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• W2761360410 abstract "•MYC and MCL1 increase cancer stem cells in chemotherapy-resistant TNBC•MYC and MCL1 cooperatively promote mtOXPHOS, which in turn induces HIF-1α•MCL1 induces CSCs independent of its BH3-dependent, anti-apoptotic function•Inhibition of HIF1-α abolishes CSC enrichment in chemotherapy-resistant TNBC Most patients with advanced triple-negative breast cancer (TNBC) develop drug resistance. MYC and MCL1 are frequently co-amplified in drug-resistant TNBC after neoadjuvant chemotherapy. Herein, we demonstrate that MYC and MCL1 cooperate in the maintenance of chemotherapy-resistant cancer stem cells (CSCs) in TNBC. MYC and MCL1 increased mitochondrial oxidative phosphorylation (mtOXPHOS) and the generation of reactive oxygen species (ROS), processes involved in maintenance of CSCs. A mutant of MCL1 that cannot localize in mitochondria reduced mtOXPHOS, ROS levels, and drug-resistant CSCs without affecting the anti-apoptotic function of MCL1. Increased levels of ROS, a by-product of activated mtOXPHOS, led to the accumulation of HIF-1α. Pharmacological inhibition of HIF-1α attenuated CSC enrichment and tumor initiation in vivo. These data suggest that (1) MYC and MCL1 confer resistance to chemotherapy by expanding CSCs via mtOXPHOS and (2) targeting mitochondrial respiration and HIF-1α may reverse chemotherapy resistance in TNBC. Most patients with advanced triple-negative breast cancer (TNBC) develop drug resistance. MYC and MCL1 are frequently co-amplified in drug-resistant TNBC after neoadjuvant chemotherapy. Herein, we demonstrate that MYC and MCL1 cooperate in the maintenance of chemotherapy-resistant cancer stem cells (CSCs) in TNBC. MYC and MCL1 increased mitochondrial oxidative phosphorylation (mtOXPHOS) and the generation of reactive oxygen species (ROS), processes involved in maintenance of CSCs. A mutant of MCL1 that cannot localize in mitochondria reduced mtOXPHOS, ROS levels, and drug-resistant CSCs without affecting the anti-apoptotic function of MCL1. Increased levels of ROS, a by-product of activated mtOXPHOS, led to the accumulation of HIF-1α. Pharmacological inhibition of HIF-1α attenuated CSC enrichment and tumor initiation in vivo. These data suggest that (1) MYC and MCL1 confer resistance to chemotherapy by expanding CSCs via mtOXPHOS and (2) targeting mitochondrial respiration and HIF-1α may reverse chemotherapy resistance in TNBC. Triple negative breast cancer (TNBC) comprises ∼15% of all invasive breast cancers. TNBC is a subtype lacking expression of the estrogen receptor (ER), progesterone receptor (PR), and ERBB2 amplification (Carey et al., 2010Carey L. Winer E. Viale G. Cameron D. Gianni L. Triple-negative breast cancer: disease entity or title of convenience?.Nat. Rev. Clin. Oncol. 2010; 7: 683-692Crossref PubMed Scopus (634) Google Scholar). Due to the lack of known targetable molecular drivers in TNBC, cytotoxic chemotherapy is widely used in these patients. Many patients with TNBC develop resistance and relapse after adjuvant chemotherapy, ultimately succumbing to metastatic disease (Liedtke et al., 2008Liedtke C. Mazouni C. Hess K.R. André F. Tordai A. Mejia J.A. Symmans W.F. Gonzalez-Angulo A.M. Hennessy B. Green M. et al.Response to neoadjuvant therapy and long-term survival in patients with triple-negative breast cancer.J. Clin. Oncol. 