OMA1 reprograms metabolism under hypoxia to promote colorectal cancer development
2020; Springer Nature; Volume: 22; Issue: 1 Linguagem: Inglês
10.15252/embr.202050827
ISSN1469-3178
AutoresZhida Wu, Meiling Zuo, Ling Zeng, Kaisa Cui, Bing Liu, Chaojun Yan, Li Chen, Jun Dong, Fugen Shangguan, Wanglai Hu, He He, Bin Lü, Zhiyin Song,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoArticle13 December 2020free access Transparent process OMA1 reprograms metabolism under hypoxia to promote colorectal cancer development Zhida Wu Zhida Wu orcid.org/0000-0002-5251-3188 Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, ChinaThese authors contributed equally to this work Search for more papers by this author Meiling Zuo Meiling Zuo Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, ChinaThese authors contributed equally to this work Search for more papers by this author Ling Zeng Ling Zeng Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Kaisa Cui Kaisa Cui Wuxi Cancer Institute, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, China Laboratory of Cancer Epigenetics, Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, China Search for more papers by this author Bing Liu Bing Liu Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Chaojun Yan Chaojun Yan Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Li Chen Li Chen Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Jun Dong Jun Dong Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Fugen Shangguan Fugen Shangguan Attardi Institute of Mitochondrial Biomedicine, School of Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China Search for more papers by this author Wanglai Hu Wanglai Hu School of Basic Medical Science, Anhui Medical University, Hefei, Anhui, China Search for more papers by this author He He He He Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Bin Lu Bin Lu Attardi Institute of Mitochondrial Biomedicine, School of Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China Search for more papers by this author Zhiyin Song Corresponding Author Zhiyin Song [email protected] orcid.org/0000-0002-5003-081X Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Zhida Wu Zhida Wu orcid.org/0000-0002-5251-3188 Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, ChinaThese authors contributed equally to this work Search for more papers by this author Meiling Zuo Meiling Zuo Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, ChinaThese authors contributed equally to this work Search for more papers by this author Ling Zeng Ling Zeng Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Kaisa Cui Kaisa Cui Wuxi Cancer Institute, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, China Laboratory of Cancer Epigenetics, Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, China Search for more papers by this author Bing Liu Bing Liu Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Chaojun Yan Chaojun Yan Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Li Chen Li Chen Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Jun Dong Jun Dong Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Fugen Shangguan Fugen Shangguan Attardi Institute of Mitochondrial Biomedicine, School of Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China Search for more papers by this author Wanglai Hu Wanglai Hu School of Basic Medical Science, Anhui Medical University, Hefei, Anhui, China Search for more papers by this author He He He He Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Bin Lu Bin Lu Attardi Institute of Mitochondrial Biomedicine, School of Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China Search for more papers by this author Zhiyin Song Corresponding Author Zhiyin Song [email protected] orcid.org/0000-0002-5003-081X Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China Search for more papers by this author Author Information Zhida Wu1, Meiling Zuo1, Ling Zeng1, Kaisa Cui2,3, Bing Liu1, Chaojun Yan1, Li Chen1, Jun Dong1, Fugen Shangguan4, Wanglai Hu5, He He1, Bin Lu4 and Zhiyin Song *,1 1Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, Hubei, China 2Wuxi Cancer Institute, Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu, China 3Laboratory of Cancer Epigenetics, Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, China 4Attardi Institute of Mitochondrial Biomedicine, School of Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China 5School of Basic Medical Science, Anhui Medical University, Hefei, Anhui, China *Corresponding author. Tel: +86 027 