Molecular Target For Gene Therapy Research Articles

CircRNA expression profiling of the rat thalamus in temporomandibular joint chronic inflammatory pain

Orofacial pain (OFP) induced by temporomandibular disorders (TMDs) is prevalent, affecting approximately 4.6 % of the population. One specific type of TMD is temporomandibular osteoarthritis (TMJOA), a common degenerative disease that significantly impacts patients’ quality of life. Differentially expressed circular RNAs (DEcircRNAs) in the thalamus, which serves as a relay station in the orofacial pain transmission pathway, may play a crucial role and serve as potential target markers for inflammation and the progression of inflammatory pain in TMJOA. The aim of this study was to investigate the expression profile of circRNAs in the thalamus of TMJOA. We obtained the circRNA expression profile from the thalamus of a rat model of TMJOA through high-throughput sequencing (HT-seq) and further validated their expression using reverse transcription real-time polymerase chain reaction (RT-qPCR), followed by bioinformatics analysis of the expression data. A total of 425 circRNAs (DESeq2 p- value < 0.05, |log2FoldChange| > 0.0) were identified as significantly differentially expressed by RNA-Seq, comprising 188 up-regulated and 237 down-regulated circRNAs. After validation via RT-qPCR, we employed miRanda software to predict the binding sites of miRNAs for the identified circRNAs to further explore the functions of DEcircRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that DEcircRNAs were primarily enriched in pathways and functions related to synapse development, protein signaling and modification, ’Circadian entertainment’, the ’MAPK signaling pathway’, and ’Glutamatergic synapse’. These findings suggest that DEcircRNAs in the thalamus play a significant role in the progression of TMJOA and may serve as promising candidate molecular targets for gene therapy.

Bioinformatics Analysis and Screening of Potential Target Genes Related to the Lung Cancer Prognosis.

Several genes have been validated as molecular targets for gene therapy in lung cancer. We screened target genes that affect survival of patients with lung cancer. Data on gene expression in normal lung tissues/lung adenocarcinoma (LUAD) samples were acquired from Genotype-Tissue Expression (GTEx)/The Cancer Genome Atlas (TCGA) databases and merged to expand the sample size, followed by differential analysis of the merged expression data and acquisition of differentially expressed genes. Survival and simple Cox analyses were used to screen for genes affecting LUAD survival. Protein-protein interaction/multivariable Cox analyses were utilized, and a risk model was established. Candidate genes expression levels in cancer/paracancerous tissues of lung cancer patients, and BEAS-2B/A549/HCC95 cells were measured by RT-qPCR/Western blot. Survival analysis of candidate genes was conducted in LUAD samples collected from TCGA. Among 947 genes differentially expressed in LUAD, 151 were correlated with patient survival, and 116 might act as risk factors for LUAD. The 7 identified candidate genes (TOP2A, TK1, KIF4A, ANLN, KIF2C, ASF1B, CCNB1) were high-risk genes playing possible roles in LUAD. These genes were differentially expressed in lung cancer and were associated with TNM stages (III - IV)/differentiation grade/lymph node metastasis/distant metastasis, which affected lung cancer patient survival. P2A, TK1, KIF4A, ANLN, KIF2C, ASF1B and CCNB1 were highly-expressed in LUAD/lung squamous cell carcinoma (LUSC) and correlated with LUAD patient survival. This study contributes to better understanding of the prognostic regulation mechanism in LUAD and the screening of target genes for clinical treatment.

