WO2014052305A2

WO2014052305A2 - Method for treating breast cancer using mitochondrial poisons that interfere with over-expressed mitochondrial proteins

Info

Publication number: WO2014052305A2
Application number: PCT/US2013/061381
Authority: WO
Inventors: Michael P. Lisanti
Original assignee: Thomas Jefferson University
Priority date: 2012-09-26
Filing date: 2013-09-24
Publication date: 2014-04-03
Also published as: WO2014052305A3

Abstract

Here, we set out to test the novel hypothesis that increased mitochondrial biogenesis in epithelial cancer cells would "fuel" enhanced tumor growth. For this purpose, we generated MDA-MB-231 cells (a triple-negative human breast cancer cell line) over-expressing PGC- lα and MitoNEET, which are established molecules that drive mitochondrial biogenesis and increased mitochondrial oxidative phosphorylation (OXPHOS). Interestingly, both PGC- lα and MitoNEET increased the abundance of OXPHOS protein complexes, conferred autophagy-resistance under conditions of starvation, and increased tumor growth by up to ~3-fold. However, this increase in tumor growth was independent of neo-angiogenesis, as assessed by immuno-staining and quantitation of vessel-density using CD31 antibodies. Quantitatively similar increases in tumor growth were also observed by over-expression of PGC- Ι β and POLRMT in MDA-MB-231 cells, which are also responsible for mediating increased mitochondrial biogenesis. Thus, we propose that increased mitochondrial "power" in epithelial cancer cells oncogenically promotes tumor growth, by conferring autophagy-resistance. As such, PGC- lα, PGC- Ιβ, mitoNEET, and POLRMT should al l be considered as tumor promoters or "metabolic oncogenes". Our results are consistent with numerous previous clinical studies showing that metformin (a weak mitochondrial "poison") prevents the onset of nearly al l types of human cancers in diabetic patients. Therefore, metformin (a Complex I inhibitor), and other mitochondrial inhibitors, should be developed as novel anti-cancer therapies, targeting mitochondrial metabolism in cancer cells.

Description

METHOD FOR TREATING BREAST CANCER USING MITOCHONDRIAL POISONS THAT INTERFERE WITH OVER-EXPRESSED MITOCHONDRIAL PROTEINS

Ahmed F. Salem^1,2'³, Diana Whitaker- enezes ''², Anthony Howell Fcdcrica Sotgia ^>:' ^ and Michael P. Lisanti¹'²'"'⁵*

The Jefferson Stem Cell Biology and Regenerative Medicine Center

departments of Stem Cell Biology & Regenerative Medicine and Cancer Biology Kimmcl Cancer Center; Thomas Jefferson University, Philadelphia, PA USA

department of Molecular Drug Evaluation, Division of Biochemistry, National Organization for Drug Control and Research, Giza, Egypt

''Manchester Breast Centre & Breakthrough Breast Cancer Research Unit; Palerson Institute for Cancer Research; School of Cancer; Enabling Sciences and Technology; Manchester Academic Health Science Centre; University of Manchester, U

⁵ Present Address:

University of Manchester, Breakthrough Breast Cancer Research Unit, Manchester, UK

* Corresponding Authors.

Fed erica Sotgia: fsotgiaiaigmail.com

ichael P. Lisanti: michaclp isai i{¾ rnail.com

Key Words: cancer metabolism, mitochondrial biogenesis, oxidative phosphorylation, OXPHOS, autophagy resistance, angiogcnesis, two compartment tumor metabolism Introduction

Mitochondria are not only the power-house of the cel l, but they also play a critical role in the metabolism of proliferating cells.¹ Despite these known function(s) in cel l growth and division, the role of mitochondria in carcinogenesis has been largely ignored for many years, because of the "Warburg Hypothesis".

Warburg's Hypothesis, also known as "aerobic glycolysis", postu lates that tumor cells shut down their oxidative mitochondrial metabolism and depend only on glycolysis, even under oxygen-rich conditions.² This hypothesis discounts the fact that glycolysis produces a very low-yield of ATP, while cancer cells have high-energy demands for ATP. This implies that cancer cel ls cannot really depend solely on the glycolytic pathway to produce their energy. Utilizing the end products of glycolysis via oxidative mitochondrial metabolism (the TCA/citric acid cycle and oxidative phosphorylation (OXPHOS)) would be a much more efficient method of provid ing energy to cancer cells, which are in desperate need of ATP molecules.

