Glucose starvation-induced oxidative stress causes mitochondrial dysfunction and apoptosis via Prohibitin 1 upregulation in human breast cancer cells

Journal Pre-proof Glucose starvation induced upregulation of Prohibitin 1 via ROS generation causes mitochondrial dysfunction and apoptosis in breast cancer cells Ganesh Kumar Raut, Moumita Chakrabarti, Deepika Pamarthy, Manika Pal Bhadra PII:

S0891-5849(19)31155-4

DOI:

https://doi.org/10.1016/j.freeradbiomed.2019.09.020

Reference:

FRB 14420

To appear in:

Free Radical Biology and Medicine

Received Date: 11 July 2019 Revised Date:

12 September 2019

Accepted Date: 20 September 2019

Please cite this article as: G.K. Raut, M. Chakrabarti, D. Pamarthy, M.P. Bhadra, Glucose starvation induced upregulation of Prohibitin 1 via ROS generation causes mitochondrial dysfunction and apoptosis in breast cancer cells, Free Radical Biology and Medicine (2019), doi: https://doi.org/10.1016/ j.freeradbiomed.2019.09.020. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

Graphical abstract

Highlights •

Glucose starvation sensitizes the breast cancer cells to death via mitochondrial dysfunction

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Increase in ROS and PHB1 level under GS in the mitochondria leads to intrinsic apoptotic cell death

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PHB1 and DRP1 are dissociated under GS condition, causing decrease in MMP, release of cytochrome c

•

Upregulated PHB1 inhibits breast cancer cells migration and invasion under GS condition

Glucose starvation induced upregulation of Prohibitin 1 via ROS generation causes mitochondrial dysfunction and apoptosis in breast cancer cells Ganesh Kumar Raut1,2, Moumita Chakrabarti1,2, Deepika Pamarthy1,2, and Manika Pal Bhadra1,2* 1

Applied Biology Division, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad, 500007, Telangana State, India. 2

Academy of Scientific and Innovative Research, Aruna asaf ali Marg, New Delhi, 110025, India.

*To whom correspondence should be addressed: Dr. Manika Pal Bhadra, Applied Biology Division, CSIR – Indian institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad – 500007, Telangana, India. Tel. +91-9848960101, Email: [email protected]

Abstract In recent years there has been an upsurge in research focusing on reprogramming cancer cells through understanding of their metabolic signatures. Alterations in mitochondrial bioenergetics and impaired mitochondrial function may serve as effective targeting strategies especially in triple-negative breast cancers (TNBC) where hormone receptors and endocrine therapy are absent. Glucose starvation (GS) of MDA-MB-231 and MCF-7 breast cancer cells showed decrease in mitochondrial Oxygen Consumption Rate (OCR), which was rescuable to control level through addition of exogenous antioxidant N-Acetyl Cysteine (NAC). Mechanistically, GS led to increase in mitochondrial ROS and upregulation of the pleiotropic protein, Prohibitin 1 (PHB1), leading to its dissociation from Dynamin-related protein 1 (DRP1), perturbance of mitochondrial membrane potential and triggering of the apoptosis cascade. PHB1 also reduced the invasive and migratory potential of both cell lines. We emphasize that glucose starvation remarkably sensitized the highly glycolytic metastatic TNBC cell line, MDA-MB-231 to apoptosis and decreased its migratory potential. Based on our findings, additional TNBC cell lines can be evaluated and a nutritional paradigm be proposed for anticancer therapy.

Graphical abstract

Highlights •

Glucose starvation sensitizes the breast cancer cells to death via mitochondrial dysfunction

•

Increase in ROS and PHB1 level under GS in the mitochondria leads to intrinsic apoptotic cell death

•

PHB1 and DRP1 are dissociated under GS condition, causing decrease in MMP, release of cytochrome c

•

Upregulated PHB1 inhibits breast cancer cells migration and invasion under GS condition

Keywords Apoptosis/ Dynamin-related protein 1 (DRP1)/ Glucose starvation (GS)/ Oxidative stress/ Prohibitin 1 (PHB1) Abbreviation 2-Deoxy-D-glucose (2-DG); Dynamin-related protein 1, (DRP1); Glucose starvation, (GS); N-acetyl-L-cysteine, (NAC); Oxygen consumption rate, (OCR); Prohibitin 1 (PHB1); Reactive oxygen species, (ROS)

1. Introduction Breast cancer is one of the most common cancers in women and second leading cause of death. The projected number of new breast cancer cases in the United States for the year 2019 are 271,270, with total deaths estimated to be 42,260 [1]. Triple Negative Breast Cancers (TNBCs) are a subtype that lack expression of the estrogen receptor (ER), the progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). They occur at a younger age with higher rate of reoccurrence and poor clinical outcomes [2]. Targeted therapies are currently unavailable due to lack of hormone receptors and treatment is limited to surgery, radiation, and chemotherapy. Approximately 15–20% of all breast cancers are triple negative, defined by specific gene-expression signatures and metabolic profiles. Evaluating the role of cellular growth and survival pathways in conjunction with the understanding of nutritional parameters, and metabolic profiling may provide therapeutic insights which can be used to tailor treatment plans for TNBCs. Metabolic reprogramming and aberrant gene regulation are hallmarks of cancer [3]. Cancer cells adapt to adverse tumor microenvironments by altering their nutritional requirements and gene expression, to maintain cellular fitness for tumor growth and progression. An attractive nutraceutical therapeutic approach involves the identification of cross-talk between the de-regulated gene expression and altered cellular metabolism. Nutritional perturbations and metabolic insults are recognized by master controllers of the cell, the mitochondria, which are central organelles that coordinate the regulation of cellular metabolic networks and signaling pathways leading to tumor growth. Recent studies have shown that mitochondria are key players in the development of cancer and can be a target for anticancer therapy [4-8]. They play a vital role in activation of signaling pathways, bioenergetic and biosynthesis activities that are required for tumorigenesis. The role of mitochondrial metabolism is increasingly being considered as a potential area for cancer therapy. Prohibitin (PHB) proteins have attracted great attention due to their multiple functions in mitochondria. The first prohibitin (PHB1) was identified as an anti-proliferative protein in mammalian cells [9]. Prohibitin1 is an evolutionarily conserved pleiotropic protein belonging to a family containing the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain that plays vital role in cell division through tumor suppressor and cell cycle regulation activities [10]. At the subcellular level, PHB1 is predominantly localized in the mitochondrial inner membrane complexed to PHB2 [11]. Prohibitins modulate multiple functions based on cellular localization and cell type. In the mitochondria, PHBs act as a regulator of mitochondrial

morphogenesis and apoptosis while in other regions they help in nuclear transcription and plasma membrane lipid scaffold [12,13]. In spite of diverse biological roles, their exact function in cancer is still unclear [14]. Initial studies reported PHB as a tumor suppressor [15]. PHB1 deficiency promotes cancer cell growth and decreases cell apoptosis [16-18], though few reports showed that downregulation of PHB resulted in increase of cancer cell apoptosis [19-25]. Increased PHB levels have been found in cancer cells, sensitizing them to apoptosis [26, 27]. Several studies reported the implication of PHB in various cancers. PHB protein and mRNA expression studies showed its increase in gastric cancer [28], prostate cancer [29], high-grade breast cancer [30], papillary thyroid cancer [31], bladder cancer [32], esophageal squamous cell carcinoma [33] and colorectal carcinoma [34]. As key players in mitochondria, PHBs are being studied as potential targets for various therapeutic applications [35, 36]. Damaged mitochondria release ROS, which lead to inflammatory processes promoting tumorigenesis and aging. Prohibitins have been found to be involved in mitochondrial biogenesis, mitochondrial pathways, and mitochondrial apoptosis. Proteins such as PHB1 and DRP1, which are tightly associated mitochondrial membrane-bound proteins, are required for mitochondrial fission [37]. Mitophagy is a process through which damaged or unwanted mitochondria are selectively removed by cells. This ensures the protection of cells against harmful effects of damaged mitochondria and is an essential part of eukaryotic developmental process, including the uniparental inheritance of mtDNA [38,39]. In the current study, this is the first report to our knowledge where we have shown glucose starvation induced upregulation of Prohibitin 1. Our mechanistic study showed initiation of mitochondrial apoptosis through PHB1 and DRP1 dissociation in a highly metastatic, high glycolytic, triple negative breast cancer cell line, MDA-MB-231 and similar effect, but to a lesser extent in MCF-7 cell line, which is not as invasive and has low glycolytic potential. Mitochondrial ROS production, followed by cytochrome c release and activation of other pro-apoptotic proteins led to triggering of the intrinsic mitochondrial apoptosis pathway. Further, we have evidenced that glucose starvation mediated PHB1 upregulation disrupted mitochondrial membrane potential and homeostasis, and also reduced the invasive, and migratory properties of the TNBC cell line, MDA-MB-231, to a greater extent. Additional studies are needed for testing other panels of TNBC cell lines, to understand the effect of glucose starvation on mitochondrial metabolism and PHB mediated signaling pathways, as well as to elucidate the post-translational modifications of PHBs that might cause expression and translocation of PHB proteins into various cellular compartments.

