New signaling pathways from cancer progression modulators to mRNA expression of matrix metalloproteinases in breast cancer cells

We observed previously that each of seven cancer progression inhibitors suppresses the mRNA expression of some matrix metalloproteinases (MMPs), but stimulates that of others, in breast cancer cells. In the present study we tested the effect of overexpressing other cancer modulators on MMP expression. The MMPs tested are MMP1, MMP2, MMP7, MMP13, MMP14, MMP16, MMP19, and MMP25. The proteins that were overexpressed are cancer inhibitors (NME, DRG1, IL10), enhancers (SOD2, FAK, IL17, and CREB), and proteins that suppress cancer progression in cells of some cancers and promote it in others (FUT1, integrin beta3, serpin E1, TIAM1, and claudin 4). Unexpectedly, all of them only lowered MMP mRNA expression, mainly of MMP16, MMP2, and MMP13, in breast cancer cells. Signaling from SOD2 uncoupled the accumulation of two MMP16 mRNA splice variants, suggesting signaling to a late step in MMP16 mRNA accumulation, such as MMP16 mRNA stabilization or late mRNA processing. Signaling that modulates MMP expression differed widely among the total population of MDA-MB-231 cells and single-cell progenies cloned from that population. It also differed substantially between cells of two metastatic breast basal adenocarcinomas, MDA-MB-231 and MDA-MB-468. The present study detected 37 new signaling pathways from cancer progression modulators located upstream of MMP mRNA expression in human breast cancer cells. Our siRNA-induced MMP knockdown data support the interpretation that signaling from MMP19, MMP1, MMP7, MMP12, MMP14, and MMP11 each stimulates the mRNA expression of other MMPs in breast cancer cells.     

Aβ1–42 oligomer‐induced leakage in an in vitro blood–brain barrier model is associated with up‐regulation of RAGE and metalloproteinases,and down‐regulation of tight junction scaffold proteins

Accumulating evidence indicates that abnormal deposition of amyloid‐β (Aβ) peptide in the brain is responsible for endothelial cell damage and consequently leads to blood–brain barrier (BBB) leakage. However, the mechanisms underlying BBB disruption are not well described. We employed an monolayer BBB model comprising bEnd.3 cell and found that BBB leakage was induced by treatment with Aβ_1–42, and the levels of tight junction (TJ) scaffold proteins (ZO‐1, Claudin‐5, and Occludin) were decreased. Through comparisons of the effects of the different components of Aβ_1–42, including monomer (Aβ_1–42‐Mono), oligomer (Aβ_1–42‐Oligo), and fibril (Aβ_1–42‐Fibril), our data confirmed that Aβ_1–42‐Oligo is likely to be the most important damage factor that results in TJ damage and BBB leakage in Alzheimer's disease. We found that the incubation of bEnd.3 cells with Aβ_1–42 significantly up‐regulated the level of receptor for advanced glycation end‐products (RAGE). Co‐incubation of a polyclonal antibody to RAGE and Aβ_1–42‐Oligo in bEnd.3 cells blocked RAGE suppression of Aβ_1–42‐Oligo‐induced alterations in TJ scaffold proteins and reversed Aβ_1–42‐Oligo‐induced up‐regulation of RAGE, matrix metalloproteinase (MMP)‐2, and MMP‐9. Furthermore, we found that these effects induced by Aβ_1–42‐Oligo treatment were effectively suppressed by knockdown of RAGE using small interfering RNA (siRNA) transfection. We also found that GM 6001, a broad‐spectrum MMP inhibitor, partially reversed the Aβ_1–42‐Oligo‐induced inhibitor effects in bEnd.3 cells. Thus, these results suggested that RAGE played an important role in Aβ‐induced BBB leakage and alterations of TJ scaffold proteins, through a mechanism that involved up‐regulation of MMP‐2 and MMP‐9.

     

TGF-beta induces formation of F-actin cores and matrix degradation in human breast cancer cells via distinct signaling pathways

Transforming growth factor beta regulates many biological processes including cell motility and invasion. Podosomes are specialized F-actin rich structures found in normal cells, such as osteoclasts and macrophages. Tumor cells often form related structures called invadopodia that are thought to promote invasion and metastasis. Here we show that human breast cancer cells organize F-actin rich structures in response to transforming growth factor beta that colocalize with areas of extracellular matrix degradation. We further show that organizing the complex of proteins needed to form these structures requires signaling through phosphatidylinositide 3-kinase and Src kinase, while activating the proteases involved in degradation of extracellular matrix requires extracellular signal-regulated kinase signaling, and that each of these pathways is activated by transforming growth factor beta in CA1D human breast cancer cells.     