Recently, we have directly shown that that oxidative mitochondrial activity is fact induced in cancer cells, when they are co-cultured with fibroblasts.^3"5 As a consequence, we have proposed a new hypothesis, termed "Two-Compartment Tumor Metabolism" to resolve the Warburg paradox.^3"5 Our previous studies also indicated that cancer cells are able to recruit stromal cells by releasing reactive oxygen species (ROS) into their local micro-environment. As a result, cancer-associated fibroblasts (CAFs) activate autophagic signaling pathways, in order to recycle their proteins and organelles, such as mitochondria (mitophagy). Without functional mitochondria, CAFs become glycolytic and secrete L-lactate and ketone bodies, which are then taken-up by nearby cancer cells.⁶ Subsequently, these energy-rich nutrients are "burned" via oxidative mitochondrial metabolism in the cancer cells, to produce energy and anabolic products.

MitoNEET is an integral membrane protein component of the outer m itochondrial membrane that was found to be co-localized with pioglitazone.⁷ It was named based on its m itochondrial localization and the presence of a unique amino acid sequence Asn-G lu-Glu-Thr (NEET) at its C-terminus.⁷ Recently, m itoNEET has been demonstrated to be a regulator of m itochondrial respiration.⁸ Wiley et al showed that mitochondria isolated from M itoN EET-nul l murine hearts displayed lower oxidative capacity.⁸ Furthermore, members of the peroxisome proliferator-activated receptor-γ coactivators- 1 (PGC- 1 ) family play a central role in cellular and systemic metabolism.⁹' ¹⁰ The family consists of PGC- l a, PGC- Ι β and the PGC-1 -related co-activator (PRC). All three proteins share a unique co-activator LXXLL motif.¹⁰' " Their role in mitochondrial biogenesis is via the regulation of nuclear respiratory factor- 1 (NRF- 1 ), NRF-2 and ERRa (estrogen-related receptor a). PGC- l a and PGC- 1 β proteins share high similarity in structure and function, " although PGC- 1 β demonstrates a higher coupling respiration, due to differences in proton leakage. "

Another important molecule for mitochondrial function and biogenesis is mitochondrial RNA polymerase, POLMRT.^{1 2} POLRMT is responsible for the transcription of the 1 3 subunits of the OXPHOS complexes and acts as a RNA primase for mitochondrial DNA replication.¹³' ¹⁴ POLRMT requires transcription factor B2M (TFB2M) and transcription factor AM (TFAM) for mitochondrial RNA transcription.¹⁴

Here, we examined the role of mitochondrial biogenesis and function in breast cancer tumor growth. Using a preclinical animal model, we determined how the over-expression of mitoNEET, PGC- l a, PGC- 1 β and POLMRT in epithelial breast cancer cells (MDA-MB-23 1 ) affects tumor growth. Remarkably, over-expression of these key molecules that drive mitochondrial biogenesis promoted tumor growth in nude mice. However, this increased growth was clearly independent of tumor vascularization. Our results demonstrate that mitochondrial biogenesis in epithelial cancer cells promotes breast cancer tumor growth, l ikely via increased energy production and nutrient reserves, which confers autophagy-resistance.

Thus, we have identified oxidative mitochondria! metabol ism as a new "druggable" therapeutic target for the development of novel anti-cancer agents.

In addition, our findings directly support clinical studies demonstrating that metformin (a mitochondrial "poison" and Complex I inh ibitor) prevents the onset of nearly al l types of human cancers in diabetic patients (reviewed with in As a consequence, metformin, and other mitochondrial inhibitors, should be used to target mitochondrial metabolism in epithelial cancer cells. Results

Mitochondrial OXPHOS is increased in breast cancer cells, during over-expression of PGC-l a and mitoNEET.

Recently, we have suggested that increased mitochondrial function in cancer cells may directly promote tumor growth.³ Here, to test this hypothesis more directly, we stably over-expressed PGC-l a and MitoNEET in MDA-MB-23 1 cells, a human triple-negative breast cancer cell line. Then, immuno-blot analysis was used to confirm the successful over-expression of both proteins (Fig. 1 A and B).