In summary, a comprehensive program that looks at cancer metabolic and signaling pathways along with genomic, proteomic, metabolomic and molecular approaches can expand our knowledge on the role of diet and nutritional regimens, opening doors for precision medicine strategies, geared especially towards treating refractory TNBCs.

2. Materials and methods 2.1 Cell culture and transfection Human breast cancer cell lines (MDA-MB-231 and MCF-7) and normal HEK293T cell line were purchased from American Type Culture Collection (ATCC, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% filtered fetal bovine serum (FBS) and 100 units/ml penicillin/streptomycin (Sigma). All cells were maintained at 37 °C in a 5% CO2 atmosphere. For glucose starvation, cells were washed twice with PBS and further incubated in DMEM without glucose for 24 h. siRNA and PHB1 construct were transfected into cells using Lipofectamine 3000 (Invitrogen). For each well of 6-wells plate, transfection reagents were prepared by mixing 4 µl of Lipofectamine 3000/46 µl of Opti-MEM (Life Technologies) to 1.5 µl (100 nM) of siRNA/48.5 µl Opti-MEM at room temperature (RT) for 30 minutes before adding to plate. After 6 h transfection, 0.5 ml of fresh medium was added to plates for all cell types. Cells were harvested 36 h post-transfection for western analysis, FACS or other assays. PHB1 was cloned in pCMV-FLAG-Tag 1 vector for overexpression purpose. siRNA for PHB1 was designed to target the sequence of PHB1 (Table 1).

2.2 Observation of cells morphology by phase contrast microscope 3x105/well MDA-MB-231 and MCF-7 cells were cultured in six-well plates and treated with or without glucose for 24 h. Cell morphology was observed under OLYMPUS CKX41 phase contrast microscope and photographed with a Digital camera (ProgResC3).

2.3 Western blot analysis Cell lysates were incubated in RIPA buffer (Sigma) for 30 minutes on ice. Supernatant fractions were recovered by centrifugation (12000 rpm, 20 min, 4°C), and the protein concentrations of the lysates were determined using Bradford protein assay. Samples were prepared with 2-mercaptoethanol and denatured by heating at 95°C for 5 min. 40 µg of protein per lane was loaded in 10% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to polyvinyldine difluoride (PVDF) membrane (Merck Life Science Pvt.

Ltd.) and blocked at room temperature for 2 h in TBS + 0.1% Tween-20 (TBST) containing 5% skim milk (purchased from Santa-Cruz Biotechnology) followed by washes with TBST for 15 min. Hybridization with primary antibody at recommended dilution was conducted; anti Bax (1:1000), anti Bcl2 (1:1000), anti cytochrome c (1:1000) (Abbiotech), anti Ncadherin (1:1000), anti MMP-9 (1:1000), anti Snail1 (1:1000), anti β-actin (1:1000), anti pDRP1 (1:1000), anti PHB1 (1:1000) (purchased from Cell Signaling Technology), DRP1 (1:1000) (Abcam, USA), overnight at 4°C. The blots were further incubated with horseradish peroxides (HRP) conjugated secondary antibody at room temperature for 1 h in dark. Protein bands were visualized after staining with chemiluminescence luminol (BIO-RAD) and visualizing under ChemiDoc imaging system (BIO-RAD). Band intensities of the western blot were analyzed using image j software.

2.4 Trypan blue exclusion assay Trypan blue assay was carried out to assess the effect of Glucose starvation on the viability of breast cancer cell. Briefly, 0.5 × 105 cells/ml was seeded into cell culture dishes. For cell death study, several pharmacological inhibitors were employed. 2mM 3-methyladenine (3MA) (Sigma, M9281) was used to study autophagy, 100µM necrostatin-1 (Sigma, N9037 ) for necroptosis analysis, 2 µM ferrostatin-1 (Sigma, SML0583) to check ferroptosis, 10mM caspase inhibitor (Sigma, SCP0093) to inhibit caspase-8/9 and 1 mM PARP1 inhibitor 1 (3ABA) (CALBIOCHEM, Cat# 165350). For other experiments involving ROS scavenging by NAC, briefly the same protocol was followed excepting that cells were treated with 5mM NAC for 24 h under GS. After 24 h, cells were trypsinized, washed and resuspended in PBS containing 0.4% trypan blue (Gibco). Then viable cells were counted using haemocytometer. Cell viability graph was plotted after experiments were performed in triplicate.

2.5 MitoSOX staining and confocal microscopy MDA-MB-231 cells were seeded onto the cover slip and incubated for 24 h following which media was removed and grown in glucose free medium for 12 and 24 h. Cells were observed under confocal and phase contrast microscope. For MitoSOX RED staining, cells were seeded and incubated with glucose free medium for 24 h followed by staining with 5 µM MitoSOX RED (Life technologies, M36008) and incubation at 37 °C for 10 minutes. Cover slip was placed on slide and observed under confocal microscope (OLYMPUS 1X81, FLUOVIEW).

2.6 Flow cytometry analysis of apoptosis Annexin V FITC Apoptosis Detection Kit (Takara) was used for flow cytometry analysis for apoptosis. PHB1 knockdown and over expressed cells were exposed to glucose-free medium for 24 h. Cells were trypsinized and washed with cell culture grade PBS. Cells were washed with binding buffer followed by addition of 5 µL of Annexin V FITC and 10 µL of propidium iodide and assessed for apoptosis. Samples were incubated at 37 °C for 15 minutes and subjected to FACS analysis.

2.7 Mitochondrial membrane potential Mitochondrial membrane potential (∆Ψm) was quantified by staining with the cationic dye JC-1 (PK-CA-707-70011). JC-1 accumulates in the mitochondria of healthy cells and fluoresces red (590 nm). When the ∆Ψm collapses, JC-1 uptake is limited to the cytoplasm where it fluoresces green (527 nm). Cells were transfected with siPHB1 and pCMV-FLAGTag1-PHB1 construct followed by treatment with or without glucose for 12 and 24 h. Cells were thereafter stained with JC-1 (10 µg/ml) and incubated at 37 °C in dark for 20 minutes. Stained cells were rinsed twice with JC-1 staining buffer and analyzed by FACS. The green and red fluorescence intensity ratio was calculated as ∆Ψm.

