Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells.

Cancer aggressiveness may result from the selective pressure of a harsh nutrient-deprived microenvironment. Here we illustrate how such conditions promote chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Glucose or glutamine withdrawal resulted in a 5- to 10-fold protective effect with chemotherapy treatment. PDAC xenografts were less sensitive to gemcitabine in hypoglycemic mice compared with hyperglycemic mice. Consistent with this observation, patients receiving adjuvant gemcitabine (n = 107) with elevated serum glucose levels (HgbA1C > 6.5%) exhibited improved survival. We identified enhanced antioxidant defense as a driver of chemoresistance in this setting. ROS levels were doubled in vitro by either nutrient withdrawal or gemcitabine treatment, but depriving PDAC cells of nutrients before gemcitabine treatment attenuated this effect. Mechanistic investigations based on RNAi or CRISPR approaches implicated the RNA binding protein HuR in preserving survival under nutrient withdrawal, with or without gemcitabine. Notably, RNA deep sequencing and functional analyses in HuR-deficient PDAC cell lines identified isocitrate dehydrogenase 1 (IDH1) as the sole antioxidant enzyme under HuR regulation. HuR-deficient PDAC cells lacked the ability to engraft successfully in immunocompromised mice, but IDH1 overexpression in these cells was sufficient to fully restore chemoresistance under low nutrient conditions. Overall, our findings highlight the HuR-IDH1 regulatory axis as a critical, actionable therapeutic target in pancreatic cancer. Cancer Res; 77(16); 4460-71. ©2017 AACR.

IsomiR expression profiles in human lymphoblastoid cell lines exhibit population and gender dependencies.

For many years it was believed that each mature microRNA (miRNA) existed as a single entity with fixed endpoints and a 'static' and unchangeable primary sequence. However, recent evidence suggests that mature miRNAs are more 'dynamic' and that each miRNA precursor arm gives rise to multiple isoforms, the isomiRs. Here we report on our identification of numerous and abundant isomiRs in the lymphoblastoid cell lines (LCLs) of 452 men and women from five different population groups. Unexpectedly, we find that these isomiRs exhibit an expression profile that is population-dependent and gender-dependent. This is important as it indicates that the LCLs of each gender/population combination have their own unique collection of mature miRNA transcripts. Moreover, each identified isomiR has its own characteristic abundance that remains consistent across biological replicates indicating that these are not degradation products. The primary sequences of identified isomiRs differ from the known miRBase miRNA either at their 5´-endpoint (leads to a different 'seed' sequence and suggests a different targetome), their 3´-endpoint, or both simultaneously. Our analysis of Argonaute PAR-CLIP data from LCLs supports the association of many of these newly identified isomiRs with the Argonaute silencing complex and thus their functional roles through participation in the RNA interference pathway.

Argonaute CLIP-Seq reveals miRNA targetome diversity across tissue types.

To date, analyses of individual targets have provided evidence of a miRNA targetome that extends beyond the boundaries of messenger RNAs (mRNAs) and can involve non-Watson-Crick base pairing in the miRNA seed region. Here we report our findings from analyzing 34 Argonaute HITS-CLIP datasets from several human and mouse cell types. Investigation of the architectural (i.e. bulge vs. contiguous pairs) and sequence (Watson-Crick vs. G:U pairs) preferences for human and mouse miRNAs revealed that many heteroduplexes are "non-canonical" i.e. their seed region comprises G:U and bulge combinations. The genomic distribution of miRNA targets differed distinctly across cell types but remained congruent across biological replicates of the same cell type. For some cell types intergenic and intronic targets were more frequent whereas in other cell types mRNA targets prevailed. The findings suggest an expanded model of miRNA targeting that is more frequent than the standard model currently in use. Lastly, our analyses of data from different cell types and laboratories revealed consistent Ago-loaded miRNA profiles across replicates whereas, unexpectedly, the Ago-loaded targets exhibited a much more dynamic behavior across biological replicates.

Donor splice-site mutation in CUL4B is likely cause of X-linked intellectual disability.

