Minnkyong Lee

Phenotype-driven chemical screening in zebrafish for compounds that inhibit collective cell migration identifies multiple pathways potentially involved in metastatic invasion

ABSTRACT In the last decade, high-throughput chemical screening has become the dominant approach for discovering novel compounds with therapeutic properties. Automated screening using in vitro or cultured cell assays have yielded thousands of candidate drugs for a variety of biological targets, but these approaches have not resulted in an increase in drug discovery despite major increases in expenditures. In contrast, phenotype-driven screens have shown a much stronger success rate, which is why we developed an in vivo assay using transgenic zebrafish with a GFP-marked migrating posterior lateral line primordium (PLLp) to identify compounds that influence collective cell migration. We then conducted a high-throughput screen using a compound library of 2160 annotated bioactive synthetic compounds and 800 natural products to identify molecules that block normal PLLp migration. We identified 165 compounds that interfere with primordium migration without overt toxicity in vivo. Selected compounds were confirmed in their migration-blocking activity by using additional assays for cell migration. We then proved the screen to be successful in identifying anti-metastatic compounds active in vivo by performing orthotopic tumor implantation assays in mice. We demonstrated that the Src inhibitor SU6656, identified in our screen, can be used to suppress the metastatic capacity of a highly aggressive mammary tumor cell line. Finally, we used CRISPR/Cas9-targeted mutagenesis in zebrafish to genetically validate predicted targets of compounds. This approach demonstrates that the migrating PLLp in zebrafish can be used for large-scale, high-throughput screening for compounds that inhibit collective cell migration and, potentially, anti-metastatic compounds. Summary: We have developed a phenotype-driven screen for identifying new inhibitors of collective cell migration and demonstrated the screen can successfully identify compounds active in vivo and potentially new pathways for targeting cancer metastasis.

A Systems Genetics Approach Identifies CXCL14, ITGAX, and LPCAT2 as Novel Aggressive Prostate Cancer Susceptibility Genes

Although prostate cancer typically runs an indolent course, a subset of men develop aggressive, fatal forms of this disease. We hypothesize that germline variation modulates susceptibility to aggressive prostate cancer. The goal of this work is to identify susceptibility genes using the C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of neuroendocrine prostate cancer. Quantitative trait locus (QTL) mapping was performed in transgene-positive (TRAMPxNOD/ShiLtJ) F2 intercross males (n = 228), which facilitated identification of 11 loci associated with aggressive disease development. Microarray data derived from 126 (TRAMPxNOD/ShiLtJ) F2 primary tumors were used to prioritize candidate genes within QTLs, with candidate genes deemed as being high priority when possessing both high levels of expression-trait correlation and a proximal expression QTL. This process enabled the identification of 35 aggressive prostate tumorigenesis candidate genes. The role of these genes in aggressive forms of human prostate cancer was investigated using two concurrent approaches. First, logistic regression analysis in two human prostate gene expression datasets revealed that expression levels of five genes (CXCL14, ITGAX, LPCAT2, RNASEH2A, and ZNF322) were positively correlated with aggressive prostate cancer and two genes (CCL19 and HIST1H1A) were protective for aggressive prostate cancer. Higher than average levels of expression of the five genes that were positively correlated with aggressive disease were consistently associated with patient outcome in both human prostate cancer tumor gene expression datasets. Second, three of these five genes (CXCL14, ITGAX, and LPCAT2) harbored polymorphisms associated with aggressive disease development in a human GWAS cohort consisting of 1,172 prostate cancer patients. This study is the first example of using a systems genetics approach to successfully identify novel susceptibility genes for aggressive prostate cancer. Such approaches will facilitate the identification of novel germline factors driving aggressive disease susceptibility and allow for new insights into these deadly forms of prostate cancer.

