Epithelial-mesenchymal transition (EMT) is a complex process in which epithelial cells acquire the characteristics of invasive mesenchymal cells. EMT has been implicated in cancer progression and metastasis as well as the formation of many tissues and organs during development. Epithelial cells undergoing EMT lose cell-cell adhesion structures and polarity, and rearrange their cytoskeletons. Several oncogenic pathways such as transforming growth factor (TGF) -β, Wnt, and Notch signaling pathways, have been shown to induce EMT. These pathways have activated transcription factors including Snail, Slug, and the ZEB family which work as transcriptional repressors of E-cadherin, thereby making epithelial cells motile and resistant to apoptosis. Mounting evidence shows that EMT is associated with cell invasion and tumor progression.In this review, we summarize the characteristic features of EMT, pathways leading to EMT, and the role of EMT in cell invasion. Three topics are addressed in this review: (1) Definition of EMT, (2) Signaling pathways leading to EMT, (3) Role of EMT in cell invasion. Understanding the role of EMT in cell invasion will provide valuable information for establishing strategies to develop anti-metastatic therapeutics which modulate malignant cellular processes mediated by EMT.

Increased expression and/or activation of H-Ras are often associated with tumor aggressiveness in breast cancer. Previously, we showed that H-Ras, but not N-Ras, induces MCF10A human breast epithelial cell invasion and migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. In an attempt to determine the sequence requirement directing the divergent phenotype induced by H-Ras and N-Ras with a focus on the induction of human breast cell invasion, we investigated the structural and functional relationships between H-Ras and N-Ras using domain-swap and site-directed mutagenesis approaches. Here, we report that the hypervariable region (HVR), consisting of amino acids 166 to 189 in H-Ras, determines the invasive/migratory signaling program as shown by the exchange of invasive phenotype by swapping HVR sequences between H-Ras and N-Ras. We also demonstrate that the H-Ras-specific additional palmitoylation site at Cys184 is not responsible for the signaling events that distinguish between H-Ras and N-Ras. Importantly, this work identifies the C-terminal HVR, especially the flexible linker domain with two consecutive proline residues Pro173 and Pro174, as a critical domain that contributes to activation of H-Ras and its invasive potential in human breast epithelial cells. The present study sheds light on the structural basis for the Ras isoform-specific invasive program of breast epithelial cells, providing information for the development of agents that specifically target invasion-related H-Ras pathways in human cancer.

Elevated expression and aberrant activation of Ras have been implicated in breast cancer aggressiveness. H-Ras, but not N-Ras, induces breast cell invasion. A crucial link between lipid rafts and H-Ras function has been suggested. This study sought to identify the lipid raft protein(s) responsible for H-Ras-induced tumorigenicity and invasiveness of breast cancer. We conducted a comparative proteomic analysis of lipid raft proteins from invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein flotillin-1 as an important regulator of H-Ras activation and breast cell invasion. Flotillin-1 was required for epidermal growth factor-induced activation of H-Ras, but not that of N-Ras, in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Flotillin-1 knockdown inhibited the invasiveness of MDA-MB-231 and Hs578T TNBC cells in vitro and in vivo. In xenograft mouse tumor models of these TNBC cell lines, we showed that flotillin-1 played a critical role in tumor growth. Using human breast cancer samples, we provided clinical evidence for the metastatic potential of flotillin-1. Membrane staining of flotillin-1 was positively correlated with metastatic spread (p 5 0.013) and inversely correlated with patient disease-free survival rates (p 5 0.005). Expression of flotillin-1 was associated with H-Ras in breast cancer, especially in TNBC (p < 0.001). Our findings provide insight into the molecular basis of Ras isoform-specific interplay with flotillin-1, leading to tumorigenicity and aggressiveness of breast cancer.Breast cancer is one of the most frequent causes of death in women.1 Among breast cancers, triple-negative breast cancers (TNBCs) lacking estrogen receptor (ER), progesterone receptor (PR) and HER2 are associated with poor prognosis and tumor aggressiveness. 2 There are no effective drugs for this type of cancer at present.3 Cancer metastasis is an important cause of death in breast cancer patients. 4 Tumor invasion and metastasis are often associated with enhanced synthesis of matrix metalloproteinases (MMPs). [5][6][7]

In an attempt to identify biomarkers and/or therapeutic targets for malignant breast cancer, the present study showed a comparative proteome profiling of invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein DS-20, a crucial regulator of H-Ras activation, as a potential marker for invasive breast cancer. DS-20 (amino acid residues 1-38) interacted with H-Ras (residues 166-189) in lipid rafts and their interaction was important for H-Ras activation. DS-20 knockdown inhibited H-Ras activation and invasion in Hs578T triple-negative breast cancer cells (TNBC) and T24 bladder carcinoma cells in which H-Ras is endogenously activated, suggesting a crucial role of DS-20 in the invasive program which relies on the activation of H-Ras. We further showed that DS-20 was required for epidermal growth factor-induced H-Ras activation, but not that of N-Ras, in MDA-MB-231 TNBC cells. Intravasation of MDA-MB-231 cells treated with shRNA DS-20 in the chick chorioallantoic membrane model was markedly reduced, indicating that DS-20 is required for an invasive capacity in vivo. In a xenograft mice tumor model, DS-20 was essential for in vivo tumor aggressiveness of Hs578T cells, suggesting DS-20 as a potential target for the treatment of breast cancer. Using human breast cancer samples, we provide clinical evidence for the tumorigenic potential of DS-20 and its association with H-Ras. Taken together, our findings provide a new insight into the molecular basis of Ras isoform-specific interplay with plasma membrane leading to cell invasion. Citation Format: Hae-Young Yong, Eun-Sook Kim, Minsoo Koh, Hwajin Son, You Rim Jeon, Jin-Sun Hwang, Myeong-Ok Kim, Yujin Cha, Wahn Soo Choi, Dong-Young Noh, Kyung-Min Lee, Ki-Bum Kim, Jae-Seon Lee, Hyung Joon Kim, Hong-Hee Kim, Eun Joo Kim, So Yeon Park, Hyeong-Reh Choi Kim, Aree Moon. Identification of a lipid raft protein that is required for H-Ras activation and breast cancer aggressiveness. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5263. doi:10.1158/1538-7445.AM2014-5263