Zhenze Zhao

miR-337-3p and Its Targets STAT3 and RAP1A Modulate Taxane Sensitivity in Non-Small Cell Lung Cancers

NSCLC (non-small cell lung cancer) often exhibits resistance to paclitaxel treatment. Identifying the elements regulating paclitaxel response will advance efforts to overcome such resistance in NSCLC therapy. Using in vitro approaches, we demonstrated that over-expression of the microRNA miR-337-3p sensitizes NCI-H1155 cells to paclitaxel, and that miR-337-3p mimic has a general effect on paclitaxel response in NSCLC cell lines, which may provide a novel adjuvant strategy to paclitaxel in the treatment of lung cancer. By combining in vitro and in silico approaches, we identified STAT3 and RAP1A as direct targets that mediate the effect of miR-337-3p on paclitaxel sensitivity. Further investigation showed that miR-337-3p mimic also sensitizes cells to docetaxel, another member of the taxane family, and that STAT3 levels are significantly correlated with taxane resistance in lung cancer cell lines, suggesting that endogenous STAT3 expression is a determinant of intrinsic taxane resistance in lung cancer. The identification of a miR-337-3p as a modulator of cellular response to taxanes, and STAT3 and RAP1A as regulatory targets which mediate that response, defines a novel regulatory pathway modulating paclitaxel sensitivity in lung cancer cells, which may provide novel adjuvant strategies along with paclitaxel in the treatment of lung cancer and may also provide biomarkers for predicting paclitaxel response in NSCLC.

miR-93-directed downregulation of DAB2 defines a novel oncogenic pathway in lung cancer.

The disabled homolog 2 (DAB2) gene was recently identified as a tumor suppressor gene with its expression downregulated in multiple cancer types. The role of DAB2 in lung tumorigenesis, however, is not fully characterized, and the mechanisms of DAB2 dysregulation in lung cancer are not defined. Here we show that low DAB2 levels in lung tumor specimens are significantly correlated with poor patient survival, and that DAB2 overexpression significantly inhibits cell growth in cultured lung cancer cells, indicating its potent tumor suppressor function. We next identify that microRNA miR-93 functions as a potent repressor of DAB2 expression by directly targeting the 3′UTR of the DAB2 mRNA. Using in vitro and in vivo approaches, we demonstrate that miR-93 overexpression has an important role in promoting lung cancer cell growth, and that its oncogenic function is primarily mediated by downregulating DAB2 expression. Our clinical investigations further indicate that high tumor levels of miR-93 are correlated with poor survival of lung cancer patients. The correlations of both low DAB2 and high miR-93 expression levels with poor patient survival strongly support the critical role of the miR-93/DAB2 pathway in determining lung cancer progression.

Genetic Mutation of p53 and Suppression of the miR-17∼92 Cluster Are Synthetic Lethal in Non–Small Cell Lung Cancer due to Upregulation of Vitamin D Signaling

Lung cancer is the leading cause of cancer-related fatalities. Recent success developing genotypically targeted therapies, with potency only in well-defined subpopulations of tumors, suggests a path to improving patient survival. We used a library of oligonucleotide inhibitors of microRNAs, a class of posttranscriptional gene regulators, to identify novel synthetic lethal interactions between miRNA inhibition and molecular mechanisms in non-small cell lung cancer (NSCLC). Two inhibitors, those for miR-92a and miR-1226*, produced a toxicity distribution across a panel of 27 cell lines that correlated with loss of p53 protein expression. Notably, depletion of p53 was sufficient to confer sensitivity to otherwise resistant telomerase-immortalized bronchial epithelial cells. We found that both miR inhibitors cause sequence-specific downregulation of the miR-17∼92 polycistron, and this downregulation was toxic only in the context of p53 loss. Mechanistic studies indicated that the selective toxicity of miR-17∼92 polycistron inactivation was the consequence of derepression of vitamin D signaling via suppression of CYP24A1, a rate-limiting enzyme in the 1α,25-dihydroxyvitamin D3 metabolic pathway. Of note, high CYP24A1 expression significantly correlated with poor patient outcome in multiple lung cancer cohorts. Our results indicate that the screening approach used in this study can identify clinically relevant synthetic lethal interactions and that vitamin D receptor agonists may show enhanced efficacy in p53-negative lung cancer patients.

