Ann F. Chambers

Epigenetic mapping and functional analysis in a breast cancer metastasis model using whole-genome promoter tiling microarrays

IntroductionBreast cancer metastasis is a complex, multi-step biological process. Genetic mutations along with epigenetic alterations in the form of DNA methylation patterns and histone modifications contribute to metastasis-related gene expression changes and genomic instability. So far, these epigenetic contributions to breast cancer metastasis have not been well characterized, and there is only a limited understanding of the functional mechanisms affected by such epigenetic alterations. Furthermore, no genome-wide assessments have been undertaken to identify altered DNA methylation patterns in the context of metastasis and their effects on specific functional pathways or gene networks.MethodsWe have used a human gene promoter tiling microarray platform to analyze a cell line model of metastasis to lymph nodes composed of a poorly metastatic MDA-MB-468GFP human breast adenocarcinoma cell line and its highly metastatic variant (468LN). Gene networks and pathways associated with metastasis were identified, and target genes associated with epithelial–mesenchymal transition were validated with respect to DNA methylation effects on gene expression.ResultsWe integrated data from the tiling microarrays with targets identified by Ingenuity Pathways Analysis software and observed epigenetic variations in genes implicated in epithelial–mesenchymal transition and with tumor cell migration. We identified widespread genomic hypermethylation and hypomethylation events in these cells and we confirmed functional associations between methylation status and expression of the CDH1, CST6, EGFR, SNAI2 and ZEB2 genes by quantitative real-time PCR. Our data also suggest that the complex genomic reorganization present in cancer cells may be superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes.ConclusionThis is the first whole-genome approach to identify genome-wide and gene-specific epigenetic alterations, and the functional consequences of these changes, in the context of breast cancer metastasis to lymph nodes. This approach allows the development of epigenetic signatures of metastasis to be used concurrently with genomic signatures to improve mapping of the evolving molecular landscape of metastasis and to permit translational approaches to target epigenetically regulated molecular pathways related to metastatic progression.

Multi-Platform Whole-Genome Microarray Analyses Refine the Epigenetic Signature of Breast Cancer Metastasis with Gene Expression and Copy Number

Background We have previously identified genome-wide DNA methylation changes in a cell line model of breast cancer metastasis. These complex epigenetic changes that we observed, along with concurrent karyotype analyses, have led us to hypothesize that complex genomic alterations in cancer cells (deletions, translocations and ploidy) are superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes observed in breast cancer metastasis. Methodology/Principal Findings We undertook simultaneous high-resolution, whole-genome analyses of MDA-MB-468GFP and MDA-MB-468GFP-LN human breast cancer cell lines (an isogenic, paired lymphatic metastasis cell line model) using Affymetrix gene expression (U133), promoter (1.0R), and SNP/CNV (SNP 6.0) microarray platforms to correlate data from gene expression, epigenetic (DNA methylation), and combination copy number variant/single nucleotide polymorphism microarrays. Using Partek Software and Ingenuity Pathway Analysis we integrated datasets from these three platforms and detected multiple hypomethylation and hypermethylation events. Many of these epigenetic alterations correlated with gene expression changes. In addition, gene dosage events correlated with the karyotypic differences observed between the cell lines and were reflected in specific promoter methylation patterns. Gene subsets were identified that correlated hyper (and hypo) methylation with the loss (or gain) of gene expression and in parallel, with gene dosage losses and gains, respectively. Individual gene targets from these subsets were also validated for their methylation, expression and copy number status, and susceptible gene pathways were identified that may indicate how selective advantage drives the processes of tumourigenesis and metastasis. Conclusions/Significance Our approach allows more precisely profiling of functionally relevant epigenetic signatures that are associated with cancer progression and metastasis.

Gene signatures of breast cancer progression and metastasis.

Breast cancer is a heterogeneous disease. Patient outcome varies significantly, depending on prognostic features of patients and their tumors, including patient age, menopausal status, tumor size and histology, nodal status, and so on. Response to treatment also depends on a series of predictive factors, such as hormone receptor and HER2 status. Current treatment guidelines use these features to determine treatment. However, these guidelines are imperfect, and do not always predict response to treatment or survival. Evolving technologies are permitting increasingly large amounts of molecular data to be obtained from tumors, which may enable more personalized treatment decisions to be made. The challenge is to learn what information leads to improved prognostic accuracy and treatment outcome for individual patients.