X-linked intellectual disability is the most common form of cognitive disability in males. Syndromic intellectual disability encompasses cognitive deficits with other medical and behavioral manifestations. Recently, a large family with a novel form of syndromic X-linked intellectual disability was characterized. Eight of 24 members of the family are male and had cognitive dysfunction, short stature, aphasia, skeletal abnormalities, and minor anomalies. To identify the causative gene(s), we performed exome sequencing in three affected boys, both parents, and an unaffected sister. We identified a haplotype consisting of eight variants located in cis within the linkage region that segregated with affected members in the family. Of these variants, two were novel. The first was at the splice-donor site of intron 7 (c.974+1G>T) in the cullin-RING ubiquitin ligase (E3) gene, CUL4B. This variant is predicted to result in failure to splice and remove intron 7 from the primary transcript. The second variant mapped to the 3'-UTR region of the KAISO gene (c.1127T>G). Sanger sequencing validated the variants in these relatives as well as in three affected males and five carriers. The KAISO gene variant was predicted to create a binding site for the microRNAs miR-4999 and miR-4774; however, luciferase expression assays failed to validate increased targeting of these miRNAs to the variant 3'-UTR. This SNP may affect 3'-UTR structure leading to decreased mRNA stability. Our results suggest that the intellectual disability phenotype in this family is caused by aberrant splicing and removal of intron 7 from CUL4B gene primary transcript.

HuR posttranscriptionally regulates WEE1: implications for the DNA damage response in pancreatic cancer cells.

HuR (ELAV1), an RNA-binding protein abundant in cancer cells, primarily resides in the nucleus, but under specific stress (e.g., gemcitabine), HuR translocates to the cytoplasm in which it tightly modulates the expression of mRNA survival cargo. Here, we demonstrate for the first time that stressing pancreatic ductal adenocarcinoma (PDA) cells by treatment with DNA-damaging anticancer agents (mitomycin C, oxaliplatin, cisplatin, carboplatin, and a PARP inhibitor) results in HuR's translocation from the nucleus to the cytoplasm. Importantly, silencing HuR in PDA cells sensitized the cells to these agents, whereas overexpressing HuR caused resistance. HuR's role in the efficacy of DNA-damaging agents in PDA cells was, in part, attributed to the acute upregulation of WEE1 by HuR. WEE1, a mitotic inhibitor kinase, regulates the DNA damage repair pathway, and therapeutic inhibition of WEE1 in combination with chemotherapy is currently in early phase trials for the treatment of cancer. We validate WEE1 as a HuR target in vitro and in vivo by demonstrating (i) direct binding of HuR to WEE1's mRNA (a discrete 56-bp region residing in the 3' untranslated region) and (ii) HuR siRNA silencing and overexpression directly affects the protein levels of WEE1, especially after DNA damage. HuR's positive regulation of WEE1 increases γ-H2AX levels, induces Cdk1 phosphorylation, and promotes cell-cycle arrest at the G2-M transition. We describe a novel mechanism that PDA cells use to protect against DNA damage in which HuR posttranscriptionally regulates the expression and downstream function of WEE1 upon exposure to DNA-damaging agents.

The human platelet: strong transcriptome correlations among individuals associate weakly with the platelet proteome.

BACKGROUND: For the anucleate platelet it has been unclear how well platelet transcriptomes correlate among different donors or across different RNA profiling platforms, and what the transcriptomes' relationship is with the platelet proteome. We profiled the platelet transcriptome of 10 healthy young males (5 white and 5 black) with no notable clinical history using RNA sequencing and by Affymetrix microarray. RESULTS: We found that the abundance of platelet mRNA transcripts was highly correlated across the 10 individuals, independently of race and of the employed technology. Our RNA-seq data showed that these high inter-individual correlations extend beyond mRNAs to several categories of non-coding RNAs. Pseudogenes represented a notable exception by exhibiting a difference in expression by race. Comparison of our mRNA signatures to a publicly available quantitative platelet proteome showed that most (87.5%) identified platelet proteins had a detectable corresponding mRNA. However, a high number of mRNAs that were present in the transcriptomes of all 10 individuals had no representation in the proteome. Spearman correlations of the relative abundances for those genes represented by both an mRNA and a protein showed a weak (~0.3) connection. Further analysis of the overlapping and non-overlapping platelet mRNAs and proteins identified gene groups corresponding to distinct cellular processes. CONCLUSIONS: The results of our analyses provide novel insights for platelet biology, show only a weak connection between the platelet transcriptome and proteome, and indicate that it is feasible to assemble a platelet mRNA-ome that can serve as a reference for future platelet transcriptomic studies of human health and disease.

