Molecular profiling of functional interactions between pre-osteoblastic and breast carcinoma cells.

The relationships between cancer cells and the microenvironment play a critical role in cancer growth and development. The bone stroma consists of mesenchymal stem cells and mature osteoblasts that promote cancer growth. Yet it is not completely understood what are the molecular processes guiding cancer cells progression to the bone. In this study, a coculture assay and subsequent gene profiling arrays were used to compare the gene expression profile of a pre-osteoblastic (PO) cell line (MBA-15) with that of a mammary adenocarcinoma (DA3) cells. After coculture, cells were separated by magnetic beads based on the expression of CD326 antigen. RNA was purified and hybridized on gene expression array. The gene expression pattern changes were followed by qRT-PCR. We demonstrate that cocultured DA3 cells express elevated levels of genes that regulate growth and responses to both hormonal stimulus and wounding, as well as reduced expression of genes related to lipid metabolism. Also, cocultured PO cells showed reduced expression of cell junction genes. The study presents a simplified model system, composed of PO and mammary cancer cells, that potentially mimics the molecular interactions in the tumor microenvironment which contribute to tumor progression.

Endothelial pro-atherosclerotic response to extracellular diabetic-like environment: possible role of thioredoxin-interacting protein.

BACKGROUND. High blood and tissue concentrations of glucose and advanced glycation end-products (AGEs) are thought to play an important role in the development of vascular diabetic complications. Therefore, the impact of extracellular AGEs and different glucose concentrations was evaluated by studying the gene expressions and the underlying cellular pathways involved in the development of inflammatory pro-atherosclerotic processes observed in cultured endothelial cells. METHODS. Fresh human umbilical vein cord endothelial cells (HUVEC) were treated in the presence of elevated extracellular glucose concentrations (5.5-28 mmol/l) with and without AGE-human serum albumin (HSA). Affymetrix GeneChip(R) Human Gene 1.0 ST arrays were used for gene expression analysis (total 20 chips). Genes of interest were further validated using real-time PCR and western blot techniques. RESULTS. Microarray analysis revealed significant changes in some gene expressions in the presence of the different stimuli, suggesting that different pathways are involved. Six genes were selected for validation as follows: thioredoxin-interacting protein (TXNIP), thioredoxin (TXN), nuclear factor of kappa B (NF-kappaB), interleukin 6 (IL6), interleukin 8 (IL8) and receptor of advanced glycation end-products (RAGE). Interestingly, it was found that the association of AGEs together with the highest pathophysiological concentration of glucose (28 mmol/l) diminished the expression of these specific genes, excluding TXN. CONCLUSIONS. In the present model that mimics a diabetic environment, the relatively short-term experimental conditions used showed an unexpected blunting action of AGEs in the presence of the highest glucose concentration (28 mmol/l). The interactive cellular pathways involved in these processes should be further investigated.

Isp7 is a novel regulator of amino acid uptake in the TOR signaling pathway.

TOR proteins reside in two distinct complexes, TOR complexes 1 and 2 (TORC1 and TORC2), that are central for the regulation of cellular growth, proliferation, and survival. TOR is also the target for the immunosuppressive and anticancer drug rapamycin. In Schizosaccharomyces pombe, disruption of the TSC complex, mutations in which can lead to the tuberous sclerosis syndrome in humans, results in a rapamycin-sensitive phenotype under poor nitrogen conditions. We show here that the sensitivity to rapamycin is mediated via inhibition of TORC1 and suppressed by overexpression of isp7(+), a member of the family of 2-oxoglutarate-Fe(II)-dependent oxygenase genes. The transcript level of isp7(+) is negatively regulated by TORC1 but positively regulated by TORC2. Yet we find extensive similarity between the transcriptome of cells disrupted for isp7(+) and cells mutated in the catalytic subunit of TORC1. Moreover, Isp7 regulates amino acid permease expression in a fashion similar to that of TORC1 and opposite that of TORC2. Overexpression of isp7(+) induces TORC1-dependent phosphorylation of ribosomal protein Rps6 while inhibiting TORC2-dependent phosphorylation and activation of the AGC-like kinase Gad8. Taken together, our findings suggest a central role for Isp7 in amino acid homeostasis and the presence of isp7(+)-dependent regulatory loops that affect both TORC1 and TORC2.

Transcriptional profiling identifies genes induced by hepatocyte-derived extracellular matrix in metastatic human colorectal cancer cell lines.

