Inflammatory Breast Cancer Tumor Research Articles

P2-02-01: A Novel Inflammatory Breast Cancer-Specific Oncogene, Tazarotene-Induced Gene 1, Promotes Tumorigenicity and Invasiveness through the Receptor Tyrosine Kinase Axl.

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer and is highly metastatic. The prognosis of patients with IBC is poor, and effective standard therapies for IBC are limited because the molecular mechanisms underlying the pathogenesis of IBC remain unknown. We recently found that tazarotene-induced gene 1 (TIG1) expression is significantly higher in IBC cell lines than in non-IBC cell lines. In both IBC and non-IBC data sets, estrogen receptor-negative/HER2−negative samples had significantly higher expression of TIG1 than did other clinical subtypes (estrogen receptor-positive/HER2−negative and HER2−positive). Therefore, we hypothesized that TIG1 plays an important role in the malignant process of IBC. In these studies, we determined the biological function of TIG1 in IBC cells and elucidated the molecular mechanism by which TIG1 regulates the invasiveness of IBC cells. Methods: TIG1 expression in SUM149 and KPL-4 IBC cells was stably knocked down, and the effects of this knockdown on in vitro cell proliferation, migration, and invasion were analyzed. The effects of restoring TIG1 expression on TIG1-silencing IBC cells were also examined. To determine the tumorigenic activity of TIG1 in vivo, TIG1 stable-knockdown SUM149 cells and control shRNA-transfected cells were implanted into the mammary fat pads of athymic nude mice, and tumor growth was monitored. The receptor tyrosine kinase Axl, a potential functional partner of TIG1, was identified using DNA microarray analysis. The interaction between TIG1 and Axl in IBC cells was examined using immunoprecipitation and confocal microscopy assays. The signaling pathway in IBC cells in which TIG1 participates was also investigated. Results: Knockdown of TIG1 expression in IBC cells reduced their proliferation, migration, and invasion in vitro. Also, silencing of TIG1 dramatically inhibited IBC tumor growth in a xenograft model. Moreover, restoring TIG1 expression rescued the proliferation, motility, and invasiveness of TIG1-silenced IBC cells. Most importantly, we identified Axl as a functional partner of TIG1 by showing that TIG1 interacted with and stabilized Axl in IBC cells. TIG1 regulated the invasiveness of IBC cells through mediation of the Axl signaling pathway. In SUM149 cells, TIG1 depletion decreased Axl expression, which led to downregulation of expression of matrix metalloproteinase-9, a molecule required for Axl-mediated invasion, and inactivation of nuclear factor-kB, ultimately leading to decreased invasiveness of IBC cells. Conclusion: Our results identified TIG1 as an oncogenic gene that contributes to the tumorigenic and metastatic properties of IBC. Our data also linked TIG1 with the key tumorigenic gene Axl in IBC cells. Further studies designed to establish TIG1 as a therapeutic target in the treatment of patients with IBC are under way. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-01.

Over-expression of Translation Initiation Factor eIF4G Confers Robust Radioresistance to the Cancer Stem Cell Population in Inflammatory Breast Cancer

Over-expression of Translation Initiation Factor eIF4G Confers Robust Radioresistance to the Cancer Stem Cell Population in Inflammatory Breast Cancer

