Proteins from the Bcl-2 family play an essential role in the regulation of apoptosis. However, they also possess cell death-unrelated activities that are less well understood. This prompted us to study apoptosis-unrelated activities of the Bax and Bak, pro-apoptotic members of the Bcl-2 family. We prepared Bax/Bak-deficient human cancer cells of different origin and found that while respiration in the glioblastoma U87 Bax/Bak-deficient cells was greatly enhanced, respiration of Bax/Bak-deficient B lymphoma HBL-2 cells was slightly suppressed. Bax/Bak-deficient U87 cells also proliferated faster in culture, formed tumours more rapidly in mice, and showed modulation of metabolism with a considerably increased NAD+/NADH ratio. Follow-up analyses documented increased/decreased expression of mitochondria-encoded subunits of respiratory complexes and stabilization/destabilization of the mitochondrial transcription elongation factor TEFM in Bax/Bak-deficient U87 and HBL-2 cells, respectively. TEFM downregulation using shRNAs attenuated mitochondrial respiration in Bax/Bak-deficient U87 as well as in parental HBL-2 cells. We propose that (post)translational regulation of TEFM levels in Bax/Bak-deficient cells modulates levels of subunits of mitochondrial respiratory complexes that, in turn, contribute to respiration and the accompanying changes in metabolism and proliferation in these cells.

1st Faculty of Medicine Charles University Prague Czech Republic

1st Faculty of Medicine Institute of Pathological Physiology Charles University Prague Czech Republic

Faculty of Science Charles University Prague Czech Republic

Institute of Biotechnology Czech Academy of Sciences Vestec Prague Czech Republic

Institute of Experimental Medicine Czech Academy of Sciences Prague Czech Republic

Institute of Molecular Genetics Czech Academy of Sciences Prague Czech Republic

School of Pharmacy and Medical Science Griffith University Southport QLD Australia

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Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257 PubMed PMC

Gross A, McDonnell JM, Korsmeyer SJ (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13:1899–1911 PubMed

Kale J, Osterlund EJ, Andrews DW (2018) BCL-2 family proteins: changing partners in the dance towards death. Cell Death Differ 25:65–80 PubMed

Hardwick JM, Soane L (2013) Multiple functions of BCL-2 family proteins. Cold Spring Harb Perspect biol. https://doi.org/10.1101/cshperspect.a008722 PubMed DOI PMC

Gross A, Katz SG (2017) Non-apoptotic functions of BCL-2 family proteins. Cell Death Differ 24:1348–1358 PubMed PMC

Morris JL, Gillet G, Prudent J, Popgeorgiev N (2021) Bcl-2 family of proteins in the control of mitochondrial calcium signalling: An old chap with new roles. Int J Mol Sci. https://doi.org/10.3390/ijms22073730 PubMed DOI PMC

Chong SJF, Marchi S, Petroni G, Kroemer G, Galluzzi L, Pervaiz S (2020) Noncanonical cell fate regulation by Bcl-2 proteins. Trends Cell Biol 30:537–555 PubMed

Eckenrode EF, Yang J, Velmurugan GV, Foskett JK, White C (2010) Apoptosis protection by Mcl-1 and Bcl-2 modulation of inositol 1,4,5-trisphosphate receptor-dependent Ca2+ signaling. J Biol Chem 285:13678–13684 PubMed PMC

White C, Li C, Yang J et al (2005) The endoplasmic reticulum gateway to apoptosis by Bcl-X(L) modulation of the InsP3R. Nat Cell Biol 7:1021–1028 PubMed PMC

Nutt LK, Pataer A, Pahler J et al (2002) Bax and bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores. J Biol Chem 277:9219–9225 PubMed

Zong WX, Li C, Hatzivassiliou G et al (2003) Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis. J Cell Biol 162:59–69 PubMed PMC

Oakes SA, Scorrano L, Opferman JT et al (2005) Proapoptotic BAX and BAK regulate the type 1 inositol trisphosphate receptor and calcium leak from the endoplasmic reticulum. Proc Natl Acad Sci USA 102:105–110 PubMed

Hirata H, Lopes GS, Jurkiewicz A, Garcez-do-Carmo L, Smaili SS (2012) Bcl-2 modulates endoplasmic reticulum and mitochondrial calcium stores in PC12 cells. Neurochem Res 37:238–243 PubMed