PGC- 1 a is a master transcription factor that stimulates mitochondrial biogenesis and respiratory function.⁸ Moreover, mitoNEET, a mitochondrial outer membrane protein, regulates mitochondrial oxidative phosphorylation.⁸ Interestingly, mitoNEET over-expressing MDA-MB- 231 cells displayed elevated PGC-l expression. As expected, immuno-blotting against various OXPHOS components showed that these mitochondrial complexes (I, III, IV, and V) were increased in both PGC- l a and MitoNEET-over-expressing MDA-MB-231 cells (Fig. IB).

PGC-l a and mitoNEET confer autophagy-resistance in breast cancer cells.

Next, we wanted to test if PGC- l a and mitoNEET will reduce autophagy in cancer cells, as they do in skeletal muscles.¹⁶ Interestingly, PGC- l and mitoNEET both protected cancer cells from autophagy, as Beclin- 1 and Cathepsin B (markers of autophagy) were not up-regulated under conditions of starvation (Fig. 2).

PGC-1 a and mitoNEET increase tumor growth in vivo, independently of angiogenesis.

Traditionally, altered mitochondrial metabolism was considered as a secondary effect of proliferation. Here, we wanted to test if increased mitochondrial biogenesis and OXPHOS could directly promote tumor growth. PGC- 1 a and mitoNEET-overexpressing MDA cells were injected in nude mice. Tumor volumes were then measured at various time points post-injection.

Tumors in which PGC-l a and mitoNEET were over-expressed were significantly larger, as compared to control tumors (Fig. 3A). At 3 weeks post-injection, PGC- l a tumor volumes and weights were on average approximately 3-fold larger in than control tumors, which harbored the expression vector alone. Moreover, MitoNEET tumor volumes and weight were 2.9- and 2.25- fold larger than control tumors, respectively (Fig. 3B).

To examine the possibility that the observed increase in tumor growth was due to increased angiogenesis, we immuno-stained tumor sections with CD31 , a well-established marker used to quantitate vessel density. Interestingly, PGC- Ι α tumors did not show any significant differences in vessel number, while mitoNEET tumors displayed decreased tumor vascularization (Fig. 3C). These results imply that the increased tumor growth caused by PGC- Ι α and/or mitoNEET over- expression is independent of tumor vascularization.

PGC-ip and POLRMT also promote breast cancer tumor growth.

Given the growth-promoting effects of PGC- Ι and mitoNEET, we decided to also study the functional effects of PGC- Ι β, and, POLRMT, a mitochondrial RNA polymerase,¹⁷ on breast cancer tumor growth.

Using lentiviral vectors, MDA-MB-231 cells were stably transduced to over-express either PGC- 1 β or POLRMT. Successful over-expression was evident by immuno-blotting for total PGC- Ι β or POLRMT (Fig. 4). To test if over-expression of PGC- Ι β or POLRMT increases tumor growth, we subcutaneously injected PGC- Ι β or POLRMT-over-expressing MDA-MB-231 cells into the flanks of nude mice. The tumor growth curves for PGC- Ι β and POLRMT show a significant increase in tumor size (Fig. 5A). At 3 weeks post-injection, PGC- Ι β and POLRMT tumor volumes were 2-fold larger than control tumors, which harbored the vector alone (Fig. 5B).

Finally, we examined if this observed increase in tumor growth is related to increased tumor angiogenesis. In fact, these tissue sections stained for CD31 displayed a significant decrease in vascularization in PGC- Ι β and POLRMT tumors (Fig 5C). Thus, these data show that enhanced tumor growth induced by PGC- Ι β and POLRMT is not due to increases in neo-vascularization. Discussion

Traditionally, the "Warburg Effect" has been used to describe cancer metabolism, whereby cancer cells become more dependent on glycolysis and less dependent on OXPHOS to produce their ATP.² However, this theory does not resolve the issue that cancer cells could not possibly depend solely on glycolysis, which has an extremely low yield of ATP.

Recently, we and other researchers have high-l ighted the crucial role of mitochondrial metabolism in tumorgenesis.^18-21 In this study, we demonstrate the effect of elevated mitochondrial biogenesis and metabolism on tumor growth, using pre-clinical animal models. This was achieved by stably transducing MDA-MB-23 1 breast cancer cells to overexpress several positive regulators of mitochondrial biogenesis, including PGC- l a, PGC- Ι β, mitoNEET, and POLRMT. Interestingly, PGC- l a and mitoN EET induced mitochondrial OXPHOS complexes and protected cancer cells from autophagy. Moreover, PGC- l a, PGC- Ι β, mitoN EET, and POLRMT over-expression all significantly increased tumor growth in nude mouse xenografts.

