IGFBP2 potentiates nuclear EGFR-STAT3 signaling.

Insulin-like growth factor binding protein 2 (IGFBP2) is a pleiotropic oncogenic protein that has both extracellular and intracellular functions. Despite a clear causal role in cancer development, the tumor-promoting mechanisms of IGFBP2 are poorly understood. The contributions of intracellular IGFBP2 to tumor development and progression are also unclear. Here we present evidence that both exogenous IGFBP2 treatment and cellular IGFBP2 overexpression lead to aberrant activation of epidermal growth factor receptor (EGFR), which subsequently activates signal transducer and activator of transcription factor 3 (STAT3) signaling. Furthermore, we demonstrate that IGFBP2 augments the nuclear accumulation of EGFR to potentiate STAT3 transactivation activities, via activation of the nuclear EGFR signaling pathway. Nuclear IGFBP2 directly influences the invasive and migratory capacities of human glioblastoma cells, providing a direct link between intracellular (and particularly nuclear) IGFBP2 and cancer hallmarks. These activities are also consistent with the strong association between IGFBP2 and STAT3-activated genes derived from The Cancer Genome Atlas database for human glioma. A high level of all three proteins (IGFBP2, EGFR and STAT3) was strongly correlated with poorer survival in an independent patient data set. These results identify a novel tumor-promoting function for IGFBP2 of activating EGFR/STAT3 signaling and facilitating EGFR accumulation in the nucleus, thereby deregulating EGFR signaling by two distinct mechanisms. As targeting EGFR in glioma has been relatively unsuccessful, this study suggests that IGFBP2 may be a novel therapeutic target.

Dissection of signaling pathways in fourteen breast cancer cell lines using reverse-phase protein lysate microarray.

Signal transduction pathways play a crucial role in breast cancer development, progression, and response to different therapies. A major problem in breast cancer therapy is the heterogeneity among different tumor types and cell lines commonly used in preclinical studies. To characterize the signaling pathways of some of the commonly used breast cancer cell lines and dissect the relationship among a number of pathways and some key genetic and molecular events in breast cancer development, such as p53 mutation, ErbB2 expression, and estrogen receptor (ER)/progesterone receptor (PR) status, we performed pathway profiling of 14 breast cancer cell lines by measuring the expression and phosphorylation status of 40 different cell signaling proteins with 53 specific antibodies using a protein lysate array. Cluster analysis of the expression data showed that there was close clustering of phosphatidylinositol 3-kinase, Akt, mammalian target of rapamycin (mTOR), Src, and platelet-derived growth factor receptor beta (PDGFRbeta) in all of the cell lines. The most differentially expressed proteins between ER- and PR-positive and ER- and PR-negative breast cells were mTOR, Akt (pThr308), PDGFRbeta, PDGFRbeta (pTyr751), panSrc, Akt (pSer473), insulin-like growth factor-binding protein 5 (IGFBP5), Src (pTyr418), mTOR (pSer2448), and IGFBP2. Many apoptotic proteins, such as apoptosis-inducing factor, IGFBP3, bad, bax, and cleaved caspase 9, were overexpressed in mutant p53-carrying breast cancer cells. Hexokinase isoenzyme 1, ND2, and c-kit were the most differentially expressed proteins in high and low ErbB2-expressing breast cancer cells. This study demonstrated that ER/PR status, ErbB2 expression, and p53 status are major molecules that impact downstream signaling pathways.

Monitoring of cDNA microarray with common primer target and hybridization specificity with selected targets.

Academic researchers are increasingly producing and using cDNA microarrays. Their quality and hybridization specificity are crucial in determining whether the generated data are accurate and interpretable. Here, we describe two methods of monitoring microarray production, the sustainability of DNA attachment, and the specificity of hybridization. The first method consists of labeling an oligonucleotide, which is one of the primers used to amplify all cDNA probes on the array (except for beta-actin and GAPDH) with fluorescent dye and hybridize it to the cDNA microarray. Attachment of the cDNAs on the array after the hybridization procedure was monitored by visualizing fluorescent signals from the spots on the array. In the second method, two selected DNA targets, beta-actin and GAPDH, were labeled with fluorescent dye to hybridize to the cDNA array. Hence, hybridization specificity was demonstrated by obtaining fluorescent signals solely from the genes corresponding to the target.

Comparative evaluation of laser-based microarray scanners.

Laboratories use different laser-based scanners to scan microarray images. To assess whether results from different scanners are comparable, and thus whether data from different laboratories can be compared, we scanned the same microarray slide with three commercial scanners that use different imaging techniques. After the acquisition of the microarray images produced by the three scanners, the images were quantified using a single imaging software package and protocol. The results were compared, and we found that the data obtained from the three scanners were comparable and that the variations caused by the use of different instruments were negligible, in spite of the fact that the scanners were based on different optical imaging techniques.

Sequence verification as quality-control step for production of cDNA microarrays.

To generate cDNA arrays in our core laboratory, we amplified about 2300 PCR products from a human, sequence-verified cDNA clone library. As a quality-control step, we sequenced the PCR products immediately before printing. The sequence information was used to search the GenBank database to confirm the identities. Although these clones were previously sequence verified by the company, we found that only 79% of the clones matched the original database after handling. Our experience strongly indicates the necessity to sequence verify the clones at the final stage before printing on microarray slides and to modify the gene list accordingly.