The milieu of the liver, and in particular hepatocyte-derived extracellular matrix (hECM), is a critical factor regulating development of liver metastases of colorectal cancer (CRC) cells. The present study has investigated genes altered by hECM in CRC cells and particularly by heparan sulfate chains of hepatocyte proteoglycans. Gene profiling analysis shows that after 2 days on hECM, 226 genes are up-regulated more than 2-fold in strongly metastatic SM cells, including genes involved in growth arrest and apoptosis, signal transduction, cell migration, proliferation, communication and angiogenesis, with activation of the erbB signaling network and p53 effectors. Genes down-regulated by hECM include genes involved in lipogenesis and the S phase of the cell cycle. Further studies exploring the kinetics of gene expression after 4 and 7 days culture on hECM show induction of EGF family members and of stem cell markers. In particular, hECM, but not collagen, increases mRNA expression of HB-EGF and colon stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5). Expression of these genes is not induced by hECM depleted of the heparan sulfate chains of proteoglycans. Lastly, a specific cell population positive for cancer stem cell (CSC) markers LGR5, epCAM and CD133, but negative for CD44, appears after 7 days culture on hECM, a population which is reduced by 50 % in cells grown on heparan sulfated-depleted hECM. Collectively, the data suggest that hECM induces growth factors and receptors regulating proliferation of metastatic CRC in the liver and offers a growth advantage for specific populations expressing CSC markers.

Pax6 is required for normal cell-cycle exit and the differentiation kinetics of retinal progenitor cells.

The coupling between cell-cycle exit and onset of differentiation is a common feature throughout the developing nervous system, but the mechanisms that link these processes are mostly unknown. Although the transcription factor Pax6 has been implicated in both proliferation and differentiation of multiple regions within the central nervous system (CNS), its contribution to the transition between these successive states remains elusive. To gain insight into the role of Pax6 during the transition from proliferating progenitors to differentiating precursors, we investigated cell-cycle and transcriptomic changes occurring in Pax6 (-) retinal progenitor cells (RPCs). Our analyses revealed a unique cell-cycle phenotype of the Pax6-deficient RPCs, which included a reduced number of cells in the S phase, an increased number of cells exiting the cell cycle, and delayed differentiation kinetics of Pax6 (-) precursors. These alterations were accompanied by coexpression of factors that promote (Ccnd1, Ccnd2, Ccnd3) and inhibit (P27 (kip1) and P27 (kip2) ) the cell cycle. Further characterization of the changes in transcription profile of the Pax6-deficient RPCs revealed abrogated expression of multiple factors which are known to be involved in regulating proliferation of RPCs, including the transcription factors Vsx2, Nr2e1, Plagl1 and Hedgehog signaling. These findings provide novel insight into the molecular mechanism mediating the pleiotropic activity of Pax6 in RPCs. The results further suggest that rather than conveying a linear effect on RPCs, such as promoting their proliferation and inhibiting their differentiation, Pax6 regulates multiple transcriptional networks that function simultaneously, thereby conferring the capacity to proliferate, assume multiple cell fates and execute the differentiation program into retinal lineages.

MicroRNA expression signatures in intraductal papillary mucinous neoplasm of the pancreas.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMN) represent a spectrum of tumors that range from low-grade (LG) dysplastic tumors to invasive cancer. Identification of IPMN at high risk for malignant transformation is important for the prevention and early treatment of pancreatic cancer. The roles of microRNA expression in the development of IPMN have not been extensively evaluated. METHODS: Expression patterns of 846 human microRNAs (miRNAs) was analyzed using microRNA microarray in 55 tissues, including LG IPMN (n = 10), moderate-grade (MG) IPMN (n = 5), high-grade (HG) IPMN (n = 5), invasive cancer with IPMN (IPMC; n = 10), pancreatic ductal adenocarcinoma without IPMN (PDA; n = 5), LG IPMN extracted from specimens that contain IPMC (LG_Ca; n = 10), and normal pancreatic tissues (n = 10). RESULTS: Fourteen miRNAs were differentially expressed in all IPMN tissues compared with normal pancreatic tissue. Expression level of 3 miRNAs was proportional to dysplasia level. Hierarchical clustering demonstrated grouping of 2 IPMN subgroups: LG and MG IPMN verses HG IPMN and IPMC. Expression of 15 miRNAs was significantly different between these groups. LG_Ca tissues clustered with the HG IPMC group, and 12 miRNAs were differentially expressed in LG_Ca, HG lesions, and IPMC compared with LG lesions. The expression patterns of selected miRNAs were validated using quantitative reverse-transcription real-time polymerase chain reaction. Hierarchical clustering demonstrated microRNA expression profile in IPMC was significantly different from PDA, suggesting that different pathways are involved in these cancer types. CONCLUSION: This study demonstrates that miRNAs are involved in the development and progression of IPMN. We identified potential targets for diagnosis, prognostication, and treatment of IPMN.

