Alternative Splicing In Breast Cancer Research Articles

Alternative splicing in breast cancer

Alternative splicing allows a gene to produce multiple proteins and is the main source of human proteome diversity. Aberrant regulation of alternative splicing produces proteins with different structures and can lead to altered protein function, inactivation, or deleterious variants, which can have severe effects on normal cellular function, leading to many diseases, including cancer. Alternative splicing abnormalities are often found in diseases, so alternative splicing is highly correlated with diseases and treatment. According to relevant statistics, breast cancer is an important factor that threatens women’s lives and health. A common sign of tumors in humans is abnormal alternative splicing. An increasing amount of evidence shows that abnormal alternative splicing plays an important role in the occurrence and development of breast cancer. At the same time, in the related treatment of breast cancer, alternative splicing can lead to the failure of existing treatment methods and induce drug resistance. Currently, targeted therapy is still the key strategy for breast cancer treatment. In this review, we discuss the five classifications of alternative splicing and aberrant alternative splicing in breast cancer. The role of alternative splicing in the drug resistance of breast cancer is also discussed. Evidence has shown that aberrant alternative splicing can lead to drug resistance to current therapies. Because splicing factors are involved in many aspects of cancer, splicing factors may provide a new strategy for cancer treatment and a powerful tool for progress in the fight against breast cancer. Antisense oligonucleotides form a double-stranded structure by complementary pairing with mRNA, which prevents the binding of mRNA and ribosomes, thus inhibiting protein synthesis. Therefore, after finding effective targets, antisense oligonucleotides can be applied in gene therapy, drug research and development, biological research, and other fields to improve therapeutic effects.

Substrate stiffness regulates focal adhesion kinase alternative splicing in breast cancer.

Altered alternative splicing is considered as a hallmark of cancer. In fact, alternatively splice protein variants can differentially influence cellular processes such as cell migration and invasion. We have shown previously that increased matrix stiffness in tumors regulates alternative splicing events through increased cell contractility. Interestingly, the focal adhesion kinase (FAK), a protein central to matrix stiffness mechanosensing in focal adhesion, has several isoforms that promote tumor progression. In this context, we hypothesized stiffness-mediated alternative splicing could regulate the expression of FAK splice variants and influence tumor progression. We first investigated FAK mRNA splicing as a function of matrix stiffness using RNAseq. Our data identified stiffness-mediated alternative splicing events in FAK exon 4 exclusion (FAK4-). RT-PCR data from in vitro experiments confirmed FAK4- expression was modulated by substrate stiffness, reaching a maximum at around 1, 5 and 10 kPa in MCF10a, MCF7 and MDA-MB-231 cells respectively. Of note, FAK4- expression at these stiffnesses was reduced upon contractility inhibition. To investigate FAK4- clinical relevance, we characterized FAK4- expression as function of the tumor stage as a proxy of tumor stiffness in patients (TCGA). Notably, we found that patients with higher proportions of FAK4- had significantly worst survival odds than patient with lower FAK4- expression. Critically, FAK4- overexpression resulted in fewer but larger FAs, suggesting a shift in FA dynamics. Taken together, our data revealed FAK alternative splicing is regulated by matrix stiffness both in patients and in vitro. Moreover, the stiffness at which the maximum FAK4- expression is observed is higher in the more invasive MDA-MB-231 cells. Overall, our results show FAK splicing is modulated by tumor mechanics, raising the possibility of an existing interplay between stiffness-driven alternative splicing and FAK-mediated mechanoregulation.

Alternative splicing in breast cancer

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Abstract PD15-01: Snord67 promotes lymph node metastasis through U6-mediated alternative splicing in breast cancer