2008; 26: 1275-1281Crossref PubMed Scopus (2083) Google Scholar, Yu et al., 2013Yu K.D. Zhu R. Zhan M. Rodriguez A.A. Yang W. Wong S. Makris A. Lehmann B.D. Chen X. Mayer I. et al.Identification of prognosis-relevant subgroups in patients with chemoresistant triple-negative breast cancer.Clin. Cancer Res. 2013; 19: 2723-2733Crossref PubMed Scopus (119) Google Scholar). Previous studies have proposed that a rare population of cancer cells, referred to as cancer stem-like cells (CSCs) or tumor-initiating cells (TICs), exhibit self-renewal capabilities and resistance to chemotherapy (Beck and Blanpain, 2013Beck B. Blanpain C. Unravelling cancer stem cell potential.Nat. Rev. Cancer. 2013; 13: 727-738Crossref PubMed Scopus (601) Google Scholar). This property of CSCs contributes to colonization of cancer cells at distant metastatic sites despite adjuvant chemotherapy (Clevers, 2011Clevers H. The cancer stem cell: premises, promises and challenges.Nat. Med. 2011; 17: 313-319Crossref PubMed Scopus (1472) Google Scholar). Consistent with this notion, patients with TNBC whose tumors express CSC markers exhibit a worse outcome (Yu et al., 2013Yu K.D. Zhu R. Zhan M. Rodriguez A.A. Yang W. Wong S. Makris A. Lehmann B.D. Chen X. Mayer I. et al.Identification of prognosis-relevant subgroups in patients with chemoresistant triple-negative breast cancer.Clin. Cancer Res. 2013; 19: 2723-2733Crossref PubMed Scopus (119) Google Scholar). In a previous study, we demonstrated that TNBCs remaining in the breast following neoadjuvant chemotherapy (NAC) harbor amplification of MYC (54%) and MCL1 (35%) (Balko et al., 2014Balko J.M. Giltnane J.M. Wang K. Schwarz L.J. Young C.D. Cook R.S. Owens P. Sanders M.E. Kuba M.G. Sánchez V. et al.Molecular profiling of the residual disease of triple-negative breast cancers after neoadjuvant chemotherapy identifies actionable therapeutic targets.Cancer Discov. 2014; 4: 232-245Crossref PubMed Scopus (330) Google Scholar). In that study, 83% of MYC-amplified tumors exhibited co-amplification of MCL1, suggesting that MYC and MCL1 may cooperatively contribute to chemotherapy resistance in TNBC. MYC is a proto-oncogene that encodes a transcription factor associated with cancer cell-cycle progression, proliferation, apoptosis, and biosynthesis (Dang, 2012Dang C.V. MYC on the path to cancer.Cell. 2012; 149: 22-35Abstract Full Text Full Text PDF PubMed Scopus (2106) Google Scholar, Li et al., 2005Li F. Wang Y. Zeller K.I. Potter J.J. Wonsey D.R. O’Donnell K.A. Kim J.W. Yustein J.T. Lee L.A. Dang C.V. Myc stimulates nuclearly encoded mitochondrial genes and mitochondrial biogenesis.Mol. Cell. Biol. 2005; 25: 6225-6234Crossref PubMed Scopus (443) Google Scholar). Myeloid cell leukemia-1 (MCL1) is an anti-apoptotic Bcl-2 family protein that prevents apoptosis by suppressing cytochrome c release through association with pro-apoptotic Bcl-2 family proteins such as BID, BIM, PUMA, and NOXA (Chen et al., 2005Chen L. Willis S.N. Wei A. Smith B.J. Fletcher J.I. Hinds M.G. Colman P.M. Day C.L. Adams J.M. Huang D.C. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function.Mol. Cell. 2005; 17: 393-403Abstract Full Text Full Text PDF PubMed Scopus (1515) Google Scholar, Opferman et al., 2003Opferman J.T. Letai A. Beard C. Sorcinelli M.D. Ong C.C. Korsmeyer S.J. Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1.Nature. 2003; 426: 671-676Crossref PubMed Scopus (674) Google Scholar, Shimazu et al., 2007Shimazu T. Degenhardt K. Nur-E-Kamal A. Zhang J. Yoshida T. Zhang Y. Mathew R. White E. Inouye M. NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition.Genes Dev. 2007; 21: 929-941Crossref PubMed Scopus (113) Google Scholar). Herein we show that MYC and MCL1 are overexpressed in TNBCs after chemotherapy and also in claudin-low TNBC cell lines where they contribute to tumor initiation and maintenance of CSCs. We also show that breast CSCs predominantly relied on mitochondrial oxidative phosphorylation (mtOXPHOS), whose activation is enhanced by both MYC and MCL1. This revealed a