Luanne Lukes

Bromodomain 4 activation predicts breast cancer survival

Previous work identified the Rap1 GTPase-activating protein Sipa1 as a germ-line-encoded metastasis modifier. The bromodomain protein Brd4 physically interacts with and modulates the enzymatic activity of Sipa1. In vitro analysis of a highly metastatic mouse mammary tumor cell line ectopically expressing Brd4 demonstrates significant reduction of invasiveness without altering intrinsic growth rate. However, a dramatic reduction of tumor growth and pulmonary metastasis was observed after s.c. implantation into mice, implying that activation of Brd4 may somehow be manipulating response to tumor microenvironment in the in vivo setting. Further in vitro analysis shows that Brd4 modulates extracellular matrix gene expression, a class of genes frequently present in metastasis-predictive gene signatures. Microarray analysis of the mammary tumor cell lines identified a Brd4 activation signature that robustly predicted progression and/or survival in multiple human breast cancer datasets analyzed on different microarray platforms. Intriguingly, the Brd4 signature also almost perfectly matches a molecular classifier of low-grade tumors. Taken together, these data suggest that dysregulation of Brd4-associated pathways may play an important role in breast cancer progression and underlies multiple common prognostic signatures.

Metastasis Predictive Signature Profiles Pre-exist in Normal Tissues

Previous studies from our laboratory have demonstrated that metastatic propensity is significantly influenced by the genetic background upon which tumors arise. We have also established that human gene expression profiles predictive of metastasis are not only present in mouse tumors with both high and low metastatic capacity, but also correlate with genetic background. These results suggest that human metastasis-predictive gene expression signatures may be significantly driven by genetic background, rather than acquired somatic mutations. To test this hypothesis, gene expression profiling was performed on inbred mouse strains with significantly different metastatic efficiencies. Analysis of previously described human metastasis signature gene expression patterns in normal tissues permitted accurate categorization of high or low metastatic mouse genotypes. Furthermore, prospective identification of animals at high risk of metastasis was achieved by using mass spectrometry to characterize salivary peptide polymorphisms in a genetically heterogeneous population. These results strongly support the role of constitutional genetic variation in modulation of metastatic efficiency and suggest that predictive signature profiles could be developed from normal tissues in humans. The ability to identify those individuals at high risk of disseminated disease at the time of clinical manifestation of a primary cancer could have a significant impact on cancer management.

Caffeine suppresses metastasis in a transgenic mouse model: a prototype molecule for prophylaxis of metastasis.

A significant fraction of cancer patients have occult disseminated tumors at the time of primary diagnosis, which usually progress to become clinically relevant lesions. Since the majority of cancer mortality is associated with metastatic disease, the ability to inhibit the growth of the secondary tumors would significantly reduce cancer-related morbidity and mortality. We have investigated whether caffeine, which has been shown to suppress tumor cell invasiveness and experimental metastasis, can suppress metastasis in a spontaneous transgene-induced mammary tumor model. Chronic exposure to caffeine prior to the appearance of palpable mammary tumors significantly reduced both tumor burden and metastatic colonization. However, when caffeine exposure began after the appearance of frank tumors, caffeine suppressed metastasis without changing primary tumor burden. The means by which caffeine suppressed metastatic activity may be associated with inhibition of malignant transformation of mammary epithelial cells, inhibition of conversion of dormant tumor cells to micrometastases, micrometastases to macrometastases, or inhibition of tumor cell adhesion and motility. Gene and protein expression patterns resulting from caffeine treatment showed that metastasis suppression may be associated with up-regulation the mRNA expression of multiple extracellular matrix genes, including Fbln1, Bgn, Sparc, Fbn1, Loxl1, Col1a1, Col3a1, Col5a1, Col5a2, Col5a3, Col6a1, Col6a2, and Col6a3. These data suggested that caffeine or other methyl xanthine derivatives may improve the clinical outcome in patients prior to and following the diagnosis of metastatic disease, and could potentially reduce the morbidity and mortality associated with disseminated tumors.