2.8 Measurement of mitochondrial respiration MDA-MB-231 cells were cultured in XF24 cell culture microplates (Agilent Seahorse) at a density of 20,000 cells/well. After an overnight incubation, cells were washed and subjected to glucose starvation with and without NAC (5mM). Oxygen consumption rate was measured over 100 minutes in XF base medium supplemented with individual glutamine (2 mM), glucose (10 mM), sodium pyruvate (1 mM) followed by the sequential addition of oligomycin (complex V inhibitor, 2.0 µM), FCCP (proton gradient uncoupler, 2.0 µM), as well as rotenone and antimycin A (complex I inhibitor, 2.0 µM) as indicated. Experiment was carried out in triplicate. The XF mito stress test report generator automatically calculate the XF cell mito stress test parameters from Wave data that have been exported to Excel. 2.9 Sub-cellular fractionation For sub-cellular fractionation study, MDA-MB-231 and MCF-7 cells were harvested by scraping and washed with cold PBS. Cells were centrifuged at 200g for 7 minutes and pellet resuspended in 400 µl of STM buffer containing 250 mM sucrose, 50 mM Tris–HCl pH 7.4, 5 mM MgCl2, protease and phosphatase inhibitor cocktails and homogenized for 1 minute on

ice using a tightfitting Teflon pestle attached to a homogenizer (REMI Motor, India) set to 600–1000 rpm. The homogenate was decanted into a centrifuge tube and maintained on ice for 30 minutes, vortexed at maximum speed for 15 seconds and further centrifuged at 800 g for 15 minutes. The pellet was kept on ice, the supernatant was used for subsequent isolation of mitochondrial and cytosolic fractions. The washed pellet was resuspended in 500 µl NET buffer (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.5 M NaCl, 0.2 mM EDTA, 20% glycerol, 1% Triton X-100, protease and phosphatase inhibitors) using a pipette to triturate until homogeneous. Pellet was vortexed at maximum speed for 15 seconds and incubated on ice for 30 minutes, this fraction contained the nuclei. Cytosolic and mitochondrial fractions were extracted from supernatant by centrifugation at 800 g for 10 minutes. The supernatant was saved and the pellet was discarded. Supernatant was then centrifuged at 11000 g for 10 minutes and the supernatant containing cytosol was stored at -80°C. The pellet was again resuspended in 200 µl STM buffer and centrifuged at 11000 g for 10 minutes. Mitochondrial pellet was resuspended in 100 µl SOL buffer (50 mM Tris HCl pH 6.8, 1 mM EDTA, 0.5% TritonX100, 1X protease and phosphatase inhibitors) and stored at -80°C. 2.10 Immunofluorescence Staining To identify the expression level and sub-cellular localization of PHB1 and p-DRP1, cells were seeded on cover slips and incubated with glucose free medium for 24 h followed by incubation with 100nM Mitotracker green (Life technologies, M7514), for 30 minutes at 37 °C. Cells were washed and fixed with 4% PFA for 30 minutes at 37 °C, and permeabilized with 0.1% Triton X-100 at 4 °C for 10 min. Further, cells were incubated in blocking solution (5% BSA) for 1 h and incubated with primary PHB1 and DRP1 antibody (1:100 and 1:400 dilution respectively) (ab75766) and p-DRP1 (Ser616, CST-3455) for 4 h at room temperature. Cells were incubated with secondary Cy3 and Cy5-conjugated goat anti-rabbit antibody (1:100 dilutions, Jackson Immuno Research Laboratories Inc.). Images were acquired with OLYMPUS 1X81, FLUOVIEW confocal microscope. The excitation and emission wavelengths for Mitotracker green were 490/516 nm; Cy3-conjugate 550/570 nm; and for Cy5-conjugate, 650/670 nm respectively.

2.11 Immunoprecipitation Cells were trypsinized and washed in PBS and extracts prepared by solubilizing 107 cells in 1 ml of cell lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris-Cl at pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 2.5 mM pyrophosphate, 1 mM glycerol phosphate and

protease inhibitor mixture) for 10 min at 4 °C. After brief sonication, lysates were cleared by centrifugation at 15000 × g for 10 min at 4 °C. Cell extract was immunoprecipitated with 6 µg of antibodies against PHB1, DRP1, and p-DRP1, (Abcam and CST), and incubated with 100 µl of protein A-agarose for 12 h at 4 °C by continuous inversion. Immunocomplexes were pelleted, washed four times, boiled in Laemmli buffer and analyzed by western blotting.

2.12 Wound healing assays. Cell migration was carried out using classical wound healing assays. Breast cancer cells (MDA-MB-231 and MCF-7) were seeded in six-well plates and incubated to 90% confluence. Wounds were created on the monolayer of cells by means of sterile 20 µl pipette tips and washed twice to remove non-adherent cells and cultured in absence of glucose for 0 and 12 h followed by refeeding the cells with glucose for 12 h. Cell migration was monitored under phase-contrast microscope and fluorescence microscope.

2.13 Transwell cell migration and invasion assay Cell migration and invasion were measured using a Transwell chamber (24-well insert) with 8 µm PET membrane (BRAND plates Insert system, Germany) without Matrigel. MDA-MB231 cells subjected to transfection, glucose starvation and refed were trypsinized, and a cell suspension was prepared. Approximately 100 µl of cell suspension was added to the upper chamber containing serum free medium at a density of 2×105 cells/ml. The lower chamber was filled with 750 µl medium containing 10% FBS. After 16 h, the migrated cells were fixed in 3.7 % paraformaldehyde for 2 minutes followed by 100% methanol for 20 min at room temperature, and the non-migrated cells were removed using cotton swabs. Subsequently, cells on the lower surface of the membrane were stained with 0.1% crystal violet for 20 minutes and were observed under microscope and counted in 5 random fields. For invasion assays, the chamber was coated with Matrigel (Corning, Bedford, MA, USA) and 2×105 cells/ml was seeded on the upper chamber. The other procedures were employed in the same manner as the migration assay.

2.14 Statistical analysis All statistical analysis was performed with unpaired two tailed Student’s t-tests, and all data are expressed as means and SEM. for at least three independent experiments, unless otherwise indicated.

3. Results: 3.1 Glucose starvation sensitizes breast cancer cells to apoptosis A highly glycolytic triple negative breast cancer cell line, MDA-MB-231 and a low glycolytic cell line, MCF-7 were subject to GS and morphological changes were observed under the microscope. Compared to control cells, glucose-starved cells were rounded, shrunken in size, and significant cell death was noted (Fig 1A and S1A). In order to determine the type of cell death, several inhibitors were assessed for their effect on MDA-MB-231 cells in GS condition (Fig S1 B). Cells were cultured in the presence of inhibitors for 24 h and % of cell viability was determined by Trypan blue assay. Cells treated with different concentrations of inhibitors of autophagy (3-Methyladenine), ferroptosis (ferrostatin-1), and necroptosis (necrostatin-1) did not prevent cell death. Moreover, cells treated with caspase-8/9 (AcLETD-CHO) and PARP1 (3-Aminobenzamide) inhibitors rescued the cells from death, suggesting that the type of cell death caused by GS is potentially mediated by apoptosis. Western blot analysis of protein lysates derived from control and GS cultures showed the expression of pro-apoptotic and anti-apoptotic factors, such as Bim, Bid, Bax, etc. (Fig 1B and S1C). Further, cell fractionation experiments showed high level of cytochrome c in cytosol under glucose starvation, compared to mitochondrial compartment (Fig 1C, 1D). Correspondingly, increase of caspase 3 and caspase 6/9 activities were observed, indicating involvement of the intrinsic apoptotic cell death pathway, triggered by glucose starvation (Fig 1E and 1F, S1E). In order to exclude the role of autophagy, expression of autophagic marker protein, LC-3 was checked. Western blots from glucose starved cell lysates and control samples (Fig S1D) showed no change in LC-3 expression between control and glucose starved cultures, indicating that cell death was due to apoptosis, mediated by cytochrome c release and translocation into cytosol, followed by caspase machinery activation. As apoptosis was more predominant in MDA-MB-231 cells, we sought to investigate whether the apoptotic response was dependent on glucose availability or due to inhibition of glycolysis. Cells were treated with 2-DG, a nontoxic analog of glucose that competes with glucose and inhibits glucose metabolism, creating a chemically induced glucose deprivation environment. Cells treated with 2-DG did not undergo cell death (Fig 1G & 1H), indicating that availability of glucose, and not a metabolite derived from glycolysis, is responsible for inducing apoptosis. We further investigated whether other sugar sources such as mannose and fructose could inhibit cell death. MDA-MB-231 cells were grown in media containing fructose or mannose instead of glucose for same interval of time. No significant death was observed, suggesting the role of metabolic reprogramming and potential usage of alternate

metabolic pathways to sustain in absence of glucose, given that this cell line is highly glycolytic (Fig 1I & 1J).

Figure 1. Glucose starvation (GS) sensitizes breast cancer cells to apoptosis. A

MDA-MB-231 cells under Phase contrast microscope. GS cells showing rounded,

shrunken morphology compared to control cells with normal media. (scale bar: 100µm) B

Western blot analysis showing expression levels of pro-apoptotic proteins (Bim, Bid,

Bax, Caspase-3 and Apaf-1) and anti-apoptotic proteins (Bcl-2) after 24 h of GS. C,D

Fractionation experiment (n=2) showing accumulation of cytochrome c in cytosol

after 24 h of GS. E,F

Graphs representing caspase 3 and caspase 6/9 activity in MDA-MB-231 cells.