Abstract P6-14-11: Exosome-mediated Circ-CRSP1 promotes tumor proliferation and metastasis through stabilizing ELAVL1 protein and suppresses anti-tumor immune response in regulating the progression of Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) is one of the most malignant subtype of breast cancer. Lacking targetable molecular drivers, chemotherapy is almost the only effective systematic treatment for TNBC. Compared with other types of breast cancer, TNBC shows a higher long-term recurrence rate and worse prognostic outcome, and its main cause of death is tumor metastasis. Therefore, there is an urgent clinical need to explore underlying molecular mechanisms and find novel targets for therapeutic intervention, as well as biomarkers for early clinical diagnosis. Materials &amp; Methods: To explore the signal pathways and target molecules related to tumor immunity by bioinformatics analysis of tissue samples [TNBC (n = 55), Her2 (n = 39), Luminal B (n = 30), Luminal A (n = 29), healthy (n = 11)]. Co-culture experiment was conducted to study the effect of TNBC-derived exosome-mediated molecules on the proliferation and migration of breast cancer. qRT-PCR assay was used to confirm the expression level of Circ-CRSP1 in tumor tissues, breast cancer cells, exosomes of breast cancer cells and serum exosomes of breast cancer patients. Overexpression and knockout experiments investigated the role of Circ-CRSP1 in promoting the proliferation and migration of TNBC cells. Protein molecular docking technique was used to predict the possible targeting binding protein ELAVL1, and molecular experiments and nude mice model were used to study the mechanism of Circ-CRSP1-ELAVL1 complex in tumor immunity. Results: TNBC-derived exosome-mediated molecular transmission promotes the proliferation and migration of breast cancer, and may promote distant metastasis of breast cancer by changing the tumor microenvironment (TME) before metastasis. Bioinformatics analysis of TNBC tumor samples confirmed that TNBC is strongly associated with tumor immune-related biological processes and signal pathways. Circ-CRSP1 is highly expressed in TNBC tumor tissues, TNBC cells, exosome derived from TNBC cells and serum exosomes of TNBC patients, and is related to the poor clinical prognosis of TNBC patients. Overexpression of Circ-CRSP1 enhanced the proliferation and migration ability of TNBC cells, and the expression levels of EMT and cell-cycle related genes altered. Circ-CRSP1 binds to protein ELAVL1 at the physical level. The target gene of Circ-CRSP1-ELAVL1 complex is involved in the immune response, and the overexpression of Circ-CRSP1 significantly down-regulates the expression level of immune-related genes. Conclusion: Exosome-mediated Circ-CRSP1 promotes the transport of ELAVL1 from nucleus to cytoplasm through targeted binding with ELAVL1, and then regulates cytoplasmic proliferation and metastasis and inhibits the stability and translation of anti-tumor immune-related gene mRNA, thus finally promotes the proliferation, invasion and metastasis of TNBC. This study can further understand the mechanism of circRNA in the growth and metastasis of TNBC from the theoretical level, further explore the feasibility of Circ-CRSP1 as a molecular target for gene therapy, and provide theoretical basis and pre-clinical data for Circ-CRSP1 in serum exosomes as a high risk screening index and prognostic marker for TNBC patients. Citation Format: Sujin Yang, Jinhai Tang. Exosome-mediated Circ-CRSP1 promotes tumor proliferation and metastasis through stabilizing ELAVL1 protein and suppresses anti-tumor immune response in regulating the progression of Triple-Negative Breast Cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-11.

High BTBD7 expression positive is correlated with SLUG-predicted poor prognosis in hormone receptor-negative breast cancer.

BackgroundHormone receptor-negative breast cancer (HRNBC), which includes triple-negative breast cancer (TNBC) and human epidermal growth factor receptor 2 (HER-2) overexpressing breast cancer, is prone to metastasis and has a poor prognosis. BTB/POZ domain-containing protein 7 (Btbd7) is thought to regulate SLUG and the epithelial-mesenchymal transition (EMT) process. However, the role of Btbd7 in HRNBC is unclear.MethodsExpression of BTBD7 and SLUG in HRNBC tumor tissue and normal adjacent tissue (NAT) as well as breast cancer cells were characterized by immunohistochemistry and immunofluorescence. MDA-MA-231 cells was transfected with BTBD7 siRNA and detected by qRT-PCR and western blot. Expression levels of Slug and EMT related proteins were detected western blot analysis. cell invasion assays were used to analyse cell invasion ability of MDA-MA-231. GO and KEGG analyses was used to analysis the gene function.ResultsThe total positive rate of BTBD7 expression in HRNBC tumor tissue was 66.7%, which was higher than that in NAT (52.1%) and benign breast lesion tissues (20%). Co-expression of SLUG and BTBD7 proteins could be found in HRNBC tissue and MDA-MA-231 cells. BTBD7 silencing significantly up-regulated the epithelial marker E-cadherin, down-regulated the mesenchymal markers α-SMA and SLUG and suppressed the invasion abilities of MDA-MA-231 cells. GO and KEGG analyses based on 322 DEGs showed that BTBD7 may be associated with generic transcription in breast cancer.ConclusionsThe study data indicated that BTBD7 was inversely associated with SLUG expression. Higher BTBD7 was associated with poor clinicopathologic features and prognosis in HRNBC patients. BTBD7 silencing inhibited EMT through regulation of SLUG expression. BTBD7 might act as a potential molecular target for gene therapy in HRNBC patients.

