Km Boley

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.

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

Genomic and Proteomic Pathway Mapping Reveals Signatures of Mesenchymal-Epithelial Plasticity in Inflammatory Breast Cancer

1.1 Inflammatory breast cancer as a distinct clinicopathologic entity There are several clinically distinct types of breast cancer, which include early stage breast cancer, locally advanced breast cancer (LABC) and metastatic breast cancer. The most rare but lethal form of LABC is inflammatory breast cancer (IBC) (reviewed in 1). This type of breast cancer accounts for an estimated 25% of all breast cancers in the United States and up to 20% of all breast cancers globally (2-4). Although primary IBC is less commonly diagnosed than other types of breast cancer, IBC is responsible for a disproportionate number of breast cancer-related deaths that occur each year world-wide due to its propensity to rapidly metastasize. (2-4). Women diagnosed with IBC have a significantly shorter median survival time (~ 2.9 years) than women with either LABC (~ 6.4 years) or non-LABC breast cancer (>10 years). The clinical diagnosis of IBC is based on the combination of the physical appearance of the affected breast, a careful medical history, physical examination, and pathological findings from a skin biopsy and/or needle or core

Abstract P6-12-14: Histone deacetylase inhibitor suberoylanilide hydroxamic acid targets breast cancer stem cells and inhibits metastasis

Introduction : Inflammatory breast cancer (IBC) is a distinct and aggressive subtype of locally advance breast cancer associated with increased aldehyde dehydrogenase 1 (ALDH1) positive cancer stem cells (CSCs). IBC is associated with a poor survival rate (40% 5-year survival), with few therapeutic strategies identified that effectively blocked the growth and metastasis of IBC. Our previous in vitro studies revealed that the pan-histone deacetylase inhibitor Suberoylanilide Hydroxamic Acid (SAHA) effectively targets self-renewal of IBC tumor cells. Materials and Methods : The IBC cell line, SUM149 was tagged with the Luciferase gene (SUM149-LUC) and their in vivo growth in immune compromised mice tested in both orthotopic and metastatic settings. Results : SUM149-LUC rapidly develop primary tumors and metastatic lesions at a variety of locations including lung, brain, bone, liver, reproductive organs and adrenal glands. SAHA effectively blocked growth of SUM149 primary tumors and inhibited metastasis, which was synergistic with the microtubule stabilizer, Paclitaxel. The therapeutic efficacy of SAHA was associated with a significant decrease in ALDH1+ CSCs populations. Conclusions : Collectively, these results suggest that strategies of combining agents such as SAHA that target CSCs with Paclitaxel that targets the bulk of proliferating tumor cells warrant further investigation for their potential effectiveness in IBC patients, who have the lowest survival of breast cancer patients and have few therapeutic options. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-14.

Inflammatory Mediators as Therapeutic Targets for Inflammatory Breast Cancer

The molecular signature of inflammatory breast cancer (IBC) includes activation of target genes of the nuclear factor-kappa B (NF-κB) transcription factor. These NF-κB target genes are differentially activated in IBC tumors and primarily produce pro-inflammatory mediators such as the chemokine interleukin-8 (IL-8), the lipid mediator prostaglandin E2, the chemokine receptor CXCR4 and its ligand partner CXCL12, and the axis defined by IL-6/Janus kinases and signal tranducer and activator of transcription 3 (STAT3). While these genes are known to regulate innate immune responses, they also are critically important to survival of tumor cells and to metastatic progression. Ongoing research is defining the roles of these inflammatory mediators and associated signaling pathways in breast cancer, in general, and in IBC. Some of these studies have evaluated pharmacological and biological agents that effectively target these pro-inflammatory mediators and have led to development of new therapeutics that may effectively abrogate IBC growth and metastasis. In summary, this chapter reviews the inflammatory mediators that have been identified as part of the molecular fingerprint of IBC and describes new evidence for the potential for inhibitors of these mediators to target specific populations of cells within IBC tumors that contribute to tumor initiation and metastatic progression.

Abstract 5342: FC-IBC-02: A new in vitro-in vivo model of inflammatory breast cancer (IBC)

Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast can[[Unsupported Character - Codename s]]cer and it is associated with a poor prognosis in spite of appropriate multidisciplinary treatments. The disease is characterized by peculiar molecular and clinical features and usually affects younger patients, mostly under the age of 50 years at diagnosis. IBC has shown the capacity to spread early, primarily through lymphatic channels and secondarily through blood vessels causing the inflammatory signs and development of early metastasis. The high incidence of metastatic disease at presentation, the persistence of residual disease after induction chemotherapy associated with significant risk of disease recurrence immediately surgery strongly support our hypothesis that these patients develop micro metastatic disease early in the disease course (Cristofanilli et al., Cancer 2007;110:1436-44). Moreover, the lower survival rate of IBC patients may be due to the early activation of metastatic process associated with a peculiar phenotype of cancer stem cells (CSCs) (Cristofanilli et al., Oncologist 2003;8:141-8). Although IBC research has been ongoing for over 15 years, very few molecular alterations have been associated specifically with IBC and few preclinical models are currently available to evaluate the peculiar biology of IBC. The majority of IBC studies have been performed using the cell lines SUM149 and SUM 190 which were developed from the primary tumor and, KPL-4 isolated from the pleural effusion of IBC patients. Although both the SUM149 and KPL-4 injected into immuno- compromised mice form primary tumors, there are currently only two in vivo xenograft models of IBC, the Mary-X and the WIBC-9 models that recapitulate the tumor emboli that are the signature of IBC in humans. We have developed another IBC model, FC-IBC-02, derivate from the pleural effusion of a 49 years old IBC triple negative patient. Tumor cells from the pleural effusion were ER(-) Pgr(-) and Her2(-) and strongly positive for tetraspanin CD151 and E-cadherin. Cells from the pleural effusion of this patient were growth under non-adherent conditions in serum-free mammary epithelial growth medium (MEGM). These tumor spheroids have Aldhehyde Dehydrogenase 1 (ALDH-1) activity and characteristics of cancer stem cells (CSC) and they rapidly developed tumors when they were injected in the mammary fat pad of SCID mice. These tumors were poorly differentiated carcinomas ER-negative PgR-negative Her2-negative and the mice developed micro metastasis in the lungs. In contrast, cells isolated under adherent conditions were unable to produce tumor in SCID mice. In summary, our studies demonstrated that IBC is a disease enriched for cells with a stem cell phenotype and these cells are highly tumorigenic. IBC may represent an ideal model to evaluate stem cell targeting therapeutic modalities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5342. doi:1538-7445.AM2012-5342

Abstract P4-06-03: Zinc Finger Nuclease Genome Engineering Reveal Multiple Functions of Secretory Leukocyte Peptidase Inhibitor in Regulating Pleuripotency of Cancer Stem Cells in Inflammatory Breast Cancer

Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal variant of this disease and is known to be enriched for cells with a cancer stem cell phenotype. IBC is characterized by the presence of cell aggregates, defined as tumor emboli, that metastasize into skin and chest wall. The only documented biomarker of tumor emboli is the surface glycoprotein, E-cadherin. We previously demonstrated that IBC tumor emboli express the alarm anti-protease, secretory leukocyte peptidase inhibitor (SLPI), a metastasis related gene highly expressed in IBC patient tumors. Since the function of SLPI in IBC is unknown, the present studies used zinc finger technology to knockout (KO) copies of SLPI in the SUM149 IBC cell line to define the role of SLPI in IBC. Materials and Methods: Using CompoZr zinc finger nuclease (ZFN) technology (Sigma-Aldrich), SLPI KO cell lines were generated by disrupting all alleles (3) in exon 1 of SUM149 cells derived from an IBC primary tumor with a high CD44+cell population. The target-specific ZFNs bound DNA at a sequence-specific location and created double strand breaks repaired by non-homologous end joining, resulting in deletions at the SLPI locus. Single cell SLPI KO clones were isolated and serially passaged to establish stable cell lines. Functional assays were used to assess proliferation, invasion, tube formation and clonogenicity. Global transcriptional profiling was performed to identify genes and signaling pathways directly regulated by SLPI. Results: SUM149 SLPI KO clones did not produce SLPI protein and had a significantly slower turn-over time of 75 hrs compared with 24 hrs in SUM149 wild type clones. Loss of SLPI blocked invasion by 50%, and completely inhibited formation of tube-like structures, an activity defined as vasculogenic mimicry, characteristic of IBC. The loss of only 1 SLPI allele resulted in the inability of SUM149 cells to grow as anchorage independent clones in soft agar, commonly used as a predictor of in vivo tumorigenicity. SLPI directly regulated expression of multiple genes within the embryonic stem cell pleuripotency canonical pathway, including WNT, Frizzled, PDK-1, platelet derived growth factor receptor and sphingosine-1-phosphate receptor. Studies are underway to determine the specific role of SLPI in IBC tumor growth and metastasis. Conclusions: Our previous studies demonstrated that SLPI is expressed by IBC tumor emboli and can be used as a biomarker of tumor emboli in IBC core and skin punch biopsies. SLPI was found to not only regulate critical functional activities of IBC tumor cells but also to directly regulate genes within pathways critically important to maintenance of pleuripotency of tumors with a cancer stem cell phenotype. Collectively, these studies demonstrate the power and utility of the zinc finger technology, which enables the interrogation of tumor cells to discern the direct function and role of specific genes of interest. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-03.

