NAF-1 and mitoNEET are central to human breast cancer proliferation by maintaining mitochondrial homeostasis and promoting tumor growth

Abstract

Mitochondria are emerging as important players in the transformation process of cells, maintaining the biosynthetic and energetic capacities of cancer cells and serving as one of the primary sites of apoptosis and autophagy regulation. Although several avenues of cancer therapy have focused on mitochondria, progress in developing mitochondria-targeting anticancer drugs nonetheless has been slow, owing to the limited number of known mitochondrial target proteins that link metabolism with autophagy or cell death. Recent studies have demonstrated that two members of the newly discovered family of NEET proteins, NAF-1 (CISD2) and mitoNEET (mNT; CISD1), could play such a role in cancer cells. NAF-1 was shown to be a key player in regulating autophagy, and mNT was proposed to mediate iron and reactive oxygen homeostasis in mitochondria. Here we show that the protein levels of NAF-1 and mNT are elevated in human epithelial breast cancer cells, and that suppressing the level of these proteins using shRNA results in significantly reduced cell proliferation and tumor growth, decreased mitochondrial performance, uncontrolled accumulation of iron and reactive oxygen in mitochondria, and activation of autophagy. Our findings highlight NEET proteins as promising mitochondrial targets for cancer therapy.

Keywords: Cisd1; Cisd2; MCF7; MDA-MB-231; Miner1.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Protein expression, cellular proliferation and mitochondrial function of mNT and NAF-1 in human epithelial breast cancer cells. (A) Accumulation of mNT and NAF-1 protein in three different human epithelial breast cancer cell lines. (B) Suppression of mNT and NAF-1 protein expression in MCF-7 cells expressing shRNA against mNT (mNT−) or NAF-1 (NAF-1−). (Upper) Protein blot. (Lower) Relative levels of the respective proteins. (C) Suppression of cell growth and proliferation in mNT− or NAF-1− MCF-7 cells. (Inset) Relative change in growth rate for control, mNT(−), and NAF-1(−) at day 6. (D) Mitochondrial function (Left) and glycolytic activity (Right) in control MCF-7 cells and MCF-7 cells expressing shRNA against mNT (mNT−) or NAF-1 (NAF-1−). (E) Spare respiratory capacity (Left) and glycolytic capacity (Right) in control and mNT− or NAF-1− MCF-7 cells. NAF-1 and mNT are seen to accumulate in breast cancer cells, and their suppression via shRNA results in suppressed cell proliferation and reduced mitochondrial function. Figs. S1, S2, and S3 present similar results with MDA-231 cells. *P < 0.05; **P < 0.01.

Fig. 2.

Decreased membrane potential and overaccumulation of iron and ROS in mitochondria of cells with suppressed expression of mNT or NAF-1. (A) Images (Left) and a quantitative graph (Right) showing decreased mitochondrial membrane potential in cells with suppressed expression of mNT (mNT−) or NAF-1 (NAF-1−). Pretreatment of cells with the iron chelator DFP (50 µM) prevents the decrease in mitochondrial membrane potential (Right). (B) Images (Left) and a quantitative graph (Right) showing overaccumulation of iron in mitochondria of mNT− or NAF-1− cells. Pretreatment of cells with the iron chelator DFP (100 µM) prevents mitochondrial iron accumulation (Right). (C) Images (Upper Left), a time course (Lower Left), and a quantitative graph (Right) showing the accumulation of ROS in mitochondria of mNT− or NAF-1− cells. Pretreatment of cells with the iron chelator DFP (100 µM) prevents mitochondrial ROS accumulation (Right). The function of mNT and NAF-1 is seen to be required for maintaining mitochondrial membrane potential, as well as mitochondrial iron and ROS homeostasis in human epithelial breast cancer cells. Fig. S5 presents similar results with MDA-231 cells. *P < 0.05, **P < 0.01.

Fig. 3.

Activation of autophagy in human epithelial breast cancer cells with suppressed expression of mNT or NAF-1. (A) Three representative transmission electron microscopy (TEM) images of mitochondria from control cancer cells (Left) and cancer cells with suppressed expression of mNT (mNT−; Center) or NAF-1 (NAF-1−; Right). (B) Quantitative analysis of mitochondrial damage in the form of loss of crista (Top), abnormal elongation (Middle), and the accumulation of autophagosomes (Bottom) in TEM images from control and mNT− or NAF-1− cells. (C and D) Protein gel analysis (C) and protein blot analysis (D) showing accumulation of autophagy marker proteins in breast cancer cells with suppressed expression of mNT (mNT−) or NAF-1 (NAF-1−). Autophagy is shown to be activated in human epithelial breast cancer cells with suppressed expression of mNT or NAF-1. The arrow indicates the position of Lc3B. *P < 0.05.

Fig. 4.

NAF-1 or mNT are required to support tumor growth. Breast cancer cells (MDA-231) with or without suppressed expression of mNT (mNT−) or NAF-1 (NAF-1−) were injected s.c. into the back of female CD1 nude mice, and tumor growth was monitored over time. (A) Slower tumor growth of breast cancer cells (MDA-231) with suppressed expression of mNT (mNT−) or NAF-1 (NAF-1−) compared with control MDA-231 cells. (B) Reduction of mean tumor volume expressed as percentage of control MDA-231 in mNT− or NAF-1− MDA-231 cells. (C and D) Images of representative tumors of each group obtained at the end of the experiment. (E) Histological analyses of tumors using H&E staining showing large necrotic areas in tumors formed from the control MDA-231 cells compared with tumors formed from mNT− or NAF-1− MDA-231 cells. *P < 0.05.