Novel Gene Regulation in Normal and Abnormal Spermatogenesis.

Spermatogenesis is a complex and dynamic process which is precisely controlledby genetic and epigenetic factors. With the development of new technologies (e.g., single-cell RNA sequencing), increasingly more regulatory genes related to spermatogenesis have been identified. In this review, we address the roles and mechanisms of novel genes in regulating the normal and abnormal spermatogenesis. Specifically, we discussed the functions and signaling pathways of key new genes in mediating the proliferation, differentiation, and apoptosis of rodent and human spermatogonial stem cells (SSCs), as well as in controlling the meiosis of spermatocytes and other germ cells. Additionally, we summarized the gene regulation in the abnormal testicular microenvironment or the niche by Sertoli cells, peritubular myoid cells, and Leydig cells. Finally, we pointed out the future directions for investigating the molecular mechanisms underlying human spermatogenesis. This review could offer novel insights into genetic regulation in the normal and abnormal spermatogenesis, and it provides new molecular targets for gene therapy of male infertility.

Downregulation of histone‑lysine N‑methyltransferase EZH2 inhibits cell viability and enhances chemosensitivity in lung cancer cells

Histone-lysine N-methyltransferase EZH2 (EZH2) is the principle component of the polycomb repressive complex 2 (PRC2)/embryonic ectoderm development protein-EZH2 complex, which promotes tumorigenesis by repressing transcription of tumor suppressor genes. EZH2 is considered a key marker in several types of cancer, such as colorectal and prostate cancer. However, the molecular mechanisms and clinical value of EZH2 in lung cancer have not yet been fully investigated. The aim of the present study was to investigate the functions of EZH2 in lung cancer progression and to determine whether treatment with an EZH2 inhibitor enhanced the chemosensitivity of lung cancer cells to cisplatin (CDDP). At the logarithmic growth phase, A549 cells were treated with a small interfering (si)RNA-EZH2, and cell viability was detected using an MTT assay. The degree of apoptosis and cell cycle were detected using flow cytometry. Cell migration and invasion were detected via wound healing and Transwell Matrigel assays. According to information from the Gene Expression Omnibus database, the results of the present study demonstrated that EZH2 was upregulated in lung cancer. Furthermore, overexpression of EZH2 was associated with poor patient prognosis, while EZH2 knockdown inhibited cell viability and migration, and enhanced apoptosis and chemosensitivity in a lung cancer cell line. EZH2 knockdown and treatment of A549 cells using EZH2 inhibitor elevated the inhibitory effects of CDDP on cell viability and apoptosis. Western blot and reverse transcription-quantitative PCR analyses were performed to assess the expression levels of relative protein and mRNA, respectively, in A549 cells treated with siRNA-EZH2 or with CDDP. Overall, the results of the present study demonstrated that high EZH2 expression was associated with poor prognosis, accompanied with a potential impairment of migration and viability in lung cancer cells. These findings suggest that EZH2 may act as a candidate molecular target for gene therapy, and treatment with EZH2 inhibitor may be used to increase chemosensitivity to CDDP agents in lung cancer.

Profiling and bioinformatics analysis of differentially expressed circular RNAs in human intervertebral disc degeneration.

