Author response: Cystathionine-β-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Appendix 2 Appendix 3 Data availability References Decision letter Author response Article and author information Metrics Abstract Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display upregulated cystathionine-β-synthase (CBS) expression and enhanced uptake of exogenous cysteine, which lead to increased hydrogen sulfide (H2S) and glutathione (GSH) production, consequently protecting senescent cells from oxidative stress-induced cell death. CBS depletion allows AIS cells to escape senescence and re-enter the cell cycle, indicating the importance of CBS activity in maintaining AIS. Mechanistically, we show this restoration of proliferation is mediated through suppressing mitochondrial respiration and reactive oxygen species (ROS) production by reducing mitochondrial localized CBS while retaining antioxidant capacity of transsulfuration pathway. These findings implicate a potential tumor-suppressive role for CBS in cells with aberrant PI3K/AKT pathway activation. Consistent with this concept, in human gastric cancer cells with activated PI3K/AKT signaling, we demonstrate that CBS expression is suppressed due to promoter hypermethylation. CBS loss cooperates with activated PI3K/AKT signaling in promoting anchorage-independent growth of gastric epithelial cells, while CBS restoration suppresses the growth of gastric tumors in vivo. Taken together, we find that CBS is a novel regulator of AIS and a potential tumor suppressor in PI3K/AKT-driven gastric cancers, providing a new exploitable metabolic vulnerability in these cancers. Editor's evaluation This paper describes a new mechanism of metabolic escape from senescence. In cells undergoing senescence induced by AKT, the enzyme cystathionine β-synthase (CBS) maintains viability in the senescent state. Suppressing CBS results in senescence escape and continued proliferation, through a mechanism involving changes in mitochondrial metabolism. https://doi.org/10.7554/eLife.71929.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Hyperactivation of oncogenic pathways such as RAS/ERK or PI3K/AKT can cause cellular senescence in non-transformed cells, termed oncogene-induced senescence (Serrano et al., 1997; Zhu et al., 2020). In addition to the well-studied RAS-induced senescence (RIS), we and others have demonstrated that hyperactivation of PI3K/AKT signaling pathway causes a senescence-like phenotype, referred to as AKT-induced senescence (AIS) or PTEN loss-induced cellular senescence (Alimonti et al., 2010; Astle et al., 2012; Chan et al., 2020; Jung et al., 2019). AIS is characterized by the common senescence hallmarks including cell cycle arrest, a senescence-associated secretory phenotype (SASP), global transcriptional changes, and metabolic hyperactivity (Chan et al., 2020). Distinct from RIS, AIS does not display either p16 upregulation, a DNA damage response or senescence-associated heterochromatin foci. Instead, AIS is associated with elevated p53 expression through increased mTORC1-dependent translation and reduced human double minute 2 (HDM2) dependent destabilization (Astle et al., 2012). Disruption of the critical mechanisms that regulate maintenance of oncogene-induced senescence can lead to tumorigenesis (Braig et al., 2005; Chen et al., 2005; Collado et al., 2005). Therefore, understanding the molecular mechanisms that regulate AIS and how they are subverted will provide opportunities to identify therapeutic strategies for suppressing PI3K/AKT-driven cancer development. We identified 98 key regulators in a whole-genome siRNA AIS escape screen and validated a subset of these genes in the functional studies to confirm their role in AIS maintenance (Chan et al., 2020). Intriguingly, 11 genes were associated with the regulation of metabolism, suggesting that an altered metabolism could be integral for maintaining AIS. In particular, the cystathionine-β-synthase (CBS) was ranked as one of the top metabolic gene candidates with loss of expression leading to AIS escape (Chan et al., 2020), but how it does so is not known. CBS is an enzyme involved in the transsulfuration metabolic