possible mechanism by which MYC and MCL1 promote CSC enrichment. Further, MYC- and MCL1-induced mtOXPHOS led to elevated production of reactive oxygen species (ROS), which in turn induced HIF-1α expression. Finally, knockdown of HIF-1α and use of a HIF-1α inhibitor, each in combination with anti-cancer chemotherapy, markedly reduced drug-resistant CSCs, suggesting a novel therapeutic strategy for patients with this subtype of breast cancer. We first performed targeted-capture next-generation sequencing (NGS) on tumors from a small cohort of patients with TNBC treated with NAC. In nine patients, tumor was available from the diagnostic pre-treatment biopsy, post-NAC mastectomy specimen, and a recurrent metastasis. In nine additional patients, tumor was available from at least two of these sequential biopsies. In all tumors, a mutation in TP53 was detected. Overall, 8 of 18 (44%) cancers exhibited MYC and MCL1 co-amplification in at least one of the serial biopsies. MYC and MCL1 were co-amplified in 4 of 18 (22%) primary untreated tumors, 4 of 18 (22%) post-NAC mastectomies, and 6 of 18 (33%) metastatic recurrences. Within the cohort with all three serial biopsies, 3 of 4 tumors with both genes amplified in the metastasis also contained the co-amplification in the original diagnostic biopsy. Overall, 17 of 18 (94%) TNBCs exhibited MYC and/or MCL1 amplification in at least one of the serial biopsies (Figure 1A). These data are consistent with and extend a previous report of ours (Balko et al., 2014Balko J.M. Giltnane J.M. Wang K. Schwarz L.J. Young C.D. Cook R.S. Owens P. Sanders M.E. Kuba M.G. Sánchez V. et al.Molecular profiling of the residual disease of triple-negative breast cancers after neoadjuvant chemotherapy identifies actionable therapeutic targets.Cancer Discov. 2014; 4: 232-245Crossref PubMed Scopus (330) Google Scholar) and further suggest an association of MYC and MCL1 co-amplification with drug-resistant TNBCs with a poor outcome as well as a higher frequency of each alteration than that reported by The Cancer Genome Atlas (TCGA; MYC: 14 of 18 [77.7%] versus 44% in TCGA, p < 0.0001; MCL1: 12 of 18 [66.6%] versus 24% in TCGA, p < 0.0001; both Fisher’s exact test). A CSC phenotype is a known feature of many chemotherapy-resistant tumors (Beck and Blanpain, 2013Beck B. Blanpain C. Unravelling cancer stem cell potential.Nat. Rev. Cancer. 2013; 13: 727-738Crossref PubMed Scopus (601) Google Scholar). MDA-MB-436 and SUM159PT cells grown as mammospheres were overall less sensitive to chemotherapeutics, including paclitaxel, docetaxel, and doxorubicin, compared to those grown in adherent conditions, supporting an association of cells with tumor-initiating, stem-like capacity with drug tolerance and potential clinical resistance to anticancer chemotherapy (Figure S1A). Thus, we evaluated MYC and MCL1 protein levels in CSCs from TNBC cell lines sorted by aldehyde dehydrogenase (ALDH) activity, a typical marker of TICs (Croker et al., 2009Croker A.K. Goodale D. Chu J. Postenka C. Hedley B.D. Hess D.A. Allan A.L. High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability.J. Cell. Mol. Med. 2009; 13: 2236-2252Crossref PubMed Scopus (391) Google Scholar). ALDH+ MDA-MB-436 and SUM159PT cells displayed enhanced mammosphere formation (Figure S1B) and higher MYC and MCL1 expression compared to ALDH− cells (Figure 1B). MYC and MCL1 expression were also increased in TNBC cells grown as mammospheres compared to adherent cells (Figure 1C). A CSC-derived gene expression signature is a feature of claudin-low TNBCs that persist following chemotherapy (Creighton et al., 2009Creighton C.J. Li X. Landis M. Dixon J.M. Neumeister V.M. Sjolund A. Rimm D.L. Wong H. Rodriguez A. Herschkowitz J.I. et al.Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features.Proc. Natl. Acad. Sci. USA. 2009; 106: 13820-13825Crossref PubMed Scopus (1103) Google Scholar, Hennessy et al., 2009Hennessy B.T. Gonzalez-Angulo A.M. Stemke-Hale K. Gilcrease M.Z. Krishnamurthy S. Lee J.S. Fridlyand J. Sahin A. Agarwal R. Joy C. et al.Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics.Cancer Res. 2009; 69: 4116-4124Crossref PubMed Scopus (687) Google Scholar). Hence, we next compared MYC and MCL1 protein levels between claudin-low and non-claudin-low TNBC subgroups (Figure S1C). Claudin-low TNBC cell lines exhibited higher expression of MYC and MCL1 compared to non-claudin-low TNBC lines (Figure 1D). Breast CSCs are also marked by CD44high/CD24low (Al-Hajj et al., 2003Al-Hajj M. Wicha M.S. Benito-Hernandez A. Morrison S.J. Clarke M.F. Prospective identification of tumorigenic breast cancer cells.Proc. Natl. Acad. Sci. USA. 2003; 100: 3983-3988Crossref PubMed Scopus (8404) Google Scholar) and CD49+/EpCAM− (Visvader, 2009Visvader J.E. Keeping abreast of the mammary epithelial hierarchy and breast tumorigenesis.Genes Dev. 2009; 23: 2563-2577Crossref PubMed Scopus (425) Google Scholar) expression. The ratio of mRNA levels of CD44 to CD24 and CD49 to EpCAM (Neve et al., 2006Neve R.M. Chin K. Fridlyand J. Yeh J. Baehner F.L. Fevr T. Clark L. Bayani N. Coppe J.P. Tong F. et al.A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes.Cancer Cell. 2006; 10: 515-527Abstract Full Text Full Text PDF PubMed Scopus (2386) Google Scholar) also correlated with expression of MYC and MCL1 in TNBC cell lines (Figures 1E and S1D). In a previous report, we quantified expression of genes associated with chemotherapy resistance in matched biopsies from TNBCs before and after NAC using NanoString nCounter analysis (Bhola et al., 2013Bhola N.E. Balko J.M. Dugger T.C. Kuba M.G. Sánchez V. Sanders M. Stanford J. Cook R.S. Arteaga C.L. TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer.J. Clin. Invest. 2013; 123: 1348-1358Crossref PubMed Scopus (429) Google Scholar). Analysis of these data showed that MYC mRNA expression (Figure 1F) and MCL1 protein levels (Figure 1G) were statistically higher in TNBCs after NAC compared to before treatment. In line with these data from primary tumors, MYC and MCL1 protein levels were upregulated in SUM159PT and MDA-MB-436 cells made resistant to paclitaxel (PCTR) (Figures 1H and S1E). The PCTR cells exhibited enhanced mammosphere forming ability (Figure S1F) and an increased proportion of ALDH+ cells (Figure S1G), also suggesting chemotherapy expands drug-tolerant CSCs. Finally, following treatment with paclitaxel, ALDH activity was higher in live MDA-MB-436 cells than in apoptotic cells sorted by flow cytometry (Figure S1H). These results suggest an association between high expression of MYC and MCL1 with chemo-resistant CSCs. To investigate the role of MYC and MCL1 on maintenance of CSCs, we knocked down MYC or MCL1 using siRNAs in MDA-MB-436 and SUM159PT cells, which express high levels of both MYC and MCL1 (Figure S2A). Conversely, we stably expressed MCL1 or GFP (control) constructs or doxycycline (DOX)-inducible MYC in MDA-MB-468 cells, which express low endogenous levels of MYC and MCL1 (Figure S2B). Knockdown of MYC or MCL1 attenuated mammosphere formation (Figure 2A) and reduced the CD44hi/CD24low and ALDH+ fraction in SUM159PT and MDA-MB-436 cells (Figure 2B), respectively. Conversely, DOX-mediated induction of MYC or transduction of MCL1 induced mammosphere formation and increased the proportion of CD44hi/CD24low MDA-MB-468 cells (Figures 2C and 2D). To assess the tumorigenic potential of MYC and MCL1, we injected SUM159PT