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

  1. Haoran Zhu
  2. Keefe T Chan
  3. Xinran Huang
  4. Carmelo Cerra
  5. Shaun Blake
  6. Anna S Trigos
  7. Dovile Anderson
  8. Darren J Creek
  9. David P De Souza
  10. Xi Wang
  11. Caiyun Fu
  12. Metta Jana
  13. Elaine Sanij
  14. Richard B Pearson
  15. Jian Kang

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    Evaluation Summary:

    Kang et al. studied the role of cystathionine beta-synthase , an enzyme involved in homocysteine catabolism, in the senescent state imposed by oncogenic Akt. They find that this enzyme facilitates the acquisition of features of senescence, and is frequently silenced in tumors, whereas re-expressing it reduces cell proliferation. This manuscript is potentially of interest to cancer biologists, particularly those studying oncogene-induced senescence and mechanisms of senescence escape in cancers.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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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 (H 2 S) 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.

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  1. Version published to 10.7554/elife.71929 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    Kang et al. studied the role of cystathionine beta-synthase (CBS), an enzyme involved in homocysteine catabolism, in the senescent state stimulated by Akt. They report that Akt induces expression of CBS and other enzymes necessary to convert homocysteine into cysteine, and that blocking CBS enhances cell proliferation and reduces beta-galactosidase expression. Mechanistic studies reveal that Akt activates several markers of mitochondrial metabolism, including respiration, and that CBS silencing mitigates this change and reduces reactive oxygen species. Analysis of human gastric tumors reveals methylation of the CBS locus and reduced CBS expression relative to nonmalignant gastric mucosa. Finally, reexpressing CBS in gastric cancer cells reduces growth and Ki67 staining in xenografts. The authors conclude that CBS is a required component of the Akt-induced senescence pathway, and that reducing CBS expression is a mechanism by which some cancers suppress senescence and promote growth. Overall, the paper describes an interesting metabolic process of oncogene-induced senescence that appears selective for Akt. Few such mechanisms have been described, so a thorough exploration of CBS's role in senescence could be impactful. The authors succeed in showing that manipulating CBS expression in a limited number of models has substantial effects on senescence and growth. However, not all of the conclusions are supported by the data in the current version of the paper, the metabolic analysis of CBS's function in Akt-expressing cells is incompletely characterized, and some central aspects of the overall mechanism (particularly the relevance of CBS to mitochondrial respiration) are unexplained.

    Specific comments:

    1. CBS expression is induced upon Akt activation, but there needs to be better evidence that activity of the pathway has changed. The metabolomics results are not very convincing, as siCBS has no or minimal effects on some metabolite pools that should respond. An isotope tracing study would help here.

    We thank the reviewer for the suggestions. We performed a [3-13C] L-serine tracing analysis by LC/MS in proliferating cells, AKT-induced senescent (AIS) cells, and AIS cells with CBS knockdown in cysteine-replete and depleted conditions. The results shown in Figure 3A-3E of the revised manuscript.

    The tracer [3-13C] L-serine has been reported to incorporate into the cellular GSH pool via transsulfuration-derived cysteine (Zhu et al., 2019) (Figure A). We replaced all the serine in the culture medium with [3-13C] L-serine. After six hours of labelling, 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 B and C). We did not detect [3-13C] L-serine incorporation into cysteine and GSH in the proliferating cells, possibly due to the short time period of metabolic labelling (Figure D and E). However, AIS cells displayed a small but significant fraction of 13C labelled cysteine and GSH along with a significant increase of total levels of serine, cysteine and GSH (Figure BE), supporting the upregulation of transsulfuration pathway activity in 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 escaped cells. Notably, the abundance of cysteine and GSH (Figure D and E) was not affected by CBS depletion. 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 D and E). On the other hand, increase of cystathionine was observed under the cysteinedepleted conditions (Figure C), possibly attributed to a marked upregulation of CBS expression observed in AIS cells after cysteine deprivation (Figure 1B in the revised manuscript). This result thus suggests that cells enhance CBS-mediated transsulfuration pathway activity in response to cysteine deficiency.

    Collectively, our results indicate that cells rely on exogenous cysteine for GSH synthesis and AKT overexpression increases cysteine import and the subsequent GSH abundance which is not affected by loss of CBS.

    1. Furthermore, the AOAA experiments are hard to interpret. This drug is a promiscuous transaminase inhibitor, so its effects on cell confluency are not surprising, and it is unclear which particular aspect of metabolism is responsible for the effect. A genetic experiment silencing the relevant transaminase would be more informative.

    We agree that the pharmacological action of AOAA is not limited to suppression of the CBS/ H2S axis. It binds irreversibly to the cofactor PLP, and therefore in addition to CBS, it also inhibits other PLP-dependent enzymes such as CTH, 3-MST, and GOT1. We therefore have modified our statement in the manuscript to be “this result suggested 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.

