EZH2 knockdown suppresses the growth and invasion of human inflammatory breast cancer cells.

INTRODUCTION: Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer with the poorest survival in all types of breast cancer patients and presently therapeutic targets for IBC are very limited. Enhancer of zeste homolog 2 (EZH2) is frequently expressed in human IBC and its expression positively correlates with worse clinical outcome. However, the molecular basis for EZH2 promoting IBC has not been explored. Here, we investigated the functional role of EZH2 in IBC cells by examining the effects of its knockdown on the formation of tumor spheroids and invasion of these cells in vitro and in vivo in an orthotopic xenograft model. METHODS: SUM149 and a new IBC cell line-FC-IBC-02 derived from pleural effusion fluid of an IBC patient were used in this study. Specific knockdown of EZH2 was performed using short hairpin RNA (shRNA) specific to the human EZH2 gene. Cell growth and the formation of tumor spheroids were examined in vitro. The effects of EZH2 knockdown on IBC cell migration and invasion were examined by a Boyden chamber assay. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice. RESULTS: The results showed that EZH2 is expressed at higher levels in human IBC cell lines compared with normal human mammary epithelial cells, and the knockdown of EZH2 expression significantly suppressed cell growth and tumor spheroid formation of human IBC cells in vitro. In addition, EZH2 knockdown inhibited the migration and invasion of IBC cells. Significantly, EZH2 knockdown suppressed the angiogenesis and tumor growth of IBC cells in vivo. CONCLUSIONS: Our results provide direct evidence that EZH2 is critical for the formation of tumor spheroids and invasion of human IBC cells and could be a potential target for developing novel therapeutic strategies for human IBC

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 AMPK\uce\ub2 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\uc2\uae, Pfizer Inc), induced both cytotoxicity (IC50= 0.89 \uce\ubcM) 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. \uc2\ua9 2013 Robertson et al

Abstract LB-387: EZH2 knockdown suppresses the growth of human inflammatory breast cancer cells both in vitro and in vivo in xenograft models

Abstract PURPOSE: Inflammatory breast cancer (IBC) is a rare but very aggressive type of advanced breast cancer with a poor prognosis. Thus, there is an urgent need for novel IBC therapeutics. Enhancer of zeste homolog 2 (EZH2) is of the catalytic subunit of the polycomb repressive complex 2, which silences the expression of its target genes by generating trimethylation of lysine 27 on histone H3 (H3K27Me3). EZH2 is overexpressed in several types of cancer including IBC and its overexpression correlates with a poor prognosis in IBC patients. Here, we investigated the molecular basis by which EZH2 promotes IBC. METHODS: IBC cell lines SUM149 and FC-IBC-02 which were developed from pleural effusion of an IBC patient were used in this study. Specific knockdown of EZH2 was performed using short hairpin RNA (shRNA) specific to the human EZH2 gene. MTS and soft-agar colony formation assays were used for anchorage-dependent and -independent cell growth in vitro, respectively. xCELLigence system was used to determine the cell migration and invasion. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice. RESULTS: EZH2 was expressed at higher levels in all tested human IBC cell lines when compared with non-transformed human mammary epithelial cells. Knockdown of EZH2 caused a significant reduction in the levels of H3K27Me3 in human IBC cell lines. Notably, knockdown of EZH2 expression significantly suppressed both anchorage-dependent and -independent growth of human IBC cells in vitro due to induction of apoptosis. In addition, EZH2 knockdown inhibited the migration and invasion of IBC cells. Significantly, EZH2 knockdown suppressed the growth IBC cells in vivo in an orthotopic IBC model. CONCLUSIONS: EZH2 knockdown suppresses the growth of human IBC cells in vitro and in vivo in an orthotopic IBC model in immunodeficient mice. In addition, EZH2 knockdown inhibits the invasion of human IBC cells. Together, we conclude that EZH2 contributes to IBC by promoting proliferation and invasion of human IBC cells. These results suggest that targeting EZH2 may provide a novel therapeutic strategy for the treatment of patients with IBC. 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 LB-387. doi:1538-7445.AM2012-LB-387</jats:p