Abstract Background: While the presence of lymph node (LN) metastases in solid tumor malignancies is clinically significant and associated with increased risk for distant metastases, there is little evidence that lymphatic metastasis is distinct from hematogenous dissemination. Recent studies showed that cancer cells in LNs can directly give rise to distant metastases, but the mechanisms behind how cancer cells exit lymph nodes to establish distant metastases is still unknown. Materials and Methods: We developed an immunocompetent mouse model of lymph node metastasis using the 4T1 murine breast cancer cell line. In this model, 4T1 cells micro-surgically injected into axillary lymph nodes (AxLN) are capable of spontaneously establishing distant metastases. We then performed microarray analyses of subclones derived from MFP tumors, AxLN-injected tumors (AxLN), and lung metastases that arose from AxLN-injected tumors (AxLN-LuM) to evaluate for non-coding RNAs (ncRNAs) that were differentially expressed in AxLN tumors. Results: Microarray analyses of ncRNAs expressed in MFP, AxLN, and AxLN-LuM subclones showed that a class of ncRNAs, small nucleolar RNAs (snoRNAs), were enriched in AxLN tumors compared to MFP and AxLN-LuM. We identified the snoRNA SNORD67 as a key regulator of LN metastasis. Knockout of SNORD67 by both CRISPR and antisense oligonucleotide (ASO) resulted in significantly decreased LN tumor growth and subsequent development of distant metastases. This was at least in-part due to loss of targeted 2’-O-methylation of the small nuclear RNA U6, a component of the spliceosome. RNA-Seq analyses revealed distinct alternative splicing in SNORD67 knockouts. Using rapid autopsy breast cancer cases, we found that matched primary tumor and LN metastases revealed similar alternatively spliced genes, including several genes with previously described cancer-promoting splice variants. Conclusions: Small nucleolar RNAs (snoRNAs) have long been regarded as “housekeeping genes”, important for ribosomal biogenesis and protein synthesis. However, there is increasing evidence that this largely ignored class of non-coding RNAs (ncRNAs) also have wide-ranging, non-canonical functions in diseases, including cancer. SnoRNAs have been shown to have both oncogenic and tumor suppressor roles, yet whether snoRNAs regulate metastasis is unknown. Here we show that expression of certain snoRNAs are enriched in lymph node (LN) metastases in a micro-surgical, immune-competent mouse model of breast cancer. Moreover, our results show that SNORD67 is critical for growth of LN metastases and subsequent spread to distant metastases, and suggest that snoRNA-guided modifications of the spliceosome represent a previously unappreciated, yet targetable pathway in cancer. Citation Format: Yvonne Chao, Yinzhou Zhu, Hannah J Wiedner, Yi-Hsuan Tsai, Lily Wilkinson, Alessandro Porrello, Amanda ED Van Swearingen, Lisa Carey, Jimena Giudice, Christopher L Holley, Chad V Pecot. Snord67 promotes lymph node metastasis through U6-mediated alternative splicing in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-01.

Abstract PD15-07: Myc regulates alternative splicing through a network of RNA binding proteins in breast cancer

Abstract The oncogenic transcription factor MYC is one of the most frequently altered genes in cancer and is highly dysregulated in aggressive tumor types, including triple negative breast cancer (TNBC). Although direct targeting of MYC or its regulators in human tumors has proved difficult, the undisputed clinical relevance of this transcription factor warrants continued study for therapeutic targeting of MYC-driven processes in tumors. Recently, we and others have shown the tumorigenic capacity of MYC is critically dependent on the RNA splicing machinery and on several splicing factor RNA-binding proteins (RBPs). Dysregulation of alternative mRNA splicing, a key step in gene expression regulation, is a hallmark of cancer, and can lead to the expression of isoforms driving tumor initiation, progression or drug resistance. Understanding which component(s) of the RNA machinery are regulated by MYC, and the functions of MYC-induced spliced isoforms, may reveal new targets that circumvent prior failures of directly targeting MYC. Here, we examine MYC-induced changes in gene expression and alternative splicing through RNA-sequencing of breast cell lines and tumors. Using a mammary epithelial cell line with an inducible MYC system, we identified over 4,000 MYC-induced alternative splicing events as well as 378 MYC-induced RBPs. Co-expression network analysis of these RBPs in TCGA breast tumor RNA-seq data uncovered 8 RBP modules, four of which are highly correlated with MYC activity and highly expressed in TNBC. One of these modules is preserved across almost all other TCGA tumor types and remains highly correlated with MYC activity, suggesting a pan-cancer role of this co-expression module. We are now defining the functional role of these RBP modules in MYC-driven tumorigenesis in breast cancer models, as well as identifying their downstream oncogenic splicing targets. Our research project will define the role and regulation of alternative splicing in breast cancer, and more specifically in aggressive tumor types such as TNBC. Our findings will provide mechanistic insight into the role of MYC-driven splicing in tumor maintenance and will uncover novel therapeutic targets for MYC-active tumors. Citation Format: Laura Urbanski, Brittany Angarola, Mattia Brugiolo, Marina Yurieva, Sunghee Park, Olga Anczukow. Myc regulates alternative splicing through a network of RNA binding proteins in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-07.