Abstract P5-05-05: Tazarotene-Induced Gene 1(TIG1) Is a Novel Tumorigenic Gene of Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of advanced breast cancer. Effective standard therapies for IBC are limited and the molecular mechanisms underlying IBC are largely unknown. In a comparison of samples from patients with IBC and non-IBC, our laboratory identified TIG1 (tazarotene-induced gene 1), also known as retinoic acid receptor responder 1 (RARRES1) as the gene with the largest difference in expression between the two groups. TIG1 is specifically expressed in IBC cell lines (SUM149 and KPL-4). In addition, TIG1 has been identified as a top druggable gene for triple-negative breast cancer. Of particular interest is that TIG1 is significantly expressed in IBC ER-/HER2- patient samples when compared with non-IBC ER-/HER2- patient samples (p = 0.084).Therefore, we hypothesize that TIG1 plays an important role in the malignant process of IBC and it promotes the tumorigenesis and metastasis of IBC. In these studies we determined the tumorigenic and metastatic activities of TIG1 in IBC. Methods: TIG1 was stably knocked down in the highly TIG1-expressing IBC cell line SUM149 and the effects of knockdown on cell proliferation, migration, invasion, and growth in three-dimensional culture were analyzed in vitro. To evaluate the tumorigenic activity of TIG1 in vivo, TIG1 stable-knockdown SUM149 cells and control shRNA-transfected cells were implanted into the mammary fat pads of athymic nude mice, and tumor growth was monitored. To investigate the underlying mechanism of TIG1- mediated tumorigenesis and metastasis of IBC, the gene expression profiles of control siRNA-transfected and TIG1 siRNA-transfected SUM149 cells were compared using cDNA microarray analysis. Results: Suppressing TIG1 significantly inhibited the proliferation, migration, and invasion of IBC cells in vitro. SUM149 cells exhibit a mesenchymal-like phenotype when grown in three-dimensional culture. However, knocking down TIG1 reversed the mesenchymal-like phenotype to the epithelial phenotype in three-dimensional culture, suggesting that TIG1 might have metastatic activity in IBC cells. TIG1 knockdown dramatically inhibited IBC tumor growth in a xenograft model. Several differentially expressed genes between control siRNA-transfected and TIG1 siRNA-transfected SUM149 cells, including Decorin, GBP1, and VAV3 were identified using cDNA microarray analysis. These genes have been reported to affect a number of biological processes, including proliferation, metastasis, inflammatory response, and angiogenesis. We will focus on these candidates and determine the molecular mechanism by which TIG1 may function as a tumorigenic gene in IBC. Conclusion: Our data demonstrate that TIG1 promotes the tumorigenesis and metastasis of IBC and it may contribute to the development and aggressiveness of IBC. Our long-term goal is to develop TIG1 as a therapeutic target for this extremely aggressive type of breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-05.

Inflammatory Breast Cancer Radio-resistance and its Cancer Stem Cell Population are Oppositely Controlled by Translation Factor eIF4G

Inflammatory breast cancer (IBC) is a highly aggressive and radiation resistant breast cancer that continues to have a dismal prognosis despite aggressive multimodality therapy, which includes ionizing radiation (IR). We recently showed that the unique pathogenic properties of IBC result in part from over-expression of the translation initiation factor eIF4G, which promotes IBC tumor cell survival, formation of tumor emboli and metastasis. We also demonstrated that protein synthesis is highly regulated during IR by the DNA-damage response (DDR) pathway through mTOR.

Suberoylanilide hydroxamic acid blocks self‐renewal and homotypic aggregation of inflammatory breast cancer spheroids

Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC). Patients with IBC commonly present with skin metastasis, which are observed microscopically as tumor emboli within dermal lymphatics. These metastatic tumor cells aberrantly overexpress E-cadherin and exhibit the ability to undergo self-renewal and are highly invasive. There are no therapeutics yet identified that target the structure and functions of IBC tumor emboli. The present studies evaluated the effects of the pan-histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) using IBC tumor spheroids derived from established IBC cell lines and tumor spheroids derived from pleural effusion (PE) aspirates of patients with IBC and LABC, designated as PE-IBC and PE-LABC. Methods used are as follows: culture of IBC cells from clonal density single cells in low adherence culture conditions that promote formation of IBC tumor spheroids; clonogenic assays; cell fractionation and Western blotting; confocal microscropy; and modified Boyden chamber invasion assays. SAHA inhibited self-renewal of IBC tumor spheroids from established IBC cell lines and PE-IBC and PE-LABC, as assessed by decreased clonogenic growth. SAHA blocked homotypic aggregation of the cells that comprised the IBC tumor spheroids leading to loss of their 3-dimensional (3D) structure, which was associated with a change in location of E-cadherin protein from the plasma membrane in untreated IBC tumor spheroids to the cytoplasm of cells within IBC tumor spheroids with SAHA treatment. In addition, SAHA blocked the robust invasion exhibited by IBC tumor spheroids of established cell lines as well as by tumor spheroids derived from PE-IBC and PE-LABC. SAHA targets the integrity and biological activities of IBC tumor spheroids and may be a promising agent to evaluate for its effectiveness in treatment of IBC.

A microRNA Expression Profile Consisting of 12 microRNAs Is Associated with Inflammatory Breast Cancer.

