Complement C1q Activates Tumor Suppressor WWOX to Induce Apoptosis in Prostate Cancer Cells

Background

Tissue exudates contain low levels of serum complement proteins, and their regulatory effects on prostate cancer progression are largely unknown. We examined specific serum complement components in coordinating the activation of tumor suppressors p53 and WWOX (also named FOR or WOX1) and kinases ERK, JNK1 and STAT3 in human prostate DU145 cells.

Methodology/Principal Findings

DU145 cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated, and hyaluronan (HA) reversed the effect. Under C6-free conditions, HA induced activation of STAT3, an enhancer of metastasis. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) microscopy, it was determined that C1q destabilized adherence of WOX1-expressing DU145 cells by partial detaching and inducing formation of clustered microvilli for focal adhesion particularly in between cells. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues.

Conclusions/Significance

We conclude that complement C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation.     

Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is overexpressed in prostate cancer, but the mechanism by which MIF exerts effects on tumor cells remains undetermined. MIF interacts with its identified membrane receptor, CD74, in association with CD44, resulting in ERK 1/2 activation. Therefore, we hypothesized that increased expression or surface localization of CD74 and MIF overexpression by prostate cancer cells regulated tumor cell viability. Prostate cancer cell lines (LNCaP and DU-145) had increased MIF gene expression and protein levels compared with normal human prostate or benign prostate epithelial cells (p < 0.01). Although MIF, CD74, and CD44 variant 9 expression were increased in both androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells, cell surface of CD74 was only detected in androgen-independent (DU-145) prostate cancer cells. Therefore, treatments aimed at blocking CD74 and/or MIF (e.g., inhibition of MIF or CD74 expression by RNA interference or treatment with anti-MIF- or anti-CD74- neutralizing Abs or MIF-specific inhibitor, ISO-1) were only effective in androgen-independent prostate cancer cells (DU-145), resulting in decreased cell proliferation, MIF protein secretion, and invasion. In DU-145 xenografts, ISO-1 significantly decreased tumor volume and tumor angiogenesis. Our results showed greater cell surface CD74 in DU-145 prostate cancer cells that bind to MIF and, thus, mediate MIF-activated signal transduction. DU-145 prostate cancer cell growth and invasion required MIF activated signal transduction pathways that were not necessary for growth or viability of androgen-dependent prostate cells. Thus, blocking MIF either at the ligand (MIF) or receptor (CD74) may provide new, targeted specific therapies for androgen-independent prostate cancer.     

前列腺癌组织及PC-3细胞中STAT3及磷酸化STAT3的表达

目的　研究前列腺癌组织及前列腺癌细胞株PC- 3 中STAT3 蛋白及磷酸化STAT3 蛋白的表达。方法　常规石蜡包埋切片SABC免疫组化法检测45例前列腺癌组织、20例前列腺增生组织中STAT3 及磷酸化STAT3 表达, 细胞免疫化学法检测前列腺癌细胞株PC 3细胞STAT3及磷酸化STAT3表达。结果　STAT3在前列腺癌及前列腺增生组织表达阳性率分别为77. 8%和50. 0%, 两者间具有显著差异; 磷酸化STAT3在前列腺癌及前列腺增生组织表达阳性率分别为68. 9%和35. 0%, 两者间具显著差异(P<0 .05); PC- 3细胞中STAT3及磷酸化STAT3表达阳性。结论　STAT3蛋白在前列腺癌中高表达且持续激活, 可能与前列腺癌的发生具有密切联系。     

Production of a mouse-human chimeric monoclonal antibody to CD20 with potent Fc-dependent biologic activity

Mouse monoclonal antibody 2H7 recognizes the CD20 cell surface phosphoprotein that is expressed in normal as well as malignant B cells. CD20 may be a useful target for therapy of B cell lymphomas, since damaged normal B cells can be replaced by their antigen-negative precursors. Monoclonal antibody 2H7 is an IgG2b (kappa) immunoglobulin which cannot mediate antibody-dependent cellular cytotoxicity with human lymphocytes or complement-dependent cytotoxicity with human serum. We have now generated a chimeric 2H7 antibody by substituting the mouse constant domains of 2H7 with the human gamma 1 and kappa domains. This new antibody has the same binding specificities as 2H7 but is highly effective in mediating antibody-dependent cellular cytotoxicity with human effector cells and complement-dependent cytotoxicity with human complement.     

