Zaiming Ye

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.

Imaging and Analysis of 3D Tumor Spheroids Enriched for a Cancer Stem Cell Phenotype

Tumors that display a highly metastatic phenotype contain subpopulations of cells that display characteristics similar to embryonic stem cells. These cells exhibit the ability to undergo self-renewal; slowly replicate to retain a nucleoside analog label, leading to their definition as “label-retaining cells”; express specific surface markers such as CD44+/CD24–/low and CD133; and can give rise to cells of different lineages (i.e., they exhibit multipotency). Based on these characteristics, as well as their demonstrated ability to give rise to tumors in vivo, these cells have been defined as tumor-initiating cells (TICs), tumor-propagating cells, or cancer stem cells (CSCs). These cells are highly resistant to chemotherapeutic agents and radiation and are believed to be responsible for the development of both primary tumors and metastatic lesions at sites distant from the primary tumor. Established cancer cell lines contain CSCs, which can be propagated in vitro using defined conditions, to form 3D tumor spheroids. Because the vast majority of studies to identify cancer-associated genes and therapeutic targets use adherent cells grown in 2 dimensions on a plastic substrate, the multicellular composition of these 3D tumor spheroids presents both challenges and opportunities for their imaging and characterization. The authors describe approaches to image and analyze the properties of CSCs within 3D tumor spheroids, which can serve as the basis for defining the gene and protein signatures of CSCs and to develop therapeutic strategies that will effectively target this critically important population of cells that may be responsible for tumor progression.

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.

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.

The pan-HDAC Inhibitor Suberoylanilide Hydroxamic Acid Targets Self Renewal of Breast Cancer Stem Cells.

Background: Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC). Patients with IBC often lack estrogen receptor and present with skin metastasis characterized by the presence of tumor emboli within dermal lymphatics. These metastatic tumor cells aberrantly over-express E-cadherin and have characteristics of stem cells. Based on our initial observations that IBC cells express multiple forms of histone deacetylase (HDAC) enzymes involved in epigenetic modulation of multiple genes associated with critical functions of both embryonic stem cells and cancer cells, the present studies evaluated the effects of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on self-renewal, expression of transcription factors associated with self renewal, homotypic aggregation, and E-cadherin expression by mammospheres of SUM149 and SUM190 established IBC cell lines and of tumor cells isolated from pleural effusion fluid of patients with IBC and LABC.Methods: Low adherence culture conditions that promote mammosphere formation and clonogenic assays were used to assess effects of SAHA on self renewal. Real time PCR arrays and protein arrays were used to evaluate the effects of SAHA on transcription factors Oct4, Nanog and Sox-2 and breast cancer associated genes including E-cadherin and ER/PR. Flow cytometry was used to document stem cell markers present on breast cancer cells grown under low adherence conditions. Confocal microscopy was used to document the effects of SAHA on E-cadherin localization and homotypic aggregation by mammospheres.Results: SAHA inhibited self-renewal of second and third passage mammospheres as assessed by clonogenic assays, which was associated with loss of Oct4, Nanog and Sox-2 transcription factors associated with stem cell self renewal an pluripotency. SAHA blocked E-cadherin mRNA and protein assessed by PCR and Western blotting and visualized by confocal imaging which demonstrated that loss of E-cadherin between cells comprising mammospheres blocked homotypic aggregation, leading to lack of integrity of the 3-dimensional spheroids. Moreover, SAHA induced ER-/low mammospheres to re-express ER mRNA and protein which was associated with responsiveness to tamoxifen and conversely, SAHA inhibited ER expression in ER+breast tumor cells. The effective inhibitory concentration50 (IC50) of SAHA to induce these effects in both established cell lines and in mammospheres derived from tumor cells isolated from pleural effusion fluid of patients with IBC and LABC was in the range of 0.37-3.31 uM, which is achievable by oral administration of SAHA. Initial in vivo studies indicate that intraperitoneal injection of SAHA effectively inhibits SUM149 breast tumor xenograft growth and formation of metastatic lesions.Conclusions: SAHA promotes differentiation of IBC stem cells and blocks E-cadherin expression, which is a hallmark of IBC skin metastasis. Taken together with the observations that SAHA can re-program ER function depending upon the ER status of the breast cancer cells, this observations suggest that SAHA may provide additional therapeutic opportunities for patients with LABC. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3141.