Performance of exome sequencing for pharmacogenomics.

AIM: We present the potential false-negative rate of exome sequencing for the detection of pharmacogenomic variants. MATERIALS & METHODS: Depth of coverage of 1928 pharmacogenomically relevant variant positions was ascertained from 62 exome-sequenced samples. RESULTS: Approximately 14% of the 1928 variant locations examined had inadequate depth of coverage (<20x). The variants with inadequate coverage were predominantly located outside of protein-coding portions and included some clinically relevant variant positions, such as the warfarin VKORC1 variant. CONCLUSION: While the use of exome sequencing is becoming more prevalent in fundamental research, clinical trials and clinical use; there is a possibility of false-negative results. The possible quality issues such as false-negative rate should be considered with the use of exome sequencing.

HuR is a post-transcriptional regulator of core metabolic enzymes in pancreatic cancer.

Cancer cell metabolism differs from normal cells, yet the regulatory mechanisms responsible for these differences are incompletely understood, particularly in response to acute changes in the tumor microenvironment. HuR, an RNA-binding protein, acts under acute stress to regulate core signaling pathways in cancer through post-transcriptional regulation of mRNA targets. We demonstrate that HuR regulates the metabolic phenotype in pancreatic cancer cells and is critical for survival under acute glucose deprivation. Using three pancreatic cancer cell line models, HuR-proficient cells demonstrated superior survival under glucose deprivation when compared with isogenic cells with siRNA-silencing of HuR expression (HuR-deficient cells). We found that HuR-proficient cells utilized less glucose, but produced greater lactate, as compared with HuR-deficient cells. Acute glucose deprivation was found to act as a potent stimulus for HuR translocation from the nucleus to the cytoplasm, where HuR stabilizes its mRNA targets. We performed a gene expression array on ribonucleoprotein-immunoprecipitated mRNAs bound to HuR and identified 11 novel HuR target transcripts that encode enzymes central to glucose metabolism. Three (GPI, PRPS2 and IDH1) were selected for validation studies, and confirmed as bona fide HuR targets. These findings establish HuR as a critical regulator of pancreatic cancer cell metabolism and survival under acute glucose deprivation. Further explorations into HuR's role in cancer cell metabolism should uncover novel therapeutic targets that are critical for cancer cell survival in a metabolically compromised tumor microenvironment.

HuR's post-transcriptional regulation of Death Receptor 5 in pancreatic cancer cells.

Apoptosis is one of the core signaling pathways disrupted in pancreatic ductal adenocarcinoma (PDA). Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF)-receptor superfamily that is expressed in cancer cells. Binding of TNF-related apoptosis-inducing ligand (TRAIL) to DR5 is a potent trigger of the extrinsic apoptotic pathway, and numerous clinical trials are based on DR5-targeted therapies for cancer, including PDA. Human antigen R (HuR), an RNA-binding protein, regulates a select number of transcripts under stress conditions. Here we report that HuR translocates from the nucleus to the cytoplasm of PDA cells upon treatment with a DR5 agonist. High doses of DR5 agonist induce cleavage of both HuR and caspase 8. HuR binds to DR5 mRNA at the 5'-untranslated region (UTR) in PDA cells in response to different cancer-associated stressors and subsequently represses DR5 protein expression; silencing HuR augments DR5 protein production by enabling its translation and thus enhances apoptosis. In PDA specimens (n = 53), negative HuR cytoplasmic expression correlated with elevated DR5 expression (odds ratio 16.1, p < 0.0001). Together, these data demonstrate a feedback mechanism elicited by HuR-mediated repression of the key apoptotic membrane protein DR5.

Whole-exome sequencing of DNA from peripheral blood mononuclear cells (PBMC) and EBV-transformed lymphocytes from the same donor.