microRNA-449a functions as a tumor suppressor in neuroblastoma through inducing cell differentiation and cell cycle arrest.

microRNA-449a (miR-449a) has been identified to function as a tumor suppressor in several types of cancers. However, the role of miR-449a in neuroblastoma has not been intensively investigated. We recently found that the overexpression of miR-449a significantly induces neuroblastoma cell differentiation, suggesting its potential tumor suppressor function in neuroblastoma. In this study, we further investigated the mechanisms underlying the tumor suppressive function of miR-449a in neuroblastoma. We observed that miR-449a inhibits neuroblastoma cell survival and growth through 2 mechanisms—inducing cell differentiation and cell cycle arrest. Our comprehensive investigations on the dissection of the target genes of miR-449a revealed that 3 novel targets- MFAP4, PKP4 and TSEN15 -play important roles in mediating its differentiation-inducing function. In addition, we further found that its function in inducing cell cycle arrest involves down-regulating its direct targets CDK6 and LEF1. To determine the clinical significance of the miR-449a-mediated tumor suppressive mechanism, we examined the correlation between the expression of these 5 target genes in neuroblastoma tumor specimens and the survival of neuroblastoma patients. Remarkably, we noted that high tumor expression levels of all the 3 miR-449a target genes involved in regulating cell differentiation, but not the target genes involved in regulating cell cycle, are significantly correlated with poor survival of neuroblastoma patients. These results suggest the critical role of the differentiation-inducing function of miR-449a in determining neuroblastoma progression. Overall, our study provides the first comprehensive characterization of the tumor-suppressive function of miR-449a in neuroblastoma, and reveals the potential clinical significance of the miR-449a-mediated tumor suppressive pathway in neuroblastoma prognosis.

A high-throughput screen identifies miRNA inhibitors regulating lung cancer cell survival and response to paclitaxel.

microRNAs (miRNAs) are small RNAs endogenously expressed in multiple organisms that regulate gene expression largely by decreasing levels of target messenger RNAs (mRNAs). Over the past few years, numerous studies have demonstrated critical roles for miRNAs in the pathogenesis of many cancers, including lung cancer. Cellular miRNA levels can be easily manipulated, showing the promise of developing miRNA-targeted oligos as next-generation therapeutic agents. In a comprehensive effort to identify novel miRNA-based therapeutic agents for lung cancer treatment, we combined a high-throughput screening platform with a library of chemically synthesized miRNA inhibitors to systematically identify miRNA inhibitors that reduce lung cancer cell survival and those that sensitize cells to paclitaxel. By screening three lung cancer cell lines with different genetic backgrounds, we identified miRNA inhibitors that potentially have a universal cytotoxic effect on lung cancer cells and miRNA inhibitors that sensitize cells to paclitaxel treatment, suggesting the potential of developing these miRNA inhibitors as therapeutic agents for lung cancer. We then focused on characterizing the inhibitors of three miRNAs (miR-133a/b, miR-361-3p, and miR-346) that have the most potent effect on cell survival. We demonstrated that two of the miRNA inhibitors (miR-133a/b and miR-361-3p) decrease cell survival by activating caspase-3/7-dependent apoptotic pathways and inducing cell cycle arrest in S phase. Future studies are certainly needed to define the mechanisms by which the identified miRNA inhibitors regulate cell survival and drug response, and to explore the potential of translating the current findings into clinical applications.

