The miRNA-200 family and miRNA-9 exhibit differential expression in primary versus corresponding metastatic tissue in breast cancer

Metastases are the major cause of cancer-related deaths, but the mechanisms of the metastatic process remain poorly understood. In recent years, the involvement of microRNAs (miRNAs) in cancer has become apparent, and the objective of this study was to identify miRNAs associated with breast cancer progression. Global miRNA expression profiling was performed on 47 tumor samples from 14 patients with paired samples from primary breast tumors and corresponding lymph node and distant metastases using LNA-enhanced miRNA microarrays. The identified miRNA expression alterations were validated by real-time PCR, and tissue distribution of the miRNAs was visualized by in situ hybridization. The patients, in which the miRNA profile of the primary tumor and corresponding distant metastasis clustered in the unsupervised cluster analysis, showed significantly shorter intervals between the diagnosis of the primary tumor and distant metastasis (median 1.6 years) compared to those that did not cluster (median 11.3 years) (p<0.003). Fifteen miRNAs were identified that were significantly differentially expressed between primary tumors and corresponding distant metastases, including miR-9, miR-219-5p and four of the five members of the miR-200 family involved in epithelial-mesenchymal transition. Tumor expression of miR-9 and miR-200b were confirmed using in situ hybridization, which also verified higher expression of these miRNAs in the distant metastases versus corresponding primary tumors. Our results demonstrate alterations in miRNA expression at different stages of disease progression in breast cancer, and suggest a direct involvement of the miR-200 family and miR-9 in the metastatic process.     

Molecular mechanisms of metastasis in breast cancer--clinical applications

The metastatic cascade is a series of biological processes that enable the movement of tumor cells from the primary site to a distant location and the establishment of a new cancer growth. Circulating tumor cells (CTCs) have a crucial role in tumor dissemination. The role of CTCs in treatment failure and disease progression can be explained by their relation to biological processes, including the epithelial-to-mesenchymal transition and 'self seeding', defined as reinfiltration of the primary tumor or established metastasis by more aggressive CTCs. CTCs are a unique and heterogeneous cell population with established prognostic and predictive value in certain clinical situations. The possibility of collecting sequential blood samples for real-time monitoring of systemic-therapy efficacy presents new possibilities to evaluate targeted therapies based on the genomic profiling of CTCs and to improve the clinical management of patients by personalized therapy. Interruption of the metastatic cascade via the targeting of CTCs might be a promising therapeutic strategy.     

Multiregion whole-exome sequencing of matched primary and metastatic tumors revealed genomic heterogeneity and suggested polyclonal seeding in colorectal cancer metastasis

BackgroundDistant metastasis accounts for 90% of deaths from colorectal cancer (CRC). Genomic heterogeneity has been reported in various solid malignancies, but remains largely under-explored in metastatic CRC tumors, especially in primary to metastatic tumor evolution.Patients and methodsWe conducted high-depth whole-exome sequencing in multiple regions of matched primary and metastatic CRC tumors. Using a total of 28 tumor, normal, and lymph node tissues, we analyzed inter- and intra-individual heterogeneity, inferred the tumor subclonal architectures, and depicted the subclonal evolutionary routes from primary to metastatic tumors.ResultsCRC has significant inter-individual but relatively limited intra-individual heterogeneity. Genomic landscapes were more similar within primary, metastatic, or lymph node tumors than across these types. Metastatic tumors exhibited less intratumor heterogeneity than primary tumors, indicating that single-region sequencing may be adequate to identify important metastasis mutations to guide treatment. Remarkably, all metastatic tumors inherited multiple genetically distinct subclones from primary tumors, supporting a possible polyclonal seeding mechanism for metastasis. Analysis of one patient with the trio samples of primary, metastatic, and lymph node tumors supported a mechanism of synchronous parallel dissemination from the primary to metastatic tumors that was not mediated through lymph nodes.ConclusionsIn CRC, metastatic tumors have different but less heterogeneous genomic landscapes than primary tumors. It is possible that CRC metastasis is, at least partly, mediated through a polyclonal seeding mechanism. These findings demonstrated the rationale and feasibility for identifying and targeting primary tumor-derived metastasis-potent subclones for the prediction, prevention, and treatment of CRC metastasis.     

