signaling pathway has been known to play a major role in the pathological process of atherogenesis. Unlike high shear stress, in which the NF-B activity is transient, our earlier studies have demonstrated a persistent activation of NF-B in response to low shear stress in human aortic endothelial cells. These findings partially explained why low shear regions that exist at bifurcations of arteries are prone to atherosclerosis, unlike the relatively atheroprotective high shear regions. In the present study, we further investigated 1) the role of NF-B signaling kinases (IKK␣ and ) that may be responsible for the sustained activation of NF-B in low shear stress and 2) the regulation of these kinases by reactive oxygen species (ROS). Our results demonstrate that not only is a significant proportion of low shear-induced-kinase activity is contributed by IKK, but it is also persistently induced for a prolonged time frame. The IKK activity (both ␣ and ) is blocked by apocynin (400 M), a specific NADPH oxidase inhibitor, and diphenyleneiodonium chloride (DPI; 10 M), an inhibitor of flavin-containing oxidases like NADPH oxidases. Determination of ROS also demonstrated an increased generation in low shear stress that could be blocked by DPI. These results suggest that the source of ROS generation in endothelial cells in response to low shear stress is NADPH oxidase. The DPI-inhibitable component of ROS is the primary regulator of specific upstream kinases that determine the persistent NF-B activation selectively in low shearinduced endothelial cells. upstream B kinases; laminar shear stress; oxidative stress; atherogenesis; reactive oxygen species ENDOTHELIAL CELLS lining the vascular wall respond differentially to different flow shear regimen. Studies from the past have clearly demonstrated that regions of artery that experience low shear stress and reversing flow patterns such as arterial bifurcations and curved segments of the large elastic artery are highly predisposed to the development of atherosclerotic lesions, unlike high shear regions that are relatively resistant to the disease (2, 15). Our earlier in vitro studies (17)(18)(19) and in vivo reports from other laboratories (9) have indicated the possible involvement of NF-B signaling mechanisms in low shear lesion-prone areas. The NF-B family of transcription factors (p65 or RelA, p50, p52, Bcl-3, c-Rel and RelB) regulate many proinflammatory endothelial signals that are activated in the early stages of atheroma formation (29). NF-B family members form homo-or heterodimeric complexes that are retained in the cytoplasm in quiescent (unstimulated) cells by associating with a family of inhibitor proteins, called IBs, which include IB␣ and IB. Activation of NF-B by a variety of stimuli is dependent on the phosphorylation of site-specific serine residues such as Ser 32 and Ser 36 of IB␣ and Ser 19 and Ser 23 of IB, followed by the subsequent degradation of the IB proteins, which allows the translocation of the active form of NF-B into the nucleus to activate downs...
Hypoxia-inducible factor-1␣ (HIF-1␣The hypoxic response is mainly regulated by the hypoxiainducible factor-1 (HIF-1), 2 a basic helix-loop-helix transcription factor composed of two subunits HIF-1␣ and HIF-1 (1). HIF-1␣ forms heterodimers with HIF-1, and this complex binds to hypoxia-responsive element (HRE: 5Ј-RCGTG-3Ј) within the promoter regions of target genes. Multiple studies of HIF-1␣ and breast cancer have shown a significant association between HIF-1␣ overexpression and poor prognosis coupled to increased patient mortality (2-6). The levels of HIF-1␣ in human primary breast tumors increased with the progression of the pathologic stage (7). In a large retrospective study of 745 patients with high levels of HIF-1␣ at diagnosis, early relapse and metastatic disease were predicted (5). HIF-1␣ expression is closely linked to an aggressive phenotype in breast cancer, and HIF-1␣ expression enhanced osteolytic bone metastasis of breast cancer (8, 9). After prolonged treatment hormone-sensitive breast tumors frequently become resistant to hormonal therapy, and it was hypothesized that hypoxia may promote estrogen-independent growth. Deletion of HIF-1␣ in the mammary epithelium resulted in delayed tumor onset and retarded tumor growth as well as decreased pulmonary metastasis (10). These results suggest that HIF-1␣ is a negative prognostic factor in breast cancer progression. The HIF-1 subunit is constitutively expressed, whereas expression of HIF-1␣ is regulated by oxygen tension. HIF-1␣ protein is not detected in cells under normoxic conditions (20 -22% O 2 ) and is rapidly induced by hypoxic conditions (1-2% O 2 ). However, in the invasive carcinoma cells, including breast, steady-state HIF-1␣ expression can be detected even under normoxia. The synthesis of HIF-1␣ protein has been shown to be regulated in an O 2 -independent fashion, for example, through activation of the receptor tyrosine kinase pathways (11,12). The molecular targets of HIF-1␣ that contribute to breast tumorigenesis are under active investigation.Macrophage-stimulating protein (MSP) is the only known ligand for recepteur d'origine nantais (RON), a tyrosine kinase receptor. MSP is an 80-kDa heterodimer consisting of a 53-kDa ␣-chain and a 30-kDa -chain linked by a disulfide bond. The -chain of MSP binds to RON (13). RON is initially synthesized as a single chain precursor, 170-kDa pro-RON, which is subsequently cleaved into 40-kDa alpha chain and 150-kDa beta chain. The alpha chain is completely extracellular, whereas the beta chain traverses the cell membrane and contains the intracellular tyrosine kinase (13). The RON receptor also participates in cross-talk with other receptor tyrosine kinases such as MET and epidermal growth factor receptor. Several human tumor tissues show increased RON expression, including tumors of the breast, colon, lung, liver, kidney, ovary, stomach, pancreas, bladder, and prostate (14). Gene expression analyses indicated increase in RON expression is associated with metastatic disease. Transgenic mice that ov...