G,H

Phase contrast microscopy and Trypan blue assay were conducted for observing

morphological changes and cell viability in MDA-MB-231. Cells grown under glucose starvation, treatment with 2-DG (10 mM) or under both conditions for 24 h; I,J Phase contrast microscopy and viability studies by Trypan blue assay in MDA-MB-231 cells grown with 4.5 g/L, fructose or mannose under glucose starvation for 24 h. Glucose concentration in control cells was set at 4.5 g/L. ***p < 0.001 indicates values vs control (cells grown under normal media containing 4.5 g/L glucose) and cells vs cells grown in glucose starved media.

# # #

p < 0.001 indicates

3.2 Cell death triggered by Glucose Starvation is oxidative stress dependent Oncogenic signaling pathways and high growth rate cause tumors to produce high amounts of ROS [40]. Further, nutritional deprivation and increasing vasculature cause metabolic stress, accelerating mitochondrial ROS production and accumulation [41-42]. Based on this general understanding of tumor behavior, we expected increase of mitochondrial ROS and apoptotic cell death under glucose starvation. We detected increase in mitochondrial ROS using Mitotracker and MitoSOX fluorescence probes [43,44]. Results showed significant increase in mitochondrial superoxide in MDA-MB-231 cells grown under glucose starvation (Fig 2A). Further, flow cytometry analysis and Trypan blue assays showed that cells treated with NAC, an anti-oxidant and ROS scavenger, rescue of glucose starvation induced apoptosis (Fig 2B, 2C and 2D). This confirms the involvement of an oxidative stress induced by glucose starvation mediates cell death in breast cancer cells. Moreover, we checked whether cell death is specific to glucose starvation-induced ROS or other exogenous chemical induction; cells were treated with different concentration of hydrogen peroxide (H2O2) and glucose. We found that cells treated with H2O2 were healthy even in 100µm concentration. However, cells grown in glucose free media showed around 80% cell death (Figure S 2A and 2B). It indicates that breast cancer cell death is specific to glucose starvation and due to metabolic stress.

Figure 2. Detection of mitochondrial associated ROS generation after glucose starvation. A Mitochondrial superoxide production was determined by MitoSOX red staining in MDAMB-231 cells grown under glucose starved condition for 12 h and 24 h. Images were taken by confocal microscopy (scale bar: 20µm). Merged panel shows mitochondrial associated ROS. B,C

Microscopic images (B) and Trypan blue activity (C) of 24 h glucose starved MDA-

MB-231 cells treated with 5mM N-acetyl-L-cysteine. (scale bar: 100µm). D

Flow cytometry analysis of MDA-MB-231 cells after 24 h of glucose starvation with and

without NAC. E

Scatter plot of GS indicates degree of enriched population. Ch01, Ch02, Ch03 and Ch06

represents bright field image, FITC stained cells, FITC and PI stained cells and side scatter image of the cells respectively. (Scale bar: 20µm).

3.3 Glucose starvation increases PHB1 expression in breast cancer cells There are conflicting experimental data with regard to the involvement of prohibitin in tumorigenesis, showing a permissive action on tumor growth or alternatively acting as an oncosuppressor, depending on the cell context [45]. Recent studies have shown that it serves as

onco-suppressor, but its expression is down regulated in ER positive breast cancer patientderived samples [46]. The role of PHB in TNBCs under glucose starvation has not been investigated. As PHB is also a mitochondrial membrane-bound protein and significant ROS mediated apoptotic cell death was detected in our preliminary experiments, we sought to further investigate PHB1 protein expression in MDA-MB231 and MCF-7 cell lines under glucose starvation for 24 h (Fig 3A, 3B, and S3A). We observed a 5-fold increase in PHB1 protein expression in MDA-MB-231 cells as compared to control cells cultured in regular glucose medium for 24 h. MCF-7 cells showed about 3-fold increase in PHB1 under glucose starvation for the same time point. In order to reconfirm PHB1 protein expression, MDAMB-231 and MCF-7 cells were grown in glucose-starved medium for 0h, 12 h and 24 h respectively followed by replenishment with glucose rich medium (glucose 4.5g/L) for 12 h and 24 h. There was a dynamic change of PHB1 expression along with change of cell morphology under glucose starvation and refed condition (Fig 3C and S3B). We next confirmed the effect of NAC under GS condition on PHB1 expression. Cells grown under glucose free media containing NAC (5mM) decreases the expression of PHB1 significantly indicating that mitochondria associated ROS triggered by GS regulating the expression of

PHB1 in MDA-MB-231 (Fig S3C). Moreover, we investigated the effect of glucose starvation on PHB1 expression by conducting siRNA knockdown experiments. PHB1 knockdown under glucose starvation showed its upregulation (Fig 3D and 3E), confirming it as a metabolic sensor protein that gets activated in the mitochondria, as shown by our data from sub-cellular fractionation experiments (Fig 3F). To reconfirm PHB1 expression specific to GS, MDA-MB-231 cells were grown under 100µM of H2O2 and glucose starvation, result showed low expression of PHB1 in H2O2 condition while in response to GS, expression was higher than control (Fig S2 C and S2 D). Immunostaining showed mitochondrial localization of PHB1 protein (Fig 3G), potentially indicating its involvement in the mitochondrial intrinsic apoptosis pathway, as shown from our initial data.

Figure 3. Glucose starvation increases prohibitin 1 expression in breast cancer cells. A

PHB1 expression in MDA-MB-231 cells after 24 h of GS.

B,C Dynamic expression of PHB1, cells first grown in glucose free medium followed by grown in glucose-rich medium for durations indicated. Phase contrast images show changes in cells morphology upon refeeding. (scale bar: 100µm). D,E Western blot and histogram are showing the effect of PHB1 knockdown under glucose starvation condition. F

PHB1 upregulation upon GS in the mitochondrial fraction. HSP60 was used as internal

loading control for mitochondria and actin was used for whole cell lysate (crude) as internal loading control. G

Immunofluorescence staining of MDA-MB-231cells using Anti PHB1 antibody (Red)

and Mitotracker green (Green) indicating clear upregulation of PHB1 in mitochondria upon GS. Merged images show a clear colocalisation of PHB1 and Mitotracker green (Yellow). (scale bar: 10µm). Results of three independent experiments are expressed as the mean ± SE. *p < 0.05, **p < 0.01 and ***p < 0.001. In all cases, cells grown in normal media were taken as controls.

3.4 Role of PHB1 is pivotal in Glucose starvation induced apoptosis Prohibitin is known to be involved in different cellular functions including metabolism, proliferation, apoptosis and aging [47]. Mitochondrial prohibitin controls stabilization of the mitochondrial genome, morphology, biogenesis and intrinsic apoptosis pathway. Annexin V FITC based FACS analysis of cells grown under glucose starvation and control cells grown in glucose supplemented media showed increased apoptosis under GS (Fig 4A). Glucose starved MDA-MB-231 cells showed nearly 80% cell death, whereas cells in normal medium supplemented with glucose showed minimal apoptosis ~ 4%. We then conducted western blot analysis of protein lysates from the same experimental setup, to confirm the expression of apoptotic (Bax and Bid) and antiapoptotic (Bcl-2) markers. Pro-apoptotic markers, such as Bax, Bid were increased in cells exposed to glucose starvation while the expression of the same proteins were decreased in PHB1 knockdown cells. In order to reconfirm, cells were transfected with PHB1 construct and post-transfection, cells were cultured in glucose free media. Results showed increased expression of apoptotic markers in PHB1 transfected cells grown in Glucose-free medium compared with PHB1 transfected grown in glucose-rich medium (Fig 4B). The above two experimental data solidify our hypothesis that PHB1 is

involved in GS mediated apoptosis and is directed by the intrinsic apoptosis cascade, which may be potentially triggered by metabolic insult and mitochondrial injury.

Figure 4. Upregulated PHB1 by GS promotes apoptosis. A FACS analysis representing apoptotic cell death. B Western blot analysis indicating the expression of apoptotic and anti-apoptotic proteins in PHB1 knockdown and overexpression condition followed by GS for 24 h. Actin represents gel loading control.

