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Efficacy of Methionine Restriction and the PARP-inhibitor Olaparib and Their Combination on BRCA1 Mutant and Wild-type Triple-negative Breast Cancer Cell Lines

MAYUKO MIKI, SACHIKO INUBUSHI, QINGHONG HAN, SHOTARO INOUE, TOMONARI KUNIHISA, HIROKAZU TANINO and ROBERT M. HOFFMAN
Anticancer Research April 2025, 45 (4) 1367-1372; DOI: https://doi.org/10.21873/anticanres.17522

Abstract

Background/Aim: Triple-negative breast cancer (TNBC) is a recalcitrant disease. The present study examined the efficacy of methionine restriction and the poly ADP-ribose polymerase (PARP)-inhibitor olaparib on BRCA1/2 wild-type and BRCA1 mutated TNBC cell lines.

Materials and Methods: The human BRCA1/2 wild-type cell line MDA-MB-231, and BRCA1-mutant cell lines MDA-MB-436 and HCC1937 were used to examine sensitivity to recombinant methioninase (rMETase) or a methionine-free medium or to olaparib or the combination of a methionine-free medium and olaparib. Cell viability was examined using the WST-8 reagent as well as by direct cell counting after Hoechst 33342 staining.

Results: MDA-MB-231 was sensitive to a methionine-free medium and rMETase and resistant to olaparib. The combination of olaparib and a methionine-free medium was not synergistic on MDA-MB-231 cells. MDA-MB-436 cells were not sensitive to a methionine-free medium but were sensitive to olaparib and rMETase. The combination of olaparib and a methionine medium was not synergistic in MDA-MB-436 cells. HCC1937 cells were sensitive to a methionine-free medium, partially sensitive to rMETase, partially resistant to olaparib, and sensitive to the combination of a methionine-free medium and olaparib. All three cell lines were sensitive to rMETase, with MDA-MB-436 being the most sensitive.

Conclusion: Methionine restriction and olaparib showed synergistic efficacy on the BRCA1-mutant TNBC cell line HCC1937. The BRCA1-mutant cell line MDA-MB-436 was most sensitive to rMETase. The BRCA1/2 wild-type TNBC cell line MDA-MB-231 was sensitive to a methionine-free medium but resistant to olaparib. Therefore, methionine restriction has clinical potential for BRCA1/2 wild-type and BRCA1-mutant olaparib-resistant and -sensitive TNBC.

Keywords:

Introduction

Triple-negative breast cancer (TNBC) has a high frequency of BRCA1 gene mutations and is characterized by high malignancy and poor prognosis (1). BRCA1/2 mutations inhibit homologous-recombination DNA repair (HR), resulting in difficulty repairing DNA double-strand breaks (DSBs). Poly ADP-ribose polymerase (PARP) inhibitors inhibit the repair of single-strand breaks (SSBs) and cause irreparable DNA damage in HR-deficient cells as accumulated SSBs progress to DSBs during replication. This selectively kills BRCA1/2 mutant cells via a mechanism of synthetic lethality (2, 3).

The PARP inhibitor olaparib is a molecular-targeted drug that inhibits tumors with homologous-recombination deficiency and is used for breast-cancer patients with BRCA1/2 mutations. However, sensitivity to PARP inhibitors varies among cells and patients with BRCA1/2 mutations, and resistance to PARP inhibitors have become a recalcitrant clinical problem. Thus, it is critical to overcome resistance to PARP inhibitors (4, 5).

Cancer cells produce normal or greater-than-normal amounts of methionine from homocysteine but require exogenous methionine to survive, which is termed methionine addiction or the Hoffman effect (6). Normal cells do not require exogenous methionine for survival when grown on homocysteine. Methionine addiction is thought to be caused by an increased methionine requirement in cancer cells due to excessive transmethylation reactions (6-14).

Recombinant methioninase (rMETase), an enzyme that degrades extracellular methionine, targets the methionine addiction of cancer (11). Previous studies have demonstrated the efficacy of rMETase in preclinical models and patients with breast cancer (15, 16).

Methionine restriction (MR) causes cancer cells to arrest in the S/G2 phase of the cell cycle and induces apoptosis. Many chemotherapeutic agents targeting the S/G2 phase of the cell cycle have synergistic efficacy against cancer cells when combined with MR (17). In contrast, normal mammalian cells proliferate under MR (9).

The purpose of the present study was to investigate the efficacy of MR and olaparib, a PARP inhibitor, and their combination on PARP inhibitor-resistant and -sensitive TNBC cell lines with and without a BRCA1 gene mutation.

Materials and Methods

Cell culture. The BRCA1/2-wild-type TNBC cell line MDA-MB-231, and the BRCA1-mutant TNBC cell lines MDA-MB-436 and HCC1937 were used in the present study. The cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (Nichirei Biosciences Inc., Tokyo, Japan) and 100 IU/ml penicillin/streptomycin (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37°C under a 5% CO2 atmosphere.

Efficacy of rMETase and olaparib. MDA-MB-231, MDA-MB-436, and HCC1937 cells were seeded in 96-well plates at a density of 1,000 to 3,000 cells per well and cultured overnight. Each cell line was then treated with olaparib (AZD2281, Selleck, Houston, TX, USA) (0 μM to 500 μM) or rMETase (0 units/ml to 8 units/ml). Cell proliferation was measured using the WST-8 reagent (Dojindo, Kumamoto, Japan) on days 1, 2, 4, 6, and 8. Each concentration was analyzed in eight replicate wells.

