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Energy metabolism drugs block triple negative breast metastatic cancer cell phenotype Silvia Cecilia Pacheco-Velazquez, Diana Xochiquetzal Robledo-Cadena, Ileana HernándezReséndiz, Juan Carlos Gallardo-Pérez, Rafael Moreno-Sánchez, and Sara Rodríguez-Enríquez Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00015 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 13, 2018

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Molecular Pharmaceutics 1

Energy metabolism drugs block triple negative breast metastatic cancer cell phenotype Silvia Cecilia Pacheco-Velázquez†, Diana Xochiquetzal Robledo-Cadena†, Ileana Hernández-Reséndiz†, Juan Carlos Gallardo-Pérez†, Rafael Moreno-Sánchez† and Sara Rodríguez-Enríquez†,‡* † Departamento de Bioquímica, Instituto Nacional de Cardiología, México ‡ Laboratorio de Medicina Traslacional, Instituto Nacional de Cancerología, México

*Author for correspondence: Sara Rodríguez-Enríquez Ph.D. Departamento de Bioquímica Instituto Nacional de Cardiología Juan Badiano No. 1. Col Sección XVI Tlalpan, México City E-mail: [email protected]; [email protected]

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ABSTRACT To establish alternative targeted therapies against triple negative (TN) breast cancer, (1) the energy metabolism and (2) the sensitivity of cell growth, migration and invasiveness towards metabolic, canonical and NSAID inhibitors was analyzed in MDA-MB-231 and MDA-MB468, two TN metastatic breast cancer cell lines, under both normoxia (21% O2) and hypoxia (0.1% O2). For comparative purposes, analysis was also carried out in the less-metastatic breast MCF-7 cancer cells. Under normoxia, oxidative phosphorylation (OxPhos) was significantly higher (2-times) in MDA-MB-468 than in MDA-MB-231 and MCF-7 whereas their glycolytic fluxes and OxPhos and glycolytic protein contents were all similar. TN cancer cell lines mainly depended on OxPhos (62-75%) whereas MCF-7 cells equally depended on both pathways for ATP supply. Hypoxia for 24 h promoted a significantly increase (>20 times) in the glycolytic transcriptional master factor HIF1-α, in its target proteins GLUT-1, HKI and II and LDH-A (2-4 times) as well as in the glycolytic flux (1.3-2 times) vs. normoxia in MDA-MB-468, MDA-MB-231 and MCF-7. On the contrary, hypoxia decreased (15-60%) the contents of COXIV, 2OGDH, ND1 and ATP synthase as well as OxPhos flux (50-75 %) correlating with a high mitophagy level in the three cell lines. Under hypoxia, the three cancer cell lines mainly depended on glycolysis (70-80%). Antimitochondrial drugs (oligomycin, casiopeina II-gly, methoxy-TEA) and celecoxib, at doses used to block OxPhos, significantly decreased TN cancer cell proliferation (IC50= 2-20 µM), migration capacity (10-90%) and invasiveness (25-65%). The present data support the use of mitochondrially targeted inhibitors for the treatment of TN breast carcinoma.

KEY WORDS: Breast cancer; celecoxib; glycolysis; energy metabolism drugs; oxidative phosphorylation; metastasis.


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1. INTRODUCTION

The lack of specific chemotherapeutic treatments for the triple negative (TN) breast cancer subtype, as compared to estrogen receptor (ER)- and human epidermal growth factor receptor-2 (HER2)-positive breast cancer subtypes, has become a wide-spread public health problem that requires further research.1-3 The current TN breast cancer treatments may be based on the combination of anthracyclines plus taxanes plus antimetabolites.4, 5 However, the overall survival rates are significantly low in comparison to ER-positive patients.6 Recently, novel adjuvant mono-therapies or combination therapies have been tested in TN patients including poly (ADP-ribose) polymerase (PARP) inhibitors, platinum based-drugs, monoclonal antibodies and tyrosine kinase inhibitors.6, 7 TN patients under these other treatment schemes have shown high response (85% effectiveness) compared to the rest of the cancer subtypes (40-70%) after the first 3 months.8 However, after longer treatment (9 months) tumor recurrence in 34% of patients has emerged.1 Therefore, to improve TN patient treatment schemes a better understanding of the illness is mandatory. In this regard, therapeutic strategies based on their metabolic properties have been proposed for deterring TN breast cancer growth and metastasis.9, 10 There are several evidences indicating that high mitochondrial activity correlates with metastatic progression.11 For instance, a higher mitochondrial membrane potential (∆ψm) has been determined in TN metastatic breast cancer cell lines (BT20, MDA-MB-468, MDAMB-231, MDA-MB-436) vs. ER and progesterone receptor (PR) positive breast cancer cell lines (BT474, MCF-7, T47D, ZR751).12