cells stably transduced with shRNAs (non-targeting shRNA control [shCont] or shMYC or shMCL1, Figure S2C) in limiting dilutions into athymic mice and monitored them for tumor formation for 8 weeks. Both shMYC and shMCL1 significantly reduced tumor initiation in vivo (Figures 2E and 2F). These results suggest that MYC and MCL1 contribute to CSC enrichment and tumor-initiating capacity in TNBC. Recent evidence suggests that functional mitochondria are crucial for the maintenance of CSCs in several tumor types (Lamb et al., 2014Lamb R. Harrison H. Hulit J. Smith D.L. Lisanti M.P. Sotgia F. Mitochondria as new therapeutic targets for eradicating cancer stem cells: Quantitative proteomics and functional validation via MCT1/2 inhibition.Oncotarget. 2014; 5: 11029-11037Crossref PubMed Scopus (140) Google Scholar). Dormant pancreatic tumor cells, which exhibit CSC features, rely on mtOXPHOS for survival after oncogene ablation (Viale et al., 2014Viale A. Pettazzoni P. Lyssiotis C.A. Ying H. Sánchez N. Marchesini M. Carugo A. Green T. Seth S. Giuliani V. et al.Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function.Nature. 2014; 514: 628-632Crossref PubMed Scopus (787) Google Scholar). Inhibition of mitochondrial function by metformin eliminates pancreatic CSCs (Sancho et al., 2015Sancho P. Burgos-Ramos E. Tavera A. Bou Kheir T. Jagust P. Schoenhals M. Barneda D. Sellers K. Campos-Olivas R. Graña O. et al.MYC/PGC-1α Balance Determines the Metabolic Phenotype and Plasticity of Pancreatic Cancer Stem Cells.Cell Metab. 2015; 22: 590-605Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). However, evidence for a mitochondrial role in breast CSCs is inconclusive. Thus, we examined the mitochondrial respiratory capacity of breast CSCs. In SUM159PT and MDA-MB-436 cells, the basal and maximal respiratory capacities of their CSC fraction (ALDH+ and cells grown as mammospheres) were higher than in ALDH− and adherent cells, suggesting activated mtOXPHOS (Figure 3A). Consistent with this result, mitochondrial membrane potentials (mtMPs) were also upregulated in SUM159PT and MDA-MB-436 CSCs (Figure 3B). Next, we measured levels of ROS, which can be elevated through activation of the electron transport chain (ETC) in mitochondria. Hydrogen peroxide (H2O2) and mitochondrial superoxide levels were elevated in SUM159PT and MDA-MB-436 CSCs compared to non-CSCs, in line with a hyperactive mtOXPHOS (Figures 3C and 3D). SUM159PT and MDA-MB-436 cells were then sorted for high versus low mtMPs by the intensity of MitoTracker Red CMXRos and plated as mammospheres. Mammosphere formation was markedly higher in cells with high mtMPs compared to low mtMPs (Figure 3E). Paclitaxel-resistant SUM159PT cells, which are enriched with CSCs (Figures S1F and S1G), also exhibited elevated mtOXPHOS capacity, mtMPs, ROS levels, and mitochondrial superoxide levels compared to parental drug-sensitive cells (Figures S3A–S3D). A previous report showed that targeting mitochondria with oligomycin A, an ATP synthase inhibitor, can attenuate mammosphere formation by pancreatic cancer cells (Viale et al., 2014Viale A. Pettazzoni P. Lyssiotis C.A. Ying H. Sánchez N. Marchesini M. Carugo A. Green T. Seth S. Giuliani V. et al.Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function.Nature. 