Identification of spliceosome components pivotal to breast cancer survival

ABSTRACT Cancer cells employ alternative splicing (AS) to acquire splicing isoforms favouring their survival. However, the causes of aberrant AS in breast cancer are poorly understood. In this study, the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) data were analysed with univariate feature selection. Of 122 analysed spliceosome components, U2SURP, PUF60, DDX41, HNRNPAB, EIF4A3, and PPIL3 were significantly associated with breast cancer survival. The top 4 four genes, U2SURP, PUF60, DDX41, and HNRNPAB, were chosen for further analyses. Their expression was significantly associated with cancer molecular subtype, tumour stage, tumour grade, overall survival (OS), and cancer-specific survival in the METABRIC data. These results were verifiable using other cohorts. The Cancer Genome Atlas data unveiled the elevated expression of PUF60, DDX41, and HNRNPAB in tumours compared with the normal tissue and confirmed the differential expression of the four genes among cancer molecular subtypes, as well as the associations of U2SURP, PUF60, and DDX41 expression with tumour stage. A meta-analysis data verified the associations of U2SURP, PUF60, and HNRNPAB expression with tumour grade, the associations of PUF60, DDX41, and HNRNPAB expression with OS and distant metastasis-free survival, and the associations of U2SURP and HNRNPAB expression with relapse-free survival. Experimentally, we demonstrated that inhibiting the expression of the four genes separately suppressed cell colony formation and slowed down cell growth considerably in breast cancer cells, but not in immortal breast epithelial cells. In conclusion, we have identified U2SURP, PUF60, DDX41, and HNRNPAB are spliceosome-related genes pivotal for breast cancer survival.

The Construction of Bone Metastasis-Specific Prognostic Model and Co-expressed Network of Alternative Splicing in Breast Cancer.

BackgroundBreast cancer (BRCA) ranks among the top most common female malignancies and was regarded as incurable when combined with bone and distant metastasis. Alternative splicing events (ASEs) together with splicing factors (SFs) were considered responsible for the development and progression of tumors.MethodsDatasets including RNA sequencing and ASEs of BRCA samples were achieved from TCGA and TCGASpliceSeq databases. Then, a survival model was built including 15 overall-survival-associated splicing events (OS-SEs) by Cox regression and Lasso regression. The co-expressed SFs of each bone-and-distant-metastasis-related OS-SE were discovered by Pearson correlation analysis. Additionally, Gene Set Variation Analysis (GSVA) was performed to identify the downstream mechanisms of the key OS-SEs. Finally, the results were validated in different online platforms.ResultsA reliable survival model was established (the area under ROC = 0.856), and CIRBP was found co-expressed with FAM110B (R = 0.320, P < 0.001) associated with the fatty acid metabolism pathway.ConclusionAberrant SF, CIRBP, regulated a specific ASE, exon skip (ES) of FAM110B, during which the fatty acid metabolism pathway played an essential part in tumorigenesis and prognosis of BRCA.

Hypoxia-induced changes in intragenic DNA methylation correlate with alternative splicing in breast cancer