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Metastatic colon carcinoma also develops higher ∆ψm compared to non-metastatic colon cancer cells.13 It has been observed that high ∆ψm in colon cancer cells correlates with high levels of the canonical metastatic markers VEGF and MMP-7 as well as with enhanced invasiveness rate and chemoresistance.14 Thus, ∆ψm has been proposed as potential marker for an acquired metastatic phenotype.13 TN metastatic breast cancer biopsies exhibit elevated (20-times) levels of the key mitochondrial protein 2OGDH vs. HER2 and triple positive breast cancer biopsies.15 All these observations clearly suggest that OxPhos (the main mitochondrial function) is essential for metastatic phenotype progression. The use of non-steroidal anti-inflammatory drugs (NSAIDs) as anti-cancer drugs is widely documented for cancer-bearing patients (colorectal, mouth, esophagus, skin and breast cancers) reviewed in 16, 17 and cultured cancer cells (gastric cancer KATOIII, MKN28 and MKN45; colon cancer HT29, HCA7, RKO and SW620).18, 19 In addition to its canonical function blocking cyclooxygenase-2, NSAIDs also activate the apoptotic intrinsic pathway20 and perturb the mitochondrial function.21, 22 Therefore, to assess whether OxPhos is essential for metastasis, (i) a systematic analysis of the energy metabolism was performed in the TN breast cancer lines MDA-MB231 and MDA-MB-468 as well as ER-positive breast line MCF-7 exposed to normoxia, in order to identify the main ATP supplier. (ii) Several OxPhos and glycolytic inhibitors were tested on proliferation, migration and invasiveness of the three breast cancer cell lines. For comparative purposes, canonical anti-cancer drugs and NSAIDs were also assayed. (iii) The levels of mitochondrial and glycolytic proteins as well as pathway fluxes were also examined under hypoxia because it is the condition under which cells within solid tumors that are far away from blood vessels- develop their aggressive metastatic phenotypes.23 Metabolic studies of TN breast cancer cells under hypoxia have not been yet undertaken.


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(iv) The levels of the master transcriptional glycolytic regulator HIF-1α as well as mitophagy (mitochondrial digestion) onset regulators were also evaluated in an attempt to envision the underlying regulatory mechanisms of the metabolic re-programming for metastasis.

2. EXPERIMENTAL SECTION

Cancer cell culture Human metastatic triple negative breast (MDA-MB-231 and MDA-MB-468) cells, less metastatic ER-positive breast (MCF-7) cells 23-27, human normal epithelial breast MCF-10A cells and rodent 3T3 fibroblasts (American Type Culture Collection; Rockville, MD, USA) were cultured in Petri dishes in 20 mL Dulbecco MEM-media (Gibco, MA, USA) supplemented with 10% fetal bovine serum (Biowest, México) and 10,000 U penicillin /streptomycin (Sigma-Aldrich, MO, USA). The genotyping (INMEGEN, Mexico) of MCF-7 (13/14), MDA-MB-231 (14/14) and MDA-MB-468 (10/10) cells revealed that the three cell lines shared more than 90% of the canonic allelic markers with the ATCC original clones. The cells were incubated in 5% CO2/95% air at 37°C.28 After reaching 80–90% confluence, cells were kept under normoxia for 24 h until their use. To determine the effect of hypoxia on cellular proliferation, normoxic cancer cells (500 x103 cells) were grown in 5 mL DMEM in Petri dishes (60 x15 mm) for 120 h, and old medium of 80–90% confluent cells was replaced by fresh medium. Then, Petri dishes were placed back in the 5% CO2/95% air incubator or in a humidified hypoxia incubator chamber (Billups Rothenberg, CA, USA) under 0.1% O2, for additional 12 or 24 h at 37°C.29 Thereafter, normoxic and hypoxic cells were carefully removed with 0.25% trypsin/1 mM EDTA (Sigma-Aldrich, MO, USA) and washed with fresh KR buffer (Krebs-Ringer buffer



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contained: 125 mM NaCl, 5 mM KCl, 25 mM HEPES, 1.4 mM CaCl2, 1 mM KH2PO4, 1 mM MgCl2, pH 7.4) at 25°C. For these proliferation assays, cell viability (> 90%) was determined by using the trypan blue assay revealing less than 10% of cellular death.28

2.2.

Western blot

Both normoxic and hypoxic cells were re-suspended in 25 mM Tris-HCl buffer, pH 7.4 plus 1 mM EDTA, 5 mM DTT and 1 mM PMSF (Sigma-Aldrich, MO, USA). Samples were centrifuged at 2,500 rpm for 3.5 min and pellets were re-suspended in 25 mM HEPES, 0.4 M NaCl, 0.2 mM EDTA, 20% glycerol, pH 7.5; and further centrifuged at 10,000 rpm for 30 min. Once the protein concentration was determined by the Lowry method30, the supernatants were kept at −20°C until use.31 Samples (50 µg protein) were separated under reducing conditions by SDS- 10-12% polyacrylamide gel electrophoresis. The proteins were blotted to PVDF membranes (BioRad; Hercules, CA, USA) and Western blot analysis was performed by immunoblotting with antibodies from Santa Cruz Biotechnology (Cambridge, MA, USA): anti-α-tubulin (sc-5286), -HIF1-α (sc-13515), -GLUT1 (sc-1603), HKI (sc-46695), -HKII (sc-130358), -LDH (sc-130327), -2OGDH (sc-49589), -ND1 (sc65237), -COX-IV (sc-376731),-ATP synthase (sc-58619), -ANT (sc-11433), -vimentin (sc7557), E-cadherin (sc-8426), -MMP9 (sc-12759), -fibronectin (sc-8422), -DRAM (sc-81713), -Beclin (sc-48341),-BNIP3 (sc-56167), -Atg3 (sc-100508), -Atg7 (sc-376212), and -LAMP1 (sc-20011); and -GA (NBPZ-29940) antibody from Novus Biologicals (Littleton, CO, USA). All antibodies were used at final dilutions of 1:1000-1:2000. The hybridization bands were revealed with the corresponding secondary antibodies conjugated with horseradish peroxidase (Santa Cruz;CA, USA) and the ECL-plus detection system (Amersham; Buckinghamshire, UK). Densitometric analysis was performed using the Scion Image ACS Paragon Plus Environment

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Software (Scion Corp., Frederic, MD, USA) and normalized against α-tubulin, which corresponded to 100% intensity.