68752235; E-mail: [email protected] EMBO Reports (2021)22:e50827https://doi.org/10.15252/embr.202050827 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Many cancer cells maintain enhanced aerobic glycolysis due to irreversible defective mitochondrial oxidative phosphorylation (OXPHOS). This phenomenon, known as the Warburg effect, is recently challenged because most cancer cells maintain OXPHOS. However, how cancer cells coordinate glycolysis and OXPHOS remains largely unknown. Here, we demonstrate that OMA1, a stress-activated mitochondrial protease, promotes colorectal cancer development by driving metabolic reprogramming. OMA1 knockout suppresses colorectal cancer development in AOM/DSS and xenograft mice models of colorectal cancer. OMA1-OPA1 axis is activated by hypoxia, increasing mitochondrial ROS to stabilize HIF-1α, thereby promoting glycolysis in colorectal cancer cells. On the other hand, under hypoxia, OMA1 depletion promotes accumulation of NDUFB5, NDUFB6, NDUFA4, and COX4L1, supporting that OMA1 suppresses OXPHOS in colorectal cancer. Therefore, our findings support a role for OMA1 in coordination of glycolysis and OXPHOS to promote colorectal cancer development and highlight OMA1 as a potential target for colorectal cancer therapy. Synopsis Mitochondrial stress sensor OMA1 promotes colorectal cancer development by driving metabolic reprogramming under hypoxia. Therefore, OMA1 may be a potential target for colorectal cancer therapy. OMA1 is upregulated in human colorectal cancer and OMA1 levels correlate with poor survival. Deletion of OMA1 suppresses colorectal cancer development in AOM/DSS and xenograft mouse models. Hypoxia upregulates OMA1 levels. OMA1 is required for HIF-1α stabilization by hypoxia. OMA1 is required for the hypoxia induced increase in glycolytic flux. OMA1 deletion leads to a drop in ROS formation and accumulation of mitochondrial respiratory chain components in mouse colorectal tumors. Introduction Alteration of cellular metabolisms, particularly of energy metabolism, is considered to be a core hallmark of cancer (Ward & Thompson, 2012). Under aerobic conditions, most normal differentiated cells generate energy by mitochondrial oxidative phosphorylation (OXPHOS) and metabolize glucose to carbon dioxide through the mitochondrial tricarboxylic acid (TCA) cycle (Matthew et al, 2009). In contrast, most cancer cells rely on aerobic glycolysis to produce energy and glycolytic intermediates for the synthesis of lipids, amino acids, nucleic acids, and so on, even in the presence of enough oxygen; meanwhile, mitochondrial OXPHOS is suppressed in cancer cells due to mitochondrial dysfunction. This energy metabolic reprogramming is known as “the Warburg effect” (Matthew et al, 2009), which contributes to tumorigenesis and progression in a variety of cancers. However, recent studies have revealed that mitochondrial bioenergetic and biosynthetic states are frequently variable in cancers rather than absolutely dysfunctional (Wallace, 2012), which depends on environmental, genetic, and tissue-of-origin differences among tumors (Vyas et al, 2016). In addition, hypoxic tumor microenvironments induced the glycolytic phenotype in many cancer cells. Therefore, mitochondrial OXPHOS and glycolysis collaborate to maintain the balance of energy metabolism in cancer cells. Colorectal cancer is the third most prevalent cancer and the second most lethal malignancy in the world (Arnold et al, 2017; Bray et al, 2018). Dysfunctional mitochondria with reduced electron transport chain activity, decreased mitochondrial ATP level, increased reactive oxygen species (ROS) generation and mtDNA mutation are reported to contribute to the pathogenesis of intestinal inflammation and colorectal tumorigenesis (Polyak et al, 1998; Cunningham et al, 2016; Heller et al, 2017; Xue et al, 2017). However, functional mitochondria are also required for the transformation of colorectal cancer (Vyas et al, 2016; Baker et al, 2019). Therefore, how mitochondria coordinate glycolysis and oxidative phosphorylation is critical for colorectal tumor development, but the mechanism remains largely unknown. Hypoxia is a typical feature in the development of colorectal cancer (Wang & Semenza, 1993; Biddlestone et al, 2015). Hypoxia can stabilize the transcription factor HIF1α, which activates the expression of lots of genes involved in the inflammatory response, tumor vascularization, metastasis, and radio- or chemo-resistance (Semenza, 2004; Baba et al, 2010; Biddlestone et al, 2015; Ioannou et