The Diasporin Pathway: a tumor progression-related transcriptional network that predicts breast cancer survival

Microarray expression signature analyses have suggested that extracellular matrix (ECM) gene dysregulation is predictive of metastasis in both mouse mammary tumorigenesis and human breast cancer. We have previously demonstrated that such ECM dysregulation is influenced by hereditary germline-encoded variation. To identify novel metastasis efficiency modifiers, we performed expression QTL (eQTL) mapping in recombinant inbred mice by characterizing genetic loci modulating metastasis-predictive ECM gene expression. Three reproducible eQTLs were observed on chromosomes 7, 17 and 18. Candidate genes were identified by correlation analyses and known associations with metastasis. Seven candidates were identified (Ndn, Pi16, Luc7l, Rrp1b, Brd4, Centd3 and Csf1r). Stable transfection of the highly metastatic Mvt-1 mouse mammary tumor cell line with expression vectors encoding each candidate modulated metastasis-predictive ECM gene expression. Implantation of these cells into mice demonstrated that candidate gene ectopic expression impacts tumor progression. Gene expression analyses facilitated the construction of a transcriptional network that we have termed the ‘Diasporin Pathway’. This pathway contains the seven candidates, as well as metastasis-predictive ECM genes and metastasis suppressors. Brd4 and Rrp1b appear to form a central node within this network, which likely is a consequence of their physical interaction with the metastasis efficiency modifier Sipa1. Furthermore, we demonstrate that the microarray gene expression signatures induced by activation of ECM eQTL genes in the Mvt-1 cell line can be used to accurately predict survival in a human breast cancer cohort. These data imply that the Diasporin Pathway may be an important nexus in tumor progression in both mice and humans.

Accelerated Preclinical Testing Using Transplanted Tumors from Genetically Engineered Mouse Breast Cancer Models

Purpose: The use of genetically engineered mouse (GEM) models for preclinical testing of anticancer therapies is hampered by variable tumor latency, incomplete penetrance, and complicated breeding schemes. Here, we describe and validate a transplantation strategy that circumvents some of these difficulties. Experimental Design: Tumor fragments from tumor-bearing MMTV-PyMT or cell suspensions from MMTV-PyMT, -Her2/neu, -wnt1, -wnt1/p53+/−, BRCA1/p53+/−, and C3(1)T-Ag mice were transplanted into the mammary fat pad or s.c. into naïve syngeneic or immunosuppressed mice. Tumor development was monitored and tissues were processed for histopathology and gene expression profiling. Metastasis was scored 60 days after the removal of transplanted tumors. Results: PyMT tumor fragments and cell suspensions from anterior glands grew faster than posterior tumors in serial passages regardless of the site of implantation. Microarray analysis revealed genetic differences between these tumors. The transplantation was reproducible using anterior tumors from multiple GEM, and tumor growth rate correlated with the number of transplanted cells. Similar morphologic appearances were observed in original and transplanted tumors. Metastasis developed in >90% of mice transplanted with PyMT, 40% with BRCA1/p53+/− and wnt1/p53+/−, and 15% with Her2/neu tumors. Expansion of PyMT and wnt1 tumors by serial transplantation for two passages did not lead to significant changes in gene expression. PyMT-transplanted tumors and anterior tumors of transgenic mice showed similar sensitivities to cyclophosphamide and paclitaxel. Conclusions: Transplantation of GEM tumors can provide a large cohort of mice bearing mammary tumors at the same stage of tumor development and with defined frequency of metastasis in a well-characterized molecular and genetic background.

The Origins of Breast Cancer Prognostic Gene Expression Profiles

Recent high profile clinical trials show that microarray-based gene expression profiling has the potential to become an important tool for predicting prognosis in breast cancer. Earlier work in our laboratory using mouse models and human breast cancer populations has enabled us to show that metastasis susceptibility is an inherited trait. This same combined approach facilitated the identification of a number of candidate genes that, when dysregulated, have the potential to induce prognostic gene expression profiles in human data sets. To investigate if these gene expression signatures were of somatic or germline origin and to assess the contribution of different cell types to the induction of these signatures, we have performed a series of expression profiling experiments in a mouse model of metastatic breast cancer. These results show that both the tumor epithelium and invading stromal tissues contribute to the development of prognostic gene signatures. Furthermore, analysis of normal tissues and tumor transplants suggests that prognostic signatures result from both somatic and inherited components, with the inherited components being more consistently predictive.