pathway. CBS converts homocysteine (Hcy), a key metabolite in the transmethylation pathway, to cystathionine which is subsequently hydrolyzed by cystathionine gamma-lyase (CTH) to form cysteine, the crucial precursor for GSH production (Figure 1). CBS also catalyzes the production of H2S, a diffusible gaseous transmitter that modulates mitochondrial function and cellular bioenergetics (Szabo et al., 2013; Szabo et al., 2014; Módis et al., 2014), exerts antioxidant effects through inhibition of reactive oxygen species (ROS) generation and lipid peroxidation (Wen et al., 2013), and stimulates antioxidant production via sulfhydration of key proteins involved in antioxidant defense such as Keap1 and p66Shc (Paul and Snyder, 2012; Yang et al., 2013). Thus, CBS acts through control of Hcy, H2S, and GSH metabolism and exerts diverse biological functions including regulating DNA methylation, mitochondrial respiration, and redox homeostasis (Zhu et al., 2018). Figure 1 with 1 supplement see all Download asset Open asset Cystathionine-β-synthase (CBS) expression and transsulfuration pathway activity are elevated in AKT-induced senescence. (A) Schematic diagram illustrating that the cytoplasmic localized CBS regulates transmethylation and transsulfuration metabolic pathways, and mitochondrial localized CBS regulates oxidative phosphorylation. (B) BJ3 human skin fibroblasts expressing telomerase reverse transcriptase (BJ-TERT) cells were transduced with pBabe, myrAKT1, or HRASG12V. On day 6 post-transduction the cells were plated in either full culture medium containing 100 µM cysteine (FM) or cysteine-deficient medium (Cys-free). Western blot analysis was performed on day 10 post-transduction. Vinculin was probed as a loading control. Representative of n=3 experiments. (C) Hydrogen sulfide (H2S) production was measured by AzMC on day 14 post-transduction. Fold changes over pBabe control are presented as mean ± SEM (n=3). One sample t-test compared to the hypothetical value 1.0 was performed (NS, not significant; *p<0.05). (D) Cells were treated with aminoxyacetate (AOAA) 30 μM on day 5 post-transduction. Cell confluency measured by IncuCyte is presented as mean ± SEM (n=3). (E) Cells were cultured in the conditions as described in (B). Cell confluency measured by IncuCyte is presented as mean ± SEM (n=3–5). Statistical significance at the last time point in (D) and (E) was determined by unpaired t-test (**p<0.01; ***p<0.001). Figure 1—source data 1 Unedited immunoblots of Figure 1B. Raw images were acquired using the ChemiDoc system (Bio-Rad). https://cdn.elifesciences.org/articles/71929/elife-71929-fig1-data1-v1.pdf Download elife-71929-fig1-data1-v1.pdf Aberrant CBS expression and/or activity contributes to a wide range of diseases including hyperhomocysteinemia (Kruger, 2017) and cancer (Zhu et al., 2018). CBS plays a complex role in cancer pathogenesis having purported tumor-promoting and -suppressive roles. Activation of CBS promoted tumor growth in colon (Phillips et al., 2017; Szabo et al., 2013), ovarian (Bhattacharyya et al., 2013), breast (Sen et al., 2015), prostate (Liu et al., 2016), and lung cancers (Szczesny et al., 2016), whereas loss of CBS in glioma cells increased tumor volume in vivo (Takano et al., 2014). In addition, the function of CBS in liver cancer remains inconclusive with conflicting reports of both tumor-promoting (Jia et al., 2017; Yin et al., 2012) and -suppressive roles (Kim et al., 2009). These studies underscore the context-dependent roles of CBS in cancer development. In this study we explored the molecular mechanisms underpinning CBS’s role in maintaining AIS and how the loss of CBS promotes AIS escape. The requirement of CBS for the maintenance of AIS implicates it as a putative tumor suppressor during PI3K/AKT pathway-driven tumorigenesis. To gain insight into this, we further characterized the expression level of CBS in gastric cancer tissue samples and cells and sought to define the functional significance of CBS loss in the context of activated PI3K/AKT signaling-driven gastric cancer development. Results CBS expression and transsulfuration pathway activity are elevated in AIS To investigate