al, 2015; Balamurugan, 2016). HIF1α also transcriptionally regulates cell metabolism to meet the demands for ATP and macromolecule anabolism during colorectal cancer development (Ioannou et al, 2015). Hypoxia-induced ROS has been reported to play an important role in stabilizing and activating HIF1α (Niecknig et al, 2012; Movafagh et al, 2015). In mammalian cells, the major site of ROS production is mitochondrial electron transport chain (Liu et al, 2002). Therefore, mitochondria are highly associated with HIF-1α stability. OMA1 is an ATP-independent zinc metalloprotease located at the mitochondrial inner membrane(Käser et al, 2003), and it is a stress-sensitive mitochondrial protease, which plays a key role in mitochondrial protein quality control and metabolic homeostasis (Ehses et al, 2009; Baker et al, 2014; Bohovych et al, 2016). OMA1 is activated by self-cleavage upon mitochondrial membrane depolarization and other cellular stress. Meanwhile, activated OMA1 also leads to degradation of itself (Baker et al, 2014; Zhang et al, 2014). Moreover, OMA1 cooperates with i-AAA protease Yme1L (Yme1 like 1 ATPase) to mediate the processing and degradation of OPA1, a dynamin-like GTPase that mediates inner mitochondrial membrane fusion, cristae formation, and resistance to apoptosis (Anand et al, 2014; Varanita et al, 2015; Wai et al, 2015; Rainbolt et al, 2016). Notably, OMA1 deficiency in mouse models exhibits defective thermogenesis, diet-induced obesity (Quirós et al, 2012), yet protects against heart failure (Wai et al, 2015; Acin-Perez et al, 2018), neurodegeneration (Korwitz et al, 2016), and ischemic kidney injury (Xiao et al, 2014). Interestingly, recent studies revealed another piece of evidence that stress-activated OMA1 mediates the cleavage of DELE1, which then relays mitochondrial stress to the cytosol (Fessler et al, 2020; Guo et al, 2020). So far, the role of OMA1 in cancer is complicated, depending on the type of cancer, the disease stage, the treatment, and many other factors (Jiang et al, 2014; Alavi, 2019; Amini et al, 2019; Daverey et al, 2019), and the underlying mechanism of OMA1 in regulating tumorigenesis and progression remains largely unknown. In this study, we report that OMA1 facilitates the development of colorectal cancer by promoting the Warburg effect. We show that OMA1 knockout (KO) protects mice from colorectal cancer induced by the azoxymethane/dextran sodium sulfate (AOM/DSS) model. Our findings also suggest that OMA1-OPA1 axis promotes glycolysis by increasing ROS generation and enhancing the stability of HIF-1α in colorectal cancer cells under hypoxia. In addition, we show that OMA1 inhibits mitochondrial oxidative phosphorylation by impairing the assembly of mitochondrial respiratory chain complexes through promoting the degradations of NDUFB5, NDUFB6, NDUFA4, and COX4L1 in colorectal cancer cells in vivo and in vitro. Therefore, our study demonstrates a role of OMA1 in coordination of glycolysis and oxidative phosphorylation to promote the Warburg effect, which facilitates colorectal cancer progression. Results OMA1 is upregulated in human colorectal cancer and is activated by hypoxia It is known that mitochondrial dysfunction contributes to the development and progression of colorectal cancer (Sanchez-Pino et al, 2007). Mitochondrial protease OMA1, a mitochondrial stresses sensor, regulates mitochondrial functions and homeostasis (Ehses et al, 2009; Baker et al, 2014; Bohovych et al, 2016). Based on that, we decided to explore the relationship between OMA1 and colorectal cancer. We firstly investigated the role of OMA1 in patients with colorectal cancer. We performed bioinformatics analysis on public datasets of human colorectal cancer samples. Results indicated that the mRNA expression of OMA1 was significantly elevated in tumor samples compared with normal samples (using datasets from NCBI’s Gene Expression Omnibus: GSE21510) (Fig 1A). Next, we analyzed the correlation between OMA1 expression and patient survival using public databases (from NCBI’s Gene Expression Omnibus: GSE17537). Consistently, high level of OMA1 mRNA expression was significantly correlated with shorter survival time (P = 0.0180) (Fig 1B). Since OPA1 can be degraded or cleaved at the S1 site by OMA1, we performed Western blotting to analyze the processing of OPA1 in colorectal cancer specimens. Compared with the adjacent normal tissues, the colorectal tumors displayed remarkably increased OPA1 degradation or processing (bands “c” and “e” are products of