The functional changes of nucleus pulposus (NP) cells are considered to be the initiating factors of intervertebral disc degeneration (IDD), and the differentially expressed circRNAs in NP cells may play an important role in the process of IDD. To identify circular RNAs (circRNAs) associated with human IDD, we isolated the NP cells from human degenerated and non-degenerated intervertebral disc and identified NP cells by microscopy and cell proliferation. CircRNA microarray expression profiles were obtained from NP cells of degenerated and non-degenerated intervertebral disc and further validated by quantitative reverse transcription PCR (qRT-PCR). The expression data were analyzed by bioinformatics. Microarray analysis identified 7294 circRNAs differentially expressed in degenerated human IDD NP cells. Among them, 3724 circRNAs were up-regulated and 3570 circRNAs were down-regulated by more than 2 folds. After validating by qRT-PCR, we predicted the possible miRNAs of the top dysregulated circRNAs using TargetScan, and miRanda. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the most modulated circRNAs regulate the viability, degradation, apoptosis and oxidative stress in NP cells, and the possible mechanism underlying IDD was discussed. These results revealed that circRNAs may play a role in IDD and might be a promising candidate molecular target for gene therapy.

Down-regulated REIC expression in lung carcinogenesis: a molecular target for gene therapy.

REIC (Reduced Expression in Immortalized Cells) gene is down-regulated in immortalized cells, compared with the normal parental counterparts. Its encoding protein could inhibit colony formation, tumor growth, and induce apoptosis. To investigate the roles of REIC expression in lung cancer, we examined REIC expression in lung cancer cells and tissues by RT-PCR or Western blot, and observed the effects of both recombinant REIC exposure and REIC overexpression on the aggressive phenotypes of lung cancer cells. It was found that the demethylation of REIC promoter by 5-Aza-dC could reserve its mRNA expression in lung cancer cells (P<0.05). There was a lower REIC mRNA expression in lung cancer than that in matched normal tissue (P<0.05). Recombinant REIC treatment enhanced the proliferation of lung cancer cells (P<0.05), but versa for REIC overexpression (P<0.05). Both recombinant REIC treatment and REIC overexpression induced apoptosis, and inhibited the migration and invasion of SQ-5 and KJ cells (P<0.05). Immunohistochemically, there was a positive correlation between REIC and Caspase-3 expression in lung cancer (P<0.05). According to Kaplan-Meier plotter, REIC mRNA overexpression was found to positively correlate with overall, progression-free and post- progression survival rates of lung cancer patients (P<0.05), even stratified by sex, histological subtyping, grading, TNM staging, chemotherapy, radiotherapy, or smoking. These findings suggested that the down-regulated REIC expression might be involved in lung carcinogenesis due to its promoter methylation. Both recombinant REIC exposure and REIC overexpression might reverse the aggressive phenotypes of lung cancer cells. REIC may be employed as a potential target of gene therapy for lung cancer.

BTBD7 silencing inhibited epithelial- mesenchymal transition (EMT) via regulating Slug expression in human salivary adenoid cystic carcinoma.

Epithelial-mesenchymal transition (EMT) is a complicated process that has been implicated in cancer progression and metastasis as well as the formation of many tissues and organs. BTB/POZ domain-containing protein 7 (BTBD7) is reported to regulate transcriptional factors and involved in the process of invasion and metastasis of some malignant tumors. Additionally, our preliminary studies have confirmed that BTBD7 expression was significantly correlated with Slug expression and poor prognosis of primary salivary adenoid cystic carcinoma (SACC). On this basis, this study further investigated function of BTBD7 in the invasion and metastasis of SACC in vitro, which may be a possible target of gene therapy in the future. The expression of BTBD7 and Slug were both examined in SACC-LM and SACC-83 cell lines by immunofluorescence staining. High invasive SACC-LM cells were transfected with BTBD7 siRNA and the expression levels of BTBD7 and Slug were detected in both gene and protein levels by qRT-PCR and western blot analysis. Assays were performed to survey cell migration, invasion and proliferation capabilities with BTBD7 silencing. BTBD7 and Slug proteins were detected in SACC-LM and SACC-83 cell lines. BTBD7 silencing down-regulated the expression of Slug and MMP9 meanwhile up-regulated the expression of E-cadherin in SACC-LM cells, the migration and invasion abilities of cells were obviously suppressed but with no influence on cell proliferation. BTBD7 silencing inhibited EMT through regulation of Slug expression in SACC-LM cells and might act as a potential molecular target for gene therapy of SACC.

Effect of PPM1H on malignant phenotype of human pancreatic cancer cells.

