Molecular Target For Tumor Therapy Research Articles

Multi-omics reveals the role of ENO1 in bladder cancer and constructs an epithelial-related prognostic model to predict prognosis and efficacy

α-Enolase (ENO1) is a crucial molecular target for tumor therapy and has emerged as a research hotspot in recent decades. Here, we aimed to explore the role of ENO1 in bladder cancer (BLCA) and then construct a signature to predict the prognosis and treatment response of BLCA. Firstly, we found ENO1 was highly expressed in BLCA tissues, as verified by IHC, and was associated with poor prognosis. The analysis of the tumor immune microenvironment by bulk RNA-seq and scRNA-seq showed that ENO1 was associated with CD8+ T-cell exhaustion. Additionally, the results in vitro showed that ENO1 could promote the proliferation and invasion of BLCA cells. Then, the analysis of epithelial cells (ECs) revealed that ENO1 might promote BLCA progression by metabolism, the cell cycle and some carcinogenic pathways. A total of 249 hub genes were obtained from differentially expressed genes between ENO1-related ECs, and we used LASSO analysis to construct a novel signature that not only accurately predicted the prognosis of BLCA patients but also predicted the response to treatment for BLCA. Finally, we constructed a nomogram to better guide clinical application. In conclusion, through multi-omics analysis, we found that ENO1 was overexpressed in bladder cancer and associated with poor prognosis, CD8+ T-cell exhaustion and epithelial heterogeneity. Moreover, the prognosis and treatment of patients can be well predicted by constructing an epithelial-related prognostic signature.

Consideration of SHP-1 as a Molecular Target for Tumor Therapy.

Abnormal activation of receptor tyrosine kinases (RTKs) contributes to tumorigenesis, while protein tyrosine phosphatases (PTPs) contribute to tumor control. One of the most representative PTPs is Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1), which is associated with either an increased or decreased survival rate depending on the cancer type. Hypermethylation in the promoter region of PTPN6, the gene for the SHP-1 protein, is a representative epigenetic regulation mechanism that suppresses the expression of SHP-1 in tumor cells. SHP-1 comprises two SH2 domains (N-SH2 and C-SH2) and a catalytic PTP domain. Intramolecular interactions between the N-SH2 and PTP domains inhibit SHP-1 activity. Opening of the PTP domain by a conformational change in SHP-1 increases enzymatic activity and contributes to a tumor control phenotype by inhibiting the activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT3) pathway. Although various compounds that increase SHP-1 activation or expression have been proposed as tumor therapeutics, except sorafenib and its derivatives, few candidates have demonstrated clinical significance. In some cancers, SHP-1 expression and activation contribute to a tumorigenic phenotype by inducing a tumor-friendly microenvironment. Therefore, developing anticancer drugs targeting SHP-1 must consider the effect of SHP-1 on both cell biological mechanisms of SHP-1 in tumor cells and the tumor microenvironment according to the target cancer type. Furthermore, the use of combination therapies should be considered.

Downregulation of ST3GAL5 is associated with muscle invasion, high grade and a poor prognosis in patients with bladder cancer.

In patients with bladder cancer (BC), the association between ST3 β-galactoside α-2,3-sialyltransferase 5 (ST3GAL5) expression and clinical outcomes, particularly regarding muscle-invasive disease, high tumor grade and prognosis, remain unknown. In the present study, the expression of ST3GAL5 and its association with clinical outcomes in patients with BC was analyzed using various public bioinformatics databases. The difference in ST3GAL5 expression between BC and healthy bladder tissues was also evaluated using data from the Oncomine database, The Cancer Genome Atlas and Gene Expression Omnibus database. The differences in ST3GAL5 expression between muscle invasive BC (MIBC) and non-muscle invasive BC (NMIBC), and high- and low-grade BC were also analyzed. Furthermore, genes that were positively co-expressed with ST3GAL5 in patients with BC were identified from the intersection between the Oncomine, Gene Expression Profiling Interactive Analysis 2 and UALCAN databases. Enrichment analysis by Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Reactome pathway enrichment analyses and a gene-concept network was performed using R package. Gene set enrichment analysis was also performed to assess the signaling pathways influenced by the high and low expression of ST3GAL5 in BC. The results indicated that ST3GAL5 expression was significantly lower in BC tissues compared with normal bladder tissues (P<0.05). Furthermore, ST3GAL5 expression in MIBC and high-grade BC was significantly lower compared with NMIBC and low-grade BC (P<0.05), respectively. The results from Kaplan-Meier survival analysis result demonstrated that ST3GAL5 downregulation was associated with poor survival in patients with BC (P<0.05). Taken together, these findings suggested that ST3GAL5 may be considered as an anti-oncogene in BC, could represent a potential predictive and prognostic biomarker for BC and may be a molecular target for tumor therapy.