Galiellalactone inhibits stem cell-like ALDH-positive prostate cancer cells

Galiellalactone is a potent and specific inhibitor of STAT3 signaling which has been shown to possess growth inhibitory effects on prostate cancer cells expressing active STAT3. In this study we aimed to investigate the effect of galiellalactone on prostate cancer stem cell-like cells. We explored the expression of aldehyde dehydrogenase (ALDH) as a marker for cancer stem cell-like cells in different human prostate cancer cell lines and the effects of galiellalactone on ALDH expressing (ALDH+) prostate cancer cells. ALDH+ subpopulations were detected and isolated from the human prostate cancer cell lines DU145 and long-term IL-6 stimulated LNCaP cells using ALDEFLUOR® assay and flow cytometry. In contrast to ALDH− cells, ALDH+ prostate cancer cells showed cancer stem cell-like characteristics such as increased self-renewing and colony forming capacity and tumorigenicity. In addition, ALDH+ cells showed an increased expression of putative prostate cancer stem cell markers (CD44 and integrin α2β1). Furthermore, ALDH+ cells expressed phosphorylated STAT3. Galiellalactone treatment decreased the proportion of ALDH+ prostate cancer cells and induced apoptosis of ALDH+ cells. The gene expression of ALDH1A1 was downregulated in vivo in galiellalactone treated DU145 xenografts. These findings emphasize that targeting the STAT3 pathway in prostate cancer cells, including prostate cancer stem cell-like cells, is a promising therapeutic approach and that galiellalactone is an interesting compound for the development of future prostate cancer drugs.     

Serpin B3/B4, activated by STAT3, promote survival of squamous carcinoma cells

Persistently activated STAT3 contributes to cell survival in many different human cancers. Cancer cell secretion of IL-6 is a frequent basis for persistent STAT3 activation; we show that antibodies against IL-6 or gp-130, the signaling unit of the IL-6 receptor, can abruptly remove persistently activated STAT3 causing prompt disappearance of cysteine proteases of serpin B3/B4 mRNAs, known as squamous cell carcinoma antigens 1 and 2. STAT3 occupies the promoter of serpin B3/B4 before removal and siRNA removal of B3/B4 mRNA caused cell death in HN13 head and neck cancer cells. Thus persistently activated STAT3 is a required part of the continuous activation of B3/B4 genes, which protects tumor cells from dying.     

HBXIP upregulates CD46, CD55 and CD59 through ERK1/2/NF-κB signaling to protect breast cancer cells from complement attack

Hepatitis B X-interacting protein (HBXIP) is able to enhance migration of breast cancer cells. However, the role of HBXIP in regulation of complement-dependent cytotoxicity (CDC) in breast cancer is not understood. Here, we report that HBXIP contributes to protecting breast cancer cells from CDC by upregulating membrane-bound complement regulatory protein (mCRPs), including CD46, CD55 and CD59. We found that HBXIP upregulated mCRPs through activating p-ERK1/2/NF-κB. Interestingly, the knockdown of CD59 was able to block the HBXIP-enhanced breast tumor growth in animal. Thus, we conclude that HBXIP upregulates CD46, CD55 and CD59 through p-ERK1/2/NF-κB signaling to protect breast cancer from CDC.     

A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell–expressed antigen.     

