Suppressing mitochondrial inner membrane protein (IMMT) inhibits the proliferation of breast cancer cells through mitochondrial remodeling and metabolic regulation

Abstract

Metabolic reprogramming is widely recognized as a hallmark of malignant tumors, and the targeting of metabolism has emerged as an appealing approach for cancer treatment. Mitochondria, as pivotal organelles, play a crucial role in the metabolic regulation of tumor cells, and their morphological and functional alterations are intricately linked to the biological characteristics of tumors. As a key regulatory subunit of mitochondria, mitochondrial inner membrane protein (IMMT), plays a vital role in degenerative diseases, but its role in tumor is almost unknown. The objective of this research was to investigate the roles that IMMT play in the development and progression of breast cancer (BC), as well as to elucidate the underlying biological mechanisms that drive these effects. In this study, it was confirmed that the expression of IMMT in BC tissues was significantly higher than that in normal tissues. The analysis of The Cancer Genome Atlas (TCGA) database revealed that IMMT can serve as an independent prognostic factor for BC patients. Additionally, verification in clinical specimens of BC demonstrated a positive association between high IMMT expression and larger tumor size (> 2 cm), Ki-67 expression (> 15%), and HER-2 status. Furthermore, in vitro experiments have substantiated that the suppression of IMMT expression resulted in a reduction in cell proliferation and alterations in mitochondrial cristae, concomitant with the liberation of cytochrome c, but it did not elicit mitochondrial apoptosis. Through Gene Set Enrichment Analysis (GSEA) analysis, we have predicted the associated metabolic genes and discovered that IMMT potentially modulates the advancement of BC through its interaction with 16 metabolic-related genes, and the changes in glycolysis related pathways have been validated in BC cell lines after IMMT inhibition. Consequently, this investigation furnishes compelling evidence supporting the classification of IMMT as prognostic marker in BC, and underscoring its prospective utility as a novel target for metabolic therapy.

Keywords: Breast cancer; Metabolic reprogramming; Mitochondria remodeling; Mitochondrial inner membrane protein (IMMT); Prognostic marker.

Figure 1

IMMT is highly expressed in BC and is related to poor prognosis. (A) An analysis of IMMT expression in TIMER2.0 database between tumor tissues and normal tissues (*P < 0.05, **P < 0.01 and ***P < 0.001). (B) Based on the TCGA database, IMMT is expressed in BC and normal breast tissues. (C) The expression of IMMT in paired BC and normal breast tissues based on TCGA database. (D) Kaplan–Meier survival analysis of BC patients based on IMMT mRNA expression levels. (E) Prognostic significance of IMMT in BC patients assessed by multivariate Cox analysis. (F–H) This is supported by Kaplan–Meier analysis, which indicates that heightened IMMT levels are linked to poorer outcomes in terms of overall survival (OS), distant metastasis-free survival (DMFS) and relapse-free survival (RFS).

Figure 2

The high expression of IMMT in BC tissues is significantly related to adverse prognosis. (A) Analysis of IMMT protein expression levels in normal breast tissue and tumor tissue through the HPA database. (B) Expression level of IMMT in BC tissues. (C) According to the Mann–Whitney test, IMMT exhibits higher expression levels in tumor tissues that are ER-negative and HER-2 positive; assessment by the Kruskal Wallis test indicates significant differences in IMMT expression across various histological grades and molecular subtypes.

Figure 3

The proliferation of BC cells is inhibited in the absence of IMMT. (A) This is supported by the expression of IMMT protein in various types of BC cell lines. Mean ± SD (n = 3). (B) The immunofluorescence localization of IMMT in SK-BR-3 cells. (C,D) CCK-8 assays revealed suppressed proliferation in SK-BR-3 and MDA-MB-436 cells upon knockdown of IMMT. Mean ± SD (n = 3 to 4). (E,F) The decrease in colony counts of SK-BR-3 and MDA-MB-436 cells transfected with si-IMMT compared to the si-Ctrl group. (G,H) This was confirmed through Western blot analysis of proliferation-related proteins following si-IMMT transfection. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 4

The depletion of IMMT results in alterations to the morphology of mitochondria. (A) SK-BR-3 cells were observed with laser confocal after IMMT-KD to observe Mito-Tracker staining. (B) The alteration of mitochondrial dynamic proteins subsequent to the deletion of IMMT was evaluated through immunoblotting analysis. (C) SK-BR-3 cells transfected with si-IMMT were analyzed by transmission electron microscope, and the number and area of mitochondria were analyzed and counted (the red arrows indicate mitochondria). Data are presented as mean ± SEM, *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 5

IMMT deficiency leads to the release of cytochrome c, but does not induce mitochondrial apoptosis. (A) TUNEL was used to detect apoptosis after treating cells with si-IMMT for 48 h, the positive control was treated with 10 nM paclitaxel for 24 h. (B) The expression of apoptosis-related proteins was detected by Western-blot. (C,D) Following the knockdown of the IMMT, the localization of cytochrome c was examined using a fluorescence microscope (the red arrow indicates cytochrome c) and laser confocal microscope. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01.

Figure 6

Gene set enrichment analysis (GSEA) based on the expression of IMMT in BC patients from TCGA database. (A) GSEA results show significant enrichment of metabolic pathways in BC patients with high expression of IMMT. Seven metabolic-related pathways enriched in IMMT high expression group NES, normalized enrichment score. (B) The interaction network between IMMT and metabolic-related proteins was generated using STRING. (C) Correlation analysis between IMMT and six interacting molecules: ACO2, PAFAH1B1, PDHA1, DGUOK, PIK3CA, and PIK3CB. (D) Interaction networks of ACO2 and PDHA1 with mitochondrial dynamics-related proteins (MFN2, MFN1, MFF, Drp1, OPA1). (E) Western blot detected glycolysis related indexes after IMMT-KD. Data are presented as mean ± standard error of the mean, *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 7

The present study investigates the 3D molecular docking interaction between IMMT and its related ligands. (A–F) The binding mode of the IMMT-ACO2, IMMT-PAFAH1B1, IMMT-PDHA1, IMMT-DGUOK, IMMT-PIK3CA, and IMMT-PIK3CB complexes is analyzed, with IMMT represented in wheaten and the target protein in blue. Hydrogen bonding and hydrophobic interactions are respectively denoted by yellow and wine red dotted lines. (G) The overall survival of 6 IMMT-associated metabolic genes in breast cancer patients were analyzed with GEPIA.