The tumor microenvironment is marked by gradients in the level of oxygen and nutrients, with oxygen levels reaching a minimum at the core of the tumor, a condition known as tumor hypoxia. Mediated by members of the HIF family of transcription factors, hypoxia leads to a more aggressive tumor phenotype by transactivation of several genes as well as reprogramming of pre-mRNA splicing. Intragenic DNA methylation, which is known to affect alternative splicing in cancer, could be one of several reasons behind the changes in splicing patterns under hypoxia. Here, we have tried to establish a correlation between intragenicDNA methylation and alternative usage of exons in tumor hypoxia. First, we have generated a customhypoxia signature consisting of 34 genes that are upregulated under hypoxia and are direct targets of HIF-1α. Using this gene expression signature, we have successfully stratified publicly available breast cancer patient samples into hypoxia positive and hypoxia negative groups followed by mining of differentially spliced isoforms between these groups. The Hypoxia Hallmark signature from MSigDB was also used independently to stratify the same tumor samples into hypoxic and normoxic.We found that 821 genes were showing differential splicing between samples stratified using a custom signature, whereas, 911 genes were showing differential splicing between samples stratified using the MSigDB signature. Finally, we performed multiple correlation tests between the methylation levels (β) of microarray probes located within 1 kilo base pairs of isoform-specific exons using those exons' expression levels in the same patient samples in which the methylation level was recorded. We found that the expression level of one of the exons ofDHX32 and BICD2 significantly correlated with the methylation levels, and we were also able to predict patient survival (p-value: 0.02 for DHX32 and 0.0024 for BICD2). Our findings provide new insights into the potential functional role of intragenic DNA methylation in modulating alternative splicing during hypoxia.

Aberrant alternative splicing in breast cancer.

Alternative splicing is critical for human gene expression regulation, which plays a determined role in expanding the diversity of functional proteins. Importantly, alternative splicing is a hallmark of cancer and a potential target for cancer therapeutics. Based on the statistical data, breast cancer is one of the top leading causes of cancer-related deaths in women worldwide. Strikingly, alternative splicing is closely associated with breast cancer development. Here, we seek to provide a general review of the relationship between alternative splicing and breast cancer. We introduce the process of alternative splicing and its regulatory role in cancers. In addition, we highlight the functions of aberrant alternative splicing and mutations of splicing factors in breast cancer progression. Moreover, we discuss the role of alternative splicing in cancer drug resistance and the potential of being targets for cancer therapeutics.

Abstract 1423: Integrative analysis predicts lncRNA regulating gene alternative splicing in breast cancer

Abstract Background: Non-coding region occupies 98% of the whole human genome and plays a regulatory role for protein-coding genes. About 95% of the protein-coding genes undergo alternative splicing, however, limited understandings for the complexity of alternative splicing (AS) mechanisms. Recent studies reported that some aberrant alternative splicing are associated with cancer development. Cancer-specific AS could be regulated by long non- coding RNAs (lncRNAs) directly or indirectly through other intermediate molecules . LncRNAs regulate gene splicing either by binding to their splicing factor proteins or competing with miRNAs to influence their targeted genes. Therefore, it is critically important to develop an effective computational framework capturing the lncRNAs regulatory mechanism in the process of AS from heterogeneous molecular relationships. Method: To model the complicated regulation mechanism in splicing event, we developed a integrative analysis framework based on machine learning algorithms. We firstly identified breast cancer-specific lncRNAs and AS genes by edgeR differential gene analysis from TCGA RNAseq tumor (n=1101) /normal (n=139) samples. Then we built co-expression network based on Spearman correlation pairwise distances from these cancer-specific lncRNAs and AS. We ranked top lncRNAs regulating alternative splicing in tumorigenesis by an optimized random walk multi-graphic method from the integrative networks of co-expression network, publically curated epigenetic network (e.g. ENCODE) and protein-protein interaction network (e.g. STRING). Linear regression analysis further refined individual candidate lncRNA regulation relating to their most correlated target splicing genes. The secondary structure of binding lncRNAs to potential splicing genes was predicted by support vector machine (SVM) algorithm to confirm the most conserved targets. Results: We identified 496 lncRNAs and 418 coding genes with alternative splicing isoforms associated with breast cancer. Pathway analysis predicted the functions of 14 candidate lncRNAs potentially regulating AS, are associated with cell migration, cell cycle progression, and more. We detected a high confidence lncRNA MALAT1 that regulates PKM gene alternative splicing, exclusion of either exon 9 or 10, in breast cancer tumorigenesis, which confirms the predictive strength of our proposed method. Conclusion: Understanding cancer-specific splicing machinery is therapeutically crucial in targeting the molecules that can influence the splicing process. We developed a network-based integrative framework to predict lncRNA regulations on breast cancer-specific alternative splicing as the potential therapeutic targets. Citation Format: Yunyun Zhou. Integrative analysis predicts lncRNA regulating gene alternative splicing in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1423.

Alternative Splicing in Breast Cancer and the Potential Development of Therapeutic Tools.

