Xiuye Ma

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.

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.

5,10b-Ethanophenanthridine amaryllidaceae alkaloids inspire the discovery of novel bicyclic ring systems with activity against drug resistant cancer cells

Plants of the Amaryllidaceae family produce a large variety of alkaloids and non-basic secondary metabolites, many of which are investigated for their promising anticancer activities. Of these, crinine-type alkaloids based on the 5,10b-ethanophenanthridine ring system were recently shown to be effective at inhibiting proliferation of cancer cells resistant to various pro-apoptotic stimuli and representing tumors with dismal prognoses refractory to current chemotherapy, such as glioma, melanoma, non-small-cell lung, esophageal, head and neck cancers, among others. Using this discovery as a starting point and taking advantage of a concise biomimetic route to the crinine skeleton, a collection of crinine analogues were synthetically prepared and evaluated against cancer cells. The compounds exhibited single-digit micromolar activities and retained this activity in a variety of drug-resistant cancer cell cultures. This investigation resulted in the discovery of new bicyclic ring systems with significant potential in the development of effective clinical cancer drugs capable of overcoming cancer chemotherapy resistance.

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 3545: A high-throughput screen identifies microRNAs regulating lung cancer cell survival and response to paclitaxel

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction. microRNAs (miRNAs) are a family of small non-coding RNAs that post-transcriptionally regulate gene expression mainly by decreasing mRNA levels of target genes. Recently, numerous studies have shown the important roles of miRNAs in regulating various cellular physiological and pathological pathways including cell differentiation, proliferation and even tumorigenesis. A large number of miRNAs are indicated to be involved in multiple pathways that are critical for lung cancer, which is the leading cause of adult cancer deaths. Intriguingly, some miRNAs sensitize lung cancer to microtubule-targeting agents, such as paclitaxel. All of these highlight the potential application of miRNA-targeted oligos as next-generation therapeutic agents. Aims and Methods. We aim to identify novel functional miRNAs in lung cancer. Using a high-throughput screening platform containing a library of chemically synthesized miRNAs, we systematically identify miRNAs that reduce lung cancer cell survival or sensitize cancer cells to paclitaxel. The top candidate miRNAs are validated and their mechanisms are further investigated. Results and Conclusions. We show that miR-195 significantly inhibits the viability of lung cancer cells in vitro, and that transfection of miR-195 into cancer cells results in G1 phase arrest and decreased cell invasion. Intriguingly, miR-195 does not show strong inhibitory effects on viability of immortalized human bronchial epithelial cells (HBECs). Analysis of miRNA expression data from The Cancer Genome Atlas (http://cancergenome.nih.gov) indicates lower expression of miR-195 in lung cancer tumors compared to normal tissues. Merging expression with clinical annotation shows that higher expression of miR-195 in lung cancer patients is associated with longer survival. These results indicate that miR-195 is a tumor suppressor miRNA and that miR-195 may be an important factor in determining lung cancer progression and prognosis. This observation is consistent with previous studies that have shown a tumor suppressor function of miR-195 in breast cancer, bladder cancer and hepatocellular carcinoma. Overall, the inhibitory effects on lung cancer cell growth and the correlation between expression and survival indicate the involvement of miR-195 in critical pathways for lung cancer. We have identified additional miRNAs that sensitize lung cancer cells to paclitaxel, and which define a potential regulatory pathway modulating paclitaxel sensitivity. Future studies are needed to identify the targets of these miRNAs, elucidate their mechanisms of action, and clarify how they are regulated in lung cancer cells. Citation Format: Xiaojie Yu, Zhenze Zhao, Xiuye Ma, Liqin Du, Alexander Pertsemlidis. A high-throughput screen identifies microRNAs regulating lung cancer cell survival and response to paclitaxel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3545. doi:10.1158/1538-7445.AM2014-3545

Abstract 466: LMO1 is a novel oncogene in neuroendocrine lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction. The aim of the study is to characterize the role of LMO1 in tumorigenesis of neuroendocrine lung cancer. LMO1 is a transcription factor that belongs to the LMO protein family containing the cysteine-rich LIM domains. LMO1 has been demonstrated to function as an important oncogene in the development of T-lineage leukemia and lymphoma. A recent study in neuroblastoma suggests that aberrant overexpression of LMO1 may play a critical role in the development of neuroendocrine cancers. The role of LMO1 in neuroendocrine lung cancer, however, was not investigated. Experimental Approaches. By analyzing a large panel of cell lines including lung cancer cell lines and normal lung epithelial cells, we compared the relative expression of LMO1 in different subtypes of lung cancers, and examined the correlation of LMO1 expression levels with neuroendocrine differentiation markers. Using in vitro approaches, we investigated the function of LMO1 in regulating lung cancer cell growth. To investigate the clinical significance of LMO1 dysregulation in lung cancer patients, we analyzed the correlation of LMO1 tumor levels with lung cancer patient survival. Results. We show that LMO1 is significantly overexpressed in small cell lung cancer (SCLC), an aggressive and major subtype of neuroendocrine lung cancers, relative to non-small cell lung cancer (NSCLC) and normal lung cells. In addition, in NSCLC cells, LMO1 mRNA levels are significantly correlated with expression of neuroendocrine differentiation markers. Functional in vitro investigations indicate that LMO1 overexpression significantly promotes growth of cultured lung cancer cells, suggesting its oncogenic function in lung cancer. More strikingly, our investigations of two independent lung cancer patient populations indicate that high tumor LMO1 mRNA level is an independent predictor of poor patient survival. Conclusions. Altogether, our findings strongly suggest that LMO1 is a neuroendocrine-specific oncogene in lung cancer that plays an important role in determining the cancer aggressiveness. Further studies are certainly warranted to define the mechanisms underlying the oncogenic function of LMO1 in neuroendocrine lung cancer and to further evaluate the clinical significance of LMO1 as a diagnostic and prognostic marker for neuroendocrine lung cancers in prospective studies. Citation Format: Zhenze Zhao, Xiuye Ma, Xiaojie Yu, Tzu-Hung Hsiao, Yidong Chen, Milind Suraokar, Ignacio Wistuba, John D. Minna, Alexander Pertsemlidis, Liqin Du. LMO1 is a novel oncogene in neuroendocrine lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 466. doi:10.1158/1538-7445.AM2014-466