Hydrophobically Modified let-7b miRNA Enhances Biodistribution to NSCLC and Downregulates HMGA2 In Vivo

MicroRNAs (miRNAs) have increasingly been shown to be involved in human cancer, and interest has grown about the potential use of miRNAs for cancer therapy. miRNA levels are known to be altered in cancer cells, including in non-small cell lung cancer (NSCLC), a subtype of lung cancer that is the most prevalent form of cancer worldwide and that lacks effective therapies. The let-7 miRNA is involved in the regulation of oncogene expression in cells and directly represses cancer growth in the lung. let-7 is therefore a potential molecular target for tumor therapy. However, applications of RNA interference for cancer research have been limited by a lack of simple and efficient methods to deliver oligonucleotides (ONs) to cancer cells. In this study, we have used in vitro and in vivo approaches to show that HCC827 cells internalize hydrophobically modified let-7b miRNAs (hmiRNAs) added directly to the culture medium without the need for lipid formulation. We identified functional let-7b hmiRNAs targeting the HMGA2 mRNA, one of the let-7 target genes upregulated in NSCLC, and show that direct uptake in HCC827 cells induced potent and specific gene silencing in vitro and in vivo. Thus, hmiRNAs constitute a novel class of ONs that enable functional studies of genes involved in cancer biology and are potentially therapeutic molecules.

Kruppel-Like Factor 4: From Physiological Functions to Tumor Therapy

Kruppel-like factor 4 (KLF4) is a zinc finger transcription factor that can regulate diverse cellular physiological functions such as cell growth, death, differentiation, and migration. Recent studies have suggested that KLF4 can act as either a suppressor or oncogene for different types of cancers, ranging from solid tumors to leukemia, by regulating target genes involved in tumor cell proliferation, survival, metastasis, invasiveness, and the constitution of the tumor microenvironment. In most cancer types, KLF4 has shown the ability to inhibit tumor progression, thus raising the possibility that it may be a novel target for multiple types of cancer, including T-cell acute lymphoblastic leukemia, which lacks targeted drugs in clinical practice. A better understanding of the role of KLF4 in tumorigenesis will facilitate the application of this potential molecular target for tumor therapy.

Molecular mechanism and application of translationally controlled tumor protein in tumor targeting therapy

Translationally controlled tumor protein (TCTP) is a small protein highly conserved in a variety of eukaryotic species. TCTP is overexpressed in various tumor cells and has been implicated in the regulation of cellular processes including apoptosis, DNA repair and drug resistance. By reviewing the recent progress of TCTP research, TCTP is becoming an important regulator of DNA repair and a new molecular target for tumor therapy. Key words: DNA damage; Drug resistance, neoplasm; Translationally controlled tumor protein

MicroRNA let-7: Regulation, single nucleotide polymorphism, and therapy in lung cancer.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are highly evolutionarily conserved noncoding small RNAs, which were first identified in Caenorhabditis elegans. There are 1100 or more miRNAs have been identified in Homo sapiens. Let-7 miRNA is involved in the regulation of gene expression in cells. Several novel factors and feedback loops involved in the regulation of the synthesis of let-7 have been identified and additional let-7 target genes have been found. Let-7 has also been shown to be significantly correlated with the occurrence and development of cancer and the results of preliminary studies suggest that it is involved in the regulation of oncogenic pathways in numerous types of tumors, such as, LC. As let-7 is a potential molecular target for tumor therapy, a mass of studies have been conducted focus on the relationship between let-7 and LC. With the mechanism becoming revealed, more and more groups are looking for the anti-tumor role of let-7 family in anti-tumor therapy development. In this review, we summarize the newest investigations on let-7 and LC, the regulation of let-7 and its targets gene have been discussed, and the attempts for new therapy for LC have also been summarized.

Hypoxia-induced miR-424 decreases tumor sensitivity to chemotherapy by inhibiting apoptosis.

