Coordinated Upregulation of Mitochondrial Biogenesis and Autophagy in Breast Cancer Cells: The Role of Dynamin Related Protein-1 and Implication for Breast Cancer Treatment

Overactive mitochondrial fission was shown to promote cell transformation and tumor growth. It remains elusive how mitochondrial quality is regulated in such conditions. Here, we show that upregulation of mitochondrial fission protein, dynamin related protein-1 (Drp1), was accompanied with increased mitochondrial biogenesis markers (PGC1α, NRF1, and Tfam) in breast cancer cells. However, mitochondrial number was reduced, which was associated with lower mitochondrial oxidative capacity in breast cancer cells. This contrast might be owing to enhanced mitochondrial turnover through autophagy, because an increased population of autophagic vacuoles engulfing mitochondria was observed in the cancer cells. Consistently, BNIP3 (a mitochondrial autophagy marker) and autophagic flux were significantly upregulated, indicative of augmented mitochondrial autophagy (mitophagy). The upregulation of Drp1 and BNIP3 was also observed in vivo (human breast carcinomas). Importantly, inhibition of Drp1 significantly suppressed mitochondrial autophagy, metabolic reprogramming, and cancer cell viability. Together, this study reveals coordinated increase of mitochondrial biogenesis and mitophagy in which Drp1 plays a central role regulating breast cancer cell metabolism and survival. Given the emerging evidence of PGC1α contributing to tumor growth, it will be of critical importance to target both mitochondrial biogenesis and mitophagy for effective cancer therapeutics.


Introduction
Metabolic and nutrient homeostasis is critical for cellular function and human health. Mitochondrial alterations have been implicated in a variety of human metabolic diseases including cancer [1][2][3]. A hallmark of cancer metabolism is the switch from oxidative phosphorylation (OXPHOS) to robust glycolysis, and deficits of OXPHOS have been associated with malignancy and cancer cell growth [4][5][6]. As such, increasing efforts have been made to explore cancer therapeutics by targeting mitochondria and the metabolic switch [4,5,7,8].
As the organelles where OXPHOS takes place, mitochondria are under tight content and quality control via the triad of de novo mitochondrial biogenesis, mitochondrial dynamics (fusion and fission), and mitochondrial autophagy (mitophagy) [9,10]. Frequent fusion and fission facilitate the exchange of proteins, mtDNA, and metabolites to maintain mitochondrial integrity [9][10][11][12]. Dysregulation of the dynamic processes impairs mitochondrial function and has been reported in cancer, diabetes, and neurodegenerative diseases [9,10,13]. For instance, mitochondrial fission is upregulated due to Drp1 activation or overexpression in different types of tumors or cancers [14][15][16][17][18]. In breast cancer, activation of Drp1 promotes mitochondrial fragmentation and facilitates cancer cell migration and invasion; by contrast, ablation of Drp1 in breast cancer cells leads to mitochondria elongation and dampens their metastatic ability [14]. However, important questions remain as to how Drp1 dysregulation may interact with mitochondrial biogenesis and mitophagy and how the interactions affect mitochondrial quality control and are related to metabolic reprograming in breast cancer cells.
In this study, we found that upregulation of Drp1 was associated with reduced mitochondrial oxidative capacity in breast cancer cells. Surprisingly, mitochondrial content or number was reduced despite elevated regulators that promote mitochondrial biogenesis (i.e., PGC1 , NRF1, and Tfam) in breast cancer cells. These changes may arise from augmented mitophagy that removes mitochondria from cancer cells. Importantly, inhibition of Drp1 attenuated mitophagy and reversed the metabolic reprogramming, which reduced breast cancer cell viability. Our data demonstrate for the first time that the Drp1-mitophagy axis plays a key role in mitochondrial turnover and metabolic reprogramming for cell survival in breast cancer.

Electron
Microscopy. The electron microscopy was performed as described previously [13,26]. Briefly, the cells were trypsinized at 80%-90% confluence, washed with PBS, and then fixed in the mix of glutaraldehyde (2.5%), formaldehyde (4.4%), and picric acid (2.75%) in 0.05% sodium cacodylate buffer at 4 ∘ C overnight. Cells were rinsed in cacodylate buffer twice and incubated for 1 h at 4 ∘ C in 1% (v/v) OsO4/1% (w/v) K 4 Fe(CN) 6 in cacodylate buffer at pH 7.4, followed by rinsing in cacodylate buffer and then in distilled water. The cells were stained with 1% (w/v) aqueous uranyl acetate for 2 h at 4 ∘ C, washed with distilled water, and dehydrated through increased graded ethanol series and embedded. Ultrathin sections were stained with lead citrate and underwent imaging analysis with electron microscopy [13].

