Review
doi: 10.1016/j.redox.2022.102331.
Epub 2022 May 10.
Emerging roles of cystathionine β-synthase in various forms of cancer
Affiliations
- PMID: 35618601
- PMCID: PMC9168780
- DOI: 10.1016/j.redox.2022.102331
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Review
Emerging roles of cystathionine β-synthase in various forms of cancer
Redox Biol.
2022 Jul.
Abstract
The expression of the reverse transsulfuration enzyme cystathionine-β-synthase (CBS) is markedly increased in many forms of cancer, including colorectal, ovarian, lung, breast and kidney, while in other cancers (liver cancer and glioma) it becomes downregulated. According to the clinical database data in high-CBS-expressor cancers (e.g. colon or ovarian cancer), high CBS expression typically predicts lower survival, while in the low-CBS-expressor cancers (e.g. liver cancer), low CBS expression is associated with lower survival. In the high-CBS expressing tumor cells, CBS, and its product hydrogen sulfide (H2S) serves as a bioenergetic, proliferative, cytoprotective and stemness factor; it also supports angiogenesis and epithelial-to-mesenchymal transition in the cancer microenvironment. The current article reviews the various tumor-cell-supporting roles of the CBS/H2S axis in high-CBS expressor cancers and overviews the anticancer effects of CBS silencing and pharmacological CBS inhibition in various cancer models in vitro and in vivo; it also outlines potential approaches for biomarker identification, to support future targeted cancer therapies based on pharmacological CBS inhibition.
Keywords: Angiogenesis; Colon cancer; Hydrogen sulfide; Mitochondria; Signalling; Stemness.
Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.
Conflict of interest statement
None.
Figures
Structure and function of CBS. A) The organization of CBS. B) Crystal structure of the Δ516-525 human CBS homodimer (PDB# 4COO ). Human CBS is architecturally organized in three regions: the Bateman module, the catalytic domain and the heme-binding domain. The engineered hCBS Δ516-525 is catalytically identical to the full-length native enzyme even if it lacks a loop consisting of 10 amino acid residues from the C-terminal regulatory domain. hCBS Δ516-525 forms dimers, rather then tetramers or higher order oligomers typical of the full-length CBS, that are colored in green and orange, respectively. The PLP and the heme cofactors are shown in sticks. The inset represents a zoom-in view into the catalytic (PLP) and regulatory (heme) sites. The PLP forms an internal aldimine intermediate via the Schiff base bond with the amino group of Lys119, while the heme is coordinated by Cys52 and His65. Figures were generated with PyMol 2.5. C) Key biochemical reactions catalyzed by hCBS. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
CBS is upregulated in human colon cancer and correlates with worse clinical prognosis. A) Formalin-fixed paraffin-embedded sections of normal colonic mucosa, hyperplastic polyp and adenocarcinoma, stained with CBS antibodies showing a gradual increase in CBS expression corresponding to the severity of the disease. The panel was adapted from data published in Ref. [48]). B). CBS expression in human colon cancer biopsies compared to normal surrounding tissue. Representative western blots and summary of expression data are shown (mean ± SEM). Arbitrary relative densitometry units were normalized with β-actin using image analysis software. ∗p < 0.05 T (tumor) vs. N (normal surrounding tissue); n = 15. The panel was redrawn from data presented in Ref. [49]. C) Effect of the CBS/CSE inhibitor AOAA (1 mM) and the CSE inhibitor PAG (3 mM) on H2S production in homogenates of a colorectal cancer and patient-matched normal colonic tissue. Data are presented as mean ± SEM of 3 independent experiments. ∗p < 0.05 shows significant inhibition of H2S production. The panel was redrawn from data presented in Ref. [6]. D) Representative images of CBS immunohistochemical staining in different-stage tumor tissues (n = 90) in the microarray. Immunohistochemical score for CBS in different-stage colon tumor tissues in the microarray. (stage 1 = 28 cases; stage 2 = 28 cases; stage 3 = 21 cases; stage 4 = 13 cases). Data are presented as mean ± SEM of at least 3 independent experiments. ∗p < 0.05 shows significantly higher expression of CBS in tumors, compared to adjacent normal tissues. The panel was redrawn from data presented in Ref. [52]. E) CBS mRNA expression in 52 colon cancer tissues and paired adjacent normal colon tissues showing that CBS in tumor tissue is higher than in the surrounding normal tissue in the majority of the patients studied. The panel was redrawn from data presented in Ref. [50]. F) Survival curve showing the impact of CBS expression on overall survival in colon cancer from COAD dataset. p < 0.05 reflects higher survival rate in low-CBS-expressor patients. The panel was redrawn from data presented in Ref. [55]. G) Survival curve showing the impact of CBS expression on overall survival in colon cancer from TCGA-OV dataset. ∗p < 0.05 reflects higher survival rate in low-CBS-expressor patients. The panel was redrawn from data presented in Ref. [52].