Chemotherapy resistance of tumor cells is a big challenge. Adaption to hypoxia is an essential cellular response that is controlled by the master oxygen-sensitive transcription factor HIF1 (hypoxia-inducible factor 1). The mechanism by which tumor cells acquire resistance to chemotherapy under hypoxic conditions is not fully understood. In this study, we found that hypoxia induces miR-424 expression and that miR-424 in turn suppresses the level of PDCD4 protein, a tumor suppressor that is involved in apoptosis, by targeting its 3′ untranslated region. Functionally, miR-424 overexpression decreases the sensitivity of cancer cells (HCT116 and A375) to doxorubicin (Dox) and etoposide. In contrast, the inhibition of miR-424 enhanced apoptosis and increased the sensitivity of cancer cells to Dox. In a xenograft tumor model, miR-424 overexpression promoted tumor growth following Dox treatment, suggesting that miR-424 promotes tumor cell resistance to Dox. Furthermore, miR-424 levels are inversely correlated with PDCD4 expression in clinical breast cancer samples. These results suggest that miR-424 is a potential molecular target for tumor therapy.

Abstract A239: T-type calcium channels as a novel molecular target for tumor therapy.

Abstract T-type Ca2+ channels are a group of low voltage-gated calcium channels which seem to be crucial for embryonic or stem cell proliferation and differentiation; however, they could be also aberrantly expressed in several human tumors. Since calcium entry is required for smooth transition through the cell cycle, it has been hypothesized that the inhibition of T-type Ca2+ channels blocks cells in G0/G1 phase of cell cycle, while a release from inhibition leads to increased number of cells entering cell cycle and thus increased susceptibility to conventional antitumor therapy. In this study we investigated the effects of T-type channel inactivation, or downregulation, on cell cycle progression, cell death and survival, and resistance to radiotherapy (RT). The experiments were conducted using several cancer cell lines expressing T-type Ca2+ channels (glioblastoma, colon and prostate) and selective and structurally unrelated calcium channels antagonists, including Mibefradil, a drug that is currently undergoing clinical trial for its antitumor properties, and is planned to be tested as radiosensitizing agent in recurrent gliobastoma tumors. The effects of T-type Ca2+ channel inhibition on cell cycle progression, cell death and the expression/activation of cell cycle regulated proteins were assessed. Finally, the combination treatments including T-type channel antagonist and radiotherapy were assayed. We show that T-type Ca2+ channels are important for cell cycle progression and resistance to RT, and that their inhibition leads to enhanced cell death and reduced survival. Our study demonstrates that one of the earliest events evoked by T-type channel inhibition is a deregulation of pro-survival pathway PI3K/Akt/mTOR and activation of pro-apoptotic stress-activated p38MAPK pathway. The results support the idea of T-type Ca2+ channel as a novel molecular target for antitumor therapy and provide a rational and plausible biological mechanism responsible for the effects induced by T-type Ca2+ channel antagonists in cancer cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A239. Citation Format: Barbara Dziegielewska, Nicholas C.K. Valerie, Amol S. Hosing, James M. Larner, David L. Brautigan, Lloyd S. Gray, Jaroslaw Dziegielewski. T-type calcium channels as a novel molecular target for tumor therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A239.

Inhibition of T-type calcium channels disrupts Akt signaling and promotes apoptosis in glioblastoma cells

Glioblastoma multiforme (GBM) are brain tumors that are exceptionally resistant to both radio- and chemotherapy regimens and novel approaches to treatment are needed. T-type calcium channels are one type of low voltage-gated channel (LVCC) involved in embryonic cell proliferation and differentiation; however they are often over-expressed in tumors, including GBM. In this study, we found that inhibition of T-type Ca2+ channels in GBM cells significantly reduced their survival and resistance to therapy. Moreover, either T-type selective antagonists, such as mibefradil, or siRNA-mediated knockdown of the T-type channel alpha subunits not only reduced cell viability and clonogenic potential, but also induced apoptosis. In response to channel blockade or ablation, we observed reduced phosphorylation of Akt and Rictor, suggesting inhibition of the mTORC2/Akt pathway. This was followed by reduction in phosphorylation of anti-apoptotic Bad and caspases activation. The apoptotic response was specific for T-type Ca2+ channels, as inhibition of L-type Ca2+ channels did not induce similar effects. Our results implicate T-type Ca2+ channels as distinct entities for survival signaling in GBM cells and suggest that they are a novel molecular target for tumor therapy.

