Craig N. Giroux

Meiosis-Specific DNA Double-Strand Breaks Are Catalyzed by Spo11, a Member of a Widely Conserved Protein Family

Meiotic recombination in S. cerevisiae is initiated by double-strand breaks (DSBs). In certain mutants, breaks accumulate with a covalently attached protein, suggesting that cleavage is catalyzed by the DSB-associated protein via a topoisomerase-like transesterase mechanism. We have purified these protein-DNA complexes and identified the protein as Spo11, one of several proteins required for DSB formation. These findings strongly implicate Spo11 as the catalytic subunit of the meiotic DNA cleavage activity. This is the first identification of a biochemical function for any of the gene products involved in DSB formation. Spo11 defines a protein family with other members in fission yeast, nematodes, and archaebacteria. The S. pombe homolog, rec12p, is also known to be required for meiotic recombination. Thus, these findings provide direct evidence that the mechanism of meiotic recombination initiation is evolutionarily conserved.

Identification of the Orphan G Protein-coupled Receptor GPR31 as a Receptor for 12-(S)-Hydroxyeicosatetraenoic Acid

Hydroxy fatty acids are critical lipid mediators involved in various pathophysiologic functions. We cloned and identified GPR31, a plasma membrane orphan G protein-coupled receptor that displays high affinity for the human 12-lipoxygenase-derived product 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE). Thus, GPR31 is named 12-(S)-HETE receptor (12-HETER) in this study. The cloned 12-HETER demonstrated high affinity binding for 12-(S)-[3H]HETE (Kd = 4.8 ± 0.12 nm). Also, 12-(S)-HETE efficiently and selectively stimulated GTPγS coupling in the membranes of 12-HETER-transfected cells (EC50 = 0.28 ± 1.26 nm). Activating GTPγS coupling with 12-(S)-HETE proved to be both regio- and stereospecific. Also, 12-(S)-HETE/12-HETER interactions lead to activation of ERK1/2, MEK, and NFκB. Moreover, knocking down 12-HRTER specifically inhibited 12-(S)-HETE-stimulated cell invasion. Thus, 12-HETER represents the first identified high affinity receptor for the 12-(S)-HETE hydroxyl fatty acids.

Transforming Properties of 8p11-12 Amplified Genes in Human Breast Cancer

Amplification of the 8p11-12 region has been found in about 15% of human breast cancers and is associated with poor prognosis. Earlier, we used genomic analysis of copy number and gene expression to perform a detailed analysis of the 8p11-12 amplicon to identify candidate oncogenes in breast cancer. We identified 21 candidate genes and provided evidence that three genes, namely, LSM-1, TC-1, and BAG4, have transforming properties when overexpressed. In the present study, we systematically investigated the transforming properties of 13 newly identified 8p11-12 candidate oncogenes in vitro. WHSC1L1, DDHD2, and ERLIN2 were most potently transforming oncogenes based on the number of altered phenotypes expressed by the cells. WHSC1L1 contains a PWWP-domain that is a methyl-lysine recognition motif involved in histone code modification and epigenetic regulation of gene expression. Knockdown of WHSC1L1 in 8p11-12-amplified breast cancer cells resulted in profound loss of growth and survival of these cells. Further, we identified several WHSC1L1 target genes, one of which is iroquois homeobox 3 gene (IRX3), a member of the Iroquois homeobox transcription factor family.

Genes Associated with Prostate Cancer Are Differentially Expressed in African American and European American Men

Background: Despite more aggressive screening across all demographics and gradual declines in mortality related to prostate cancer (PCa) in the United States, disparities among populations persist. A substantial proportion of African American men (AAM) have a higher overall incidence, earlier age of onset, increased proportion of clinically advanced disease, and increased bone metastases and mortality from PCa compared to European American men (EAM). Limited early evidence indicates that underlying causes for disparities may be observed in tumor-specific gene expression programs. Methods: This study used microarray-based methods to measure expression levels for 517 genes that were previously associated with PCa in archived formalin-fixed paraffin embedded (FFPE) specimens; testing the hypothesis that gene expression features of functional consequence to cancer distinguish PCa from AAM and EAM. A t test was conducted comparing AAM to EAM expression levels for each probe on the array. Results: Analysis of 639 tumor samples (270 AAM, 369 EAM) showed that 95 genes were overexpressed specifically in PCa from AAM relative to EAM and 132 were overexpressed in PCa from EAM relative to AAM. Furthermore, systems-level analyses highlight the relevant signaling pathways and functions associated with the EAM- or AAM-specific overexpressed gene sets, for example, inflammation and lipid metabolism. Conclusions: Results here bring further understanding to the potential for molecular differences for PCa in AAM versus EAM. Impact: The results support the notion that therapeutic benefits will be realized when targeted treatments are designed to acknowledge and address a greater spectrum of PCa subtypes and molecular distinctions. Cancer Epidemiol Biomarkers Prev; 22(5); 891–7. ©2013 AACR.

