Metastasis Susceptibility Gene Research Articles

Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer.

Patients with ER-negative breast cancer have the worst prognosis of all breast cancer subtypes, often experiencing rapid recurrence or progression to metastatic disease shortly after diagnosis. Given that metastasis is the primary cause of mortality in most solid tumors, understanding metastatic biology is crucial for effective intervention. Using a mouse systems genetics approach, we previously identified 12 genes associated with metastatic susceptibility. Here, we extend those studies to identify Resf1, a poorly characterized gene, as a novel metastasis susceptibility gene in ER- breast cancer. Resf1 is a large, unstructured protein with an evolutionarily conserved intron-exon structure, but with poor amino acid conservation. CRISPR or gene trap mouse models crossed to the Polyoma Middle-T antigen genetically engineered mouse model (MMTV-PyMT) demonstrated that reduction of Resf1 resulted in a significant increase in tumor growth, a shortened overall survival time, and increased incidence and number of lung metastases, consistent with patient data. Furthermore, an analysis of matched tail and primary tissues revealed loss of the wildtype copy in tumor tissue, consistent with Resf1 being a tumor suppressor. Mechanistic analysis revealed a potential role of Resf1 in transcriptional control through association with compound G4 quadruplexes in expressed sequences, particularly those associated with ribosomal biogenesis. These results suggest that loss of Resf1 enhances tumor progression in ER- breast cancer through multiple alterations in both transcriptional and translational control.

Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.

Aicardi-Goutières syndrome gene Rnaseh2c is a metastasis susceptibility gene in breast cancer.

Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. We found that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity, and immunophenotyping and RNA-sequencing analysis revealed engagement of the T cell-mediated adaptive immune response. Furthermore, the cGAS-Sting pathway was not activated in the metastatic cancer cells used in this study, suggesting that the mechanism of immune response in breast cancer is different from the mechanism proposed for Aicardi-Goutières Syndrome, a rare interferonopathy caused by RNase H2 mutation. These results suggest an important novel, non-enzymatic role for RNASEH2C during breast cancer progression and add Rnaseh2c to a panel of genes we have identified that together could determine patients with high risk for metastasis. These results also highlight a potential new target for combination with immunotherapies and may contribute to a better understanding of the etiology of Aicardi-Goutières Syndrome autoimmunity.

Abstract 2850: A systems genetics approach reveals Rnaseh2c-immune response axis that affects metastasis severity in breast cancer

Abstract Metastatic breast cancer is a devastating disease with a 5-year survival rate of only 26%. This is due to a lack of effective therapies against established metastases and an inability to identify high risk patients who would benefit from specific adjuvant therapies to prevent metastatic progression. We have shown in mouse models that spontaneously arising tumors metastasize with different severity based on the mouse genetic background. Using systems genetics approaches we have identified genes correlated with metastasis and survival in both mice and humans. Rnaseh2c was identified as a novel candidate metastasis susceptibility gene. This gene encodes a scaffolding subunit of the Ribonuclease H2 enzyme which removes ribonucleotides misincorporated into the DNA. Experimentally modulating Rnaseh2c expression in a murine mammary cancer cell line resulted in significant changes in pulmonary metastasis, confirming this gene as a metastasis modifier. Mutations in Rnaseh2c are known to cause Aicardi-Goutieres Syndrome, a neurological autoinflammatory disorder that overlaps clinically with congenital viral infections and the autoimmune disease Systemic Lupus Erythematosus. Given this, we hypothesized that altered expression of Rnaseh2c in breast cancer cells affects metastasis by engaging the immune system. To investigate immune system involvement, we analyzed metastasis in immunocompromised mice. T cell deficiency ablated the effect of reduced Rnaseh2c expression on metastasis, revealing for the first time an Rnaseh2c-immune response axis in metastasis. Gene ontology pathway analysis following mRNA-sequencing of Rnaseh2c knockdown and overexpression tumors revealed that 20% of the genes with altered expression are involved in immune system-related pathways, including T cell signaling and antigen presentation. Furthermore, genes with significant changes included Type I interferons, T cell markers, and immune regulators. These results confirm that Rnaseh2c is a novel metastasis modifier gene and validate our hypothesis that the immune system is mediating the effect of Rnaseh2c on metastasis. This mechanism highlights a potential new target for combination with immune modulatory therapies to combat this devastating disease and adds to a panel of genes we identified that together could determine patients with high risk for metastasis. Citation Format: Sarah Deasy, Kent Hunter. A systems genetics approach reveals Rnaseh2c-immune response axis that affects metastasis severity 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 2850. doi:10.1158/1538-7445.AM2017-2850

The Circadian Rhythm Gene Arntl2 Is a Metastasis Susceptibility Gene for Estrogen Receptor-Negative Breast Cancer.