Alternative splicing is a key molecular mechanism now considered as a hallmark of cancer that has been associated with the expression of distinct isoforms during the onset and progression of the disease. The leading cause of cancer-related deaths in women worldwide is breast cancer, and even when the role of alternative splicing in this type of cancer has been established, the function of this mechanism in breast cancer biology is not completely decoded. In order to gain a comprehensive view of the role of alternative splicing in breast cancer biology and development, we summarize here recent findings regarding alternative splicing events that have been well documented for breast cancer evolution, considering its prognostic and therapeutic value. Moreover, we analyze how the response to endocrine and chemical therapies could be affected due to alternative splicing and differential expression of variant isoforms. With all this knowledge, it becomes clear that targeting alternative splicing represents an innovative approach for breast cancer therapeutics and the information derived from current studies could guide clinical decisions with a direct impact in the clinical advances for breast cancer patients nowadays.

Abstract 4512: The landscape of hypoxia-driven alternative splicing in breast cancer

Abstract Tumor hypoxia is generally associated with poor patient outcome and resistance to therapy. Hypoxia has an impact on multiple pathways inside the cell and a widespread effect on phenotype. Here we determined the changes in transcript architecture that arise as result of alternative splicing in hypoxic cells. A panel of breast cancer cell lines that include the Luminal A and B, HER2+, Triple-Negative subcategories grown in hypoxic and normoxic conditions were subjected for deep RNA-sequencing. Depending on the cell lines we found between 23- 927 splicing events with 3-45% of them also present in the differentially expressed fraction of genes. This points to an extensive isoform switching control independently of the transcriptional control of the genes themselves. The splicing events appear on genes responsible for mRNA processing (splicing, nuclear export and catabolism) as well as initiation of translation. Members of the SR family of proteins involved in RNA splicing, appear to be potential regulators of the biogenesis of the hypoxia specific splicing events. The differentially spliced exons are frequently part of both the coding and noncoding isoforms of a given gene potentially indicating a switch between these two types under hypoxia. Ten of the hypoxia specific splicing events were also found to be associated with the hypoxia status of primary breast cancer samples taken from The Cancer Genome Atlas (TCGA) database. Altogether, these data demonstrate the important role of hypoxia in driving alternative splicing events in breast cancer. Citation Format: Hani Choudhry, Spyridon Oikonomopoulos, Peng Yu, Cristina Ivan, Mircea Ivan, Adrian L. Harris, Jiannis Ragoussis. The landscape of hypoxia-driven alternative splicing in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4512. doi:10.1158/1538-7445.AM2017-4512

Abstract 3372: Differential exon usage in the HER2 subtype of breast cancer identified with RNA-seq and proteomic data

