CDK12 regulates alternative last exon mRNA splicing and promotes breast cancer cell invasion

Tien, Jerry F.; Mazloomian, Alborz; Cheng, Soo‐Chen; Hughes, Christopher S.; Chow, Christalle C T; Canapi, Leanna T.; Oloumi, Arusha; Trigo-Gonzalez, Genny; Bashashati, Ali; Xu, James; Chang, Vicky C.; Shah, Sohrab P.; Aparicio, Samuel; Morin, Gregg B.

doi:10.1093/nar/gkx187

Cited by 127 publications

(131 citation statements)

References 86 publications

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“…SNRNP70, a component of the U1 snRNP complex was identified as a potential phosphorylation substrate of CDK12/13 in our study; hence, it is quite possible that its decreased phosphorylation could partly account for the increased usage of alternate polyadenylation sites in DDR and other long genes. This could also explain why in contrast to findings in other studies implicating CDK12 in splicing regulation 32,43 , CDK12 inhibition did not lead to major splicing alterations, most likely because transcription was terminated well before it reached the 3′ splice sites 53 .…”

Section: Inhibition Of Cdk12 By Thz531 Results In Increased Transcripcontrasting

confidence: 58%

“…Because previous studies point to a role for CDK12 in splicing regulation 10,32,43,44 , we determined whether aberrant splicing could explain the elongation defect seen with THZ531 treatment. Analysis of the nascent transcriptomic data showed that in general, there was a paucity of significantly altered splicing events following THZ531 treatment.…”

Section: Cdk12/13 Inhibition Induces Minimal Splicing Alterations In mentioning

confidence: 99%

“…1e). This result, plus the fact that CDK12 and 13 have been implicated in pre-mRNA processing 10,31,32 where observable changes are likely to occur within minutes or hours, indicated that further analysis of steady-state RNA would not be sufficient to fully characterize alterations in the tightly coupled Pol II transcription and RNA processing processes or…”

mentioning

confidence: 99%

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CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation

Krajewska

Dries

Grassetti

et al. 2018

Preprint

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damage response (DDR) and mRNA processing genes. Yet, the mechanism(s) by which it achieves this selectivity remains unclear. Using a highly selective CDK12/13 inhibitor, THZ531, and nascent RNA sequencing, we show that CDK12 inhibition results in gene length-dependent elongation defects, leading to premature cleavage and polyadenylation (PCPA) as well as loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlated with an increased proportion of intronic polyadenylation sites, a feature that was especially prominent among DDR genes. Finally, phosphoproteomic analysis indicated that pre-mRNA processing factors, including those involved in PCPA, are direct phosphotargets of CDKs 12 and 13. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition due primarily to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites. 5 CDK12 inhibition preferentially affects DDR genes. CDK12 inhibition has been shown to affect the expression of genes involved in the DDR 5,6,10 . To determine whether similar effects are produced by our selective inhibitor in NB cells, we analyzed the gene expression profiles of cells treated with and without THZ531 for 6 hr., a time point at which there were little or no confounding effects due to cell cycle changes ( Supplementary Fig. 1e).Unlike the effects seen with THZ1 17 , predominantly an inhibitor of CDK7 with some activity against CDK12/13 28 , we failed to observe a complete and global transcriptional shutdown in THZ531-treated NB cells; instead, only 57.4% of the transcripts were downregulated (n=10,707), with 0.35% (n=66) upregulated [false discovery rate (FDR) <0.05] (Supplementary Fig. 2a; Supplementary Table 1). Consistent with earlier studies 15,16 , THZ531 led to significant downregulation of both transcription-associated and DDR genes ( Fig. 1c, Supplementary Fig. 2b, c), the latter of which were primarily associated with homologous recombination (HR) repair and are crucial for the maintenance of genome stability, including BRCA1, BARD1 and RAD51 29 (Fig. 1c, Supplementary Fig. 2c, d). To determine whether these effects were due to inhibition of CDK12 or 13, we depleted the expression of each kinase individually in NB cells, and in keeping with prior studies 6,10,30 , observed selective downregulation of DDR genes with CDK12 and not CDK13 knockdown (KD) (Supplementary Fig. 2e). Additionally, the expression of DDR genes was not affected in Kelly E9R THZ531-resistant cells, further implicating the selective role of CDK12 in regulating the DDR (Fig. 1d). Consistent with these observations, THZ531 also led to increased DNA damage with elevated γ-H2AX levels (Fig. 1e) and decreased radiationinduced RAD51 foci, indicating defects in DNA repair (Fig. 1f). Thus, our findings indicate that DDR genes are selectively affected by THZ531 and that such regulation is driven predominantly by CDK12.CDK12/13 inhibition with TH...

