuzumab; RU486, mifepristone.
Figure 2.
Figure 2.
Effect of Pg, RU486, TTZ and their combinations on the expression and phosphorylation levels of PR isoforms. (A) Representative image of the western blot analysis of pPR-B ser345, PR-B and PR-A expression in BT474 cells treated with Pg, RU486, TTZ alone or in combination. Densitometric analysis of (B) PR-A, (C) PR-B and (D) pPR-B ser345. Data are presented as the mean ± SD of three independent experiments (n=3). *P<0.05. T144, control cells; V, vehicle (DMSO); Pg, progesterone; PR, progesterone receptor; PR-A, PR isoform A; PR-B, PR isoform B; pPR-B ser345, phosphorylated-PR in serine residue 345; TTZ, trastuzumab; RU486, mifepristone.
Figure 3.
Figure 3.
Effect of Pg, RU486, TTZ and their combinations on the expression of cyclin D1, CDK4 and p27Kip1. (A) Representative image of the western blot analysis of cyclin D1, CDK4 and p27Kip1 expression in BT474 cells treated with Pg, RU486, TTZ alone or in combination. Densitometric analysis of (B) CDK4, (C) cyclin D1 and (D) p27Kip1. Data are presented as the mean ± SD of three independent experiments (n=3). *P<0.05 vs. T144 or as indicated. T144, control cells; V, vehicle (DMSO); Pg, progesterone; TTZ, trastuzumab; RU486, mifepristone.
Figure 4.
Figure 4.
Effect of a PR-siRNA on the protein expression levels of PR isoforms. (A) Representative image of the western blot analysis of PR-B and PR-A protein expression levels in BT474 cells post-transfection. Densitometric analysis of (B) PR-A and (C) PR-B. Data are presented as the mean ± SD of three independent experiments (n=3). *P<0.05. PR, progesterone receptor; PR-A, PR isoform A; PR-B, PR isoform B; siRNA, small interfering RNA; CT, transfection control; Ctrl, untransfected cells.
Figure 5.
Figure 5.
Effect of Pg, RU486, TTZ and their combinations on the viability of PR-silenced BT474 cells. Data are presented as the mean ± SD of three independent experiments performed in triplicate (n=9). *P<0.05, **P<0.01 and ***P<0.001 vs. T144 or as indicated. T144, control cells; V, vehicle (DMSO); Pg, progesterone; TTZ, trastuzumab; RU486, mifepristone.
Figure 6.
Figure 6.
Effect of Pg, RU486, TTZ and their combinations on the expression and phosphorylation levels of HER2 and Akt in BT474 cells. (A) Representative western blot analysis of the protein expression levels of pHER2, HER2, pAkt and Akt in BT474 cells treated with Pg, RU486, TTZ alone or in combination. Densitometric analysis of (B) HER2, (C) pHER2, (D) Akt and (E) pAkt. Data are presented as the mean ± SD of three independent experiments (n=3). *P<0.05 and **P<0.01 vs. T144 or as indicated. T144, control cells; V, vehicle (DMSO); Pg, progesterone; TTZ, trastuzumab; HER2, human epidermal growth factor receptor-2; pHER2, phosphorylated HER2; pAkt, phosphorylated Akt; RU486, mifepristone.
Figure 7.
Figure 7.
Inhibitory effect of TTZ on the viability of BT474 cells is interfered with by Pg, E2 and by their combination to a greater extent. Data are presented as the mean ± SD of three independent experiments performed in triplicate (n=9). *P<0.05, **P<0.01 and ***P<0.001 vs. T144 or as indicated. T144, control cells; V, vehicle (DMSO); Pg, progesterone; TTZ, trastuzumab; E2, estradiol.
Figure 8.
Figure 8.
