Progesterone modulates cell growth via integrin αvβ3-dependent pathway in progesterone receptor-negative MDA-MB-231 cells

Abstract

Progesterone (P₄) plays a pivotal role in regulating the cancer progression of various types, including breast cancer, primarily through its interaction with the P₄ receptor (PR). In PR-negative breast cancer cells, P₄ appears to function in mediating cancer progression, such as cell growth. However, the mechanisms underlying the roles of P₄ in PR-negative breast cancer cells remain incompletely understood. This study aimed to investigate the effects of P₄ on cell proliferation, gene expression, and signal transduction in PR-negative MDA-MB-231 breast cancer cells. P₄-activated genes, associated with proliferation in breast cancer cells, exhibit a stimulating effect on cell growth in PR-negative MDA-MB-231 cells, while demonstrating an inhibitory impact in PR-positive MCF-7 cells. The use of arginine-glycine-aspartate (RGD) peptide successfully blocked P₄-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation, aligning with computational models of P₄ binding to integrin αvβ3. Disrupting integrin αvβ3 binding with RGD peptide or anti-integrin αvβ3 antibody altered P₄-induced expression of proliferative genes and modified P₄-induced cell growth in breast cancer cells. In conclusion, integrin αvβ3 appears to mediate P₄-induced ERK1/2 signal pathway to regulate proliferation via alteration of proliferation-related gene expression in PR-negative breast cancer cells.

Keywords: Breast cancer; Integrin αvβ3; Progesterone; Progesterone receptor; Proliferation.

Fig. 1

P₄ induces different growth patterns in PR-positive MCF-7 and PR-negative MDA-MB-231 breast cancer cells. (A) Serum-starved cells were left unstimulated or stimulated with different concentrations of P₄ (10⁻⁸ to 10⁻⁵ M) for 72 h or 120 h. The cells were then subjected to the cell viability assay. Data are represented normalized to the unstimulated group of each cell line and presented as the mean ± standard deviation of triplicate cultures in three independent experiments. *P < 0.05, ***P < 0.001 compared to the unstimulated group. (B) Serum-starved cells were left unstimulated or stimulated with 10⁻⁵ M P₄ for 72 h. The cells were then subjected to the flow cytometric analysis. The presented histograms and bar graphs show the percentage of cell populations in each cell cycle phase, as measured by DNA content stained with PI. The blue area represents the G0/G1 fraction, the dark yellow area represents the S fraction, and the green area represents the G2/M fraction. Similar results were obtained in three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

P₄ regulates gene expression in PR-positive MCF-7 and PR-negative MDA-MB-231 breast cancer cells. (A, B) Serum-starved cells were left unstimulated (━) or stimulated with different concentrations of P₄ (10⁻⁷ and 10⁻⁵ M) for 24 h. The cells were lysed and the mRNAs extracted from cell lysates were subjected to the reverse transcription reaction. The mRNA expression of PCNA, CCND1, MMP-9, PD-L1, integrin αv, integrin β3, integrin β5, and β-actin, as a loading control, was quantified by qRT-PCR. The mRNA expression of these genes was normalized to that of β-actin. The quantitative values were expressed as relative mRNA levels by defining the amounts of gene expression in unstimulated group as 1. Data are represented as the mean ± standard deviation of triplicate cultures in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the unstimulated group.

Fig. 3

Predicted docking poses of P₄ bound at the cRGD-binding site of integrin αvβ3. (A, B) Docking models 1 and models 2 of P₄ are respectively colored in blue and white, and the free binding energies are anticipated to be −6.7 -and −6.5 kcal/mol, respectively. (C) Superimpositions of binding models for modes 1 (blue) and 2 (white) mapped into cRGD peptide (purple) of αvβ3 integrin subunits. (D, E) Binding mode 1 and mode 2 of P₄ are illustrated within integrin αvβ3 and their corresponding 2D interaction plots by the BIOVIA Discovery Studio Visualizer (http://accelrys.com). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Blocking of integrin αvβ3 activity down-regulates P₄-induced integrin β3 expression in MDA-MB-231 cells. Serum-starved cells were left unpretreated or pretreated with anti-integrin αvβ3 antibody (2 μg/ml) or the RGD peptide (500 nM) for 1 h and then were left unstimulated or stimulated with10⁻⁵ M P₄ for 72 h. The cells were then lysed and cell lysates were subjected to Western blotting for the detection of the indicated integrin β3, integrin β5, and GAPDH, as a loading control (These original blot images are provided in the Supplementary file). Similar results were obtained in three independent experiments. The quantitative results were expressed as fold increase by defining the amounts of the indicated detected proteins in untreated cells, where the absence of P₄ stimulation was considered as 1. Data are represented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to untreated cells, where the absence of P₄ stimulation; ^#P < 0.05,^##P < 0.01 compared to the P₄-stimulated unpretreated cells.