BACKGROUND: The creation of lymphoblastoid cell lines (LCLs) through Epstein-Barr virus (EBV) transformation of B-lymphocytes can result in a valuable biomaterial for cell biology research and a renewable source of DNA. While LCLs have been used extensively in cellular and genetic studies, the process of cell transformation and expansion during culturing may introduce genomic changes that may impact their use and the interpretation of subsequent genetic findings. RESULTS: We performed whole exome sequencing on a tetrad family using DNA derived from peripheral blood mononuclear cells (PBMCs) and LCLs from each individual. We generated over 4.7 GB of mappable sequence to a 125X read coverage per sample. An average of 19,354 genetic variants were identified. Comparison of the two DNA sources from each individual showed an average concordance rate of 95.69%. By lowering the variant calling parameters, the concordance rate between the paired samples increased to 99.82%. Sanger sequencing of a subset of the remaining discordant variants did confirm the presence of de novo mutations arising in LCLs. CONCLUSIONS: By varying software stringency parameters, we identified 99% concordance between DNA sequences derived from the two different sources from the same donors. These results suggest that LCLs are an appropriate representation of the genetic material of the donor and suggest that EBV transformation can result in low-level generation of de novo mutations. Therefore, use of PBMC or early passage EBV-transformed cells is recommended. These findings have broad-reaching implications, as there are thousands of LCLs in public biorepositories and individual laboratories.

CoAIMs: a cost-effective panel of ancestry informative markers for determining continental origins.

BACKGROUND: Genetic ancestry is known to impact outcomes of genotype-phenotype studies that are designed to identify risk for common diseases in human populations. Failure to control for population stratification due to genetic ancestry can significantly confound results of disease association studies. Moreover, ancestry is a critical factor in assessing lifetime risk of disease, and can play an important role in optimizing treatment. As modern medicine moves towards using personal genetic information for clinical applications, it is important to determine genetic ancestry in an accurate, cost-effective and efficient manner. Self-identified race is a common method used to track and control for population stratification; however, social constructs of race are not necessarily informative for genetic applications. The use of ancestry informative markers (AIMs) is a more accurate method for determining genetic ancestry for the purposes of population stratification. METHODOLOGY/PRINCIPAL FINDINGS: Here we introduce a novel panel of 36 microsatellite (MSAT) AIMs that determines continental admixture proportions. This panel, which we have named Continental Ancestry Informative Markers or CoAIMs, consists of MSAT AIMs that were chosen based upon their measure of genetic variance (F(st)), allele frequencies and their suitability for efficient genotyping. Genotype analysis using CoAIMs along with a Bayesian clustering method (STRUCTURE) is able to discern continental origins including Europe/Middle East (Caucasians), East Asia, Africa, Native America, and Oceania. In addition to determining continental ancestry for individuals without significant admixture, we applied CoAIMs to ascertain admixture proportions of individuals of self declared race. CONCLUSION/SIGNIFICANCE: CoAIMs can be used to efficiently and effectively determine continental admixture proportions in a sample set. The CoAIMs panel is a valuable resource for genetic researchers performing case-control genetic association studies, as it can control for the confounding effects of population stratification. The MSAT-based approach used here has potential for broad applicability as a cost effective tool toward determining admixture proportions.

Churchill regulates cell movement and mesoderm specification by repressing Nodal signaling.

BACKGROUND: Cell movements are essential to the determination of cell fates during development. The zinc-finger transcription factor, Churchill (ChCh) has been proposed to regulate cell fate by regulating cell movements during gastrulation in the chick. However, the mechanism of action of ChCh is not understood. RESULTS: We demonstrate that ChCh acts to repress the response to Nodal-related signals in zebrafish. When ChCh function is abrogated the expression of mesodermal markers is enhanced while ectodermal markers are expressed at decreased levels. In cell transplant assays, we observed that ChCh-deficient cells are more motile than wild-type cells. When placed in wild-type hosts, ChCh-deficient cells often leave the epiblast, migrate to the germ ring and are later found in mesodermal structures. We demonstrate that both movement of ChCh-compromised cells to the germ ring and acquisition of mesodermal character depend on the ability of the donor cells to respond to Nodal signals. Blocking Nodal signaling in the donor cells at the levels of Oep, Alk receptors or Fast1 inhibited migration to the germ ring and mesodermal fate change in the donor cells. We also detect additional unusual movements of transplanted ChCh-deficient cells which suggests that movement and acquisition of mesodermal character can be uncoupled. Finally, we demonstrate that ChCh is required to limit the transcriptional response to Nodal. CONCLUSION: These data establish a broad role for ChCh in regulating both cell movement and Nodal signaling during early zebrafish development. We show that chch is required to limit mesodermal gene expression, inhibit Nodal-dependant movement of presumptive ectodermal cells and repress the transcriptional response to Nodal signaling. These findings reveal a dynamic role for chch in regulating cell movement and fate during early development.