    We further analysed the data from the AIS-escape siRNA screen and presented these data in Figure 3-figure supplement 1A.

    We found that except CBS, siRNA knockdown of other genes involved in the transsulfuration pathway did not significantly affect AIS cell numbers (robust Z score < 2). Therefore, it is likely that AIS escape in cysteine-replete conditions by loss of CBS is through a transsulfuration/transmethylation pathway-independent mechanism.

    3)The GC/MS data in Fig. 3L are misleading, as the range on the color scale goes from FDR of 0.0504 to 0.0498. Also, the authors claim that CBS regulates the malate-aspartate shuttle, but no mechanism is proposed and this is not intuitive.

    The altered activity of malate-aspartate shuttle is only based on the changes in glutamate and aspartate levels, as measured by GC-MS metabolomics analysis. We agree that these metabolite changes are not sufficient to support the specificity of malate-aspartate shuttle being involved in CBS-mediated metabolic alterations. Therefore, for clarity we have decided to remove this figure and the relevant text from the manuscript.

    1. CBS's role in modulating mitochondrial function is complicated, but its ability to sustain OxPhos and ROS seem to underlie its effects on AIS. The key unanswered question is how CBS promotes OxPhos in these models.

    To determine the mechanisms underlying the increased oxidative phosphorylation and ROS in CBS deficient cells, we investigated the mitochondrial localization of CBS. In addition to the immunofluorescent data showing localization of CBS in the mitochondria (Figure 4A), in the revised manuscript, we generated lentiviral expression vectors encoding wild type CBS and N-terminal and C-terminally truncated mutants. We showed that, consistent with a previous study (Teng et al., PNAS 2013), the C-terminal CBSD2 motif is required for CBS mitochondrial localization (Figure 4C-4F). We then reconstituted CBS-depleted AIS escaped cells with wild type CBS or a C-terminally truncated mutant (Δ468-551). Expression of wild type CBS prevented AIS escape while cells expressing the truncation mutant still escaped from AIS (Figure 4G and 4H), demonstrating that mitochondrial localization of CBS is required to maintain AIS. Consistent with these findings, the Seahorse analysis showed that reconstitution with wild type CBS rescued basal OCR and ATP production levels in CBSdepleted AIS cells. In contrast, AIS cells expressing C-terminally truncated CBS protein failed to restore basal OCR and ATP production. Collectively, our results support the concept that AKT overexpression promotes CBS translocation to mitochondria, increases oxidative phosphorylation and ROS production to sustain the senescence state.

    Reviewer #3 (Public Review):

    In the manuscript by Zhu, Haoran et al., titled "Cystathionine-β-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation", the authors investigated the contributions of cystathionine-β-synthase (CBS) to AKT-induced senescence (AIS) and the potential mechanisms which drove these phenotypes. The authors showed that AKT hyperactivation (using myristoylated AKT) promoted H2S production and treatment with a compound (AOAA) that blocked H2S production, reduced proliferation, and promoted senescence in cells with hyperactivated AKT, compared to normally proliferating cells or cells that have expressed other oncogenes (i.e., HRAS). Next, they used genetic approaches (both knockdown of CBS and rescue experiments with reexpression of CBS in CBS-knockdown cells) to clearly demonstrate that CBS was required for AIS and loss of CBS promoted AIS-escape. The authors then extended these findings to patient tumors and in vivo systems. They found reduced CBS expression in gastric cancer samples compared to matched normal samples and that the reduced expression was due to hypermethylation of DNA encoding CBS. Finally, they found that CBS functions as a tumor suppressor in gastric cancer cells by showing that depletion of CBS promoted colony formation, and overexpression of CBS blocked tumor growth in vivo. This is a very strong study with relevance to numerous research fields. However, a major weakness of the study is the proposed mechanism by which CBS functions in AIS-escape, as the data are largely not supported by the mechanistic conclusions.

    1. In Figure 1, the authors show that AIS cells are unaffected by cysteine depletion and conclude, "Furthermore, cysteine deprivation potently increased the expression levels of CBS and CTH in AIS cells (Figure 1B) and did not affect the survival of AIS cells, consistent with increased cysteine synthesis due to elevated CBS expression being critical for cell viability (Figure 1E)". Although the authors show in Fig. 3F that cysteine levels are elevated in AIS cells compared to control cells in cystine-replete media, they do not measure cysteine synthesis via the transsulfuration pathway in AIS and control cells in cystine-replete and cystine-depleted media.

    Please see our response to the question #1 from the Reviewer 1.

