The oxidoreductase CLIC4 is required to maintain mitochondrial function and resistance to exogenous oxidants in breast cancer cells
2022; Elsevier BV; Volume: 298; Issue: 9 Linguagem: Inglês
10.1016/j.jbc.2022.102275
ISSN1083-351X
AutoresHeba Al Khamici, Vanesa C. Sanchez, Hualong Yan, Christophe Cataisson, Aleksandra M. Michalowski, Howard H. Yang, Luowei Li, Maxwell P. Lee, Jing Huang, Stuart H. Yuspa,
Tópico(s)ATP Synthase and ATPases Research
ResumoThe chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H2O2 as a ROS-inducing agent. In intact cells, H2O2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H2O2-induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H2O2-treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells. The chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H2O2 as a ROS-inducing agent. In intact cells, H2O2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H2O2-induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H2O2-treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells. Reactive oxygen species (ROS) can be produced through enzymatic reactions by NADPH oxidases, lipoxygenase, cytochrome P450, and cyclooxygenase (1Leone A. Roca M.S. 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We postulated that CLIC4 could participate in regulating the oxidative environment in cancer cells. Further, the soluble fraction of CLIC4 might serve as a protective mechanism for cellular and mitochondrial damage generating ROS in response to exogenous toxins such as oxidants or certain anticancer drugs (41Singha B. Harper S.L. Goldman A.R. Bitler B.G. Aird K.M. Borowsky M.E. et al.CLIC1 and CLIC4 complement CA125 as a diagnostic biomarker panel for all subtypes of epithelial ovarian cancer.Sci. Rep. 2018; 8: 14725Crossref PubMed Scopus (26) Google Scholar, 42Faraji F. Pang Y.L. Walker R.C. Borges R.N. Yang L. Hunter K.W. Cadm1 is a metastasis susceptibility gene that suppresses metastasis by modifying tumor interaction with the cell-mediated immunity.PLoS Genet. 2012; 8e1002926Crossref PubMed Scopus (58) Google Scholar, 43Nagashima K. Zheng J.W. Parmiter D. Patri A.K. Biological tissue and cell culture specimen preparation for TEM nanoparticle characterization.Methods Mol. 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Physiol. 2020; 11: 96Crossref PubMed Scopus (57) Google Scholar). Herein, we used murine breast cancer cells proficient or deficient in CLIC4 to study the influence of oxidative stress and breast cancer redox homeostasis to shed light on the role of CLIC4 that contributes to the growth and viability of those cancer cells. While the oxidoreductase activity of CLIC4 was established with recombinant protein, similar properties have not been confirmed in cells. To address this issue, we used murine breast cancer cell line 6DT1 cells (47Yang Y. Yang H.H. Hu Y. Watson P.H. Liu H. Geiger T.R. et al.Immunocompetent mouse allograft models for development of therapies to target breast cancer metastasis.Oncotarget. 2017; 8: 30621-30643Crossref PubMed Scopus (54) Google Scholar) and stable CLIC4-deficient 6DT1 clones (sg# 1 and sg# 2) that were isolated using CRISPR. First, we investigated the localization of CLIC4 in WT 6DT1 cells and found that CLIC4 is widely expressed in cells without treatment and abundant in mitochondria. Adding 500 nM of H2O2 to cells increased the expression of CLIC4 in 6DT1 cells (Fig. 1A). The intrinsic ROS was measured using CellROX green or CM-H2DCFDA. Confocal imaging results of whole cell projection showed that untreated CLIC4 KO 6DT1 cells (CLIC4-KO) displayed higher fluorescence for CellROX green than WT cells (Fig. 1B), while fluorescent intensity was similar for both genotypes 24 h after treatment with 1 μM of H2O2. After incubation with CM-H2DCFDA, fluorescence-activated cell sorting (FACS) analysis (Fig. 1C) confirmed that ROS is elevated in untreated CLIC4-KO cells at baseline level. The mean fluorescence intensity of three independent repeats also showed that treating the 6DT1 cells with H2O2 increased the level of ROS in CLIC4-WT but not the CLIC4-KO 6DT1 cells, as quantified in Figure 1D. The ROS disparities among the genotypes disappeared when cells were