Pre-clinical Models Of Inflammatory Breast Cancer Research Articles

Abstract 3232: CEP-37440, a highly selective and potent dual inhibitor of ALK and FAK1 inhibits the proliferation of inflammatory breast cancer cells

Abstract Inflammatory breast cancer (IBC) is a very aggressive type of advanced breast cancer with a poor prognosis. Due to its propensity to rapidly metastasize, IBC is responsible for a disproportionate number of breast cancer-related deaths; 15% of all breast cancer deaths result from this disease. Approximately 30% of IBC patients have distant metastases at time of diagnosis, in contrast to 5% of patients with non-IBC breast cancers. Currently, there is no adequate adjuvant therapy to reduce the risk of recurrence and mortality in IBC patients. We recently found that FAK1, also known as PTK2, is amplified, up-regulated and phosphorylated (active) in some inflammatory breast cancer cells [1]. The focal adhesion kinase FAK1 is a cytoplasmic tyrosine kinase that localizes to focal adhesions, and controls a number of cell pathways including proliferation, viability and survival. Activated FAK1 is absent or low in normal tissue and benign neoplasms and up-regulated in invasive and metastatic tumors. In this study, we investigated a new dual FAK1/ALK inhibitor, CEP-37440, in vitro and in vivo using preclinical models of IBC. We found that 300 nM CEP-37440 was able to decrease the proliferation of the triple negative FC-IBC02 cell line and, 1000 nM CEP-37440 was able to complete inhibit the proliferation of these cells. SUM190 showed a 50% decreased in proliferation when these cells were treated with 1000 nM CEP-37440. Genes involved in functions such as cellular growth and proliferation, cell cycle, cell death and survival and cellular movement and cell-to cell signaling and interaction were differentially regulated by CEP-37440 in sensitive IBC cell lines. Among them, TGFβ1 and MMP3 were down-regulated and DKK3, CAV1 and TFPI2 were up-regulated by CEP-37440. In FC-IBC02 orthotopic breast cancer xenograft models, CEP-37440 was able to reduce the growth of the primary tumor and inhibit the development of spontaneous metastases in brain. In conclusion, CEP-37440 was highly effective in reducing the proliferation in vitro and in vivo of IBC cells that expressed high levels of phospho-FAK (Tyr397).

Abstract P3-06-39: Anaplastic lymphoma kinase (ALK) protein overexpression is not a feature of inflammatory breast cancer

Abstract Recent studies suggest that anaplastic lymphoma kinase (ALK) could serve as a therapeutic target for inflammatory breast cancer (IBC) and that strategies targeting ALK should be considered for evaluation in clinical trials. Reverse phase protein arays revealed activation of the ALK receptor tyrosine kinase and biochemically-linked downstream signalling molecules in pre-clinical models of IBC. ALK genetic abnormalities have been reported in 80% (20/25) of IBC patients with a high prevalence of ALK alterations in basal-like IBC (Robertson F. et al., 2013). However, ALK protein expression has not been studied in human IBC samples. Immunohistochemical detection of the ALK protein is increasingly being used as a screening tool to test samples for ALK rearrangements. The biological premise is that the genetic abnormality of the ALK gene leads to overexpression of the ALK protein and therefore overactivity of the ALK tyrosine kinase; since this kinase is the target of crizotinib, it makes sense to assess the drug target directly. Formalin fixed paraffin embedded tissue samples from pre-treatment surgical biopsies of 79 consecutive IBC patients were immunohistochemically (IHC) tested for ALK protein over-expression using a validated IHC test (NCL-ALK, clone 5A4) with a sensitivity of 93% and a specificity of 100% when the Vysis ALK-FISH break apart test is used as a gold standard. Stained tissue sections were evaluated using the IHC histoscore proposed by Thunnissen E. et al., 2012, assessing both the proportion of tumor cells showing cytoplasmic staining and the staining intensity. Cytoplasmic staining of appendiceal ganglion cells was used as an on-slide external positive control; weak to moderate cytoplasmic staining of macrophages was used as an internal positive control. In 75 of the 79 tissue samples none of the tumor cells showed any ALK immunoreactivity. One tumor showed moderate cytoplasmic staining in a few cells (<1%, score 2+); ALK (2p23) FISH showed no rearrangement. This tumor was hormone receptor negative and HER2 negative. Three tumors showed minimal cytoplasmic staining in a few cells (<1%, score 1+). These results demonstrate that ALK protein overexpression is not a feature of IBC. Although genetic abnormalities of ALK without protein overexpression are not excluded, our results show that ALK IHC cannot be used to identify IBC patients eligible for enrollment in clinical trials evaluating ALK targeted therapeutics. Citation Format: Cecile Colpaert, Melike Marsan, Peter Vermeulen, Luc Dirix, Steven Van Laere, Inflammatory Breast Cancer International Consortium. Anaplastic lymphoma kinase (ALK) protein overexpression is not a feature of inflammatory breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-39.