The objective of this study was to investigate the effect of silencing gene protein phosphatase 1H (PPM1H) on malignant phenotype of human pancreatic cancer cell line BxPC-3. In order to explore the function of PPM1H in pancreatic cancer cells, real-time PCR and western blotting were used to detect the expression of PPM1H in different pancreatic cancer cell lines. Human pancreatic cancer cell line BxPC-3 was treated with 10ng/ml TGF-β1 and 200ng/ml BMP2 for 72h, respectively, and the mRNA and protein expression levels of PPM1H and EMT-related markers (E-cadherin, vimentin) were detected by real-time PCR and western blotting, respectively. Using exogenous RNA interference technology to silence the PPM1H gene, the expression of PPM1H and EMT-related markers at mRNA and protein levels were detected by real-time PCR and western blotting. The cell migration and invasion were measured using Transwell assays. Finally, cell counting kit-8 (CCK-8) and flow cytometry were used to determine the effect of PPM1H on cell proliferation and apoptosis of BxPC-3 cells. The expression levels of PPM1H in all of the examined pancreatic cancer cell lines (BxPC-3, MIA-PACA2, PANC-1, SW1990, PANC-03.27) were lower than that of normal pancreatic ductal epithelial cells (HPDE6-C7) at both mRNA and protein levels. Both TGF-β1 and BMP2 treatment induced EMT and downregulation of PPM1H in BxPC-3 cells. By using RNA interference to transiently knock down PPM1H expression in BxPC-3 cells, we found that the expression of E-cadherin was downregulated while vimentin was up-regulated. The data suggested that silencing PPM1H gene can induce EMT in BxPC-3 cells. In addition, Transwell migration assays showed that silencing PPM1H gene can promote the invasion and metastasis of BxPC-3 cells. Cell proliferation and apotosis detection demonstrated that silencing PPM1H gene can promote the proliferation and inhibit apoptosis of BxPC-3 cells. In conclusion, PPM1H is aberrantly expressed in human pancreatic cancer cell lines and can be downregulated when EMT is induced by cytokine stimulation. Silencing PPM1H gene can induce EMT in BxPC-3 cells, and promote the invasion and metastasis of BxPC-3 cells. Moreover, silencing PPM1H gene can promote the proliferation and inhibit apoptosis of BxPC-3 cells. PPM1H may be a new tumor-suppressor factor for pancreatic cancer and provides new insight into molecular targets for gene therapy of pancreatic cancer.

Viral Etiology Relationship between Human Papillomavirus and Human Breast Cancer and Target of Gene Therapy

ObjectiveTo explore the viral etiology of human breast cancer to determine whether there are novel molecular targets for gene therapy of breast cancer and provide evidence for the research of gene therapy and vaccine development for breast cancer. MethodsPCR was used to screen HPV16 and HPV18 oncogenes E6 and E7 in the SKBR3 cell line and in 76 paraffin embedded breast cancer tissue samples. RNA interference was used to knock down the expression of HPV18 E6 and E7 in SKBR3 cells, then the changes in the expression of cell-cycle related proteins, cell viability, colony formation, metastasis, and cell cycle progression were determined. ResultsHPV18 oncogenes E6 and E7 were amplified and sequenced from the SKBR3 cells. Of the patient samples, 6.58% and 23.68% were tested to be positive for HPV18 E6 and HPV18 E7. In the cell culture models, the knockdown of HPV18 E6 and E7 inhibited the proliferation, metastasis, and cell cycle progression of SKBR3 cell. The knockdown also clearly affected the expression levels of cell cycle related proteins. ConclusionHPV was a contributor to virus caused human breast cancer, suggesting that the oncogenes in HPV were potential targets for gene therapy of breast cancer.

Anti-proliferation effects of Twist gene silencing in gastric cancer SGC7901 cells

To study the role of Twist gene in gastric cancer by gene silencing, including the potential of induction of apoptosis, cell cycle arrest, and proliferation inhibition in human malignant gastric SGC7901 cells. The expression level of Twist in gastric cancer samples was measured by immunohistochemistry. The effects of Twist gene silencing were detected at both mRNA and protein levels by RT-PCR and Western blot. We also evaluated the cell proliferation and apoptosis by CCK-8 assay and flow cytometry. We determined the activity of caspase-3 and caspase-9 with a caspase activity assay kit. Cell cycle distribution was analyzed by flow cytometry. Cell migration and invasion ability was evaluated by wound scratch assay and Boyden chamber assay. Twist protein was highly expressed in gastric cancer samples. Twist gene silencing significantly induced apoptosis, cell cycle arrest at G0/G1 phase, proliferation inhibition, and reduced the ability of migration and invasion in human gastric cancer SGC7901 cells. Meanwhile, both caspase-3 and caspase-9 were activated. The Twist gene could serve as a potential molecular target for gene therapy of gastric cancer with targeted small interfering RNA.