the mechanisms by which CBS contributes to AIS maintenance, we first evaluated CBS expression and activity in several non-transformed cells with hyperactivated AKT. An increase of CBS protein expression was observed in BJ3 human skin fibroblasts expressing telomerase reverse transcriptase (BJ-TERT) (Figure 1B) and IMR90 human fetal lung fibroblasts (Figure 1—figure supplement 1A) overexpressing myristoylated (myr)-AKT1. In BJ-TERT and human mammary epithelial cells (HMEC), overexpressing AKT1E17K, a clinically relevant activated mutant form of AKT1 in multiple cancer types including breast cancer and ovarian cancer (Carpten et al., 2007), also enhanced CBS protein expression (Figure 1—figure supplement 1B). However, AKT hyperactivation did not affect CBS mRNA expression (Figure 1—figure supplement 1C), suggesting a post-transcriptional regulatory mechanism underpinning increased CBS protein expression. We hypothesized that the increased CBS expression in AKT-hyperactivated cells was associated with upregulation of the transsulfuration pathway activity and cysteine metabolism. We thus examined the expression levels of CTH, a key enzyme in the transsulfuration pathway and xCT, the Xc- amino acid antiporter responsible for the uptake of cystine (an oxidized form of cysteine) (Figure 1A). Both CTH and xCT were upregulated in AIS cells compared to proliferating control cells, suggesting an elevated cysteine synthesis via the transsulfuration pathway and cysteine uptake (Figure 1B). In contrast, senescent cells expressing HRASV12 cells exhibited a moderate increase of CBS and CTH expression levels. The expression level of xCT was also slightly upregulated during RIS, albeit to a lesser extent than during AIS, in line with the finding that upregulation of xCT facilitates RAS-mediated transformation (Lim et al., 2019). To assess transsulfuration pathway activity, we measured H2S production. A significant increase in transsulfuration pathway activity was observed in BJ-TERT fibroblasts upon AKT but not HRAS hyperactivation (Figure 1C), suggesting that activation of transsulfuration pathway is a specific cellular response to constitutive activation of AKT. Inhibition of H2S production by aminoxyacetate (AOAA) (Szabo, 2016) impaired cell proliferation (Figure 1D) and increased SA-βGal activity (Figure 1—figure supplement 1D) of BJ-TERT cells overexpressing myrAKT1. This result suggests that H2S, the major metabolite downstream of the transsulfuration pathway, has a protective effect on AIS cells although the actions of AOAA on other PLP-dependent enzymes cannot be excluded (Asimakopoulou et al., 2013; Hellmich et al., 2015; Szabo et al., 2013). Cysteine starvation has been reported to induce necrosis and ferroptosis in cancer cells (Chen et al., 2017). Since the transsulfuration pathway mediates de novo cysteine synthesis, an increase in transsulfuration pathway activity may support the survival of AIS cells upon cysteine limitation. Consistent with our hypothesis, cysteine deprivation potently increased the expression of CBS and CTH in AIS cells (Figure 1B) and did not affect the survival of AIS cells (Figure 1E), indicating that increased cysteine level in AIS cells due to elevated transsulfuration pathway activity is critical for cell viability. Depletion of CBS promotes escape from AIS While our results suggest a protective role of transsulfuration pathway for the survival of AIS cells, our AIS siRNA screen showed CBS loss enabled cells to escape from AIS evidenced by an increase in cell numbers. To validate the function of CBS in AIS maintenance, we depleted CBS using two independent small hairpin RNAs (shRNAs) in AIS cells (Figure 2A). Loss of CBS in AIS cells significantly decreased the proportion of cells with SA-βGal activity, increased EdU incorporation, and enhanced colony formation, demonstrating an essential role of CBS in AIS maintenance (Figure 2B). To further confirm the on-target specificity of the knockdown, we generated BJ-TERT cells expressing a doxycycline-inducible CBS shRNA and an shRNA-resistant 4-OHT-inducible estrogen receptor (ER)-tagged CBS fusion (Figure 2C and Figure 2—figure supplement 1A). Upon