Activation of Pro-uPA Is Critical for Initial Escape from the Primary Tumor and Hematogenous Dissemination of Human Carcinoma Cells

Urokinase-type plasminogen activator (uPA) and plasmin have long been implicated in cancer progression. However, the precise contributions of the uPA/plasmin system to specific steps involved in cancer cell dissemination have not been fully established. Herein, we have used a highly disseminating variant of the human PC-3 prostate carcinoma cell line, PC-hi/diss, as a prototype of aggressive carcinomas to investigate the mechanisms whereby pro-uPA activation and uPA-generated plasmin functionally contribute to specific stages of metastasis. The PC-hi/diss cells secrete and activate significant amounts of pro-uPA, leading to efficient generation of plasmin in solution and at the cell surface. In a mouse orthotopic xenograft model, treatment with the specific pro-uPA activation-blocking antibody mAb-112 significantly inhibited local invasion and distant metastasis of the PC-hi/diss cells. To mechanistically examine the uPA/plasmin-mediated aspects of tumor cell dissemination, the anti-pro-uPA mAb-112 and the potent serine protease inhibitor, aprotinin, were used in parallel in a number of in vivo assays modeling various rate-limiting steps in early metastatic spread. Our findings demonstrate that, by generating plasmin, activated tumor-derived uPA facilitates early stages of PC-hi/diss dissemination, specifically the escape from the primary tumor and tumor cell intravasation. Moreover, through a series of in vitro and in vivo analyses, we suggest that PC-hi/diss-invasive escape and dissemination may be enhanced by cleavage of stromal fibronectin by uPA-generated plasmin. Together, our findings point to inhibition of pro-uPA activation at the apex of the uPA/plasmin cascade as a therapy-valid approach to control onset of tumor escape and ensuing metastatic spread.     

A study of tumor heterogeneity in a case with breast cancer

Tumor heterogeneity has been suggested based on clinical and pathological findings. Several clinical findings can be explained by tumor evolution during progression and metastasis. We herein report a case of metastatic breast cancer indicated tumor heterogeneity by clinical findings and a genomic analysis. A 64-year-old woman with advanced breast cancer was treated with primary chemotherapy, to which primary tumor responded. After a 6 month treatment pause, lung, liver, and skin metastases developed and her serum tumor markers were elevated. None of those serum markers had been elevated before the treatment, despite the large tumor burden. Notably, there was discordance in the expression of human epidermal growth factor receptor 2 (HER2) between the primary tumor and metastatic skin lesions, with the former being negative and the latter positive. A genomic analysis was performed by in-house Breast Cancer Panel, which consisted of 53 pre-selected genes. Twenty-three somatic mutations were found in primary breast tumor and 7 in the skin metastasis. None of these 30 genes matched. However, the cell-free (cf) DNA in the plasma taken at the time of skin metastasis contained 10 mutations, 7 from the primary lesion and 3 from the metastasis. These data indicate that the clonal changes or tumor heterogeneity was shown in two solid tumors by clinical and the result of a genomic analysis. Of particular interest was that cell-free DNA could be a powerful tool to look into these dynamic changes.     

The Antitumorigenic Function of EGFR in Metastatic Breast Cancer is Regulated by Expression of Mig6

