CDO1 Human

What is CDO1 and what is its primary biochemical function?

CDO1 is a non-heme structured, iron-containing metalloenzyme that catalyzes the conversion of cysteine to cysteine sulfinate, playing a key role in taurine biosynthesis . As a metabolic enzyme, CDO1 represents the first step in the major catabolic pathway for cysteine, ultimately leading to the production of hypotaurine (HTAU) and taurine . The conversion pathway involves CDO1-mediated oxidation of cysteine to cysteine sulfinic acid (CSA), which can then be decarboxylated to form HTAU.

The catalytic activity of CDO1 depends on its iron-containing active site, and mutations affecting this site (such as Y157F) render the enzyme catalytically inactive . CDO1's enzymatic function places it at a critical junction in cellular sulfur metabolism, influencing not only taurine production but also potentially affecting glutathione synthesis and cellular redox status.

How does CDO1 expression vary across human tissues?

CDO1 expression shows considerable tissue specificity, with liver demonstrating particularly high expression levels. The liver is considered one of the highest CDO1-expressing tissues and is responsible for supplying taurine to the body . Lung tissue also expresses CDO1, though typically at lower levels than liver. In experimental models, researchers have observed that physiological CDO1 protein levels in mouse lung are substantially lower than in liver, suggesting tissue-specific regulation of this enzyme .

This differential expression pattern may relate to the varying metabolic requirements of different tissues, with high-CDO1 tissues potentially requiring enhanced capacity for cysteine catabolism or taurine production.

What metabolic pathways intersect with CDO1 activity?

CDO1 sits at a crucial intersection of several metabolic pathways:

• Taurine biosynthesis pathway: CDO1 catalyzes the first step in converting cysteine to taurine via CSA and HTAU intermediates .

• Glutathione synthesis: CDO1 competes with glutathione synthesis for the available cysteine pool. Experimental evidence shows that Cdo1 depletion in mouse models modestly increases both total cysteine and glutathione levels, demonstrating this metabolic competition .

• Sulfite generation: In certain cell types, particularly those with high GOT1 (glutamic-oxaloacetic transaminase 1) expression and low CSAD (cysteine sulfinic acid decarboxylase) expression, CDO1-derived CSA can lead to sulfite (SO₃²⁻) production .

• NADPH-dependent processes: CDO1 activity can affect cellular NADPH/NADP+ ratios, subsequently impacting NADPH-dependent reactions such as reductive carboxylation and proline synthesis .

These interconnected pathways highlight CDO1's potential to influence multiple aspects of cellular metabolism beyond simple cysteine catabolism.

What experimental evidence supports CDO1's role as a tumor suppressor?

Multiple lines of experimental evidence support CDO1's tumor suppressor function:

• Growth inhibition in vitro: Forced expression of CDO1 in cancer cell lines (including HCT116, DLD-1, KYSE30, MCF-7, NUGC3, and H1431) significantly suppresses cell growth. In HCT116 and DLD-1 colorectal cancer cells, CDO1 expression reduced growth to 42% and 27% of control cells, respectively .

• Colony formation suppression: CDO1 expression dramatically reduces colony-forming ability in multiple cancer cell lines. Control HCT116 and DLD-1 cells formed 200±45.3 and 300±35.3 colonies respectively, while CDO1-transfected cells formed only 20±12.2 and 80±11.3 colonies .

• Inhibition of anchorage-independent growth: CDO1-expressing cancer cell clones showed remarkably decreased colony formation in soft agar compared to control cells, indicating reduced transformation potential .

• Invasion suppression: Cell invasion assays demonstrated that CDO1 expression significantly decreased the invasive capacity of both HCT116 and DLD-1 colorectal cancer cells .

• In vivo tumor growth inhibition: In mouse xenograft models, DLD-1 cells stably expressing CDO1 showed significantly reduced tumor growth compared to control cells. At 4 weeks post-injection, tumors from CDO1-expressing cells were significantly smaller (989±408.1 mm³) than control tumors (2,091±119.2 mm³, p=0.009) .

