Yu-Hung Huang

Claudin-3 gene silencing with siRNA suppresses ovarian tumor growth and metastasis

Claudin-3 (CLDN3) is a tight junction protein that is overexpressed in 90% of ovarian tumors. Previous in vitro studies have indicated that CLDN3 overexpression promotes the migration, invasion, and survival of ovarian cancer cells. Here, we investigated the efficacy of lipidoid-formulated CLDN3 siRNA in 3 different ovarian cancer models. Intratumoral injection of lipidoid/CLDN3 siRNA into OVCAR-3 xenografts resulted in dramatic silencing of CLDN3, significant reduction in cell proliferation, reduction in tumor growth, and a significant increase in the number of apoptotic cells. Intraperitoneal injection of lipidoid-formulated CLDN3 siRNA resulted in a substantial reduction in tumor burden in MISIIR/TAg transgenic mice and mice bearing tumors derived from mouse ovarian surface epithelial cells. Ascites development was reduced in CLDN3 siRNA-treated mice, suggesting the treatment effectively suppressed metastasis. Toxicity was not observed after multiple i.p. injections. Importantly, treatment of mice with nonimmunostimulatory 2′-OMe modified CLDN3 siRNA was as effective in suppressing tumor growth as unmodifed siRNA. These results suggest that lipidoid-formulated CLDN3 siRNA has potential as a therapeutic for ovarian cancer.

Rapid Optimization of Gene Delivery by Parallel End-modification of Poly(β-amino ester)s

Poly(β-amino ester)s are cationic degradable polymers that have significant potential as gene delivery vectors. Here we present a generalized method to modify poly(β-amino ester)s at the chain ends to improve their delivery performance. End-chain coupling reactions were developed so that polymers could be synthesized and tested in a high-throughput manner, without the need for purification. In this way, many structural variations at the polymer terminus could be rapidly evaluated. End-modification of the terminal amine structure of a previously optimized poly(β-amino ester), C32, significantly enhanced its in vitro transfection efficiency. In vivo, intraperitoneal (IP) gene delivery using end-modified C32 polymers resulted in expression levels over one order of magnitude higher than unmodified C32 and jet-polyethylenimine (jet-PEI) levels in several abdominal organs. The rapid end-modification strategy presented here has led to the discovery of many effective polymers for gene delivery and may be a useful method to develop and optimize cationic polymers for gene therapy.

Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells.

Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the mRNA-binding protein HuR (ELAVL1) is elevated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR’s role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeted inhibition of HuR may be a novel, promising approach to the treatment of PDA.

Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth

Growing evidence shows that cancer cells use mRNA-binding proteins and miRNAs to posttranscriptionally regulate signaling pathways to adapt to harsh tumor microenvironments. In ovarian cancer, cytoplasmic accumulation of mRNA-binding protein HuR (ELAVL1) is associated with poor prognosis. In this study, we observed high HuR expression in ovarian cancer cells compared with ovarian primary cells, providing a rationale for targeting HuR. RNAi-mediated silencing of HuR in ovarian cancer cells significantly decreased cell proliferation and anchorage-independent growth, and impaired migration and invasion. In addition, HuR-depleted human ovarian xenografts were smaller than control tumors. A biodistribution study showed effective tumor-targeting by a novel Cy3-labeled folic acid (FA)-derivatized DNA dendrimer nanocarrier (3DNA). We combined siRNAs against HuR with FA-3DNA and found that systemic administration of the resultant FA-3DNA-siHuR conjugates to ovarian tumor-bearing mice suppressed tumor growth and ascites development, significantly prolonging lifespan. NanoString gene expression analysis identified multiple HuR-regulated genes that function in many essential cellular and molecular pathways, an attractive feature of candidate therapeutic targets. Taken together, these results are the first to demonstrate the versatility of the 3DNA nanocarrier for in vivo-targeted delivery of a cancer therapeutic and support further preclinical investigation of this system adapted to siHuR-targeted therapy for ovarian cancer.