Abstract Background: Heterogeneity between different breast cancer tumors plays an important role in patients’ survival outcomes, and such tumor heterogeneity unveiled by transcriptome and proteome are often in disagreement. We hypothesize that alternative splicing of mRNA could explain heterogeneity within intrinsic breast cancer subtypes. Methods: Cases used in this study were drawn from the Clinical Breast Care Project where breast cancer patients were consented using an IRB-approved protocol. Fifty breast tumors were selected and processed by laser microdissection. RNA and protein were extracted from tissues using the Illustra triplePrep kit (GE Healthcare). Paired-end mRNA sequencing was performed using the Illumina HiSeq platform. Paired-end reads were preprocessed using PRINSEQ and splice-aligned to the genome using GSNAP software. Gene counts were measured using HTSeq while Exon counts used the DEXSeq. Differential expression was called at 10% false discovery rate. Proteome Discoverer with Byonic node was used for analyzing the quantitative global proteomics dataset, and we were able to quantitate 8600 proteins. All other analyses were performed using Perl and R. Results: The number of sequencing reads for the 50 cases ranged from 60 million to 410 million reads. After preprocessing, an average of 93% of reads was mapped, and 36,700 genes were identified among 50 tumor samples. PAM50 algorithm was used for intrinsic subtype calls, yielding 16 Basal, 8 Her2+, 19 Luminal A (LA) and 7 Luminal B tumors. Unsupervised clustering of the 8 Her2+ cases using the PAM50 genes and highly varying proteins resulted in different clustering patterns, with the latter clustering 6 of the 8 Her2+ cases with two distinct 3-case sub-clusters. Between these two sub-clusters, there were 10 differentially expressed (DEX) genes and 25 DEX proteins, but none of the 25 proteins were mapped to the 10 DEX genes. This motivated us to investigate the DEX exons, and we found 7,076 DEX exons between the Her2+ sub-clusters, and 9 of the 25 DEX proteins were matched to genes bearing DEX exons. For comparison, we performed the same analyses between similarly clustered Basal and LA sub-clusters. Even though the number of DEX genes between Basal (8) and LA (18) sub-clusters were comparable to that between the Her2+ sub-clusters, DEX exons were much lower for both subtypes (616 &amp; 157), and none of the DEX proteins (3 &amp; 7) mapped to DEX exons or genes for either subtype. Conclusions: Our findings imply that there is more proteomic-level heterogeneity in the Her2+ subtype than in Basal and LA subtypes, which could be due to alternative usage of exons (alternative splicing). If such heterogeneity is associated with patients’ survival outcomes then our results will further stress the importance of alternative splicing in breast cancer. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Format: Praveen Kumar Raj Kumar, Tao Liu, Lori A. Sturtz, Albert Kovatich, Marina A. Gritsenko, Vladislav A. Petyuk, Brenda Deyarmin, Viswanadham Sridhara, James Craig, Jason E. McDermott, Anil K. Shukla, Ronald J. Moore, Matthew E. Monroe, Bobbie-Jo M. Webb-Robertson, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Leonid Kvecher, Jianfang Liu, Jennifer Kane, Jennifer Melley, Stella Somiari, Joji Iida, Stephen C. Benz, Justin Golovato, Shahrooz Rabizadeh, Patrick Soon-Shiong, Richard D. Smith, Richard J. Mural, Karin D. Rodland, Craig D. Shriver, Hai Hu. Differential exon usage in the HER2 subtype of breast cancer identified with RNA-seq and proteomic data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3372. doi:10.1158/1538-7445.AM2017-3372

Alternative Splicing in Adhesion- and Motility-Related Genes in Breast Cancer.

Breast cancer is the most common tumor and the second leading cause of cancer death among woman, mainly caused by the metastatic spread. Tumor invasiveness is due to an altered expression of adhesion molecules. Among them, semaphorins are of peculiar interest. Cancer cells can manipulate alternative splicing patterns to modulate the expression of adhesion- and motility-related molecules, also at the isoform level. In this study, combining RNA-Sequencing on MCF-7 to targeted experimental validations—in human breast cell lines and breast tumor biopsies—we identified 12 new alternative splicing transcripts in genes encoding adhesion- and motility-related molecules, including semaphorins, their receptors and co-receptors. Among them, a new SEMA3F transcript is expressed in all breast cell lines and breast cancer biopsies, and is translated into a new semaphorin 3F isoform. In silico analysis predicted that most of the new putative proteins lack functional domains, potentially missing some functions and acquiring new ones. Our findings better describe the extent of alternative splicing in breast cancer and highlight the need to further investigate adhesion- and motility-related molecules to gain insights into breast cancer progression.

SRSF1-Regulated Alternative Splicing in Breast Cancer

Splicing factor SRSF1 is upregulated in human breast tumors, and its overexpression promotes transformation of mammary cells. Using RNA-seq, we identified SRSF1-regulated alternative splicing (AS) targets in organotypic three-dimensional MCF-10A cell cultures that mimic a context relevant to breast cancer. We identified and validated hundreds of endogenous SRSF1-regulated AS events. De novo discovery of the SRSF1 binding motif reconciled discrepancies in previous motif analyses. Using a Bayesian model, we determined positional effects of SRSF1 binding on cassette exons: binding close to the 5' splice site generally promoted exon inclusion, whereas binding near the 3' splice site promoted either exon skipping or inclusion. Finally, we identified SRSF1-regulated AS events deregulated in human tumors; overexpressing one such isoform, exon-9-included CASC4, increased acinar size and proliferation, and decreased apoptosis, partially recapitulating SRSF1's oncogenic effects. Thus, we uncovered SRSF1 positive and negative regulatory mechanisms, and oncogenic AS events that represent potential targets for therapeutics development.

