Callie R. Merry

Emerging Roles for Long Non-Coding RNAs in Cancer and Neurological Disorders

The recent discovery of thousands of long non-coding (lnc)RNAs in the human genome has prompted investigation of the potential roles of these molecules in human biology and medicine. Indeed, it is now well documented that many lncRNAs are involved in key biological processes, including dosage compensation, genomic imprinting, chromatin regulation, alternative splicing of pre-mRNA, nuclear organization; and potentially many other biological processes, which are yet to be elucidated. Recently, a number of studies have also reported that lncRNAs are dysregulated in a number of human diseases, including several cancers and neurological disorders. Although many of these studies have fallen short of implicating lncRNAs as causative, they suggest potential roles that warrant further in depth investigations. In this review, we discuss the current state of knowledge regarding the roles of lncRNAs in cancer and neurological disorders, and suggest potential future directions in this rapidly emerging field.

Targeting the checkpoint kinase Chk1 in cancer therapy

A paramount objective of the eukaryotic cell division cycle is to overcome numerous internal and external insults to faithfully duplicate the genetic information once per every cycle. This is carried out by elaborate networks of genome surveillance signaling pathways, termed replication checkpoints. Central to replication checkpoints are two protein kinases, the upstream kinase ATR, and its downstream target kinase, Chk1. When the DNA replication process is interrupted, the ATR-Chk1 pathway transmits signals to delay cell cycle progression, and to maintain fork viability so that DNA duplication can resume after the initial damage is corrected. Previous studies showed that replicative stress not only activated Chk1, but also triggered the ubiquitin-dependent destruction of Chk1 in cultured human cells. In a recent study, we identified the F-box protein, Fbx6, as the mediator that regulates Chk1 ubiquitination and degradation in both normally cycling cells and during replication stress. We further showed that expression levels of Chk1 and Fbx6 exhibited an overall inverse correlation in both cultured cancer cell lines and in breast tumor tissues, and that defects in Chk1 degradation, for instance, due to reduced expression of Fbx6, rendered tumor cells resistant to anticancer treatment. Here we highlight those findings and their implications in the replication checkpoint and cellular sensitivity to cancer therapies.

DNMT1-associated long non-coding RNAs regulate global gene expression and DNA methylation in colon cancer.

The cancer epigenome exhibits global loss of DNA methylation, which contributes to genomic instability and aberrant gene expression by mechanisms that are yet to be fully elucidated. We previously discovered over 3300 long non-coding (lnc)RNAs in human cells and demonstrated that specific lncRNAs regulate gene expression via interactions with chromatin-modifying complexes. Here, we tested whether lncRNAs could also associate with DNA methyltransferases to regulate DNA methylation and gene expression. Using RIP-seq, we identified a subset of lncRNAs that interact with the DNA methyltransferase DNMT1 in a colon cancer cell line, HCT116. One lncRNA, TCONS_00023265, which we named DACOR1 (DNMT1-associated Colon Cancer Repressed lncRNA 1), shows high, tissue-specific expression in the normal colon (including colon crypts) but was repressed in a panel of colon tumors and patient-derived colon cancer cell lines. We identified the genomic occupancy sites of DACOR1, which we found to significantly overlap with known differentially methylated regions (DMRs) in colon tumors. Induction of DACOR1 in colon cancer cell lines significantly reduced their ability to form colonies in vitro, suggesting a growth suppressor function. Consistent with the observed phenotype, induction of DACOR1 led to the activation of tumor-suppressor pathways and attenuation of cancer-associated metabolic pathways. Notably, DACOR1 induction resulted in down-regulation of Cystathionine β-synthase, which is known to lead to increased levels of S-adenosyl methionine-the key methyl donor for DNA methylation. Collectively, our results demonstrate that deregulation of DNMT1-associated lncRNAs contributes to aberrant DNA methylation and gene expression during colon tumorigenesis.