expressing myrAKT1, these cells underwent AIS, as indicated by a significant increase in SA-βGal-positive cells and decrease in EdU-positive cells and, consistent with the finding in the AIS escape siRNA screen, CBS depletion induced by doxycycline decreased SA-βGal activity, and increased EdU incorporation in AIS cells (Figure 2D). Importantly, simultaneously expressing ER-CBS prevented senescence escape of CBS-depleted cells, confirming the on-target specificity of the knockdown and the critical role of CBS in maintaining AIS (Figure 2D and Figure 2—figure supplement 1B). Modulation of CBS expression in proliferating control cells did not affect the percentage of SA-βGal- and EdU-positive cells (Figure 2D), suggesting that the effect of CBS depletion on cell proliferation is specific for AIS cells. Similar to the findings in BJ-TERT cells, AKT1 hyperactivation also caused senescence in IMR-90 lung fibroblasts (Figure 2—figure supplement 1C). CBS knockdown in AIS cells significantly suppressed SA-βGal staining and enhanced EdU incorporation but not in proliferating cells. Knockdown of CBS in BJ-TERT cells with constitutive RAS activation did not affect colony formation, suggesting CBS has a specific regulatory role for AIS but not RIS maintenance (Figure 2E and Figure 2—figure supplement 1D). Figure 2 with 1 supplement see all Download asset Open asset Depletion of cystathionine-β-synthase (CBS) promotes escape from AKT-induced senescence. (A and B) BJ3 human skin fibroblasts expressing telomerase reverse transcriptase (BJ-TERT) cells expressing myrAKT1 were transduced with pGIPZ-shCBS or non-silencing small hairpin RNA (shRNA) control (shCtrl) on day 6 post-transduction of myrAKT1. (A) Western blot analysis was performed on day 8 post-transduction of shRNA. Representative of n=2 experiments. (B) Images and quantification of the percentage of cells with positive staining for SA-βGal activity and EdU incorporation on day 8 post-transduction of shRNA, as well as colony formation assay on day 14 post-transduction of shRNA. Data is presented as mean ± SEM (n=3). One-way analysis of variance (ANOVA) with Holm-Šídák’s multiple comparisons was performed (**p<0.01; ***p<0.001). Relative colony area normalized to shCtrl group is presented as mean ± SEM (n=3) and one sample t-test compared to the hypothetical value 1.0 was performed (C–D) BJ-TERT cells expressing doxycycline-inducible CBS shRNA#1 and 4-OHT-inducible CBS were transduced with pBabe or myrAKT1, treated with ±doxycycline (1 μg/ml)±4 OHT (20 nM) on day 5 post-transduction and analyzed on day 14 post-transduction. (C) Western blot analysis of CBS expression. Actin was probed as a loading control. (D) The percentage of cells with positive staining for SA-βGal activity or EdU proliferation marker incorporation is presented as mean ± SEM (n=3). Two-way ANOVA with Holm-Šídák’s multiple comparisons was performed (NS, not significant; ****p<0.0001). (E) BJ-TERT cells expressing doxycycline-inducible CBS shRNA were transduced with HRASG12V, treated with doxycycline (1 μg/ml) on day 5 post-transduction and colony formation assay analyzed on day 14 post-transduction. Relative colony area normalized to doxycycline-untreated group is presented as mean ± SEM (n=3) and one-sample t-test compared to the hypothetical value 1.0 was performed (NS, not significant). (F) BJ-TERT cells expressing doxycycline-inducible shCBS or control shREN were transduced with pBabe or myrAKT1 and then treated with doxycycline (1 μg/ml) on day 5 post-transduction. Western blot analysis was performed on day 14 post-transduction. Vinculin was probed as a loading control. Representative of n=2–4 experiments. Figure 2—source data 1 Unedited immunoblots of Figure 2A. Raw images were acquired using the ChemiDoc system (Bio-Rad). https://cdn.elifesciences.org/articles/71929/elife-71929-fig2-data1-v1.pdf Download elife-71929-fig2-data1-v1.pdf Figure 2—source data 2 Unedited immunoblots of Figure 2C. Raw images were acquired using the ChemiDoc system (Bio-Rad). https://cdn.elifesciences.org/articles/71929/elife-71929-fig2-data2-v1.pdf Download elife-71929-fig2-data2-v1.pdf Figure 2—source data 3 Unedited immunoblots