CBS is upregulated in human ovarian cancer and correlates with worse clinical prognosis. A) Immunohistochemical staining of a tissue microarray of epithelial ovarian cancer samples. Representative images are shown of none (i), weak (ii), moderate (iii), and (iv) strong staining. The panel was redrawn from data presented in Ref. [7]. B) CBS overexpression in the late stages and grades of ovarian cancer. The panel was redrawn from data presented in Ref. [7]. C) Survival curve showing the impact of CBS expression in ovarian cancer on overall survival from Atlas database (https://www.proteinatlas.org ). The panel was redrawn from data presented in Ref. [61]. D) Survival curve showing the impact of CBS expression in ovarian serous cystic adenocarcinoma on overall survival from OV dataset. ∗p < 0.05 reflects higher survival rate in low-CBS-expressor patients. The panel was redrawn from data presented in Ref. [55].
Patient survival rates, as a function of CBS mRNA levels in the primary tumor, in several types of cancer. A-T) Data were obtained from the Atlas database (https://www.proteinatlas.org ). ∗p < 0.05, ∗∗p < 0.01, reflect significant differences in patient survival between the high- and low-CBS expressor groups.
CBS is upregulated in human breast cancer and correlates with worse clinical prognosis. A) Immunohistochemical staining of a tissue microarray of 60 human breast cancer samples. Data are presented as mean ± SD of CBS staining. ∗p < 0.05 shows significantly higher tumoral CBS levels compared to normal tissue. The panel was redrawn from data presented in Ref. [62]. B) Representative western blot, detecting CBS in-patient derived breast cancer tissues and matched normal breast tissues (n = 5). The panel was redrawn from data presented in Ref. [63]. C) Cystathionine levels in patient derived breast tumor and normal breast tissues (n = 5). Data are represented as mean ± SEM from three independent experiments, ∗p < 0.05. The panel was redrawn from data presented in Ref. [63]. D) CBS overexpression in basal-like breast cancer. Correlation of CBS mRNA and protein levels in 45 tumors from the Oslo 2 cohort. The panel was redrawn from data presented in Ref. [64]. E) Survival curve showing the impact of CBS expression on overall survival in invasive breast carcinoma from the BRCA dataset showing a trend for better survival in low-CBS-expressing patients. The panel was redrawn from data presented in Ref. [55]. F) Survival curves showing the impact of CBS expression on overall survival in breast cancer from Atlas database (https://www.proteinatlas.org ). ∗p < 0.05 shows significantly better survival in low-CBS-expressing patients.
CBS is downregulated in human hepatocellular carcinoma and correlates with better clinical prognosis. A, B) Relative CBS expression in hepatocellular carcinoma tumor versus peritumor tissues from TCGA and GSE14520 database, respectively. Data are shown as mean ± SEM, ∗p < 0.05, ∗∗p < 0.01 showing better survival in high-CBS expressor patients. The panel was redrawn from data presented in Ref. [87]. C) Western blot analysis of CBS protein levels in 28 hepatocellular carcinoma tumor tissues and paired peritumor tissues. Data, shown as mean ± SEM, show a significant downregulation of CBS in hepatocellular carcinoma ∗∗p < 0.01. The panel was redrawn from data presented in Ref. [87]. D) Survival curve showing the impact of CBS expression on overall survival in hepatocellular carcinoma. ∗∗p < 0.01 reflects better patient survival in high-CBS-expressing patients. The panel was redrawn from data presented in Ref. [84]. E) Survival curve showing the impact of CBS expression on overall survival in hepatocellular carcinoma from the LIHC dataset. ∗p < 0.05 reflects better patient survival in high-CBS-expressing patients. The panel was redrawn from data presented in Ref. [55]. F) Survival curve showing the impact of CBS expression on overall survival in hepatocellular carcinoma from Atlas database (https://www.proteinatlas.org ). ∗∗∗p < 0.001 reflects better patient survival in high-CBS-expressing patients. G) Survival curve showing the impact of CBS expression on overall survival in hepatocellular carcinoma from TCGA database. p < 0.05 reflects the patient survival statistics. The panel was redrawn from data presented in Ref. [87].