Breast cancer mortality is primarily due to metastasis rather than primary tumors, yet relatively little is understood regarding the etiology of metastatic breast cancer. Previously, using a mouse genetics approach, we demonstrated that inherited germline polymorphisms contribute to metastatic disease, and that these single nucleotide polymorphisms (SNPs) could be used to predict outcome in breast cancer patients. In this study, a backcross between a highly metastatic (FVB/NJ) and low metastatic (MOLF/EiJ) mouse strain identified Arntl2, a gene encoding a circadian rhythm transcription factor, as a metastasis susceptibility gene associated with progression, specifically in estrogen receptor-negative breast cancer patients. Integrated whole genome sequence analysis with DNase hypersensitivity sites reveals SNPs in the predicted promoter of Arntl2. Using CRISPR/Cas9-mediated substitution of the MOLF promoter, we demonstrate that the SNPs regulate Arntl2 transcription and affect metastatic burden. Finally, analysis of SNPs associated with ARNTL2 expression in human breast cancer patients revealed reproducible associations of ARNTL2 expression quantitative trait loci (eQTL) SNPs with disease-free survival, consistent with the mouse studies.

The Clock Is Ticking: Countdown to Metastases.

Metastases cause more than 90% of the morbidity and mortality associated with human cancers. Gene expression signatures associated with cancer progression and metastasis serve as unique tools to assist in patient diagnosis, prognosis, and treatment. Various types of signatures have been identified, ranging from those that are tumor-intrinsic or specific to a particular cancer subtype [1] to genes associated with a specific clinical outcome (e.g., “poor prognosis gene signature”) [2], as well as to genes associated with the development of metastatic lesions [3]. Of those genes that drive cancer processes, some may function by acting on the primary tumor—causing rise of metastatic lesions—or at the metastatic site to promote colonization, survival, and incorporation of surrounding stroma. Identification of metastasis susceptibility genes is thus key for prediction of cancer risk and metastatic relapse.

Abstract IA21: An integrated genome-wide systems genetics screen for breast cancer metastasis susceptibility genes

Abstract Metastasis remains the primary cause of patient morbidity and mortality in solid tumors and is due to the action of a large number of tumor-autonomous and non-autonomous factors. Hundreds or thousands of genes are thought to be associated with metastasis however how many of these genes contribute etiologically to tumor progression is not currently known. Identification of the genes contributing mechanistically to the metastatic processes will not only deepen our understanding of how metastases occur, but also provide novel targets for either preventing their formation or combatting their life-threatening effects. Previously our laboratory demonstrated that inbred mice of distinct phylogenetic lineages possess distinct propensity for metastatic disease, indicating that inherited susceptibility for metastasis exists and that polymorphisms can both mark and functionally effect genes within the metastatic cascade. Here we report the results of a genome-wide integrated strategy to identify novel metastasis susceptibility candidate genes and molecular pathways which implicates a number of transcriptional regulators and suggests cell-mediated immunity is an important determinant. The strategy described integrates meiotic genetic screens with epigenetic control of gene expression and three dimensional chromatin structure analyses to identify genes, molecular and cellular pathways likely to be important in metastatic disease. Moreover, the analysis identified novel or FDA-approved drugs as potentially useful for anti-metastatic therapy. Further explorations implementing this strategy may therefore provide a variety of information for clinical applications in the control and treatment of advanced neoplastic disease. Citation Format: Kent W. Hunter, Ling Bai, Howard H. Yang, Ying Hu, Anjali Shukla, Ngoc-Han Ha, Anthony Doran, Farhoud Faraji, Natalie Goldberger, Maxwell P. Lee, Thomas Keane. An integrated genome-wide systems genetics screen for breast cancer metastasis susceptibility genes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr IA21.