Abstract P5-07-05: Oncogene PELP1 regulates alternative splicing in breast cancer through PRMT6

Abstract Background: Alternative splicing (AS) is essential in regulating gene expression and protein diversity; however, dysregulation leads to cancer development with tumor-specific splice variants that act by disrupting a tumor suppressor function. Protein arginine methylation is a modification involved in signal transduction, pre-RNA metabolism and transcriptional activation and deregulation of arginine methylase expression or functions can contribute to cancer progression via alternate splicing. PELP1 is a proto-oncogene, whose expression is associated with higher histological grade and shorter breast cancer specific survival, and PELP1 localization is a determinant of hormone sensitivity. This study focuses on examining the role of PELP1 in regulating gene expression and its possible role in alternative splicing. Methods: ZR75 and MCF7 cells stably expressing PELP1-shRNA or overexpressing PELP1 were used in RNASeq and ChipSeq analysis. Gene differential expression lists were generated using DEseq and PELP1 regulated pathways were analyzed using Ingenuity Pathway Analysis (IPA). Cell proliferation, migration and ERE reporter gene assays were used to test the role of PRMT6 in PELP1's oncogenic functions. Immunoprecipitation, confocal microscopy, ChIP, and GST pull down assays were used to demonstrate PELP1-PRMT6 interactions. Exon specific RTqPCR assays and CD44 minigene reporter assays were used to demonstrate PELP1's role in splicing. Results: RNA- and ChIP-sequencing results showed that the ERa coregulator PELP1 is a novel regulator of alternative splicing in breast cancer through the modulation of several genes involved in splicing including protein arginine methyltransferase 6 (PRMT6). PRMT6 plays a key role in coupling of transcription and alternative splicing by functioning as a transcriptional coactivator that can also regulate alternative splicing in a hormone independent manner. We discovered the novel complex of PELP1 and PRMT6 by co-immunoprecipitation. Biochemical assays revealed that PELP1 binds RNA, colocalizes with the splicing factor SC35 and promotes alternative splicing in reporter gene assays. PELP1 regulates the exon inclusion:skipping ratio of alternatively spliced exons in endogenous vascular endothelial growth factor (VEGF). PELP1's regulation of VEGF splicing is affected by the status of PRMT6. Interestingly, PRMT6 is needed for optimal oncogenic functions of PELP1. Further PELP1 has the potential to regulate the histone methyltransferase activity of PRMT6 and chromatin immunoprecipitation showed that PELP1 affects the enzyme recruitment and enrichment of the histone modification H3R2me2 at estrogen responsive genes. Conclusions: Our findings show a role of PELP1-PRMT6 axis in ERα-mediated RNA splicing and their deregulation has implications for targeted therapeutics in ERα-driven breast cancer. This work was supported by the NIH grant CA095681 and NIH F31 Fellowship 1F31CA173909-01A1. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-05.

Exon-Level Microarray Analyses Identify Alternative Splicing Programs in Breast Cancer

Protein isoforms produced by alternative splicing (AS) of many genes have been implicated in several aspects of cancer genesis and progression. These observations motivated a genome-wide assessment of AS in breast cancer. We accomplished this by measuring exon level expression in 31 breast cancer and nonmalignant immortalized cell lines representing luminal, basal, and claudin-low breast cancer subtypes using Affymetrix Human Junction Arrays. We analyzed these data using a computational pipeline specifically designed to detect AS with a low false-positive rate. This identified 181 splice events representing 156 genes as candidates for AS. Reverse transcription-PCR validation of a subset of predicted AS events confirmed 90%. Approximately half of the AS events were associated with basal, luminal, or claudin-low breast cancer subtypes. Exons involved in claudin-low subtype-specific AS were significantly associated with the presence of evolutionarily conserved binding motifs for the tissue-specific Fox2 splicing factor. Small interfering RNA knockdown of Fox2 confirmed the involvement of this splicing factor in subtype-specific AS. The subtype-specific AS detected in this study likely reflects the splicing pattern in the breast cancer progenitor cells in which the tumor arose and suggests the utility of assays for Fox-mediated AS in cancer subtype definition and early detection. These data also suggest the possibility of reducing the toxicity of protein-targeted breast cancer treatments by targeting protein isoforms that are not present in limiting normal tissues.