2.3.

OxPhos and glycolysis fluxes under normoxia and hypoxia

For glycolysis flux, cancer cells (2 mg protein/mL) were incubated in Krebs-Ringer buffer as described elsewhere.32 Glycolysis was started by adding 5 mM external glucose (SigmaAldrich, MO, USA) and cellular samples were collected after 0 and 10 min incubation at 37°C under smooth orbital shaking. At the indicated times, cells were rapidly mixed with 3% (w/v) cold perchloric acid and centrifuged. The supernatants were neutralized with 1N KOH/100 mM Tris. To rule out lactate production by glycogen degradation and glutaminolysis, cells were also incubated with 2-deoxyglucose (2-DG, 10 mM) or rotenone (5 µM), respectively (Sigma-Aldrich, MO, USA). Lactate was determined by a standard method with lactate dehydrogenase (Roche, Mannheim, Germany) following the NADH formation at 340 nm.33 For total oxygen consumption and OxPhos flux, cancer cells (2–5 mg protein/mL) were incubated at 37°C in air-saturated Krebs-Ringer medium plus 5 mM glucose. Under this near-physiological condition, the Crabtree effect is included in which external glucose exerts a slight inhibitory effect on cellular respiration.34 To distinguish between the oxygen consumption by mitochondria and non-mitochondrial sources,35,36 cells were incubated with 5 µM oligomycin (Sigma-Aldrich, MO, USA), a potent, specific and permeable inhibitor of the mitochondrial ATP synthase. The OxPhos rate (i.e., the rate of oligomycin-sensitive oxygen consumption) was determined by using a Clark-type electrode as previously described37 in a high-resolution respirometer (Oroboros Instruments, Innsbruck, Austria) at 37°C.



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The contribution of OxPhos and glycolysis to cellular ATP supply was determined, respectively, from the oligomycin-sensitive respiration rate multiplied by the ATP/O ratio that corresponds to 2.538; and from the rate of lactate production assuming a stoichiometry of 1 mol of ATP produced per 1 mol of lactate produced.

2.4.

Proliferation assay in the presence of metabolic inhibitors and canonical anti-cancer drugs All the canonical and metabolic inhibitors were dissolved in 70% ethanol/30% DMSO.

The maximal amount of ethanol/DMSO used was less than 10% of the final volume in the well and did not affect proliferation rate and cellular viability (>95%). For proliferation in the presence of drugs, cancer cells (MDA-MB-231, MDA-MB-468, MCF-7) and non-cancer cells (MCF-10A breast, 3T3 fibroblasts) were grown in 96-well plates at 2 x104 cells/well under normoxia (21% O2). After 24 h, glycolytic (2-deoxyglucose, iodoacetate, gossypol; SigmaAldrich, MO, USA); mitochondrial (CasII-gly, casiopeina-II gly; oligomicin; M-TEA, Methoxytocopheryl oxyacetic acid; α-TEA, α-tocopheryloxyacetic acid; α-TOS, α-tocopherol succinate), canonical (paclitaxel, doxorubicin, cisplatin; Sigma-Aldrich, MO, USA) or noncanonical (celecoxib, sulindac, simvastatin; Sigma-Aldrich, MO, USA) drugs were added and cells cultured for additional 24 h. CasII-gly was kindly donated by Dr. Lena Ruiz from Instituto de Química, UNAM-México. α-TEA, M-TEA and α-TOS were kindly donated by Dr. Emmanuel T. Akporiaye from Providence Portland Medical Center, Portland, OR, USA. Inhibitor concentrations used were 0.01, 0.1, 10, 100 µM and in some cases 1, 2 and 10 mM. Effect of inhibitors on cancer cell proliferation was determined by the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich, MO, USA) assay as it is described elsewhere.39 ACS Paragon Plus Environment

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2.5.

Therapeutic index ratio (TI)

TI values were determined by dividing the IC50 value (concentration of drug required to inhibit by 50% cellular growth) for non-cancer cells and the IC50 value for cancer cells.40-42 A TI > 3 indicates that the exposure to the drug results in no toxicity for normal cell but produces the desired effect in cancer cells.40-42

2.6.

Migration assays

MDA-MB-231 and MDA-MB-468 cells were grown in complete DMEM medium in multiwellplates (5 ×105 cells/well) under normoxia (21% O2). After reaching 80-90% confluence, the cell culture was wounded by using a plastic tip (wound healing assay), washed twice with 37°C PBS (155 mM NaCl, 1.5 mM KH2PO4 and 2.7 mM NaH2PO4, pH 7.2) buffer and incubated with fresh non-serum DMEM with either CasII-gly (0.01, 0.1, 1, 10 µM), oligomycin (0.01, 0.1, 1 or 10 µM), iodoacetate, doxorubicin or celecoxib (0.1, 1, 10, 100 µM). Images of the cellular migration were taken at 0 and 24 h with an inverted microscope (Zeiss; Thornwood, NY, USA). For each experiment, cellular migration distance from the border to the center of the Petri dish was measured with a graduated reticule (Zeiss; Thornwood, NY, USA).43 At the end of the migration process, attached cells (7-10x106 cells/mL; viability > 90%) were rinsed with saline buffer, gently collected and re-suspended in 25 mM Tris-HCl buffer, pH 7.4 plus 1 mM EDTA, 5 mM DTT and 1 mM PMSF (Sigma-Aldrich, MO, USA). The content of migration proteins was determined in 50 µg cell protein samples separated under reducing conditions by SDS- 10-12% polyacrylamide gel electrophoresis, further transferred to PVDF membranes and immunoblotted with the respective antibodies. ACS Paragon Plus Environment


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2.7.