Abstract P2-05-04: Comparative expression profiling of patient samples and preclinical models of inflammatory breast cancer reveals gene signatures of epithelial plasticity and suppression of TGFb signaling

Abstract Introduction: Genome-wide expression profiling of samples from patients with and without Inflammatory Breast Cancer (IBC) has revealed novel insights into the biology of IBC. The present study was undertaken to compare these novel insights with data obtained from all available preclinical IBC models including 2 new models that we have recently developed that recapitulate the characteristics of IBC including retention of E-cadherin, formation of tumor emboli and encircling lymphoangiogenesis Materials and Methods: Five replicates of 7 preclinical IBC models (SUM149, SUM190, FC-IBC-01, FC-IBC-02, MDA-IBC-03, KPL-4, and Mary-X) were profiled using Affymetrix HGU133plus2 GeneChips. Using a nearest shrunken centroid algorithm, each expression profile was classified according to an IBC-specific signature identified in patient samples. Available expression profiles were further queried for expression patterns related to Epithelial-to-Mesenchymal Transition (EMT), TGFβ-signaling and IBC-specific patterns of transcription factor activation. Results: Application of our IBC-specific signature (posterior probabilities exceeded 0.50 in at least 4/5 replicates) revealed that out of 7 pre-clinical models of IBC, 3 of these robustly classified as IBC (FC-IBC-01, FC-IBC-02, and KPL-4). All preclinical IBC models were characterized by retention of E-Cadherin expression, absence of ZEB1 expression, attenuated expression of specific components of the TGFβ pathway (TGFβR2, SMAD3, SMAD7, and TGFβ1), and ambiguous activation patterns of several transcription factors involved in regulating cellular plasticity and cell fate decisions (Up in IBC: NR4A2, RARB/RXRA, PTX3, GSC2, and ZEB1; Down in IBC: SOX10, PAX5, and SMAD2). For each of the molecular alterations described above, Z-scores greater than 2 were achieved in at least 4/5 replicates. Conclusions: The observations that we have made using IBC patient tumor samples with regards to EMT, cell plasticity and TGFβ-signaling are corroborated in pre-clinical models of IBC using current analytic approaches, despite the fact that expression patterns of the majority of preclinical models of IBC deviate from the IBC-specific expression patterns observed in patient samples. Our data suggest that despite their highly invasive nature, IBC cancer cells retain an epithelial cell phenotype characterized by E-cadherin expression and loss of ZEB1 which appears to be mediated by, amongst others, attenuated TGFβ-signaling. This study strengthens our hypothesis that cancer cells from IBC exhibit cohesive invasion, and invade as a unit, possibly explaining the presence of florid tumour emboli which is a primary characteristic observed in IBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-05-04.

Presence of anaplastic lymphoma kinase in inflammatory breast cancer

Although Inflammatory Breast Cancer (IBC) is recognized as the most metastatic variant of locally advanced breast cancer, the molecular basis for the distinct clinical presentation and accelerated program of metastasis of IBC is unknown. Reverse phase protein arrays revealed activation of the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and biochemically-linked downstream signaling molecules including JAK1/STAT3, AKT, mTor, PDK1, and AMPKβ in pre-clinical models of IBC. To evaluate the clinical relevance of ALK in IBC, analysis of 25 IBC patient tumors using the FDA approved diagnostic test for ALK genetic abnormalities was performed. These studies revealed that 20/25 (80%) had either increased ALK copy number, low level ALK gene amplification, or ALK gene expression, with a prevalence of ALK alterations in basal-like IBC. One of 25 patients was identified as having an EML4-ALK translocation. The generality of gains in ALK copy number in basal-like breast tumors with IBC characteristics was demonstrated by analysis of 479 breast tumors using the TGCA data-base and our newly developed 79 IBC-like gene signature. The small molecule dual tyrosine kinase cMET/ALK inhibitor, Crizotinib (PF-02341066/Xalkori®, Pfizer Inc), induced both cytotoxicity (IC50 = 0.89 μM) and apoptosis, with abrogation of pALK signaling in IBC tumor cells and in FC-IBC01 tumor xenograft model, a new IBC model derived from pleural effusion cells isolated from an ALK+ IBC patient. Based on these studies, IBC patients are currently being evaluated for the presence of ALK genetic abnormalities and when eligible, are being enrolled into clinical trials evaluating ALK targeted therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-497) contains supplementary material, which is available to authorized users.