Platinum-resistance in ovarian cancer cells is mediated by IL-6 secretion via the increased expression of its target cIAP-2.

Ovarian carcinoma patients are initially responsive to platinum-based therapy, but eventually become refractory to treatment due to the development of platinum chemoresistance. Elevated levels of interleukin-6 (IL-6) in the sera and ascites of these patients predict poor clinical outcome. Our goal was to analyze the interaction between cisplatin and cisplatin-resistant ovarian cancer cells, and to identify means of circumventing platinum resistance. We studied ovarian carcinoma cell lines and cells drawn from ovarian carcinoma patients. Gene array analyses were performed on ovarian carcinoma cells upon treatment with cisplatin, and the results were validated by ELISA and Western blotting (WB). Cytotoxicity assays were performed on anti-IL-6 Ab-, IL-6-, and cellular inhibitor of apoptosis 2 (cIAP-2) siRNA-treated cells, following cisplatin addition. Our results revealed a highly significant increase in IL-6 and cIAP-2 mRNA and protein levels upon treatment with cisplatin. WB analysis of cisplatin-treated cells exhibited decreased cIAP-2 expression level following anti-IL-6 Ab addition. Furthermore, IL-6 by itself, significantly increased cIAP-2 levels in ovarian carcinoma cells. Finally, cytotoxicity assays showed sensitization to cisplatin following the addition of IL-6 and cIAP-2 inhibitors. In conclusion, cisplatin treatment of ovarian carcinoma cells upregulates IL-6 and cIAP-2 levels while their inhibition significantly sensitizes them to cisplatin. Here, we present cIAP-2 as a novel inducer of platinum resistance in ovarian carcinoma cells, and suggest an axis beginning with an encounter between cisplatin and these cells, mediated sequentially by IL-6 and cIAP-2, resulting in cisplatin resistance. Consequently, we propose that combining IL-6/cIAP-2 inhibitors with cisplatin will provide new hope for ovarian carcinoma patients by improving the current treatment.

Genome-wide transcriptomic variations of human lymphoblastoid cell lines: insights from pairwise gene-expression correlations.

AIMS: Human lymphoblastoid cell lines (LCLs) are a rich resource of information on human interindividual genomic, transcriptomic, proteomic and phenomic variations, and are therefore gaining popularity for pharmacogenomic studies. In the present study we demonstrate that genome-wide transcriptomic data from a small LCL panel from unrelated individuals is sufficient for detecting pairs of genes that exhibit highly correlated expression levels and may thus convey insights about coregulated genes. MATERIALS & METHODS: RNA samples were prepared from LCLs representing 12 unrelated healthy adult female Caucasian donors. Transcript levels were determined with the Affymetrix Human Gene arrays. Expression-level correlations were searched using Partek(®) Genomics Suite™ and the R environment. Sequences of detected correlated gene pairs were compared for shared conserved 3´-UTR miRNA binding. RESULTS: Most of the approximately 33,000 transcripts covered by the Affymetrix arrays showed closely similar expression levels in LCLs from unrelated donors. However, the expression levels of some transcripts showed large inter-individual variations. When comparing the expression levels of each of the top 1000 genes showing the largest interindividual expression variations against the others, two sets containing 156 and 4438 correlated gene pairs with false-discovery rates of 0.01 and 0.05 were detected, respectively. Similar analysis of another gene-expression data set from LCLs (GSE11582) indicated that 61 and 39% of identified pairs matched the pairs detected from our transcriptomic data, respectively. Shared conserved 3´-UTR miRNA binding sites were noted for 14-17% of the top 100 gene pairs, suggesting that regulation by miRNA may contribute to their coordinated expression. CONCLUSION: Probing genome-wide transcriptomic data sets of LCLs from unrelated individuals may detect coregulated genes, adding insights on cellular regulation by miRNAs.

Genome-wide miRNA expression profiling of human lymphoblastoid cell lines identifies tentative SSRI antidepressant response biomarkers.

AIM: Over 30% of patients with major depression do not respond well to first-line treatment with selective serotonin reuptake inhibitors (SSRIs). Using genome-wide expression profiling of human lymphoblastoid cell lines (LCLs) CHL1 was identified as a tentative SSRI sensitivity biomarker. This study reports on miRNAs implicated in SSRI sensitivity of LCLs. METHODS: Eighty LCLs were screened from healthy adult female individuals for growth inhibition by paroxetine. Eight LCLs exhibiting high or low sensitivities to paroxetine were chosen for genome-wide expression profiling with miRNA microarrays. RESULTS: The miRNA miR-151-3p had 6.7-fold higher basal expression in paroxetine-sensitive LCLs. This corresponds with lower expression of CHL1, a target of miR-151-3p. The additional miRNAs miR-212, miR-132, miR-30b*, let-7b and let-7c also differed by >1.5-fold (p < 0.05) between the two LCL groups. CONCLUSION: The potential value of these miRNAs as tentative SSRI response biomarkers awaits validation with lymphocyte samples of major depression patients.