Expression and regulation of the zinc finger transcription factor Churchill during zebrafish development.

During gastrulation dynamic cell movements establish the germ layers and shape the body axis of the vertebrate embryo. The zinc finger protein Churchill (chch) has been proposed to be a key regulator of these movements. We examined the expression pattern of chch in zebrafish and studied the regulation of chch by FGF signaling. We observed zygotic expression of chch during early cleavage stages. Two lines of evidence demonstrate that chch is zygotically expressed prior to the mid-blastula transition. First, blocking transcription during early cleavage stages represses chch expression. Second, endogenous levels of chch transcripts increase between 1-cell and 16-cell embryos. chch remains widely expressed during blastula and gastrula stages but scattered cells express higher levels of chch. By somitogenesis, chch is expressed in the ventral-most cells of the embryo adjacent to the yolk. In addition, transcripts are also observed in superficial cells on the surface of the yolk, in presumptive mucous cells and keratinocytes. By 30 hpf transcripts are observed in anterior neural tissue and ventral cells adjacent to the yolk. Over the next three days chch expression is indistinct until 4 dpf when we observe expression in the pharynx and gut. We show that activation of FGF signaling during gastrulation is sufficient to induce chch expression. In addition, we demonstrate that blocking FGF signaling between the 4-cell and shield stages represses chch expression.

Chordin, FGF signaling, and mesodermal factors cooperate in zebrafish neural induction.

The ectoderm gives rise to both neural tissue and epidermis. In vertebrates, specification of the neural plate requires repression of bone morphogenetic protein (BMP) signaling in the dorsal ectoderm. The extracellular BMP antagonist Chordin and other signals from the dorsal mesoderm play important roles in this process. We utilized zebrafish mutant combinations that disrupt Chordin and mesoderm formation to reveal additional signals that contribute to the establishment of the neural domain. We demonstrate that fibroblast growth factor (FGF) signaling accounts for the additional activity in neural specification. Impeding FGF signaling results in a shift of ectodermal markers from neural to epidermal. However, following inhibition of FGF signaling, expression of anterior neural markers recovers in a Nodal-dependent fashion. Simultaneously blocking, Chordin, mesoderm formation, and FGF signaling eliminates neural marker expression during gastrula stages. We observed that FGF signaling is required for chordin expression but that it also acts via other mechanisms to repress BMP transcription during late blastula stages. Activation of FGF signaling was also able to repress BMP transcription in the absence of protein synthesis. Our results support a model in which specification of anterior neural tissue requires early FGF-mediated repression of BMP transcript levels and later activities of Chordin and mesodermal factors.

A transcription map of the 6p22.3 reading disability locus identifying candidate genes.

BACKGROUND: Reading disability (RD) is a common syndrome with a large genetic component. Chromosome 6 has been identified in several linkage studies as playing a significant role. A more recent study identified a peak of transmission disequilibrium to marker JA04 (G72384) on chromosome 6p22.3, suggesting that a gene is located near this marker. RESULTS: In silico cloning was used to identify possible candidate genes located near the JA04 marker. The 2 million base pairs of sequence surrounding JA04 was downloaded and searched against the dbEST database to identify ESTs. In total, 623 ESTs from 80 different tissues were identified and assembled into 153 putative coding regions from 19 genes and 2 pseudogenes encoded near JA04. The identified genes were tested for their tissue specific expression by RT-PCR. CONCLUSIONS: In total, five possible candidate genes for RD and other diseases mapping to this region were identified.

A detailed physical map of the 6p reading disability locus, including new markers and confirmation of recombination suppression.

Loci for several complex disorders have been genetically linked to markers located telomeric of the HLA class I region of the major histocompatibility complex on 6p21.3-22. However, this same region has been characterized by a large interval of recombination suppression with the potential to greatly complicate precise localization of these risk loci. Furthermore, a paucity of markers and physical mapping data has confounded precise localization of the boundaries of linkage and recombination suppression. In order to create a more detailed marker map of this region and define these boundaries we generated a minimal tiling pathway of BACs, PACs, and cosmids and a multiplexed panel of 29 short tandem repeat markers spanning 10 Mb. In addition to providing precise marker order and distances, the pathway and marker panel frame an inversion of recombination frequency that has distorted the resolution of linkage studies for 6p loci such as reading disability and others, and that should be accounted for in the design of future studies.