Abstract P5-04-05: E-Cadherin is Required for In Vivo Growth of Inflammatory Breast Cancer: Importance of Mesenchymal-Epithelial Transition (MET) and Role of HIF1α

Background: Inflammatory breast cancer (IBC) is the most aggressive and deadly form of breast cancer and is phenotypically distinct from other forms of locally advanced breast cancer. The 5-year survival for IBC patients (40%) has not improved even with the implementation of multi-disciplinary care and targeted therapies (e.g. anti-HER2 or anti-estrogen therapies). Lymph node involvement is common at first diagnosis and the accelerated metastasis program that characterizes IBC account for a disproportionate number of the 40,000 women who will die from breast cancer. The most common molecular marker for IBC is the cell adhesion surface glycoprotein, E-Cadherin. Classically, E-Cadherin is thought to be a tumor suppressor and its loss is associated with the epithelial-mesenchymal transition (EMT) and acquisition of an invasive phenotype. The importance of EMT in metastasis is well accepted in numerous tumor models although the presence of EMT in patient samples is rarely demonstrated. Given the propensity of IBC to metastasize, one would expect a strong EMT signature in IBC cell lines, however the persistent robust expression of E-Cadherin in IBC patient tumors and in IBC cell lines, with a lack of expression of ZEB1, a transcriptional repressor of E-cadherin and diminished expression of genes within the transforming growth factor beta signaling pathway, suggests otherwise. Methods: To further define the role of E-Cadherin in IBC, we used the SUM149 IBC cell line to manipulate E-Cadherin via shRNA knockdown and by overexpression of ZEB1 a known transcriptional repressor of E-cadherin. Results: While the lack of E-Cadherin or ZEB1 overexpression in SUM149 cells led to an EMT phenotype in vitro, E-Cadherin was required for in vivo growth of SUM149 tumor cells. Gene profiling identified novel E-Cadherin signaling pathways including an hypoxia gene signature through HIF1a as well as cytokine responsive pathways. The requirement of HIF-1a for in vivo growth of SUM149 cells was confirmed by shRNA knockdown of HIF-1a. Conclusions: The absolute requirement for E-Cadherin and maintenance of the mesenchymal-epithelial transition in IBC tumor growth is demonstrated. Furthermore, a novel functional link between E-Cadherin and HIF-1a and in their critical role in survival of IBC tumors is identified for the first time and warrants further investigation. Supported in part by The Susan G Komen Organization Promise Grant KG081287 (FMR and MC) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-05.

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

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.

P1-02-03: The Reciprocal Roles of E-Cadherin and ZEB1 Demonstrate the Mesenchymal-Epithelial Transition as a Primary Characteristic of Inflammatory Breast Cancer.

Background: Inflammatory breast cancer (IBC) is a rare but very aggressive form of breast cancer. IBC is characterized by nests of tightly aggregated cells, defined as tumor emboli, that exhibit characteristics of cancer stem cells (CSCs). IBC tumor emboli express E-cadherin which is required to maintain their integrity and our recent evidence demonstrates that expression of E-cadherin by tumor emboli is associated with lack of ZEB1 expression, a transcriptional repressor of E-cadherin. This is at odds with the current hypothesis that metastatic progression is associated with the process of epithelial mesenchymal transition (EMT), with loss of E-cadherin and gain of transcription factors including ZEB1, acquisition of CSC characteristics and enhanced invasive capabilities. Materials and Methods: shRNA knockdown and over-expression methods, real time PCR arrays, western blotting, and in vitro assays to evaluate proliferation, invasion, growth in soft agar and clonogenicity and in vivo animal studies were used. Results: Expression of E-cadherin was reduced by shRNA and ZEB1 was expressed in SUM149 IBC tumor cells. Numerous EMT-related genes were upregulated with loss of E-cadherin and gain of ZEB1, including N-cadherin and vimentin. However, there were marginal differences in the in vitro parameters of proliferation, Matrigel invasion and anchorage independent growth in soft agar between SUM149-shECad or SUM149-ZEB1 clones and their respective vector control cells. The loss of E-cadherin and gain of ZEB1 altered the morphology of SUM149 cells when cultured under low adherence conditions permissive for the enrichment of CSC, exhibiting a reversion in grape-like morphology to more well defined spheres, which was accompanied by increased clonogenicity in both SUM149-shECad and SUM149-ZEB1 cells. The loss of E-cadherin and the gain of ZEB1 significantly inhibited tumor growth of cells injected in the mammary fat pad of NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice. Tumor volume at 56 days for E-cadherin vector control cells was 771.9 mm 3 +/− 185.6 compared to shECadherin tumors, which was 13.6 mm 3 +/− 7.2. Tumor volume of ZEB1 vector control tumors was 346.1 mm 3 +/− 96 compared to volume of ZEB1 expressing tumors, which was 21.5 mm 3 +/− 7.2.Conclusions: E-cadherin with lack of ZEB1 expression in IBC is consistent with a mesenchymal-epithelial transition (MET), consistent with the retention of the epithelial phenotype while maintaining a program of rapid metastasis and colonization of lymph nodes and distant organ sites. Furthermore, we demonstrate that the E-cadherin-ZEB1 axis is critical for the in vivo growth of IBC tumor cells. Although SUM149 cells are fully capable of undergoing an EMT process, which is under negative regulation by E-cadherin, the process of EMT does not drive in vivo tumor growth in IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-03.