Lentivirus Mediated GOLPH3 shRNA Inhibits Growth and Metastasis of Esophageal Squamous Cancer

To investigate the role of Golgi phosphoprotein 3 (GOLPH3) in tumour growth and metastasis of esophageal squamous cancer. A lentiviral shRNA-vector was utilized to stably knockdown GOLPH3 in Eca-109 esophageal squamous cancer cells. mRNA transcription and protein expression of GOLPH3 were examined by real-time quantitative PCR and Western blotting, respectively. Cell proliferation activity was assessed by MTT assay and invasion and migration potentials by matrigel invasion and transwell motility assays. Stable knockdown in the GOLPH3 cell line was established. PD-A gene expression was significantly suppressed by lentivirus-mediated RNAi, which resulted in reducing the capacity for cell proliferation, migration, invasion and adhesion in vitro. In vivo, GOLPH3 depletion resulted in inhibition of tumour growth, with stable decrease in the expression of GOLPH3 in tumor xenografts. Our findings suggest that lentivirus mediated silencing of the GOLPH3 gene has a significant anti-tumour effect on esophageal squamous cancer in vitro and in vivo. In addition, the results indicate that GOLPH3 might be an effective molecular target for gene therapy in esophageal squamous cancer.

Regulation of cell proliferation and migration in gallbladder cancer by zinc finger X-chromosomal protein

Gallbladder carcinoma (GBC) is one of the mostly aggressive and fatal malignancies. However, little is known about the oncogenic genes that contributed to the development of GBC. Zinc finger X-chromosomal protein (ZFX) was a novel member of the Krueppel C2H2-type zinc-finger protein family and its down-regulation led to impaired cell growth in human laryngeal squamous cell carcinoma. Here, we aim to investigate the function of ZFX in GBC cell proliferation and migration. Loss of function analysis was performed on GBC cell line (GBC-SD) using lentivirus-mediated siRNA against ZFX. The proliferation, in vitro tumorigenesis (colony-formation) ability as well as cell migration was significantly suppressed after GBC-SD cells which were infected with ZFX-siRNA-expressing lentivirus (Lv-shZFX). Our finding suggested that ZFX promoted the growth and migration of GBC cells and could present a potential molecular target for gene therapy of GBC.

Abstract 3917: Regulatory role of ROBO-1, a novel tumor suppressor on Androgen receptor and Wnt signaling during castration-resistant prostate cancer development: A novel molecular target for gene therapy