PGC- l a and PGC- Ι β are transcriptional co-activators that have a central role in maintaining metabolic pathways, such as oxidative metabolism, energy homeostasis and glucose and lipid metabolism in normal cells.²² Recently, PGC- 1 β knock-down in tamoxifen-resistant MCF-7 cells resulted in a cell l ine that was sensitive to tamoxifen-treatment.²³ Sim i larly, Sh iota et al . showed

24

that silencing of PGC- l a inhibited LNCaP prostate cancer cell prol iferation .

It is worth pointing out that PGC-1 family members induce superoxide dismutase (SOD), catalase (CAT), and glutathione-peroxidase (GPX), all critical protein anti-oxidants.²⁵' ²⁶ Th is action protects cancer cel ls against elevated mitochondrial ROS. Alternatively, PGC- l has been shown to inhibit ROS by inducing uncoupling proteins, which leak protons across the inner membrane of mitochondria to lower their membrane potential, eventually decreasing ROS produced by the mitochondria. Interestingly, this anti-oxidant role can protect cancer cel ls from self-released ROS, al lowing them to activate neighboring CAEs, via oxidative stress.

Interestingly, type 2 d iabetic patients suffer from oxidative stress due to reduced mitochondrial mass and oxidative capacity in skeletal muscle.^27"29 Pioglitazone, a thiazolidinedione member which is used as an insulin-sensitizing drug for type 2 d iabetes, improves mitochondrial respiration in type 2 diabetic patients.²⁹ Classically, thiazolidinedione family members are thought to be agonists for peroxisomal proliferator-activated receptor (PPAR)-y expression, which in turn induces the expression of PGC- l a.³⁰

Recently, pioglitazone was found to co-localize with an iron-containing outer mitochondrial membrane protein, mitoNEET,⁷ It is possible that piogl itazone induces mitochondrial respiration through mitoNEET binding as mitoNEET-null mice demonstrated lower mitochondrial capacity.⁸' ²⁹ Accordingly, our results demonstrate that mitoNEET-overexpressing cancer cel ls exhibited an increase in mitochondrial complexes I, I I I, IV, V and a decrease in autophagy. Strikingly, mitoNEET tumors were ~3-fold larger than control tumors. Thus, we established a novel role for mitoNEET in promoting tumor growth. These results provide evidence that mitochondrial metabol ism and OXPHOS are driving factors for cancer growth and are not a a side-effect of increased cellular proliferation.

Moreover, POLMRT is a mitochondrial bacteriophage-related RNA polymerase which is needed for the transcription and replication of mtDNA.^{3 1} ' ³² POLRMT is responsible for transcription of several OXPHOS subunits. Interestingly, nuclear factor N RF- 1 , which is regulated by PGC- 1 family members, stimulates POLRMT to increase m itochondrial replication. ' ⁴ Moreover, POLRMT depletion affects mtDNA replication.³² Our results demonstrate that POLRMT-over- expressing cancer cel ls drive a 2-fold increase in tumor growth in nude m ice. This growth was independent of tumor angiogenesis. Thus, our current studies are consistent with a new role for POLRMT as a tumor promoter.

Finally, we observed that over-expression of PGC- l a and mitoNEET, which induce mitochondrial biogenesis, conferred autophagy-resistance under conditions of starvation. In addition, over-expression of PGC- l a and mitoNEET increased tumor growth by ~3-fold. Similarly, loss of Beclin- 1 , a required autophagy gene and tumor suppressor, is known to confer autophagy-resistance in cancer cells and increase tumor growth ^33"35. Thus, autophagy-resistance in cancer cells -- mediated by either i) increased mitochondrial power or ii) genetic loss of required autophagy genes— has the same net effect, to increase tumor growth rates. In conclusion, we studied several molecules that induce mitochondrial biogenesis in cancer cel ls. We have shown that all four of these molecules promote tumor growth. Thus, we propose that increased mitochondrial biogenesis functions as an "oncogene" or tumor promoter in cancer cells. Induced oxidative mitochondrial metabolism would allows cancer cells to more efficiently utilize imported L-lactate, ketone bodies, glutamine, and fatty acids, wh ich are all produced by neighboring activated CAFs or related stromal cells. Thus, targeting cancer cell m itochondria with mitochondrial "poisons" could halt this metabol ic-coupling, effectively shutting-down the "engine" or "powerhouse" driving tumor growth.