Energy metabolism evaluation after the migration assay

After assessing cellular migration under normoxia (21% O2) in the presence of different drugs, TN cells were carefully detached from the Petri dishes and washed once with fresh KR buffer at room temperature. Both OxPhos and glycolysis fluxes were determined as described above.

2.8 Invasiveness assays MDA-MB-231 and MDA-MB-468 cells were incubated in free-serum DMEM in the absence or presence of CasII-gly, oligomycin (0.01, 0.1 and 1 µM), M-TEA, celecoxib, iodoacetate, or doxorubicin (0.1. 1 and 10 µM) for 24 h under normoxia (21% O2). Afterwards, cells were washed, re-suspended in free-serum DMEM medium and placed in the upper compartment of 96-multiwell Boyden chambers (Merck Millipore, MA, USA) at a final concentration of 5x104 cells/well and 37°C. Boyden chamber lower compartment was filled with free-serum DMEM. After 24 h, the number of cells collected in the lower chamber compartment was determined with 60 nM calcein-AM after 60 min incubation. Fluorescence was detected at 485 nm excitation and 520 nm emission in a microplate reader (NunclonTM, Roskilde, Denmark).23

2.9. Mitophagy evaluation by confocal microscopy under normoxia and hypoxia Normoxic cancer cells (3 x105 cells) were grown in glass bottom microwell 35-mm Petri dishes (MatTek; Ashland, MA, USA) in DMEM medium for 24 h and then the old medium was replaced by fresh medium. Cells were either returned to a normoxic incubator or ACS Paragon Plus Environment

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placed in a humidified hypoxia incubator chamber (Billups Rothenberg, CA, USA) under 0.1% O2, for additional 24 h at 37°C.29 To assess mitophagy (i.e., mitochondrial digestion by lysosomes), TN cells were incubated with MitoTracker Green (MTG, 0.5 µM) and LysoTracker Red (LTR, 0.5 µM) for 20 min in DMEM medium at 37°C, to label mitochondria and lysosomes, respectively. Cell fluorescence images were collected every 1 to 2 min with a Zeiss LSM 510 meta inverted laser scanning confocal microscope (Carl Zeiss; Oberkochen, Germany) using 63X oil 1.4 N.A. planapochromat objective lens. LTR λexcitation of 543 nm was provided by a helium/neon laser and λemission of 560 nm was used to collect the dye signal. MTG λexcitation of 488 nm was provided by an argon laser and λemission of 500550 nm was used to collect the dye signal. Laser excitation energy was attenuated 1000fold to minimize photobleaching and photodamage.44 The fluorescence signal of MTG- and LTR-loaded cells was analyzed with the J-Image software (NIH; Maryland, USA) and the MitoTracker/LysoTracker ratio was calculated for each sample (MDA-MB-231 and MDA-MB-468). Ten-fifteen different areas from each image (at least 35 cells) were used for statistical analysis.45-47

2.10. Data analysis Experiments were performed at least with three independent cell preparations (n).48 Data shown represent mean ± standard deviation (SD). Student´s t test and ANOVA/post hoc Scheffe49, 50 analyses with P values < 0.05 or lower were used to determine statistical significance.

3. RESULTS



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3.1. Triple negative breast cancer cell growth

Metastatic MDA-MB-231 and MDA-MB-468 cells showed a doubling time of 41 ± 3 h and 40 ± 2 h, respectively (Fig. 1) in normoxia, whereas less-metastatic MCF-7 cells exhibited a lower doubling time of 28 ± 5 h, indicating that MCF-7 cells have a significantly higher proliferation rate than TN breast cancer cells. The doubling times determined for the three cell lines were within the values reported by other groups.51, 52 Severe hypoxia arrested proliferation of the three cell lines, although high viabilities (>90%) were preserved (Fig. 1). The inhibitory effect of hypoxia on the metastatic and less- metastatic cell growth has also been widely described for other cancer cell lines.29, 53-55

3.2 Contents of HIF-1α and energy metabolism proteins Under normoxia, the glycolytic and OxPhos protein contents were similar among the three breast cancer cell lines (Figs. 2A and 2B). Also, the transcriptional factor HIF-1α level was negligible (Fig. 2A) as it has been reported for breast, cervix, and colorectal cancer cell lines in normoxia.29, 47, 56 On the contrary, the hypoxic condition allowed the HIF1-α stabilization (up to 20 times higher level vs. normoxia) in the three breast cancer cells (Fig. 2A). As expected, HIF1-α activation promoted significant increases (2-4 times) in its glycolytic protein targets57 GLUT1, HKI, HKII and LDH-A in the metastatic TN cells vs. normoxic conditions (Fig. 2A). Hypoxia also promoted decreased (40-70%) contents of mitochondrial proteins compared to normoxia, except for GA-L (glutaminase type-L) which remained unaltered (Fig. 2B).

