Phd in MORPHOGENESIS AND TISSUE ENGINEERING

Thesis title: Long non-coding RNA MALAT1-dependent TPX2 protein controls the expression of cancer stem cell markers in breast cancer cells

Abstract MALAT1 is a long non-coding RNA (lncRNA), which co-localizes with several transcription factors as well as with pre-mRNA processing factors in the nuclear speckles, domains enriched in pre-mRNA splicing factors that are located in the interchromatin regions of the nucleoplasm of mammalian cells. MALAT1 plays a critical role in coordinating transcriptional and post-transcriptional gene regulation, impinging on proliferation and motility of cancer cells. In breast cancer, MALAT1 has been related to chemoresistance, metastatic dissemination and poor survival. Aimed at deciphering post-transcriptional regulatory mechanisms driven by MALAT1 in breast cancer, here we profiled the transcripts associated to lncRNA MALAT1 by performing ChIRP-RNAseq in MDA-MB-468 cells. This analysis allowed identifying a strong enrichment of MALAT1 in the internal exonic regions of two mRNAs, TPX2 and PLK2, encoding proteins involved in the control of mitotic spindle checkpoint. We assessed that silencing of MALAT1 causes a significant reduction of TPX2 and PLK2 expression in breast cancer cells. Consistent with the previous observations showing that MALAT1 interacts with its target RNAs through protein intermediates, we here identified, through ribonucleoprotein immunoprecipitation (RIP) experiments, that splicing factor SRSF1 is the mediator of the interaction between lncRNA MALAT1 and the mRNAs of TPX2 and PLK2. Accordingly, silencing of SRSF1 by siRNA transfection leads to reduced TPX2 and PLK2 expression levels. Moreover, blocking of SRSF1 through the use of a drug (SRPIN340) inhibiting SRPK1 kinase, responsible for SRSF1 phosphorylation and activation, leads to reduced TPX2 and PLK2 mRNA levels. We further show that the activation of SRPK1>SRSF1 axis relies on the protein complex enclosing mutant p53 and ID4 proteins, previously reported as regulators of SRSF1 protein. Exogenous expression of mutant p53R273H and ID4 proteins in p53-null cells indeed causes the interaction between SRPK1 and SRSF1, as well as theirphosphorylation. Accordingly, silencing of p53 or ID4 in cells carrying endogenous mutant p53 leads to reduced TPX2 and PLK2 expression levels. Analysis of the TCGA dataset of breast cancer allowed assessing that SRPK1 is strictly associated to TPX2 expression, but not to that of PLK2. Functionally, both silencing TPX2 by siRNA transfection and blocking SRPK1 activity by using SRPIN340 lead to the downregulation of the expression of cancer stem cell (CSC) markers, as CD44, ALDH1A1 and KLF5. Accordingly, treatment with SRPIN340 reduces the CD44+/CD24- population of CSCs. In conclusion, this study revealed a novel molecular network whereby lncRNA MALAT1 and splicing factor SRSF1 control the expression of spindle-checkpoint regulator TPX2 in breast cancer cells expressing mutant p53 and ID4 proteins. Importantly, this regulation may be blocked by inhibitors of the upstream kinase SRPK1, opening to novel potential treatment strategies for breast cancer.