Fig. 5

Blocking of Integrin αvβ3 activity affects P₄-induced gene expressions in MDA-MB-231 cells. (A–F) Serum-starved cells were left unpretreated or pretreated with anti-integrin αvβ3 antibody (2 μg/ml) or the RGD peptide (500 nM) for 1 h and then were left unstimulated or stimulated with10⁻⁵ M P₄ for 72 h. The cells were lysed and the mRNAs extracted from cell lysates were subjected to the reverse transcription reaction. The mRNA expression of integrin αv, integrin β3, integrin β5, CCND1, p21, PCNA, and β-actin, as a loading control, was quantified by qRT-PCR. The mRNA expression of these genes was normalized to that of β-actin. The quantitative values were expressed as relative mRNA levels by defining the amounts of gene expression in untreated cells, where the absence of P₄ stimulation as 1. Data are represented as the mean ± standard deviation of triplicate cultures in three‐independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to untreated cells, where the absence of P₄ stimulation; ^##P < 0.01,^###P < 0.001 compared to the P₄-stimulated unpretreated cells.

Fig. 6

P₄-induced ERK1/2 activation is integrin αvβ3-dependent in MDA-MB-231 breast cancer cells. (A, B) Cells were seeded on a cover glass and starved for 48 h. Different combinations of treatment were described in the Confocal microscopy section of Materials and Methods. Cells then were fixed for confocal microscopy. The cells were fixed, permeabilized and immunostained with antibodies against integrin αvβ3 (red color) and p-ERK1/2 (green color). The merge image shows colocalization (yellow color) of these two proteins. Nuclei were counterstained with DAPI (blue color). Accumulation of p-ERK1/2 in the nucleus was showed as white arrows. The right panel (B) shows a zoom-in image of the left panel (A) to present cells in a more focused manner. (C) Quantification of the number of p-ERK1/2 accumulation in the nucleus. Data are represented as the mean ± standard deviation of triplicate cultures in three‐independent experiments. *P < 0.05, **P<0.01, ***P < 0.001 compared to untreated cells, where the absence of P₄ stimulation; ^#P < 0.01 compared to the P₄-stimulated unpretreated cells. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

P₄ regulates signal transduction protein profiles in MDA-MB-231 cells. Serum-starved cells were left unpretreated or pretreated with anti-integrin αvβ3 antibody (2 μg/ml) or the RGD peptide (500 nM) for 1 h and then were left unstimulated or stimulated with 10⁻⁵ M P₄ for 72 h. The cells were then lysed and cell lysates were subjected to Western blotting for the detection of the indicated p-FAK (Y397), FAK, p-ERK1/2 (T202/Y204), ERK1/2, and GAPDH, as a loading control (These original blot images are provided in the Supplementary file). Similar results were obtained in three independent experiments. The quantitative results were expressed as fold increase by defining the amounts of the indicated detected proteins in untreated cells, where the absence of P₄ stimulation was considered as 1. Data are represented as the mean ± SD of three independent experiments. *P < 0.05, ***P < 0.001 compared to untreated cells, where the absence of P₄ stimulation; ^#P < 0.05 compared to the P₄-stimulated unpretreated cells.

Fig. 8

Blocking of RGD binding site affects P₄-induced cell growth in MDA-MB-231 cells. (A, B) Serum-starved cells were left unpretreated or pretreated with anti-integrin αvβ3 antibody (2 μg/ml) or the RGD peptide (500 nM) for 1 h and then were left unstimulated or stimulated with 10⁻⁵ M P₄ for 72 h. (A) The cells were then subjected to the cell viability assay. Data are represented normalized to the untreated cells, where the absence of P₄ stimulation and presented as the mean ± standard deviation of triplicate cultures in three‐independent experiments. ***P < 0.001 compared to untreated cells, where the absence of P₄ stimulation; ^###P < 0.001, compared to the P₄-stimulated unpretreated cells. (B) The cells were then subjected to the flow cytometric analysis. The bar graphs represent the percentage of cell populations in each cell cycle phase, as measured by DNA content stained with PI. Similar results were obtained in three‐independent experiments.

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