Inflammatory breast cancer (IBC): clues for targeted therapies

Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development and poor prognosis. Since there are few preclinical models of IBC, there is a general lack of understanding of the complexity of the disease. Recently, we have developed a new model of IBC derived from the pleural effusion of a woman with metastatic secondary IBC. FC-IBC02 cells are triple negative and form clusters (mammospheres) in suspension that are strongly positive for E-cadherin, β-catenin and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. FC-IBC02 cells expressed stem cell markers and some, but not all of the characteristics of cells undergoing epithelial mesenchymal transition (EMT). Breast tumor FC-IBC02 xenografts developed quickly in SCID mice with the presence of tumor emboli and the development of lymph node and lung metastases. Remarkably, FC-IBC02 cells were able to produce brain metastasis in mice on intracardiac or intraperitoneal injections. Genomic studies of FC-IBC02 and other IBC cell lines showed that IBC cells had important amplification of 8q24 where MYC, ATAD2 and the focal adhesion kinase FAK1 are located. MYC and ATAD2 showed between 2.5 and 7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6–4 copies in IBC cells. Also, CD44 was amplified in triple-negative IBC cells (10–3 copies). Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. These results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-013-2600-4) contains supplementary material, which is available to authorized users.

New Therapeutic Targets in Inflammatory Breast Cancer

Inflammatory breast cancer (IBC) is the most lethal variant of locally advanced breast cancer. Although recognized as a distinct clinical entity, there have been few advances in the development of pre-clinical models of IBC, and a lack of IBC-specific therapeutic targets translated into clinical utility to increase overall survival, which is currently 40 % at three years. By use of newly developed pre-clinical models of IBC and patient tumor tissues, E-cadherin, anaplastic lymphoma kinase (ALK), and HSP90 have been identified as targets relevant to IBC that are matched by therapeutics that are either currently in clinical trials or will be tested in clinical trials within the next year. These exciting results illustrate the advances that have been made in recent years in defining the molecular basis of IBC as a distinct disease and the significant strides made in identifying more effective strategies for treatment of patients with IBC.

Genomic Profiling of Pre-Clinical Models of Inflammatory Breast Cancer Identifies a Signature of Epithelial Plasticity and Suppression of TGFβ Signaling