Proanthocyanidins from the American Cranberry (Vaccinium macrocarpon) inhibit matrix metalloproteinase‐2 and matrix metalloproteinase‐9 activity in human prostate cancer cells via alterations in multiple cellular signalling pathways

Prostate cancer is one of the most common cancers in the Western world, and it is believed that an individual's diet affects his risk of developing cancer. There has been an interest in examining phytochemicals, the secondary metabolites of plants, in order to determine their potential anti‐cancer activities in vitro and in vivo. In this study we document the effects of proanthocyanidins (PACs) from the American Cranberry (Vaccinium macrocarpon) on matrix metalloproteinase (MMP) activity in DU145 human prostate cancer cells. Cranberry PACs decreased cellular viability of DU145 cells at a concentration of 25 µg/ml by 30% after 6 h of treatment. Treatment of DU145 cells with PACs resulted in an inhibition of both MMPs 2 and 9 activity. PACs increased the expression of TIMP‐2, a known inhibitor of MMP activity, and decreased the expression of EMMPRIN, an inducer of MMP expression. PACs decreased the expression of PI‐3 kinase and AKT proteins, and increased the phosphorylation of both p38 and ERK1/2. Cranberry PACs also decreased the translocation of the NF‐κB p65 protein to the nucleus. Cranberry PACs increased c‐jun and decreased c‐fos protein levels. These results suggest that cranberry PACs decreases MMP activity through the induction and/or inhibition of specific temporal MMP regulators, and by affecting either the phosphorylation status and/or expression of MAP kinase, PI‐3 kinase, NF‐κB and AP‐1 pathway proteins. This study further demonstrates that cranberry PACs are a strong candidate for further research as novel anti‐cancer agents. J. Cell. Biochem. 111: 742–754, 2010. © 2010 Wiley‐Liss, Inc.     

The role of microRNAs regulating the expression of matrix metalloproteinases (MMPs) in breast cancer development,progression, and metastasis

MicroRNAs (miRNAs) regulate several biological and physiological processes in mammalian cells, including cellular proliferation, differentiation, apoptosis, and metabolism. Recent studies have confirmed the alteration of them during the cancer development. Matrix metalloproteinases (MMPs), belonging to the large family of proteases, have also been demonstrated to play crucial roles in tissue remodeling, and to support cancer progression and metastasis. There are several known miRNAs which regulate the MMP family and their expression. The expression profiles of miRNAs involved in MMP regulation, change during cancer progression, and metastasis. The present review focuses on important miRNAs capable of targeting MMPs through direct and indirect interactions during the breast cancer development, progression, and metastasis.     

Apoptotic and autophagic pathways with relevant small‐molecule compounds,in cancer stem cells

Accumulating evidence demonstrates existence of cancer stem cells (CSCs), which are suspected of contributing to cancer cell self‐renewal capacity and resistance to radiation and/or chemotherapy. Including evasion of apoptosis and autophagic cell death, CSCs have revealed abilities to resist cell death, making them appealing targets for cancer therapy. Recently, molecular mechanisms of apoptosis and of autophagy in CSCs have been gradually explored, comparing them in stem cells and in cancer cells; distinct expression of these systems in CSCs may elucidate how these cells exert their capacity of unlimited self‐renewal and hierarchical differentiation. Due to their proposed ability to drive tumour initiation and progression, CSCs may be considered to be potentially useful pharmacological targets. Further, multiple compounds have been verified as triggering apoptosis and/or autophagy, suppressing tumour growth, thus providing new strategies for cancer therapy. In this review, we summarized regulation of apoptosis and autophagy in CSCs to elucidate how key proteins participate in control of survival and death; in addition, currently well‐studied compounds that target CSC apoptosis and autophagy are selectively presented. With increasing attention to CSCs in cancer therapy, researchers are now trying to find responses to unsolved questions as unambiguous as possible, which may provide novel insight into future anti‐cancer regimes.     

