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1.
Am J Pathol ; 183(3): 831-40, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23830872

ABSTRACT

Integrin α7 (ITGA7) is a tumor-suppressor gene that is critical for suppressing the growth of malignant tumors; however, the mechanisms allowing ITGA7 to suppress the growth of cancer cells remain unclear. Herein, we show that ITGA7 binds to tissue inhibitor of metalloproteinase 3 (TIMP3) in prostate cancer cells. The ITGA7-TIMP3 binding led to a decreased protein level of tumor necrosis factor α, cytoplasmic translocation of NF-κB, and down-regulation of cyclin D1. These changes led to an accumulation of cells in G0/G1 and a dramatic suppression of cell growth. Knocking down TIMP3 or ITGA7/TIMP3 binding interference largely abrogated the signaling changes induced by ITGA7, whereas a mutant ITGA7 lacking TIMP3 binding activity had no tumor-suppressor activity. Interestingly, knocking down ITGA7 ligand laminin ß1 enhanced ITGA7-TIMP3 signaling and the downstream tumor-suppressor activity, suggesting the existence of a counterbalancing role between extracellular matrix and integrin signaling. As a result, this report demonstrates a novel and critical signaling mechanism of ITGA7, through the TIMP3/NF-κB/cyclin D1 pathway.


Subject(s)
Antigens, CD/metabolism , Integrin alpha Chains/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Tissue Inhibitor of Metalloproteinase-3/metabolism , Amino Acid Motifs , Cell Line, Tumor , Cell Proliferation , Cyclin D1/metabolism , Down-Regulation , Humans , Laminin/metabolism , Male , Protein Binding , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/metabolism
2.
Am J Pathol ; 183(1): 153-9, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23665349

ABSTRACT

Previous studies from our laboratory have found glypican 3 (GPC3) as a negative regulator of growth. CD81 was found to be a binding partner for GPC3, and its expression and co-localization with GPC3 increased at the end of hepatocyte proliferation. However, the mechanisms through which these two molecules might regulate liver regeneration are not known. We tested the hypothesis that GPC3 down-regulates the hedgehog (HH) signaling pathway by competing with patched-1 for HH binding. We found decreased GPC3-Indian HH binding at peak proliferation in mice followed by increase in glioblastoma 1 protein (effector of HH signaling). We performed a yeast two-hybrid assay and identified hematopoietically expressed homeobox (Hhex, a known transcriptional repressor) as a binding partner for CD81. We tested the hypothesis that Hhex binding to CD81 keeps it outside the nucleus. However, when GPC3 binds to CD81, CD81-Hhex binding decreases, resulting in nuclear translocation of Hhex and transcriptional repression. In support of this, we found decreased GPC3-CD81 binding at hepatocyte proliferation peak, increased CD81-Hhex binding, and decreased nuclear Hhex. GPC3 transgenic mice were used as an additional tool to test our hypothesis. Overall, our data suggest that GPC3 down-regulates cell proliferation by binding to HH and down-regulating the HH signaling pathway and binding with CD81, thus making it unavailable to bind to Hhex and causing its nuclear translocation.


Subject(s)
Cell Proliferation , Glypicans/metabolism , Hedgehog Proteins/metabolism , Hepatocytes/metabolism , Homeodomain Proteins/metabolism , Liver Regeneration/physiology , Tetraspanin 28/metabolism , Transcription Factors/metabolism , Animals , Biomarkers/metabolism , Blotting, Western , Down-Regulation , Hepatocytes/physiology , Immunohistochemistry , Male , Mice , Mice, Transgenic
3.
Acta Crystallogr Sect E Struct Rep Online ; 68(Pt 10): o2828, 2012 Oct 01.
Article in English | MEDLINE | ID: mdl-23125634

ABSTRACT

In the title compound, C(16)H(16)N(4)O(3)·CH(3)OH, the aromatic rings form a dihedral angle of 0.4 (2)°. The nitro group is twisted from the attached benzene ring by 7.5 (2)°. In the crystal, N-H⋯O and O-H⋯O hydrogen bonds link alternating hydrazone and methanol mol-ecules into chains in [100]. The crystal packing exhibits π-π inter-actions between aromatic rings from neighbouring chains [centroid-centroid distances = 3.734 (3) and 3.903 (3) Å].

