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Sequential Accumulation of 'Driver' Pathway Mutations Induces the Upregulation of Hydrogen-Sulfide-Producing Enzymes
Kelly Ascenção1, Nahzli Dilek1, Karim Zuhra1
1Chair of Pharmacology, Faculty of Science and Medicine, University of Fribourg, 1700 Fribourg, Switzerland.
Abstract:
Recently, a CRISPR-Cas9 genome-editing system was developed with introduced sequential 'driver' mutations in the WNT, MAPK, TGF-β, TP53 and PI3K pathways into organoids derived from normal human intestinal epithelial cells. Prior studies have demonstrated that isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, as well as the oncogene KRAS, assumed more proliferative and invasive properties in vitro and in vivo. A separate body of studies implicates the role of various hydrogen sulfide (H2S)-producing enzymes in the pathogenesis of colon cancer. The current study was designed to determine if the sequential mutations in the above pathway affect the expression of various H2S producing enzymes. Western blotting was used to detect the expression of the H2S-producing enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), as well as several key enzymes involved in H2S degradation such as thiosulfate sulfurtransferase/rhodanese (TST), ethylmalonic encephalopathy 1 protein/persulfide dioxygenase (ETHE1) and sulfide-quinone oxidoreductase (SQR). H2S levels were detected by live-cell imaging using a fluorescent H2S probe. Bioenergetic parameters were assessed by Extracellular Flux Analysis; markers of epithelial-mesenchymal transition (EMT) were assessed by Western blotting. The results show that the consecutive mutations produced gradual upregulations in CBS expression-in particular in its truncated (45 kDa) form-as well as in CSE and 3-MST expression. In more advanced organoids, when the upregulation of H2S-producing enzymes coincided with the downregulation of the H2S-degrading enzyme SQR, increased H2S generation was also detected. This effect coincided with the upregulation of cellular bioenergetics (mitochondrial respiration and/or glycolysis) and an upregulation of the Wnt/β-catenin pathway, a key effector of EMT. Thus sequential mutations in colon epithelial cells according to the Vogelstein sequence are associated with a gradual upregulation of multiple H2S generating pathways, which, in turn, translates into functional changes in cellular bioenergetics and dedifferentiation, producing more aggressive and more invasive colon cancer phenotypes.
Insights
Sequential mutations in colon cancer organoids increase hydrogen sulfide (H2S) production by upregulating H2S-generating enzymes. This leads to enhanced cellular bioenergetics and promotes more aggressive, invasive colon cancer phenotypes.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Cancer development involves sequential acquisition of driver mutations in key signaling pathways like WNT, MAPK, TP53, and PI3K.
- Hydrogen sulfide (H2S) producing enzymes are implicated in colon cancer pathogenesis.
- Previous studies show mutations in APC, SMAD4, TP53, and KRAS promote proliferative and invasive properties in intestinal organoids.
Purpose of the Study:
- To investigate the effect of sequential driver mutations on the expression of H2S-producing and degrading enzymes in human intestinal organoids.
- To determine if these mutations influence H2S levels, cellular bioenergetics, and epithelial-mesenchymal transition (EMT) markers.
Main Methods:
- CRISPR-Cas9 genome editing was used to introduce sequential mutations into human intestinal epithelial organoids.
- Western blotting assessed expression of H2S-producing enzymes (CBS, CSE, 3-MST) and degrading enzymes (TST, ETHE1, SQR).
- Live-cell imaging measured H2S levels, Extracellular Flux Analysis evaluated bioenergetics, and Western blotting assessed EMT markers.
Main Results:
- Sequential mutations led to gradual upregulation of H2S-producing enzymes (CBS, CSE, 3-MST), including a truncated CBS form.
- Advanced organoids showed decreased expression of the H2S-degrading enzyme SQR, correlating with increased H2S generation.
- Elevated H2S levels coincided with enhanced cellular bioenergetics and Wnt/β-catenin pathway activation, a key EMT driver.
Conclusions:
- Sequential mutations mimicking the Vogelstein model in colon epithelial cells progressively upregulate H2S-generating pathways.
- Increased H2S production contributes to altered cellular bioenergetics and dedifferentiation.
- These molecular and functional changes promote more aggressive and invasive colon cancer phenotypes.