Abstract Introduction and Objectives: Prostatic epithelial cells while progressing from premalignant stages to malignant stages (hormone-sensitive and castration-resistance phenotypes) undergo a lot of phenotypical changes which include increased clonal expansion; and modulation in cytoskeleton and cell adhesion characteristics. During organogenesis, the Roundabout-1 (ROBO-1) regulates cell proliferation, migration and adhesion. We for the first time (1) established the mechanismαbased role of ROBO1 tumor suppressor gene in the development of castrationαresistant CaP (CRPC) and (2) determined its significance as potential target for gene therapy. Methods: Immunoblot analysis, real time PCR and immunohistochemistry was used to analyze human prostatic tissues and cell lines for ROBO1. ROBOα1 was (i) overαexpressed by transfecting ROBO1αexpressing plasmid (ii) knocked down by employing vectorαbased shRNA. ROBO1αsuppressed and ROBO1αoverexpressing cells were tested for proliferation, clonogenicity, sensitivity towards chemotherapeutic agents, androgen receptor (AR)α binding to target genes, AR and Tcfαtranscriptional activities by employing 3[H]thymidine uptake, softαagar, luciferase reporter and ChIP assays. Results: We provide evidence that mRNA and protein levels of ROBO1 are lost in (i) CRPC cells, ii) prostatic tissues of NKx3.1/Pten mouse model and (ii) human prostatic tissues during the progression of disease from early stage (low) to late stage (highαGleason score). We show that Loss of ROBO-1 was observed to increase (i) AR transcriptional activity, (ii)Tcf-transcriptional activity (marker of Wnt signaling) (iii) accumulation of β-catenin and (iv) Akt activity and (v) proliferation of CRPC cells. Importantly, CRPC cells (which survived therapy) exhibited high potential to proliferate after repopulation when ROBO-1 was silenced. We provide compelling evidence that under ablation conditions ROBO1 is lost in some malignant CaP cells which results in a subtype that is castration-resistant. We show that ROBO-1 is inactivated due to epigenetic changes in CRPC cells under castration-conditions. ROBO-1-deficient CRPC cells were more chemoresistant and exhibited increased AR-activity than ROBO-1-expressing counterparts. ROBO1αexpressing CRPC cells exhibited reduced tumorigenicity as compared to ROBO-1-silenced CRPC cells. Finally, reactivation of ROBO-1 by gene therapy resulted in decreased AR/Wnt signaling and sensitization of CRPC cells to chemotherapy. Conclusions: We suggest that inactivation of ROBO1 during androgen ablation treatment could be one of the possible mechanisms involved in the recurrence of disease in CaP patients. Therefore ROBO-1 offers a potential to be exploited as a molecular target for gene therapy to treat CRPC condition in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3917. doi:1538-7445.AM2012-3917

Anti-Inflammatory Gene Therapy for Cardiovascular Disease

Inflammation in the vascular wall is an essential hallmark during the development of atherosclerosis, for which major leukocytes infiltrated in the lesions are monocytes/macrophages. Therefore, monocyte chemoattractant protein-1 (MCP-1) and its primary receptor CC chemokine receptor 2 (CCR2) are feasible molecular targets for gene therapy to inhibit monocyte/macrophage-mediated inflammation in atherogenesis. A mutant MCP-1 that lacks N-terminal 7 amino acids (7ND) has been shown to heterodimerize with native MCP-1, bind to CCR2 and block MCP-1-mediated monocyte chemotaxis by a dominant-negative manner. Gene therapy using intramuscular transfection with plasmid DNA encoding 7ND showed inhibitory effects on atherosclerosis in hypercholesterolemic mice, and neointima formation after vascular injury in animal models. Bare metal stents for coronary intervention were coated with multiple thin layers of biocompatible polymer with 7ND plasmid. The 7ND gene-eluting stent inhibited macrophage infiltration surrounding stent struts and in-stent neointima formation in rabbit femoral arteries and cynomolgus monkey iliac arteries. Finally, the authors describe new application of 7ND plasmid encapsulated in polymer nanoparticle (NP) that functions as gene delivery system with unique in vivo kinetics. NP-mediated 7ND gene delivery inhibited MCP-1-induced chemotaxis of mouse peritoneal macrophage ex vivo, which may be applicable for the treatment of atherosclerotic cardiovascular disease. In conclusion, anti-inflammatory gene therapy targeting MCP-1/CCR2 signal, with a novel NP-mediated gene delivery system, is a potent therapeutic strategy for the treatment of cardiovascular diseases.

The Potential of Osteopontin as a Therapeutic Target for Human Colorectal Cancer

ObjectiveOsteopontin (OPN), a phosphorylated glycoprotein, is involved in tumor progression and metastasis. Previously, we have reported that high OPN mRNA expression level possessed clinicopathological or prognostic significance in human colorectal cancer (CRC). The aim of this study is to investigate whether OPN can serve as a novel molecular target for CRC therapy. Material and MethodsWestern Blot assay was performed to detect the expression of OPN protein in 18 CRC and corresponding nontumor colon tissue samples. RNA interference (RNAi) was employed to knockdown endogenous OPN expression in CRC cell line (LoVo). MTT, colony formation, and tumorigenicity assays were performed to analyze the effect of OPN downregulation on the in vitro and in vivo proliferation of CRC cells. Wound healing and Matrigel invasion assays were performed to analyze the effect of OPN downregulation on migration and invasion of CRC cells. A clonogenic cell survival assay after radiation was performed to analyze the effect of OPN downregulation on the radiosensitivity of CRC cells. ResultsThe relative level of OPN protein expression in CRC tissues was significantly higher than that in corresponding nontumor colon tissues (P < 0.05). We found that RNAi-mediated OPN downregulation could inhibit not only in vitro proliferation but also in vivo tumorigenicity of CRC cells. In addition, OPN downregulation could suppress in vitro invasion capacity and enhance in vitro radiosensitivity of CRC cells, which might be associated with decreased levels of MMP-2 and -9 expression. ConclusionRNAi-targeting OPN could inhibit proliferation, invasion and enhance radiosensitivity of human CRC cells. Therefore, OPN could serve as a novel molecular target for gene therapy of CRC.