Abstract B30: Rnaseh2c is a candidate metastasis susceptibility gene in breast cancer

Abstract Breast cancer is a leading cause of cancer-related female deaths in the United States, the majority of which are due to distant metastases. Elucidating novel genes that drive metastasis is critical to improving patient outcome. Our laboratory has extensively shown that germline variants impact an individual's susceptibility for metastasis, and we have used this to identify novel metastasis modifier genes. Here we applied a mouse model of spontaneous metastasis to a mouse breeding scheme to screen for strain-specific haplotypes associated with metastatic phenotype. Additional filtering of the haplotype-containing genes was performed for those that were highly expressed in tumors. This screen identified Rnaseh2c, a scaffolding subunit of the Ribonuclease H2 complex which degrades RNA from DNA:RNA heteroduplexes such as those generated during replication and transcription, thus helping maintain genomic stability. Though mutations in RNASEH2C are known to cause the neurological autoinflammatory disorder Aicardi-Goutières Syndrome (AGS), it has not previously been linked to cancer. Given the known pathology of AGS, we hypothesize that differential expression of Rnaseh2c in tumor cells alters tumor progression and metastasis potentially via interaction with the immune system. We directly assessed the effect of tumor cell Rnaseh2c expression on metastasis using in vivo metastasis assays. We implanted into the mammary fat pad tumor cells expressing shRNAs that reduced Rnaseh2c expression and observed significantly fewer pulmonary metastases and reduced tumor burden. To complement this approach, we performed the same metastasis assay with cells overexpressing exogenous Rnaseh2c. In agreement with our initial result, overexpression generated significantly more metastases without affecting primary tumor size, confirming Rnaseh2c as a metastasis modifier. The results thus far validate our approach of employing germline variation in mice to identify novel metastasis modifier genes. Current and future work will focus on elucidating the mechanism by which Rnaseh2c expression impacts metastasis. Specifically, we will determine where Rnaseh2c functions in the metastatic cascade, whether modulated expression disrupts Rnase H2 complex formation, and whether the immune system is involved in mediating this gene's effect on metastasis. Elucidating the mechanism by which this gene acts can contribute to both the development of novel metastasis biomarkers as well as potential therapeutic targets. Citation Format: Sarah Deasy, Kent Hunter. Rnaseh2c is a candidate metastasis susceptibility gene in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B30.

Post-transcriptional Control of Tumor Cell Autonomous Metastatic Potential by CCR4-NOT Deadenylase CNOT7.

Accumulating evidence supports the role of an aberrant transcriptome as a driver of metastatic potential. Deadenylation is a general regulatory node for post-transcriptional control by microRNAs and other determinants of RNA stability. Previously, we demonstrated that the CCR4-NOT scaffold component Cnot2 is an inherited metastasis susceptibility gene. In this study, using orthotopic metastasis assays and genetically engineered mouse models, we show that one of the enzymatic subunits of the CCR4-NOT complex, Cnot7, is also a metastasis modifying gene. We demonstrate that higher expression of Cnot7 drives tumor cell autonomous metastatic potential, which requires its deadenylase activity. Furthermore, metastasis promotion by CNOT7 is dependent on interaction with CNOT1 and TOB1. CNOT7 ribonucleoprotein-immunoprecipitation (RIP) and integrated transcriptome wide analyses reveal that CNOT7-regulated transcripts are enriched for a tripartite 3’UTR motif bound by RNA-binding proteins known to complex with CNOT7, TOB1, and CNOT1. Collectively, our data support a model of CNOT7, TOB1, CNOT1, and RNA-binding proteins collectively exerting post-transcriptional control on a metastasis suppressive transcriptional program to drive tumor cell metastasis.

Defining the Influence of Germline Variation on Metastasis Using Systems Genetics Approaches.