• Growth acceleration with CDO1 knockdown: Conversely, siRNA-mediated knockdown of CDO1 in CDO1-expressing cell lines (HEK293 and HepG2) resulted in increased cell growth, further supporting CDO1's growth-inhibitory function .

How does CDO1 expression affect cancer cell metabolism and proliferation?

CDO1 expression impacts cancer cell metabolism and proliferation through several mechanisms:

• Metabolic alteration: CDO1 expression in cancer cells leads to increased conversion of cysteine to CSA and HTAU, potentially diverting cysteine from other metabolic pathways .

• Reduction in NADPH/NADP+ ratio: CDO1 expression decreases cellular NADPH/NADP+ ratios, particularly affecting non-small cell lung cancer (NSCLC) cells with high NRF2 activity .

• Impairment of NADPH-dependent processes: The reduced NADPH availability in CDO1-expressing cells impairs NADPH-dependent metabolic reactions, including:

  • Glutamine-dependent reductive carboxylation, shown by decreased M+5 citrate production from ¹³C₅-glutamine

  • Proline synthesis from glutamate

  • Potentially other NADPH-requiring biosynthetic processes

• Sulfite generation in specific cellular contexts: In cells with high GOT1 expression and low CSAD expression (common in NSCLC cell lines), CDO1 activity leads to sulfite production, which can form adducts with cysteine and potentially cause protein sulfitolysis .

These metabolic changes collectively contribute to CDO1's antiproliferative effects, particularly in cancer cells with elevated cysteine levels.

Is CDO1 selectively toxic to certain cancer cell types?

Yes, CDO1 shows selective toxicity toward cancer cells with specific metabolic characteristics:

• Cells with high intracellular cysteine: CDO1's antiproliferative effects are strongly correlated with intracellular cysteine levels. Cancer cells with elevated cysteine accumulation (such as NRF2/KEAP1 mutant cells) are particularly vulnerable to CDO1 expression .

• NRF2-high cells: NRF2 HIGH cancer cells accumulate more CDO1 protein than NRF2 LOW cells and are more sensitive to CDO1-mediated growth inhibition. In isogenic A549 cells, CDO1 expression significantly impaired the proliferation of NRF2-expressing cells while having no effect on NRF2 knockout cells .

• Cells with altered CSA metabolism: Cancer cells with high GOT1 and low CSAD expression (which favors sulfite production over taurine synthesis) may be more sensitive to CDO1-mediated toxicity .

This selective toxicity makes CDO1 particularly interesting as a potential therapeutic vulnerability in cancers with these metabolic characteristics.

How is CDO1 expression regulated at the epigenetic level?

CDO1 expression is tightly regulated through epigenetic mechanisms, with promoter methylation playing a crucial role:

• Promoter methylation: The CDO1 promoter exhibits differential methylation between normal and cancer tissues. Hypermethylation of the CDO1 promoter in cancer leads to transcriptional silencing .

• Demethylating agent response: Treatment of colorectal cancer cell lines with 5-aza-2'-deoxycytidine (a DNA methyltransferase inhibitor) restores CDO1 expression, confirming the role of methylation in CDO1 silencing .

• Methylation-independent protein regulation: While transcription is regulated by promoter methylation, CDO1 protein levels are additionally regulated by a post-translational mechanism linked to intracellular cysteine levels. High intracellular cysteine promotes CDO1 protein stabilization through reduced ubiquitination and degradation .

This dual regulation (transcriptional control via methylation and post-translational control via cysteine levels) allows for precise modulation of CDO1 activity in response to both epigenetic changes and metabolic status.

What techniques are used to study CDO1 promoter methylation?

Researchers employ several techniques to study CDO1 promoter methylation:

• Differential expression profiling: Comparing RNA expression profiles of cancer cell lines before and after treatment with 5-aza-2'-deoxycytidine to identify genes whose expression is restored by demethylation .

• Methylation-specific PCR: Using primers specific for methylated or unmethylated DNA sequences to assess the methylation status of the CDO1 promoter in clinical samples .