4.
Am J Pathol ; 181(2): 463-71, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22683311

ABSTRACT

Cellular Stress Response 1 (CSR1) is a tumor suppressor gene that is located at 8p21, a region that is frequently deleted in prostate cancer as well as a variety of human malignancies. Previous studies have indicated that the expression of CSR1 induces cell death. In this study, we found that CSR1 interacts with X-linked Inhibitor of Apoptosis Protein (XIAP), using yeast two-hybrid screening analyses. XIAP overexpression has been found in many human cancers, and forced expression of XIAP blocks apoptosis. Both in vitro and in vivo analyses indicated that the C-terminus of CSR1 binds XIAP with high affinity. Through a series of in vitro recombinant protein-binding analyses, the XIAP-binding motif in CSR1 was determined to include amino acids 513 to 572. Targeted knock-down of XIAP enhanced CSR1-induced cell death, while overexpression of XIAP antagonized CSR1 activity. The binding of CSR1 with XIAP enhanced caspase-9 and caspase-3 protease activities, and CSR1-induced cell death was dramatically reduced on expression of a mutant CSR1 that does not bind XIAP. However, a XIAP mutant that does not interact with caspase-9 had no impact on CSR1-induced cell death. These results suggest that cell death is induced when CSR1 binds XIAP, preventing the interaction of XIAP with caspases. Thus, this study may have elucidated a novel mechanism by which tumor suppressors induce cell death.


Subject(s)
Caspase 3/metabolism , Caspase 9/metabolism , Heat-Shock Proteins/metabolism , Scavenger Receptors, Class A/metabolism , X-Linked Inhibitor of Apoptosis Protein/metabolism , Amino Acid Motifs , Cell Death/radiation effects , Cell Line, Tumor , Enzyme Activation/radiation effects , Gene Knockdown Techniques , Heat-Shock Proteins/chemistry , Humans , Male , Protein Binding/radiation effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Scavenger Receptors, Class A/chemistry , Ultraviolet Rays
5.
J Biol Chem ; 287(20): 16890-902, 2012 May 11.
Article in English | MEDLINE | ID: mdl-22461624

ABSTRACT

Expression of glutathione peroxidase 3 (GPx3) is down-regulated in a variety of human malignancies. Both methylation and deletion of GPx3 gene underlie the alterations of GPx3 expression in prostate cancer. A strong correlation between the down-regulation of GPx3 expression and progression of prostate cancer and the suppression of prostate cancer xenografts in SCID mice by forced expression of GPx3 suggests a tumor suppression role of GPx3 in prostate cancer. However, the mechanism of GPx3-mediated tumor suppression remains unclear. In this report, GPx3 was found to interact directly with p53-induced gene 3 (PIG3). Forced overexpression of GPx3 in prostate cancer cell lines DU145 and PC3 as well as immortalized prostate epithelial cells RWPE-1 increased apoptotic cell death. Expression of GPx3(x73c), a peroxidase-negative OPAL codon mutant, in DU145 and PC3 cells also increased cell death. The induced expression of GPx3 in DU145 and PC3 cells resulted in an increase in reactive oxygen species and caspase-3 activity. These activities were abrogated by either knocking down PIG3 or mutating the PIG3 binding motif in GPx3 or binding interference from a peptide corresponding to PIG3 binding motif in GPx3. In addition, UV-treated RWPE-1 cells underwent apoptotic death, which was partially prevented by knocking down GPx3 or PIG3, suggesting that GPx3-PIG3 signaling is critical for UV-induced apoptosis. Taken together, these results reveal a novel signaling pathway of GPx3-PIG3 in the regulation of cell death in prostate cancer.


Subject(s)
Apoptosis , Glutathione Peroxidase/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins/metabolism , Signal Transduction , Amino Acid Motifs , Animals , Cell Line, Tumor , Gene Knockdown Techniques , Glutathione Peroxidase/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Mice , Mice, SCID , Neoplasm Transplantation , Prostatic Neoplasms/genetics , Protein Binding/genetics , Protein Binding/radiation effects , Proto-Oncogene Proteins/genetics , Transplantation, Heterologous , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ultraviolet Rays
6.
J Biol Chem ; 283(33): 22709-22, 2008 Aug 15.
Article in English | MEDLINE | ID: mdl-18482983