Cancer is estimated to be responsible for 8 million deaths worldwide and over half a million deaths every year in the United States. The majority of cancer-related deaths in solid tumors is directly associated with the effects of metastasis. While the influence of germline factors on cancer risk and development has long been recognized, the contribution of hereditary variation to tumor progression and metastasis has only gained acceptance more recently. A variety of approaches have been used to define how hereditary variation influences tumor progression and metastasis. One approach that garnered much early attention was epidemiological studies of cohorts of cancer patients, which demonstrated that specific loci within the human genome are associated with a differential propensity for aggressive tumor development. However, a powerful, and somewhat underutilized approach has been the use of systems genetics approaches in transgenic mouse models of human cancer. Such approaches are typically multifaceted, and involve integration of multiple lines of evidence derived, for example, from genetic and transcriptomic screens of genetically diverse mouse models of cancer, coupled with bioinformatics analysis of human cancer datasets, and functional analysis of candidate genes. These methodologies have allowed for the identification of multiple hereditary metastasis susceptibility genes, with wide-ranging cellular functions including regulation of gene transcription, cell proliferation, and cell-cell adhesion. In this chapter, we review how each of these approaches have facilitated the identification of these hereditary metastasis modifiers, the molecular functions of these metastasis-associated genes, and the implications of these findings upon patient survival.

GNL3 and SKA3 are novel prostate cancer metastasis susceptibility genes.

Prostate cancer (PC) is very common in developed countries. However, the molecular determinants of PC metastasis are unclear. Previously, we reported that germline variation influences metastasis in the C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of PC. These mice develop prostate tumors similar to a subset of poor outcome, treatment-associated human PC tumors. Here, we used TRAMP mice to nominate candidate genes and validate their role in aggressive human PC in PC datasets and cell lines. Candidate metastasis susceptibility genes were identified through quantitative trait locus (QTL) mapping in 201 (TRAMP × PWK/PhJ) F2 males. Two metastasis-associated QTLs were identified; one on chromosome 12 (LOD = 5.86), and one on chromosome 14 (LOD = 4.41). Correlation analysis using microarray data from (TRAMP × PWK/PhJ) F2 prostate tumors identified 35 metastasis-associated transcripts within the two loci. The role of these genes in susceptibility to aggressive human PC was determined through in silico analysis using multiple datasets. First, analysis of candidate gene expression in two human PC datasets demonstrated that five candidate genes were associated with an increased risk of aggressive disease and lower disease-free survival. Second, four of these genes (GNL3, MAT1A, SKA3, and ZMYM5) harbored SNPs associated with aggressive tumorigenesis in the PLCO/CGEMS GWAS of 1172 PC patients. Finally, over-expression of GNL3 and SKA3 in the PC-3 human PC cell line decreased in vitro cell migration and invasion. This novel approach demonstrates how mouse models can be used to identify metastasis susceptibility genes, and gives new insight into the molecular mechanisms of fatal PC.

A Genome-Wide Scan Identifies Variants in NFIB Associated with Metastasis in Patients with Osteosarcoma.

Metastasis is the leading cause of death in patients with osteosarcoma, the most common pediatric bone malignancy. We conducted a multistage genome-wide association study of osteosarcoma metastasis at diagnosis in 935 osteosarcoma patients to determine whether germline genetic variation contributes to risk of metastasis. We identified an SNP, rs7034162, in NFIB significantly associated with metastasis in European osteosarcoma cases, as well as in cases of African and Brazilian ancestry (meta-analysis of all cases: P = 1.2 × 10(-9); OR, 2.43; 95% confidence interval, 1.83-3.24). The risk allele was significantly associated with lowered NFIB expression, which led to increased osteosarcoma cell migration, proliferation, and colony formation. In addition, a transposon screen in mice identified a significant proportion of osteosarcomas harboring inactivating insertions in Nfib and with lowered NFIB expression. These data suggest that germline genetic variation at rs7034162 is important in osteosarcoma metastasis and that NFIB is an osteosarcoma metastasis susceptibility gene. Metastasis at diagnosis in osteosarcoma is the leading cause of death in these patients. Here we show data that are supportive for the NFIB locus as associated with metastatic potential in osteosarcoma.