Numerous studies by our lab and others demonstrate that epidermal growth factor receptor (EGFR) plays critical roles in primary breast cancer (BC) initiation, growth and dissemination. However, clinical trials targeting EGFR function in BC have lead to disappointing results. In the current study we sought to identify the mechanisms responsible for this disparity by investigating the function of EGFR across the continuum of the metastatic cascade. We previously established that overexpression of EGFR is sufficient for formation of in situ primary tumors by otherwise nontransformed murine mammary gland cells. Induction of epithelial-mesenchymal transition (EMT) is sufficient to drive the metastasis of these EGFR-transformed tumors. Examining growth factor receptor expression across this and other models revealed a potent downregulation of EGFR through metastatic progression. Consistent with diminution of EGFR following EMT and metastasis EGF stimulation changes from a proliferative to an apoptotic response in in situ versus metastatic tumor cells, respectively. Furthermore, overexpression of EGFR in metastatic MDA-MB-231 BC cells promoted their antitumorigenic response to EGF in three dimensional (3D) metastatic outgrowth assays. In line with the paradoxical function of EGFR through EMT and metastasis we demonstrate that the EGFR inhibitory molecule, Mitogen Induced Gene-6 (Mig6), is tumor suppressive in in situ tumor cells. However, Mig6 expression is absolutely required for prevention of apoptosis and ultimate metastasis of MDA-MB-231 cells. Further understanding of the paradoxical function of EGFR between primary and metastatic tumors will be essential for application of its targeted molecular therapies in BC.Abbreviations: Mig6, mitogen-induced gene-6; BC, breast cancer; EGFR, epidermal growth factor receptor; Mig6-EBR, EGFR binding region of Mig6; TGF-β, transforming growth factor-β; WT, wild type; NMuMG, normal murine mammary gland cells; NME, NMuMG cells transformed by EGFR overexpression; CCLE, Cell Line Encyclopedia; MET, mesenchymal-epithelial transition     

Dietary fat and breast cancer metastasis by human tumor xenografts

Human breast cancer cell lines growing as xenografts in athymic nude mice have been used to examine the effects of dietary fat and fatty acids on tumor progression. The estrogen independent MDA-MB-435 cell line has the advantage that it metastasizes consistently to the lungs and forms quantifiable secondary nodules when injected into the mammary fat pads. With these breast cancer cells, the stimulating effects of polyunsaturated omega-6 fatty acids on both primary tumor growth and metastasis were demonstrated; in contrast, the long-chain omega-3 fatty acids were inhibitory. The model can also be adapted to examine dietary fatty acids, and inhibitors of their metabolism, as experimental adjuvant therapy after surgical excision of the primary tumors. Unfortunately, estrogen dependent human breast cancer cells do not metastasize, or do so rarely, in nude mice; in consequence, it is not possible to use the model to study estrogen-fatty acid interactions on the metastatic process. In addition to metastasis from a primary location, intravenous injection of MDA-MB-435 cells into the nude mouse host, particularly when combined with studies using Matrigel-based in vitro invasion assays, permits further dissection of the steps in the metastatic cascade which are influenced by dietary fatty acids. The results obtained by these several approaches have demonstrated distinct roles for the cyclooxygenase and lipoxygenase-mediated products of omega-6 fatty acid metabolism, and suggest new approaches to experimental breast cancer therapy.     

NM23, an example of a metastasis suppressor gene

Metastasis suppressor genes - unlike tumor suppressor genes - are defined by their capacity to control metastatic dissemination in vivo without affecting growth of the primary tumor. The first of these metastasis suppressor genes, NM23, was identified in 1988. Since then, expression of NM23 has been studied widely in human tumor cohorts, often with contradictory results. Not only is NM23 overexpressed in most human solid tumors when compared to healthy tissues, but also low expression of NM23 correlates with metastasis and poor clinical prognosis in the advanced stages of a number of epithelial cancer types, including melanoma, breast, colon, and liver carcinoma. This does not hold true, however, for other cancer types such as neuroblastoma and hematological malignancies, in which high NM23 expression correlates with more aggressive disease. Genetic alterations in the NM23 gene - loss of heterozygosity, spontaneous mutations and polymorphisms - are rarely found in tumors; thus, the metastatic potential of tumor cells is probably affected by NM23 protein levels. Three lines of evidence demonstrate the anti-metastatic activity of NM23: first, overexpression of NM23 in metastatic cell lines reduces their metastatic potential in xenograft models; second, the incidence of lung metastases is elevated in NM23 knockout mice prone to develop hepatocellular carcinoma, and, third, silencing NM23 by RNA interference confers a "metastatic phenotype" on non-invasive human epithelial liver and colon cancer cell lines. It appears that NM23 is crucial for inhibiting invasive migration, so acting at early stages of metastatic dissemination. The mechanistic basis of the metastasis suppressor function of NM23 and its regulated expression still remains obscure, however. Reactivation of expression of the endogenous NM23 gene in tumor cells, or stimulation of the pathways it controls, constitutes a promising avenue for anti-metastatic therapy.     