Integrated cross-species transcriptional network analysis of metastatic susceptibility

Metastatic disease is the proximal cause of mortality for most cancers and remains a significant problem for the clinical management of neoplastic disease. Recent advances in global transcriptional analysis have enabled better prediction of individuals likely to progress to metastatic disease. However, minimal overlap between predictive signatures has precluded easy identification of key biological processes contributing to the prometastatic transcriptional state. To overcome this limitation, we have applied network analysis to two independent human breast cancer datasets and three different mouse populations developed for quantitative analysis of metastasis. Analysis of these datasets revealed that the gene membership of the networks is highly conserved within and between species, and that these networks predicted distant metastasis free survival. Furthermore these results suggest that susceptibility to metastatic disease is cell-autonomous in estrogen receptor-positive tumors and associated with the mitotic spindle checkpoint. In contrast, nontumor genetics and pathway activities-associated stromal biology are significant modifiers of the rate of metastatic spread of estrogen receptor-negative tumors. These results suggest that the application of network analysis across species may provide a robust method to identify key biological programs associated with human cancer progression.

Allelic variation and differential expression of the mSIN3A histone deacetylase complex gene Arid4b promote mammary tumor growth and metastasis.

Accumulating evidence suggests that breast cancer metastatic progression is modified by germline polymorphism, although specific modifier genes have remained largely undefined. In the current study, we employ the MMTV-PyMT transgenic mouse model and the AKXD panel of recombinant inbred mice to identify AT–rich interactive domain 4B (Arid4b; NM_194262) as a breast cancer progression modifier gene. Ectopic expression of Arid4b promoted primary tumor growth in vivo as well as increased migration and invasion in vitro, and the phenotype was associated with polymorphisms identified between the AKR/J and DBA/2J alleles as predicted by our genetic analyses. Stable shRNA–mediated knockdown of Arid4b caused a significant reduction in pulmonary metastases, validating a role for Arid4b as a metastasis modifier gene. ARID4B physically interacts with the breast cancer metastasis suppressor BRMS1, and we detected differential binding of the Arid4b alleles to histone deacetylase complex members mSIN3A and mSDS3, suggesting that the mechanism of Arid4b action likely involves interactions with chromatin modifying complexes. Downregulation of the conserved Tpx2 gene network, which is comprised of many factors regulating cell cycle and mitotic spindle biology, was observed concomitant with loss of metastatic efficiency in Arid4b knockdown cells. Consistent with our genetic analysis and in vivo experiments in our mouse model system, ARID4B expression was also an independent predictor of distant metastasis-free survival in breast cancer patients with ER+ tumors. These studies support a causative role of ARID4B in metastatic progression of breast cancer.

Comparative sequence analysis in eight inbred strains of the metastasis modifier QTL candidate gene Brms1