Abstract Introduction: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with a dismal clinical outcome. In the past we showed that IBC is characterized by a specific gene expression pattern. In this study, we investigate if microRNAs (miRNAs) are responsible for the specific IBC gene signature.Materials and methods: 377 miRNAs were profiled in 20 IBC tumor samples and 50 non-stage matched nIBC tumour samples using the human MicroRNA A Array Set version 2.0 (Applied Biosystems). The IBC and nIBC study populations are matched for tumor grade and ER status. All miRNAs with Ct-values less than 35 in 25% of the samples were included for analysis. Data normalization was performed relative to the median miRNA expression level per sample and expression values were log2-transformed. Principal component analysis (PCA) was performed to identify metagenes of miRNAs, associated with the IBC phenotype. Relevant metagenes were compared with prognostic/predictive gene signature-classifications (wound healing response, invasiveness gene signature, 70-gene prognostic signature, genomic grade index, recurrence score, HOXB13/IL17RB-expression ratio and the molecular breast cancer subtypes). Data were validated by analysing relevant miRNA target gene sets on an IBC/nIBC gene expression data set.Results: Using PCA, we identified a metagene associated with the IBC phenotype (Hazard Ratio: 4.500; P=0.011). This metagene was also significantly associated with the Luminal B phenotype, the IBC signature and with an elevated HOXB13/IL17RB-expresion ratio (range Rs = 0.302 – 0.366; P<0.05). MiRNAs significantly (FDR<0.1) associated with this metagene were: miR29A, miR544, miR574-3p, miR548C-3p, miR451, miR548B-5p, miR24, miR28-5p, miR137, miR302B, miR148A and miR302A (range Rs: -0.367 – 0.307). Cluster analysis using this miRNA signature identified two sample clusters, one containing 70% of the IBC samples and a second one containing 64% of the nIBC samples (Pearson X2; P=0.021). Using gene set enrichment analysis for miR24-, miR28-, miR29A-, miR137-, miR148A-, miR302A- and miR302B target gene sets, we confirmed the differential expression for the above mentioned miRNAs (P<0.01) except for miR28. Using Oncomine analyses we were able to associate the identified miRNAs with NFkB activation (miR29, miR148), steroid receptor signalling (miR302A, miR302B, and miR137), cell adhesion (miR29), stem cell signalling (miR137, miR28) and regulation of the extracellular matrix composition (miR29, miR148).Discussion: Through PCA we identified a miRNA signature associated with IBC. The miRNA signature was partly validated on the gene expression level. Interestingly, most identified miRNAs regulate biological processes previously associated with IBC through gene expression analysis, confirming our results also at the biological level. The association of the IBC-specific miRNA-metagene with the Luminal B phenotype and elevated HOXB13/IL17RB-expression ratios confirms our data that ER+ IBC samples, which constitute the majority of the present IBC study population, generally belong to the Luminal B subtype and are associated with resistance to endocrine therapy. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6119.

Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Reverses Mesenchymal to Epithelial Phenotype and Inhibits Metastasis in Inflammatory Breast Cancer

Inflammatory breast cancer (IBC) is a rare but aggressive type of advanced breast cancer. Epidermal growth factor receptor (EGFR) expression is an independent poor prognostic factor in IBC. The purpose of this study was to determine the effect on IBC tumorigenicity and metastasis of blocking the EGFR pathway. IBC cell lines, which express high level of EGFR, were treated with EGFR small interfering RNA and with the EGFR tyrosine kinase inhibitor erlotinib. The role of EGFR in IBC cell proliferation, motility, invasiveness, and change of the expression levels of epithelial-mesenchymal transition markers was examined. The role of extracellular signal-regulated kinase (ERK)-1/2 in erlotinib activity was also studied. The activity of erlotinib in tumor growth and metastasis was examined in an orthotopic xenograft model of IBC. Erlotinib inhibited proliferation and anchorage-independent growth of IBC cells, and this inhibition was ERK dependent. Erlotinib inhibited cell motility and invasiveness and reversed the mesenchymal phenotype of IBC cells to epithelial phenotype in three-dimensional culture. Erlotinib dramatically inhibited IBC tumor growth in a xenograft model. Interestingly, erlotinib inhibited spontaneous lung metastasis, even at a low dose that had no significant effect on primary tumor growth. These erlotinib-treated tumors were converted to epithelial phenotype from mesenchymal phenotype. The EGFR pathway is involved in tumor growth and metastasis of IBC. Targeting EGFR through the ERK pathway may represent an effective therapeutic approach to suppress tumorigenicity and prevent metastasis in EGFR-expressing IBC.