• Bisulfite sequencing: For detailed mapping of methylated cytosines within the CDO1 promoter region .

• Correlation analysis: Comparing CDO1 mRNA expression levels with promoter methylation status across multiple samples to establish the relationship between methylation and gene silencing .

These approaches have collectively established CDO1 promoter methylation as a common event in human carcinogenesis and a potential biomarker for cancer detection.

Which cancer types show aberrant CDO1 expression or methylation?

CDO1 silencing through promoter methylation has been observed across multiple cancer types:

• Colorectal cancer (CRC): CDO1 promoter is differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues .

• Breast cancer: Significant difference in CDO1 promoter methylation frequency between primary normal and tumor tissues .

• Esophageal cancer: Exhibits differential CDO1 methylation compared to normal esophageal tissue .

• Lung cancer: NSCLC cells, particularly those with NRF2/KEAP1 mutations, often show altered CDO1 regulation .

• Bladder cancer: Shows significant differences in CDO1 methylation between tumor and normal tissues .

• Gastric cancer: Demonstrates aberrant CDO1 methylation compared to normal stomach tissue .

Across these cancer types, CDO1 downregulation at both mRNA and protein levels is consistently observed, suggesting that CDO1 silencing may be a common event in human carcinogenesis .

How does CDO1 function as a metabolic liability for cancer cells?

CDO1 functions as a metabolic liability for cancer cells through several mechanisms:

• Competition for cysteine: CDO1 diverts cysteine from other metabolic pathways, potentially limiting availability for critical processes like glutathione synthesis in cells highly dependent on cysteine .

• NADPH depletion: CDO1 activity leads to decreased NADPH/NADP+ ratios, impairing NADPH-dependent processes essential for cancer cell metabolism and proliferation .

• Cysteine-dependent growth impairment: CDO1's antiproliferative effects are cysteine-dependent. When cysteine uptake is inhibited (e.g., with erastin) or under low cysteine conditions, CDO1 expression decreases and its growth-inhibitory effects are lost .

• Selective impact on detached conditions: CDO1 expression significantly impairs the ability of cancer cells to grow in soft agar, suggesting a particular vulnerability under detached conditions. This is potentially relevant to metastatic spread, as detached cells rely more heavily on IDH1-dependent reductive carboxylation for NADPH generation in mitochondria .

• Sulfite production: In certain cellular contexts (high GOT1, low CSAD), CDO1 activity leads to sulfite generation, which may have additional toxic effects through protein modification .

These mechanisms collectively explain why cancer cells often silence CDO1 expression as they evolve toward more aggressive phenotypes.

What experimental models are suitable for studying CDO1 function?

Several experimental models have proven valuable for investigating CDO1 function:

• Cell line models:

  • Isogenic systems: Genetically identical cells differing only in CDO1 expression. Examples include:

    • CRISPR/Cas9-engineered Keap1 WT and Keap1 R554Q MEFs with or without Cdo1

    • Isogenic NRF2 knockout A549 cells with or without CDO1 expression

  • Stable expression systems: Cancer cell lines with doxycycline-inducible CDO1 expression, allowing controlled study of CDO1's effects

  • Endogenous CDO1 expression: H1581 cells naturally express CDO1 and can be engineered for CDO1 knockout

• Functional assays:

  • Cell proliferation assays: MTT assays to measure growth inhibition

  • Colony formation assays: To assess long-term proliferative capacity

  • Soft agar assays: To measure anchorage-independent growth

  • Invasion assays: To assess effects on cancer cell invasion

  • Metabolic tracing: Using isotope-labeled metabolites (e.g., ¹³C₆-cystine) to track metabolic fates

• In vivo models:

  • Xenograft mouse models: Subcutaneous injection of CDO1-expressing or control cancer cells into nude mice to assess tumor growth in vivo

  • Tissue analysis: Comparison of CDO1 expression between normal tissues and cancer tissues from patients

These diverse models allow for comprehensive investigation of CDO1's biochemical functions, antiproliferative effects, and potential as a therapeutic target or biomarker.