Abstract 2: Naked CanScript, an 18-base pair sequence, has tumor cell-specific promoter activity in vivo: Implications for targeted gene therapy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The MSLN gene encodes mesothelin and is highly active in several human malignancies including ovarian, pancreatic, bronchial, gastroesophageal, cervical, endometrial, and biliary carcinomas. The high cancer cell-specific activity of MSLN is attributable to an 18-bp upstream enhancer, CanScript, which selectively elevates transcription upon binding of TEF-1, a transcription enhancer factor, to a MCAT motif. It has previously been shown that three concatemerized copies of CanScript produce a 30-fold enhancement over a SV40 minimal promoter in MSLN-expressing cancer cells (Cancer Res. 2007 Oct 1;67(19):9055-65). To determine whether incorporation of CanScript into gene constructs may have application in enhancing activity of promoters used in cancer-targeting gene therapy strategies, we generated several luciferase reporter gene constructs having different cell type-specific promoters (MSLN, HE-4, PSA) and different copy numbers of CanScript (1X, 2X, 3X, 6X). We transfected multiple cell lines, including MSLN+ (ovarian and pancreatic) and MSLN− (liver, lung, and prostate) tumor cells, with these DNA constructs, and measured luciferase activity 48 hrs later. To replicate an in vivo therapeutic setting, we used nanoparticles to deliver the constructs to the peritoneal space of healthy and ovarian-tumor bearing mice, and measured gene expression in tumors and multiple organs by optical imaging. Surprisingly, we determined that an 18-bp CanScript sequence activates gene expression in a cell-specific manner in the absence of any other promoter sequence, and that amplification of CanScript in combination with either the MSLN or HE4 promoter sequences, i.e., two gene promoters that are highly active in ovarian cancer cells, significantly enhances their activity proportional to the number of copies of CanScript, in MSLN+ cells, including MSLN+ HE4− pancreatic cells, but has little effect in MSLN− cells. These results support the use of CanScript when increased, specific gene expression is desired to improve therapeutic efficacy in tumor cells in which MSLN is highly active. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2.

Insights from HuR biology point to potential improvement for second-line ovarian cancer therapy

This retrospective study aimed to investigate the role that an RNA-binding protein, HuR, plays in the response of high-grade serous ovarian tumors to chemotherapeutics. We immunohistochemically stained sections of 31 surgically-debulked chemo-naïve ovarian tumors for HuR and scored the degree of HuR cytoplasmic staining. We found no correlation between HuR intracellular localization in tumor sections and progression free survival (PFS) of these patients, 29 of whom underwent second-line gemcitabine/platin combination therapy for recurrent disease. Ribonucleoprotein immunoprecipitation (RNP-IP) analysis of ovarian cancer cells in culture showed that cytoplasmic HuR increases deoxycytidine kinase (dCK), a metabolic enzyme that activates gemcitabine. The effects of carboplatin treatment on HuR and WEE1 (a mitotic inhibitor) expression, and on cell cycle kinetics, were also examined. Treatment of ovarian cancer cells with carboplatin results in increased HuR cytoplasmic expression and elevated WEE1 expression, arresting cell cycle G2/M transition. This may explain why HuR cytoplasmic localization in chemo-naïve tumors is not predictive of therapeutic response and PFS following second-line gemcitabine/platin combination therapy. These results suggest treatment of recurrent ovarian tumors with a combination of gemcitabine, carboplatin, and a WEE1 inhibitor may be potentially advantageous as compared to current clinical practices.

CanScript, an 18-Base pair DNA sequence, boosts tumor cell-specific promoter activity: implications for targeted gene therapy.