Effects of IKAP/hELP1 deficiency on gene expression in differentiating neuroblastoma cells: implications for familial dysautonomia.

Familial dysautonomia (FD) is a developmental neuropathy of the sensory and autonomous nervous systems. The IKBKAP gene, encoding the IKAP/hELP1 subunit of the RNA polymerase II Elongator complex is mutated in FD patients, leading to a tissue-specific mis-splicing of the gene and to the absence of the protein in neuronal tissues. To elucidate the function of IKAP/hELP1 in the development of neuronal cells, we have downregulated IKBKAP expression in SHSY5Y cells, a neuroblastoma cell line of a neural crest origin. We have previously shown that these cells exhibit abnormal cell adhesion when allowed to differentiate under defined culture conditions on laminin substratum. Here, we report results of a microarray expression analysis of IKAP/hELP1 downregulated cells that were grown on laminin under differentiation or non-differentiation growth conditions. It is shown that under non-differentiation growth conditions, IKAP/hELP1 downregulation affects genes important for early developmental stages of the nervous system, including cell signaling, cell adhesion and neural crest migration. IKAP/hELP1 downregulation during differentiation affects the expression of genes that play a role in late neuronal development, in axonal projection and synapse formation and function. We also show that IKAP/hELP1 deficiency affects the expression of genes involved in calcium metabolism before and after differentiation of the neuroblastoma cells. Hence, our data support IKAP/hELP1 importance in the development and function of neuronal cells and contribute to the understanding of the FD phenotype.

Genome-wide expression profiling of human lymphoblastoid cell lines identifies CHL1 as a putative SSRI antidepressant response biomarker.

AIMS: Selective serotonin reuptake inhibitors (SSRIs) are the most commonly used class of antidepressants for treating major depression. However, approximately 30% of patients do not respond sufficiently to first-line antidepressant drug treatment and require alternative therapeutics. Genome-wide studies searching for SSRI response DNA biomarkers or studies of candidate serotonin-related genes so far have given inconclusive or contradictory results. Here, we present an alternative transcriptome-based genome-wide approach for searching antidepressant drug-response biomarkers by using drug-effect phenotypes in human lymphoblastoid cell lines (LCLs). MATERIALS & METHODS: We screened 80 LCLs from healthy adult female individuals for growth inhibition by paroxetine. A total of 14 LCLs with reproducible high and low sensitivities to paroxetine (seven from each phenotypic group) were chosen for genome-wide expression profiling with commercial microarrays. RESULTS: The most notable genome-wide transcriptome difference between LCLs displaying high versus low paroxetine sensitivities was a 6.3-fold lower (p = 0.0000256) basal expression of CHL1, a gene coding for a neuronal cell adhesion protein implicated in correct thalamocortical circuitry, schizophrenia and autism. The microarray findings were confirmed by real-time PCR (36-fold lower CHL1 expression levels in the high paroxetine sensitivity group). Several additional genes implicated in synaptogenesis or in psychiatric disorders, including ARRB1, CCL5, DDX60, DDX60L, ENDOD1, ENPP2, FLT1, GABRA4, GAP43, MCTP2 and SPRY2, also differed by more than 1.5-fold and a p-value of less than 0.005 between the two paroxetine sensitivity groups, as confirmed by real-time PCR experiments. CONCLUSION: Genome-wide transcriptional profiling of in vitro phenotyped LCLs identified CHL1 and additional genes implicated in synaptogenesis and brain circuitry as putative SSRI response biomarkers. This method might be used as a preliminary tool for searching for potential depression treatment biomarkers.

Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro.

BACKGROUND: Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells. METHODOLOGY/PRINCIPAL FINDING: Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening.

Targeted genomic capture and massively parallel sequencing to identify genes for hereditary hearing loss in Middle Eastern families.