of Figure 2F. Raw images were acquired using the ChemiDoc system (Bio-Rad). https://cdn.elifesciences.org/articles/71929/elife-71929-fig2-data3-v1.pdf Download elife-71929-fig2-data3-v1.pdf To determine the mechanisms by which CBS depletion causes escape, we examined the impact on key senescence hallmarks. While loss of CBS released AIS cells from cell cycle arrest, knockdown of CBS did not significantly change the mRNA and protein expression level of several key SASP-related genes including IL1A, IL1B, IL6, and IL8, which are upregulated during AIS (Astle et al., 2012; Chan et al., 2020; Figure 2—figure supplement 1E and F). Given the p53 and Retinoblastoma protein (Rb) pathways predominantly control senescence-mediated cell cycle arrest, we examined signaling downstream of AKT activation in the presence and absence of CBS (Figure 2F). Consistent with our previous findings, p53 and its downstream target p21 were upregulated during AIS, but depletion of CBS had no effect on these levels. Furthermore, total Rb, a key regulator of the G1/S phase transition, and its inhibitory phosphorylated form at serine 807/811 were markedly suppressed in AIS and partially rescued upon CBS knockdown. Cyclin A, which mediates S to G2/M phase cell cycle progression, was also upregulated upon depleting CBS in AIS cells. These results demonstrate that CBS depletion can restore the proliferation of cells that have undergone AIS in a p53-independent manner. Depletion of CBS in AIS cells does not affect cysteine and GSH abundance in cysteine-replete conditions CBS is the key enzyme regulating the transsulfuration and transmethylation pathways. By analysis of the data from the AIS escape siRNA screen, we found that except CBS, siRNA knockdown of other genes involved in the transsulfuration and transmethylation pathway did not significantly affect AIS cell numbers (robust Z score <2, Figure 3—figure supplement 1A). Therefore, it is likely that AIS escape in cysteine-replete conditions upon CBS loss is through a transsulfuration/transmethylation pathway-independent mechanism. In addition, knockdown of CBS did not affect the expression levels of CTH and xCT when BJ-TERT cells were grown in cysteine-replete culture medium (Figure 3—figure supplement 1B). To explore metabolic alterations that are associated with CBS-mediated AIS maintenance, we performed gas chromatography mass spectrometry (GC/MS)-based untargeted metabolomics. AIS (myrAKT1-shCtrl), AIS-escaped (myrAKT1-shCBS), and control proliferating cells (pBabe-shCtrl) displayed distinct metabolic profiles, as indicated by principal component analysis (Figure 3—figure supplement 1C and D). AIS cells showed increased abundance of serine, glycine, and glutamate, the metabolites involved in transsulfuration pathway and GSH synthesis (Figure 3—figure supplement 1F). Loss of CBS did not affect abundance of these metabolites but resulted in an increased level of Hcy, suggesting loss of CBS causes an accumulation of the upstream substrate Hcy (Figure 3—figure supplement 1F). To further determine the activity of the transsulfuration pathway in AIS cells and the impact of CBS loss, we performed a stable isotope tracing assay using serine labeled at the third carbon position ([3–13C] L-serine) followed by liquid chromatography mass spectrometry (LC/MS) after thiol derivatization with N-ethylmaleimide (Figure 3A). This tracer has been reported to incorporate into the cellular GSH pool via transsulfuration-derived cysteine (Zhu et al., 2019). We replaced all the serine in the culture medium with [3–13C] L-serine. After 6 hr of labeling, a substantial fraction of [3–13C] L-serine was detected intracellularly and in the cystathionine pool in proliferating (pBabe-siOTP), AIS (myrAKT1-siOTP), and AIS-escaped (myrAKT1-siCBS) cells (Figure 3B and C). We did not detect [3–13C] L-serine incorporation into cysteine and GSH in proliferating cells, possibly due to the short time period of metabolic labeling (Figure 3D and E). However, AIS cells displayed a small but significant fraction of 13C labeled cysteine and GSH along with a significant increase of total levels of serine, cysteine, and GSH (Figure 3B-E), indicating