    1. The metabolic changes presented in Figure 3 are unclear. The authors state, "Depletion of CBS in AIS cells increases GSH metabolism in cysteine-replete condition", but it is not clear what "GSH metabolism" means, especially for the AIS-related phenotypes. Further, the authors appear to use "GSH metabolism" interchangeable with GSH synthesis; in the Discussion, they state, "In this study we uncovered another mechanism of AKT-mediated ROS detoxification by upregulation of transsulfuration pathway activity and enhancing glutathione and H2S synthesis (Fig.4H)." These conclusions are not supported by the findings presented in Figure 3 that show GSH levels are unchanged between control, AIS, and CBSdepleted AIS cells. While the authors show an increased abundance of the GSH precursor gamma-glutamylcysteine and the GSH catabolic product cysteinylglycine, how CBS would alter these metabolites are unclear. Additionally, they show that H2S levels are unaffected by CBS depletion, which further confounds the conclusions.

    To determine the transsulfuration pathway activity in AIS cells and the effect of CBS loss, we performed a stable isotope tracing assay followed by LC-MS assay using [3-13C] L-serine. Please see our response to the question #1 from the Reviewer 1.

  3. eLife

    Evaluation Summary:

    Kang et al. studied the role of cystathionine beta-synthase , an enzyme involved in homocysteine catabolism, in the senescent state imposed by oncogenic Akt. They find that this enzyme facilitates the acquisition of features of senescence, and is frequently silenced in tumors, whereas re-expressing it reduces cell proliferation. This manuscript is potentially of interest to cancer biologists, particularly those studying oncogene-induced senescence and mechanisms of senescence escape in cancers.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Kang et al. studied the role of cystathionine beta-synthase (CBS), an enzyme involved in homocysteine catabolism, in the senescent state stimulated by Akt. They report that Akt induces expression of CBS and other enzymes necessary to convert homocysteine into cysteine, and that blocking CBS enhances cell proliferation and reduces beta-galactosidase expression. Mechanistic studies reveal that Akt activates several markers of mitochondrial metabolism, including respiration, and that CBS silencing mitigates this change and reduces reactive oxygen species. Analysis of human gastric tumors reveals methylation of the CBS locus and reduced CBS expression relative to nonmalignant gastric mucosa. Finally, re-expressing CBS in gastric cancer cells reduces growth and Ki67 staining in xenografts. The authors conclude that CBS is a required component of the Akt-induced senescence pathway, and that reducing CBS expression is a mechanism by which some cancers suppress senescence and promote growth. Overall, the paper describes an interesting metabolic process of oncogene-induced senescence that appears selective for Akt. Few such mechanisms have been described, so a thorough exploration of CBS's role in senescence could be impactful. The authors succeed in showing that manipulating CBS expression in a limited number of models has substantial effects on senescence and growth. However, not all of the conclusions are supported by the data in the current version of the paper, the metabolic analysis of CBS's function in Akt-expressing cells is incompletely characterized, and some central aspects of the overall mechanism (particularly the relevance of CBS to mitochondrial respiration) are unexplained.

    Specific comments:

    1. CBS expression is induced upon Akt activation, but there needs to be better evidence that activity of the pathway has changed. The metabolomics results are not very convincing, as siCBS has no or minimal effects on some metabolite pools that should respond. An isotope tracing study would help here.

    2. Furthermore, the AOAA experiments are hard to interpret. This drug is a promiscuous transaminase inhibitor, so its effects on cell confluency are not surprising, and it is unclear which particular aspect of metabolism is responsible for the effect. A genetic experiment silencing the relevant transaminase would be more informative.

    3. The GC/MS data in Fig. 3L are misleading, as the range on the color scale goes from FDR of 0.0504 to 0.0498. Also, the authors claim that CBS regulates the malate-aspartate shuttle, but no mechanism is proposed and this is not intuitive.

    4. CBS's role in modulating mitochondrial function is complicated, but its ability to sustain OxPhos and ROS seem to underlie its effects on AIS. The key unanswered question is how CBS promotes OxPhos in these models.

  5. eLife

    Reviewer #2 (Public Review):

    This work evaluates the role of cystathione beta-synthase (CBS) as a regulatory tumor suppressor in AKT-activated tumors. The authors previously conducted a genome-wide siRNA screen for genes whose removal promotes escape from AKT-induced senescence (AIS), in which CBS was identified as top hit. Here, they validate the role of CBS in AIS escape in a number of cell lines, probe the mechanism of escape through metabolomic and transcriptomic analysis, and examine CBS levels in human gastric tumor samples. Overall, the presence of CBS loss in human samples is well-validated, and its impact on tumorigenesis is presented in a xenograft model. However, the authors' conclusions regarding the biological mechanism are not well-supported by the presented data and techniques.

