Investigating the regulation of alternative splicing by Tra2 proteins and RBMX in triple negative breast cancer cells

PhD ThesisAlternative splicing is a key process that enables the expression of a large number of proteins from a limited number of genes. Regulation of alternative splicing has been directly linked with the hallmarks of cancer. Human Transformer 2-α and -β are two homologous RNA binding proteins involved in the regulation of alternative splicing that are overexpressed in many different cancers. Previous work suggests Tra2β interacts closely with RBMX, encoded by the gene RBMX, to regulate the splicing of key pathologically related genes such as SMN2. The objective of this study is to investigate the role of alternative splicing regulators in breast cancer in order to further understand their biological function. Firstly, I utilise previously obtained RNAseq data from the double knockdown of Tra2α and Tra2β in the MDA-MB-231 breast cancer cell line to identify an extended number of genes regulated by Tra2 proteins using recently developed bioinformatics pipelines. The analysis revealed a range of alternative events including intron retentions, alternative 3’ and 5’ splice sites and exon repression events, suggesting different mechanisms of splice site regulation by Tra2 proteins. I experimentally validated the results of the RNAseq analysis by PCR. Subsequent Gene Ontology enrichment analysis of Tra2-regulated targets reveals several genes involved in the cellular response to DNA damage. I employed western immunoblotting to investigate the protein expression changes in these genes and was able to detect short protein isoforms expressed for MBD4 and XPA. Subsequently, nuclear fractionation and immunofluorescence microscopy of Tra2 depleted cells showed cytoplasmic localisation of the short isoforms for both MBD4 and XPA. In order to determine the role of the alternative isoforms regulated by Tra2 proteins, antisense oligonucleotide targeting exon 5 of BRIP1, exon 3 of CHEK1, and exon 5 of XPA were transfected into MDA-MB-231 cells. Expression of the short XPA isoform using antisense oligonucleotides results in an increase of γH2AX and reduced cell viability. This data suggests Tra2 proteins sustain the DNA integrity and protect the cells from death by ensuring the expression of correctly spliced DNA repair genes. In order to compare the role of Tra2 proteins with the commonly associated splicing regulator RBMX, I knocked down the expression of RBMX in MDA-MB-231 cells and obtained RNAseq data. Bioinformatics analysis was able to identify many commonly regulated alternative events, but also some novel isoforms regulated by RBMX alone. RNAseq data also unveiled an interesting autoregulatory mechanism for the RBMX gene, whereby the expression of RBMX is regulated by alternative splicing of the 3’ UTR. Subsequent examination of Upf1 knockdown cells showed stabilisation of the alternating isoform of RBMX indicating it is targeted by nonsense mediated decay. Future work into the role of RBMX regulated targets similar to the work done for Tra2-regulated genes could reveal a complete and clear picture of the function of RBMX in breast cancer cells

SUPPA2:fast, accurate, and uncertainty-aware differential splicing analysis across multiple conditions

Supplementary methods. (PDF 315 kb

ST6GAL1-mediated aberrant sialylation promotes prostate cancer progression.

Aberrant glycosylation is a universal feature of cancer cells, and cancer-associated glycans have been detected in virtually every cancer type. A common change in tumour cell glycosylation is an increase in α2,6 sialylation of N-glycans, a modification driven by the sialyltransferase ST6GAL1. ST6GAL1 is overexpressed in numerous cancer types, and sialylated glycans are fundamental for tumour growth, metastasis, immune evasion, and drug resistance, but the role of ST6GAL1 in prostate cancer is poorly understood. Here, we analyse matched cancer and normal tissue samples from 200 patients and verify that ST6GAL1 is upregulated in prostate cancer tissue. Using MALDI imaging mass spectrometry (MALDI-IMS), we identify larger branched α2,6 sialylated N-glycans that show specificity to prostate tumour tissue. We also monitored ST6GAL1 in plasma samples from >400 patients and reveal ST6GAL1 levels are significantly increased in the blood of men with prostate cancer. Using both in vitro and in vivo studies, we demonstrate that ST6GAL1 promotes prostate tumour growth and invasion. Our findings show ST6GAL1 introduces α2,6 sialylated N-glycans on prostate cancer cells and raise the possibility that prostate cancer cells can secrete active ST6GAL1 enzyme capable of remodelling glycans on the surface of other cells. Furthermore, we find α2,6 sialylated N-glycans expressed by prostate cancer cells can be targeted using the sialyltransferase inhibitor P-3FAX -Neu5Ac. Our study identifies an important role for ST6GAL1 and α2,6 sialylated N-glycans in prostate cancer progression and highlights the opportunity to inhibit abnormal sialylation for the development of new prostate cancer therapeutics. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland

ST6GAL1-mediated aberrant sialylation promotes prostate cancer progression

Aberrant glycosylation is a universal feature of cancer cells, and cancer-associated glycans have been detected in virtually every cancer type. A common change in tumour cell glycosylation is an increase in α2,6 sialylation of N-glycans, a modification driven by the sialyltransferase ST6GAL1. ST6GAL1 is overexpressed in numerous cancer types, and sialylated glycans are fundamental for tumour growth, metastasis, immune evasion, and drug resistance, but the role of ST6GAL1 in prostate cancer is poorly understood. Here, we analyse matched cancer and normal tissue samples from 200 patients and verify that ST6GAL1 is upregulated in prostate cancer tissue. Using MALDI imaging mass spectrometry (MALDI-IMS), we identify larger branched α2,6 sialylated N-glycans that show specificity to prostate tumour tissue. We also monitored ST6GAL1 in plasma samples from >400 patients and reveal ST6GAL1 levels are significantly increased in the blood of men with prostate cancer. Using both in vitro and in vivo studies, we demonstrate that ST6GAL1 promotes prostate tumour growth and invasion. Our findings show ST6GAL1 introduces α2,6 sialylated N-glycans on prostate cancer cells and raise the possibility that prostate cancer cells can secrete active ST6GAL1 enzyme capable of remodelling glycans on the surface of other cells. Furthermore, we find α2,6 sialylated N-glycans expressed by prostate cancer cells can be targeted using the sialyltransferase inhibitor P-3FAX-Neu5Ac. Our study identifies an important role for ST6GAL1 and α2,6 sialylated N-glycans in prostate cancer progression and highlights the opportunity to inhibit abnormal sialylation for the development of new prostate cancer therapeutics

Upregulation of GALNT7 in prostate cancer modifies O-glycosylation and promotes tumour growth.

Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O-glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O-glycosylation as an important driver of prostate cancer progression

Upregulation of GALNT7 in prostate cancer modifies O-glycosylation and promotes tumour growth

Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O-glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O-glycosylation as an important driver of prostate cancer progression

SUPPA2: fast, accurate, and uncertainty-aware differential splicing analysis across multiple conditions

Despite the many approaches to study differential splicing from RNA-seq, many challenges remain unsolved, including computing capacity and sequencing depth requirements. Here we present SUPPA2, a new method that addresses these challenges, and enables streamlined analysis across multiple conditions taking into account biological variability. Using experimental and simulated data, we show that SUPPA2 achieves higher accuracy compared to other methods, especially at low sequencing depth and short read length. We use SUPPA2 to identify novel Transformer2-regulated exons, novel microexons induced during differentiation of bipolar neurons, and novel intron retention events during erythroblast differentiation.This work was supported by the MINECO and FEDER with grants BIO2014-52566-R and BIO2017-85364-R, by AGAUR with grants SGR2014-1121 and SGR2017-1020, by BBSRC (BB/P006612/1), and by Breast Cancer Now (2014NovPR355). GH is a BBSRC-funded PhD student

Additional file 1: of SUPPA2: fast, accurate, and uncertainty-aware differential splicing analysis across multiple conditions

Figures S1–S6. (PDF 3939 kb

Sialic acid blockade inhibits the metastatic spread of prostate cancer to bone

Background Bone metastasis is a common consequence of advanced prostate cancer. Bisphosphonates can be used to manage symptoms, but there are currently no curative treatments available. Altered tumour cell glycosylation is a hallmark of cancer and is an important driver of a malignant phenotype. In prostate cancer, the sialyltransferase ST6GAL1 is upregulated, and studies show ST6GAL1-mediated aberrant sialylation of N-glycans promotes prostate tumour growth and disease progression. Methods Here, we monitor ST6GAL1 in tumour and serum samples from men with aggressive prostate cancer and using in vitro and in vivo models we investigate the role of ST6GAL1 in prostate cancer bone metastasis. Findings ST6GAL1 is upregulated in patients with prostate cancer with tumours that have spread to the bone and can promote prostate cancer bone metastasis in vivo. The mechanisms involved are multi-faceted and involve modification of the pre-metastatic niche towards bone resorption to promote the vicious cycle, promoting the development of M2 like macrophages, and the regulation of immunosuppressive sialoglycans. Furthermore, using syngeneic mouse models, we show that inhibiting sialylation can block the spread of prostate tumours to bone. Interpretation Our study identifies an important role for ST6GAL1 and α2-6 sialylated N-glycans in prostate cancer bone metastasis, provides proof-of-concept data to show that inhibiting sialylation can suppress the spread of prostate tumours to bone, and highlights sialic acid blockade as an exciting new strategy to develop new therapies for patients with advanced prostate cancer