Macrophage-stimulating protein (MSP)2 is the only known ligand for RON (recepteur d'origine nantais). MSP is an 80 kDa heterodimer consisting of a 53 kDa ␣ chain and a 30-kDa  chain linked by a disulfide bond. The  chain of MSP binds to RON. MSP belongs to the plasminogen-prothrombin gene family (1, 2). MSP gene knock-out in mice is not lethal, indicating that MSP is not required for embryonic development and growth (3). Besides macrophages, MSP is expressed in a variety of epithelial cells. RON is initially synthesized as a single chain precursor, 170-kDa pro-RON, which is subsequently cleaved into 40-kDa ␣ chain and 150-kDa  chain. The ␣ chain is completely extracellular, whereas the  chain traverses the cell membrane and contains the intracellular tyrosine kinase (1). The C-terminal of RON regulates its kinase activity (4). RON forms either homodimers or heterodimers with other receptors such as c-Met and epidermal growth factor receptor (5-7). In addition to macrophages, RON is also expressed in multiple epithelial cells both malignant and nonmalignant. Homozygous deletion of RON was embryonically lethal. However, RON heterozygous mice mature normally except for an inappropriate inflammatory response (8, 9). The RON protein is regulated through c-Cbl ubiquitin ligase binding to phosphorylated RON leading to endocytosis and the subsequent degradation of RON (10).Abnormal expression of RON was reported in various cancers of epithelial origin. However, fibroblasts do not express RON. RON is moderately expressed in normal colorectal mucosa but significantly elevated in a majority of primary human colorectal adenocarcinoma samples (11). RON protein accumulation was reported to induce autophosphorylation of RON tyrosine kinase receptor and transduces signals that regulate tumorigenic activities of colon cancer cells (12). In nonsmall cell lung cancer cell lines, RON overexpression was reported in a majority of the cell lines examined. In addition, these cell lines expressed high levels of MSP ligand (13). The combination of RON overexpression and activation by MSP leads to increased invasion and resistance to apoptosis. These tumors supported by either autocrine or paracrine effects may acquire a survival advantage because of increased activation of the RON receptor by the local secretion of MSP.Altered RON expression was noticed in bladder and ovarian cancers. RON expression was positively associated with tumor size, stage, and grade in bladder carcinomas (14). The majority of ovarian carcinoma samples showed up-regulation in RON expression with a mix of cytoplasmic and membrane staining (5). Co-expression of MSP with RON was observed in ovarian carcinomas, providing a selective growth advantage and subsequent tumor progression. RON overexpression and not mutations is associated with head and neck squamous cell carcinomas (15). Normal breast cells and benign lesions (adenomas and papillomas) express relatively low levels of RON. However, RON is highly expressed in tumor specimens (1). Further, increased RON e...