CBS knockdown suppresses tumor growth and tumor angiogenesis in colon and ovarian cancer. Effects of CBS knockdown on colon cancer xenografts: A) tumor growth rate from shNT (nontargeting shRNA control) and CBS knockdown (shCBS) groups. Data are shown as mean ± SEM from n = 6 mice, ∗p < 0.05 compared to shRNA control. The panel was redrawn from data presented in Ref. [6]. B) Photomicrographs of representative sections (10 μm) from shNT and shCBS xenografts showing CD31-positive blood vessels (brown). Arrows indicate larger vessels and bracket indicates areas of necrosis with shCBS xenograft. The panel was redrawn from data presented in Ref. [6]. C) CD31-positive blood vessel density quantification in HCT116 tumor xenografts. Data are shown as mean ± SEM from n = 6 mice, ∗∗p < 0.01 compared to shRNA control. The panel was redrawn from data presented in Ref. [6]. D) Average area of metastatic foci in liver sections from control, control + bevacizumab (control + Bev), CBS knockdown (CBS-KD), CBS knockdown + bevacizumab (CBS-KD + Bev) groups. Data are shown as mean ± SEM from n = 5 mice, ∗p < 0.05 vs. the control group, #p < 0.05 vs. the group of mice implanted with WT cells and treated with Bev. The panel was redrawn from data presented in Ref. [52]. E) Representative images of CD31 immunohistochemical staining in liver tissue sections from control, control + Bev, CBS-KD, CBS-KD + Bev groups. The panel was redrawn from data presented in Ref. [52]. F) CD31-positive blood vessel density quantification in liver tissue sections from control, control + bevacizumab (control + Bev), CBS knockdown (CBS-KD), CBS knockdown + bevacizumab (CBS-KD + Bev) groups. Data are shown as mean ± SEM from n = 5 mice, ∗p < 0.05 vs. the control group. The panel was redrawn from data presented in Ref. [52]. Effect of CBS knockdown on chemoresistant ovarian cancer xenografts: G) tumor growth rate from Sc-siRNA, Sc-siRNA + cisplatin (Sc-siRNA + CIS), CBS siRNA, CBS siRNA + cisplatin (CBS siRNA + CIS) groups. Data are shown as mean ± SD from n = 10 mice, ∗p < 0.05, ∗∗p < 0.01 compared to siRNA control, #p < 0.05 vs. the group of mice implanted with siRNA control cells and treated with cisplatin. The panel was redrawn from data presented in Ref. [7]. H) Representative images of CD31 immunohistochemical staining in tumor tissue sections from Sc-siRNA, Sc-siRNA + CIS, CBS siRNA, CBS siRNA + CIS groups. The panel was redrawn from data presented in Ref. [7]. I) CD31-positive blood vessel density quantification in tumor tissue sections from Sc-siRNA, Sc-siRNA + CIS, CBS siRNA, CBS siRNA + CIS groups. Data are shown as mean ± SD from n = 4 mice, ∗∗p < 0.01 compared to siRNA control. The panel was redrawn from data presented in Ref. [7]. Effects of CBS knockdown on basal-like breast cancer xenografts: J) tumor growth rate from Cal51 control and shCBS groups. Data are shown as mean ± SD from n = 8 mice, ∗p < 0.05, compared to control. The panel was redrawn from data presented in Ref. [64]. K) Representative images of CD31 immunohistochemical staining in tumor tissue sections from HCC1143 control and shCBS groups. The panel was redrawn from data presented in Ref. [64]. L) CD31-positive blood vessel density quantification in tumor tissue sections from HCC1143 control and shCBS groups. The values were determined by Image J analysis from the figures shown in K). Data are shown as mean ± SEM, ∗∗p < 0.01 compared to control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Pharmacological inhibition of CBS causes the regression of established tumors in tumor-bearing mouse models. A) Effect of CBS inhibitor (AOAA) in tumor growth rate of NCM356 cell line overexpressing CBS xenografts. Data are shown as mean ± SEM of n = 10 animals per group; ∗p < 0.05 shows significant difference between tumor size between the indicated two time points. The figure was redrawn from data presented in Ref. [48]. B) Effect of CBS inhibitor (YD0171) in tumor growth rate of HCT116 xenografts. Data are shown as mean ± SEM of n = 8 animals per group; ∗p < 0.05 shows significant difference between tumor size between the indicated two time points. The figure was redrawn from data presented in Ref. [155]. C) Effect of CBS inhibitor (AOAA) in tumor growth rate of A549 xenografts. Data are shown as mean ± SEM of n = 5 animals per group; ∗p < 0.05 shows significant difference between tumor size between the indicated two time points. The figure was redrawn from data presented in Ref. [69].