Gene therapy protocols for the treatment of cancer often employ gene promoter sequences that are known to be over-expressed in specific tumor cell types relative to normal cells. These promoters, while specific, are often weakly active. It would be desirable to increase the activity of such promoters, while at the same time retain specificity, so that the therapeutic gene is more robustly expressed. Using a luciferase reporter DNA construct in both in vitro cell transfection assays and in vivo mouse tumor models, we have determined that in the absence of any other DNA sequence, a previously identified 18-base pair enhancer sequence called CanScript, lying upstream of the MSLN gene, has ~25% of the promoter activity of CAG, a very strong non-specific promoter/enhancer, in tumor cells in which MSLN is highly expressed. Furthermore, tandem repeat copies of CanScript enhance transcription in a dose-dependent manner and, when coupled with promoter sequences that are active in tumor cells, increase promoter activity. These findings suggest that the incorporation of CanScript into gene constructs may have application in enhancing activity of promoters used in cancer-targeting gene therapy strategies, thereby improving therapeutic efficacy.

Abstract 3542: Inhibition of HuR effectively suppresses ovarian tumor growth in mice

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Ovarian cancer is the most deadly gynecologic cancer. To address the clinical need for a more effective therapy than currently exists, this investigation focused on targeting a post-transcriptional (RNA to protein) regulatory RNA-binding protein, HuR (Human antigen R, aka ELAVL1). HuR regulates the expression of multiple genes known to function in tumor cell survival and in drug resistance. It is abundant and hyper-functional in ovarian cancer cells as compared to normal cells, thus providing a therapeutic window. We investigated the effect of inhibiting HuR expression in ovarian tumor cells in culture and in ovarian tumors in mice. We generated A2780 and OVCAR5 cells that constitutively express either shHuR or a control shRNA, and assayed cell proliferation and the ability of these cells to produce colonies in a soft agar. Cell proliferation in both shRNA-expressing cell lines was significantly reduced compared to control shRNA-expressing cells (19% and 11% in A2780 and OVCAR5, respectively; p<0.005). A2780-shRNA cells produced 65% fewer colonies in soft agar a compared to parental A2780 cells and control shRNA-expressing cells. To evaluate the effect of HuR suppression on tumor growth, C57BL/6 female mice were injected intraperitoneally with ID8-luc murine ovarian cancer cells; mice developed tumors throughout the peritoneal cavity. Approximately 4 weeks following cell injection, tumor-bearing mice were optically imaged to establish baseline biolumiscence, an indicator of ID8-luc-derived tumor load. A novel CpG-free DNA dendrimer vector, 3DNA, was derivatized with folate, aiming for tumor-targeted siHuR delivery upon systemic administration to mice. Mice were treated twice a week for four weeks (retro-orbital injection) with one of three 3DNA formulations or with 0.9% saline (n = 5-6/group). The 3DNA formulations were: 1) Folate-derivatized 3DNA/siHuR; 2) 3DNA/siHuR; and 3) Folate-derivatized 3DNA/control siRNA. Tumor response to treatment was assessed once a week by optical imaging. Using baseline tumor load as the comparator, tumor growth was suppressed ∼3-fold in mice treated for 4 weeks with folate-derivatized 3DNA/siHuR, the highest suppression of all treatment groups, including non-folate derivatized 3DNA/siHuR (p = 0.01), folate-derivatized 3DNA/control siRNA (p = 0.05), and 0.9% saline (p = 0.04). No overt toxicity was observed. A biodistribution study in which CY3-labeled folate-derivatized 3DNA was systemically administered resulted in very high fluorescence in ovarian tumors with little to no fluorescence in normal tissues, thus supporting the tumor-targeting potential of folate and the enhanced therapeutic effect observed in folate-derivatized 3DNA/siHuR treated mice. These results support further pursuit of HuR-targeted therapy as a complementary approach to current, active therapies for ovarian cancer for overcoming drug resistance and inhibiting tumor growth. Citation Format: Janet A. Sawicki, Yu-Hung Huang, Jonathan R. Brody, Robert C. Getts, Kelly Rhodes, Jackie Gerhart. Inhibition of HuR effectively suppresses ovarian tumor growth in mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3542. doi:10.1158/1538-7445.AM2015-3542

Abstract 5133: The RNA-binding protein HuR facilitates proliferation and metastasis in human pancreatic ductal adenocarcinoma