Carvalho AC, Sharpe J, Rosenstock TR, Teles AF, Youle RJ, Smaili SS (2004) Bax affects intracellular Ca2+ stores and induces Ca2+ wave propagation. Cell Death Differ 11:1265–1276 PubMed

Youle RJ, Karbowski M (2005) Mitochondrial fission in apoptosis. Nat Rev Mol Cell Biol 6:657–663 PubMed

Brooks C, Wei Q, Feng L et al (2007) Bak regulates mitochondrial morphology and pathology during apoptosis by interacting with mitofusins. Proc Natl Acad Sci USA 104:11649–11654 PubMed PMC

Karbowski M, Norris KL, Cleland MM, Jeong SY, Youle RJ (2006) Role of Bax and Bak in mitochondrial morphogenesis. Nature 443:658–662 PubMed

Um HD (2016) Bcl-2 family proteins as regulators of cancer cell invasion and metastasis: a review focusing on mitochondrial respiration and reactive oxygen species. Oncotarget 7:5193–5203 PubMed

Chen ZX, Pervaiz S (2007) Bcl-2 induces pro-oxidant state by engaging mitochondrial respiration in tumor cells. Cell Death Differ 14:1617–1627 PubMed

Eliseev RA, Malecki J, Lester T, Zhang Y, Humphrey J, Gunter TE (2009) Cyclophilin D interacts with Bcl2 and exerts an anti-apoptotic effect. J Biol Chem 284:9692–9699 PubMed PMC

Chen ZX, Pervaiz S (2010) Involvement of cytochrome c oxidase subunits Va and Vb in the regulation of cancer cell metabolism by Bcl-2. Cell Death Differ 17:408–420 PubMed

Lagadinou ED, Sach A, Callahan K et al (2013) BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells. Cell Stem Cell 12:329–341 PubMed PMC

Kane DJ, Sarafian TA, Anton R et al (1993) Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science 262:1274–1277 PubMed

Imahashi K, Schneider MD, Steenbergen C, Murphy E (2004) Transgenic expression of Bcl-2 modulates energy metabolism, prevents cytosolic acidification during ischemia, and reduces ischemia/reperfusion injury. Circul Res 95:734–741

Alavian KN, Li H, Collis L et al (2011) Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase. Nat Cell Biol 13:1224–1233 PubMed PMC

Li M, Wang D, He J, Chen L, Li H (2020) Bcl-XL: a multifunctional anti-apoptotic protein. Pharmacol Res 151:104547 PubMed

Chen YB, Aon MA, Hsu YT et al (2011) Bcl-xL regulates mitochondrial energetics by stabilizing the inner membrane potential. J Cell Biol 195:263–276 PubMed PMC

Lucantoni F, Salvucci M, Düssmann H, Lindner AU, Lambrechts D, Prehn JHM (2021) BCL(X)L and BCL2 increase the metabolic fitness of breast cancer cells: a single-cell imaging study. Cell Death Differ 28:1512–1531 PubMed

Lee KM, Giltnane JM, Balko JM et al (2017) MYC and MCL1 cooperatively promote chemotherapy-resistant breast cancer stem cells via regulation of mitochondrial oxidative phosphorylation. Cell Metabol 26:633-647e637

Carter BZ, Mak PY, Tao W et al (2022) Targeting MCL-1 dysregulates cell metabolism and leukemia-stroma interactions and resensitizes acute myeloid leukemia to BCL-2 inhibition. Haematologica 107:58–76 PubMed

Huang CR, Yang-Yen HF (2010) The fast-mobility isoform of mouse Mcl-1 is a mitochondrial matrix-localized protein with attenuated anti-apoptotic activity. FEBS Lett 584:3323–3330 PubMed

Perciavalle RM, Stewart DP, Koss B et al (2012) Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration. Nat Cell Biol 14:575–583 PubMed PMC

Wang X, Bathina M, Lynch J et al (2013) Deletion of MCL-1 causes lethal cardiac failure and mitochondrial dysfunction. Genes Dev 27:1351–1364 PubMed PMC

Boohaker RJ, Zhang G, Carlson AL, Nemec KN, Khaled AR (2011) BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells. Am J Physiol Cell Physiol 300:C1466-1478 PubMed PMC

Kim EM, Park JK, Hwang SG et al (2014) Nuclear and cytoplasmic p53 suppress cell invasion by inhibiting respiratory complex-I activity via Bcl-2 family proteins. Oncotarget 5:8452–8465 PubMed PMC