3.5 Glucose starvation induced PHB1 upregulation causes collapse in mitochondrial membrane potential Glucose starvation mediated metabolic reprogramming and adaptation to new signaling pathways to survive the metabolic insult potentially leads to disruption in mitochondrial homeostasis, architecture and number. As prohibitin is a mitochondrial membrane associated protein, we expected a change in mitochondrial membrane potential (∆Ψm) mediated by PHB1 upregulation under GS condition. We evaluated the mitochondrial membrane potential in MDA-MB-231 cells grown under GS and normal media. Cells were stained with Mitotracker red dye (∆Ψm dependent, 100 nM) and cationic dye JC-1 (10µg/ml) and intensity of dye was observed via confocal imaging (Fig 5A & 5B). CCCP (Carbonyl cyanide m-chlorophenylhydrazone), a mitochondrial uncoupling agent, which reduces mitochondrial membrane potential, was taken as positive control. Fluorescence intensity was further measured by flow cytometry and a reduction was seen in cells grown under GS, for 12 and 24 h compared to control cells grown in normal media. Cells having siRNA mediated knockdown of PHB1 as well as overexpression of PHB1 grown under GS also showed reduced membrane potential as compared to the cells grown in normal medium (Fig 5C & 5D). Overall the results showed that PHB1 regulated mitochondrial membrane potential under GS condition.

Figure 5. GS-induced PHB1 upregulation reduces mitochondrial membrane potential. A

Confocal images of MDA-MB-231 cells stained with Mitotracker red (∆Ψm

dependent dye). Intensity of Mitotracker red reflects the level of ∆Ψm. (scale bar: 20 µm). B

Confocal images of cells stained with JC-1 dye for 30 mins. Red fluorescence

indicates aggregates with high ∆Ψm and green fluorescence indicates monomers with low ∆Ψm. (scale bar: 100 µm). C

Flow cytometry results of ∆Ψm measurements of cells stained with JC-1.

D

Histograms representing the red/green fluorescence ratio (high ∆Ψm/ low ∆Ψm)

produced by cells and recorded by Flow cytometry. Cells treated with CCCP served as positive control. Cells were transfected with siPHB1 and CMV-FLAG Tag1s-PHB1 construct followed by GS for 12 h.

3.6 Glucose starvation impaired Mitochondrial Respiration in MDA-MB-231 cells In order to investigate the mitochondrial metabolism in response to glucose starvation, we measured the oxygen consumption rate (OCR) in glucose starvation condition alone and treated with NAC using Seahorse Metabolic Analyzer. OCR was measured during the sequential addition of oligomycin, FCCP, as well as rotenone and antimycin A to acquire the basal OCR, ATP synthase-dependent OCR, maximum respiratory capacity, and nonmitochondrial respiration [48]. There was significant reduction of OCR and individual parameters under glucose starvation condition compared to control. However, cells treated with NAC under glucose starvation condition showed clear increase of OCR (similar to control) compared to GS condition (Fig 6 A-H). These data demonstrate that ROS generated by glucose starvation caused mitochondrial dysfunction, however NAC is rescuing the mitochondrial dependent metabolism which meets the tumor cell demand to sustain their proliferation.

Figure 6: Glucose starvation impaired mitochondrial respiration in MDA-MB-231 (A) Schematic of mitochondrial stress test. (B) Oxygen consumption rate (OCR) and (C,D,E,F,G and H) individual parameters for basal respiration, proton leak, maximal respiration, spare respiratory capacity, non-mitochondrial respiration and ATP production.

3.7 Elucidation of mechanism of glucose starvation mediated apoptotic response by pDRP1 and its subsequent dissociation from PHB1 Mitochondrial fragmentation is crucial for apoptosis [49-50]. Upon confirmation of PHB1 role in GS mediated decrease in mitochondrial membrane potential, we dissected the mechanism by which PHB1 and other effectors are involved in mitochondrial fragmentation. Previous studies revealed that down-regulation of DRP1 delays mitochondrial fragmentation, cytochrome c release, caspase activation, and cell death [49-53]. Based on our findings that PHB1 mediated mitochondrial apoptosis under GS, we further investigated the role of DRP1 (Dynamin-related protein 1), which is a mitochondrial fission protein involved in the regulation of mitochondrial division and apoptosis [49, 54]. Confocal imaging showed that MDA-MB-231 and MCF-7 cell lines have fragmented mitochondria under GS (Fig 7A and S4A). Fission machinery of the mitochondrial outer membrane is composed of Dynaminrelated protein1 (DRP1) [55-56]. Fragmented mitochondria often appear in the early steps of cell death associated with permeabilization of the mitochondrial outer membrane, which induces the release of inter membrane space-stored proapoptotic factors, such as Cytochrome c and Apoptosis-Inducing Factor (AIF) [57-60]. In an effort to explore the mechanism of induction of apoptosis via involvement of mitochondria under glucose starvation, we evaluated the expression of p-DRP1 (ser616) protein in both whole cell lysate and cellular fractions of MDA-MB-231 cell line grown in glucose starvation condition. p-DRP1 expression was increased after 12 h and 24 h of GS but showed a decrease in expression when the cells were refed with glucose (Fig 7B). Further, cellular fractionation studies showed clear localization of DRP1 and p-DRP1 in the mitochondria under glucose starved condition (Fig 7C and S4B). Moreover, p-DRP1 expression level and its colocalization with PHB1 in the mitochondria were also confirmed by immunostaining experiment (Fig 7D).These observations led us to investigate possible interactions between PHB1, DRP1 and p-DRP1. Immunoprecipitation studies were carried out with cells grown under normal media or glucose starved media. Both MDA-MB-231 and MCF-7 cells grown under normal media conditions showed a clear association of PHB1 with DRP1. Interestingly both cell lines under GS showed disruption in the interaction between PHB1 and DRP1 proteins (Fig 7E and S4C). An immunoprecipitation study carried out with control HEK293T cells grown under normal media showed no interaction between PHB1 and DRP1 (Fig 7F). This indicates that tumor cells sustain under normal conditions with increased mitochondrial bioenergetics through PHB1 and DRP1 interaction. In summary, our data suggest that breast cancer cells upon GS caused disruption of PHB1 and DRP1 interaction which sensitizes cells towards apoptosis

through reduction in mitochondrial membrane potential and mitochondrial fragmentation respectively.

Figure 7. Mitochondrial morphology and interaction studies between PHB1 and DRP1 in MDA-MB-231 cells after glucose starvation. A

Confocal microscopy images of MDA-MB-231 showing fragmented morphology of

mitochondria after 24 h of GS. (scale bar: 10µm). Inset shows a magnified view of the fragmented mitochondria. B

Western blot showing the expression level of p-DRP1 under GS and refed conditions

for 12 and 24 h in MDA-MB-231 cell line. C

Cellular Fractionation blots indicating activation of p-DRP1 in mitochondria in

response to GS. D

Expression level and colocalization of DRP1 and PHB1 in MDA-MB-231 cells and

analysed by confocal microscopy. Panels 1, 2 and 3 are showing the expression level of pDRP1 in control and starved cells (4, 5 and 6 panels). Signals detected for the mitotracker (1

and 4, green) and p-DRP1 (2 and 5, blue pseudocolor). Panels 7 and 8 are the same as in panels 4 and 5. Panel 9 shows the merged image (purple) and colocalization of p-DRP1 (blue pseudocolor) and PHB1 (panel 10, red) to the mitochondria. (Scale bar: 10 µm). E

IP blot indicating there is no interaction between PHB1 and DRP1 in MDA-MB-

231after 24 h GS F

IP blot indicating no interaction between PHB1 and DRP1 in HEK293T cells in

normal media Note: All the above experiments were performed in triplicates. 3.8 Glucose starvation-induced PHB1 inhibits breast cancer cells migration and invasion We evaluated the role of PHB1 in breast cancer cell migration and invasion under glucose starvation. Results of migration and invasion assays showed that cell migration and invasion was decreased under glucose starvation and dramatically enhanced upon refeeding in MDAMB-231 (Fig 8A, C and E). MCF-7 cell migration was also reduced confirmed by scratch assay (S5A). Over expression of PHB1 blocked the migration potential in MDA-MB-231 and MCF-7 cells as compared to empty vector transfected control whereas cells transfected with siRNA against PHB1 resulted in cell migration (Fig 8B, D, F and S5B). To reconfirm the results of migration assay, we further analyzed the expression of metastatic protein markers such as N-cadherin, MMP 9 and Snail1, which were found to be downregulated under GS but in case of PHB1 knockdown, the same migratory markers were upregulated. In case of overexpression of PHB1, their levels were downregulated, confirming that indeed PHB1 inhibits cell migration in breast cancer cells (Fig 8G). In addition, we observed differential expression of migratory marker proteins, under GS and refeeding conditions (Fig 8H).