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

Gene Signatures of Inflammatory Breast Cancer: Epithelial Plasticity and a Cancer Stem Cell Phenotype

Inflammatory breast cancer (IBC) is the most lethal variant of locally advanced breast cancer and carries with it a very low survival rate of 40 % at 5 years. IBC does not present as a lump but rather mimics characteristics of an inflammation that first appears as swelling of the breast, with edema, redness, and common lymph node involvement. The physical changes in the breast are associated with the presence of nests of aggregated tumor cells, defined as tumor emboli that are encircled by lymphatic vessels, effectively blocking lymphatic drainage. Little is understood about IBC, in part due to the lack of preclinical models that recapitulate its distinct characteristics. This chapter provides an overview of our studies that have profiled all available preclinical models of IBC, including two new models recently developed, to elucidate the molecular underpinnings of this lethal variant of breast cancer. Our studies demonstrate that IBC is enriched for cells that express CD44+ and CD133+ and have aldehyde dehydrogenase-1 (ALDH1) activity, supporting a cancer stem cell/tumor initiating phenotype, associated with a very high metastatic potential to multiple distant organ sites. IBC has a distinct gene signature including E-cadherin expression with associated loss of expression of ZEB1, a transcriptional repressor of E-cadherin. IBC is also characterized by loss of expression of genes within the transforming growth factor-beta (TGFβ) signaling pathway, which is permissive for cohesive invasion by IBC tumor emboli. Taken together, these studies suggest that IBC is a very distinct variant of breast cancer characterized by epithelial plasticity, enrichment of a stem cell phenotype, and cohesive invasion as an adaptive survival mechanism, consistent with the definition of IBC as the most metastatic variant of breast cancer.

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 P6-10-04: The Presence of Anaplastic Lymphoma Kinase Recapitulates Formation of Breast Tumor Emboli with Encircling Lymphovasculogenesis

Background: Genetic abnormalities in the anaplastic lymphoma kinase (ALK) gene result in activation of signaling pathways including Akt, mTor, and JAK/Stat3. ALK has been shown to be a primary oncogenic driver in a variety of human tumors, including both hematologic malignancies such as anaplastic large cell lymphoma, as well as solid tumors including neuroblastoma, non-small cell lung cancer, myofibroblastic tumors and most recently, high grade serous ovarian carcinoma. While only ∼3% of all breast cancers have been reported to have ALK genetic abnormalities, our studies revealed that inflammatory breast cancer (IBC), the most lethal variant of breast cancer, is characterized by prevalent ALK gene amplification with activated ALK signaling. The present studies investigated the role of ALK in breast cancer by expressing full-length wild type ALK in MCF-7 cells. Materials and Methods: Clones of MCF-7 breast cancer cells expressing wild type ALK or non-target vector were produced by lentivirus infection and selection of single cell clones. MCF-7 ALK clones were evaluated using live cell and phase contrast imaging, immunofluorescent staining with confocal imaging, gene profiling, phospho-protein array analysis, western blot and ELISA validation. In vivo studies were performed by injection of MCF-7 ALK clones into using NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice using IACUC approved animal protocols. Results: When cultured on plastic substrates, MCF-7 ALK clones formed tumor cell aggregates instead of monolayer cultures, and when cultured as tumor spheroids under non-adherent 3D conditions had a distinct cellular phenotype with significantly greater clonogenicity than either non-target vector MCF-7 clones or the parental cell line. Whole transcriptome analysis, with validation using protein arrays, western blots and ELISA analysis revealed that the presence of ALK up-regulated phospho-src. In vivo studies revealed that ALK expressing MCF-7 clones formed tumor emboli that were enwrapped by dermal lymphatic vessels, essentially recapitulating the phenotype of IBC tumor emboli that exhibit encircling lymphovasculogenesis. Enforced expression of wild type ALK in another breast cancer cell line resulted in similar formation of tumor emboli. Discussion: These studies provide first time evidence for the association between full length ALK and formation of highly invasive tumor emboli enwrapped by lymphatic vessels, which is a primary characteristic of IBC. These studies, taken together with discovery of the prevalence of ALK gene amplification in IBC patients, indicate that ALK represents an important therapeutic target for IBC, with the availability of new ALK targeted therapies to evaluate as single agents and in combinations with other agents that may effectively target IBC tumor emboli that we have now linked to ALK and which represent the metastatic lesion of this lethal variant of breast cancer. Funding by Susan G. Komen Organization Promise Grant KG081287 (FMR and MC). Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-10-04.

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.