Metastasis-Associated Protein Ribosomal RNA Processing 1 Homolog B (RRP1B) Modulates Metastasis through Regulation of Histone Methylation

Overexpression of ribosomal RNA processing 1 homolog B (RRP1B) induces a transcriptional profile that accurately predicts patient outcome in breast cancer. However, the mechanism by which RRP1B modulates transcription is unclear. Here, the chromatin-binding properties of RRP1B were examined to define how it regulates metastasis-associated transcription. To identify genome-wide RRP1B-binding sites, high-throughput ChIP-seq was performed in the human breast cancer cell line MDA-MB-231 and HeLa cells using antibodies against endogenous RRP1B. Global changes in repressive marks such as histone H3 lysine 9 trimethylation (H3K9me3) were also examined by ChIP-seq. Analysis of these samples identified 339 binding regions in MDA-MB-231 cells and 689 RRP1B-binding regions in HeLa cells. Among these, 136 regions were common to both cell lines. Gene expression analyses of these RRP1B-binding regions revealed that transcriptional repression is the primary result of RRP1B binding to chromatin. ChIP-reChIP assays demonstrated that RRP1B co-occupies loci with decreased gene expression with the heterochromatin-associated proteins, tripartite motif-containing protein 28 (TRIM28/KAP1), and heterochromatin protein 1-α (CBX5/HP1α). RRP1B occupancy at these loci was also associated with higher H3K9me3 levels, indicative of heterochromatinization mediated by the TRIM28/HP1α complex. In addition, RRP1B upregulation, which is associated with metastasis suppression, induced global changes in histone methylation. Implications: RRP1B, a breast cancer metastasis suppressor, regulates gene expression through heterochromatinization and transcriptional repression, which helps our understanding of mechanisms that drive prognostic gene expression in human breast cancer. Mol Cancer Res; 12(12); 1818–28. ©2014 AACR.

Necdin is a breast cancer metastasis suppressor that regulates the transcription of c-Myc

Metastasis is the primary cause of death in breast cancer. Earlier studies using a mammary tumorigenesis mouse model identified Necdin (Ndn) as a germline modifier of metastasis. Differential expression of Ndn induces a gene-expression signature that predicts prognosis in human breast cancer. Additionally, a non-synonymous germline single nucleotide polymorphism (T50C; V17A) in Ndn distinguishes mouse strains with differing metastatic capacities. To better understand how hereditary factors influence metastasis in breast cancer, we characterized NDN-mediated transcription. Haplotype analysis in a well-characterized breast cancer cohort revealed that NDN germline variation is associated with both NDN expression levels and patient outcome. To examine the role of NDN in mammary tumor metastasis and transcriptional regulation, mouse mammary tumor cell lines stably over-expressing either the wildtype 50T or variant 50C Ndn allele were generated. Cells over-expressing Ndn 50T, but not Ndn 50C, exhibited significant decrease in cell invasiveness and pulmonary metastases compared to control cells. Transcriptome analyses identified a 71-gene expression signature that distinguishes cells over-expressing the two Ndn allelic variants. Furthermore, ChIP assays revealed c-Myc, a target gene of NDN, to be differentially regulated by the allelic variants. These data demonstrate that NDN and the T50C allele regulate gene expression and metastasis efficiency.

SIN3A and SIN3B differentially regulate breast cancer metastasis

SIN3 corepressor complexes play important roles in both normal development and breast cancer. Mammalian cells have two paralogs of SIN3 (SIN3A and SIN3B) that are encoded by distinct genes and have unique functions in many developmental processes. However, specific roles for SIN3A and SIN3B in breast cancer progression have not been characterized. We generated stable knockdown cells of SIN3 paralogs individually and in combination using three non-overlapping shRNA. Stable knockdown of SIN3B caused a significant decrease in transwell invasion through Matrigel and decreased the number of invasive colonies when grown in a 3D extracellular matrix. Conversely, stable knockdown of SIN3A significantly increased transwell invasion and increased the number of invasive colonies. These results were corroborated in vivo in which SIN3B knockdown significantly decreased and SIN3A knockdown increased experimental lung metastases. RNA sequencing was used to identify unique targets and biological pathways that were altered upon knockdown of SIN3A compared to SIN3B. Additionally, we analyzed microarray data sets to identify correlations of SIN3A and SIN3B expression with survival in patients with breast cancer. These data sets indicated that high mRNA expression of SIN3A as well as low mRNA expression of SIN3B correlates with longer relapse free survival specifically in patients with triple negative breast cancer which corresponds with our in vitro and in vivo data. These results demonstrate key functional differences between SIN3 paralogs in regulating the process of breast cancer metastasis and suggest metastasis suppressive roles of SIN3A and metastasis promoting roles of SIN3B.

