The oncogenic kinase NEK2 regulates an RBFOX2-dependent pro-mesenchymal splicing program in triple-negative breast cancer cells

Abstract

Background Triple-negative breast cancer (TNBC) is the most heterogeneous and malignant subtype of breast cancer (BC). TNBC is defined by the absence of expression of estrogen, progesterone and HER2 receptors and lacks efficacious targeted therapies. NEK2 is an oncogenic kinase that is significantly upregulated in TNBC, thereby representing a promising therapeutic target. NEK2 localizes in the nucleus and promotes oncogenic splice variants in different cancer cells. Notably, alternative splicing (AS) dysregulation has recently emerged as a featuring trait of TNBC that contributes to its aggressive phenotype. Methods To investigate whether NEK2 modulates TNBC transcriptome we performed RNA-sequencing analyses in a representative TNBC cell line (MDA-MB-231) and results were validated in multiple TNBC cell lines. Bioinformatics and functional analyses were carried out to elucidate the mechanism of splicing regulation by NEK2. Data from The Cancer Genome Atlas were mined to evaluate the potential of NEK2-sensitive exons as markers to identify the TNBC subtype and to assess their prognostic value. Results Transcriptome analysis revealed a widespread impact of NEK2 on the transcriptome of TNBC cells, with 1830 AS events that are susceptible to its expression. NEK2 regulates the inclusion of cassette exons in splice variants that discriminate TNBC from other BC and that correlate with poor prognosis, suggesting that this kinase contributes to the TNBC-specific splicing program. NEK2 elicits its effects by modulating the expression of the splicing factor RBFOX2, a well-known regulator of epithelial to mesenchymal transition (EMT). Accordingly, NEK2 splicing-regulated genes are enriched in functional terms related to cell adhesion and contractile cytoskeleton and NEK2 depletion in mesenchymal TNBC cells induces phenotypic and molecular traits typical of epithelial cells. Remarkably, depletion of select NEK2-sensitive splice-variants that are prognostic in TNBC patients is sufficient to interfere with TNBC cell morphology and motility, suggesting that NEK2 orchestrates a pro-mesenchymal splicing program that modulates migratory and invasive properties of TNBC cells. Conclusions Our study uncovers an extensive splicing program modulated by NEK2 involving splice variants that confer an invasive phenotype to TNBCs and that might represent, together with NEK2 itself, valuable therapeutic targets for this disease.

Functional Interaction Between the Oncogenic Kinase NEK2 and Sam68 Promotes a Splicing Program Involved in Migration and Invasion in Triple-Negative Breast Cancer

“…We recently found that NEK2 exerts widespread modulation of the alternative splicing program of MDA-MB-231 (GSE140803), a cell line representative of the TNBC subtype ( 25 ). A substantial fraction of the NEK2-regulated splicing events were dependent on the ability of the kinase to promote the expression of RBFOX2 ( 25 ), a splicing factor involved in the regulation of the epithelial-to-mesenchymal transition (EMT) process ( 25 ). However, other exons regulated by NEK2 lacked binding sites for RBFOX2 and were likely regulated by other splicing factors in TNBC cells.…”

“…PolyA plus RNA-seq libraries were constructed according to Illumina’s protocol and sequenced using a 100-bp single-end format on an Illumina HiSeq 2000. RNA-Seq data analysis was performed by GenoSplice technology ( ), as previously described ( 25 , 35 ), using Human FAST DB v2016_1 annotations. Results were considered statistically significant for p -values ≤0.05 and fold-changes ≥1.5.…”

“…RNA extraction, RT-PCR, and real-time PCR analysis were performed as previously described ( 25 ). All primers used are listed in Additional File 1: Supplementary Table 2 .…”

“…Total cell extracts were obtained by lysis in 50 mM HEPES, 10 mM MgCl 2 ,100 mM NaCl, 10 mM β-glycerophosphate, 2 mM EGTA, 10% (v/v) glycerol, and 1% (v/v) Triton X-100 for HEK293T and in RIPA buffer for MDA-MB-231, as described ( 25 , 41 ). Nuclear extracts from MDA-MB-231 cells for co-immunoprecipitation were obtained as described ( 42 ).…”

“…Accordingly, high NEK2 expression was correlated with rapid relapse and poor outcome in multiple cancers ( 21 ), including BC ( 24 ). In this regard, NEK2 is particularly upregulated in TNBC ( 25 ), the BC subtype displaying the poorest prognosis due to high metastatic rate and lack of targeted therapies ( 26 , 27 ). Although NEK2 oncogenic activity in cancer has been primarily associated to the promotion of genome instability and aneuploidy ( 22 , 28 – 30 ), mounting evidence suggests its implication in the pro-tumoral regulation of alternative splicing.…”