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Section: Inhibition Of Cdk12 By Thz531 Results In Increased Transcripcontrasting

confidence: 58%

Section: Cdk12/13 Inhibition Induces Minimal Splicing Alterations In mentioning

confidence: 99%

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CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation

Krajewska

Dries

Grassetti

et al. 2018

Preprint

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“…As the results show ( Figure 5D), the differentially expressed proteins were mainly involved mRNA splicing, which is usually enhanced when cancer cells go through migration or invasion. [10][11][12] In dense environments, proteins associated with cytoskeleton re-organization also increased in 4T1 cells. The cytoskeleton has roles in organelle transport, cell division, signalling, and motility, and it is closely related to cancer metastasis.…”

Section: Bioinformatic Information For Differentially Expressed Promentioning

confidence: 96%

Proteomic analysis of the effects of cell culture density on the metastasis of breast cancer cells

Guo

Mixin

Lan

et al. 2019

Cell Biochemistry & Function

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Cancer cell progression and proliferation increase cell density, resulting in changes to the tumour site, including the microenvironment. What is not known is if increased cell density influences the aggressiveness of cancer cells, especially their proliferation, migration, and invasion capabilities. In this study, we found that dense cell culture enhances the aggressiveness of the metastatic cancer cell lines, 4T1 and ZR-75-30, by increasing their proliferation, migration, and invasion capabilities. However, a less metastatic cell line, MCF-7, did not show an increase in aggressiveness, following dense cell culture conditions. We conducted a differential proteomic analysis on 4T1 cells cultured under dense or sparse conditions and identified an increase in expression for proteins involved in migration, including focal adhesion, cytoskeletal reorganization, and transendothelial migration. In contrast, 4T1 cells grown under sparse conditions had higher expression levels for proteins involved in metabolism, including lipid and phospholipid binding, lipid and cholesterol transporter activity, and protein binding.These results suggest that the high-density tumour microenvironment can cause a change in cellular behaviour, leading towards more aggressive cancers.Significance of the study: Metastasis of cancer cells is an obstacle to the clinical treatment of cancer. We found that dense cultures made metastatic cancer cells more potent in terms of proliferation, migration, and invasion. The proteomic and bioinformatic analyses provided some valuable clues for further intensive studies about the effects of cell density on cancer cell aggressiveness, which were associated with events such as pre-mRNA splicing and RNA transport, focal adhesion and cytoskeleton reorganization, ribosome biogenesis, and transendothelial migration, or associated with proteins, such as JAM-1 and S100A11. This investigation gives us new perspectives to investigate the metastasis mechanisms related to the microenvironment of tumour sites.

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“…Among the top 5% (51/1012) of our ranked genes is CDK12 (rank 40), which is impacted by multiple but non-overlapping types of alterations. Alterations within its protein kinase domain have been shown to cause dysregulation of DNA repair in cancer [114][115][116][117] . Aggregating over all alterations, we find 87 samples that have an alteration in this domain, with 64 (74%) patients having no DNA, but only a RNA alteration in the domain.…”

Section: Known and Novel Candidate Driver Genes Are Recurrently Altermentioning

confidence: 99%

Genomic basis for RNA alterations revealed by whole-genome analyses of 27 cancer types

Calabrese¹,

Davidson

Fonseca³

et al. 2017

Preprint

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We present the most comprehensive catalogue of cancer-associated gene alterations through characterization of tumor transcriptomes from 1,188 donors of the Pan-Cancer Analysis of Whole Genomes project. Using matched whole-genome sequencing data, we attributed RNA alterations to germline and somatic DNA alterations, revealing likely genetic mechanisms. We identified 444 associations of gene expression with somatic non-coding single-nucleotide variants. We found 1,872 splicing alterations associated with somatic mutation in intronic regions, including novel exonization events associated with Alu elements. Somatic copy number alterations were the major driver of total gene and allele-specific expression (ASE) variation. Additionally, 82% of gene fusions had structural variant support, including 75 of a novel class called “bridged” fusions, in which a third genomic location bridged two different genes. Globally, we observe transcriptomic alteration signatures that differ between cancer types and have associations with DNA mutational signatures. Given this unique dataset of RNA alterations, we also identified 1,012 genes significantly altered through both DNA and RNA mechanisms. Our study represents an extensive catalog of RNA alterations and reveals new insights into the heterogeneous molecular mechanisms of cancer gene alterations.

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CDK12 regulates alternative last exon mRNA splicing and promotes breast cancer cell invasion

Cited by 127 publications

References 86 publications

CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation

CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation

Proteomic analysis of the effects of cell culture density on the metastasis of breast cancer cells

Genomic basis for RNA alterations revealed by whole-genome analyses of 27 cancer types

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