Regulation of let-7 and its target oncogenes (Review)

MicroRNAs (miRNAs) are highly evolutionarily-conserved non-coding small RNAs, which were first identified in Caenorhabditis elegans. Let-7 miRNA is involved in the regulation of gene expression in cells. Several novel factors and feedback loops involved in the regulation of the synthesis of let-7 have been identified and additional let-7 target genes have been found. Let-7 has also been shown to be significantly correlated with the occurrence and development of cancer and the results of preliminary studies suggest that it is involved in the regulation of oncogenic pathways in numerous types of tumors. Let-7 is, therefore, a potential molecular target for tumor therapy. Thus, this review examined let-7 and the correlation between let-7 and oncogenic pathways in cancer.

Lysine-specific histone demethylase 1 (LSD1): A potential molecular target for tumor therapy

Lysine-specific demethylase 1 (LSD1), the first identified histone demethylase, was belonged to the superfamily of the flavin adenine dinucleotide (FAD)-dependent amine oxidases. LSD1 specifically demethylates mono- or dimethylated dimethylated histone H3 lysine4 (H3K4) and H3 lysine 9 (H3K9) via a redox process. Recently evidences showed that LSD1 played an important role in a broad spectrum of biological processes, including cell proliferation, adipogenesis, spermatogenesis, chromosome segregation and embryonic development. Furthermore, LSD1 also could promote progress of tumor by inhibiting the tumor suppressor activity of p53. To date, as a potential drug for discovering anti-tumor drugs, the medical significance of LSD1 inhibitors have been greatly appreciated. Here, we reviewed the remarkable progress being made in understanding of LSD1, mainly on its structure, basic function and medical application in tumor therapy.

Thymosin β4: A Potential Molecular Target for Tumor Therapy

Thymosin β4 (Tβ4), a 5 kDa protein, has been demonstrated to play an important role in a variety of biological activities, such as actin sequestering, cellular motility, migration, inflammation, and damage repair. Recently, several novel findings provided compelling evidence that Tβ4 played a key role in facilitating tumor metastasis and angiogenesis. It has been found that Tβ4 expressed increasingly in a number of metastatic tumors, which was associated with an increased expression of a known angiogenic factor, vascular endothelial growth factor. Thus, Tβ4 provided a potential target of opportunity for cancer management, especially for cancer metastasis therapy.

Abstract C53: Potent CDC, ADCC, and antitumor activity of human monoclonal antibodies to Sialyl Lewisa.

Abstract The carbohydrate antigen sialyl-Lewis A (sLea), also known as CA19.9, is widely expressed on epithelial tumors of the gastrointestinal tract, on breast cancer cells, and also on small cell lung cancer cells. sLea serves as a ligand for epithelial leukocyte adhesion molecules and over-expression of sLea appears to be a key event in invasion and metastasis of many tumor cells. Since tumor cells expressing sLea are highly susceptible to antibody mediated lysis mechanisms, sLea presents an attractive molecular target for tumor therapy in a minimal disease setting. Hybridomas were generated with human B cells isolated from individuals vaccinated with MSKCC's cancer vaccine. Antibody producing cells were identified by ELISA using sLea-HSA and synthetic sLea -PAA-biotin conjugates. Binding to the native antigen expressed on the cell surface was tested by FACS analysis on sLea positive DMS-79 cells and others and the biological activity was measured in CDC and ADCC assays. The binding affinity was determined by Surface Plasmon Resonance and the specificity of the carbohydrate binding was evaluated by ELISA and by glycan array analysis (CFG, Array 4.1). Recombinant antibodies were generated in CHO cells and purified on Protein A (IgG) or hydroxyapatite (IgM), respectively. We report the discovery and initial characterization of fully human antibodies that were generated from blood lymphocytes from individuals immunized with sLea -KLH vaccine. Two antibodies were selected for further studies based on the apparent high affinity, which was estimated by BiaCore at 0.14 nM for 5B1 (IgG/λ) and 0.04 nM for 7E3 (IgM/κ). Both antibodies were highly specific for Neu5Acα2–3Galβ1–3 (Fucα1–4) GlcNAcβ and did not bind to sialyl-Lewisx, Lewisa, and other related carbohydrates. Both antibodies have been expressed as fully functional human recombinant antibodies in CHO cells. Complement dependent cytotoxicity (CDC) against DMS-79 cells was approximately 60% and 70–90% for r5B1 and r7E3, respectively. Moreover, r5B1 antibodies showed approximately 50% ADCC of DMS-79 cells with human NK cells (at 5:1 ratio) and 80–90% ADCC with human peripheral blood mononuclear cells with two different blood donors (at 100:1 E/T ratio). These antibodies were tested in two xenograft models: 1) Treatment of animals with 5B1 on the day of engraftment with DMS-79 cells in a subcutaneous model completely prevented tumor growth. 2) Delayed treatment with various doses of 5B1 showed dose dependent protection up to complete cure in SCID mice engrafted (IV) with Colo205 cells. 7E3 antibodies did not show higher protection despite increased apparent affinity. The specificity and potency of 5B1 antibodies in CDC and ADCC assays translates into good activity in xenograft models. Further studies are warranted to explore the activity against additional cancer specific cell lines in animal models and to pursue preclinical development of 5B1 antibodies. Funding support by NCI #R42CA128362. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C53.