upregulation of transsulfuration pathway activity during AIS. Consistent with the role of CBS in catalyzing de novo cystathionine synthesis, a significant decrease of cystathionine abundance was observed in CBS-depleted AIS cells. Notably, the abundance of cysteine and GSH was not affected by CBS depletion (Figure 3D and E) and neither was H2S production (Figure 3F). We hypothesized that CBS-depleted cells maintained the cysteine and GSH pools via increase of cysteine uptake from the culture medium. Indeed, deprivation of cysteine from the medium markedly diminished the intracellular cysteine and GSH abundance in AIS cells (Figure 3D and E). On the other hand, increase of cystathionine was observed in the cysteine-depleted conditions (Figure 3C), possibly attributed to a marked upregulation of CBS expression observed in AIS cells after cysteine deprivation (Figure 1B). This result suggests that cells enhance CBS-mediated transsulfuration pathway activity in response to cysteine deficiency. If cells rely on exogenous cysteine to maintain cysteine and GSH pools, blocking cysteine import would impair GSH synthesis and antioxidant capacity and consequently reduce cell viability. Consistent with this hypothesis, the viability of both proliferating cells and AIS cells was decreased after treatment with erastin, an inhibitor of cystine-glutamate antiporter Xc-, and further suppressed by CBS knockdown (Figure 3—figure supplement 1F). Taken together, these results conclusively demonstrate that exogenous cysteine is the major source for GSH synthesis and AKT overexpression increases cysteine import and the subsequent GSH abundance. As the loss of CBS does not affect intracellular cysteine and GSH levels, senescence escape upon CBS depletion is mediated by the mechanisms independent of the transsulfuration pathway. Figure 3 with 1 supplement see all Download asset Open asset Depletion of cystathionine-β-synthase (CBS) in AKT-induced senescence (AIS) cells does not affect cysteine and glutathione (GSH) abundance in cysteine-replete conditions. (A) Schematic of [3–13C] serine isotope tracing. Gray circles indicate 13C carbon atoms. Clear circles indicate unlabeled carbon atoms. (B–E) BJ3 human skin fibroblasts expressing telomerase reverse transcriptase (BJ-TERT) cells were transduced with pBabe or myrAKT1. After 6 days cells were transfected with either CBS siRNA (siCBS) or control siRNA (siOTP). On day 3 post-siRNA transfection, cells were cultured in either full medium (FM) or cysteine-deficient medium (Cys-Free) for 48 hr. Six hours before harvest, the culture medium was replaced with the basal isotope labeling medium containing 400 μM [3–13C] serine. The thiol redox metabolome was assessed by targeted liquid chromatography mass spectrometry (LC/MS). The abundances of labeled and unlabeled metabolites including (B) serine, (C) cystathionine, (D) NEM-Cys, and (E) NEM-GSH normalized with cell number are presented as mean ± SEM (n=4). Statistical significance was determined by one-way analysis of variance (ANOVA) with Holm-Šídák’s multiple comparisons (for the total metabolite levels, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; and for the M+1 metabolite levels, ##p<0.01; ###p<0.001; ####p<0.0001 compared to pBabe-siNTP_FM cells). (F) BJ-TERT cells expressing doxycycline-inducible shCBS were transduced with myrAKT1, and treated with doxycycline (1 μg/ml) on day 5 post-transduction. Hydrogen sulfide (H2S) production was measured by AzMC on day 14 post-transduction. Fold changes over doxycycline-untreated group are presented as mean ± SEM (n=3). One sample t-test compared to the hypothetical value 1.0 was performed (NS, not significant). (G and H) Gene set enrichment analysis of RNA-seq data showing downregulation of hallmark of oxidative phosphorylation and reactive oxygen species pathways in myrAKT1-shCBS cells compared with myrAKT1-shCtrl cells. To investigate the transsulfuration pathway-independent molecular mechanisms underlying CBS-mediated AIS maintenance, we characterized the transcriptomic changes upon depleting CBS during AIS. Differential gene expression analysis of AIS-escaped cells (AIS-shCBS) compared with