Figure 8. Effect of GS in breast cancer cell migration. A

Wound healing (I and II), Transwell migration (III) and invasion assays (IV) in MDA-

MB-231 cells after 12h of GS and refeeding. Fluorescence images (II) of wound healing were taken using GFP transgenic MDA-MB-231 cell line. B

MDA-MB-231 cells were subjected to transfection with empty vector (EV), siRNA

against PHB1 (siPHB1) and PHB1 construct (Ovx-PHB1). (Scale bar: 100µm). Histograms (C D, E and F) were calculated by counting cells of five random fields of a transwell chamber under phase contrast microscope. G, H Western blot showing the expression of proteins associated with migration of cancer cells under indicated conditions and time points. 4. Discussion Cancer cells adjust to growing nutritional demands by various mechanisms [61-62]. The metabolic features of cancer cells and pathways through which they replenish their nutritional needs are altered compared to that of normal cells, due to metabolic reprogramming [63]. Most of the reprogramming depends on utilizing mitochondria as chief biosynthesis organelles. Recent research studies showed that mitochondrial metabolism is vital for tumor growth [64] and targeting their metabolic pathways is an emerging area of research. Glucose is one of the most abundant sources of energy in cancer cells and targeting mitochondrial metabolism is an alternate strategy that can be used to slow down or inhibit cancer cell growth [65-67]. We used 2-Deoxy-D-glucose (2-DG), a synthetic, nonmetabolizable glucose analogue that blocks glycolysis and results showed growth of tumor cells, reflecting their metabolic flexibility to escape glucose dependency to other metabolic pathways through reprogramming. Recently, antioxidant usage has shown conflicting evidence, causing suppression of cancer initiation in some cases, or increase of tumor progression in others [68-69]. In the present study, exogenous supplementation of NAC rescued cell death triggered by GSinduced ROS and promoted breast cancer cell proliferation. Further, mitochondrial bioenergetics was analysed by Seahorse platform, which confirmed the decrease of mitochondrial OCR under GS; however NAC treatment recovered the OCR to near normal levels, reflecting that mitochondrial homeostasis is required for tumor cells to proliferate. In summary, based on our findings we propose a novel approach of depriving cancer cells of glucose, through starvation, which elicits cellular and mitochondrial responses to the metabolic insult, leading to our exploration of this non-drug based strategy for anti-cancer

therapy. This approach has been tested in our lab using MDA-MB-231 and MCF-7 breast cancer cell lines, the former being highly glycolytic, hormone receptor negative and latter being a low glycolytic, hormone receptor positive cell line. Our results showed that glucose starvation led to an increase of mitochondrial-associated ROS and upregulation of the mitochondrial protein, Prohibitin 1, leading both cell lines towards apoptosis, albeit a higher response in MDA-MB-231. The result was further confirmed by knockdown and overexpression of PHB1. Elsewhere, siRNA-mediated silencing of PHB1 expression was found to increase breast cancer cell proliferation [70]. This confirms the tumor suppressive action of PHB1. Mechanistic studies further showed the involvement of mitochondrial protein DRP1 which is an interacting partner with PHB1, and GS triggers its dissociation from PHB1. As a consequence of this dissociation, reduction of mitochondrial membrane potential, release of cytochrome c, and other factors caused caspase activation, and apoptosis. PHB1 upregulation also inhibited the invasive and migratory activities of the TNBC cell line, MDA-MB-231 to a larger extent compared to MCF-7. These preliminary findings may provide useful clues to propose nutritional strategies for understanding the metabolic behavior and signaling pathways in other TNBC cell lines, which is the need of the hour due to lack of targeted therapies for this subtype of cancers. Further, understanding the role of mitochondrial bioenergetics with changing nutritional requirements in primary cell cultures established from patient-derived tissues as well as in vivo modeling in rodents may enable researchers to develop personalized medicine strategies. Other combinatorial therapies such as radiation, chemotherapy and surgery may be explored alongside the proposed nutritional therapy, which may further enhance the anti-cancer potential. Conflict of interest The authors declare no conflicts of interest related to this work Author contributions GKR: Designed and performed overall experiments, analyzed the data and wrote the manuscript. MC: Helped in writing the manuscript and maintaining the cell cultures for the experiments and performing the microscopy experiments. DP: Helped in reviewing data and writing the manuscript. MPB: Conceived the idea, designed experiments and wrote the manuscript. All authors approved the manuscript.

Acknowledgements We thank Dr. Ramakrishna Sistla for gifting MDA-MB-231/GFP cells and Y. Suresh for his assistance with FACS analysis. GKR, MC and DP thank ICMR and CSIR for fellowship support. GKR, MC and DP thank Academy of Scientific and Innovative Research (AcSIR) for Ph.D. registration. We acknowledge CSIR-IICT for evaluating the manuscript and providing with communication number IICT/Pubs./2019/230. Funding: This work was supported by a grant from Indian Council of Medical Research [ICMR, GAP 0268].

References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2019, CA: a cancer journal for clinicians 69(1) (2019) 7-34. [2.] W.D. Foulkes, I.E. Smith, J.S. Reis-Filho, Triple-negative breast cancer, New England journal of medicine 363(20) (2010) 1938-1948. [3.] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, cell 144(5) (2011) 646-674. [4] Y. Zhang, J.-M. Yang, Altered energy metabolism in cancer: a unique opportunity for therapeutic intervention, Cancer biology & therapy 14(2) (2013) 81-89. [5] D.C. Wallace, Mitochondria and cancer, Nature Reviews Cancer 12(10) (2012) 685. [6] A. Schulze, A.L. Harris, How cancer metabolism is tuned for proliferation and vulnerable to disruption, Nature 491(7424) (2012) 364. [7] V. Shoshan-Barmatz, D. Mizrachi, VDAC1: from structure to cancer therapy, Frontiers in oncology 2 (2012) 164. [8] S. Fulda, L. Galluzzi, G. Kroemer, Targeting mitochondria for cancer therapy, Nature reviews Drug discovery 9(6) (2010) 447. [9] J.K. McClung, D.B. Danner, D.A. Stewart, J.R. Smith, E.L. Schneider, C.K. Lumpkin, R.T. Dell'Orco, M.J. Nuell, Isolation of a cDNA that hybrid selects antiproliferative mRNA from rat liver, Biochemical and biophysical research communications 164(3) (1989) 13161322. [10] R. Nadimpalli, N. Yalpani, G.S. Johal, C.R. Simmons, Prohibitins, stomatins, and plant disease response genes compose a protein superfamily that controls cell proliferation, ion channel regulation, and death, Journal of Biological Chemistry 275(38) (2000) 29579-29586. [11] E. Ikonen, K. Fiedler, R.G. Parton, K. Simons, Prohibitin, an antiproliferative protein, is localized to mitochondria, FEBS letters 358(3) (1995) 273-277.