2014; 514: 628-632Crossref PubMed Scopus (787) Google Scholar). Treatment of TNBCs with oligomycin A reduced the oxygen consumption rate (OCR) and mammosphere formation by SUM159PT and MDA-MB-436 cells (Figures S3E and S3F). Similarly, metformin, a mitochondrial complex I inhibitor, reduced mammosphere formation and limited tumor initiation (Figures S3G and S7L). Moreover, PCTR cells and SUM159PT cells grown as mammospheres exhibited a statistically higher inhibition of OCR when treated with etomoxir, an inhibitor of fatty acid oxidation, compared to cells treated with BPTES and UK5099, suggesting a higher bioenergetic reliance on fatty acid oxidation in TNBC CSCs (Figures S3H and S3I). These results suggest that TNBC CSCs exhibit hyperactive mtOXPHOS, which in turn maintains their self-renewal capacity. Previous studies have shown that MYC directly regulates expression of genes involved in mitochondrial biogenesis (Li et al., 2005Li F. Wang Y. Zeller K.I. Potter J.J. Wonsey D.R. O’Donnell K.A. Kim J.W. Yustein J.T. Lee L.A. Dang C.V. Myc stimulates nuclearly encoded mitochondrial genes and mitochondrial biogenesis.Mol. Cell. Biol. 2005; 25: 6225-6234Crossref PubMed Scopus (443) Google Scholar) and affects metabolic processes required for mitochondrial respiration (Stine et al., 2015Stine Z.E. Walton Z.E. Altman B.J. Hsieh A.L. Dang C.V. MYC, Metabolism, and Cancer.Cancer Discov. 2015; 5: 1024-1039Crossref PubMed Scopus (715) Google Scholar). To examine whether MYC contributes to mtOXPHOS in TNBC, we determined the OCR in MDA-MB-468 cells stably expressing DOX-inducible MYC. Induction of MYC by DOX increased basal and maximal mitochondrial respiratory capacities in MDA-MB-468 cells (Figure 4A, left). Conversely, knockdown of MYC by siRNA reduced mitochondrial respiratory capacities in MDA-MB-436 and SUM159PT cells (Figure 4A, middle and right). Ablation of MYC with siRNA also inhibited mitochondrial biogenesis in MDA-MB-468 cells as assessed by the number of mitochondria measured by transmission electron microscopy (TEM; Figure 4B). Both the mitochondrial DNA content and mass were also reduced upon MYC knockdown in MDA-MB-436 and SUM159PT cells. Conversely, expression of DOX-inducible MYC increased the mitochondrial DNA content and mass in MDA-MB-468 cells (Figures 4C and 4D). Further, induction of MYC upregulated ROS production in TNBC cells (Figure 4E). Finally, the inhibitory effect of oligomycin A on mammosphere formation was much more potent in MDA-MB-468 cells expressing DOX-inducible MYC compared to uninduced cells, suggesting that a MYC-dependent tumor-initiating capacity relies on mtOXPHOS (Figure 4F). These results imply that MYC potentiates mtOXPHOS and the subsequent enrichment of CSCs through its role in mitochondrial biogenesis. Despite the established anti-apoptotic role of MCL1, knockdown of MCL1 with siRNA did not induce apoptosis in MDA-MB-436 and SUM159PT cells (Figure S4A). Thus, we speculated that the anti-CSC effect of MCL1 knockdown (Figure 2) might be due to a mechanism independent of its anti-apoptotic function. When associated with the outer mitochondrial membrane (OMM), MCL1 serves as an anti-apoptotic protein, whereas inside the mitochondrial matrix, MCL1 stimulates mitochondrial respiration (Perciavalle et al., 2012Perciavalle R.M. Stewart D.P. Koss B. Lynch J. Milasta S. Bathina M. Temirov J. Cleland M.M. Pelletier S. Schuetz J.D. et al.Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration.Nat. Cell Biol. 2012; 14: 575-583Crossref PubMed Scopus (295) Google Scholar). Stable overexpression of MCL1 in MDA-MB-468 cells upregulated OCR, mtMPs, and ROS levels. Conversely, transfection of MCL1 siRNA into MDA-MB-436 and SUM159PT cells reduced OCR, mtMPs, and ROS levels (Figures 5A–5C ). During the respiratory phase, mitochondria appear elongated and their cristae compartments are enlarged; in the glycolytic phase, however, the cristae appear round and fragmented (Alirol and Martinou, 2006Alirol E. Martinou J.C. Mitochondria and cancer: is there a morphological connection?.Oncogene. 