Recepteur d' origine nantais (RON)2 , a tyrosine kinase receptor, for macrophage-stimulating protein (MSP) was reported to be overexpressed in various cancers of epithelial origin (1, 2). Activation of the RON receptor in breast cancer cells is linked to tamoxifen resistance (3). Further, gene expression analyses suggested that increases in RON expression are associated with metastatic disease. Transgenic mice that overexpress a wild type or constitutively active RON receptor in the mammary epithelium induced mammary transformation and associated with a high degree of metastasis (4). Aberrant RON expression in human breast cancer is associated with an aggressive cancer phenotype with decreased disease-free survival time in patients and an increase in breast cancer metastasis (5). These studies demonstrated that RON overexpression can be a causative factor for metastatic breast cancer. Nrf2 (NF-E2-related factor 2) belongs to the cap n collar subfamily of basic leucine zipper family of transcription factors (6). Its role in chemoprevention as the inducer of several hundred cytoprotective genes, which contain one or more antioxidant response elements (ARE: 5Ј-TGACnnnGC-3Ј) is well documented (7). Nrf2-null mice are susceptible to carcinogen-induced cancer development (8 -12). Nrf2 activity is regulated by keap1 (kelch-like ECHassociated protein 1). Keap1 serves as an adapter protein for interaction of cul3-based E3-ubiquitin ligase complex with Nrf2 leading to continuous ubiquitination of Nrf2 and its proteasomal degradation (13). Activators of Nrf2 like sulforaphane (SFN), an isothiocyanate, have been shown to modify the keap1 protein leading to the dissociation of Nrf2-keap1 complex resulting in the escape of Nrf2 from proteasomal degradation (14). Nrf2 was reported to be depleted in breast cancer cell lines (15). The data presented in this report indicate depletion of Nrf2 is prevalent in breast tumors and is associated with overexpression of RON. We also report that Nrf2 binds a closely related cis-element (5Ј-TGA(C/G)TCA-3Ј) to ARE but displays a novel function as an inhibitor of oncogene RON and invasion of carcinoma cells. SFN induced Nrf2 and blocked RON expression in invasive carcinoma cells from various tumor types. Consequently, our studies identified a novel functional role for Nrf2 as a "repressor" of an oncogene and RON kinase as one of the potential molecular targets of SFN, which mediates the antitumor effects of SFN. EXPERIMENTAL PROCEDURESCell Culture-Cell lines were obtained from American Type Culture Collection (ATCC). Sulforaphane was purchased from LKT Laboratories.Immunohistochemical Analysis-Breast cancer metastasis tumor microarray (60 samples) was purchased from IMGENEX (catalogue number: IMH-364). Immunohistochemical analysis using RON and Nrf2 antibodies was performed by our institution pathology core facility.Generation of Stable Cell Lines-Scramble shRNA and shRNA keap1 and Nrf2 expression plasmids were purchased from Oregene. We have generated keap1 knock-down clones for characteriza...
We developed a transgenic mouse line that expresses the codon-optimized Flp recombinase under the control of the MMTV promoter in luminal epithelial cells of the mammary gland. In this report, we demonstrate the versatile applicability of the new MMTV-Flp strain to manipulate genes in a temporally and spatially controlled manner in the normal mammary gland, in luminal-type mammary tumors that overexpress ERBB2, and in a new KRAS-associated mammary cancer model. Although the MMTV-Flp is expressed in a mosaic pattern in the luminal epithelium, the Flp-mediated activation of a mutant KrasG12D allele resulted in basal-like mammary tumors that progressively acquired mesenchymal features. Besides its applicability as a tool for gene activation and cell lineage tracing to validate the cellular origin of primary and metastatic tumor cells, we employed the MMTV-Flp transgene together with the tamoxifen-inducible Cre recombinase to demonstrate that the combinatorial action of both recombinases can be used to delete or to activate genes in established tumors. In a proof-of-principle experiment, we conditionally deleted the JAK1 tyrosine kinase in KRAS-transformed mammary cancer cells using the dual recombinase approach and found that lack of JAK1 was sufficient to block the constitutive activation of STAT3. The collective results from the various lines of investigation showed that it is, in principle, feasible to manipulate genes in a ligand-controlled manner in neoplastic mammary epithelial cells, even when cancer cells acquire a state of cellular plasticity that may no longer support the expression of the MMTV-Flp transgene.
The Cancer Genome Atlas (TCGA) of a pancreatic cancer cohort identified high MST1R (RON tyrosine kinase receptor) expression correlated with poor prognosis in human pancreatic cancer. RON expression is null/minimal in normal pancreas but elevates from pan‐in lesions through invasive carcinomas. We report using multiple approaches RON directly regulates HIF‐1α, a critical driver of genes involved in cancer cell invasion and metastasis. RON and HIF‐1α are highly co‐expressed in the 101 human PDAC tumors analyzed and RON expression correlated with HIF‐1α expression in a subset of PDAC cell lines. knockdown of RON expression in RON positive cells blocked HIF‐1α expression, whereas ectopic RON expression in RON null cells induced HIF‐1α expression suggesting the direct regulation of HIF‐1α by RON kinase receptor. RON regulates HIF‐1α through an unreported transcriptional mechanism involving PI3 kinase‐mediated AKT phosphorylation and Sp1‐dependent HIF‐1α promoter activity leading to increased HIF‐1α mRNA expression. RON/HIF‐1α modulation altered the invasive behavior of PDAC cells. A small‐molecule RON kinase inhibitor decreased RON ligand, MSP‐induced HIF‐1α expression, and invasion of PDAC cells. Immunohistochemical analysis on RON knockdown orthotopic PDAC tumor xenograft confirmed that RON inhibition significantly blocked HIF‐1α expression. RON/HIF‐1α co‐expression also exists in triple‐negative breast cancer cells, a tumor type that also lacks molecular therapeutic targets. This is the first report describing RON/HIF‐1α axis in any tumor type and is a potential novel therapeutic target.
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