OPA1 cleavage by OMA1) (Fig 1C), suggesting that OMA1 is activated in colorectal tumors (Fig 1C) and probably influenced by the tumor microenvironments. And since one of the most pervasive element of solid tumor microenvironments is hypoxia (Harris, 2002), we later tested whether OMA1 is activated by hypoxia. Control and OMA1 KO HCT116 cells were treated with or without hypoxia (1% O2) for the indicated time. Consistent with previous works that OPA1 processing is enhanced in hypoxia (An et al, 2013; MacVicar et al, 2019), our Western blotting data showed that OPA1 was processed and degraded in control cells; however, this was inhibited in OMA1 KO cells (Fig 1D), suggesting that OMA1 is activated by hypoxia to regulate OPA1 processing and degradation. Along with that, OPA1 KO MEFs stably expressing OPA1 isoform 1, which can only be processed by OMA1, displayed increased cleavage of OPA1 isform1 after hypoxia (Fig 1E), further confirming that OMA1 is activated by hypoxia. Moreover, our RT–PCR assays revealed that hypoxia induced a distinct increased expression of GLUT1, but did not affect OMA1, YME1L, and OPA1 in all three cell lines we used, i.e., HCT116, HT29, and SW48 cells (Appendix Fig S1A–C), which indicates that OMA1 is activated post-translationally. In addition, hypoxia can lead to the reduction of mitochondrial membrane potential (Solaini et al, 2010), which also contribute to the activation of OMA1. Figure 1. OMA1 is upregulated in human colorectal cancer and is activated by hypoxia Relative levels of OMA1 mRNA in colorectal cancer tissues (n = 105) compared with adjacent normal tissues (n = 43) from colorectal cancer patients were shown (using the GEO dataset GSE21510). The data are presented as mean ± SEM, and statistical significance was determined by a Mann–Whitney test. ***P < 0.001. Kaplan–Meier curves were constructed to analyze and compare between patients with high and low levels of OMA1 in colorectal cancer samples from the GEO dataset GSE17537. “Low” indicates patients with OMA1 mRNA levels less than the median. “High” indicates patients with OMA1 mRNA levels greater than the median. Statistical analysis was performed using log-rank tests, n = 55, P = 0.0180. The lysates of tumors (T) and adjacent normal (N) tissues from colorectal cancer patients were analyzed by Western blotting with antibodies against OPA1 or Tubulin. The “c, d and e” bands of OPA1 indicate cleaved OPA1 bands. Tubulin was used as a loading control. Control and OMA1 KO HCT116 cells were cultured in hypoxia (1% O2) for 0 (normoxia), 24, or 48 h. Cell lysates were then assessed by Western blotting with antibodies against OPA1, OMA1, or β-Actin. β-Actin was used as a loading control. Representative immunoblots were from n = 3 independent experiments. OPA1-null mouse embryonic fibroblasts (MEFs) expressing control (empty vector) or OPA1 isoform 1 were cultured in normoxia or hypoxia (1% O2) for 24 h. Cell lysates were analyzed by Western blotting with anti-OPA1 or anti-Tubulin antibody. Representative immunoblots were from n = 3 independent experiments. WT, OMA1 KO, and OMA1 KO HCT116 cells expressing WT-OMA1(OMA1-Flag) or proteolytic inactive OMA1(E324Q-Flag) were cultured in hypoxia (1% O2) for 0, 24, or 48 h, and the cell lysates were assessed by Western blot with antibodies against OMA1, and β-Actin. β-Actin was used as a loading control (representative data from three independent experiments). The asterisk indicates a nonspecific band. The relative protein levels were evaluated by densitometry analysis using ImageJ software and were quantified for the ratio of OMA1-Flag/β-Actin or E324Q-Flag/β-Actin in hypoxia for 0, 24 or 48 h (n = 3 independent experiments). The data are presented as mean ± SD. Download figure Download PowerPoint Additionally, our studies showed that hypoxia-activated OMA1 may in turn lead to the degradation and cleavage of OMA1 (Fig 1D, F, and G). Nevertheless, the degradation of OMA1-E324Q (proteolytic inactive OMA1) was largely inhibited in OMA1 KO HCT116 cells stably expressing OMA1-E324Q under hypoxia, indicating that OMA1 may undergo auto-proteolytic cleavage and auto-degradation, consisting with the previous reports (Baker et al, 2014; Jiang et al, 2014; Zhang et al, 2014; Simula et al, 2020), but further experiments are needed to prove this. Furthermore, OMA1-E324Q was still mildly reduced in hypoxia (48 h) (Fig 1F and G), suggesting that OMA1 can be degraded or cleaved by some other proteases in hypoxia. Our findings are consistent with the recent