3.3 Metabolic fluxes ACS Paragon Plus Environment

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Under normoxia, the OxPhos, glycolysis and glutaminolysis fluxes were similar between MDA-MB-231 and MCF-7 cells (Figs. 3A and 3B), whereas both total oxygen consumption and OxPhos were significantly higher (2.2 times) in MDA-MB-468 vs. MDAMB-231 (Fig. 3B). This last observation indicated that non-mitochondrial O2-consuming processes are greater in MDA-MB-468 cells. OxPhos was the principal ATP supplier (6275%) in both TN cells, whereas for MCF-7 cells both OxPhos and glycolysis equally contributed (50%) to the ATP supply (Fig. 3C). Severe hypoxia (0.1% atmospheric O2) for 24 h drastically impaired total oxygen consumption, OxPhos and mitochondrial membrane potential in the metastatic TN cells (Fig. 3B and Table 1), correlating with a strong suppression of the OxPhos proteins content (Fig. 2B). On the other hand, hypoxia also increased glycolysis (1.3-2 times) and glutaminolysis (1.7-2.6 folds) compared to normoxia in metastatic and less-metastatic cells (Fig. 3B). However, this glycolysis enhancement was more evident in MCF-7 and MDA-MB-231 than in MDA-MB-468. As a consequence of OxPhos impairment and glycolysis activation induced by hypoxia, the principal ATP supplier for the three cancer cell lines became glycolysis (70-80%) (Fig. 3C).

3.4 Mitophagy activation in TN cells exposed to severe hypoxia Lowering in the OxPhos protein contents induced by prolonged and severe hypoxia (0.1 % O2 for 24 h) correlated with a marked increase (3-25 times) in several autophagy proteins (DRAM, Beclin, LAMP1, Atg3) in MDA-MB-231 vs. normoxia (Fig. 4A). In contrast, hypoxia did not affect the contents of the autophagy proteins in MDA-MB-468 (except for a ACS Paragon Plus Environment


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decreased Beclin and increased BNIP3) and MCF-7 cells (except for DRAM, Beclin and LAMP1, which exhibited null or negligible levels). High autophagy protein contents in MDAMB-231 cells under hypoxia matched with a significant decrease in the mitochondria content (>50%) and significant increase in the lysosomes content (2-times) leading to mitochondrial digestion or mitophagy (Fig. 4B). Furthermore, mitophagy was apparent after 12 h under hypoxia (Fig. S1) and progressively increased after 24 h (Fig. 4B), indicating that hypoxia stimulated mitophagy as also reported for MDA-MB-157 (TN breast cancer) and leukemia human cells THP-1.58, 59 However, mitophagy was also clearly observed in MDA-MB-468 (Fig. 4B) and MCF-7 cells (Fig. S2) indicating that, independently of the metastatic potential, hypoxia activates mitophagy in breast cancer cells. Thus, a correlation between metastasis and mitophagy was not found either, whereas mitophagy activation did correlate with a decreased OxPhos flux.

3.5 Effect of energy-metabolism inhibitors on TN breast cancer cell proliferation, migration and invasiveness under normoxia Once the principal ATP supplier was identified in TN cells (Fig. 3C), mitochondrial, glycolytic and NSAIDs inhibitors were assayed on ATP-depending processes such as cellular proliferation (Table 2), migration (Fig. 5; Fig. S3) and invasiveness (Fig. 6), and compared to canonical anti-cancer drugs. The levels of the migration and invasiveness proteins markers (E-cadherin, vimentin) were similar to reported patterns. 60-62 As mitochondrial inhibitors, CasII-gly, oligomycin and vitamin E derivatives (α-TOS, α-TEA and M-TEA) were used. CasII-gly in the 1-10 µM range is a potent inhibitor of several Krebs cycle dehydrogenases63, 64 and has no apparent effect on non-tumor cell growth28 and DNA integrity;65 oligomycin is the classical, potent and highly specific inhibitor of the ATP ACS Paragon Plus Environment

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synthase and α-TOS, α-TEA and M-TEA are considered respiratory complex II inhibitors.66 To inhibit glycolysis, 2-deoxyglucose (2DG), iodoacetate (IAA) and gossypol (Goss) were used. These inhibitors mainly affect the activity of the glycolytic enzymes hexosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase. 67, 68 CasII-gly was the most potent drug to inhibit both TN and MCF-7 cell proliferation followed by the canonical anti-cancer drug doxorubicin (Table 2). Interestingly, the NSAID celecoxib also showed a potent inhibitory effect on the TN and MCF-7 cell growth (Table 2). Although CasII-gly and celecoxib at the doses used showed lower effects on breast MCF10A and 3T3 fibroblast proliferation (Table 2), their therapeutic index ratios of 3-4 and 4-100 (Table S1), respectively indicated that CasII-gly was more toxic than celecoxib for noncancer cells limiting its clinical use. Doxorubicin was more toxic to all the human cells lines (Tables 2 and S1) including the non-malignant cells, whereas simvastatin (IC50= 12 µM) also showed high potency against MDA-MB-231 cells but was also highly toxic for human cells (Table S1). IAA and cisplatin were more potent against MDA-MB-468 cells. On the other hand, α-TEA, α-TOS and paclitaxel were more potent against HR-positive MCF-7 than TN cells, MCF-10A cells or 3T3 fibroblasts. In order to establish the relationship between OxPhos and the ATP-dependent processes of metastatic cancer cells, celecoxib and the classical OxPhos inhibitor oligomycin were also assayed on TN cell migration and invasiveness. Although M-TEA and CasII-gly showed high toxicity against non-cancer MCF-10A cells and 3T3 fibroblasts (Table S1), these drugs were also assayed on TN cell migration and invasiveness because they significantly affected cancer cell proliferation at low doses. α-TEA and α-TOS were not assayed because their relatively high concentrations required to affect cancer cell