Human Monoclonal Antibodies to Sialyl-Lewisa (CA19.9) with Potent CDC, ADCC, and Antitumor Activity

The carbohydrate antigen sialyl-Lewis(a) (sLe(a)), also known as CA19.9, is widely expressed on epithelial tumors of the gastrointestinal tract and breast and on small-cell lung cancers. Since overexpression of sLe(a) appears to be a key event in invasion and metastasis of many tumors and results in susceptibility to antibody-mediated lysis, sLe(a) is an attractive molecular target for tumor therapy. We generated and characterized fully human monoclonal antibodies (mAb) from blood lymphocytes from individuals immunized with a sLe(a)-KLH vaccine. Several mAbs were selected based on ELISA and FACS including two mAbs with high affinity for sLe(a) (5B1 and 7E3, binding affinities 0.14 and 0.04 nmol/L, respectively) and further characterized. Both antibodies were specific for Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ as determined by glycan array analysis. Complement-dependent cytotoxicity against DMS-79 cells was higher (EC(50) 0.1 μg/mL vs. 1.7 μg/mL) for r7E3 (IgM) than for r5B1 (IgG1). In addition, r5B1 antibodies showed high level of antibody-dependent cell-mediated cytotoxicity activity on DMS-79 cells with human NK cells or peripheral blood mononuclear cells. To evaluate in vivo efficacy, the antibodies were tested in a xenograft model with Colo205 tumor cells engrafted into SCID (severe combined immunodeficient mice) mice. Treatment during the first 21 days with four doses of r5B1 (100 μg per dose) doubled the median survival time to 207 days, and three of five animals survived with six doses. On the basis of the potential of sLe(a) as a target for immune attack and their affinity, specificity, and effector functions, 5B1and 7E3 may have clinical utility.

Abstract B122: Human monoclonal antibodies to sialyl-Lewis-A with potent CDC and ADCC activity

Abstract The carbohydrate antigen sialyl-Lewis A (sLea) is widely expressed on epithelial tumors of the gastrointestinal tract, on breast cancer cells, and also on small cell lung cancer cells, but is expressed minimally or not at all on normal tissues. sLea serves as a ligand for epithelial leukocyte adhesion molecules and higher expression of sLea was observed in patients with greater node involvement. Since over-expression of sLea appears to be a key event in invasion and metastasis of many tumor cells and tumor cells expressing sLea are highly susceptible to antibody mediated lysis mechanisms, sLea presents an attractive molecular target for tumor therapy in a minimal disease setting. Here we report the discovery and initial characterization of fully human antibodies that were generated from blood lymphocytes from individuals immunized with sLea vaccine at MSKCC. Antibodies were identified by ELISA using sLea-HSA conjugates and binding to the native antigen was verified by FACS analysis on DMS-79 cells. Two antibodies were selected for further studies based on the apparent high affinity, which was estimated by Biacore at 0.14 nM for 5B1 (IgG/λ) and 0.04 nM for 7E3 (IgM/κ). These antibodies did not bind to sialyl-Lewis X, Lewis A and other related carbohydrates. Both antibodies have been expressed as fully functional human recombinant antibodies in CHO cells. Complement dependent cytotoxicity (CDC) against DMS-79 cells was approximately 60% and 70–90% for r5B1 and r7E3, respectively. Moreover, r5B1 antibodies showed approximately 50% ADCC of DMS-79 cells with human NK cells (at 5:1 ratio) and 80–90% ADCC with human peripheral blood mononuclear cells with two different blood donors (at 100:1 ratio). These results are very encouraging and we believe that further studies to scale up and test these antibodies in various tumor challenge models are warranted. Since sLea is widely expressed on human cancers, such antibodies could eventually find utility in approximately half of the new cancer cases occurring each year. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B122.