Identification of genomic signatures in circulating tumor cells from breast cancer

Levels of circulating tumor cells (CTCs) in blood have prognostic value in early and metastatic breast cancer. CTCs also show varying degrees of concordance with molecular markers of primary tumors they originate from. It is expected that individual cells reflect the heterogeneity and evolution of tumor cells as they acquire new functions and differential responses to chemotherapy. However, a degree of commonality is also plausible, highlighting alterations that allow tumor cells to perform CTC‐defining activities such as invasion and intravasation. Using a matched tumor‐normal approach, we performed high‐resolution copy number profiling of CTCs from breast cancer to identify occult changes occurring during progression to metastasis. We identified a signature of recurrent gain in CTCs, consisting of 90 minimal common regions (MCRs) of copy number gain. These were predominantly found across chromosome 19 and were identified at low frequencies (3–4%) in 787 primary breast carcinomas examined. CTC genomic signatures clustered into two groups independent of subtype: a dormancy‐related signature with 16 MCRs (AKT2, PTEN, CADM2); and a tumor‐aggressiveness related signature with 358 MCRs (ANGPTL4, BSG, MIR‐373). There were two MCRs in common between the groups on 19q13 and 21q21, containing genes involved in resistance to anoikis, TGFβ‐signaling and metastasis (TFF3, LTBP4, NUMBL). Furthermore, a region harboring the ERBB2 gene was gained in a majority of patients. Regions 20q13 and 15q24 were associated with distant metastasis. The distinctiveness of CTC signatures highlights cell populations with different functional or metastatic potential. Such novel targets could help to specifically identify and block dissemination.     

The estrogen-regulated anterior gradient 2 (AGR2) protein in breast cancer: a potential drug target and biomarker

Initially discovered as an estrogen-responsive gene in breast cancer cell lines, anterior gradient 2 (AGR2) is a developmentally regulated gene belonging to the protein disulfide isomerase (PDI) gene family. Developmentally, AGR2 is expressed in the mammary gland in an estrogen-dependent manner, and AGR2 knockout and overexpression mouse models indicate that the gene promotes lobuloalveolar development by stimulating cell proliferation. Although AGR2 overexpression alone seems insufficient for breast tumorigenesis in mice, several lines of investigations suggest that AGR2 promotes breast tumorigenesis. Overexpression of AGR2 in several breast cancer cell lines increases cell survival in clonogenic assays and cell proliferation, whereas AGR2 loss of function leads to decreased cell cycle progression and cell death. In addition, AGR2 was shown to promote metastasis of breast epithelial cells in an in vivo metastasis assay. As a PDI, AGR2 is thought to be involved in the unfolded protein response that alleviates endoplasmic reticulum stress. Since cancer has to overcome proteotoxic stress due to excess protein production, AGR2 may be one of many pro-survival factors recruited to assist in protein folding or degradation or both. When AGR2 is secreted, it plays a role in cellular adhesion and dissemination of metastatic tumor cells. In breast cancer, AGR2 expression is associated with estrogen receptor (ER)-positive tumors; its overexpression is a predictor of poor prognosis. The AGR2 gene is directly targeted by ER-alpha, which is preferentially bound in tumors with poor outcome. Whereas aromatase inhibitor therapy decreases AGR2 expression, tamoxifen acts as an agonist of AGR2 expression in ER-positive tumors, perhaps contributing to tamoxifen resistance. AGR2 is also overexpressed in a subset of ER-negative tumors. Furthermore, AGR2 expression is associated with the dissemination of metastatic breast cancer cells and can be used as a marker to identify circulating tumor cells and metastatic cells in sentinel lymph nodes. In conclusion, AGR2 is a promising drug target in breast cancer and may serve as a useful prognostic indicator as well as a marker of breast cancer metastasis.     

Local treatment of metastatic cancer--killing the seed or disturbing the soil?

