Nigel P.S. Crawford

Mechanisms of metastasis

Metastasis is an enormously complex process that remains to be a major problem in the management of cancer. The fact that cancer patients might develop metastasis after years or even decades from diagnosis of the primary tumor makes the metastatic process even more complex. Over the years many hypotheses were developed to try to explain the inefficiency of the metastatic process, but none of these theories completely explains the current biological and clinical observations. In this review we summarize some of the proposed models that were developed in attempt to understand the mechanisms of tumor dissemination and colonization as well as metastatic progression.

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.

Differential microRNA expression tracks neoplastic progression in inflammatory bowel disease-associated colorectal cancer.

One of the most serious complications faced by patients with inflammatory bowel disease (IBD) is the potential development of colorectal cancer (CRC). There is a compelling need to enhance the accuracy of cancer screening of IBD patients. MicroRNAs (miRNAs) are small nonprotein‐coding RNAs that play important roles in CRC oncogenesis. In this study, we report differential miRNA expression in IBD patients with associated CRC from non‐neoplastic tissue to dysplasia and eventually cancer. In addition, we identify and examine the role of dysregulated miRNAs in the TP53 pathway. In our CD patients, six miRNAs were upregulated from non‐neoplastic tissue to dysplasia, but downregulated from dysplasia to cancer (miR‐122, miR‐181a, miR‐146b‐5p, let‐7e, miR‐17, miR‐143) (P < 0.001). Six differentially expressed miRNAs affected the TP53 pathway (miR‐122, miR‐214, miR‐372, miR‐15b, let‐7e, miR‐17) (P < 0.001). Using two human colon cancer cell lines (HT‐29 and HCT‐116), E2F1, an upstream regulator of TP53, was downregulated in both cell lines transfected with let‐7e (P < 0.05) as well as in HCT‐116 cells transfected with miR‐17 (P < 0.05). Additionally, cyclin G, a cell‐cycle regulator miR‐122 target was downregulated in both cell lines (P < 0.05). Unique differentially expressed miRNAs were observed in CD‐associated CRC progression. Six of these miRNAs had a tumorigenic effect on the TP53 pathway; the effect of three of which was studied using cell lines. Hum Mutat 33:551–560, 2012.

The Future of Mouse QTL Mapping to Diagnose Disease in Mice in the Age of Whole-Genome Association Studies*

Genome-wide association analysis is emerging as a powerful tool to define novel genes and molecular pathways involved in susceptibility to human complex disorders. However, in spite of recent successes, this approach is not without its limitations, the most notable of which is inconsistent phenotype penetrance due to varied environmental exposures. Mouse models do, however, circumvent some of these drawbacks by allowing for a much higher degree of control over genetic variation and environmental exposure, and although their application to human complex genetics is not always straightforward, they do serve as a powerful means of complementing observations in human populations. Mouse quantitative trait locus mapping has proven a successful, yet technically demanding method for defining trait susceptibility. In this review, we focus upon recent advances that are both reducing the technical burden traditionally associated with quantitative trait locus mapping, and enhancing the applicability of these approaches to human disease.

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.

Germ Line Polymorphism in Metastatic Progression

Somatic genetic analysis of tumors and metastases has yielded a plethora of information regarding genes associated with cancer progression. However, somatic alterations in tumor cells are only one source of variability. Genetic polymorphism may also play a significant role in person-to-person variability in metastasis frequency, raising the intriguing possibility that some individuals could be predisposed to secondary tumor development. The identification and characterization of these polymorphisms may have significant implications for the development of tailored treatment or prevention of recurrent disease.

Deletion of the Proline-Rich Region of the Murine Metastasis Susceptibility Gene Brd4 Promotes Epithelial-to-Mesenchymal Transition- and Stem Cell-Like Conversion

The bromodomain-containing chromatin-modifying factor BRD4 is an inherited susceptibility gene for breast cancer progression and metastasis, but its functionality in these settings has yet to be explored. Here we show that deletion of either of the BRD4 bromodomains had modest effects on the metastatic suppression ability of BRD4. In contrast, expression of the natural short isoform of BRD4 that truncates the protein after the SEED domain restored progression and metastatic capacity. Unexpectedly, deletion of the proline-rich region induced mesenchymal-like conversion and acquisition of cancer stem cell-like properties, which are mediated by the carboxy-terminal P-TEFb binding domain. Deletion of this proline-rich region also induced a gene expression signature that predicted poor outcome in human breast cancer data sets and that overlapped G3 grade human breast tumors. Thus our findings suggest that BRD4 may be altering the predisposition of tumors to undergo conversion to a more de-differentiated or primitive state during metastatic progression.

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.

WISP1/CCN4: A Potential Target for Inhibiting Prostate Cancer Growth and Spread to Bone

Prostate cancer (PC) is a leading cause of death in men however the factors that regulate its progression and eventual metastasis to bone remain unclear. Here we show that WISP1/CCN4 expression in prostate cancer tissues was up-regulated in early stages of the disease and, further, that it correlated with increased circulating levels of WISP1 in the sera of patients at early stages of the disease. WISP1 was also elevated in the mouse prostate cancer model TRAMP in the hypoplastic diseased tissue that develops prior to advanced carcinoma formation. When the ability of anti-WISP1 antibodies to reduce the spread of PC3-Luc cells to distant sites was tested it showed that twice weekly injections of anti-WISP1 antibodies reduced the number and overall size of distant tumors developed after intracardiac (IC) injection of PC3-Luc cells in mice. The ability of antibodies against WISP1 to inhibit growth of PC3-Luc cancer cells in mice was also evaluated and showed that twice weekly injections of anti-WISP1 antibodies reduced local tumor growth when examined in xenografts. To better understand the mechanism of action, the migration of PC3-Luc cells through membranes with or without a Matrigel™ barrier showed the cells were attracted to WISP1, and that this attraction was inhibited by treatment with anti-WISP1 antibodies. We also show the expression of WISP1 at the bone-tumor interface and in the stroma of early grade cancers suggested WISP1 expression is well placed to play roles in both fostering growth of the cancer and its spread to bone. In summary, the up-regulation of WISP1 in the early stages of cancer development coupled with its ability to inhibit spread and growth of prostate cancer cells makes it both a potential target and an accessible diagnostic marker for prostate cancer.