ABSTRACT

The ubiquitously expressed Src tyrosine kinases (c-Src, c-Yes, and c-Fyn) regulate intestinal cell growth and differentiation. Src activity is also elevated in the majority of malignant and premalignant tumors of the colon. The development of fibroblasts with the three ubiquitously expressed kinases deleted (SYF cells) has identified the role of Src proteins in the regulation of actin dynamics associated with increased cell migration and invasion. Despite this, unexpectedly nothing is known about the role of the individual Src kinases on intestinal cell cytoskeleton and/or cell migration. We have previously reported that villin, an epithelial cell-specific actin-modifying protein that regulates actin reorganization, cell morphology, cell migration, cell invasion, and apoptosis, is tyrosine-phosphorylated. In this report using the SYF cells reconstituted individually with c-Src, c-Yes, c-Fyn, and wild type or phosphorylation site mutants of villin, we demonstrate for the first time the absolute requirement for c-Src in villin-induced regulation of cell migration. The other major finding of our study is that contrary to previous reports, the nonreceptor tyrosine kinase, Jak3 (Janus kinase 3), does not regulate phosphorylation of villin or villin-induced cell migration and is, in fact, not expressed in intestinal epithelial cells. Further, we identify SHP-2 and PTP-PEST (protein-tyrosine phosphatase proline-, glutamate-, serine-, and threonine-rich sequence) as negative regulators of c-Src kinase and demonstrate a new function for these phosphatases in intestinal cell migration. Together, these data suggest that in colorectal carcinogenesis, elevation of c-Src or down-regulation of SHP-2 and/or PTP-PEST may promote cancer metastases and invasion by regulating villin-induced cell migration and cell invasion.


Subject(s)
Cell Movement/physiology , Protein-Tyrosine Kinases/metabolism , CSK Tyrosine-Protein Kinase , Caco-2 Cells/physiology , Cell Line, Tumor , Cytoskeletal Proteins/metabolism , HeLa Cells , Humans , Janus Kinase 3/metabolism , Neoplasm Invasiveness , Phosphorylation , src-Family Kinases
7.
Clin Cancer Res ; 8(2): 607-14, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11839683

ABSTRACT

It has been shown that expression of the RIalpha subunit of cyclic AMP (cAMP)-dependent protein kinase is enhanced in human cancer cell lines, primary tumors, and cells after transformation. Using an antisense strategy, we have shown that RIalpha has a role in neoplastic cell growth in vitro and in vivo. In the present study, we have investigated the sequence- and target-specific effects of exogenous RIalpha antisense oligodeoxynucleotides (ODNs) and endogenous antisense gene on tumor growth, apoptosis, and cAMP signaling in androgen-insensitive prostate cancer cells, both in vitro and in nude mice. Here, we show that an RIalpha antisense, RNA/DNA mixed backbone ODN exerts a reduction in RIalpha expression at both the mRNA and protein levels, up-regulation of both the RIIbeta subunit of cAMP-dependent protein kinase or protein kinase A and c-AMP-phosphodiesterase IV expression, and inhibition of cell growth. Growth inhibition was accompanied by changes in cell morphology and the appearance of apoptotic nuclei. In addition, Bcl-2 hyperphosphorylation; increase in the proapoptotic proteins Bax, Bak, and Bad; and Bad hypophosphorylation occurred in the antisense-treated cells. These effects of exogenously supplied antisense ODN mirrored those induced by endogenous antisense gene overexpression. The RIalpha antisense ODNs, which differed in sequence or chemical modification, promoted a sequence- and target-specific reduction in RIalpha protein levels and inhibited tumor growth in nude mice. These results demonstrate that in a sequence-specific manner, RIalpha antisense, via efficient depletion of the growth stimulatory molecule RIalpha, induces growth inhibition, apoptosis, and phenotypic (cell morphology) changes, providing an innovative approach to combat hormone-insensitive prostate cancer cell growth.


Subject(s)
Carrier Proteins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Oligonucleotides, Antisense/pharmacology , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins/metabolism , Up-Regulation , Apoptosis , Blotting, Northern , Blotting, Western , Cell Nucleus/metabolism , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit , Cyclic AMP-Dependent Protein Kinases/pharmacology , Down-Regulation , Humans , Male , Phosphorylation , Signal Transduction , Time Factors , Tumor Cells, Cultured , bcl-2-Associated X Protein , bcl-Associated Death Protein
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