report that OMA1 is degraded by Yme1L in hypoxia (MacVicar et al, 2019). Therefore, OMA1 may cooperate with Yme1L to degrade OMA1 in hypoxia. These results suggest that OMA1 degradation may also act as a stop signal to prevent unlimited OMA1 activation under hypoxia, which may be a critical negative feedback to regulate OMA1 activity. Overall, our data suggest a causal link between OMA1 activation and colorectal cancer. Loss of OMA1 inhibits the development of AOM/DSS-induced colorectal cancer in mice To investigate the role of OMA1 in colorectal cancer, we generated the OMA1 knockout (KO) mouse model by deleting exon 3 of the mouse OMA1 genomic DNA (Appendix Fig S2A). The genomic DNA of WT, Oma1−/− or Oma1+/− mice were analyzed by PCR, and expression of OMA1 in different tissues was analyzed by Western blotting (Appendix Fig S2B and C). The mice were then injected intraperitoneally with 10 mg/kg AOM, followed by three cycles of 1.5% DSS treatment to induce colorectal cancer (Fig 2A). The AOM/DSS model utilizes chemical induction of DNA damage followed by repeated cycles of colonic inflammation (Thaker et al, 2012). After induction of tumorigenesis, all mice were euthanized on day 105, and intestines were removed and collected from each mouse. Compared with WT mice, OMA1 KO mice exhibited less body weight loss during DSS treatment (Fig 2B), and the length of colorectum in OMA1 KO mice was longer than that in WT mice (Fig 2C and D), indicating that OMA1 KO mice were less susceptible to DSS-induced colitis than WT mice. And as shown in Fig 2E and F, most tumors located at the distal and middle region of the intestine, and the number of colorectal tumors in OMA1 KO mice was significantly reduced compared with that of tumors in WT mice. In addition, the pathological quantification verified that the number of different tumor sizes was markedly reduced in the intestine of OMA1 KO mice (Fig 2G). These data suggest that OMA1 KO inhibits colorectal carcinogenesis. Interestingly, OMA1 KO mice intestines had a lower frequency of large tumors than control mice (Fig 2H), indicating that OMA1 KO blocked the progression of colorectal cancer. Consistently, histological analysis revealed that the adenomas in the intestines of WT mice were high-grade dysplasia and infiltrated with a greater extent of inflammation, while most of the adenomas in the intestine of OMA1 KO mice were low-grade dysplasia (Fig 2I). Moreover, after AOM/DSS treatment, OMA1 deficiency attenuates the mRNA levels of the pro-inflammatory factors including Tnfα, Il6, Cox2, and Ccl2 compared with the control mice (Fig 2J), suggesting that OMA1 regulates AOM/DSS-induced inflammation, which is associated with AOM/DSS-induced colorectal cancer. Consistently, there was a significant decrease in the proliferation rates of colorectal tumors of OMA1 KO mice, as detected by Ki-67 nuclear staining (Fig 2K). Taken together, our findings demonstrate that OMA1 deficiency suppresses colorectal cancer development in the AOM/DSS mouse model. Figure 2. Loss of OMA1 inhibits the development of AOM/DSS-induced colorectal cancer in mice A. Schematic representation of the AOM/DSS procedure. To develop colitis-associated cancer (CAC), WT (n = 11) and Oma1−/− (n = 12) mice were injected intraperitoneally with AOM (10 mg/kg) on day 0. Then, three cycles of feeding water with 1.5% DSS treatment were administered. Mice were euthanized on day 105 and all intestinal tissues, tumors, and serum were collected. After mice were euthanized, intestines were removed and flushed with cold PBS. All experiments were repeated with three independent biological replicates. B. Body weight changes of WT (n = 11) and Oma1−/− (n = 12) mice during AOM/DSS treatment. Data are presented as mean ± SEM. Statistical significance was assessed by a two-way ANOVA, **P < 0.01. C. Typical intestine images on day 105 after mice were euthanized. D. Colorectal lengths (C) of WT (n = 11 biological replicates) and Oma1−/− (n = 12 biological replicates) mice were measured. Data with error bars are presented as mean ± SEM. Statistical significance was assessed by unpaired Student’s t-test, *P < 0.05. E. Representative images of colorectal tumors from WT (left) and Oma1−/− (right) mice. F. The number of tumors in WT (n = 11) and Oma1−/− (n = 12) mice was measured. Each dot represents the tumor number of one individual mouse. Data with error bars are presented as mean ± SEM. Statistical significance was assessed by unpaired Student’s t-test, ***P < 0.001. G, H. The number of tumors