preincubated with the general antioxidant N-acetyl cysteine or NAC (Fig. 1, C and D). These results indicated that CLIC4 participated in an intrinsic antioxidant environment in untreated cancer cells, but compensatory pathways were available for protection from an extrinsic oxidative insult. To determine the functional consequences of ROS alterations, WT, KO, and CLIC4 KO reconstituted with CLIC4 6DT1 breast cancer cells were exposed to different concentrations of H2O2 and tested for cell viability (CCK-8) and cell proliferation responses (CyQuant). There was no significant baseline difference in cell viability percentage among cells that do or do not express CLIC4 over 7 days (Fig. 2A). However, 6DT1 cells treated with different concentrations of H2O2 for 24 h exhibited attenuated cell viability in a dose-dependent manner, while the loss of viability in CLIC4-KO was substantially accelerated as the concentration of H2O2 increased when compared to WT and KO cells reconstituted with CLIC4 via lentivirus (KO+CLIC4) (Fig. 2B). Similar results were observed with a second CLIC4-KO (sg# 2) clone (Fig. S1). As seen in Figure 1, the sensitivity to H2O2 is ROS mediated in all genotypes since viability was restored by incubating the cells with catalase or NAC with/without H2O2 (as shown in Fig. 2, C and D). Loss of CLIC4 also sensitized 6DT1 cells to the antiproliferative effects of increasing the concentration of H2O2 to 1 μM in comparison to the cells that express CLIC4 (WT and KO+CLIC4) as shown in Figure 2E. Postincubating the cells with catalase (Fig. 2F) or NAC (Fig. 2G) in the presence or absence of H2O2 restored proliferation of CLIC4 null cells to the same levels as the cells that express the protein. While the loss of CLIC4 leads to a pro-oxidant internal environment, we asked if CLIC4 was elevated in response to an external pro-oxidant environment. In Figure 3, A and B, increasing concentrations of H2O2 induced the expression of CLIC4 protein and transcripts in WT 6DT1 cells as well as in 4T1 breast cancer cells as in Figure 3C. The induction of CLIC4 transcripts was in response to a pro-oxidant environment as it was mitigated by cotreatment with NAC (Fig. 3B). There was little or no change of catalase, CLIC1, and GST-omega 1 proteins in WT 6DT1 cells. In contrast, in the absence of CLIC4, H2O2 elicited an increased induction in SOD1 and SOD2 proteins. Interestingly, while the DNA damage marker yH2Ax was elevated in WT cells under treatment with H2O2, it was initially higher in CLIC4-KO cells and remained higher regardless of H2O2 treatment. The compensatory increased expression of SOD1 and SOD2 proteins in CLIC4-KO cells suggests an increase in H2O2 results from the dismutation of superoxide to H2O2 and water. These results indicated that the CLIC4 response could be an essential protection for oxidative stress or H2O2-induced apoptosis in 6DT1 and 4T1 cells. While the loss of CLIC4 was associated with elevated intracellular H2O2, the source of this elevation was not yet defined. In cells, ROS is generated as a byproduct of oxidative phosphorylation by the mitochondria or by NADPH oxidases in cell membranes. Mitochondrial superoxides form from one-electron reduction of molecular oxygen (O2) in complexes I and III (48Fridovich I. Superoxide anion radical (O2-.), superoxide dismutases, and related matters.J. Biol. Chem. 1997; 272: 18515-18517Abstract Full Text Full Text PDF PubMed Scopus (1081) Google Scholar) and their accumulation in cells is highly associated with oxidative stress and redox signaling and may cause damage to proteins containing iron-sulfur clusters (48Fridovich I. Superoxide anion radical (O2-.), superoxide dismutases, and related matters.J. Biol. Chem. 1997; 272: 18515-18517Abstract Full Text Full Text PDF PubMed Scopus (1081) Google Scholar). Previous studies in isolated mitochondria subfractions from rat cardiomyocytes showed that CLIC4 was enriched in the outer mitochondrial membrane fraction with a smaller subfraction partitioning with the inner mitochondrial membrane (49Ponnalagu D. Rao S.G. Farber J. Xin W.Y. Hussain A.T. Shah K. et al.Molecular identity of cardiac mitochondrial chloride intracellular channel proteins.Mitochondrion. 