Background: Pancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer-related death in the Unites States, with an overall 5-year survival rate of Methods: Stable MiaPaCa2 cell lines were generated that produce two different shRNAs against the HuR coding sequence (labeled Mia.sh290 and Mia.sh700) in response to doxycycline (DOX) induction. HuR silencing was validated by RT-qPCR and Western blotting. Similarly, a DOX-inducible MiaPaCa2 cell line that overexpresses HuR cDNA (Mia.HuR) was generated and validated. Sustained ∼50% knockdown and 1.5-2-fold overexpression were achieved, respectively. Following characterization of HuR expression in these cell lines under ±DOX conditions, we performed a 7-day cell proliferation (PicoGreen) assay and assessed anchorage-independence over a 4-week period by soft agar colony formation assay. Metastatic potential was measured by Matrigel invasion and in vitro scratch test assays. Results: Upon DOX induction, Mia.sh290 and Mia.sh700 cell lines showed significantly reduced cell proliferation over 7 days (22% and 15% reduction compared to non-induced in sh290 and sh700 respectively, p Conclusion: Taken together with our previous findings, our work demonstrates that manipulation of HuR expression affects key characteristics of tumorigenesis (i.e. proliferation, invasion, and migration). These findings, along with ongoing in vivo work, continue to support the notion that targeting HuR in PDA cells may be a promising therapeutic strategy. Citation Format: Masaya Jimbo, Fernando F. Blanco, Brad Screnci, Gabriela Cosma, Vitali Alexeev, Yu-Hung Huang, Jordan M. Winter, Charles J. Yeo, Janet A. Sawicki, Jonathan R. Brody. The RNA-binding protein HuR facilitates proliferation and metastasis in human pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5133. doi:10.1158/1538-7445.AM2015-5133

Abstract 5630: Nanotherapeutic silencing of HuR: Targeting a tumor-promoting response in ovarian cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL HuR is a RNA-binding protein that post-transcriptionally regulates genes involved in the normal cellular response to cancer-associated stressors (e.g., DNA damage, nutrient depletion, hypoxia, and therapeutic agents). When triggered by stress, HuR translocates from the nucleus to the cytoplasm where it potently influences translation of key tumor promoting mRNAs by mRNA stabilization and direct facilitation of translation. Previously, it has been shown that HuR expression is a prognostic marker in ovarian cancers. Thus, we tested the effects of manipulating HuR expression levels on ovarian tumor growth characteristics and tested the hypothesis that silencing HuR through delivery of an HuR siRNA would be effective in suppressing the growth of ovarian tumors. Following treatment of A2780 ovarian cancer cells in culture with an adenovirus containing the HuR coding sequence, HuR expression was increased by ∼40% above control cells as determined by qRT-PCR. When these cells were plated in soft agar, the Adeno-HuR-treated cells markedly enhanced anchorage independent growth properties compared to control cells treated with an Adeno-GFP virus. When OvCAR-3 xenografts in nude mice were injected with lipidoid-siHuR formulated nanoparticles (2 injections/wk, 7 mg/kg/injection), HuR expression was reduced by 80% as determined by immunoblot analysis of resected tumors, and tumor growth was suppressed 2.6-fold after 3 weeks compared to control tumors (siLuciferase- and PBS-treated) (p = 0.0004). After 4 weeks of treatment, all control mice were sacrificed due to large tumor size. siHuR-treated mice continued to receive 2 injections/week for an additional 4 weeks during which time tumor size was maintained at 4-5X the baseline size. In our patient cohort, we also detected HuR activation (i.e., cytoplasmic HuR positivity) in twenty-four of thirty-four patients (71%), providing evidence that the majority of patients have activated HuR. Mechanistically, in A2780 ovarian cancer cells, we detected HuR binding to tumor-promoting target mRNAs including dCK, DR5, and HuR itself. These data provide evidence that silencing HuR, even as a monotherapeutic strategy, may be a promising therapeutic approach for the treatment of ovarian cancer. Studies combining HuR inhibition with other chemotherapies (including gemcitabine), and studies exploring targeting strategies, are ongoing. This work was supported by the Marsha Rivkin Center for Ovarian Cancer Research. 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 5630. doi:1538-7445.AM2012-5630