Cyclooxygenase‐2 up‐regulates CCR7 expression via AKT‐mediated phosphorylation and activation of Sp1 in breast cancer cells

Up‐regulation of cyclooxygenase‐2 (COX‐2) is frequently found in human cancers and is significantly associated with tumor metastasis. Our previous results demonstrate that COX‐2 and its metabolite prostaglandin E2 (PGE2) stimulate the expression of CCR7 chemokine receptor via EP2/EP4 receptors to promote lymphatic invasion in breast cancer cells. In this study, we address the underlying mechanism of COX‐2/PGE2‐induced CCR7 expression. We find that COX‐2/PGE2 increase CCR7 expression via the AKT signaling pathway in breast cancer cells. Promoter deletion and mutation assays identify the Sp1 site located at the −60/−57 region of CCR7 gene promoter is critical for stimulation. Chromatin immunoprecipitation (ChIP) assay confirms that in vivo binding of Sp1 to human CCR7 promoter is increased by COX‐2 and PGE2. Knockdown of Sp1 by shRNA reduces the induction of CCR7 by PGE2. We demonstrate for the first time that AKT may directly phosphorylate Sp1 at S42, T679, and S698. Phosphorylation‐mimic Sp1 protein harboring S42D, T679D, and S698D mutation strongly activates CCR7 expression. In contrast, change of these three residues to alanine completely blocks the induction of CCR7 by PGE2. Pathological investigation demonstrates that CCR7 expression is strongly associated with phospho‐AKT and Sp1 in 120 breast cancer tissues. Collectively, our results demonstrate that COX‐2 up‐regulates CCR7 expression via AKT‐mediated phosphorylation and activation of Sp1 and this pathway is highly activated in metastatic breast cancer. J. Cell. Physiol. 228: 341–348, 2013. © 2012 Wiley Periodicals, Inc.     

Overexpression of Par‐4 sensitizes TRAIL‐induced apoptosis via inactivation of NF‐κB and Akt signaling pathways in renal cancer cells

The prostate‐apoptosis‐response‐gene‐4 (Par‐4) is up‐regulated in prostate cells undergoing programmed cell death. Furthermore, Par‐4 protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor‐mediated cell death pathways. In this study, we investigated how Par‐4 modulates TRAIL‐mediated apoptosis in TRAIL‐resistant Caki cells. Par‐4 overexpressing cells were strikingly sensitive to apoptosis induced by TRAIL compared with control cells. Par‐4 overexpressing Caki cells treated with TRAIL showed an increased activation of the initiator caspase‐8 and the effector caspase‐3, together with an enforced cleavage of XIAP and c‐FLIP. TRAIL‐induced reduction of XIAP and c‐FLIP protein levels in Par‐4 overexpressing cells was prevented by z‐VAD pretreatment. In addition, the surface DR5 protein level was increased in TRAIL‐treated Par‐4 overexpressing cells. Interestingly, even though a deletion of leucine zipper domain in Par‐4 recovered Bcl‐2 level to basal level induced by wild type Par‐4, it partly decreased sensitivity to TRAIL in Caki cells. In addition, exposure of Caki/Par‐4 cells to TRAIL led to reduction of phosphorylated Akt levels, but deletion of leucine zipper domain of Par‐4 did not affect these phosphorylated Akt levels. In conclusion, we here provide evidence that ectopic expression of Par‐4 sensitizes Caki cells to TRAIL via modulation of multiple targets, including DR5, Bcl‐2, Akt, and NF‐κB. J. Cell. Biochem. 109: 885–895, 2010. © 2010 Wiley‐Liss, Inc.     

Bone sialoprotein stimulates focal adhesion‐related signaling pathways: Role in migration and survival of breast and prostate cancer cells