Comparative genomics has recently become a significant tool in the study of mammalian genomes. The completion of the human genome sequence and the increasing amounts of publicly available mouse and rat high throughput sequence have enabled identification of regions of DNA sequence that have been highly conserved throughout mammalian evolution and likely represent novel genes or gene regulatory elements (Dehal et al. 2001; Qiu et al. 2001). In addition to comparative sequence analysis, comparative genetic analysis has also proven to be extremely valuable. The mapping of hypertension susceptibility mapping in mouse, rat, and human identified orthologous chromosomal segments, providing strong evidence for the presence of quantitative trait loci (QTL) at these positions in all three species (Stoll et al. 2000). Identification of the evolutionary chromosome breakpoints defining these regions will likely significantly constrain the number of candidate genes by identifying a relatively few common linked loci in these regions in all three species. Comparative genome analysis also may have significant utility within inbred mouse strains. The mouse has been the primary mammalian organism for quantitative trait genetics. The large number of inbred strains, ease of breeding and genome manipulation, and expanding genomic resources have permitted genetic analysis of a plethora of phenotypes, including models of many human clinical diseases. Unlike many of the human hereditary syndromes, the genetic basis for most of these modifier genes is unlikely to be deletions or chain termination mutations. Instead, it is anticipated that single nucleotide polymorphisms (SNPs) will be responsible for the vast majority of interstrain phenotypic variation. Comparative sequence analysis within species, between inbred strains, therefore offers opportunities to identify polymorphisms that may underlie specific quantitative traits. This strategy may be most useful when a locus has been identified as affecting a particular trait in multiple inbred strains. Polymorphisms that are shared between strains that modify the phenotype, but present in strains that do not alter the trait, could then be studied in greater detail. This strategy would be particularly valuable for polymorphisms causing subtle changes in gene expression, rather than alterations in amino acid sequence. These polymorphisms include those in non-coding regions, 5 or 3 UTRs, promoter regions, or introns, which might affect transcription or mRNA processing and stability. Previously our laboratory performed a large strain survey to identify inbred strains that modified the dissemination of a transgene-induced mammary tumor (Lifsted et al. 1998). More recently we described complete genome scans of backcrosses between the mammary tumor model [FVB/N-TgN(MMTV-PyMT)634Mul] and three different inbred strains that suppressed pulmonary metastatic spread (DBA/2J, I/LnJ, NZB/B1NJ), as well as mapping results of a cross between the transgenic parent and the AKXD/Ty recombinant inbred panel (Hunter et al. 2001). Two additional experimental crosses with strains C58/J and MOLF/Ei were spot genotyped for the candidate regions determined in the initial experiments (K. Hunter, unpublished results). One of the QTL candidate regions that was reproducibly observed included a 5to 7-cM region of Chr 19 that is orthologous to the region of human 11q13 containing the breast cancer metastasis suppressor gene BRMS1 (Seraj et al., 2000). This region of mouse Chr 19 harbors the murine Brms1 (Hunter et al. 2001; Samant et al. 2001), suggesting that Brms1 might be a strong candidate for the mouse metastasis modifier gene Mtes1. To evaluate this possibility, we have sequenced the entire Brms1 locus in all seven inbred strains used in our mapping crosses to identify polymorphisms that might be responsible for the metastatic suppression. Primers were generated from the published 129/SvJ cDNA (accession # AF233580; primers available on request) and were used to PCR amplify testis cDNA. PCR products were purified with Qiagen PCR purification kits, and double-strand sequencing was performed with a Perkin-Elmer BigDye Terminator sequence kit and analysis on a Perkin-Elmer 3100 Automated Fluorescent Sequencer. Sequences were compiled and analyzed with the computer software packages PHRED and PHRAP (Gordon et al. 1998). Primers for the intronic sequences were designed from 129/SvJ BAC sequences (accession # AF368292) (Samant et al. 2001) and were used to amplify PCR products from genomic DNA for sequencing. Promoter sequences were obtained by direct sequencing of the 129/SvJ BAC to obtain high-quality sequence of the 497-bp Gcnt1–Brms1 intergenic region. Promoter primers were then designed, and genomic DNA from other strains was amplified and sequenced. The sequences are available in GenBank, accession numbers AF467879–AF467885. In total, 59 polymorphisms were identified in the eight inbred strains analyzed (Table 1). As expected, the vast majority were located in the intronic regions. Three polymorphisms were observed in exons; one in the 5 UTR (A>G in C58/J, NZB/B1NJ, MOLF/Ei, FVB/NJ), one in the 3 UTR (T>C in 129/SvJ) and a silent codon change in exon 4 (C>T in DBA/2J, MOLF/Ei). Fourteen polymorphisms were insertion/deletion (indel) events. The indels ranged from 1 to 17 basepairs in length. Two indels occurred in or immediately adjacent to short polyA or polyT sequences and are likely to have arisen by DNA polymerase stuttering. A 14-bp insertion, the result of tandem duplication of a 7-bp repeat, was observed in the Brms1 promoter in strain MOLF/Ei. The remaining indels (1–5 bp in length) have no obvious sequence features to suggest probable mechanism. Of the single base pair substitutions, 33 were transition and 12 were transversions. None Correspondence to: K.W. Hunter; E-mail: hunterk@mail.nih.gov Mammalian Genome 13, 289–292 (2002). DOI: 10.1007/s00335-001-2151-6

Abstract 2980: An integrated cross-species transcriptional network analysis of metastatic susceptibility

Metastatic disease is the proximal cause of mortality for most cancers and remains a significant problem for the clinical management of neoplastic disease. Recent advances in global transcriptional analysis have enabled better prediction of individuals likely to progress to metastatic disease. However, minimal overlap between predictive signatures has precluded easy identification of key biological processes contributing to the pro-metastatic transcriptional state. To overcome this limitation, we have applied network analysis to two independent human breast cancer data sets and three different mouse populations developed for quantitative analysis of metastasis. Analysis of these data sets revealed that the gene membership of the networks is highly conserved within and between species, and that these networks predicted distant metastasis free survival. Furthermore these results suggest that inherited susceptibility to metastatic disease is cell autonomous in ER+ tumors and associated with the mitotic spindle checkpoint. In contrast, susceptibility to metastasis is tumor non-autonomous in ER- patients and associated with an inherited difference in immune infiltration of the primary tumor. These results suggest that the application of network analysis across species may provide a robust method to identify key biological programs associated with human cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2980. doi:1538-7445.AM2012-2980