Enhancer-mediated DDIT4 activation by SMYD2-dependent H3K4me1 promotes pazopanib resistance in clear cell renal cell carcinoma.
BACKGROUND: The progression and resistance to targeted therapy, including pazopanib, frequently lead to poor prognosis in clear cell renal cell carcinoma (ccRCC) patients. However, the underlying molecular mechanisms of these processes remain unclear. METHODS: In this study, we first performed RNA-seq to identify genes that were differentially expressed in both SMYD2-knockdown and pazopanib-resistant cells, indicating their potential role in SMYD2-mediated drug resistance. We analyzed TCGA-KIRC data and 150 patient samples to identify the relationship between SMYD2 and DDIT4 expression levels, as well as the prognostic significance of DDIT4. In vitro functional assays and murine models were applied to evaluate the effects of SMYD2 and DDIT4 on tumor growth and on pazopanib resistance. CUT&Tag and chromosome conformation capture (4 C) assays were applied to identify enhancers associated with SMYD2-mediated regulation of DDIT4, while the JASPAR database was utilized to predict transcription factors involved in the enhancer regulation. CRISPR-mediated enhancer deletion and ChIP-qPCR were subsequently performed to validate the regulatory roles of the identified enhancer and the transcription factor SPI1 in DDIT4 expression. RESULTS: Our study revealed that the expression level of DDIT4 is positively correlated with SMYD2. DDIT4 is highly expressed in renal cell carcinoma and is associated with poorer survival outcomes. Further research revealed that SMYD2 regulates H3K4me1 in a DDIT4 distal enhancer (chr10:72830412-72830891), promoting the recruitment of the transcription factor SPI1, thereby activating DDIT4 expression. We found that DDIT4 promotes the proliferation, metastasis, and pazopanib resistance of ccRCC, and DDIT4 knockdown enhances drug sensitivity in both in vitro and in vivo experiments. Furthermore, the SMYD2-DDIT4 axis activates the downstream STAT3 signaling pathway, thereby promoting tumor progression. In addition, DDIT4-related prognostic features showed potential associations with patient survival and predicted drug sensitivity in computational analyses. CONCLUSIONS: Our study identifies a previously unrecognized SMYD2-enhancer-DDIT4 regulatory axis, which promotes tumor progression and pazopanib resistance in ccRCC. These findings may provide potential therapeutic implications to overcome pazopanib resistance and improve treatment outcomes in ccRCC by targeting the SMYD2-enhancer-DDIT4 axis.