Enhancer role of STAT5 in CD2 activation of IFN-gamma gene expression

IFN-gamma is an important immunoregulatory protein with tightly controlled expression in activated T and NK cells. Three potential STAT binding regions have been recognized within the IFN-gamma promoter: 1) an IL-12-mediated STAT4 binding site at -236 bp; 2) a newly identified IL-2-induced STAT5 binding element at -3.6 kb; and 3) CD2-mediated STAT1 and STAT4 binding to an intronic element in mucosal T cells. However, functional activation of these sites remains unclear. In this study we demonstrate CD2-mediated activation of the newly characterized -3.6-kb IFN-gamma STAT5 binding region. CD2 signaling of human PBMC results in activation of the -3.6-kb IFN-gamma promoter, whereas mutation of the -3.6-kb STAT5 site attenuates promoter activity. Functional activation is accompanied by STAT5A but little STAT5B nucleoprotein binding to the IFN-gamma STAT5 site, as determined by competition and supershift assays. STAT5 activation via CD2 occurs independent of IL-2. Western and FACS analysis shows increased phospho-STAT5 following CD2 signaling. AG490, a tyrosine kinase inhibitor affecting Jak proteins, inhibits CD2-mediated IFN-gamma mRNA expression, secretion, and nucleoprotein binding to the IFN-gamma STAT5 site in a dose-dependent fashion. This report is the first to describe CD2-mediated activation of STAT5 and supports STAT5 involvement in regulation of IFN-gamma expression.     

Protein kinase Cdelta signaling downstream of the EGF receptor mediates migration and invasiveness of prostate cancer cells

Tumor progression to the invasive phenotype occurs secondary to upregulated signaling from growth factor receptors that drive key cellular responses like proliferation, migration, and invasion. We hypothesized that Protein kinase Cdelta (PKCdelta)-mediated transcellular contractility is required for migration and invasion of prostate tumor cells. Two invasive human prostate cancer cell lines, DU145 cells overexpressing wildtype human EGFR (DU145WT) and PC3 cells, were studied. PKCdelta is overexpressed in these cells relative to normal prostate epithelial cells, and is activated downstream of EGFR leading to cell motility via modulation of myosin light chain activity. Abrogation of PKCdelta using Rottlerin and specific siRNA significantly decreased migration and invasion of both cell lines in vitro. Both PKCdelta and phosphorylated PKCdelta protein levels were higher in human prostate cancer tissue relative to normal donor prostate as assessed by Western blotting and immunohistochemistry. Thus, we conclude that PKCdelta inhibition can limit migration and invasion of prostate cancer cells.     

p53 regulates the expression of the tumor suppressor gene maspin

Maspin has been shown to inhibit tumor cell invasion and metastasis in breast tumor cells. Maspin expression was detected in normal breast and prostate epithelial cells, whereas tumor cells exhibited reduced or no expression. However, the regulatory mechanism of maspin expression remains unknown. We report here a rapid and robust induction of maspin expression in prostate cancer cells (LNCaP, DU145, and PC3) and breast tumor cells (MCF7) following wild type p53 expression from an adenovirus p53 expression vector (AdWTp53). p53 activates the maspin promoter by binding directly to the p53 consensus-binding site present in the maspin promoter. DNA-damaging agents and cytotoxic drugs induced endogenous maspin expression in cells containing the wild type p53. Maspin expression was refractory to the DNA-damaging agents in cells containing mutant p53. These results, combined with recent studies of the tumor metastasis suppressor gene KAI1 and plasminogen activator inhibitor 1 (PAI1), define a new category of molecular targets of p53 that have the potential to negatively regulate tumor invasion and/or metastasis.     