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proliferation. For comparison, the glycolytic inhibitor IAA and the canonical anti-cancer drug doxorubicin were also tested. CasII-gly (0.1-1 µM) was able to significantly decreased the content (15-90%) of several proteins associated with the metastatic progression vs. non treated cells (Figs. 5A and 5B) whereas 10 µM doxorubicin, IAA (except for E-cadherin) or celecoxib did not significantly affect them. Oligomycin significantly decreased migration (70 %) and invasiveness (25%) processes in both TN cells (Figs. 5, 6 and S2) at the same doses required to abolish OxPhos flux (Table 3). Thus, the sensitivity to oligomycin was migration > OxPhos > invasiveness. Similarly, celecoxib (10 µM) and CasII-gly (1 µM) decreased migration by 60-90% (Figs. 5C and S2), invasiveness by 25-50 % (Fig. 6) and OxPhos (flux and protein contents) by 20-90% in both TN cells. Celecoxib or CasII-gly affected the mitochondrial function without an apparent effect on glycolysis (Table 3, Figs. 7A and 7B) indicating a tight relationship between OxPhos and the metastasis process at the transcriptional/translational level. At 10 µM, IAA, doxorubicin and M-TEA were also able to decrease TN cellular migration (Fig. 5C) and invasiveness profiles (Fig. 6).

4. DISCUSSION

4.1. Limited cell growth of triple negative cells under normoxia Clear differences between proliferation rates of TN and HR-positive carcinoma cells were observed under normoxia, in agreement with other reports.51, 52 The lower growth rate observed in TN cells may be associated to (a) the low cyclin CDH1 content found in TN cells


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vs. MCF-769 or (b) the relative quiescence induced by the over-expression of several stemness-associated genes.70

4.2.

Dependence of triple negative breast cancer cells to mitochondrial ATP: normoxic condition The present study demonstrates that TN breast cancer cells are predominantly

oxidative under normoxic conditions; therefore, anti-mitochondrial therapy may result advantageous as an alternative treatment. However, our results contrasted those reported by Pelicano et al.,12 who determined that several TN breast cancer cell lines (BT20, MDAMB-468, MDA-MB-231 and MDA-MB-436) showed 2 times higher lactate production and lower (50-75%) mitochondrial respiration vs. ER and PR-positive breast cancer cell lines. These authors concluded that glycolysis predominates for the ATP supply under normoxic conditions in TN cancer cells. On this issue, it is worth clarifying that a high glycolytic rate does not ensure that glycolysis is the principal ATP supplier, which is a common assumption in the cancer biology field. Reviewed in 71, 72-74 To solidly conclude that TN cells depend on glycolysis, the ATP contribution from each energy pathway must be determined.73, 74 In addition, the glycolytic and OxPhos fluxes should be rigorously determined by using, respectively, a glycolytic inhibitor to discard glucogenolysis and glutaminolysis,47, 75 and oligomycin to discard cellular oxygen consumption non-linked to ATP synthesis.28, 29, 76 The intracellular ATP concentration determined by Pelicano et al.,12 was similar in both TN and MCF-7 cells, which was in contrast to other reports where ATP content is higher (30%) in TN cells vs. MCF-7 cells.77 Furthermore, the mitochondrial membrane potential determined by Pelicano et al.,12 was clearly greater (75%) in TN cells than in MCF-7 cells whereas the potency of glycolytic ACS Paragon Plus Environment


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inhibitors on cell growth was similar for both cancer types. These observations do not support their conclusion that TN breast cancer cells have a predominant glycolytic metabolism. In marked contrast, the proteomic, kinetomic and fluxomic data of TN cell energy metabolism presented here consistently showed that OxPhos but not glycolysis predominated for the ATP supply of TN cells under normoxic conditions. On the other hand, MDA-MB-468 cells showed greater OxPhos flux and lower glycolytic flux than MDA-MB-231 cells; others have also reported greater glycolysis in MDAMB-231 cells than in MDA-MB-468 cells.12, 78 Differences in energy metabolism between both TN cells cannot be related to the absence of hormone or Her2-receptors, but they could be associated with their tissue of origin (epidermal or mesenchymal) and the differential activation of signaling pathways of the cell cycle or epithelial mesenchymal transition.3

4.3.

Hypoxia arrests triple negative and ER-positive cell cancer growth The accelerated growth of solid tumors leads to episodes of hypoxia.79-83 Hence,

analyses of proliferation rate and energy metabolism in TN breast cancer cells were extended to this condition. Hypoxia impaired proliferation of both TN and MCF-7 cells, as also reported for different cancer cell lines (cervix HeLa, non-small cell lung H1299, prostate PC3, breast MDA-MB-231).29, 67-69, 84 The mechanisms associated with the cell cycle arrest induced by hypoxia may include: (a) apoptosis onset,85 although a high (>95%, data not shown) viability of the remaining hypoxic-resistant cells was observed which does not favor this explanation; (b) a decrease in the transcription of proliferation and cell cycle genes29 and/or increase in cell


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cycle HIF-1α-mediated inhibitors p21 and p27;86 and (c) a severe blocking of OxPhos29, 87 as also demonstrated here for TN and ER-positive carcinomas (cf. Fig. 3B).

