Natalie Goldberger

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.

An integrated systems genetics screen reveals the transcriptional structure of inherited predisposition to metastatic disease

Metastasis is the result of stochastic genomic and epigenetic events leading to gene expression profiles that drive tumor dissemination. Here we exploit the principle that metastatic propensity is modified by the genetic background to generate prognostic gene expression signatures that illuminate regulators of metastasis. We also identify multiple microRNAs whose germline variation is causally linked to tumor progression and metastasis. We employ network analysis of global gene expression profiles in tumors derived from a panel of recombinant inbred mice to identify a network of co-expressed genes centered on Cnot2 that predicts metastasis-free survival. Modulating Cnot2 expression changes tumor cell metastatic potential in vivo, supporting a functional role for Cnot2 in metastasis. Small RNA sequencing of the same tumor set revealed a negative correlation between expression of the Mir216/217 cluster and tumor progression. Expression quantitative trait locus analysis (eQTL) identified cis-eQTLs at the Mir216/217 locus, indicating that differences in expression may be inherited. Ectopic expression of Mir216/217 in tumor cells suppressed metastasis in vivo. Finally, small RNA sequencing and mRNA expression profiling data were integrated to reveal that miR-3470a/b target a high proportion of network transcripts. In vivo analysis of Mir3470a/b demonstrated that both promote metastasis. Moreover, Mir3470b is a likely regulator of the Cnot2 network as its overexpression down-regulated expression of network hub genes and enhanced metastasis in vivo, phenocopying Cnot2 knockdown. The resulting data from this strategy identify Cnot2 as a novel regulator of metastasis and demonstrate the power of our systems-level approach in identifying modifiers of metastasis.

An Integrated Genome-Wide Systems Genetics Screen for Breast Cancer Metastasis Susceptibility Genes

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. Here we report the results of a genome-wide integrated strategy to identify novel metastasis susceptibility candidate genes and molecular pathways in breast cancer metastasis. This analysis implicates a number of transcriptional regulators and suggests cell-mediated immunity is an important determinant. 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.

Genetic Background May Contribute to PAM50 Gene Expression Breast Cancer Subtype Assignments

Recent advances in genome wide transcriptional analysis have provided greater insights into the etiology and heterogeneity of breast cancer. Molecular signatures have been developed that stratify the conventional estrogen receptor positive or negative categories into subtypes that are associated with differing clinical outcomes. It is thought that the expression patterns of the molecular subtypes primarily reflect cell-of-origin or tumor driver mutations. In this study however, using a genetically engineered mouse mammary tumor model we demonstrate that the PAM50 subtype signature of tumors driven by a common oncogenic event can be significantly influenced by the genetic background on which the tumor arises. These results have important implications for interpretation of “snapshot” expression profiles, as well as suggesting that incorporation of genetic background effects may allow investigation into phenotypes not initially anticipated in individual mouse models of cancer.

A systems biology approach to defining metastatic biomarkers and signaling pathways

Metastasis is the final stage of cancer and the primary cause of mortality for most solid malignancies. This terminal phase of cancer progression has been investigated using a variety of high‐throughput technologies (i.e., gene expression arrays, array comparative genomic hybridization (aCGH), and proteomics) to identify prognostic expression profiles and better characterize the metastatic process. For decades, the predominant model for the metastatic process has been the ‘progression model’, yet recent microarray results tend to support an inherent metastatic capability within primary tumors. Moreover, studies using a highly metastatic transgenic mammary tumor model suggest that germline polymorphisms are significant determinants of metastatic efficiency. Likewise, a strong concordance of survival has been observed between family members with cancer, further supporting the link between genetic inheritance and survival. In addition, chromosomal aberrations and signaling pathways related to metastatic capacity have been identified by array comparative genomic hybridization (aCGH) and proteomic studies, respectively. Lastly, carcinoma enzyme activity profiles using activity‐based proteomics (ABPP), may be more clinically useful than expression‐based proteomics for certain cancers. Most importantly, the application of these high‐throughput techniques should expedite the search for additional biomarkers, germline polymorphisms, and expression signatures with greater prognostic value. Copyright

Gene expression changes during hormonal therapy for prostate cancer reveal candidate diagnostic and drug targets

Gene expression changes during hormonal therapy for prostate cancer reveal candidate diagnostic and drug targets

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

Abstracts: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX 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 B20: Breast cancer progression suppressor miR-290 targets breast cancer biomarker Arid4b

Several microRNAs (miRNAs) have been classified as metastasis regulators in breast cancer, yet few studies have examined how germline variations may promote metastasis via miRNA dysregulation. To explore this concept, 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. All mammary tumors in the F1 progeny were evaluated by miRNA microarray and correlated with metastatic index to produce miR-290 (containing mature miR-290-3p and miR-290-5p) as one of the top candidates. Further analysis showed miR-290-3p was downregulated in normal lung and breast tissue from highly metastatic AKR/J strains versus less metastatic DBA/2J strains, thereby supporting a germline component to miR-290 regulation. Next, when miR-290 was ectopically expressed in Mvt-1 and 6DT1, and orthotopically injected into FVB/N mice, a significant reduction in both mammary tumor burden and the number of pulmonary metastases was observed. Computational analysis identified breast cancer biomarker Arid4b as a top target of miR-290-3p, which was confirmed by luciferase reporter assay. In conclusion, these results suggest germline genetic changes may reduce miR-290 expression, in turn increasing Arid4b expression, to create a predisposition towards breast cancer progression. Citation Format: Natalie Goldberger, Renard Walker, Chang Hee Kim, Kent Hunter. Breast cancer progression suppressor miR-290 targets breast cancer biomarker Arid4b [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B20.

Abstract 3988: Identification of miR-132 and miR-290-3p as putative tumor and metastasis suppressor microRNAs in breast cancer

Several microRNAs (miRNAs) have been classified as regulators of breast cancer metastasis [1], yet additional unidentified miRNAs may also exist that regulate metastasis. Most interesting, we propose that germline genetic changes may exist which create a predisposition towards developing breast cancer metastasis through the dysregulation of miRNAs. Thus, to identify novel miRNAs associated with a germline predisposition towards breast cancer metastasis, highly metastatic FVB/N-TgN (MMTV-PyMT) mice were crossed with 25 AKXD RI (AKR/J × DBA/2J) strains to produce F1 progeny displaying primary mammary tumors with varying degrees of pulmonary metastasis. The miRNA expression levels within all mammary tumors were evaluated using a miRNA microarray and correlated with the metastatic index for each strain. The analysis produced 27 candidate miRNAs possessing a significant p-value (p 1. Hurst, D.R., M.D. Edmonds, and D.R. Welch, Metastamir: the field of metastasis-regulatory microRNA is spreading. Cancer Res, 2009. 69(19): p. 7495-8. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3988. doi:10.1158/1538-7445.AM2011-3988