Abstract Study background: Inflammatory Breast Cancer (IBC) is the most metastatic variant of breast cancer. Although IBC is recognized as a distinct variant of breast cancer, the molecular basis for the rapid progression of IBC remains largely undefined, in part due to the lack of preclinical models that recapitulate the human disease as well as a lack of comprehensive analysis of the preclinical models of IBC that are available. Methods: All available 7 pre-clinical models of IBC, including 2 new models, FC-IBC01 and FC-IBC02 developed from pleural effusion, were used to identify genes and molecular pathways that are selectively altered compared to non IBC breast tumor models. Laser capture micro dissection of biopsy tissue from core biopsy and skin punch biopsies were also analyzed by whole transcriptome analysis. Results: Whole transcriptome analysis defined 7 pre-clinical models of IBC as being within either the triple negative or ErbB2/Her-2 expressing subtypes, similar to the prevalence of these subtypes of breast cancer observed in IBC patients. Comparative analysis of the FC-IBC01, FC-IBC02 and Mary-X models of IBC demonstrated that each of these recapitulate the formation of tumor emboli with encircling lymphovasculogenesis. The majority (6/7) of the pre-clinical models of IBC express CDH1, which encodes for E-cadherin, which was associated with a loss of ZEB1, a transcriptional repressor of E-cadherin. The lack of ZEB1 expression was validated in a limited set of 4 skin punch biopsy samples from IBC patients that were isolated by laser capture micro-dissection, demonstrating concordance with loss of ZEB1 in pre-clinical models of IBC. Expression of other transcription factors involved in acquisition of a cancer stem cell phenotype, including Snai1, which encodes for Snail, SNAI2, which encodes for Slug and TWIST1, was retained in pre-clinical models of IBC. Maintenance of E-cadherin in pre-clinical models of IBC was associated with a loss of genes within the transforming growth factor beta (TGFβ signaling pathway, with expression of SMAD6, a known repressor of TGFβ. This is similar to a recent study reporting the persistence of E-cadherin and loss of TGFβ signaling in IBC patient tumors based on gene profiling of 3 independent data sets. Conclusion: The present studies provide first time comparison of gene signatures of 7 pre-clinical models of IBC, including our 2 newly developed pre-clinical models, FC-IBC01 and FC-IBC02, that recapitulate formation of tumor emboli with encircling lymphatic vessels, similar to that observed in biopsy tissues of IBC patients. We demonstrate that E-cadherin expression was associated with both loss of ZEB1 and diminished expression of multiple genes within the TGFβ signaling pathway, with retention of expression of transcription factors and surface markers consistent with maintenance of a cancer stem cell phenotype, as has been reported to be a characteristic of IBC tumors. Collectively, these observations provide first time characterization of the molecular signatures of all available pre-clinical models of IBC, and suggest that IBC has a signature of epithelial plasticity, with characteristics of their ability to undergo the mesenchymal to epithelial reverting transition. The loss of genes within the TGFβ signaling is also consistent with the tight cell: cell aggregation of IBC tumor cells within tumor emboli that exhibit “cohesive invasion”. The new pre-clinical models of IBC that recapitulate the human disease will serve as useful tools to accelerate our understanding of the molecular underpinnings and therapeutic targets of IBC as the most lethal form of breast cancer.

P4-03-06: Development and Comparative Characterization of Metastasis in Newly Developed Pre-Clinical Models of Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer. It is associated with a poor survival rate (40% 5-year survival) despite appropriate multidisciplinary care. For such an aggressive type of cancer, IBC has been understudied, in part due to the lack of adequate numbers of cell lines and mouse models that recapitulate the human disease. To expand our understanding of IBC, we have obtained all of the previously developed and characterized IBC cell lines and models including Mary-X, SUM149, SUM190, KPL-4, MDA-IBC-3 and have developed two new IBC models, designated as FC-IBC01 and FC-IBC02, using tumor cells derived from pleural effusion of IBC patients. Materials and Methods: Each of these IBC cell lines has been luciferase (LUC)-tagged, allowing the growth of orthotopic injection or subcutaneous implantation to be evaluated by bioluminescent imaging (BLI). Alternatively, the LUC-tagged IBC cells can by injected via either intra-cardiac or intravenous route of delivery, which promotes rapid tumor colonization, resulting in both visceral and skeletal metastasis. Growth of IBC tumors can then be monitored immediately using BLI, thus eliminating the lag time needed for the physical detection of palpable tumors. BLI imaging also allows for monitoring of the kinetics and location of development of metastatic lesions. Whole transcriptome analysis was performed on IBC cell lines and xenograft tissues to define the heterogeneity of IBC as a distinct variant of breast cancer Results: These models have allowed us to identify micro-metastatic foci in multiple sites distant from the IBC primary tumor in each of these models of IBC and allow the quantitation of anti-tumor and anti-metastatic effects of targeted therapeutics as single agents as well as the potential synergy of combinations of agents. As an example, injection of LUC-tagged IBC cell lines such as SUM149-Luc, into the left ventricle of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice allows the metastatic tumor burden to be monitored longitudinally by whole animal BLI, which can be validated at necropsy and by immunohistochemical analysis. Whole transcriptome analysis of pre-clinical models of IBC reflect the molecular subtypes observed in IBC patients, with the majority of IBC models being of the basal like, luminal B and Her2 amplified. Discussion: First time analysis of known and newly developed pre-clinical models of IBC allows a more complete analysis of IBC as a distinct variant of breast cancer. Furthermore, these approaches allow rapid evaluation of the promising targeted therapeutics identified based on whole transcriptome analysis of both IBC patient tumors and pre-clinical models developed from IBC patients. We believe that this extensive collection of LUC-tagged IBC cell lines is an invaluable tool for IBC research since the cell lines encompass the broad spectrum of IBC heterogeneity. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-03-06.

