www.oncotarget.com Oncotarget, 2019, Vol. 10, (No. 57), pp: 6021-6037 Review Splicing regulatory factors in breast cancer hallmarks and disease progression Esmee Koedoot1, Liesanne Wolters1, Bob van de Water1 and Sylvia E. Le Dévédec1 1Division of Drug Discovery and Safety, LACDR, Leiden University, Leiden, The Netherlands Correspondence to: Sylvia E. Le Dévédec, email: [email protected] Keywords: hallmarks of cancer; breast cancer; alternative splicing; splice factors; RNA sequencing Received: April 23, 2019 Accepted: August 29, 2019 Published: October 15, 2019 Copyright: Koedoot et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT By regulating transcript isoform expression levels, alternative splicing provides an additional layer of protein control. Recent studies show evidence that cancer cells use different splicing events to fulfill their requirements in order to develop, progress and metastasize. However, there has been less attention for the role of the complex catalyzing the complicated multistep splicing reaction: the spliceosome. The spliceosome consists of multiple sub-complexes in total comprising 244 proteins or splice factors and 5 associated RNA molecules. Here we discuss the role of splice factors in the oncogenic processes tumors cells need to fulfill their oncogenic properties (the so-called the hallmarks of cancer). Despite the fact that splice factors have been investigated only recently, they seem to play a prominent role in already five hallmarks of cancer: angiogenesis, resisting cell death, sustaining proliferation, deregulating cellular energetics and invasion and metastasis formation by affecting major signaling pathways such as epithelial-to-mesenchymal transition, the Warburg effect, DNA damage response and hormone receptor dependent proliferation. Moreover, we could relate expression of representative genes of four other hallmarks (enabling replicative mortality, genomic instability, avoiding immune destruction and evading growth suppression) to splice factor levels in human breast cancer tumors, suggesting that also these hallmarks could be regulated by splice factors. Since many splice factors are involved in multiple hallmarks of cancer, inhibiting splice factors might provide a new layer of oncogenic control and a powerful method to combat breast cancer progression. INTRODUCTION (AS) and provides an essential layer of post-transcriptional regulation that only recently received much attention from During gene transcription, a pre-mature messenger the research community. In particular, it is becoming clear RNA (pre-mRNA) molecule is generated that requires that tumor cells benefit greatly from this flexible regulatory further processing to a mature in a mRNA molecule that process since many specific isoforms have been identified will be translated into a protein. In this maturation step, the as promoting and supporting neoplastic transformation, introns are usually removed and the exons are ligated. This tumor growth and progression. Many reports have linked process, called splicing, is one of the post-transcriptional AS to up-regulation of proto-oncogenes, deregulated cell processes essential for RNA translation into functional division, increased survival, altered metabolism, onset of proteins and requires the activity of the splice factors. angiogenesis, increased invasion and metastasis in different Besides simple intron removal and exon coupling, the cancer types including breast cancer [5–8]. activity of those splice factors enable that multiple protein Splicing is a complex multistep process catalyzed isoforms can be translated out of one pre-mRNA transcript by the spliceosome, a large, dynamic, multicomponent by selective incorporation of pre-mRNA parts in the mature complex consisting of five small nuclear ribonucleoproteins mRNA transcript [1–4]. This is called alternative splicing (snRNPs) U1, U2, U4, U5 and U6 and many associated www.oncotarget.com 6021 Oncotarget proteins. In the human spliceosome, the 141 core factors suppressed intron or exon inclusion. Those deregulatory are highly abundant and/or are specifically associated with events in splicing have been shown to play a prominent the U1, U2, U5, U4/U6 snRNPs, or the U4/U6. U5 tri- role in breast cancer. snRNP [9, 10]. The auxiliary splice factors that are not part of the core spliceosome regulate AS and are less abundant Altered expression, activity and localization of when co-purified with the core spliceosome members splice factors [9, 10]. Splice factors are highly diverse considering both function and structure. For example, hnRNPs are Altered expression characterized by a RNA Recognition