4.4.

Dependence of triple negative breast cancer to glycolytic ATP: hypoxic condition OxPhos is severely impaired by hypoxia through well known mechanisms including

(a) decrease in the transcription and activities of several (PDH, 2OGDH, COXIV, ATPS) enzymes;29, 88-91 (b) enhancement in the mitochondrial ROS levels exerting harmful effects on mtDNA. As mtDNA encodes subunits of OxPhos enzymes,92 the oxidative stressinduced mtDNA damage may in turn deter OxPhos flux.29 (c) Mitophagy, which was activated by prolonged and severe hypoxia93 (0.1% O2) in the three assayed breast cancer cell lines (cf. Fig. 4, Fig. S1 and S2), and it has been described for the same cancer cell lines (MCF-7, MDA-MB-231) and others (ZR75 , HeLa),29, 47, 94 can also limit OxPhos. The lack of correlation between autophagy protein contents (Fig. 4A) and their function, i.e. mitophagy progression (Fig.4B) may be due to the presence of covalent regulation mechanisms reviewed in 95, 96 in Atgs (5, 3, 7, 9, 12), BNIP and beclin, which may modify their activity by phosphorylation, O-GlcNAcylation or ubiquitination.97, 98 Then, the use of protein levels as the only marker of a biological function should be interpreted with caution. Hypoxia favors glycolytic metabolism through the stabilization of HIF-1α, a transcriptional factor that regulates the transcription of genes involved in several survival processes such as angiogenesis, cell proliferation and erythropoiesis99 as well as glycolysis.57 Indeed, the enhanced glycolytic rates of TN and MCF-7 cells under hypoxia positively correlated with the substantial increased contents of the HIF-1α-targets GLUT-1 and HK-I and -II, the main tumor glycolysis controlling steps.32 In consequence, under hypoxia, glycolysis became the principal ATP supplier. ACS Paragon Plus Environment


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4.5.

Mitochondrial metabolism as target of breast cancer triple negative-subtype under normoxia Clinical combination treatment of breast cancer with tamoxifen and/or trastuzumab

plus chemotherapy canonical drugs (taxol, cisplatin) is currently used. However, the success rate of these treatments is poor particularly for metastatic breast cancer.100 Targeting signal pathways should take into account that they have high plasticity which facilitates the surge of compensatory mechanisms for survival.101, 102 Therefore, other more suitable targets should be proposed and tested. In the present study, TN cell energy metabolism is proposed as drug target because ATP supply is essential for any cell function and particularly for proliferation, migration and invasiveness. Thus, the identification of the principal ATP supplier appears relevant for developing specific anti-TN therapy. Certainly, inhibition of tumor energy metabolism may also affect healthy cells with high proliferative rates.65, 103 However, growth of non-cancer cells as well as tumor glycolysis has shown to be fairly resistant to low micromolar doses of mitochondrially-targeted drugs such as CasII-gly and vitamin E derivatives.28, 65, 77, 103 In addition, there is a loss of growth, metastasis and invasiveness potential in tumor cells o

lacking mitochondria (ρ cells)104, 105 as well as in metastatic cells cultured in the presence of the ATP synthase inhibitor oligomycin (Figs. 5C, 6 and S2).11 Then, glycolytic inhibitors (2DG, gossypol) were not able to diminish TN cell growth (Table 2; see also12). Unfortunately, oligomycin as well as other mitochondrial respiratory chain inhibitors severely affect normal cells (see Table S1).106 However, tumor cells and mitochondria develop biochemical properties for which drugs can be designed to specifically target OxPhos and mitochondria. For instance, tumor mitochondria may have higher inner ACS Paragon Plus Environment

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membrane electrical potential (up to 180 mV, negative inside), favoring the accumulation of lipophilic cations reviewed in 107 such as CasII-gly. Once inside the mitochondrial matrix, CasIIgly may block the mitochondrial function by inhibiting pyruvate-, 2OG- and succinatedehydrogenases at low micromolar concentrations.63 CasII-gly did block proliferation and OxPhos of TN cells at doses similar to those used against other oxidative cancer cell lines.108-110 However, CasII-gly may also affect non-cancer cells with apparent moderate potency (Table S1). In turn, vitamin E-derivatives such as α-TOS show greater potency for cancer cells because non-cancer cells presumably have higher esterase activity than cancer cells releasing vitamin E from its succinyl moiety whereas cancer cells preserve longer the prooxidant α-TOS.109 Vitamin E derivatives with ether bond instead of ester bond such as aTEA and M-TEA cannot be hydrolyzed by esterases and remain intact. Both the succinyl and α-tocopheryl moieties interact with succinate dehydrogenase inhibiting its activity and inducing OxPhos depression and increased ROS production.66, 111 Due to their lower antioxidant capacity,112 cancer cells become more susceptible to the enhanced oxidative stress. Furthermore, the more acidic microenvironment generated by cancer cells favors the accumulation of vitamin E-derivatives in tumors; some vitamin E-derivatives may also have a positively charged moiety further increasing their accumulation into cancer mitochondria.109 Interestingly, celecoxib a third generation NSAID also showed high potency for blocking TN cell proliferation, migration and invasiveness with no apparent effect on noncancer cells (c.f. Table S1). Potent deleterious effects by celecoxib on gastric and colorectal cancers have also been documented.18, 19 These celecoxib effects seem not to be associated to cyclooxygenase 2 inhibition but rather to its interaction with mitochondria



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acting as OxPhos uncoupler and respiratory chain inhibitor20, 21 prompting ROS production.22, 113

4.6.