Mitochondrial Oxidative
Capacity. Mitochondrial oxidative capacity was measured with Oxygen Consumption Rate Assay Kits (Cayman Chemical), and parameters including oxygen consumption rate (OCR), mitochondrial ATP turnover, respiration control ratio, coupling efficiency (CE), max respiration rate, and spare respiration capacity (SRC) were analyzed as described previously [33].

Measurement of Mitochondrial Membrane Potential
(ΔΨm). The ΔΨm value was measured with fluorescent carbocyanine dye JC1 that enters the mitochondria and exhibits red fluorescence (FL) in a ΔΨm dependent manner [13]. JC1 retained in the cytosol displays green FL. The cells were stained with JC1 (2 M) in culture media for 30 min and washed with cold PBS 5 times, and the fluorescence was recorded with a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek), set at 488 nm excitation and 530 nm (green) and 585 nm (red) emission [34]. The ratio of red FL against green FL reflects ΔΨm of the mitochondria [13,34].
2.6. Glycolytic Activity. The glycolytic activity was analyzed with Glycolysis Cell-Based Assay Kits (Cayman Chemical) according to the manufacturer's instructions. Briefly, the cells were maintained or treated as described above until the day before measurement, when the cells were switched to regular media with no FBS for 20 hours in a 5% CO 2 incubator at 37 ∘ C, and the media were taken to measure glycolytic activity.

Immunohistochemistry Analysis of Human Breast Tissues.
Archive paraffin blocks of breast cancer tissues and adjacent normal tissues from human breasts were used for immunohistochemical analysis using a rabbit polyclonal Drp1 antibody (NB110-55237, Novus Biologicals) and a mouse monoclonal anti-BNIP3 antibody (ab10433, Abcam) by following the manufacturers' instruction. Hematoxylin was used for counterstaining. Slides were examined with a Nikon Eclipse 80i microscope.

Statistical Analyses.
All results are expressed as means ± SEM and are analyzed by analysis of variance (ANOVA) to determine values; < 0.05 was considered statistically significant.

Transcript and Protein Levels of Drp1
Were Upregulated in Breast Cancer. Activation or overexpression of Drp1 protein has been implicated in oncogenic pathways, tumor growth, and metastatic process [14][15][16][17][18]. To determine whether gene expression of Drp1 is dysregulated, we analyzed the transcript level of DNM1L (encoding Drp1) in 529 human breast cancer tissues and 61 normal breast tissue entities, which showed significant upregulation of DNM1L in breast cancer tissues (Figure 1(a)). Immunohistochemistry (IHC) analysis of normal breast tissues and invasive carcinomas revealed strong staining of Drp1 in the cancer tissues but not in normal breast tissues (Figures 1(b) and 1(c)). Western blot analysis of Drp1 protein in breast cancer MDA-MB-231 cells indicated 5.1-fold ( < 0.001) elevation in comparison with nontumorigenic human breast MCF10A cells (Figures 1(d) and 1(e)). Interestingly, mitochondrial fusion protein Mfn1 was downregulated by 60% ( < 0.01), which was associated with a significant reduction of Mfn1 gene expression; however, Mfn2 mRNA and protein were unchanged (Figures 1(d) and These results suggest that breast cancer adopts selective mitochondrial dynamics which favor fission over fusion by upregulating Drp1 but downregulating Mfn1.

Mitochondrial Content and Oxidative Capacity Were Reduced in Breast Cancer Cells.
To determine how dysregulated mitochondrial dynamics might affect mitochondrial content and quality in breast cancer cells, we employed electron microscopy to study the ultrathin sections of the cells. Figure  2, the mitochondria in MDA-MB-231 cells had lower electron density in the matrix than in MCF-10A cells, indicative of metabolically less active or unhealthy mitochondria [13,36,37]. In line with upregulated Drp1 and downregulated Mfn1 (Figure 1), the mitochondria were smaller or more fragmented in MDA-MB-231 cancer cells (supplemental Figure  2). Moreover, the mitochondrial number in MDA-MB-231 cells was reduced by 34% when compared with MCF-10A cells (a median value of 25.5 versus 38.5 mitochondria/cell, resp., < 0.05) (Figures 2(a)-2(c)). The reduced mitochondrial content was further validated by a significant decrease in the protein level of VDAC (82%, < 0.001), a biomarker of mitochondrial content [38] (Figures 2(d) and 2(e)).

Mitochondrial Biogenesis Was Enhanced in Breast Cancer
Cells. The reduced mitochondrial number prompted us to examine whether mitochondrial biogenesis was downregulated. Unexpectedly, the key activators of mitochondrial biogenesis, PGC1 and its downstream targets NRF1 and Tfam [39][40][41], were upregulated by 2.9-fold ( < 0.05), 4.8fold ( < 0.001), and 2.1-fold ( < 0.05), respectively, in MDA-MB-231 cells when compared with MCF-10A cells (Figures 3(a) and 3(b)). The paradoxical reduction of mitochondrial number versus increased mitochondrial biogenesis and fission suggests that breast cancer cells may adopt an overactive process of mitochondrial turnover.