Abstract 4138: Integrating SNPs, epigenetics and transcriptomics to better understand the inherited predisposition to breast cancer metastasis

Abstract Breast cancer is the most frequent cancer and the second leading cause of cancer mortality in women. The vast majority of breast cancer mortality is due to metastatic disease since the primary tumor can be relatively easily surgically resected. More comprehensive understanding of biology of metastasis is therefore clearly warranted to unveil novel metastasis-associated molecules and cellular processes that might be targeted for clinical intervention. Previously we demonstrated that like cancer incidence, metastatic progression has a significant inherited component. Using mouse complex trait mapping strategies we have been isolating metastasis susceptibility genes from backcross analysis. However, studies suggest that many of these QTL peaks are the result of the contribution of multiple genes, suggesting that causal genes are not being identified. To increase our ability to detect causal genes we have implemented a new integrated strategy. Whole genome sequencing of appropriate high or low metastatic mouse strains is performed to identify variants, which are then filtered for those within DNAse hypersensitive sites (DHS), based on the hypothesis that most inherited phenotypes are due to expression differences rather than missense variants. The genes associated with polymorphic DHS are then screened through mouse and human tumor expression databases to identify those genes associated with development of metastatic disease. This strategy was able to identify genes previously associated with metastatic breast cancer in both mouse and humans and new genes have also been validated. The results further suggest that inherited variation in cellular mediated immune response may be an important contributor to metastatic disease. Citation Format: Anjali Shukla, Ling Bai, Howard Yang, Anthony Doran, Ying Hu, Thomas Geiger, Maxwell Lee, Thomas Keane, Kent W. Hunter. Integrating SNPs, epigenetics and transcriptomics to better understand the inherited predisposition to breast cancer metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4138. doi:10.1158/1538-7445.AM2015-4138

Abstract 2261: DLGAP5, MAT1A, SKA3, and ZMYM5 are novel susceptibility genes for aggressive prostate cancer

Abstract Prostate cancer is a common disease with approximately 233,000 men estimated to be diagnosed in the United States alone in 2014. Yet, it is usually an indolent disease with only 13% of patients succumbing to prostate cancer, and the molecular determinants of aggressive prostate cancer remain unclear. Previously, we reported that germline variation influences disease aggressiveness in the C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model. These mice develop neuroendocrine prostate tumors similar to a subset of human prostate tumors associated with poor outcomes. Here, we used a multifaceted approach to identify candidate genes for susceptibility to aggressive prostate tumorigenesis and metastasis using the TRAMP mouse model. Candidate prostate cancer metastasis susceptibility genes were identified through quantitative trait locus (QTL) mapping in 201 (TRAMP x PWK/PhJ) F2 males. Two aggressive disease QTLs were identified; one for lymph node metastasis burden on chromosome 12 (LOD = 5.86) and one for distant metastasis-free survival on chromosome 14 (LOD = 4.41). Correlation analysis using microarray data derived from 27 (TRAMP x PWK/PhJ) F2 prostate tumors identified 35 metastasis-associated transcripts within the two loci. The role of these genes in susceptibility to aggressive human prostate cancer was analyzed in two different datasets. First, logistic regression and survival analyses in human prostate cancer gene expression datasets demonstrated that the expression levels of 5 of the 35 candidate genes was associated with both an increased risk of aggressive disease and a poorer disease-free survival. Second, four of these genes - DLGAP5, MAT1A, SKA3, and ZMYM5 - harbored SNPs associated with aggressive tumorigenesis in the PLCO/CGEMS GWAS cohort of 1,172 prostate cancer patients. This approach, novel to the prostate cancer field, demonstrates how mouse models can be used to identify aggressive disease susceptibility genes, and gives new insight into the molecular mechanisms of aggressive disease. Citation Format: Minnkyong Lee, Kendra A. Williams, Ying Hu, Jonathan Andreas, Shashankkumar J. Patel, Suiyuan Zhang, Nigel PS Crawford. DLGAP5, MAT1A, SKA3, and ZMYM5 are novel susceptibility genes for aggressive prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2261. doi:10.1158/1538-7445.AM2015-2261

RRP1B is a metastasis modifier that regulates the expression of alternative mRNA isoforms through interactions with SRSF1