of different sizes (diameter) in each mouse (G) and the distribution of different tumor sizes (H) were shown (WT n = 11 biological replicates, and Oma1−/− n = 12 biological replicates). Results are shown as mean ± SEM. P-values were calculated by unpaired Student’s t-test, **P < 0.01, ***P < 0.001. I. Histological analysis of colorectal tumors of WT and Oma1−/− mice was shown by hematoxylin and eosin (H&E) staining. J. Relative mRNA expression levels of pro-inflammatory genes (Tnfα, Il6, Cox2, and Ccl2) in colorectal homogenates of WT and OMA1_KO mice treated with AOM/DSS (n = 4–5 per genotype). The data represent the mean ± SEM, n = 3 independent experiments, and statistical significance was determined by a two-tailed Student’s t-test. *P < 0.05, **P < 0.01. K. The colorectal tissues of WT and Oma1−/− mice were stained with Ki67. Download figure Download PowerPoint Then, we evaluated the role of OMA1 in colorectal tumor growth in nude mice. We developed a mouse xenograft model by subcutaneously injecting control, OMA1 KO HCT116 cells into nude mice, respectively. Consistent with AOM/DSS-induced colorectal cancer mouse model, OMA1 KO significantly suppressed HCT116 xenograft tumor growth (Fig EV1A and B), and the tumor weight of OMA1 KO HCT116 at the endpoint was also significantly reduced compared with that of control (Fig EV1C), suggesting that OMA1 depletion suppresses HCT116 xenograft tumor development in nude mice. Click here to expand this figure. Figure EV1. Loss of OMA1 significantly reduced colorectal tumor growth in nude mice and OPA1 deficiency makes cells more dependent on glycolysis for survival A–C. Control or OMA1 KO HCT116 cells (5 × 106) were injected into the flanks of nude mice. Mice were euthanized and photographed at day 16 (A). Representative photographs of tumors in nude mice were performed (A) and tumor weights were analyzed (C). The tumor sizes were measured at an interval of 6 days after injection and calculated as = width2 × length/2. Xenograft tumor growths of control or OMA1 KO HCT116 tumors in nude mice were shown (B). Data are presented as the mean ± SEM, n = 5 biological replicates. Scale bars, 2 cm. P-values were calculated by two-way ANOVA of variance; *P < 0.05, **P < 0.01. All experiments were repeated with three independent biological replicates. D. Control and OPA1 KO HCT116 cells were treated with glucose deprivation (galactose) or the glycolytic inhibitor (2-Deoxy-D-glucose, 2-DG, 10 mM) for 24 h. Cell deaths were quantified by cell counting with trypan blue exclusion. Error bars represent the mean ± SD (n = 3, 300 cells per independent experiment), statistical significance was assessed by a two-way ANOVA of variance, ***P < 0.001. E. OPA1-null MEFs expressing control (empty vector) or OPA1 isoform 1 were cultured with galactose or 2-DG for 48 h. Cell death was quantified by cell counting with trypan blue exclusion. Error bars represent the mean ± SD (n = 3, 300 cells per independent experiment), statistical significance was assessed by a two-way ANOVA of variance, ***P < 0.001. Download figure Download PowerPoint In a summary, our findings show that OMA1 promotes colorectal cancer development in mice. OMA1-OPA1 axis promotes glycolysis under hypoxia Metabolic reprogramming plays a critical role in tumorigenesis, while glycolysis, but not OXPHOS, contributes mainly to ATP production in cancer cells (Matthew et al, 2009; Ward & Thompson, 2012). Based on that, we later investigated the role of OMA1 in energy metabolism. Technologically, we evaluated the effect of OMA1 depletion on the energy metabolism of colorectal cancer cells under normoxic and hypoxic conditions. Results showed that no significant differences of glycolytic metabolism between control and OMA1 KO HCT116 cells exposed to normoxia (Fig 3A and B), and consistent with previous studies, hypoxia promoted glycolysis in all HCT116 cells, characterized by increased glucose uptake and lactate production (Fig 3A and B). However, compared with control cells, OMA1 KO showed significantly decreased levels of glucose uptake and lactate production under hypoxic conditions, while expression of OMA1-Flag in OMA1 KO cells recovered the levels of glucose uptake and lactate production (Fig 3A and B), suggesting that OMA1 promotes glycolysis under hypoxia. Considering that OMA1 may be involved in tumor bioenergetics, we analyzed the glycolytic activity of these cells by performing real-time analysis of the extracellular acidification rate (ECAR) with or without tr
Referência(s)