2006; 25: 4706-4716Crossref PubMed Scopus (133) Google Scholar). Knockdown of MCL1 with siRNA modulated mitochondria from an elongated shape to a round shape, indicative of an inactive respiratory phenotype (Figure 5D). In addition, the inhibitory effect of oligomycin A on mammosphere formation was more potent against MDA-MB-468 cells stably overexpressing MCL1 compared to control cells, suggesting that self-renewal capacity potentiated by MCL1 also relies on mtOXPHOS (Figure 5E). Metabolome analysis of SUM159PT cells showed that ablation of MCL1 with siRNA reduced the level of TCA cycle metabolites, including citrate, isocitrate, succinate, fumarate, and malate, compared to control siRNA, suggesting a role of MCL1 in the aerobic oxidation of mitochondrial fuels (Figure 5F). The localization of murine MCL1 within the mitochondrial matrix is mediated by its N-terminal mitochondrial targeting sequence (MTS) (Huang and Yang-Yen, 2010Huang C.R. Yang-Yen H.F. The fast-mobility isoform of mouse Mcl-1 is a mitochondrial matrix-localized protein with attenuated anti-apoptotic activity.FEBS Lett. 2010; 584: 3323-3330Crossref PubMed Scopus (30) Google Scholar), and this localization is causally associated with its role in mitochondrial respiration (Perciavalle et al., 2012Perciavalle R.M. Stewart D.P. Koss B. Lynch J. Milasta S. Bathina M. Temirov J. Cleland M.M. Pelletier S. Schuetz J.D. et al.Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration.Nat. Cell Biol. 2012; 14: 575-583Crossref PubMed Scopus (295) Google Scholar). Hence, we next conducted an in silico analysis of the human MCL1 protein sequence to examine a probability of mitochondrial export (Claros and Vincens, 1996Claros M.G. Vincens P. Computational method to predict mitochondrially imported proteins and their targeting sequences.Eur. J. Biochem. 1996; 241: 779-786Crossref PubMed Scopus (1371) Google Scholar). In this analysis, amino acids 1–44 of human MCL1 were highly predicted to play a role in mitochondrial export (0.8364; Figure S4B). The role of the N-terminal domain of murine MCL1 in its proper mitochondrial localization was previously reported (Perciavalle et al., 2012Perciavalle R.M. Stewart D.P. Koss B. Lynch J. Milasta S. Bathina M. Temirov J. Cleland M.M. Pelletier S. Schuetz J.D. et al.Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration.Nat. Cell Biol. 2012; 14: 575-583Crossref PubMed Scopus (295) Google Scholar). To test whether the localization of MCL1 in the mitochondrial matrix is required to induce CSCs, we designed a human MCL1 mutant without the N-terminal MTS (MCL1-ΔMTS; Figure S4C). Deletion of the MCL1 MTS impaired its ability to localize in the mitochondrial matrix (Figures S4D and S4E), resulting in reduced mitochondrial respiratory capacities in MDA-MB-468 cells (Figure 5G). Similarly, expression of MCL1-ΔMTS diminished mtMPs and ROS production, suggesting mitochondrial localization is required for the ability of MCL1 to regulate mtOXPHOS (Figures S4F and S4G). Further, CD44hi/CD24low cells and mammosphere formation were reduced in MDA-MB-468 cells expressing MCL1-ΔMTS compared to wild-type MCL1 (Figures 5H and 5I). Finally, deletion of the MTS attenuated enrichment of CSCs, but did not affect the anti-apoptotic activity of MCL1 (Figure S4H). To further investigate the discordance between the mitochondrial and anti-apoptotic actions of MCL1, we used the BH3-mimetic small-molecule MCL1 inhibitor VU0659158 (Lee et al., 2017Lee T. Bian Z. Zhao B. Hogdal L.J. Sensintaffar J.L. Goodwin C.M. Belmar J. Shaw S. Tarr J.C. Veerasamy N. et al.Discovery and biological characterization of potent myeloid cell leukemia-1 inhibitors.FEBS Lett. 2017; 591: 240-251Crossref PubMed Scopus (44) Google Scholar). Since this drug specifically binds the BH3-binding