Relapse-Free Survival in Breast Cancer Patients Is Associated with a Gene Expression Signature Characteristic for Inflammatory Breast Cancer

We hypothesize that a gene expression profile characteristic for inflammatory breast cancer (IBC), an aggressive form of breast cancer associated with rapid cancer dissemination and poor survival, might be related to tumor aggressiveness in non-IBC (nIBC). RNA from 17 IBC samples and 40 nIBC samples was hybridized onto Affymetrix chips. A gene signature predictive of IBC was identified and applied onto 1,157 nIBC samples with survival data of 881 nIBC samples. Samples were classified as IBC-like or nIBC-like. The IBC signature classification was compared with the classifications according to other prognostically relevant gene signatures and clinicopathologic variables. In addition, relapse-free survival (RFS) was compared by the Kaplan-Meyer method. Classification according to the IBC signature is significantly (P < 0.05) associated with the cell-of-origin subtypes, the wound healing response, the invasive gene signature, the genomic grade index, the fibroblastic neoplasm signature, and the 70-gene prognostic signature. Significant associations (P < 0.01) were found between the IBC signature and tumor grade, estrogen receptor status, ErbB2 status, and patient age at diagnosis. Patients with an IBC-like phenotype show a significantly shorter RFS interval (P < 0.05). Oncomine analysis identified cell motility as an important concept linked with the IBC signature. We show that nIBC carcinomas having an IBC-like phenotype have a reduced RFS interval. This suggests that IBC and nIBC show comparable phenotypic traits, for example augmented cell motility, with respect to aggressive tumor cell behavior. This observation lends credit to the use of IBC to study aggressive tumor cell behavior.

Persistent E-Cadherin Expression in Inflammatory Breast Cancer

E-cadherin is a transmembrane glycoprotein that mediates epithelial cell-to-cell adhesion. Because loss of E-cadherin expression results in disruption of cellular clusters, it has been postulated that E-cadherin functions as a tumor suppressor protein. The role of E-cadherin in inflammatory breast cancer (IBC), a distinct and highly aggressive form of breast cancer, is largely unknown. The aim of our study was to elucidate whether E-cadherin expression contributes to the development and progression of the IBC phenotype and to investigate any differences in E-cadherin expression between IBC and stage-matched non-IBC. Forty-two breast cancer cases (20 IBC and 22 non-IBC) were identified. Strict and well-accepted criteria were used for the diagnosis of IBC. Clinical and pathologic features were studied, and formalin-fixed, paraffin-embedded tissue sections were immunostained for E-cadherin, estrogen and progesterone receptors (ER and PR, respectively), and HER2/neu. Statistical analysis was performed using Fisher's exact test. All IBC uniformly expressed E-cadherin, whereas 15 of the 22 (68%) of the non-IBC expressed the protein (P = .006). Intralymphatic tumor emboli in the IBC cases were also all E-cadherin positive. Two IBC tumors demonstrated invasive lobular histology, and both cases were positive for E-cadherin. Of the non-IBC cases, three were invasive lobular carcinomas, and all were positive for E-cadherin. No association was found between E-cadherin expression and ER, PR status, or HER2/neu overexpression. Our study demonstrates that there is a strong association between E-cadherin expression and IBC and suggests that E-cadherin may be involved in the pathogenesis of this form of advanced breast cancer. In our study, we demonstrate that circulating IBC tumor cells strongly express E-cadherin, thereby providing an important exception to the positive association between E-cadherin loss and poor prognosis in breast cancer.

RhoC GTPase Overexpression Modulates Induction of Angiogenic Factors in Breast Cells

Inflammatory breast cancer (IBC) is a distinct and aggressive form of locally advanced breast cancer. IBC is highly angiogenic, invasive, and metastatic at its inception. Previously, we identified specific genetic alterations of IBC that contribute to this highly invasive phenotype. RhoC GTPase was overexpressed in 90% of archival IBC tumor samples, but not in stage-matched, non-IBC tumors. To study the role of RhoC GTPase in contributing to an IBC-like phenotype, we generated stable transfectants of human mammary epithelial cells overexpressing the RhoC gene, and studied the effect of RhoC GTPase overexpression on the modulation of angiogenesis in IBC. Levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-6 (IL-6), and interleukin-8 (IL-8) were significantly higher in the conditioned media of the HME-RhoC transfectants than in the untransfected HME and HME-β-galactosidase control media, similar to the SUM149 IBC cell line. Inhibition of RhoC function by introduction of C3 exotransferase decreased production of angiogenic factors by the HME-RhoC transfectants and the SUM149 IBC cell line, but did not affect the control cells. These data support the conclusion that overexpression of RhoC GTPase is specifically and directly implicated in the control of the production of angiogenic factors by IBC cells.