Stage of Breast Cancer Progression Influences Cellular Response to Activation of the WNT/Planar Cell Polarity Pathway

Planar cell polarity (PCP) signaling has been shown in different studies to either promote or inhibit the malignancy of breast cancer. Using the 21T cell lines, which were derived from an individual patient and represent distinct stages of progression, we show that the prototypical PCP ligand, WNT5A, is expressed highest in 21MT-1 cells (invasive mammary carcinoma) and lowest in 21PT (atypical ductal hyperplasia) and 21NT (ductal carcinoma in situ) cells. Overexpression of WNT5A decreased spherical colony formation and increased invasion and in vivo extravasation only in 21NT cells; whereas overexpression increased migration of both 21PT and 21NT cells. WNT5A overexpression also increased RHOA expression of both cell lines and subsequent RHOA knockdown blocked WNT5A-induced migration, but only partially blocked WNT5A-induced invasion of 21NT cells. PCP can signal through VANGL1 to modulate AP-1 target genes (e.g. MMP3) and induce invasion. VANGL1 knockdown inhibited WNT5A-induced invasion of 21NT cells, but had no effect on WNT5A-induced migration of either 21PT or 21NT cells. WNT5A-induced MMP3 expression was seen only in 21NT cells, an effect that was VANGL1 dependent, but independent of AP-1. We thus provide evidence that PCP signaling can act in a context dependent manner to promote breast cancer progression.

Human 21T breast epithelial cell lines mimic breast cancer progression in vivo and in vitro and show stage-specific gene expression patterns

Early breast cancer progression involves advancement through specific morphological stages including atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (IMC), although not necessarily always in a linear fashion. Observational studies have examined genetic, epigenetic and gene expression differences in breast tissues representing these stages of progression, but model systems which would allow for experimental testing of specific factors influencing transition through these stages are scarce. The 21T series cell lines, all originally derived from the same patient with metastatic breast cancer, have been proposed to represent a mammary tumor progression series. We report here that three of the 21T cell lines indeed mimic specific stages of human breast cancer progression (21PT-derived cells, ADH; 21NT-derived cells, DCIS; 21MT-1 cells, IMC) when grown in the mammary fat pad of nude mice, albeit after a year. To develop a more rapid, readily manipulatable in vitro assay for examining the biological differences between these cell lines, we have used a 3D Matrigel system. When the three cell lines were grown in 3D Matrigel, they showed characteristic morphologies, in which quantifiable aspects of stage-specific in vivo behaviors (ie, differences in acinar structure formation, cell polarization, colony morphology, cell proliferation, cell invasion) were recapitulated in a reproducible fashion. Gene expression profiling revealed a characteristic pattern for each of the three cell lines. Interestingly, Wnt pathway alterations are particularly predominant in the early transition from 21PTci (ADH) to 21NTci (DCIS), whereas alterations in expression of genes associated with control of cell motility and invasion phenomena are more prominent in the later transition of 21NTci (DCIS) to 21MT-1 (IMC). This system thus reveals potential therapeutic targets and will provide a means of testing the influences of identified genes on transitions between these stages of pre-malignant to malignant growth.

Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel-Lindau (VHL)-deficiency, resulting in pseudohypoxic, angiogenic and glycolytic tumours. Hydrogen sulfide (H2S) is an endogenously-produced gasotransmitter that accumulates under hypoxia and has been shown to be pro-angiogenic and cytoprotective in cancer. It was hypothesized that H2S levels are elevated in VHL-deficient ccRCC, contributing to survival, metabolism and angiogenesis. Using the H2S-specific probe MeRhoAz, it was found that H2S levels were higher in VHL-deficient ccRCC cell lines compared to cells with wild-type VHL. Inhibition of H2S-producing enzymes could reduce the proliferation, metabolism and survival of ccRCC cell lines, as determined by live-cell imaging, XTT/ATP assay, and flow cytometry respectively. Using the chorioallantoic membrane angiogenesis model, it was found that systemic inhibition of endogenous H2S production was able to decrease vascularization of VHL-deficient ccRCC xenografts. Endogenous H2S production is an attractive new target in ccRCC due to its involvement in multiple aspects of disease.

Concurrent Neoadjuvant Chemotherapy and Radiation Therapy in Locally Advanced Breast Cancer