Prostate cancer susceptibility gene HIST1H1A is a modulator of androgen receptor signaling and epithelial to mesenchymal transition

In 2018, approximately 165,000 new prostate cancer (PC) cases will be diagnosed, and over 29,000 men will succumb to PC in the U.S. alone. The means of assessing outcome in the clinic are inaccurate, and there is a pressing need to more precisely identify men at risk of aggressive PC. We previously identified HIST1H1A as a susceptibility gene for aggressive PC. HIST1H1A encodes H1.1, a member of the linker histone family that is involved in chromatin organization and compaction. To understand the molecular basis of aggressive PC, we have characterized how germline variation modulates susceptibility to neuroendocrine differentiation, which is a form of aggressive PC. Immunohistochemistry studies revealed that HIST1H1A is over-expressed in normal human prostate tissue compared to prostate adenocarcinoma. Functional characterization of HIST1H1A in prostate LNCaP cells indicated that HIST1HA over-expression increased cell growth, as well as the expression of neuroendocrine and epithelial-to-mesenchymal markers in vitro. Assay for Transposase-Accessible Chromatin (ATAC-seq), which is used to assess chromatin compaction and thus the transcriptional availability of individual genomic regions, demonstrated that H1.1 plays a prominent role in modulating Wnt signaling pathway genes, which are implicated in prostate tumorigenesis. These results demonstrate that HIST1H1A is a modulator of aggressive PC susceptibility.

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

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

Abstract 123: The breast cancer metastasis suppressor RRP1B modulates metastasis through regulation of histone methylation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ribosomal RNA Processing 1 Homolog B (Rrp1b) is a germline breast cancer metastasis suppressor that regulates transcription to induce a highly prognostic gene expression signature. Although RRP1B interacts with chromatin- and mRNA splicing-associated factors, the mechanisms by which RRP1B modulates metastasis-associated gene expression are unclear. In this study, we examined the chromatin-binding properties of RRP1B to determine the mechanism by which it regulates metastasis-associated gene transcription. Genome-wide chromatin binding sites for endogenous RRP1B were defined using chromatin immunoprecipitation-sequencing (ChIP-seq) in Hela and MDA-MB-231 cells. ChIP-seq analysis of these samples identified 689 RRP1B binding regions in Hela and 339 binding regions in MDA-MB-231 cells. Among these, 136 regions were common to both cell lines. The expression of 40 genes located at or near these binding regions was examined by qPCR in MDA-MB-231 cells stably over-expressing RRP1B. These experiments demonstrated that RRP1B binding is primarily associated with transcriptional repression. Co-occupancy of RRP1B with the heterochromatin-associated proteins TRIM28 and HP1α at these loci were observed through ChIP-reChIP assays. Interaction between RRP1B and TRIM28 and HP1α was confirmed through co-immunoprecipitation. TRIM28 and HP1α have been shown to induce trimethylation of histone H3 at the lysine-9 position (H3K9me3) via the histone methyltransferase, SETDB1. Increases in H3K9me3 are typically associated with heterochromatinization and transcriptional repression. Given that RRP1B interacts with both TRIM28 and HP1α, and binds at common chromatin regions with these factors, we hypothesized that RRP1B is also implicated in increasing H3K9me3 levels at these loci. To examine the effect of RRP1B on H3K9me3, ChIP assays were performed in MDA-MB-231 cells stably over-expressing RRP1B and control cells with an antibody against H3K9me3. ChIP-qPCR demonstrated that chromatin regions that displayed co-occupancy of RRP1B with TRIM28 and HP1α have increased levels of H3K9me3 compared to that of the control. Additionally, ChIP-seq for H3K9me3 in cells stably over-expressing RRP1B demonstrated that RRP1B causes a global increase in H3K9me3 levels compared to control. These data collectively demonstrate that RRP1B regulates gene expression at the transcriptional level through an association with the transcriptional repressors TRIM28 and HP1α. Furthermore, the prominent mechanism that the metastasis suppressor RRP1B uses to regulate transcription is through epigenetic modifications, such as histone methylation. Citation Format: Minnkyong Lee, Amy M. Dworkin, Jens Lichtenberg, Shashank J. Patel, Derek Gildea, David M. Bodine, Nigel PS Crawford. The breast cancer metastasis suppressor RRP1B modulates metastasis through regulation of histone methylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 123. doi:10.1158/1538-7445.AM2014-123

