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RNA N6-METHYLADENOSINE ERASER ALKBH5 DRIVES STEMNESS AND CHEMORESISTANCE OF COLORECTAL CANCER THROUGH A M6A-FAM84A-Β-CATENIN AXIS
Date
May 20, 2024
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Background: RNA N6-methyladenoine (m6A) modification plays multifaceted roles in cancer, including tumor initiation, progression and therapy resistance. Nevertheless, the role of aberrant m6A in cancer stem cells (CSCs) remains unclear. Here we aim to unveil the function, mechanism and translational significance of m6A eraser AlkB homologue 5 (ALKBH5) in colorectal cancer (CRC) CSCs.
Methods: Expression of ALKBH5 and CSC markers in CRC patients was assessed by qPCR (n=160) and immunohistochemistry on tissue microarrays (n=156). Effect of ALKBH5 on CRC stemness and chemoresistance was studied in colon stem cell-specific ALKBH5 knockin (Alkbh5KILgr5-CreER) and knockout (Alkbh5KOLgr5-CreER) mice, colorectal CSCs and patient-derived organoids (PDOs). RNA-seq, m6A-seq and Ribo-seq were performed to identify ALKBH5 targets. Immunoprecipitation-Mass Spectrometry (IP-MS) was conducted to probe FAM84-binding proteins. Vesicle-like nanoparticles (VNPs)-encapsulated si-ALKBH5 were used to target ALKBH5 .
Results: ALKBH5 mRNA and protein expression positively correlated with CSC markers (LGR5, CD133) in CRC patients (all P<0.01). ALKBH5 overexpression in colorectal CSCs and PDOs enhanced self-renewal capacity and CSC marker expression, whereas ALKBH5 knockdown exerted opposite effects. In vivo limiting dilution assay validated ALKBH5 loss impaired tumorigenicity of CSCs. Importantly, colon stem cell-specific ALKBH5 knockin mice showed exacerbated azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC with higher tumor multiplicity and load (P<0.01). In contrast, AOM/DSS-induced CRC was attenuated in Alkbh5KOLgr5-CreER mice (P<0.01), inferring a role for ALKBH5 in promoting cancer-initiating capacity in colorectal CSCs. Integrative RNA-seq, m6A-seq and Ribo-seq identified FAM84A as a target of ALKBH5 in CSCs. ALKBH5 binds and demethylates m6A-modified FAM84A, causing FAM84A mRNA decay and reduced FAM84A mRNA and protein expression. FAM84A overexpression abolished ALKBH5-induced self-renewal and CSC markers, while FAM84A knockout restored stemness in ALKBH5-depleted CSCs, confirming that FAM84A is a functional target of ALKBH5 in CSCs. Mechanistically, we revealed that FAM84A interacts with β-catenin to induce ubiquitination and degradation of β-catenin by recruiting β-catenin destruction complex components (e.g. Axin2, GSK-3β). Moreover, ALKBH5 overexpression elicited fluorouracil (5-FU) and oxaliplatin resistance in CSCs and PDOs, implying ALKBH5 as a therapeutic target. Targeting ALKBH5 by knockout or VNPs-siALKBH5 exerted synergistic effects with 5-FU or oxaliplatin to trigger tumor regression in CSCs- and PDOs-derived xenografts in mice, and in ALKBH5-KO mice.
Conclusion: We identified an ALKBH5-m6A-FAM84A-β-catenin axis in driving CRC stemness and chemoresistance. ALKBH5 is a potential therapeutic target in overcoming chemoresistance in CRC patients.
Methods: Expression of ALKBH5 and CSC markers in CRC patients was assessed by qPCR (n=160) and immunohistochemistry on tissue microarrays (n=156). Effect of ALKBH5 on CRC stemness and chemoresistance was studied in colon stem cell-specific ALKBH5 knockin (Alkbh5KILgr5-CreER) and knockout (Alkbh5KOLgr5-CreER) mice, colorectal CSCs and patient-derived organoids (PDOs). RNA-seq, m6A-seq and Ribo-seq were performed to identify ALKBH5 targets. Immunoprecipitation-Mass Spectrometry (IP-MS) was conducted to probe FAM84-binding proteins. Vesicle-like nanoparticles (VNPs)-encapsulated si-ALKBH5 were used to target ALKBH5 .
Results: ALKBH5 mRNA and protein expression positively correlated with CSC markers (LGR5, CD133) in CRC patients (all P<0.01). ALKBH5 overexpression in colorectal CSCs and PDOs enhanced self-renewal capacity and CSC marker expression, whereas ALKBH5 knockdown exerted opposite effects. In vivo limiting dilution assay validated ALKBH5 loss impaired tumorigenicity of CSCs. Importantly, colon stem cell-specific ALKBH5 knockin mice showed exacerbated azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC with higher tumor multiplicity and load (P<0.01). In contrast, AOM/DSS-induced CRC was attenuated in Alkbh5KOLgr5-CreER mice (P<0.01), inferring a role for ALKBH5 in promoting cancer-initiating capacity in colorectal CSCs. Integrative RNA-seq, m6A-seq and Ribo-seq identified FAM84A as a target of ALKBH5 in CSCs. ALKBH5 binds and demethylates m6A-modified FAM84A, causing FAM84A mRNA decay and reduced FAM84A mRNA and protein expression. FAM84A overexpression abolished ALKBH5-induced self-renewal and CSC markers, while FAM84A knockout restored stemness in ALKBH5-depleted CSCs, confirming that FAM84A is a functional target of ALKBH5 in CSCs. Mechanistically, we revealed that FAM84A interacts with β-catenin to induce ubiquitination and degradation of β-catenin by recruiting β-catenin destruction complex components (e.g. Axin2, GSK-3β). Moreover, ALKBH5 overexpression elicited fluorouracil (5-FU) and oxaliplatin resistance in CSCs and PDOs, implying ALKBH5 as a therapeutic target. Targeting ALKBH5 by knockout or VNPs-siALKBH5 exerted synergistic effects with 5-FU or oxaliplatin to trigger tumor regression in CSCs- and PDOs-derived xenografts in mice, and in ALKBH5-KO mice.
Conclusion: We identified an ALKBH5-m6A-FAM84A-β-catenin axis in driving CRC stemness and chemoresistance. ALKBH5 is a potential therapeutic target in overcoming chemoresistance in CRC patients.
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