Identification of mRNAs and lincRNAs associated with lung cancer progression using next-generation RNA sequencing from laser micro-dissected archival FFPE tissue specimens

OBJECTIVES Adenocarcinoma in situ (AIS) is an intermediate step in the progression of normal lung tissue to invasive adenocarcinoma. However, molecular mechanisms underlying this progression remain to be fully elucidated due to challenges in obtaining fresh clinical samples for downstream analyses. Formalin fixation and paraffin embedding (FFPE) is a tissue preservation system widely used for long-term storage. Until recently, challenges in working with FFPE precluded using new RNA sequencing technologies (RNA-seq), which would help clarify key pathways in cancer progression. Also, isolation techniques including laser-capture micro-dissection provide the ability to select histopathologically distinct tissues, allowing researchers to study transcriptional variations between tightly juxtaposed cell and tissue types. MATERIALS AND METHODS Utilizing these technologies and new alignment tools we examined differential expression of long intergenic non-coding RNAs (lincRNAs) and mRNAs across normal, AIS and invasive adenocarcinoma samples from six patients to identify possible markers of lung cancer progression. RESULTS RNA extracted and sequenced from these 18 samples generated an average of 198 million reads per sample. After alignment and filtering, uniquely aligned reads represented an average 35% of the total reads. We detected differential expression of a number of lincRNAs and mRNAs when comparing normal to AIS, or AIS to invasive adenocarcinoma. Of these, 5 lincRNAs and 31 mRNAs were consistently up- or down-regulated from normal to AIS and more so to invasive carcinoma. We validated the up-regulation of two mRNAs and one lincRNA by RT-qPCR as proof of principle. CONCLUSION Our findings indicate a potential role of not only mRNAs, but also lincRNAs in the progression to invasive adenocarcinoma. We anticipate that these findings will lay the groundwork for future experimental studies of candidate RNAs from FFPE to identify their functional roles in lung cancer.

Transcriptome-wide identification of mRNAs and lincRNAs associated with trastuzumab-resistance in HER2-positive breast cancer

Approximately, 25–30% of early-stage breast tumors are classified at the molecular level as HER2-positive, which is an aggressive subtype of breast cancer. Amplification of the HER2 gene in these tumors results in a substantial increase in HER2 mRNA levels, and consequently, HER2 protein levels. HER2, a transmembrane receptor tyrosine kinase (RTK), is targeted therapeutically by a monoclonal antibody, trastuzumab (Tz), which has dramatically improved the prognosis of HER2-driven breast cancers. However, ~30% of patients develop resistance to trastuzumab and recur; and nearly all patients with advanced disease develop resistance over time and succumb to the disease. Mechanisms of trastuzumab resistance (TzR) are not well understood, although some studies suggest that growth factor signaling through other receptors may be responsible. However, these studies were based on cell culture models of the disease, and thus, it is not known which pathways are driving the resistance in vivo. Using an integrative transcriptomic approach of RNA isolated from trastuzumab-sensitive and trastuzumab-resistant HER2+ tumors, and isogenic cell culture models, we identified a small set of mRNAs and lincRNAs that are associated with trastuzumab-resistance (TzR). Functional analysis of a top candidate gene, S100P, demonstrated that inhibition of S100P results in reversing TzR. Mechanistically, S100P activates the RAS/MEK/MAPK pathway to compensate for HER2 inhibition by trastuzumab. Finally, we demonstrated that the upregulation of S100P appears to be driven by epigenomic changes at the enhancer level. Our current findings should pave the path toward new therapies for breast cancer patients.

Characterization of a Novel Two-Component System in Burkholderia cenocepacia

AbstractTwo-component systems are important regulatory systems that allow bacteria to adjust to environmental conditions, and in some bacteria are used in pathogenesis. We identified a novel two-component system in Burkholderia cenocepacia, an opportunistic pathogen that causes pneumonia in cystic fibrosis (CF) patients. The putative operon encodes BceS, a sensor kinase, and BceR, a response regulator. Our studies indicated that the bceR mutant showed a statistically significant decrease in protease, swimming motility, and quorum sensing when compared to the wild-type, but there was no significant difference in phospholipase C activity, swarming, and biofilm formation. In addition, the mutant showed a statistically significant reduction in virulence compared to the wild-type using the alfalfa plant model. Examination of the Burkholderia cepacia complex (a group of organisms that are phenotypically similar, but genotypically distinct) revealed that this system is prevalent in B. ambifaria, B. multivorans, B. vietnamiensis and B. dolosa. Interestingly, all these organisms have been associated with CF patients. The collective results indicate that BceSR influences various activities important in Burkholderia physiology and possibly pathogenesis. This information could be important in the design of novel therapeutics for Burkholderia infections.

Diverse functions and mechanisms of mammalian long noncoding RNAs

Long noncoding RNAs are becoming increasingly appreciated as major players in gene regulation. They have been reported to play diverse roles in many biological processes. Here, we discuss their discovery, features, and known functions in cells. While not comprehensive, this chapter should serve to illustrate the power and promise of studying long noncoding RNAs.