A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells

MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3′UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation.

miR-195 targets cyclin D3 and survivin to modulate the tumorigenesis of non-small cell lung cancer

AbstractmiR-195 has recently been reported to function as a tumor suppressor in various cancers, including non-small cell lung cancer (NSCLC). However, the mechanisms by which miR-195 represses the tumorigenesis of NSCLC cells are not fully understood. We performed a high-throughput screen using an miRNA mimic library and confirmed the identification of miR-195 as a tumor suppressor in NSCLC. We demonstrated that overexpression or induced expression of miR-195 in lung tumors slows tumor growth and that repression of miR-195 accelerates tumor growth. In addition, we found that knockout of miR-195 promotes cancer cell growth. We demonstrated that miR-195 targets cyclin D3 to cause cell cycle arrest at the G1 phase and that miR-195 targets survivin to induce apoptosis and senescence in NSCLC cells. Overexpression of cyclin D3 or survivin reverses the effects of miR-195 in NSCLC cells. Through the analysis of data from The Cancer Genome Atlas, we confirmed that the expression of miR-195 is lower in tumors than in adjacent normal tissues and that low expression of miR-195 is associated with poor survival in both lung adenocarcinoma and squamous cell carcinoma patients. Specifically, we found that BIRC5, which codes for survivin, is upregulated in both adenocarcinoma and squamous cell carcinoma tissues and that high expression of BIRC5 is associated with poor survival in adenocarcinoma, but not squamous cell carcinoma. In addition, the ratio of miR-195 level to BIRC5 level is associated with both recurrence-free and overall survival in lung adenocarcinoma. Our results suggest that the miR-195/BIRC5 axis is a potential target for treatment of lung adenocarcinoma specifically, and NSCLC in general.

LMO1 functions as an oncogene by regulating TTK expression and correlates with neuroendocrine differentiation of lung cancer

LMO1 encodes a protein containing a cysteine-rich LIM domain involved in protein–protein interactions. Recent studies have shown that LMO1 functions as an oncogene in several cancer types, including non-small cell lung cancer (NSCLC). However, the function of LMO1 in other histological subtypes of lung cancer, such as small cell lung cancer (SCLC), was not investigated. In analyzing the expression of LMO1 across a panel of lung cell lines, we found that LMO1 expression levels were significantly and dramatically higher in SCLC cells, an aggressive neuroendocrine subtype of lung cancer, relative to NSCLC and normal lung cells. In NSCLC cells, LMO1 mRNA levels were significantly correlated with expression of neuroendocrine differentiation markers. Our in vitro investigations indicated that LMO1 had the general property of promoting cell proliferation in lung cancer cells representing different histological subtypes, suggesting a general oncogenic function of LMO1 in lung cancer. In investigating the clinical relevance of LMO1 as an oncogene, we found that a high tumor level of the LMO1 mRNA was an independent predictor of poor patient survival. These results suggest that LMO1 acts as an oncogene, with expression correlated with neuroendocrine differentiation of lung cancer, and that it is a determinant of lung cancer aggressiveness and prognosis. By combining gene expression correlations with patient survival and functional in vitro investigations, we further identified TTK as mediating the oncogenic function of LMO1 in lung cancer cells.