Roles of H2S in the stimulation of cellular bioenergetics in cancer cells. H2S can serve as a direct electron donor into the mitochondrial electron transport chain at the level of Complex II via SQR (Pathway #1), but also, at low concentrations, it can serve as an electron donor at Complex IV (Pathway #2). H2S can inhibit mitochondrial cAMP phosphodiesterases (as PDE2A), and thereby stimulate intramitochondrial cAMP-dependent protein kinases, which can phosphorylate and thus further activate the electron transport chain (Pathway #3). In addition, H2S can sulfhydrate LDH-A, resulting in its activation (Pathway #4). H2S can also directly stimulate the activity of ATP synthase via the sulfhydration of specific cysteines (Pathway #5). H2S can regulate mitochondrial dynamics (fusion/fission) to maintain the mitochondrial pool in its most effective state (Pathway #6). H2S can also increase mitochondrial mass via the stimulation of mitochondrial biogenesis (Pathway #7). Additional mechanisms underlying mitochondrial “stabilization” may be simply related to the general antioxidant role of H2S (Pathway #8). Another mitochondrial protective mechanism is related to the stimulation of mitochondrial DNA repair (Pathway #12). H2S may also support the cancer cell metabolism via the stimulation of glycolysis, in part through GAPDH and PKM2 sulfhydration and their consequent activation (Pathway #9). H2S may also stimulate the uptake of glucose and its utilization (Pathway #10), and lipid uptake and its utilization (Pathway #11) into the cells.
The various roles of H2S in the stimulation of cells signaling in cancer cells. H2S can stimulate the PI3K/Akt pathway through: (a) stimulation of Akt via induction of the phosphorylation of its active site (Ser493) (probably via the activation of intermediary kinases); (b) inhibition of Phosphatase and Tensin homolog (PTEN) - an essential counterregulatory enzyme of the PI3K pathway - via sulfhydration of Cys124 or Cys71 and (c) inhibition of the activity of PTP1B (another counterregulatory enzyme in the PI3K pathway) via sulfhydration at Cys215 (Pathway #1). All of these actions result in increased PIP3 levels. H2S can stimulate ERK1/2 pathway through: ERK1/2 phosphorylation; RAF phosphorylation that, in turn, can phosphorylate MEK1; sulfhydration of MEK1 (at Cys341). MEK1 activation, in turn, leads to the phosphorylation of ERK1/2 and translocation of ERK1/2 into the nucleus to stimulate ERK1/2 mediated downstream signaling; KATP channels opening and PKC activation resulting in downstream ERK1/2 activation (Pathway #2). H2S can stimulate mTOR pathway through: mTOR phosphorylation; AMPK phosphorylation via sulfhydration of liver kinase B1 (LKB1) leading to phosphorylation at Ser428 and/or to the activation of calcium/calmodulin-activated protein kinase 2 (CaMKK2), the phosphorylate AMPK can, in turn, phosphorylate mTOR resulting in downstream mTOR activation (Pathway #3). The PI3K/Akt, ERK1/2 and mTOR pathways activation result in the activation of both proliferative and cytoprotective downstream pathways.
Various roles of H2S as a cytoprotective factor in cancer cells. H2S can lead to the upregulation of various antioxidant systems. H2S can stimulate Nrf2/ARE pathway through: sulfhydration of Keap1 (at Cys151), that in turn, undergoes a conformational change which leads to the dissociation of Nrf2 from the Keap1-Cul3-RBX1 E3 ligase complex. Subsequently, the free Nrf2 translocates into the nucleus to induce a global change in gene expression, which includes the upregulation of a host of antioxidant genes and enzyme systems. H2S can stimulate PI3K/Akt, ERK1/2 and mTOR pathways, already discussed in Fig. 10. H2S can stimulate NF-kB pathway through: sulfhydration of NF-kB at Cys38. H2S can stimulate HSP90 pathway. The activation of these pathways confers general cytoprotection and cancer cell resistance to chemotherapeutic agents induced cytotoxicity. However, in the context of chemotherapeutic agents, additional, more specific mechanisms may also be involved in the protection provided by H2S. H2S can stimulate STAT3/Akt/Bcl-2 pathway through Akt activation. H2S can also promote the upregulation of P-glycoprotein (P-gp) and thymidylate synthetase (TYMS).