Kim EM, Jung CH, Song JY, Park JK, Um HD (2018) Pro-apoptotic Bax promotes mesenchymal-epithelial transition by binding to respiratory complex-I and antagonizing the malignant actions of pro-survival Bcl-2 proteins. Cancer Lett 424:127–135 PubMed

Vercellino I, Sazanov LA (2022) The assembly, regulation and function of the mitochondrial respiratory chain. Nat Rev Mol Cell Biol 23:141–161 PubMed

Tan AS, Baty JW, Dong LF et al (2015) Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA. Cell Metabol 21:81–94

Chiu HY, Loh AHP, Taneja R (2022) Mitochondrial calcium uptake regulates tumour progression in embryonal rhabdomyosarcoma. Cell Death Dis 13:419 PubMed PMC

Lindsten T, Ross AJ, King A et al (2000) The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol Cell 6:1389–1399 PubMed PMC

Scorrano L, Oakes SA, Opferman JT et al (2003) Bax and bak regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science 300:135–139 PubMed

Brayer S, Joannes A, Jaillet M et al (2017) The pro-apoptotic bax protein influences cell growth and differentiation from the nucleus in healthy interphasic cells. Cell Cycle 16:2108–2118 PubMed PMC

Wang C, Youle RJ (2012) Predominant requirement of bax for apoptosis in HCT116 cells is determined by Mcl-1’s inhibitory effect on bak. Oncogene 31:3177–3189 PubMed

Zhang L, Yu J, Park BH, Kinzler KW, Vogelstein B (2000) Role of BAX in the apoptotic response to anticancer agents. Science 290:989–992 PubMed

Deng J, Gutierrez LG, Stoll G et al (2021) Paradoxical implication of BAX/BAK in the persistence of tetraploid cells. Cell Death Dis 12:1039 PubMed PMC

Bomba-Warczak E, Edassery SL, Hark TJ, Savas JN (2021) Long-lived mitochondrial cristae proteins in mouse heart and brain. J Cell Biol. https://doi.org/10.1083/jcb.202005193 PubMed DOI PMC

Takeuchi A, Kim B, Matsuoka S (2015) The destiny of ca(2+) released by mitochondria. J Physiol Sci 65:11–24 PubMed

Minczuk M, He J, Duch AM et al (2011) TEFM (c17orf42) is necessary for transcription of human mtDNA. Nucleic Acids Res 39:4284–4299 PubMed PMC

Hillen HS, Parshin AV, Agaronyan K et al (2017) Mechanism of transcription anti-termination in human mitochondria. Cell 171:1082–1093 PubMed PMC

Posse V, Shahzad S, Falkenberg M, Hallberg BM, Gustafsson CM (2015) TEFM is a potent stimulator of mitochondrial transcription elongation in vitro. Nucleic Acids Res 43:2615–2624 PubMed PMC

Yu H, Xue C, Long M et al (2018) TEFM enhances transcription elongation by modifying mtRNAP Pausing dynamics. Biophys J 115:2295–2300 PubMed PMC

Jiang S, Koolmeister C, Misic J et al (2019) TEFM regulates both transcription elongation and RNA processing in mitochondria. EMBO Rep 20:e48101 PubMed PMC

Fei ZY, Wang WS, Li SF et al (2020) High expression of the TEFM gene predicts poor prognosis in hepatocellular carcinoma. J Gastrointest Oncol 11:1291–1304 PubMed PMC

Li S, Wang W, Zi J et al (2020) Elevated expression of mitochondrial transcription elongation factor (TEFM) predicts poor prognosis in low grade glioma-an analysis of the cancer genome atlas (TCGA) dataset. Transl Cancer Res 9:3610–3622 PubMed PMC

Wan L, Wang Y, Zhang Z et al (2021) Elevated TEFM expression promotes growth and metastasis through activation of ROS/ERK signaling in hepatocellular carcinoma. Cell Death Dis 12:325 PubMed PMC

Van Haute L, O’Connor E, Diaz-Maldonado H et al (2023) TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease. Nat Commun 14:1009 PubMed PMC

Eischen CM, Rehg JE, Korsmeyer SJ, Cleveland JL (2002) Loss of bax alters tumor spectrum and tumor numbers in ARF-deficient mice. Cancer Res 62:2184–2191 PubMed

Mrozek A, Petrowsky H, Sturm I et al (2003) Combined p53/Bax mutation results in extremely poor prognosis in gastric carcinoma with low microsatellite instability. Cell Death Differ 10:461–467 PubMed