RRP1B (ribosomal RNA processing 1 homolog B) was first identified as a metastasis susceptibility gene in breast cancer through its ability to modulate gene expression in a manner that can be used to accurately predict prognosis in breast cancer. However, the mechanism(s) by which RRP1B modulates gene expression is currently unclear. Many RRP1B binding candidates are involved in alternative splicing, a mechanism of gene expression regulation that is increasingly recognized to be involved in cancer progression and metastasis. One such target is SRSF1 (SF2/ASF), an essential splicing regulator that also functions as an oncoprotein. Earlier studies demonstrated that splicing and transcription occur concurrently and are coupled processes. Given that RRP1B regulates transcriptional activity, we hypothesized that RRP1B also regulates the expression of alternative mRNA isoforms through its interaction with SRSF1. Interaction between RRP1B and SRSF1 was verified by co-immunoprecipitation and co-immunofluorescence. Treatment of cells with transcriptional inhibitors significantly increased this interaction, demonstrating that the association of these two proteins is transcriptionally regulated. To assess the role of RRP1B in the regulation of alternative isoform expression, RNA-seq data were generated from control and Rrp1b-knockdown cells. Knockdown of Rrp1b induced a significant change in isoform expression in over 600 genes compared to control cell lines. This was verified by qRT-PCR using isoform-specific primers. Pathway enrichment analyses identified cell cycle and checkpoint regulation to be those most affected by Rrp1b knockdown. These data suggest that RRP1B suppresses metastatic progression by altering the transcriptome through its interaction with splicing regulators such as SRSF1.

Abstract 4590: RRP1B, a hereditary metastasis modifier, mediates transcription and splicing to regulate mRNA expression.

Abstract RRP1B (ribosomal RNA processing 1 homolog B) was first identified as a metastasis susceptibility gene in breast cancer by its association with the altered expression of extracellular matrix genes, which are common factors in metastasis predictive gene expression signatures. A non-synonymous polymorphism in RRP1B (1421C>T; 436P>L) is associated with metastasis-free survival in multiple breast cancer cohorts, representing over 2,000 patients. These data collectively demonstrate the clinical importance of RRP1B in tumor progression in breast cancer. Yet, the mechanism through which RRP1B alters the expression of survival-associated genes is unknown. Previous studies have shown that many RRP1B binding candidates are involved in alternative splicing, a method of gene expression regulation that is increasingly recognized to be involved in cancer progression and metastasis. One such target is SRSF1 (SF2/ASF), an essential splicing regulator that also functions as an oncogene. Since RRP1B regulates transcriptional activity, we hypothesized that RRP1B also regulates the expression of alternative mRNA isoforms through its interaction with SRSF1. Interaction between RRP1B and SRSF1 was verified by co-immunoprecipitation and co-immunofluorescence. Earlier studies demonstrated that transcription and mRNA splicing are co-dependent in which each process affects the other through either physical interaction or kinetic regulation. Given that RRP1B interacts with both chromatin-associated factors and splicing regulators, we examined whether RRP1B acts as a mediator between the two sets of machinery. Treatment of cells with transcriptional inhibitors significantly increased the interaction between RRP1B and SRSF1, demonstrating that the association of these two proteins is transcriptionally modulated. In addition, immunoprecipitation assays showed that RRP1B interacts with RNA polymerase II. To assess the role of RRP1B in the regulation of alternative isoforms, we generated RNA-sequencing data from control and Rrp1b-knockdown cells. Knockdown of Rrp1b induced a significant change in isoform expression in over 600 genes compared to control cell lines. This was verified by qRT-PCR using isoform-specific primers. Pathway enrichment analyses identified genes involved in cell cycle and checkpoint regulation to be those most affected by Rrp1b knockdown. Also, knockdown of Rrp1b increased in vitro cell invasiveness and in vivo metastasis, confirming the role of RRP1B as a metastasis suppressor. Based on these findings, we propose that RRP1B regulates metastatic progression by altering the transcriptome through its interaction with transcriptional machinery as well as splicing regulators such as SRSF1. To the best of our knowledge, this is the first study to show that a metastasis susceptibility gene is directly involved in regulating alternative isoform expression. Citation Format: Minnkyong Lee, Derek Gildea, Niraj S. Trivedi, NISC Comparative Sequencing Program, Tyra G. Wolfsberg, Nigel P.S. Crawford. RRP1B, a hereditary metastasis modifier, mediates transcription and splicing to regulate mRNA expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4590. doi:10.1158/1538-7445.AM2013-4590