Clinical utility of direct mutation testing for congenital nephrogenic diabetes insipidus in families.

OBJECTIVE: To ascertain the clinical scenarios in which genetic testing for congenital nephrogenic diabetes insipidus (NDI) by direct detection of mutations might prove valuable, and to assess the use of automated sequencing for testing. METHODS: We reviewed NDI cases referred to our research laboratory for enrollment in our study of mutations in the AVPR2 gene that is disrupted in the X-linked form of the disease. We selected 5 cases that illustrate the value of genetic testing in different clinical situations. Clinical information was obtained from the patient's personal physicians and the patients' families. Direct automated fluorescent DNA sequencing of AVPR2 gene amplification product was used to identify disease-associated mutations in patients. The presence or absence of mutations in family members was then established by using automated sequencing, restriction enzyme analysis, or both. RESULTS: In 2 of the 5 selected cases, the diagnosis of a genetic form of NDI was confirmed by mutation analysis in a sporadic case of an affected boy. In 2 cases, a suspected diagnosis of X-linked NDI was confirmed in an affected girl. In 4 of the cases, 1 or more unaffected female relatives were determined to carry or not to carry the disease-associated gene. In 2 cases, testing of the newborn child of a known or suspected carrier confirmed the clinical suspicion of affected status and justified proactive therapy. In 4 of the 5 cases, the mode of inheritance was not clear from the family history and was established as X-linked by the testing. Assay for restriction sites changed by disease-associated mutations agreed with the automated sequencing results. CONCLUSIONS: We conclude that direct mutation analysis in patients suspected of NDI and in selected family members is indicated. The results of testing can confirm a clinical diagnosis of disease, which may otherwise be difficult to make in girls. It can further establish the mode of inheritance, unambiguously distinguish carriers from noncarriers, and justify special observation or treatment of newborns at risk, thereby averting dehydration and the attendant complications. We also conclude that, with proper controls, automated sequencing is the preferred method of testing, because it is sufficiently robust, sensitive, and adaptable for this short gene with a large variety of causative mutations.

AVPR2 variants and V2 vasopressin receptor function in nephrogenic diabetes insipidus.

BACKGROUND: The AVPR2 gene encodes the type 2 vasopressin receptor, a member of the vasopressin/oxytocin receptor subfamily of G protein-coupled receptors. Disruption of AVPR2 causes X-linked congenital nephrogenic diabetes insipidus (NDI), yet the functional significance of most gene sequence variations found in association with NDI has not been proven. The large number of naturally occurring AVPR2 mutations constitutes a model system for studying the structure-function relationship of G protein-coupled receptors. This analysis can be aided by examining amino acid sequence variation and conservation among evolutionarily disparate members of the subfamily. METHODS: Twenty-five new NDI patients were evaluated by DNA sequencing for mutations in AVPR2. Receptors encoded by eighteen NDI alleles were tested for physiologic signaling activity in response to varying concentrations of arginine vasopressin (AVP) in a sensitive cell culture assay. Seventeen amino acid sequences from the vasopressin/oxytocin receptor subfamily were aligned and conserved residues were identified and correlated with the locations of NDI associated variations. RESULTS: Twenty-four variant alleles were found among the 25 new patients. Thirteen had no prior family history of expressed NDI. All 18 of the NDI-associated AVPR2 alleles tested for function demonstrated diminished response to stimulation with AVP. Twelve failed to respond at all, whereas six signaled only at high AVP concentrations. Evolutionarily conserved residues clustered in the transmembrane domains and in the first and second extracellular loops, and NDI-associated missense mutations appeared mostly in the conserved domains. CONCLUSIONS: Sporadic cases are frequent and they usually represent the X-linked rather than the autosomal form of NDI. Genetic and functional testing can confirm this in individual cases. Mutations in this study affecting ligand binding domains tend to retain partial signaling in vitro, whereas those that introduce a charged residue in a transmembrane domain are inactive. The minimal partial signaling observed in cultured cells is unlikely to correlate with clinically significant urine concentrating ability. Other AVPR2 mutations with milder effects on receptor function probably exist, but may not be expressed clinically as typical NDI.

Identification of two classes of transcriptional regulator genes in the cyanobacterium Synechococcus sp. strain PCC 7942.

We designed a strategy to isolate and characterize response regulator genes from the cyanobacterium Synechococcus sp. strain PCC 7942 based on the premise that cyanobacterial response regulators would bear strong similarity to their counterparts from other eubacteria. Two response regulator genes, srrA and srrB, were isolated from Synechococcus and found to encode proteins similar to the OmpR subclass of response regulators. Disruption of either gene by insertional mutagenesis did not produce an obvious phenotype and did not affect the accumulation of psbAII mRNA under high-light conditions, indicating that these gene products are not involved in mediating the well characterized standard- to high-light transition response of photosystem II genes in this cyanobacterium. Analysis of the chromosomal region adjacent to srrA revealed the presence of another presumptive transcriptional activator gene. This gene, named lrrA, belongs to the lysR family. Attempts to disrupt lrrA or an adjacent ORF (orfG) were not successful, suggesting that these genes are important for the growth of Synechococcus.