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Splicing factors control triple-negative breast cancer cell mitosis through SUN2 interaction and sororin intron retention

ABSTRACT: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic opportunities. Recently, splicing factors have gained attention as potential targets for cancer treatment. We performed an RNAi screen targeting 244 individual splicing factors to systematically evaluate their role in TNBC cell proliferation. We identified nine splicing factors, including SNRPD2, SNRPD3 and NHP2L1, of which depletion inhibited proliferation in two TNBC cell lines by deregulation of sister chromatid cohesion (SCC) via increased sororin intron 1 retention and down-regulation of SMC1, MAU2 and ESPL1. Protein-protein interaction analysis of SNRPD2, SNRPD3 and NHP2L1 identified that seven out of the nine identified splicing factors belong to the same spliceosome complex including novel componentSUN2 that was also critical for efficient sororin splicing. Finally, sororin transcript levels are highly correlated to various proliferation markers in BC patients, suggesting that deregulating sororin levels through targeting of the relevant splicing factors might be a potential strategy to treat TNBC

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ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Román González-Prieto

LAB HEAD: Alfred C.O. Vertegaal

PROVIDER: PXD013968 | Pride | 2021-03-07

REPOSITORIES: Pride

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Splicing factors control triple-negative breast cancer cell mitosis through SUN2 interaction and sororin intron retention.

Koedoot Esmee E van Steijn Eline E Vermeer Marjolein M González-Prieto Román R Vertegaal Alfred C O ACO Martens John W M JWM Le Dévédec Sylvia E SE van de Water Bob B

Journal of experimental & clinical cancer research : CR 20210301 1

<h4>Background</h4>Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic opportunities. Recently, splicing factors have gained attention as potential targets for cancer treatment. Here we systematically evaluated the role of RNA splicing factors in TNBC cell proliferation.<h4>Methods</h4>In this study, we performed an RNAi screen targeting 244 individual splicing factors to systematically evaluate their role in TNBC cell proliferation. For top ca ...[more]

PMID: 33648524

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Project description:Triple-negative breast cancer (TNBC) represents the most aggressive subtype of breast cancer, characterized by a lack of therapeutic options. Dysregulation of alternative splicing plays a pivotal role in tumorigenesis and metastasis. Serine/arginine-rich (SR) proteins, essential components of the spliceosome, undergo phosphorylation by Cdc2-like kinases (CLKs). Here we explored the impact of pharmacological inhibition of CLKs using a novel inhibitor, T-025, on TNBC cell proliferation and migration. Methods In this study, we evaluated the anti-proliferative and anti-migratory efficacy of T-025 in a spectrum of TNBC cell lines. Fluorescent reporter cell lines and flowcytometry were used to determine the effect of T-025 on cell cycle. Live-cell imaging and photobleaching experiments were conducted to determine the subnuclear localization of SRSF7-GFP and its dynamic mobility. Pulldown/MS was used to uncover the impact of T-025 on SRSF7 interactome. Deep RNA sequencing was performed to unravel the differentially expressed genes (DEGs) and alternatively spliced genes (ASGs) upon T-025 treatment. Results T-025 exhibited a potent anti-proliferative effect, particularly in highly proliferative TNBC cell lines. Treatment with T-025 induced cell cycle arrest in the G1-S phase, resulting in an increased proportion of aneuploidy cells and cells with 4N DNA. T-025 significantly inhibited cell migration in highly migratory TNBC cell lines. T-025 led to the accumulation of SRSF7-GFP at nuclear speckles, restricting its mobility. SRSF7 interacted more with nuclear-speckle-residing proteins, while less with RNA helicases and polymerases upon T-025 treatment. Enhanced interactions between SRSF7 and other phosphorylated SR proteins localized at nuclear speckles were observed. RNA sequencing uncovered multiple DEGs and ASGs upon T-025 treatment, which were significantly enriched in pathways related to cell division, RNA splicing and cell migration. Conclusions We demonstrated the pivotal role of CLKs inhibition in the proper function of SR proteins and RNA splicing in TNBC cell lines. CLKs inhibition leads to the accumulation of splicing factors at nuclear speckles and stalls their release to splicing sites, resulting in the RNA splicing reprogramming of a large number of genes involved in cell division and migration. Our findings provide evidence that CLKs inhibition could be a promising therapeutic approach for TNBC patients.

Project description:Although a nonessential amino acid in normal cells, the demand for glutamine is dramatically increased throughout malignant transformation, supporting a range of metabolic processes including mitochondrial ATP production, protein synthesis, purine and pyrimidine biosynthesis. We previously showed that triple-negative breast cancer (TNBC) cells rely on glutamine uptake by the amino acid transporter ASCT2 to sustain their unique glutamine metabolism, thereby supporting in vitro growth and in vivo tumour formation. However, it is known that TNBC cells can also utilise non-transporter mediated nutrient uptake facilitated by processes such as macropinocytosis. We examined proliferation and colony forming ability of human breast cancer cell lines after ASCT2 CRISPR/Cas9 knockout (clonal and polyclonal populations) and shRNA knockdown. Proteomics and mRNAseq analysis further examined cellular and adaptive changes to ASCT2 knockout. Cellular changes were further analysed by western blotting, with macropinocytosis examined using 70kDa dextran-FITC uptake. Metabolic changes were assessed using targeted metabolomics approaches including 13C-labelled substrate tracing and liquid chromatography coupled tandem-mass spectrometry (LC-MS/MS) to determine intracellular levels of key tricarboxylic acid (TCA) cycle intermediates, glycolytic metabolites, fatty acid precursors, nucleotides, and amino acids in human TNBC cell lines in vitro. Despite our previous data showing a significant reduction in cell growth after ASCT2 knockdown, ASCT2 knockout was well-tolerated by both TNBC and Luminal A breast cancer cell lines, with proliferation rates similar to non-targeted CRISPR/Cas9 control cells. This adaptation to knockout was not due to the high glutamine levels present in culture media, as the knockout cells could be cloned in media containing physiological 0.5 mM glutamine. Previous data have shown that TNBC cell lines can undergo constitutive macropinocytosis, and that this could be enhanced when cells are cultured in low nutrient conditions. Indeed, not only did the TNBC cell line HCC1806 undergo constitutive macropinocytosis, the amount of macropinocytosis was significantly enhanced (5-10 fold) in 5 separate ASCT2 knockout clones. By comparison, the ASCT2 knockdown cell line, which have a significant proliferation deficit, showed a modest 2-fold increase in macropinocytosis. Despite in-depth analysis of gene and protein levels by mRNAseq and proteomics, ASCT2 knockout cells did not display a significant alteration in macropinocytic gene expression, but instead showed a substantial upregulation of Ser473-Akt phosphorylation which may drive the adaptive macropinocytosis in TNBC. These data suggest that the constitutive macropinocytosis present in TNBC cell lines provides a novel resistance mechanism to strategies targeting glutamine uptake alone. Despite this adaptation, TNBC cells continue to rely on glutamine, however therapeutic targeting may need to focus on other unique TNBC metabolic pathways such as single-pass glutaminolysis, which couples glutamine and glucose metabolism together.

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