    Strengths:
    The authors validate the role of CBS loss in escape from AIS, using appropriate knockdown cell lines and rescue experiments. Interestingly, CBS's function is specific to AIS, and not other forms of senescence, in particular RAS-induced senescence. These results validate their identification of CBS in their previously published genome-wide screen.

    The authors provide strong data (Fig. 5) that CBS loss is frequent in gastric cancer tumor samples and gastric cancer cell lines; due to mutations, and promoter hypermethylation.

    The authors provide strong data (Fig. 6) that CBS loss is sufficient to promote oncogenic transformation and tumorigenesis, making use of a gastric epithelial cell line (GES-1), and AGS cancer cells (which harbor AKT activation and CBS loss). These results correlate well with the frequent loss of CBS observed in human gastric tumors (Fig. 5), and support a role for CBS as a tumor suppressor in AKT-activated gastric cancer.

    Weaknesses:
    The primary weakness of the manuscript lies within the author's exploration of mechanism: i.e., how CBS loss promotes cancer.

    First: Upon removal of CBS, the authors do not observed expected changes in levels of transsulfuration and glutathione-related metabolites: for instance, cystathionine, cysteine and glutathione, and H2S are not depleted. The manuscript does not probe or explain the underlying compensatory mechanisms for these metabolite abundances in the setting of CBS loss.

    Second: Based on the above, the authors propose a mitochondrial role for CBS in regulating CBS senescence. However, their experiments do not convincingly demonstrate the existence of mitochondrially-localized CBS: their fluorescence-based imaging is not sufficiently conclusive, and no alternative methods are used to validate mitochondrial localization of CBS.

    Third: Critically, the authors do not demonstrate that the mitochondrial form of CBS is required or sufficient for the observed effects on oxidative phosphorylation and ROS generation, as well as the escape from AIS. Without this demonstration, the proposed mechanism is not well supported.

  6. eLife

    Reviewer #3 (Public Review):

    In the manuscript by Zhu, Haoran et al., titled "Cystathionine-β-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation", the authors investigated the contributions of cystathionine-β-synthase (CBS) to AKT-induced senescence (AIS) and the potential mechanisms which drove these phenotypes. The authors showed that AKT hyperactivation (using myristoylated AKT) promoted H2S production and treatment with a compound (AOAA) that blocked H2S production, reduced proliferation, and promoted senescence in cells with hyperactivated AKT, compared to normally proliferating cells or cells that have expressed other oncogenes (i.e., HRAS). Next, they used genetic approaches (both knockdown of CBS and rescue experiments with re-expression of CBS in CBS-knockdown cells) to clearly demonstrate that CBS was required for AIS and loss of CBS promoted AIS-escape. The authors then extended these findings to patient tumors and in vivo systems. They found reduced CBS expression in gastric cancer samples compared to matched normal samples and that the reduced expression was due to hypermethylation of DNA encoding CBS. Finally, they found that CBS functions as a tumor suppressor in gastric cancer cells by showing that depletion of CBS promoted colony formation, and overexpression of CBS blocked tumor growth in vivo. This is a very strong study with relevance to numerous research fields. However, a major weakness of the study is the proposed mechanism by which CBS functions in AIS-escape, as the data are largely not supported by the mechanistic conclusions.

    1. In Figure 1, the authors show that AIS cells are unaffected by cysteine depletion and conclude, "Furthermore, cysteine deprivation potently increased the expression levels of CBS and CTH in AIS cells (Figure 1B) and did not affect the survival of AIS cells, consistent with increased cysteine synthesis due to elevated CBS expression being critical for cell viability (Figure 1E)". Although the authors show in Fig. 3F that cysteine levels are elevated in AIS cells compared to control cells in cystine-replete media, they do not measure cysteine synthesis via the transsulfuration pathway in AIS and control cells in cystine-replete and cystine-depleted media.

    2. The metabolic changes presented in Figure 3 are unclear. The authors state, "Depletion of CBS in AIS cells increases GSH metabolism in cysteine-replete condition", but it is not clear what "GSH metabolism" means, especially for the AIS-related phenotypes. Further, the authors appear to use "GSH metabolism" interchangeable with GSH synthesis; in the Discussion, they state, "In this study we uncovered another mechanism of AKT-mediated ROS detoxification by upregulation of transsulfuration pathway activity and enhancing glutathione and H2S synthesis (Fig.4H)." These conclusions are not supported by the findings presented in Figure 3 that show GSH levels are unchanged between control, AIS, and CBS-depleted AIS cells. While the authors show an increased abundance of the GSH precursor gamma-glutamylcysteine and the GSH catabolic product cysteinylglycine, how CBS would alter these metabolites are unclear. Additionally, they show that H2S levels are unaffected by CBS depletion, which further confounds the conclusions.

  7. Version published to 10.1101/2021.07.04.451041v1 on bioRxiv