AIS cells (AIS-shCtrl) revealed 404 genes were significantly upregulated (adjusted p-value < 0.05, Log2FC > 1) and 181 genes significantly downregulated (adjusted p-value < 0.05, Log2FC < –1) (Figure 3—figure supplement 1G). Gene set enrichment analysis (GSEA) using the hallmark gene sets in the molecular signatures database (MSigDB) identified that pathways involved in oxidative phosphorylation and ROS were significantly downregulated in CBS-depleted AIS cells compared to the control AIS cells (Figure 3G and H). Therefore, altered mitochondrial energy metabolism and ROS production may contribute to CBS-dependent AIS maintenance. CBS mitochondrial localization is required for AIS maintenance CBS has been reported to localize to both the cytoplasm and mitochondria and regulate mitochondrial function and ATP synthesis via H2S (Bhattacharyya et al., 2013; Panagaki et al., 2019). Consistent with this, we also observed CBS localization in the mitochondria by immunofluorescent cell staining (Figure 4A and B). AIS cells exhibited elevated mitochondrial abundance as indicated by increased intensity of MitoTracker-Deep Red staining compared to proliferating cells (Figure 4A), consistent with an increased abundance of proteins involved in the mitochondrial electron transport chain as detected by Western blotting (Figure 4—figure supplement 1A). The mitochondrial localization of CBS was further supported by Western blotting of mitochondrial extracts isolated from AIS and proliferating cells (Figure 4C). AIS cells displayed increased mitochondrial CBS abundance. To further validate CBS localization in mitochondria, we performed a protease protection assay using mitochondria isolated from cells expressing wild type CBS fused to a C-terminal FLAG tag (Figure 4D). The C-terminally FLAG-tagged CBS was present in intact mitochondria and was resistant to protease treatment and only degraded upon membrane solubilization by Triton X-100. A similar result was observed for the mitochondrial ATP synthase F1 subunit alpha ATP5A. Figure 4 with 1 supplement see all Download asset Open asset Cystathionine-β-synthase (CBS) mitochondrial localization is required for AKT-induced senescence (AIS) maintenance. (A–B) BJ3 human skin fibroblasts expressing telomerase reverse transcriptase (BJ-TERT) cells were transduced with pBabe or myrAKT1. Immunofluorescent staining showing CBS (green) and mitochondria (red) on day 10 post-transduction. The representative images are from one of two independent experiments. Scale bar = 20 μm. (B) Quantification of signal intensities using ImageJ by applying a single ROI to two color channels in the same image and extracting the plot profile. (C) Western blot analysis of CBS expression in the cytoplasmic and mitochondrial fractions isolated from BJ-TERT cells transduced with pBabe or myrAKT1. ATP5A and vinculin serve as the markers of mitochondria and cytoplasm, respectively. (D) Western blot analysis of a protease protection assay using the mitochondrial fraction isolated from BJ-TERT cells expressing C-terminal FLAG-tagged CBS. ATP5A serves as a positive control. (E) Schematic of 4-OHT-inducible plasmids expressing FLAG-tagged wild type CBS (WT) or a C-terminal regulatory domain CBSD2 truncated CBS mutant. (F) Western blot analysis of CBS expression in the cytoplasmic and mitochondrial fractions isolated from BJ-TERT cells transduced with FLAG-tagged wild type (WT) or a truncated mutant CBS after 20 nM 4-OHT induction for 3 days. ATP5A and vinculin serve as the markers of mitochondria and cytoplasm, respectively. (C), (D), and (F) are representative of at least n=3 experiments. (G–H) BJ-TERT cells expressing doxycycline-inducible CBS shRNA#2 and 4-OHT-inducible CBS wide type or a truncated mutant were transduced with myr-AKT1, treated with doxycycline (1 μg/ml)±4 OHT (20 nM) on day 5 post-transduction and analyzed on day 12 post-transduction. The percentage of cells with positive staining for (G) EdU proliferation marker incorporation or (H) SA-βGal activity is expressed as mean ± SEM (n=3). One-way analysis of variance (ANOVA) with Holm-Šídák’s multiple comparisons was performed (NS, not significant; **p<0.01; ***p<