P2-05-04: Mapping the Specific Gene Families Activated in the Lymphangiogenesis and Vasculogenic Mimicry Exhibited by Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. Although IBC is diagnosed less commonly than other types of breast cancer, it is extremely aggressive, and accounts for a disproportionate number of breast cancer related deaths annually. IBC exhibits very specific patterns of lymphangiogenesis and vasculogenic mimicry, however detailed studies of the genes and proteins involved in these angiogenic processes are lacking. This study performed whole unbiased gene transcription studies with validation by protein arrays using all available pre-clinical cell lines and in vivo xenograft models of IBC, including a new model of IBC, FC-IBC01, which exhibits lymphovascular invasion, to identify the specific pathways involved in the distinctive angiogenesis observed in IBC. Materials and Methods: Real-time quantitative RT-PCR, cDNA microarray gene profiling, immunofluorescence with confocal imaging and protein arrays were used to examine differential expression of specific angiogenic gene families including VEGFA,B,C,D, VEGF Receptor genes, and ANG/TIE genes linked to angiogenesis and lymphangiogenesis. Results: Activity of the matrix metalloproteinase, MMP-2, is required for IBC tumor cells to undergo vasculogenic mimicry (VM), which is associated with a loss of TIMP-2, a well known inhibitor of angiogenesis. Therapeutics that target MMP activity can successfully inhibit this VM. Furthermore, pre-clinical models of IBC that form IBC tumor emboli exhibit lymphovascular invasion that is associated with distinct patterns of expression of genes that encode for distinct receptor tyrosine kinases that may represent important therapeutic targets for IBC. Discussion: Identification of the distinct angiogenic pathways that are activated in IBC provides insight into the therapeutic targets that may abrogate the distinct lymphovascular invasion and vasculogenic mimicry that are linked to the aggressive metastasis of IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-04.

P4-09-08: Secretory Leukocyte Protease Inhibitor as a Differential Diagnostic Biomarker of Tumor Emboli in Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal variant of locally advanced breast cancer. Pathologically, IBC is characterized by the presence of nests of cells, defined as tumor emboli, that undergo lymphovascular invasion into the skin and chest wall and represent the metastatic lesion of IBC. Due to the distinct presentation of IBC, it is often misdiagnosed due to a lack of defined biomarkers that are specific for tumor emboli in biopsy samples of IBC patients. To date, the only validated marker that has shown to consistently identify IBC tumor emboli is the surface glycoprotein E-cadherin. Materials and Methods: With the goal of identifying genes specifically expressed by IBC tumor emboli, the present studies performed whole transcriptome analysis of tumor emboli from skin punch biopsy samples from IBC patients and tumor emboli from the Mary-X pre-clinical model of IBC which were isolated using laser capture microdissection. Results: Secretory leukocyte peptidase inhibitor (SLPI) was a gene identified as being highly expressed by tumor emboli. This gene was previously identified as a metastasis related gene, has been reported to be associated with poor prognosis in serous ovarian carcinoma and is implicated in mediating resistance to paclitaxel. Immunofluorescence staining and confocal microscopy revealed that SLPI is a robust marker of tumor emboli in skin punch biopsies from IBC patient and in Mary-X tumor emboli. SLPI differentially delineates between IBC tumor emboli and hair follicles present in the skin, which also stain with E-cadherin. An additional marker, Cytokeratin 15 (CK15), can be used in combination with SLPI to differentially identify the components of the skin that are CK15 positive, and IBC tumor emboli that reside within the skin, which are CK15 negative. Use of podoplanin antibodies, which stain lymphovascular endothelium, demonstrate that IBC tumor emboli that express SLPI protein, are completely encircled within the dermal lymphovasculature. Conclusions: These studies are among the first to identify SLPI as a potential biomarker of tumor emboli in skin punch biopsy samples from IBC patients and in pre-clinical models of IBC. Studies are ongoing to establish the molecular function and role of SLPI in mediating the survival and invasion of IBC tumor emboli. Collectively, these studies are the first to identify SLPI as a potential biomarker of IBC tumor emboli in skin punch biopsy samples. In addition to serving as a potential biomarker of tumor emboli, SLPI may also serve as a therapeutic target for development of approaches to eradicate IBC tumor emboli. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-09-08.