A novel role for flotillin-1 in H-Ras-regulated breast cancer aggressiveness.

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 = 0.013) and inversely correlated with patient disease-free survival rates (p = 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.

Silicon interposer BGA package with a Cu-filled through silicon via and a multilayer redistribution layer fabricated via electroplating.

As large-scale integrated circuit chips become smaller, conventional organic buildup substrates can no longer support them. To resolve this problem, silicon interposers with through silicon via (TSV) technology are gaining recognition as alternative solution to provide high-density interconnection, improved electrical performance due to shorter interconnection from the die to substrate for nano-scale devices. In this study, we fabricated a silicon interposer to achieve high density and high performance packages. Via holes were etched via the Bosch process using a deep reactive ion etcher and SiO2 formed with a diffusion furnace as the diffusion barrier of the Cu electrode. TSVs were filled with Cu under various electroplating conditions. After Cu filling, a Cu post was formed directly using the over-filled Cu electrode through a chemical mechanical polishing process. A double-layer redistribution layer was formed on one side of the interposer using a lift-off process. Sn-3.5% Ag solder bumps 40 µm in diameter were formed directly on the Cu post on another side of the interposer using electroplating and the reflow method.

Electroless nickel alloy deposition on SiO2 for application as a diffusion barrier and seed layer in 3D copper interconnect technology.

Electroless Ni-P films were investigated with the aim of application as barrier and seed layers in 3D interconnect technology. Different shapes of blind-via holes were fabricated with a deep reactive ion etcher and SiO2 formed on these holes as an insulating layer. The surface of the substrate has been made hydrophilic by O2 plasma treatment with 100 W of power for 20 min. Electroless Ni-P films were deposited as both a diffusion barrier and a seed layer for Cu filling process. Prior to plating, substrates were activated in a palladium chloride solution after sensitization in a tin chloride solution with various conditions in order to deposit uniform films in TSV. After the formation of the electroless barrier layer, electro Cu was plated directly on the barrier layer. Ni-P films fabricated in blind-via holes were observed by scanning electron microscope. Energy dispersive spectroscopy line scanning was carried out for evaluating the diffusion barrier properties of the Ni-P films. The electroless Ni-P layer worked well as a Cu diffusion barrier until 300 degrees C. However, Cu ions diffused into barrier layer when the annealing temperature increases over 400 degrees C.

Identification of H-Ras-specific motif for the activation of invasive signaling program in human breast epithelial cells.

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.

Epithelial-mesenchymal Transition and Cell Invasion.

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) -ß, 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.