Comparison of cell growth in methionine-free and methionine-containing media with or without olaparib. The TNBC cells were seeded into 24-well plates. The next day, the cells were washed with phosphate-buffered saline (PBS) (Fujifilm Wako Pure Chemical Industries, Ltd.). DMEM without methionine (Thermo Fisher Scientific Inc.) or DMEM with methionine, both supplemented with 10% FBS were used (18). Olaparib was used at 2 μM. All treatments were performed for 48 h. After treatment cells were stained with Hoechst 33342 solution (Dojindo) to identify viable cells then, fixed with 4% paraformaldehyde in phosphate buffer (Fujifilm Wako Pure Chemical Industries, Ltd.). Six fields were photographed with a fluorescence microscope (BZ-X800, KEYENCE, Osaka, Japan), and the number of viable cells with nuclei stained with Hoechst 33342 was counted.

Statistical analysis. One-way ANOVA with Bonferroni-Dunn correction was used to test for differences in cell counts between the two groups. Statistical analysis was performed using Statcel 4 software (OMS Publishing Inc., Tokyo, Japan).

Results

Sensitivity of TNBC cells, with and without a BRCA1 mutation, to the PARP inhibitor olaparib. BRCA1-mutant MDA-MB-436 TNBC cells were more sensitive to low concentrations of olaparib, than BRCA1-mutant TNBC HCC1937 cells. BRCA1/2 wild-type TNBC MDA-MB-231 cells were more resistant to olaparib (Figure 1).

Figure 1.
Figure 1.

Time-course efficacy of olaparib on the triple-negative breast cancer (TNBC) cell line MDA-MB-231, BRCA1/2 wild type, and BRCA1-mutant TNBC cell lines MDA-MB-436 and HCC1937. Please see Materials and Methods for details.

Sensitivity of TNBC cells with and without a BRCA1 mutation to rMETase. BRCA1/2 wild-type MDA-MB-231 cells and BRCA1-mutant HCC1937 cells were resistant to low concentrations of rMETase. BRCA1-mutant MDA-MB-436 cells were more sensitive to low concentrations of rMETase. All cell lines responded to rMETase in a concentration- and time-dependent manner (Figure 2).

Figure 2.
Figure 2.

Time-course efficacy of various concentrations of rMETase on triple-negative breast cancer cell lines described in the legend of Figure 1. Please see Materials and Methods for details.

Effect of a methionine-free medium or olaparib or their combination on the proliferation of TNBC cells. BRCA1/2 wild-type MDA-MB-231 cells were resistant to olaparib (2 μM) alone and inhibited by MR alone (p<0.01). Their combination did not produce a synergistic effect (Figure 3). BRCA1-mutant MDA-MB-436 cells were significantly inhibited by olaparib (p<0.01) which is consistent with previous reports (19, 20). MR alone did not significantly inhibit cell proliferation and the combination of MR and olaparib was not more effective than olaparib alone. The BRCA1-mutant HCC1937 cell line was inhibited by both MR and olaparib. The combination of MR and olaparib synergistically inhibited cell proliferation (p<0.01) (Figure 3).

Figure 3.
Figure 3.

Efficacy of olaparib (2 μM), a methionine-free medium, or their combination on the triple-negative breast cancer cell lines described in Figure 1. A) Hoescht 3,342-stained cells. B) Quantitation of cells in A. Please see Materials and Methods for details. MR: Methionine restriction (methionine-free medium); Ola: olaparib.

Discussion

In the present study, olaparib inhibited the growth of the BRCA1 mutant TNBC cell lines MDA-MB-436 and HCC1937 more than the BRCA1/2-wild type TNBC cell line MDA-MB-231. The combination of MR and olaparib had synergistic efficacy on the BRCA1 mutant cell line HCC1937. All three TNBC cell lines were sensitive to rMETase, at least at high concentrations.

MR alone was effective on the MDA-MB-231 BRCA1/2-wild-type cell line. Thus, MR can be effective for TNBC.

The present results, thus, suggest that MR can be effective in TNBC cell lines and was synergistically effective with olaparib on the BRCA1-mutant cell line HCC1937.

Voutsadakis et al. described a strategy of combining PARP inhibitors and other DNA-damage-response inhibitors, and we believe that MR is a potential candidate for this strategy (21).

oral rMETase (o-rMETase) plus first-line chemotherapy was highly effective in the clinic for stage IV invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) of the breast (13, 22). These results and the present report suggest that o-rMETase can be an effective agent in the clinic for TNBC with and without a BRCA mutation.

Footnotes

  • Authors’ Contributions

    MM, SI, and RMH designed the study. QH produced rMETase. MM and SI conducted all experiments. SI wrote the article and RMH revised the article. SI, TK, and HT critically read the manuscript.

  • Conflicts of Interest

    All Authors have no conflicts of interest or financial ties to disclose in relation to this study.

  • Funding

    The Authors would like to thank the Kobe University International Collaborative Research PI Training Program for providing funds.

  • Received February 12, 2025.
  • Revision received February 25, 2025.
  • Accepted February 26, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Efficacy of Methionine Restriction and the PARP-inhibitor Olaparib and Their Combination on BRCA1 Mutant and Wild-type Triple-negative Breast Cancer Cell Lines
MAYUKO MIKI, SACHIKO INUBUSHI, QINGHONG HAN, SHOTARO INOUE, TOMONARI KUNIHISA, HIROKAZU TANINO, ROBERT M. HOFFMAN
Anticancer Research Apr 2025, 45 (4) 1367-1372; DOI: 10.21873/anticanres.17522
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