Bone sialoprotein (BSP) is a secreted glycoprotein found in mineralized tissues however, BSP is aberrantly expressed in a variety of osteotropic tumors. Elevated BSP expression in breast and prostate primary carcinomas is directly correlated with increased bone metastases and tumor progression. In this study, the intracellular signaling pathways responsible for BSP‐induced migration and tumor survival were examined in breast and prostate cancer cells (MDA‐MB‐231, Hs578T and PC3). Additionally, the effects of exogenous TGF‐β1 and EGF, cytokines associated with tumor metastasis and present in high‐levels in the bone microenvironment, were examined in BSP‐expressing cancer cells. Expression of BSP but not an integrin‐binding mutant (BSP‐KAE) in tumor cell lines resulted in increased levels of α_v‐containing integrins and number of mature focal adhesions. Adhesion of cells to recombinant BSP or the expression of BSP stimulated focal adhesion kinase and ERK phosphorylation, as well as activated AP‐1‐family proteins. Activation of these pathways by BSP expression increased the expression of the matrix metalloproteinases MMP‐2, MMP‐9, and MMP‐14. The BSP‐mediated activation of the FAK‐associated pathway resulted in increased cancer cell invasion in a Matrigel‐coated Boyden‐chamber assay and increased cell survival upon withdrawal of serum. Addition of EGF or TGF‐β1 to the BSP‐expressing cell lines significantly increased ERK phosphorylation, AP‐1 activation, MMP‐2 expression, cell migration and survival compared to untreated cells expressing BSP. This study thus defines the cooperative mechanisms by which BSP can enhance specific factors associated with a metastatic phenotype in tumor cell lines, an effect that is increased by circulating TGF‐β1 and EGF. J. Cell. Biochem. 107: 1118–1128, 2009. © 2009 Wiley‐Liss, Inc.     

Nuclear matrix protein,prohibitin, was down‐regulated and translocated from nucleus to cytoplasm during the differentiation of osteosarcoma MG‐63 cells induced by ginsenoside Rg1, cinnamic acid,and tanshinone IIA (RCT)

Ginsenoside Rg1, cinnamic acid, and tanshinone IIA (RCT) are effective anticancer and antioxidant constituents of traditional Chinese herbal medicines of Ginseng, Xuanseng, and Danseng. The molecular mechanisms of anticancer effects of those constituents and their targets are unknown. Prohibitin, an inner membrane‐bound chaperone in mitochondrion involved in the regulation of cell growth, proliferation, differentiation, aging, and apoptosis, was chosen as a candidate molecular target because of its frequent up‐regulation in various cancer cells. We demonstrated that prohibitin existed in the filaments of the nuclear matrix of the MG‐63 cell and its expression was down‐regulated by the treatment of RCT using proteomic methodologies and Western blot analysis. Immunogold electro‐microscopy also found that prohibitin was localized on nuclear matrix intermediate filaments (NM‐IF) that had undergone restorational changes after RCT treatment. Prohibitin may function as a molecular chaperone that might interact with multiple oncogenes and tumor suppressor genes. We found that oncogenes c‐myc and c‐fos and tumor suppressor genes P53 and Rb were regulated by RCT as well and that these gene products co‐localized with prohibitin. Our study identified prohibitin as a molecular target of the effective anticancer constituents of Ginseng, Xuanseng, and Danseng that down‐regulated prohibitin in nuclear matrix, changed prohibtin trafficking from nucleolus to cytoplasm, and regulated several oncogenes and tumor suppressor genes. Prohibitin downregulation and cellular trafficking from nucleolus to cytoplasm indicated RCT protective roles in cancer prevention and treatment. J. Cell. Biochem. 108: 926–934, 2009. © 2009 Wiley‐Liss, Inc.     

INPP1 up‐regulation by miR‐27a contributes to the growth,migration and invasion of human cervical cancer

Inositol polyphosphate‐1‐phosphatase (INPP1) is an enzyme that is responsible for glycolysis and lipid metabolism. Here, we discovered that INPP1 expression was up‐regulated in CC tissues compared to that in adjacent normal tissues by RT‐qPCR. Inositol polyphosphate‐1‐phosphatase overexpression promoted and INPP1 knockdown suppressed cell viability, cellular migration/invasion and EMT in CC cells. To explore the mechanism of dysregulation, INPP1 was predicted to be a target of miR‐27a, and a pmiRGLO dual‐luciferase reporter assay showed that miR‐27a bound to the 3′ UTR of INPP1. RT‐qPCR revealed that miR‐27a was also up‐regulated and had a positive correlation with INPP1 expression in CC tissues. Furthermore, shR‐INPP1 could favour the malignant phenotype reversion induced by miR‐27a, suggesting that miR‐27a up‐regulates INPP1 to promote tumorigenic activities. Altogether, our findings show that the up‐regulation of INPP1 by miR‐27a contributes to tumorigenic activities and may provide a potential biomarker for CC.