Motif (RRM) domain that accommodates site-specific binding to the target RNA In two independent studies, the comparison of the typically resulting in splicing inhibition by suppressing transcriptome of human breast tumors versus matched assembly of the spliceosome [11] or attraction of snRNPs healthy tissue revealed that 10%–50% of the protein- [12, 13]. SR splice factors contain a domain consisting of coding genes have altered transcript variant expression arginine/serine repeats (RS domain) and at least one RRM levels [24, 25]. These patient data are in line with recent domain [14–16] and facilitate recruitment of the snRNPs to in vitro findings that show a significant switch in splicing the splice sites [2]. Additionally, their activity is regulated pattern during epithelial-to-mesenchymal transition through phosphorylation by SR protein kinases (SRPKs). (EMT) accompanied with a specific EMT splicing Currently almost 250 splice factors distributed over signature [26]. Interestingly, this shift in splicing pattern different classes have been identified, all playing a specific was correlated to the expression levels of specific splice role at a specific stage of the splicing process [9, 17, 18]). factors; all three studies revealed splice factor RBFOX2 Breast cancer is the most frequent type of cancer as one of the most differentially expressed between the in women with an estimation of 268,670 new cases and epithelial and mesenchymal cell state [24–26]. Moreover, 41,400 deaths in the United States in 2018 [19]. In order expression levels of MBNL1, QKI, PTBP1, ELAV1, to develop and progress, (breast) cancer cells move HNRNPC, KHDRBS1, SRSF2 and TIAR were also linked through various steps to fulfill their requirements for to the mesenchymal state [24]. By applying a splicing certain oncogenic properties. These processes – the so- motif analysis in EMT regulated alternative transcripts, called ‘hallmarks of cancer’ – have been summarized Shapiro et al. concluded that the MBNL, CELF, hnRNP, by Hanahan and Weinberg in 2000 and 2011 [20, 21] or ESRP splice factors were most likely involved in the and currently include ten processes essential for tumor EMT splicing patterns [26]. Furthermore, depletion of the development and progression. In this review, we will mesenchymal splice factor RBFOX2 or overexpression of discuss the spliceosomal changes across the different the epithelial factor ESRP in mesenchymal cells induced a hallmarks of breast cancer. Since many of the already more epithelial morphology and reduced cell motility [26]. known spliceosome target genes have already been Altogether, these data clearly suggest that splice factors reviewed extensively elsewhere [5–8], we will focus on can be in control of EMT and breast cancer progression. the role of splice factors as potential oncogenes or tumor suppressors in breast cancer. We will highlight newly Post-translational modifications and chromatin identified splice factors of which abnormal regulation is structure linked to the different hallmarks of breast cancer [21]. Next to changes in expression levels, activity For the hallmarks that have not yet been linked to splice of certain splice factors can also be regulated by post- factors expression, we identified factors strongly related translational modifications (PTMs) such as acetylation, to hallmark-specific oncogenic processes using publicly phosphorylation and ubiquitination. Strong interactions available RNA sequencing data. Finally, we discuss the between ubiquitination and the spliceosome have been clinical relevance of using splice factors as biomarkers and demonstrated and SR proteins are widely known to regulate potential targets in breast cancer therapy. the activation of other splicing factors by phosphorylation [27–29]. For example, acetylation and ubiquitination of the SPLICE FACTOR DYSREGULATION splicing factor SRSF5 has been shown to control tumor IN BREAST CANCER growth [30]. The phosphorylation status of SRSF1 and SRSF7 controls their function as only non-phosphorylated Cancer-specific splicing events are established via SR proteins were shown to facilitate the recruitment of different routes: 1) changes in expression levels, activity mRNA to nuclear export receptors [31–34]. and localization of splice factors and/or 2) mutations Moreover, the intracellular distribution can be in functional domains of splicing related proteins and/ crucial for downstream signaling events. Although most or mutations in regulatory sequences, such as enhancer/ splice factors reside in the nucleus, cytoplasmic splicing silencer sequences and branch points [22, 23]. Both has been recently shown to take