Abstract 1937: Dysregulation of lincRNAs is a major driver of aberrant epigenomes during tumorigenesis

It has now been unequivocally demonstrated that the process of tumorigenesis is driven by both genetic and epigenetic alterations, but the mechanisms of epigenetic dysregulations remain to be elucidated. Work in our laboratory has demonstrated that numerous long intergenic non-coding RNAs (lincRNAs) become dysregulated in human cancers, and a significant subset of these lincRNAs are associated with histone-modifying enzymes and DNA methyltransferases in healthy, non-tumor cells. Based on these observations, we tested an exceptionally novel hypothesis that dysregulation of chromatin-associated lincRNAs is a major mechanism driving cancer-associated aberrant epigenomes. To test this novel concept, we have performed experiments to modulate the expression of specific chromatin-associated lincRNAs in cancer cells, and assessed their impact on the epigenome. Consistent with our hypothesis, modulating the expression of lincRNAs in cancer cells resulted in massive reprogramming of their epigenomes. These epigenomic changes impacted the growth of cancer cells and global gene expression. Mapping the genomic occupancy sites of lincRNAs, and intersection of those sites with altered genomic regions in tumors, further supported direct roles of lincRNAs in modulating chromatin structure. These findings demonstrate key roles of chromatin-associated lincRNAs in suppressing tumorigenesis in normal human tissues by regulating the epigenome. Citation Format: Callie R. Merry, Megan E. Forrest, Sanford Markowitz, Ahmad M. Khalil. Dysregulation of lincRNAs is a major driver of aberrant epigenomes during tumorigenesis. [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 1937.

Abstract 3487: The tumor and circulating transcriptomes in breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Circulating biomarkers can provide a less invasive way to monitor patients and their response to therapies. Recent advances in next generation RNA sequencing have allowed for a comprehensive characterization of the transcribed genome, which extends past protein-coding genes to include non-coding RNAs, important regulators of protein-coding gene expression. While several studies have shown that circulating small non-coding RNAs, specifically microRNAs, have the potential to serve as biomarkers of cancer, little work has been done to study the role of long RNAs as circulating biomarkers. Our goal was to identify the role of mRNAs and long non-coding RNAs as biomarkers of detection and response to therapy for breast cancer. To start, we utilized samples from 13 breast cancer patients whose tumors were biopsied for research prior to and after trastuzumab treatment as part of a clinical trial. RNA was extracted from both the pre- and post-treatment biopsies for each patient. Next generation RNA-sequencing was completed and reads aligned to known mRNAs and lncRNAs. Mean expression levels were inferred and the statistical significance of differences in expression between pre- and post-treatment samples was assessed among the 11 women in the trial that achieved a pathological complete response. We then compared this to the circulating transcriptome from 10 breast cancer patients and 10 age and race matched mammography-screened controls, which provided blood samples for biomarker studies as part of a larger case-control study. RNA was extracted from the plasma on all 20 samples. Next generation RNA-sequencing was done and was aligned to known mRNAs and lncRNAs as in the tumor samples from the clinical trial. Differences in circulating RNA expression between the breast cancer patients and controls was done using a t-test. After exclusion of RNAs that were expressed in <25% of the samples, the expression level of 163 lncRNAs and 1702 mRNAs were associated with treatment (p<0.05). Of the mRNAs associated with treatment response, 9 that were expressed at higher levels before treatment were also increased in the circulation in breast cancer patients, compared to controls (p<0.05). None of the lncRNAs that were altered in response to treatment were statistically significantly differentially expressed in the circulation, although we are limited in our ability to rule this out due to sample size. Validation of these mRNAs in plasma in a larger sample of cases and controls, as well as in patients before and after treatment, is currently ongoing. Our data shows that, as expected, the expression profile of both mRNAs and lncRNAs in breast cancer tissue changes with treatment, and transcriptome profiling of RNA from the plasma suggests that many of these same RNAs are deregulated in the circulation of breast cancer patients. More work will need to be done to see if the circulating transcriptome can be used as a marker for early breast cancer diagnosis or response to treatment. Citation Format: Cheryl L. Thompson, Ahmad M. Khalil, Kristy Miskimen, Callie Merry, Lyndsay Harris. The tumor and circulating transcriptomes in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3487. doi:10.1158/1538-7445.AM2013-3487