groove and mimics the binding of pro-apoptotic BH3 family members, we hypothesized that VU0659158 would not affect MCL1 action in the mitochondrial matrix where multi-BH domain cell death effectors, such as BAX and BAK, are absent. Unlike MCL1 siRNA, treatment with VU0659158 did not attenuate mammosphere formation or the OCR in MDA-MB-436 and SUM159PT cells (Figures 5J and S4I) but still induced apoptosis as measured by cleaved caspase 3/7 activity (Figure S4J). Collectively, these data suggest that mitochondrial MCL1 contributes to CSC enrichment through enhancement of mtOXPHOS, independent of its BH3 domain-mediated anti-apoptotic function. Since we observed that ∼44% of post-treatment, drug-resistant TNBCs harbor co-amplification of MYC and MCL1 (Figure 1A), we next explored whether MYC and MCL1 cooperate to enrich CSCs. Knockdown of both MYC and MCL1 with siRNA reduced expression of both MYC and MCL1 (Figure S5A). Simultaneous knockdown of MYC and MCL1 reduced ALDH+ cells and mammosphere formation in MDA-MB-436 and SUM159PT cells more potently than knockdown of MYC or MCL1 alone (Figures 6A and 6B ). A reduction in mammosphere formation and the CD44hi/CD24low fraction was also observed in PCTR SUM159PT cells transfected with both siRNAs (Figures 6C and 6D). Conversely, we stably expressed MCL1 or GFP in MDA-MB-468 cells and then transduced them with DOX-inducible MYC (Figure S5B). Induction of MYC in MDA-MB-468 cells overexpressing MCL1 stimulated mammosphere formation and CD44hi/CD24low cells to a larger degree than in control cells expressing GFP (Figures 6E and 6F). Subsequently, simultaneous knockdown of MYC and MCL1 resulted in a more potent reduction in OCR and ROS levels compared to knockdown of MYC or MCL1 alone (Figures 6G and 6H). To generate correlative data in human tumors, we performed Gene Set Variation Analysis (GSVA) of TCGA data and found that both OXPHOS and ROS pathways are significantly upregulated in breast tumors with MYC and/or MCL1 amplifications or mRNA upregulation (Table S1 and Figure S6A). We confirmed that levels of MYC or MCL1 mRNA were elevated in those tumors with MYC and MCL1 alterations (Figure S6B). Further, the GSVA score determined using a gene set involved in OXPHOS was higher in breast tumors with MYC and MCL1 amplifications or mRNA upregulation compared to tumors with overexpression of MYC or MCL1 alone (Figure 6I). These results suggest that MYC and MCL1 cooperate to enhance mitochondrial respiration, which in turn maintains cancer cell stemness. Hypoxia is a micro-environmental state that activates hypoxia-inducible factor (HIF) signaling, promoting CSCs and tumorigenesis (Lee and Simon, 2012Lee K.E. Simon M.C. From stem cells to cancer stem cells: HIF takes the stage.Curr. Opin. Cell Biol. 2012; 24: 232-235Crossref PubMed Scopus (46) Google Scholar, Rankin and Giaccia, 2016Rankin E.B. Giaccia A.J. Hypoxic control of metastasis.Science. 2016; 352: 175-180Crossref PubMed Scopus (755) Google Scholar). As a byproduct of mtOXPHOS, ROS stabilize HIF proteins, resulting in activation of hypoxia signaling (Hwang and Lee, 2011Hwang A.B. Lee S.J. Regulation of life span by mitochondrial respiration: the HIF-1 and ROS connection.Aging (Albany NY). 2011; 3: 304-310Crossref PubMed Scopus (70) Google Scholar, Mathi" @default.
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• W2761360410 title "MYC and MCL1 Cooperatively Promote Chemotherapy-Resistant Breast Cancer Stem Cells via Regulation of Mitochondrial Oxidative Phosphorylation" @default.
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• W2761360410 doi "https://doi.org/10.1016/j.cmet.2017.09.009" @default.
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