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

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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

Abstract C21: A germline polymorphism in Necdin causes changes in gene expression and mammary tumor metastasis

Necdin (NDN) is a transcription factor that regulates gene expression by cooperating with chromatin modifying factors. Earlier studies using mouse models of mammary tumorigenesis identified Ndn as a metastasis efficiency modifier that exerts its influence at the germline level. Differential expression of Ndn induces a gene expression signature that accurately predicts prognosis in human breast cancer. Mouse Ndn contains a non-synonymous germline single nucleotide polymorphism (SNP) (T50C; V17A) that distinguishes mouse strains with differing susceptibilities to mammary tumor metastasis. The aim of this study is to characterize how germline variation in Ndn regulates metastasis-associated transcription to enhance our understanding of how hereditary factors influence metastasis in breast cancer. We hypothesize that NDN, a known transcription factor, induces metastasis-predictive gene expression by interacting with chromatin to regulate transcription, and that Ndn coding variants differ in their capacity in regulating the transcription of target genes. To confirm the role of NDN in human breast cancer, haplotype analysis was performed in a population cohort consisting of 956 breast cancer patients. These analyses revealed that a two marker SNP haplotype in linkage disequilibrium with NDN is significantly associated with survival (P=0.0002). We examined the role of NDN in mammary tumor metastasis and transcriptional regulation by using cells stably over-expressing either the wildtype 17V or the variant 17A Ndn alleles in the highly metastatic Mvt-1 and 4T1 mouse mammary tumor cell lines. Orthotopic implantation of these cells into the mammary fat pad of NU/J and BALB/cJ mice demonstrated that cells expressing the wildtype Ndn 17V allele exhibit a significant decrease in pulmonary metastases compared to cells transduced with an empty vector control. However, these metastasis suppressive properties of Ndn were consistently lost in cells ectopically expressing the variant Ndn 17A allele, demonstrating a prominent role for this SNP in regulation of metastasis efficiency. Soft agar assays confirmed these results in vitro as well as, where a two-fold decrease in cell invasiveness was observed with the wildtype Ndn 17V allele compared to the control and cells over-expressing the variant Ndn 17A allele. Furthermore, microarray analyses revealed that a 71-gene expression signature accurately distinguished cells expressing the two Ndn allelic variants demonstrating that this SNP plays a significant part in gene regulation as well. These data collectively demonstrate that Ndn functions as a germline metastasis modifier, most likely by regulating gene expression at the transcriptional level. Additionally, our work illustrates that the Ndn V17A SNP plays a significant role in both metastasis and transcriptional regulation. Our ongoing work is focused on defining the mechanism of action of Ndn V17A SNP using ChIP-seq to identify target genes and pathways responsible for the profound influence of Ndn allelic variants on metastatic efficiency. Citation Format: Minnkyong Lee, Sarah M. Beggs, Jens Lichtenberg, Sujata B. Bupp, Niraj S. Trivedi, Ying Hu, David M. Bodine, Nigel P.S. Crawford. A germline polymorphism in Necdin causes changes in gene expression and mammary tumor metastasis. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C21.