Abstract
MAF1 homolog, negative regulator of RNA polymerase III (MAF1) is a key repressor of RNA polymerase (pol) III-dependent transcription and functions as a tumor suppressor. Its expression is frequently down-regulated in primary human hepatocellular carcinomas (HCCs). However, this reduction in MAF1 protein levels does not correlate with its transcript levels, indicating that MAF1 is regulated post-transcriptionally. Here, we demonstrate that MAF1 is a labile protein whose levels are regulated through the ubiquitin-dependent proteasome pathway. We found that MAF1 ubiquitination is enhanced upon mTOR complex 1 (TORC1)-mediated phosphorylation at Ser-75. Moreover, we observed that the E3 ubiquitin ligase cullin 2 (CUL2) critically regulates MAF1 ubiquitination and controls its stability and subsequent RNA pol III-dependent transcription. Analysis of the phenotypic consequences of modulating either CUL2 or MAF1 protein expression revealed changes in actin cytoskeleton reorganization and altered sensitivity to doxorubicin-induced apoptosis. Repression of RNA pol III-dependent transcription by chemical inhibition or knockdown of BRF1 RNA pol III transcription initiation factor subunit (BRF1) enhanced HCC cell sensitivity to doxorubicin, suggesting that MAF1 regulates doxorubicin resistance in HCC by controlling RNA pol III-dependent transcription. Together, our results identify the ubiquitin proteasome pathway and CUL2 as important regulators of MAF1 levels. They suggest that decreases in MAF1 protein underlie chemoresistance in HCC and perhaps other cancers and point to an important role for MAF1 and RNA pol III-mediated transcription in chemosensitivity and apoptosis.
Highlights
MAF1 homolog, negative regulator of RNA polymerase III (MAF1) is a key repressor of RNA polymerase III– dependent transcription and functions as a tumor suppressor
RNA-Seq analysis from the Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn)3 [22], based on the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) data set for transcriptomic analysis, revealed that MAF1 transcript levels in liver tumor tissues tend to be higher compared with normal tissues (Fig. 1A)
To further determine whether the observed changes in drug resistance by RNA pol III– mediated transcription might be mediated through changes in tRNAs, we examined whether the association of cytochrome c with Apaf-1 was impaired when RNA pol III– dependent transcription was induced by decreases in MAF1 expression
Summary
Accumulating evidence supports the idea that enhanced RNA pol III– dependent transcription is necessary to drive oncogenesis [11, 12] and that alterations in the expression of specific tRNAs or tRNA derivatives are involved in proliferation, metastasis, and invasiveness of cancer cells, as well as tumor growth and angiogenesis in several malignant human tumors [13,14,15,16,17] These collective results indicate that the observed decrease in MAF1 protein expression and the resultant enhanced expression of MAF1 target genes provide an advantage to tumor cells beyond meeting the increased demand for overall protein synthesis. Our results further reveal that alterations in cellular MAF1 protein expression and RNA pol III– dependent transcription affect chemoresistance and the ability of cells to undergo apoptosis
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