Effect of Pg, E2, TTZ and their combinations on the expression and phosphorylation levels of PR isoforms. (A) Representative western blot analysis of the protein expression levels of pPR-B ser345, PR-B, pPR-A ser345 and PR-A in BT474 cells treated with Pg, E2, TTZ alone or in combination. Densitometric analysis of (B) PR-A, (C) pPR-A ser345, (D) PR-B and (E) pPR-B ser345. Data are presented as the mean ± SD of three independent experiments (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. T144 or as indicated. T144, control cells; V, vehicle (DMSO); Pg, progesterone; TTZ, trastuzumab; E2, estradiol; PR, progesterone receptor; PR-A, PR isoform A; PR-B, PR isoform B; pPR-A/B ser345, phosphorylated-PRA/B in serine residue 345.
Figure 9.
Figure 9.
Association between TTZ treatment response and the expression profile of PGR, ESR1 and their downstream genes in patients with TPBC. Normalized mRNA expression levels of PGR, ESR1, CCND1, CDK4 and CDKN1B in (A) patients with TPBC and pR or CR to therapy from the GSE22358 dataset (37) or in (B) patients with TPBC and RD or pCR from the GSE50948 dataset (38). (C) Percentage of patients from the GSE22358, GSE50948 and GSE41656 (39) datasets with varied responses to therapy for which their tumors express ESR1, PGR, ERBB2 or a combination of these genes. (D) Survival curve of patients from the GSE96058 dataset (40) with ER+/HER2+ breast cancer, low or high levels of PGR and following a treatment scheme that included TTZ. *P<0.05 vs. CR. pR, partial response; CR, complete response; PGR, progesterone receptor; ESR1, estrogen receptor 1; CCND1, cyclin D1; CDKN1B, p27Kip1; RD, residual disease; pCR, pathological complete response; ERBB2, human epidermal growth factor receptor-2; TPBC, triple-positive breast cancer; HR, hazard ratio.
Figure 10.
Figure 10.
Schematic diagram of the findings on the effect of Pg, E2, TTZ and their combinations on BT474 cells. (A) HER2/Akt-dependent proliferation pathway in tumor cells. (B) Interference of TTZ activity by Pg is associated with PR-B phosphorylation and induction of CDK4 and cyclin D1. (C) Combinatorial effect of Pg and E2 on TTZ activity is associated with PR-A dephosphorylation. GF, growth factor; Pg, progesterone; TTZ, trastuzumab; E2, estradiol; HER2, human epidermal growth factor receptor-2; PR, progesterone receptor; PR-A, PR isoform A; PR-B, PR isoform B.
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References

    1. Lv H, Yan M, Jiang Z. Recent advances in the treatment of hormone receptor-positive/human epidermal growth factor 2-positive advanced breast cancer. Ther Adv Med Oncol. 2021;13:17588359211013326. doi: 10.1177/17588359211013326. - DOI - PMC - PubMed
    1. Parise CA, Caggiano V. Differences in clinicopatholgic characteristics and risk of mortality between the triple positive and ER+/PR+/HER2-breast cancer subtypes. Cancer Causes Control. 2019;30:417–424. doi: 10.1007/s10552-019-01152-8. - DOI - PubMed
    1. Vici P, Pizzuti L, Natoli C, Gamucci T, Di Lauro L, Barba M, Sergi D, Botti C, Michelotti A, Moscetti L, et al. Triple positive breast cancer: A distinct subtype? Cancer Treat Rev. 2015;41:69–76. doi: 10.1016/j.ctrv.2014.12.005. - DOI - PubMed
    1. Tran B, Bedard PL. Luminal-B breast cancer and novel therapeutic targets. Breast Cancer Res. 2011;13:221. doi: 10.1186/bcr2904. - DOI - PMC - PubMed
    1. Knutson TP, Lange CA. Tracking progesterone receptor-mediated actions in breast cancer. Pharmacol Ther. 2014;142:114–125. doi: 10.1016/j.pharmthera.2013.11.010. - DOI - PMC - PubMed