The fact that local therapy of the primary tumor is futile in the presence of metastatic disease is almost considered axiomatic among oncologists. However, this perception is now being challenged by new laboratory and clinical data. Results from animal models have demonstrated that some primary tumors release factors that enter the circulation which, by mobilizing cells from bone marrow, render distant organs more receptive to metastasis. Clinical observations in renal, breast, and prostate cancer are all consistent with the hypothesis that treatment directed against the primary tumor might retard progression of existing metastases. This hypothesis is amenable to testing by randomized trials of local therapy to the primary site in patients with metastatic cancer.     

Primary breast tumor-derived cellular models: characterization of tumorigenic,metastatic, and cancer-associated fibroblasts in dissociated tumor (DT) cultures

Our goal was to establish primary cultures from dissociation of breast tumors in order to provide cellular models that may better recapitulate breast cancer pathogenesis and the metastatic process. Here, we report the characterization of six cellular models derived from the dissociation of primary breast tumor specimens, referred to as “dissociated tumor (DT) cells.” In vitro, DT cells were characterized by proliferation assays, colony formation assays, protein, and gene expression profiling, including PAM50 predictor analysis. In vivo, tumorigenic and metastatic potential of DT cultures was assessed in NOD/SCID and NSG mice. These cellular models differ from recently developed patient-derived xenograft models in that they can be used for both in vitro and in vivo studies. PAM50 predictor analysis showed DT cultures similar to their paired primary tumor and as belonging to the basal and Her2-enriched subtypes. In vivo, three DT cultures are tumorigenic in NOD/SCID and NSG mice, and one of these is metastatic to lymph nodes and lung after orthotopic inoculation into the mammary fat pad, without excision of the primary tumor. Three DT cultures comprised of cancer-associated fibroblasts (CAFs) were isolated from luminal A, Her2-enriched, and basal primary tumors. Among the DT cells are those that are tumorigenic and metastatic in immunosuppressed mice, offering novel cellular models of ER-negative breast cancer subtypes. A group of CAFs provide tumor subtype-specific components of the tumor microenvironment (TME). Altogether, these DT cultures provide closer-to-primary cellular models for the study of breast cancer pathogenesis, metastasis, and TME.     

Signaling pathways in breast cancer metastasis - novel insights from functional genomics

The advent of genomic profiling technology has brought about revolutionary changes in our understanding of breast cancer metastasis. Gene expression analyses of primary tumors have been used to predict metastatic propensity with high accuracy. Animal models of metastasis additionally offer a platform to experimentally dissect components of the metastasis genetic program. Recent integrated studies have synergized clinical bioinformatic analyses with advanced experimental methodology and begun to uncover the identities and dynamics of signaling programs driving breast cancer metastasis. Such functional genomics studies hold great promise for understanding the genetic basis of metastasis and improving therapeutics for advanced diseases.     

The clonal evolution of metastases from primary serous epithelial ovarian cancers

Several models of evolution from primary cancers to metastases have been proposed; but the most widely accepted is the clonal evolution model proposed for colorectal cancer in which tumors develop by a process of linear clonal evolution driven by the accumulation of somatic genetic alterations. Various other models of cancer progression and metastasis have been proposed, including parallel evolution and the same gene model. The aim of this study was to investigate the evolution of metastases from primary cancer in 22 patients diagnosed with high‐grade serous epithelial ovarian cancer. We established somatic genetic profiles based on the pattern of loss of heterozygosity, in several different regions of tumor tissue within the primary tumor and metastatic deposits from each case. Maximum parsimony tree analysis was used to examine the evolutionary relationship between the primary and metastatic samples for each patient. In addition, we investigated the extent of genetic heterogeneity within and between metastatic tumors compared with primary ovarian tumors. Our data suggest that most, if not all, metastases are clonally related to the primary tumors. However, the data oppose a single model of linear‐clonal evolution whereby a late stage clone within the primary tumor acquires additional genetic changes that enable metastatic progression. Instead, the data support a model in which primary ovarian cancers have a common clonal origin, but become polyclonal, with different clones at both early and late stages of genetic divergence acquiring the ability to progress to metastasis. © 2009 Wiley‐Liss, Inc.