Mitochondrial function is essential for TN cell migration and invasiveness The proposal that TN cells are highly dependent on the mitochondrial function under

normoxia (i.e., OxPhos contribution to cell ATP supply was higher than 50%) was further supported by the marked effect of the classical mitochondrial inhibitor oligomycin and the potent mitochondrial blockers CasII-gly, M-TEA, and celecoxib on cell migration and invasiveness. Similarly, melanoma and lung cancer cell migration and invasiveness have been successfully blocked with the mitochondrial inhibitors rotenone and dequalinium chloride114, 115 although these two drugs also severely affect normal cells.116, 117 These results clearly indicated (i) a tight relationship between OxPhos and the metastasis process at the transcriptional/translational level and (ii) functional mitochondria are needed for TN metastatic progression (Figs. 5C and Fig. 6). In contrast, only high concentrations of glycolytic inhibitors (>0.6 mM 2DG) affect the migration rate (25-50%) and invasiveness of the metastatic osteosarcoma cell lines DLM8-luc-M1 (murine), SJSA1 (human) and Abrams (canine); and the murine (4T1) and human (MDA-MB-231) mammary adenocarcinoma cells.118 However, a confounding observation was that IAA, a presumed glycolytic inhibitor, showed similar blocking potency on TN cell migration and invasiveness. Then, IAA an acylating agent may also affect mitochondrial metabolism of TN cells after 24 h incubation or TN cell migration and invasiveness depend on both energy pathways. Clearly this relevant question deserves further studies by using in vivo models of metastasis.

4.7.

Celecoxib as potential anticancer drug against ER-positive and TN cells ACS Paragon Plus Environment

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The observation that the energy profile found in TN cells is similar to that observed for ER-positive MCF-7 cells indicated that mitochondria predominate for the energy supply independently of the presence or absence of the ER receptor, i.e., the signaling pathways associated to estrogen receptor activation are not associated with the transcription factors regulating energy metabolism in breast cancer cells. This observation takes relevance because in some cases where conventional treatment against ER-positive breast cancer (i.e., tamixofen) is ineffective,119 anti-mitochondrial therapy emerges as potential alternative treatment. For HER2-positive breast cancer cells (BT-474), Pelicano et. al.,12 found a high glycolytic rate and low mitochondrial function vs. ER-positive breast cancer cells. This observation suggested that the Her2-receptor, but not the ER-receptor, could be involved in the regulation of energy metabolism. As mitochondrial metabolism may become a suitable drug target for blocking TN cell growth and metastasis, the interest in using experimental drugs and in re-purposing conventional drugs such as celecoxib has increased in recent years. Among the different drugs tested, CasII-gly showed the greatest inhibitory potency on TN cell proliferation, migration and invasiveness; however, this drug is still in the pre-clinical experimental phase and has not yet been approved for clinical trials. On the contrary, celecoxib is commonly used as analgesic and anti-inflammatory for rheumatic diseases, and hence it could undergo a faster process to be used in clinical trials for colon cancer patients16 and perhaps for other cancer patients.

CONFLICT OF INTEREST



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The authors declare no potential conflicts of interest. AUTHOR´S CONTRIBUTIONS. SRE and SCPV: conception and design; development of methodology; acquisition of data; analysis, interpretation of data and revision of manuscript. SCPV, IHR, JCGP, and DXRC: experimental design; development of methodology, statistical analysis and manuscript proof-reading. RMS and SRE: study supervision; manuscript writing; manuscript revision; grant funding procurement. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS The present work was partially supported by grants from CONACyT-México to SRE (283144), JCGP (243249) and RMS (239930 and 281428). Authors thank Dr. Ambar López Macay from Laboratorio de Enfermedades Neuromusculares, Instituto Nacional de Rehabilitación, México for technical assistance in the immunofluorescence assays. SUPPORTING INFORMATION S1. Mitophagy activation in TN breast cancer cells exposed to hypoxia (0.1% O2) by 12 h. S2. Mitophagy activation in MCF7 breast cancer cells exposed to hypoxia (0.1% O2) by 24 h. S3 Effect of energy metabolism inhibitors on migration rate of TN breast cancer cells.


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FIGURE LEGENDS Figure 1. Growth of MDA-MB-231 (■), MDA-MB-468 (●) and MCF7 (▲) cells in normoxia (full symbols) and hypoxia (Hyp, open symbols). Cells (1x 105 cells/mL) were cultured in DMEM medium at 37°C, 21% O2 and 5% CO2. After 120 h (black arrow), cells were exposed to hypoxia (0.1% O2), or they continued under normoxia, for 24 h more. Cellular viability for normoxia and hypoxia (Hyp) was 90-95%. Data shown represent the mean ± S.D. of at least 3 different preparations. Doubling time was determined with the following formula:

Where NF represents the number of cultured cells at the end of the exponential growth phase, NI represents the number of cells at the beginning of the growth curve, tF is the time at which cells were harvested and tI is the initial culture time according to McAtter and Davis.120 *P

Energy metabolism drugs block triple negative ... - ACS Publications

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