Abstract 1940: Non-coding RNA regulation of eribulin response in neuroblastoma

Introduction: Neuroblastoma (NB) is the most common cancer in infants and the most common extracranial solid tumor in children, accounting for 15% of all childhood cancer deaths. The overall prognosis for those with high-risk or relapsed disease remains poor despite the standard therapies of surgery, radiation, and high dose chemotherapy. We have previously shown that altering levels of specific microRNAs (miRNAs) can improve cellular response to microtubule-targeting agents (MTAs) and that exposure to MTAs results in dramatic changes in microRNA expression. Methods: Libraries of chemically synthesized ncRNA mimics and inhibitors, including both miRNAs and lncRNAs, are screened to identify ncRNAs that regulate neuroblastoma cell viability or sensitize neuroblastoma cells to specific microtubule-targeting agents. Candidate targets are validated using qRT-PCR, protein quantification, and luciferase reporter assays. The response of neuroblastoma cells to perturbations in candidate ncRNA levels is assessed through flow cytometric analysis of cell cycle phase distribution, density and anisotropy of microtubule bundles, and through colony formation and caspase activation assays, and validated in mouse xenograft models. Networks of miRNAs, mRNAs and lncRNAs are derived from combining complementary cellular response with potential regulatory interactions. Results: We previously reported the identification of miRNAs and lncRNAs that significantly decrease or increase neuroblastoma cell viability. Here, we extend those observations to drug response. We first demonstrate that the microtubule-targeting agent eribulin has both short-term and long-term effects, as reflected by a significant alteration in microtubule structure in response to high doses for short times and arrest at the G(2)/M phase of the cell cycle in response to low doses for long times. We next show that altering intracellular levels of ncRNAs that sensitize cells to eribulin both alters microtubule density and anisotropy and recapitulates the effect of eribulin treatment. Finally, we show that complementarity of effect between miRNAs and lncRNAs is accompanied by potential regulatory interaction. Conclusions: Taken together, our results suggest that the response of neuroblastoma cells to eribulin is mediated by a regulatory network that includes different coding and non-coding RNA species. While these RNAs may have intrinsic value as biomarkers or therapeutic agents, either individually or in combination, the vulnerabilities that they uncover may be exploited with pathway-specific perturbations. This project was supported by NIH R01 CA129632 and CPRIT Training Grant RP140105. Citation Format: Xiuye Ma, Xiaojie Yu, Harsh Patolia, Zhenze Zhao, Liqin Du, Alexander Pertsemlidis. Non-coding RNA regulation of eribulin response in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1940.

Abstract 1787: Imipramine blue: a novel NOX inhibitor as potent therapeutic agent to treat triple-negative breast cancers

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Breast cancer is the most common cancer in women and the second leading cause of cancer death in US. Triple-negative breast cancers (TNBC) are the most aggressive forms of breast cancer. Almost 60% of patients with TNBCs develop chemo-resistance, leading to early relapse and shorter survival. The family of NADPH oxidases (NOX) enzymes is more abundant source of reactive oxygen species, which generally over expressed in a wide range of cancers including breast cancer. Over expression of NOX family of proteins in the tumor cells and stroma results in the activation of several intracellular pathways that promotes neoplastic transformation. Moreover, NOX is an important metabolic enzyme that plays a critical role in supporting increased glycolysis in cancer cells by generating NAD+, a substrate for one of the key glycolytic reactions. The constitutive upregulation of glycolysis is thought to confer significant growth advantage to cancer cells leading to uncontrolled proliferation and increased invasion. This is especially true for TNBC that shows a strong association with the Warburg effect. Herein we evaluated first time the role of imipramine blue (IB), a NOX inhibitor and a derivative of the FDA approved antidepressant imipramine in TNBC cells growth and progression. Our results demonstrated that imipramine blue inhibits TNBC, MDA-MB-231, MDA-MB-468 and Bt-549 cells growth without affecting normal mammary epithelial (MCF-10A) cells. Notably, our studies revealed that imipramine blue targets multiple NOX family members, which are highly expressed in breast tumors when compared to adjacent normal tissue. Imipramine blue treatment also reduced migration and invasion and inhibited the self-renewal capability of MDA-MB-231 cells. Importantly, imipramine blue treatment significantly reduced experimental lung metastasis of MDA-MB-231 cells in athymic nude mice without induce apparent toxicity. Our gene microarray results further showed that IB treatment significantly altered an array of genes, including FOXM1, Aurora kinase A (AURAK) and Polo-like kinase 1 (PLK1), which play important roles in cancer growth and progression as well as in mediating sensitivity/resistance of paclitaxel (PTX), a chemotherapy drug that is routinely used as a first line treatment for breast cancer patients. Taken together, our findings propose NOX inhibitor imipramine blue as a novel therapeutic agent with less toxicity to treat triple negative breast cancers. Citation Format: Subapriya Rajamanickam, Panneerdoss Subbarayalu, Santhosh Timilsina, Michael T. Drake, Zhenze Zhao, Hung I Harry Chen, Yidong Chen, Jack L. Arbiser, Manjeet K K. Rao. Imipramine blue: a novel NOX inhibitor as potent therapeutic agent to treat triple-negative breast cancers. [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 1787. doi:10.1158/1538-7445.AM2015-1787