How can researchers manipulate CDO1 expression for experimental purposes?

Researchers can manipulate CDO1 expression through several approaches:

• Forced expression systems:

  • Transient transfection: Using CDO1 expression vectors for short-term studies

  • Stable expression: Establishing cell lines with constitutive or inducible CDO1 expression

    • Doxycycline-inducible lentiviral expression systems allow controlled single-copy expression of CDO1

    • Expression of catalytically inactive mutants (e.g., CDO1 Y157F) as controls to distinguish enzymatic from non-enzymatic effects

• Knockdown and knockout approaches:

  • siRNA-mediated knockdown: Transient reduction of CDO1 expression using targeted siRNAs

  • CRISPR/Cas9-mediated knockout: Complete deletion of CDO1 for studying loss-of-function effects

  • Adenoviral Cre recombinase: Used with floxed alleles for conditional knockout in specific cellular contexts

• Epigenetic modulation:

  • Demethylating agents: 5-aza-2'-deoxycytidine treatment to reverse promoter methylation and restore CDO1 expression

These methodologies allow researchers to precisely control CDO1 expression levels and timing, facilitating detailed investigation of its functions in various experimental contexts.

What analytical methods are used to study CDO1-mediated metabolic changes?

Several analytical techniques are employed to study CDO1-mediated metabolic alterations:

• Isotope tracing metabolomics:

  • ¹³C₆-cystine tracing: To track cysteine entry into various metabolic pathways, including GSH synthesis and taurine production

  • ¹³C₅-glutamine tracing: To examine effects on TCA cycle metabolism and reductive carboxylation

• Metabolite quantification:

  • Liquid chromatography-mass spectrometry (LC-MS): For quantitative analysis of metabolites like cysteine, GSH, CSA, and HTAU

  • Detection of reaction products: Methods for measuring sulfite (SO₃²⁻) and cysteine-sulfite adducts (CYS-SO₃⁻)

• Enzymatic activity assays:

  • CDO1 enzymatic activity: Measuring the conversion of cysteine to CSA

  • NADPH/NADP⁺ ratio measurement: To assess effects on cellular redox status

• Metabolic flux analysis:

  • Reductive carboxylation assessment: Measuring M+5 citrate production from ¹³C₅-glutamine

  • GSH synthesis rates: Quantifying labeled GSH production from isotope-labeled precursors

These analytical approaches provide comprehensive insights into how CDO1 expression affects cellular metabolism, particularly in cancer contexts where metabolic reprogramming is a common feature.

How does CDO1 interact with other metabolic pathways in cancer cells?

CDO1 intersects with several critical metabolic pathways in cancer cells:

• Interaction with NRF2-mediated metabolism:

  • NRF2, a key regulator of cellular redox homeostasis, promotes intracellular cysteine accumulation

  • This cysteine accumulation stabilizes CDO1 protein in NRF2-high cells

  • CDO1 then functions as a metabolic liability for these cells by affecting NADPH status

• Relationship with cystine import systems:

  • CDO1's effects are dependent on functional cystine import

  • Inhibition of cystine uptake (e.g., with erastin) abolishes CDO1 expression and its antiproliferative effects

• Impact on mitochondrial metabolism:

  • CDO1 expression impairs NADPH-dependent reductive carboxylation, which is particularly important under detached conditions

  • This suggests potential interaction with mitochondrial metabolic pathways critical for cancer cell survival during metastatic spread

• Interaction with GOT1-mediated metabolism:

  • The metabolic fate of CDO1-produced CSA depends on the relative expression of downstream enzymes

  • High GOT1/low CSAD expression (common in NSCLC) favors sulfite production

  • Knockout of GOT1 significantly lowers CDO1-dependent cystine consumption and sulfite production

These interactions highlight CDO1's position at a critical node in cancer cell metabolism, potentially explaining why its silencing provides a selective advantage during tumor progression.