BACKGROUND: Identification of genes responsible for medically important traits is a major challenge in human genetics. Due to the genetic heterogeneity of hearing loss, targeted DNA capture and massively parallel sequencing are ideal tools to address this challenge. Our subjects for genome analysis are Israeli Jewish and Palestinian Arab families with hearing loss that varies in mode of inheritance and severity. RESULTS: A custom 1.46 MB design of cRNA oligonucleotides was constructed containing 246 genes responsible for either human or mouse deafness. Paired-end libraries were prepared from 11 probands and bar-coded multiplexed samples were sequenced to high depth of coverage. Rare single base pair and indel variants were identified by filtering sequence reads against polymorphisms in dbSNP132 and the 1000 Genomes Project. We identified deleterious mutations in CDH23, MYO15A, TECTA, TMC1, and WFS1. Critical mutations of the probands co-segregated with hearing loss. Screening of additional families in a relevant population was performed. TMC1 p.S647P proved to be a founder allele, contributing to 34% of genetic hearing loss in the Moroccan Jewish population. CONCLUSIONS: Critical mutations were identified in 6 of the 11 original probands and their families, leading to the identification of causative alleles in 20 additional probands and their families. The integration of genomic analysis into early clinical diagnosis of hearing loss will enable prediction of related phenotypes and enhance rehabilitation. Characterization of the proteins encoded by these genes will enable an understanding of the biological mechanisms involved in hearing loss.

Dual requirement for Pax6 in retinal progenitor cells.

Throughout the developing central nervous system, pre-patterning of the ventricular zone into discrete neural progenitor domains is one of the predominant strategies used to produce neuronal diversity in a spatially coordinated manner. In the retina, neurogenesis proceeds in an intricate chronological and spatial sequence, yet it remains unclear whether retinal progenitor cells (RPCs) display intrinsic heterogeneity at any given time point. Here, we performed a detailed study of RPC fate upon temporally and spatially confined inactivation of Pax6. Timed genetic removal of Pax6 appeared to unmask a cryptic divergence of RPCs into qualitatively divergent progenitor pools. In the more peripheral RPCs under normal circumstances, Pax6 seemed to prevent premature activation of a photoreceptor-differentiation pathway by suppressing expression of the transcription factor Crx. More centrally, Pax6 contributed to the execution of the comprehensive potential of RPCs: Pax6 ablation resulted in the exclusive generation of amacrine interneurons. Together, these data suggest an intricate dual role for Pax6 in retinal neurogenesis, while pointing to the cryptic divergence of RPCs into distinct progenitor pools.

Genetic dissection of Pax6 dosage requirements in the developing mouse eye.

Haploinsufficiency of the transcription factor Pax6/PAX6 has been implicated in a number of congenital eye disorders in humans and mice, such as aniridia and Small-eye, which affect the development and function of the lens, cornea, anterior eye segment and neuroretina. However, the widespread distribution of Pax6/PAX6 protein within the developing and adult eye preclude the identification and direct study of the ocular tissues affected by a reduction in Pax6/PAX6 dosage. Here, we employed Cre/loxP-mediated inactivation of a single Pax6 allele in either the lens/cornea or the distal optic cup to dissect the tissue-specific sensitivity to Pax6 haploinsufficiency. Exclusive inactivation of a single Pax6 allele in the lens recapitulates the Small-eye lens and corneal defects, while only mildly affects iris morphology in a non-cell-autonomous fashion. Conversely, selective inactivation of a single Pax6 allele in the distal optic cup revealed primarily cell-autonomous dosage requirements for proper iris differentiation, with no affects on either lens or corneal morphology. Pax6 dosage within the distal optic cup is found here to influence the number of progenitors destined for the anterior ocular structures, the timing of iris muscle-cell differentiation and iris stroma development. Taken together, we genetically dissected the complex mouse Small-eye phenotype, thereby pinpointing the underlying Pax6/PAX6 haploinsufficiency to autonomous dosage requirements within the developing iris and lens/cornea tissues.

An unexpectedly high frequency of heterozygosity for alpha-thalassemia in Ashkenazi Jews.

alpha-Thalassemia is among the world's most common single gene disorders, which is most prevalent in the malaria belt. This geographic distribution has been attributed to a selective advantage of heterozygotes against this disease. Unexpectedly, we have found a high frequency of heterozygosity for deletional alpha-thalassemia (-alpha3.7) in Ashkenazi Jews (carrier frequency of 7.9%, allele frequency of 0.04). This population has resided in temperate climates for many centuries and was therefore not subjected to malarial selection pressure. In comparison, heterozygosity for beta-thalassemia, which is highly subject to malarial selection pressure, is very low (estimated <0.1%) in this group. It is possible that founder effect and genetic drift have contributed to the high frequency of deletional alpha-thalassemia in Ashkenazim, as may occur in closed populations. Alternatively, we hypothesize that positive selection pressure for an as yet unknown linked allele on chromosome 16 may be a significant factor leading to this high frequency.