[12] C. Merkwirth, T. Langer, Prohibitin function within mitochondria: essential roles for cell proliferation and cristae morphogenesis, Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1793(1) (2009) 27-32. [13] J. Han, C. Yu, R.F. Souza, A.L. Theiss, Prohibitin 1 modulates mitochondrial function of Stat3, Cellular signalling 26(10) (2014) 2086-2095. [14] S. Koushyar, W.G. Jiang, D.A. Dart, Unveiling the potential of prohibitin in cancer, Cancer letters 369(2) (2015) 316-322. [15] M. Nuell, D. Stewart, L. Walker, V. Friedman, C. Wood, G. Owens, J. Smith, E. Schneider, R. Dell'Orco, C. Lumpkin, Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells, Molecular and cellular biology 11(3) (1991) 1372-1381. [16] Y.-H. Liu, K. Peck, J.-Y. Lin, Involvement of prohibitin upregulation in abrin-triggered apoptosis, Evidence-Based Complementary and Alternative Medicine 2012 (2012). [17] N. Zhong, Y. Cui, X. Zhou, T. Li, J. Han, Identification of prohibitin 1 as a potential prognostic biomarker in human pancreatic carcinoma using modified aqueous two-phase partition system combined with 2D-MALDI-TOF-TOF-MS/MS, Tumor Biology 36(2) (2015) 1221-1231. [18] L. Zhang, Q. Ji, Z.-H. Ni, J. Sun, Prohibitin induces apoptosis in BGC823 gastric cancer cells through the mitochondrial pathway, Asian Pacific Journal of Cancer Prevention 13(8) (2012) 3803-3807. [19] K. Kasashima, E. Ohta, Y. Kagawa, H. Endo, Mitochondrial functions and estrogen receptor-dependent nuclear translocation of pleiotropic human prohibitin 2, Journal of Biological Chemistry 281(47) (2006) 36401-36410. [20] R.C. Gregory-Bass, M. Olatinwo, W. Xu, R. Matthews, J.K. Stiles, K. Thomas, D. Liu, B. Tsang, W.E. Thompson, Prohibitin silencing reverses stabilization of mitochondrial integrity and chemoresistance in ovarian cancer cells by increasing their sensitivity to apoptosis, International journal of cancer 122(9) (2008) 1923-1930. [21] Q. Wu, S. Wu, Lipid rafts association and anti-apoptotic function of prohibitin in ultraviolet B light-irradiated H a C a T keratinocytes, Experimental dermatology 21(8) (2012) 640-642. [22] S.Y. Kim, Y. Kim, H.Y. Hwang, T.-Y. Kim, Altered expression of prohibitin in psoriatic lesions and its cellular implication, Biochemical and biophysical research communications 360(3) (2007) 653-658. [23] Y. Zhang, Y. Chen, C. Qu, M. Zhou, Q. Ni, L. Xu, siRNA targeting prohibitins inhibits proliferation and promotes apoptosis of gastric carcinoma cell line SGC7901 in vitro and in vivo, Cellular and molecular biology (Noisy-le-Grand, France) 60(1) (2014) 26-32.

[24] V. Sánchez-Quiles, E. Santamaría, V. Segura, L. Sesma, J. Prieto, F.J. Corrales, Prohibitin deficiency blocks proliferation and induces apoptosis in human hepatoma cells: molecular mechanisms and functional implications, Proteomics 10(8) (2010) 1609-1620. [25] K.S. Ko, M.L. Tomasi, A. Iglesias-Ara, B.A. French, S.W. French, K. Ramani, J.J. Lozano, P. Oh, L. He, B.L. Stiles, Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice, Hepatology 52(6) (2010) 2096-2108. [26] F. Thuaud, N. Ribeiro, C.G. Nebigil, L. Désaubry, Prohibitin ligands in cell death and survival: mode of action and therapeutic potential, Chemistry & biology 20(3) (2013) 316331. [27] C. Moncunill-Massaguer, J. Saura-Esteller, A. Pérez-Perarnau, C.M. Palmeri, S. NúñezVázquez, A.M. Cosialls, D.M. González-Gironès, H. Pomares, A. Korwitz, S. Preciado, A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation, Oncotarget 6(39) (2015) 41750. [28] X. Kang, L. Zhang, J. Sun, Z. Ni, Y. Ma, X. Chen, X. Sheng, T. Chen, Prohibitin: a potential biomarker for tissue-based detection of gastric cancer, Journal of gastroenterology 43(8) (2008) 618-625. [29] R. Ummanni, H. Junker, U. Zimmermann, S. Venz, S. Teller, J. Giebel, C. Scharf, C. Woenckhaus, F. Dombrowski, R. Walther, Prohibitin identified by proteomic analysis of prostate biopsies distinguishes hyperplasia and cancer, Cancer letters 266(2) (2008) 171-185. [30] L.R. Webster, P.J. Provan, D.J. Graham, K. Byth, R.L. Walker, S. Davis, E.L. Salisbury, A.L. Morey, R.L. Ward, N.J. Hawkins, Prohibitin expression is associated with high grade breast cancer but is not a driver of amplification at 17q21. 33, Pathology 45(7) (2013) 629636. [31] A. Franzoni, M. Dima, M. D'Agostino, C. Puppin, D. Fabbro, C. Di Loreto, M. Pandolfi, E. Puxeddu, S. Moretti, M. Celano, Prohibitin is overexpressed in papillary thyroid carcinomas bearing the BRAFV600E mutation, Thyroid 19(3) (2009) 247-255. [32] T.-F. Wu, H. Wu, Y.-W. Wang, T.-Y. Chang, S.-H. Chan, Y.-P. Lin, H.-S. Liu, N.-H. Chow, Prohibitin in the pathogenesis of transitional cell bladder cancer, Anticancer research 27(2) (2007) 895-900. [33] H.-Z. Ren, J.-S. Wang, P. Wang, G.-q. Pan, J.-F. Wen, H. Fu, X.-z. Shan, Increased expression of prohibitin and its relationship with poor prognosis in esophageal squamous cell carcinoma, Pathology & Oncology Research 16(4) (2010) 515-522. [34] D. Chen, F. Chen, X. Lu, X. Yang, Z. Xu, J. Pan, Y. Huang, H. Lin, P. Chi, Identification of prohibitin as a potential biomarker for colorectal carcinoma based on proteomics technology, International journal of oncology 37(2) (2010) 355-365. [35] A. Bavelloni, M. Piazzi, M. Raffini, I. Faenza, W.L. Blalock, Prohibitin 2: At a communications crossroads, IUBMB life 67(4) (2015) 239-254.

[36] W. Wang, L. Xu, Y. Yang, L. Dong, B. Zhao, J. Lu, T. Zhang, Y. Zhao, A novel prognostic marker and immunogenic membrane antigen: prohibitin (PHB) in pancreatic cancer, Clinical and translational gastroenterology 9(9) (2018) 178. [37] L. Pernas, L. Scorrano, Mito-morphosis: mitochondrial fusion, fission, and cristae remodeling as key mediators of cellular function, Annual review of physiology 78 (2016) 505-531. [38] D.R. Green, B. Levine, To be or not to be? How selective autophagy and cell death govern cell fate, Cell 157(1) (2014) 65-75. [39] M. Redmann, M. Dodson, M. Boyer-Guittaut, V. Darley-Usmar, J. Zhang, Mitophagy mechanisms and role in human diseases, The international journal of biochemistry & cell biology 53 (2014) 127-133. [40] R.A. Cairns, I.S. Harris, T.W. Mak, Regulation of cancer cell metabolism, Nature Reviews Cancer 11(2) (2011) 85. [41] N.C. Denko, Hypoxia, HIF1 and glucose metabolism in the solid tumour, Nature Reviews Cancer 8(9) (2008) 705. [42] L.A. Sena, N.S. Chandel, Physiological roles of mitochondrial reactive oxygen species, Molecular cell 48(2) (2012) 158-167. [43] C.C. Winterbourn, The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells, Biochimica et Biophysica Acta (BBA)General Subjects 1840(2) (2014) 730-738. [44] B. Kalyanaraman, V. Darley-Usmar, K.J. Davies, P.A. Dennery, H.J. Forman, M.B. Grisham, G.E. Mann, K. Moore, L.J. Roberts II, H. Ischiropoulos, Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations, Free Radical Biology and Medicine 52(1) (2012) 1-6. [45] S. Mishra, L.C. Murphy, L.J. Murphy, The Prohibitins: emerging roles in diverse functions, Journal of cellular and molecular medicine 10(2) (2006) 353-363. [46] P. Liu, Y. Xu, W. Zhang, Y. Li, L. Tang, W. Chen, J. Xu, Q. Sun, X. Guan, Prohibitin promotes androgen receptor activation in ER-positive breast cancer, Cell Cycle 16(8) (2017) 776-784. [47] Y.-T. Peng, P. Chen, R.-Y. Ouyang, L. Song, Multifaceted role of prohibitin in cell survival and apoptosis, Apoptosis 20(9) (2015) 1135-1149. [48] M.D. Brand, D.G. Nicholls, Assessing mitochondrial dysfunction in cells, Biochemical Journal 435(2) (2011) 297-312. [49] S. Frank, B. Gaume, E.S. Bergmann-Leitner, W.W. Leitner, E.G. Robert, F. Catez, C.L. Smith, R.J. Youle, The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis, Developmental cell 1(4) (2001) 515-525