Materials and Methods

Materials. Antibodies were all purchased from commercial sources: anti-PGC-l (NBP-I- 04676, Novus), anti-mitoNEET (ab 118027, Abeam), anti-PGC-1 total (ab72230, Abeam), anti- POLRMT (HPA006366, Sigma), anti-OXPHOS (MS601, Mitoscience), anti-beta actin (A5441, Sigma), anti-Beclin-1 (Novus, NBP1 -00085) and anti-Cathepsin B (FL-339, Santa Cruz, biotechnology).

Cell culture and transfection. MDA-MB-231 cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum in a 37°C humidified atmosphere containing 5% C0₂, unless otherwise noted. Lentiviral particles were produced after 48 hours of transfecting the GeneCopoeia 293Ta lentiviral packaging cell line with lentiviral plasmids (EX-NEG-Lvl 05) (empty vector), PGC-Ια (EX-U0564-Lvl05), itoNEET (EX- V0831-Lvl05), PGC-Ιβ (EX-Mm 13777-Lv 105), and POLRMT (EX-Z7565-Lvl05) (all obtained from GeneCopoeia. Inc) using the Lenti-Pac HIV Expression Packing Kit (GeneCopoeia. Inc), according to manufacturer's recommendation. MDA-MB-231 breast cancer cells were transduced with lentivirual particles in the presence of 5 μg/ml of Polybrene (Santa Cruz Biotech). Successfully transduced cells were selected with 2 vg/m\ of puromycin.

Immuno-blotting. Cells were lysed by scraping into cell lysis buffer (10 inM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100 and 60 mM n-octyl-glucoside), containing protease inhibitors (Boehringer Mannheim, Indianapolis, IN). To induce autophagy, cells were starved for 5 hrs using Hank's Balanced Salt Solution (HBSS) supplemented with 40 mM HEPES. Samples were incubated on a rotating platform at 4°C and were then centrifuged at 12,000x g for 10 min (at 4°C) to remove insoluble debris. Protein concentrations were analyzed using the BCA reagent (Pierce, Rockford, IL). Samples were then separated by SDS-PAGE (12% acrylamide) and transferred to nitrocellulose. All subsequent wash buffers contained 10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20, which was supplemented with 5% nonfat dry milk (Carnation) for the blocking solution and 1% bovine serum albumin (Sigma) for the antibody diluent. Horseradish peroxidase-conjugated secondary antibodies were used to visualize bound primary antibodies with an ECL detection kit (Pierce). (Pierce, Rockford, IL). Animal studies. All animals studied were maintained in a pathogen-free environment/barrier facility at the Kimmel Cancer Center at Thomas Jefferson University under National Institutes of Health (NIH) guidelines. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC). Tumor cells (MDA-MB-231 ; 1 x 10⁶ cells) in 100 μΐ of sterile PBS were injected into the flanks of athymic NCr nude mice (NCRNU; Taconic Farms; 6-8 weeks of age). Mice were then sacrificed at 21 days post-injection; tumors were excised to determine their weights and volumes.

Angiogenesis quantification. Frozen sections were processed, as we described previously/⁶ Then the anti-CD3 1 antibody was incubated overnight at 4°C. Finally, the sections were counter- stained with hematoxylin for 5-10 sec, air-dried and mounted with cover -slips. To quantify tumor angiogenesis, CD31 -positive vessels were enumerated in 8- 10 fields within the central area of each tumor using a 20X objective lens and an ocular grid (0.25 mm² per field). The total numbers of vessel per unit area was calculated using Image J, and the data was represented graphically.

Statistical analysis. Statistical significance was examined by the Student's /-test. Values of p < 0.05 were considered significant. All p-values were 2-tailed (2T), unless otherwise noted.

Acknowledgements

F.S. was the recipient of a Young Investigator Award from the Breast Cancer Alliance. Funds were also contributed by the Margaret Q. Landenberger Research Foundation (to M.P.L.).