The various roles of H2S in the stimulation of EMT in cancer cells. H2S can stimulate Wnt/β-catenin pathway through: Sp3 transcription factor upregulation that, in turn, upregulate ACLY. ACLY interacts with β-catenin and may block β-catenin ubiquitination leading to its accumulation in the cytoplasm which, in turn, also translocates into the nucleus. In the nucleus, β-catenin binds to LEF and activates Twist1 and Snail1 transcription factors. H2S can stimulate PI3K/Akt pathway, which upregulates HIF1α, or induces Snail1 transcription factors expression through NF-κB. H2S can also stimulate the MEK/ERK pathway, leading to the activation of the transcription factor AP1. H2S can stimulate TGFβ pathway through: Smad2/3 activation, which, in turn, activates the transcription factor Snail1. H2S can stimulate p38MAPK pathway, either by activation of the upstream kinases MKKx which consequently phosphorylate p38MAPK or by the autophosphorylation of p38MAPK. Under hypoxic conditions H2S can induce HIF-1α expression and further up-regulate VEGFA and the transcription factor Twist1. Some of these pathways are interconnected and can be activated at the same time. Possible crosstalks between these pathways are indicated by brown arrows. The bell-shaped effect H2S should be emphasized in the above processes, meaning that these pathways can be upregulated during EMT in cancer cells, however this process can be reverted if additional H2S is added to the cells or if the H2S biosynthesis is inhibited in cancer cells. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Various roles of H2S in the stimulation of angiogenesis. H2S can stimulate intracellular Ca2+ mobilization in endothelial cells, which stimulates eNOS via the calcium-calmodulin system. H2S can activate PI3K/Akt pathway resulting in eNOS activation through phosphorylation at Ser1177. H2S can also induce a direct posttranslational modification of eNOS via sulfhydration on Cys443. This action, in turn, can further activate and stabilizes eNOS by promoting its dimerization. H2S can also stimulate the expression of eNOS mRNA, increasing eNOS protein levels. Further downstream, NO produced by eNOS binds to soluble guanylate cyclase (sGC) and induces the formation of cGMP. H2S can inhibit PDE, suppressing the degradation of cGMP and increasing cGMP levels enhancing the activation of its downstream protein kinase, PKG. H2S can open KATP channels on the endothelial cell membrane. H2S can upregulate VEGF and its receptor through HI–F1α upregulation but also through Flt induction. H2S can stimulate angiopoietin/tie system, possibly via the upregulation of Ang1, Ang2 and Tie. H2S can also stimulate various endothelial cell receptors with pro-angiogenic roles, including VEGFR, CXCR1 and VCAM.
Various tumor-supporting roles of H2S in high-CBS-expressing tumors; expected effects of CBS inhibition on these processes. Please note that these mechanisms are not present simultaneously and do not apply to all cancer types.
The bell-shaped concentration-response of H2S in cancer. H2S in the low-to-medium concentration range serves signaling functions, stimulates cellular bioenergetics, exerts cytoprotective effects, while high concentrations of H2S are generally cytotoxic. High-CBS-expressor cancer cells upregulate their H2S generation so that they are near the top of the bell-shaped H2S concentration-response curve, so that they best benefit from the supporting actions of H2S. This is beneficial for the cancer cell, but detrimental to the tumor-bearing host. Arrow #1: Inhibition of CBS-mediated H2S production in cancer cells can “take away” the stimulatory effects of H2S on cellular signaling and bioenergetics and can suppress cancer cell proliferation; this intervention, on its own, is typically not directly cytotoxic. However, CBS inhibition can synergize with the effect of chemotherapeutic agents (Arrow #2). Addition of lower concentrations of H2S to cancer cells may, in some conditions, induce a further proliferative or cytoprotective effect, by moving the cells to the “top” of the H2S concentration-response curve (Arrow #3), but donation of H2S, at higher concentrations, moves the curve to the right, where the suppressive bioenergetic and cytotoxic effects of H2S prevail (Arrow #4).
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