Extracellular matrix stiffness reduces DNA 6 ma level to facilitate colorectal cancer progression via disrupting P53 binding to CDKN1A promoter.

The extracellular matrix (ECM) forms the primary scaffold of the tumor microenvironment, with matrix stiffness serving as a critical physical cue that modulates cancer progression. However, the impact of matrix stiffness on colorectal cancer (CRC) progression remains elusive. This study aimed to elucidate the role of substrate stiffness in regulating DNA N6-methyladenine (6 mA) modifications and their association with CRC progression. We observed significantly reduced DNA 6 mA levels in CRC cells and tissues compared to normal controls, which progressively declined with advancing CRC stages. A negative correlation was identified between CRC tissue stiffness and DNA 6 mA levels. The 6 mA demethylase ALKBH1 was identified as a poor prognostic indicator in CRC and responded to increased substrate stiffness, correlating with enhanced CRC proliferation. Mechanistically, ALKBH1 mediated DNA 6 mA demethylation in response to substrate stiffening, thereby modulating gene transcription and promoting CRC tumorigenesis. Notably, ALKBH1 lost its proliferative effect in P53-knockout CRC cells, while a catalytically inactive ALKBH1 mutant suppressed oncogenesis. Furthermore, ALKBH1 diminished CDKN1A expression by impairing P53 binding to the CDKN1A promoter region. Collectively, our findings demonstrate that ALKBH1 acts as a pivotal mediator linking matrix stiffness to DNA 6 mA demethylation, critically driving CRC progression and highlighting its therapeutic potential. These results underscore the importance of DNA 6 mA modifications in CRC development and tumor response to microenvironmental cues.