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Figure Legends

Figure 1. Over-expression of PGC- l a and MitoNEET induces the expression of mitochondrial OXPHOS complexes. A, Using a lentiviral vector, we established MDA-MB-23 1 cell lines that stably over-express MitoNEET, as seen by immuno-blotting. β-actin is shown as a control for equal protein loading. B, PGC- 1 a and MitoNEET induce expression of mitochondrial OXPHOS complexes. Both PGC- 1 a and mitoNEET-over-expressing MDA-MD-23 1 cells displayed elevated PGC-l expression. Most importantly, they also showed an elevation of mitochondrial complexes (OXPHOS). β-actin is shown as a control for equal protein loading. EV, empty vector (Lv l 05).

Figure 2. PGC- l a and MitoNEET protect cancer cells against autophagy. Under conditions of starvation, PGC- l a and MitoNEET-over-expressing MDA-MB-23 1 cells fai l to up-regulate autophagy markers, such as Beclin- 1 and Cathepsin B. EV, empty vector (Lv l 05).

Figure 3. PGC- l a and MitoNEET over-expression increases tumor growth. PGC- l a and MitoNEET over-expressing MDA-MB-23 1 cells were subcutaneously injected into the flanks of nude mice.

A, Tumor volumes were measured on days 14, 19, 21 . Note that PGC- l a and MitoNEET tumors demonstrate a trend of significantly larger volumes, as compared to control tumors.

B, Tumors were excised, weighed and their volumes were measured. PGC- l a tumor volumes and weights were 3- and 2.7-fold larger, as compared to control tumors, respectively. Also, MitoNEET tumor volumes and weights were 2.9- and 2.25-fold larger, as compared to control tumors, respectively.

C, Tumor angiogenesis and vessel quantification (number of vessels per field). Frozen sections were cut and immuno-stained with anti-CD31 antibodies. Quantification of CD3 1 -positive vessels shows a non-significant reduction in angiogenesis in PGC- l tumors, while MitoNEET tumors displayed a significant reduction in CD31 -positive vessels. These results indicates that PGC- l a and MitoNEET stimulate tumor growth, but independently of neo-angiogenesis. Figure 4. Overexpression of PGC- Ι β and POLRMT. Immuno-blot analysis shows successful over-expression of PGC- Ι β and POLRMT in transduced MDA-MB-23 1 cells, β-actin is shown as a control for equal protein loading.

Figure 5. PGC- Ι β and POLRMT increase tumor growth in a pre-clinical animal model. PGC- 1 β and POLRMT over-expressing MDA-MB-231 cells were injected into the flanks of nude mice. A, Tumor growth curve. Tumor volumes were measured on days 1 1 , 14, 1 8, and 20. Note that PGC- 1 β and POLRMT tumors are significantly larger in volume, versus control tumors. B, Excised tumor volumes. PGC- 1 β and POLRMT tumors were 2.2- and 2-fold larger than control tumors. C, Tumor angiogenesis and vessel quantification (number of vessels per field). PGC- 1 β and POLRMT tumors displayed a significant reduction in tumor vascularization, showing that PGC-1 β and POLRMT increase tumor growth, independently of angiogenesis.

Figure 6. Mitochondrial biogenesis increases tumor growth. Schematic diagram highlighting that MitoNEET, PGC-1 (a- and β-isoforms) and POLRMT all drive mitochondrial biogenesis, and effectively promote tumor growth, when expressed in cancer cel ls. Thus, the therapeutic use of mitochondrial inhibitors or "poisons" (such as metformin) will inhibit tumor growth. Importantly, numerous clinical studies have now shown that metformin (a mitochondrial Compex I inhibitor) prevents the onset of nearly all types of human cancers in diabetic patients.

Definitions

A pharmaceutical composition typically comprises an active agent and a pharmaceutical ly acceptable carrier. Pharmaceutically acceptable carriers are well known to those ski lled in the art and include, but are not limited to, 0.01 -0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers can be aqueous or nonaqueous solutions, suspensions, and emu lsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as ol ive oil, and injectable organ ic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emu lsions and suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sod ium chloride, lactated Ringer's and fixed oils.

Intravenous vehicles include flu id and nutrient replenishers, electrolyte replen ishers such as Ringer's dextrose, those based on Ringer's dextrose, and the like. Fluids used commonly for i .v. administration are found, for example, in Rem ington: The Science and Practice of Pharmacy, 20.sup.th Ed., p. 808, Lippincott Williams & Wi lkins (2000). Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like.