MiRNA regulation of TRAIL expression exerts selective cytotoxicity to prostate carcinoma cells

Prostate carcinoma is the most common cancer for men and among the leading cancer-related causes. Many evidences have shown that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) potently induces apoptosis in cancer cells, and thus, is a promising biologic agent for prostate carcinoma therapy. However, TRAIL expression mediated by the current vectors lacks tumor specificity, thereby exerting cytotoxicity to normal cells. To solve this problem, we inserted miRNA response elements (MREs), miR-143 and miR-145, expression levels of which were reduced in prostate carcinoma, as well as that of miR-122, which is specifically expressed in hepatic cells, into adenoviral vectors to control TRAIL expression (Ad-TRAIL-M3). qPCR data confirmed that miR-143, miR-145, and miR-122 levels were all decreased in prostate carcinoma cell lines and prostate cancer samples from patients. Luciferase assays showed that MREs-regulated luciferase expression was potently suppressed in normal cells, but not in prostate cancer cells. Ad-TRAIL-M3, which expresses TRAIL in a MREs-regulated manner, produced high level of TRAIL and suppressed the survival of prostate cancer cells by inducing apoptosis, while Ad-TRAIL-M3 had no TRAIL expression in normal cells and thus exerted no cytotoxicity to them. The studies on PC-3 tumor xenograft in mice further confirmed that Ad-TRAIL-M3 was able to inhibit the growth of tumors and possessed high biosafety. In conclusion, we successfully generated an adenoviral vector that expresses TRAIL in miRNA-regulated mechanism. This miRNA-based gene therapy may be promising for prostate carcinoma treatment.     

Human osteocalcin: a strong promoter for nitric oxide synthase gene therapy, with specificity for hormone refractory prostate cancer

BACKGROUND: Gene therapy has been identified as a promising treatment strategy for hormone refractory prostate cancer (HRPC). We report, for the first time, the use of the human osteocalcin (hOC) promoter to control inducible nitric oxide synthase (iNOS) transgene expression in HRPC. METHODS: Human prostate carcinoma cells (PC3, DU145, LNCaP), colon cancer cells (HT29) and human microvascular endothelial cells (HMEC-1) were transfected in vitro with constitutively driven CMV/iNOS or hOC/iNOS plasmid DNA by cationic lipid vector. End points of these experiments were Western blotting, NO(.) generation using the Greiss test to measure accumulated nitrite, and clonogenic assay. RESULTS: Transfection of the hOC/iNOS plasmid increased iNOS protein and total nitrite levels in PC3 and DU145 cells, but not LNCaP or HT29. Transfection with CMV/iNOS or hOC/iNOS resulted in no additional cytotoxicity in androgen-dependent LNCaP cells or in the non-prostate cell lines. However, transfection with either construct resulted in a greatly reduced cell survival (to 10-20%) in the androgen-independent PC3 and DU145 cell lines. CONCLUSIONS: Utilising the tumour-type specific properties of the hOC promoter in tandem with the iNOS gene, we have demonstrated target cell specificity, and transgene activation, in the androgen-independent prostate cancer cell lines (PC3 and DU145), an effect absent in normal and androgen-dependent cells. Furthermore, the levels of NO(.) generated are comparable with those seen generated with constitutively (CMV)-driven iNOS. The data obtained from this study provide a basis for future development of hOC/iNOS gene therapy.     

p53-induced gene 3 mediates cell death induced by glutathione peroxidase 3

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.     

Development of a STAT3 reporter prostate cancer cell line for high throughput screening of STAT3 activators and inhibitors

STAT3 is constitutively activated in several cancers, including prostate cancer, and is therefore, a potential target for cancer therapy. DU-145 prostate cancer cells were stably co-transfected with STAT3 reporter and puromycin resistant plasmids to create a stable STAT3 reporter cell line that can be used for high throughput screening of STAT3 modulators. The applicability of this cell line was tested with two known activators and inhibitors of STAT3. As expected, EGF and IL-6 increased STAT3 reporter activity and enhanced the nuclear localization of phosphorylated STAT3 (pSTAT3); whereas Cucurbitacin I and AG490 decreased STAT3 reporter activity dose and time-dependently and reduced the localization of pSTAT3 in the nuclei of prostate cancer cells. Given the importance of STAT3 in cancer initiation and progression, the development of a stable STAT3 reporter cell line in prostate cancer cells provides a rapid, sensitive, and cost effective method for the screening of potential STAT3 modulators.