Cytokine and Cytokine Receptor Single-Nucleotide Polymorphisms Predict Risk for Non–Small Cell Lung Cancer among Women

Studies on the relationships between inflammatory pathway genes and lung cancer risk have not included African-Americans and have only included a handful of genes. In a population-based case-control study on 198 African-American and 744 Caucasian women, we examined the association between 70 cytokine and cytokine receptor single-nucleotide polymorphisms (SNPs) and risk of non–small cell lung cancer (NSCLC). Unconditional logistic regression was used to estimate odds ratios and 95% confidence intervals in a dominant model adjusting for major risk factors for lung cancer. Separate analyses were conducted by race and by smoking history and history of chronic obstructive pulmonary disease among Caucasians. Random forest analysis was conducted by race. On logistic regression analysis, IL6 (interleukin 6), IL7R, IL15, TNF (tumor necrosis factor), and IL10 SNP were associated with risk of non–small cell lung cancer among African-Americans; IL7R and IL10 SNPs were also associated with risk of lung cancer among Caucasians. Although random forest analysis showed IL7R and IL10 SNPs as being associated with risk for lung cancer among African-Americans, it also identified TNFRSF10A SNP as an important predictor. On random forest analysis, an IL1A SNP was identified as an important predictor of lung cancer among Caucasian women. Inflammatory SNPs differentially predicted risk for NSCLC according to race, as well as based on smoking history and history of chronic obstructive pulmonary disease among Caucasian women. Pathway analysis results are presented. Inflammatory pathway genotypes may serve to define a high risk group; further exploration of these genes in minority populations is warranted. (Cancer Epidemiol Biomarkers Prev 2009;18(6):1829–40)

Knock-down of amphiregulin inhibits cellular invasion in inflammatory breast cancer.

We have previously shown that SUM‐149 human breast cancer cells require an amphiregulin (AREG) autocrine loop for cell proliferation. We also demonstrated that AREG can increase epidermal growth factor receptor (EGFR) stability and promote EGFR localization to the plasma membrane. In the present studies we successfully knocked‐down AREG expression in SUM‐149 cells by lentiviral infection of AREG shRNA. In the absence of AREG expression, SUM‐149 cell growth was slowed, but not completely inhibited. Furthermore, cells infected with AREG shRNA constructs showed an increase in EGFR protein expression by Western blot. Immunofluorescence and confocal microscopy showed that following AREG knock‐down, EGFR continued to localize to the cell surface. Soft agar assays demonstrated that AREG knock‐down cells retain anchorage‐independent growth capacity. Additionally mammosphere forming assays and Adefluor staining analysis showed that knock‐down of AREG expression did not affect the expression of stem cell phenotypes. However, following AREG knock‐down, SUM‐149 cells demonstrated a dramatic decrease in their ability to invade a Matrigel matrix. Consistent with this observation, microarray analysis comparing cells infected with a non‐silencing vector to the AREG knock‐down cells, identified genes associated with the invasive phenotype such as RHOB and DKK1, and networks associated with cell motility such as integrin‐linked kinase signaling, and focal adhesion kinase signaling. AREG was also found to modulate WNT and Notch signaling in these cells. Thus, AREG functions in regulating the invasive phenotype, and we propose that this regulation may be through altered signaling that occurs when AREG activates plasma membrane localized EGFR. J. Cell. Physiol. 226: 2691–2701, 2011.

HER-2 Signaling, Acquisition of Growth Factor Independence, and Regulation of Biological Networks Associated with Cell Transformation

Activated oncogenes are the dominant drivers of malignant progression in human cancer, yet little is known about how the transformation from proto-oncogene to activated oncogene drives the expression of transformed phenotypes. An isogenic model of HER-2-mediated transformation of human mammary epithelial cells was used along with HER-2-amplified human breast cancers to investigate how HER-2 activation alters its properties as a signaling molecule and changes the networks of HER-2-regulated genes. Our results show that full oncogenic activation of HER-2 is the result of a transition in which activated HER-2 acquires dominant signaling properties that qualitatively alter the network of genes regulated by the activated oncogene compared with the proto-oncogene. Consequently, gene expression programs related to invasion, cell stress, and stemness become regulated by HER-2 in a manner not observed in nontransformed cells, even when HER-2 is overexpressed. Our results offer novel insights into biological processes that come under the control of HER-2 after it acquires full oncogenic potential.

Differential biclustering for gene expression analysis

Biclustering algorithms have been successfully used to find subsets of co-expressed genes under subsets of conditions. In some cases, microarray experiments are performed to compare the biological activities of the genes between two classes of cells, such as normal and cancer cells. In this paper, we propose DiBiCLUS, a novel Differential Biclustering algorithm, to identify differential biclusters from the gene expression data where the samples belong to one of the two classes. The genes in these differential biclusters can be positively or negatively co-expressed. We introduce two criteria for any pair of genes to be considered as a differential pair across the two classes. To illustrate the performance of the proposed algorithm, we present the experimental results of applying DiBiCLUS algorithm on synthetic and reallife datasets. These experiments show that the identified differential biclusters are both statistically and biologically significant.

Analysis of the spectrum of mutations induced by the rad3-102 mutator allele of yeast

The product of the RAD3 gene of Saccharomyces cerevisiae is required for mitotic cell viability and excision repair of UV-induced pyrimidine dimers. Certain rad3 mutant alleles (originally called rem1) increase the rates of both spontaneous mitotic recombination and mutation. The increase in mutation rates is not dependent upon the presence of the RAD6 error-prone pathway. The mutator phenotype suggests that the wild-type RAD3 gene product may be involved in the maintenance of fidelity of DNA replication in addition to its known role in excision repair. To investigate the role that RAD3 might play in mutation avoidance, we have utilized a well-characterized shuttle vector system to study the mutational spectrum occurring in rad3-102 strains and compare it to that seen in RAD3 strains. The results put constraints on the role that the rad-102 mutant gene product must play if the RAD3 protein is a component of the replication complex. Alternatively, the mutational spectrum is consistent with the hypothesis that the rad3-102 mutant protein interferes with postreplication mismatch repair.

Antioxidant Genes, Hormesis, and Demographic Longevity

Mortality rates have been observed to slow down or even decline in late life such that a small subset of the population has a significantly longer survival than does the remainder of its cohort. Th...