As used here, a "nucleic acid" can be DNA, RNA, or a variant thereof. Nucleic acids include, for example, RNAi and antisense molecules.

As used herein, "subject" shall mean any animal, such as a human, non-human primate, mouse, rat, guinea pig or rabbit.

As used herein, "treating" a subject afflicted with a breast tumor shall mean slowing, stopping or reversing the tumor's progression. In the preferred embodiment, treating a subject affl icted with a breast tumor means reversing the tumor's progression, ideal ly to the point of el imi nating the tumor itself. As used herein, a "breast tumor" may be, for example, Luminal A, Luminal B, triple

negative/basal-like, HER2-type, and ductal carcinoma in situ.

As used herein, "inhibiting the activity" of a mitochondrial protein shall mean reducing the activity and/or amount of the protein (for example, reducing the activity and/or amount of one or more of mitoNEET, PGC- l , PGC-Ι β and POLMRT).

Therapeutic agents can be administered by any known means including, for example, orally, intravenously, intramuscularly, topically and subcutaneously.

Metformin dosing: The dosage of metformin should be individualized on the basis of both effectiveness and tolerance, while not exceeding the maximum recommended daily doses. The maximum recommended daily dose of metformin is 2550 mg (25.5 mL) in adults. Doses above 2000 mg (20 mL) may be better tolerated given three times a day with meals.

Metformin should be given in divided doses with meals. Metformin should be started at a low dose, with gradual dose escalation, both to reduce gastrointestinal side effects and to permit identification of the minimum dose required for therapeutic effect.

Recommended Dosing Schedule for Adults— A recommended starting dose below 1 500 mg ( 1 mL) per day, and gradually increased dosage, is advised to minimize gastrointestinal symptoms.

The usual starting dose of metformin (metformin hydrochloride oral solution) is 500 mg (5 mL) twice a day or 850 mg (8.5 mL) once a day, given with meals. Dosage increases should be made in increments of 500 mg (5 mL) weekly or 850 mg (8.5 mL) every 2 weeks, up to a total of 2000 mg (20 mL) per day, given in divided doses. Subjects can also be titrated from 500 mg (5 mL) twice a day to 850 mg (8.5 mL) twice a day after 2 weeks.

Claims

What is claimed is:

1 . A method for treating a subject affl icted with a breast tumor comprising admin istering to the subject a therapeutically effective amount of an agent that specifically interferes with a mitochondrial protein known to be over-expressed in the cells of the subject' s breast tumor.

2. The method of claim 1 , wherein the agent interferes with the m itochondrial protein by inhibiting its activity.

3. The method of claim 2, wherein the m itochondrial protein is selected from the group consisting of mitoNEET, PGC- l a, PGC- Ι β and POLMRT.

4. The method of claim 3, wherein the mitochondrial protein is mitoNEET.

5. The method of claim 3, wherein the mitochondrial protein is PGC- l a.

6. The method of claim 3, wherein the mitochondrial protein is PGC- Ι β.

7. The method of claim 3, wherein the mitochondrial protein is POLMRT.

8. The method of claim 2, wherein the agent is an antibody, a polypeptide or a smal l molecule.

9. The method of claim 1 , wherein the agent interferes with the mitochondrial protein by inhibiting its expression.

1 0. The method of claim 9, wherein the mitochondrial protein is selected from the group consisting of mitoNEET, PGC- l a, PGC- l p and POLMRT.

1 1 . The method of claim 10, wherein the mitochondrial protein is mitoNEET.

12. The method of claim 1 0, wherein the m itochondrial protein is PGC- l .

1 3. The method of claim 1 0, wherein the mitochondrial protein is PGC- 1 β.

14. The method of claim 1 0, wherein the mitochondrial protein is POLMRT.

1 5. The method of claim 9, wherein the agent is a nucleic acid.

1 6. The method of claim 1 , wherein the subject is human.

1 7. The method of claim 1 , wherein the agent is adm inistered in the form of a pharmaceutical composition.

1 8. A method for treating a subject affl icted with a breast tumor comprising admin istering to the subject a therapeutically effective amount of metformin.

19. The method of claim 1 8, wherein the agent is administered in the form of a

pharmaceutical composition.