Growth inhibition of human ovarian cancer cells by blocking STAT3 activation with small interfering RNA

Objective Signal transducer and activator of transcription 3 (STAT3) are constitutively activated in a variety of cancers and it is a common feature of ovarian cancer. Thus, STAT3 represents a promising molecular target for tumor therapy. We applied a DNA vector-based STAT3-specific RNA interference approach which specifically blocks over-activated STAT3, to treat human ovarian cancer cells, and evaluated the cellular proliferation ability and investigated the molecular mechanisms in vitro. Study design A DNA vector-based RNA interference approach was used to knockdown STAT3 expression in human ovarian cancer cells in vitro. Results The STAT3 siRNA down-regulated the expression of cyclin D1, survivin, and VEGF in ovarian cancer cells both at transcription and translation levels. Inhibition of STAT3 and its related genes was accompanied by growth suppression and induction of apoptosis in cancer cells in vitro. Conclusions These data indicate that STAT3 signaling is a promising molecular target for ovarian cancer therapy.

Thrombospondin 2 Potentiates Notch3/Jagged1 Signaling

Extracellular thrombospondins (TSP or THBS) and the Notch family of transmembrane receptors share a role in multiple, overlapping cellular functions and participate in developmental signaling and pathological reactions to tissue injury. We demonstrate that TSP2, but not TSP1, enhances the potency of Notch3 signal transduction. In addition, TSP2 reduces cancer cell proliferation in a Notch-ligand dependent fashion. The loss of TSP2 in knock-out mice reduces Notch target gene expression. TSP2 binds directly to Notch3 and Jagged1. TSP1 also binds to Notch3 and Jagged1; however, only TSP2 augments the interaction between Notch3 and Jagged1. These studies demonstrate that the diverse functions of TSP2 may also include a role as an intermediary protein that facilitates transcellular receptor-ligand interactions.

Down-Regulation of Signal Transducer and Activator of Transcription 3 Expression Using Vector-Based Small Interfering RNAs Suppresses Growth of Human Prostate Tumor In vivo

Signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a variety of cancers and it is a common feature of prostate cancer. Thus, Stat3 represents a promising molecular target for tumor therapy. We applied a DNA vector-based Stat3-specific RNA interference approach to block Stat3 signaling and to evaluate the biological consequences of Stat3 down-modulation on tumor growth using a mouse model. To investigate the therapeutic potential of blocking Stat3 in cancer cells, three small interfering RNAs (siRNA; Stat3-1, Stat3-2, and Stat3-3) specific for different target sites on Stat3 mRNA were designed and used with a DNA vector-based RNA interference approach expressing short hairpin RNAs to knockdown Stat3 expression in human prostate cancer cells in vitro as well as in vivo. Of the three equivalently expressed siRNAs, only Stat3-3 and Stat3-2, which target the region coding for the SH2 domain and the coiled-coil domain, respectively, strongly suppressed the expression of Stat3 in PC3 and LNCaP cells. The Stat3-1 siRNA, which targeted the DNA-binding domain, exerted no effect on Stat3 expression, indicating that the gene silencing efficiency of siRNA may be dependent on the local structure of Stat3 mRNA. The Stat3 siRNAs down-regulated the expression of Bcl-2 (an anti-apoptotic protein), and cyclin D1 and c-Myc (cell growth activators) in prostate cancer cells. Inhibition of Stat3 and its related genes was accompanied by growth suppression and induction of apoptosis in cancer cells in vitro and in tumors implanted in nude mice. These data indicate that Stat3 signaling is a promising molecular target for prostate cancer therapy and that vector-based Stat3 siRNA may be useful as a therapeutic agent for treatment of prostate cancer.