[50] Y.-j. Lee, S.-Y. Jeong, M. Karbowski, C.L. Smith, R.J. Youle, Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis, Molecular biology of the cell 15(11) (2004) 5001-5011. [51] D.G. Breckenridge, M. Stojanovic, R.C. Marcellus, G.C. Shore, Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol, The Journal of cell biology 160(7) (2003) 1115-1127. [52] M. Germain, J.P. Mathai, H.M. McBride, G.C. Shore, Endoplasmic reticulum BIK initiates DRP1-regulated remodelling of mitochondrial cristae during apoptosis, The EMBO journal 24(8) (2005) 1546-1556. [53] M.J. Barsoum, H. Yuan, A.A. Gerencser, G. Liot, Y. Kushnareva, S. Gräber, I. Kovacs, W.D. Lee, J. Waggoner, J. Cui, Nitric oxide-induced mitochondrial fission is regulated by dynamin-related GTPases in neurons, The EMBO journal 25(16) (2006) 3900-3911. [54] H. Chen, D.C. Chan, Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases, Human molecular genetics 18(R2) (2009) R169R176. [55] J.A. Mears, L.L. Lackner, S. Fang, E. Ingerman, J. Nunnari, J.E. Hinshaw, Conformational changes in Dnm1 support a contractile mechanism for mitochondrial fission, Nature structural & molecular biology 18(1) (2011) 20. [56] J.E. Lee, L.M. Westrate, H. Wu, C. Page, G.K. Voeltz, Multiple dynamin family members collaborate to drive mitochondrial division, Nature 540(7631) (2016) 139. [57] X. Liu, C.N. Kim, J. Yang, R. Jemmerson, X. Wang, Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c, Cell 86(1) (1996) 147-157. [58] S.A. Susin, H.K. Lorenzo, N. Zamzami, I. Marzo, B.E. Snow, G.M. Brothers, J. Mangion, E. Jacotot, P. Costantini, M. Loeffler, Molecular characterization of mitochondrial apoptosis-inducing factor, Nature 397(6718) (1999) 441. [59] L.Y. Li, X. Luo, X. Wang, Endonuclease G is an apoptotic DNase when released from mitochondria, Nature 412(6842) (2001) 95. [60] J.-C. Martinou, R.J. Youle, Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics, Developmental cell 21(1) (2011) 92-101. [61] R.J. DeBerardinis, N. Sayed, D. Ditsworth, C.B. Thompson, Brick by brick: metabolism and tumor cell growth, Current opinion in genetics & development 18(1) (2008) 54-61. [62] E. Michalopoulou, V. Bulusu, J.J. Kamphorst, Metabolic scavenging by cancer cells: when the going gets tough, the tough keep eating, British journal of cancer 115(6) (2016) 635.

[63] P.S. Ward, C.B. Thompson, Metabolic reprogramming: a cancer hallmark even warburg did not anticipate, Cancer cell 21(3) (2012) 297-308. [64] M. Ristow, Oxidative metabolism in cancer growth, Current Opinion in Clinical Nutrition & Metabolic Care 9(4) (2006) 339-345. [65] J.-H. Cheong, E.S. Park, J. Liang, J.B. Dennison, D. Tsavachidou, C. Nguyen-Charles, K.W. Cheng, H. Hall, D. Zhang, Y. Lu, Dual inhibition of tumor energy pathway by 2deoxyglucose and metformin is effective against a broad spectrum of preclinical cancer models, Molecular cancer therapeutics 10(12) (2011) 2350-2362. [66] H. Liu, Y. Hu, N. Savaraj, W. Priebe, T. Lampidis, Hypersensitization of tumor cells to glycolytic inhibitors, Biochemistry 40(18) (2001) 5542-5547. [67] G. Cheng, J. Zielonka, B.P. Dranka, D. McAllister, A.C. Mackinnon, J. Joseph, B. Kalyanaraman, Mitochondria-targeted drugs synergize with 2-deoxyglucose to trigger breast cancer cell death, Cancer research 72(10) (2012) 2634-2644. [68] L. Lignitto, S.E. LeBoeuf, H. Homer, S. Jiang, M. Askenazi, T.R. Karakousi, H.I. Pass, A.J. Bhutkar, A. Tsirigos, B. Ueberheide, Nrf2 Activation Promotes Lung Cancer Metastasis by Inhibiting the Degradation of Bach1, Cell (2019). [69] C. Wiel, K. Le Gal, M.X. Ibrahim, C.A. Jahangir, M. Kashif, H. Yao, D.V. Ziegler, X. Xu, T. Ghosh, T. Mondal, BACH1 Stabilization by Antioxidants Stimulates Lung Cancer Metastasis, Cell (2019). [70] X. Peng, R. Mehta, S. Wang, S. Chellappan, R.G. Mehta, Prohibitin is a novel target gene of vitamin D involved in its antiproliferative action in breast cancer cells, Cancer research 66(14) (2006) 7361-7369.

Supplementary Information

Figure S 1. Glucose starvation (GS) sensitizes breast cancer cells to apoptosis.

A MCF-7 cells under Phase contrast microscope. GS cells showing rounded, shrunken morphology compared to control cells with normal media. (scale bar: 100µm). B Cell viability was measured by Trypan blue assay in MDA-MB-231 cells. First cells were treated with different types of inhibitors and grown under glucose starvation condition for 24 h. C

Western blot analysis of MCF-7 cells showing expression levels of pro-apoptotic proteins

(Cytochrome c, Bim, Bid, Bax, Caspase-3 and Apaf-1) and anti-apoptotic proteins (Bcl-2) after 24 h of GS. D

Western blot of MCF-7 cells showing the expression of autophagy marker LC-3 after

glucose starvation for 24 h E Graphs representing caspase 6/9 activity in MCF-7 cells.

Figure S 2: Analysis of glucose starvation specific cell death A

Phase contrast images of MDA-MB-231 cells grown under different concentration of

H2O2 as indicated. B

Phase contrast images of cells grown under different concentration of glucose as

indicated. Experiments were carried out in triplicate. (Scale bar: 100µm).

Fig S 3. Glucose starvation increases prohibitin 1 expression in breast cancer cells. A

Prohibitin 1 (PHB1) expression in MCF-7 cells after 24 h of glucose starvation (GS).

B

Dynamic expression of PHB1, MCF-7 cells first grown in glucose free medium followed

by grown in glucose-rich medium for durations indicated. C

Prohibitin 1 (PHB1) expression in MDA-MB-231 cells after 24 h of glucose starvation

(GS) and GS with NAC (5mM) for 24 h. D,E PHB1expression was determined for cells grown under Glucose Starvation (0.0g/L) and 100µM of H2O2.

Figure S 4. Mitochondrial morphology and interaction studies between PHB1 and DRP1 in MDA-MB-231 cells after glucose starvation. A

Confocal microscopy images of MCF-7 cells showing fragmented morphology of

mitochondria after 24 h of GS. (scale bar: 10µm). Inset shows a magnified view of the fragmented mitochondria. B

Cellular fractionation blots indicating activation of p-DRP1 in mitochondria in

response to GS. C

IP blot indicating there is no interaction between PHB1 and DRP1 in MCF-7 cells

after 24 h GS

Figure S 5. Effect of GS in breast cancer cell migration. A

Wound heal assay in MCF-7 cells after GS and refeeding.

B

Wound heal assay in MCF-7 cells transfected with empty vector (EV) and PHB1

plasmid. Histograms representing the % of wound heal area.

Table 1: siRNA for PHB1 was designed to target the sequence of PHB1 Strand

Sequence

Sense

CCCAGAAAUCACUGUGAAAdTdT

Anti-sense

UUUCACAGUGAUUUCUGGG-dTdT