Mitochondrial Autophagy Was Upregulated in Breast
Cancer. Mitochondrial autophagy (or mitophagy) plays an important role in mitochondrial turnover and quality control [42]. To test whether mitophagy is involved in mitochondrial changes in cancer cells, we analyzed the mitophagy marker BNIP3 [30,43] and found that its protein level was 7.9fold elevated ( < 0.001) in MDA-MB-231 cells when it was compared with that in MCF-10A cells (Figures 4(a) and 4(b)). By immunohistochemistry analysis, we observed dense staining of BNIP3 in invasive carcinomas, which was absent from normal breast tissues (supplemental Figure  4), suggestive of upregulated mitochondrial autophagy in breast cancer. Indeed, we observed a larger population of autophagic vacuoles that engulfed mitochondria in MDA-MB-231 cells than in MCF-10A cells by electron microscopic study (Figures 4(c) and 4(d) and supplemental Figure 2). Accordingly, the protein levels of beclin-1 and LC3-II that contribute to autophagosome formation and maturation [30,44,45] were 5.4-fold ( < 0.05) and 2.9-fold ( < 0.01) increased, respectively (Figures 4(a) and 4(b)). Measurement of LC3-II level in the absence and presence of bafilomycin A1 and leupeptin (autolysosome inhibitors) [27][28][29][30] indicated that MDA-MB-231 cells had an autophagic flux 2.3 times ( < 0.01) higher than MCF-10A cells (Figures 4(e) and 4(f)). Taken together, these results demonstrate an increased removal of mitochondria via mitophagy in breast cancer cells, which may account for the reduced mitochondrial content.

Inhibition of Drp1 Suppressed Mitochondrial Autophagy, Metabolic Reprograming, and Cancer Cell Viability.
Previous studies suggest that mitophagy is accompanied with enhanced mitochondrial fission [46,47], which prompted us to examine whether Drp1 upregulation accounted for the aug- suggesting that Mdivi-1 or Drp1 may mediate mitochondrial autophagy downstream of beclin-1 (supplemental Figure 5).

Discussion
Overactivation of mitochondrial fission was recently shown to promote tumorigenesis and cancer invasion [14][15][16][17][18]. An important question to be addressed is how mitochondrial quality control is regulated by overactive mitochondrial division. We employed bioinformatics approach analyzing 529 human breast cancer entities from The Cancer Genome Atlas (TCGA) database, and the results were validated by experimental biology in human breast cancer tissues and cell cultures. Our data suggest that breast cancer adopts a coordinated upregulation of mitophagy and mitochondrial biogenesis during the selective dynamics favoring mitochondrial fission (Figures 1-4). Moreover, we showed that upregulation of Drp1 directly contributed to augmented mitophagy, which accounted at least in part for the increased mitochondrial turnover and metabolic reprograming in breast cancer cells (Figures 4 and 5). Activation of Drp1 to promote mitochondrial fission has been well established in general. However, the evidence for the physiological role of Drp1-induced mitophagy has just started to emerge, including maintenance of functional -cells and cardio-/neuroprotections [22,[46][47][48]. To our knowledge, this is the first study investigating the triad of mitochondrial biogenesis, dynamics, and autophagy  Oxidative Medicine and Cellular Longevity in mitochondrial quality control with regard to cancer metabolism. It is noteworthy that mitochondrial biogenesis pathway via PGC1 is activated in breast cancer cells. Because a basal mitochondrial function is required for cancer cell survival and tumor growth (e.g., generating chemical building blocks for biosynthesis) [4,5], upregulation of PGC1mediated mitochondrial biogenesis may play a critical role in meeting such a requirement, particularly in the case of augmented mitochondrial turnover (mitophagy) induced by Drp1. Indeed, recent studies suggested that expression of ectopic PGC1 promoted tumor growth in an ErbB2/Neuinduced breast cancer, and elevation of PGC1 expression was correlated with a lower patient survival rate [49,50]. These findings highlight the importance of considering both mitochondrial biogenesis and mitophagy for effective cancer treatment. Indeed, targeting mitophagy or BNIP3 pathway alone was paradoxically shown to promote or suppress tumorigenesis [51][52][53][54][55][56]. This complexity and controversy may arise from the coordinated increase of mitochondrial biogenesis and mitophagy. Thus, it is of critical importance for future studies to test the effectiveness of simultaneously targeting mitophagy and mitochondrial biogenesis for breast cancer treatment.