Clinical value of signal transducers and activators of transcription 3 (STAT3) gene expression in human osteosarcoma

The dysregulation of signal transducers and activators of transcription 3 (STAT3) has been reported to be associated with tumor progression, angiogenesis and metastasis. The purpose of this study was to analyze the clinical value of STAT3 expression in human osteosarcoma. First, semi-quantitative RT-PCR was performed to detect the expression of STAT3 mRNA in normal bone tissues, chondroma tissues and osteosarcoma tissues. Then, immunohistochemistry was performed to detect the expression of STAT3 protein in 76 osteosarcoma tissues and the relationship of STAT3 protein expression with clinicopathologic factors or prognosis of osteosarcoma patients. RNA interference (RNAi) technology was employed to inhibit STAT3 expression. MTT and flow cytometric assays were performed to analyze the effect of STAT3 inhibition on proliferation and apoptosis of osteosarcoma cells. Finally, the expression of STAT3-related target genes were also determined. Results showed that osteosarcoma tissues showed significantly higher expression levels of STAT3 mRNA than normal bone or chondroma tissues ( P < 0.05). Immunohistochemistry showed that the staining of STAT3 protein was mainly located in cytoplasm of osteosarcoma cells in osteosarcoma tissue samples. The high level of STAT3 protein was associated with poor tumor differentiation and presentation of metastasis ( P = 0.039 and 0.022). Moreover, the 5-year overall and relapse-free survival rates for osteosarcoma patients with high STAT3 expression were lower than those for patients with low STAT3 expression. In addition, the status of STAT3 protein expression was an independent prognostic factor for both disease-free survival ( P = 0.0235) and overall survival ( P = 0.0032). RNAi-mediated STAT3 inhibition could induce proliferation inhibition and apoptosis enhancement in osteosarcoma cells, which might be associated with inhibition of some anti-apoptosis genes. Overall, STAT3 plays crucial roles in osteosarcoma development and might become a potential molecular target for gene therapy of human osteosarcomas.

RNAi-mediated down-regulation of α-actinin-4 decreases invasion potential in oral squamous cell carcinoma

alpha-actinin-4, originally identified as an actin-binding protein associated with cell motility, invasion, and metastasis of cancer cells, appears to be overexpressed in various human epithelial carcinomas, including colorectal, breast, esophageal, ovarian, and non-small cell lung carcinomas. The authors evaluated whether alpha-actinin-4 might be appropriate as a molecular target for cancer gene therapy. In 64 primary oral squamous cell carcinomas (OSCCs) and 10 normal oral mucosal specimens, and in seven human OSCC cell lines, alpha-actinin-4 expression was evaluated immunologically and correlations with clinicopathologic factors were examined. Overexpression of alpha-actinin-4 was detected in 38 of 64 oral squamous cell carcinomas (70%); significantly more frequently than in normal oral mucosa. The expression of alpha-actinin-4 was significantly associated with invasion potential defined by the Matrigel invasion assay. Cancer cell lines with higher alpha-actinin-4 expression had greater invasive potential. An RNAi-mediated decrease in alpha-actinin-4 expression reduced the invasion potential. These results indicated that the overexpression of alpha-actinin-4 was associated with an aggressive phenotype of OSCC. The study indicated that alpha-actinin-4 could be a potential molecular target for gene therapy by RNAi targeting for OSCC.

294. Mesothelin, a Novel Molecular Target for Gene Therapy of Cancer

294. Mesothelin, a Novel Molecular Target for Gene Therapy of Cancer