2016; 27: 6-14Crossref PubMed Scopus (56) Google Scholar). In contrast, in situ immunogold electron microscopy (EM) of human keratinocytes indicated that CLIC4 localized mainly with the inner mitochondrial membrane and cristae with occasional gold particles in the mitochondrial matrix (50Fernandez-Salas E. Suh K.S. Speransky V.V. Bowers W.L. Levy J.M. Adams T. et al.mtCLIC/CLIC4, an organellular chloride channel protein, is increased by DNA damage and participates in the apoptotic response to p53.Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (152) Google Scholar). Thus, both methods suggest that CLIC4 is associated with mitochondrial membranes, but functional studies are required to determine if the protein could participate in the regulation of mitochondrial membrane potential. Confocal microscopy using MitoSOX red (Fig. 4A) detected a substantial increase in superoxide generated in CLIC4-null relative to WT 6DT1 cells. FACS histograms of fluorescence intensity (Fig. 4B) and quantification of mean fluorescent intensity (Fig. 4C) confirmed the higher level of superoxide in CLIC4-null cells than WT at baseline, and this difference was mitigated by treatment with NAC. WT 6DT1 cells displayed increased intensity when treated with H2O2 and the fluorescence intensity decreased upon treatment with 50 μM NAC in both genotypes. Since the CLIC4-null 6DT1 cells displayed higher superoxide content, we measured the H2O2 content in cells. Using Amplite colorimetric hydrogen peroxide assay kit, the total content of H2O2 in fresh lysates of CLIC4-KO and WT cells was determined. As shown in Figure 4D, the baseline level of H2O2 in CLIC4-KO cells and those reconstituted with an empty lentiviral vector was significantly higher than the cells that express CLIC4 (WT) and CLIC4-KO cells reconstituted with CLIC4. Treatment with NAC lowered H2O2 levels in all genotypes. Treatment with exogenous H2O2 elevated the intracellular H2O2 in WT cells to the constitutive levels in CLIC4-null cells. NAC was sufficient to decrease the H2O2 levels in all genotypes. Surprisingly, no significant differences were detected in catalase activity in fresh cell lysates among all cell types (Fig. S2A). As shown in Figure 4E, CLIC4-deficient cells had lower peroxidase activity than the cells that express CLIC4 protein. After incubating the cells with 1 μM H2O2 or with both 1 μM H2O2 and 50 μM NAC for 24 h, the peroxidase activity increased specifically in CLIC4-KO cells compared to baseline. This elevation in peroxidase activity in response to H2O2 could explain the unchanged ROS and H2O2 levels in CLIC4-KO cells post-treatment with exogenous H2O2. Using ThiolTracker violet and FACS analysis (Fig. S2B), there were no significant differences detected in GSH level between CLIC4-deficient and the WT 6DT1 cells. Altogether, these results support the concept that in the absence of CLIC4, cells live in a pro-oxidant internal environment. Specific components of mitochondrial respiration were further studied using Seahorse technology. At baseline, overall mitochondrial respiration did not differ among genotypes as measured by oxygen consumption rate (OCR) (Fig. 5, A and B). In contrast, oxygen consumption of CLIC4-KO cells was higher than the WT 24 h after exposure to 1 μM H2O2. Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, an uncoupler of mitochondrial oxidative phosphorylation, maximally stimulated oxygen consumption in CLIC4-KO cells treated with H2O2, suggesting these cells maintained higher spare respiratory capacity. The dependence on CLIC4 was supported by the return to WT levels of spare capacity when the CLIC4-deficient cells were reconstituted with CLIC4 lentivirus but not the empty vector. Shutting down mitochondrial respiration by inhibiting complex I and III with rotenone and antimycin A or reconstituting CLIC4 in CLIC4-KO cells did not reveal any differences in nonmitochondrial oxygen consumption among the genotypes. This result was further confirmed by using a second CLIC4-KO clone (Fig. S3A). Extracellular acidification rate was also assayed by Seahorse to investigate the glycolytic capacity of cells
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