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Inherited Variation in miR-290 Expression Suppresses Breast Cancer Progression by Targeting the Metastasis Susceptibility Gene Arid4b

The metastatic cascade is a complex and extremely inefficient process with many potential barriers. Understanding this process is of critical importance because the majority of cancer mortality is associated with metastatic disease. Recently, it has become increasingly clear that microRNAs (miRNA) play important roles in tumorigenesis and metastasis, yet few studies have examined how germline variations may dysregulate miRNAs, in turn affecting metastatic potential. To explore this possibility, the highly metastatic MMTV-PyMT mice were crossed with 25 AKXD (AKR/J × DBA/2J) recombinant inbred strains to produce F1 progeny with varying metastatic indices. When mammary tumors from the F1 progeny were analyzed by miRNA microarray, miR-290 (containing miR-290-3p and miR-290-5p) was identified as a top candidate progression-associated miRNA. The microarray results were validated in vivo when miR-290 upregulation in two independent breast cancer cell lines suppressed both primary tumor and metastatic growth. Computational analysis identified breast cancer progression gene Arid4b as a top target of miR-290-3p, which was confirmed by luciferase reporter assay. Surprisingly, pathway analysis identified estrogen receptor (ER) signaling as the top canonical pathway affected by miR-290 upregulation. Further analysis showed that ER levels were elevated in miR-290-expressing tumors and positively correlated with apoptosis. Taken together, our results suggest miR-290 targets Arid4b while simultaneously enhancing ER signaling and increasing apoptosis, thereby suppressing breast cancer progression. This, to the best of our knowledge, is the first example of inherited differences in miRNA expression playing a role in breast cancer progression.

Abstract IA21: Using mouse models to identify breast cancer metastasis susceptibility genes.

Abstract Approximately 200,000 women are diagnosed with breast cancer and about 40,000 patients will succumb to this disease per year. The vast majority of these deaths are due to metastatic disease, rather than the primary tumor, so better understanding of the terminal stages of breast cancer is of great importance for the design of improved clinical interventions. Mouse genetic studies from our laboratory previously demonstrated that like susceptibility to the development of breast cancer, a significant inherited component exists for disseminated disease. However, despite the tremendous success of GWAS in identifying inherited variants associated with cancer etiology, significantly less success has been achieved in identifying SNPs associated with disease prognosis. This may be due in part to the complexity of the metastatic process, which involves genetic, epigenetic as well as stochastic somatic events. The ability to directly detect inherited metastasis susceptibility loci in human populations may be further complicated by clinical interventions, including surgical resection, radiation and/or adjuvant therapy which may alter or interrupt the natural progression of the disease in patient cohorts, reducing the power of detection. Therefore to complement human studies of inherited susceptibility to progression, our laboratory has employed a dominant metastatic genetically engineered mouse model and a quantitative trait analysis strategy to identify genes associated with disseminated disease. The results of these studies will be presented in this presentation. Citation Format: Kent W. Hunter. Using mouse models to identify breast cancer metastasis susceptibility genes. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr IA21.

Cadm1 Is a Metastasis Susceptibility Gene That Suppresses Metastasis by Modifying Tumor Interaction with the Cell-Mediated Immunity

Metastasis is a complex process utilizing both tumor-cell-autonomous properties and host-derived factors, including cellular immunity. We have previously shown that germline polymorphisms can modify tumor cell metastatic capabilities through cell-autonomous mechanisms. However, how metastasis susceptibility genes interact with the tumor stroma is incompletely understood. Here, we employ a complex genetic screen to identify Cadm1 as a novel modifier of metastasis. We demonstrate that Cadm1 can specifically suppress metastasis without affecting primary tumor growth. Unexpectedly, Cadm1 did not alter tumor-cell-autonomous properties such as proliferation or invasion, but required the host's adaptive immune system to affect metastasis. The metastasis-suppressing effect of Cadm1 was lost in mice lacking T cell–mediated immunity, which was partially phenocopied by depleting CD8+ T cells in immune-competent mice. Our data show a novel function for Cadm1 in suppressing metastasis by sensitizing tumor cells to immune surveillance mechanisms, and this is the first report of a heritable metastasis susceptibility gene engaging tumor non-autonomous factors.