PURPOSEnToxa0evaluate whether concurrent neoadjuvant radiation added to standard chemotherapy could increase the pathologic complete response (pCR) to treatment for locally advanced breast cancer (LABC).nnnMETHODS AND MATERIALSnThis prospective phase 2 trial recruited 32 LABC patients from 2009 to 2011. Patients received neoadjuvant every-3-weekly 5-fluorouracil (500xa0mg/m2), epirubicin (100xa0mg/m2), and cyclophosphamide (500xa0mg/m2) for 3 cycles, followed by weekly docetaxel (35xa0mg/m2) for 9 cycles. Regional radiation (45xa0Gy/25 plus 5.4xa0Gy/5) was delivered concurrently with docetaxel, then modified radical mastectomy. Patients were matched post hoc by a blinded statistician to a concurrent cohort treated with neoadjuvant chemotherapy, modified radical mastectomy, and adjuvant regional radiation.nnnRESULTSnThirty of 32 patients completed treatment. Twenty-seven were successfully matched by propensity score to 81 control patients by age, stage, and molecular subtype. The concurrent chemoradiation produced a significant increase in pCR (14% vs 22%, P<.001) but no statistically significant difference in disease-free and overall survival at 3xa0years (respectively, 69% vs 81%, P=.186, hazard ratio 0.51; and 74% vs 89%, P=.162, hazard ratio 0.46). Toxicity included 25% of patients with grade 3 pneumonitis and 25% of patients with dermatitis, and 1 death.nnnCONCLUSIONSnConcurrent neoadjuvant radiation added to radiosensitizing chemotherapy significantly improved pCR. A prospective randomized clinical trial is warranted to exploit the improved response seen with concurrent therapy but using another radio-sensitizing taxane, to better minimize treatment-related toxicity and determine its impact on overall survival.

Improved Linear Contrast-Enhanced Ultrasound Imaging via Analysis of First-Order Speckle Statistics

The linear subtraction methods commonly used for preclinical contrast-enhanced imaging are susceptible to registration errors and motion artifacts that lead to reduced contrast-to-tissue ratios. To address this limitation, a new approach to linear contrast-enhanced ultrasound (CEUS) is proposed based on the analysis of the temporal dynamics of the speckle statistics during wash-in of a bolus injection of microbubbles. In the proposed method, the speckle signal is approximated as a mixture of temporally varying random processes, representing the microbubble signal, superimposed onto spatially heterogeneous tissue backscatter in multiple subvolumes within the region of interest. A wash-in curve is constructed by plotting the effective degrees of freedom (EDoFs) of the histogram of the speckle signal as a function of time. The proposed method is, therefore, named the EDoF method. The EDoF parameter is proportional to the shape parameter of the Nakagami distribution. Images acquired at 18 MHz from a murine mammary fat pad breast cancer xenograft model were processed using gold-standard nonlinear amplitude modulation, conventional linear subtraction, and the proposed statistical method. The EDoF method shows promise for improving the robustness of linear CEUS based on reduced frame-to-frame variability compared with the conventional linear subtraction time-intensity curves. Wash-in curve parameters estimated using the EDoF method also demonstrate higher correlation to nonlinear CEUS than the conventional linear method. The conceptual basis of the statistical method implies that EDoF wash-in curves may carry information about vascular complexity that could provide valuable new imaging biomarkers for cancer research.

Progression of Early Breast Cancer to an Invasive Phenotype

Histological and molecular evidence has led to a model of breast cancer progression in which cells from the terminal duct lobular unit give rise to atypical ductal hyperplasia or atypical lobular hyperplasia, which can progress to ductal carcinoma in situ or lobular carcinoma in situ, and eventually to invasive ductal carcinoma or invasive lobular carcinoma respectively. This review will present a histomorphological and epidemiological overview of the pre-invasive stages of breast cancer progression. As there is mounting evidence that these stages are likely rough phenotypes of underlying molecular changes, current knowledge regarding changes in genetic and epigenetic features of breast cancer progression will also be discussed. Microarray and CGH-based studies will be described, which suggest that low- and high-grade breast cancers can arise from normal terminal ducts through two distinct molecular pathways. Various in vitro and in vivo models used to study the cellular and molecular changes involved in early breast cancer progression will be presented. Lastly, the specific transition from pre-invasive to invasive breast cancer will be addressed, including possible molecular predictors of the invasive phenotype and a contemporary view highlighting the involvement of the tumor microenvironment during the transition to invasive disease.

Prevention of Conversion of Tumor Dormancy into Proliferative Metastases

Late recurrences of cancer are believed to be due to dormant disease that can persist for long periods following apparently successful treatment of a primary tumor. Clinical tumor dormancy thus creates uncertainty for cancer patients and their physicians, who cannot be certain that their cancer will not recur. We have a poor understanding about which individual patients are at risk for cancer recurrence following a period of tumor dormancy. Thus, in spite of the clinical importance of tumor dormancy, much remains to be learned about the mechanisms responsible for induction of, and release from, dormancy. Here we consider the clinical problem of tumor dormancy and discuss evolving ideas of how tumor dormancy and reinitiation of growth may be regulated, both naturally in the body and therapeutically. A better understanding of mechanisms by which dormancy can be regulated may suggest new therapeutic approaches to either eliminate dormant cancer cells or promote the maintenance of dormancy.