Aliccia Bollig-Fischer

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.

Intronic Non-CG DNA hydroxymethylation and alternative mRNA splicing in honey bees

BackgroundPrevious whole-genome shotgun bisulfite sequencing experiments showed that DNA cytosine methylation in the honey bee (Apis mellifera) is almost exclusively at CG dinucleotides in exons. However, the most commonly used method, bisulfite sequencing, cannot distinguish 5-methylcytosine from 5-hydroxymethylcytosine, an oxidized form of 5-methylcytosine that is catalyzed by the TET family of dioxygenases. Furthermore, some analysis software programs under-represent non-CG DNA methylation and hydryoxymethylation for a variety of reasons. Therefore, we used an unbiased analysis of bisulfite sequencing data combined with molecular and bioinformatics approaches to distinguish 5-methylcytosine from 5-hydroxymethylcytosine. By doing this, we have performed the first whole genome analyses of DNA modifications at non-CG sites in honey bees and correlated the effects of these DNA modifications on gene expression and alternative mRNA splicing.ResultsWe confirmed, using unbiased analyses of whole-genome shotgun bisulfite sequencing (BS-seq) data, with both new data and published data, the previous finding that CG DNA methylation is enriched in exons in honey bees. However, we also found evidence that cytosine methylation and hydroxymethylation at non-CG sites is enriched in introns. Using antibodies against 5-hydroxmethylcytosine, we confirmed that DNA hydroxymethylation at non-CG sites is enriched in introns. Additionally, using a new technique, Pvu-seq (which employs the enzyme PvuRts1l to digest DNA at 5-hydroxymethylcytosine sites followed by next-generation DNA sequencing), we further confirmed that hydroxymethylation is enriched in introns at non-CG sites.ConclusionsCytosine hydroxymethylation at non-CG sites might have more functional significance than previously appreciated, and in honey bees these modifications might be related to the regulation of alternative mRNA splicing by defining the locations of the introns.

Minireview: The Molecular and Genomic Basis for Prostate Cancer Health Disparities

Despite more aggressive screening across all demographics and gradual declines in mortality related to prostate cancer (PCa) in the United States, race disparities persist. For African American men (AAM), PCa is more often an aggressive disease showing increased metastases and greater PCa-related mortality compared with European American men. The earliest research points to how distinctions are likely the result of a combination of factors, including ancestry genetics and lifestyle variables. More recent research considers that cancer, although influenced by external forces, is ultimately a disease primarily driven by aberrations observed in the molecular genetics of the tumor. Research studying PCa predominantly from European American men shows that indolent and advanced or metastatic prostate tumors have distinguishing molecular genomic make-ups. Early yet increasing evidence suggests that clinically distinct PCa from AAM also display molecular distinctions. It is reasonable to predict that further study will reveal molecular subtypes and various frequencies for PCa subtypes among diverse patient groups, thereby providing insight as to the genomic lesions and gene signatures that are functionally implicated in carcinogenesis or aggressive PCa in AAM. That knowledge will prove useful in developing strategies to predict who will develop advanced PCa among AAM and will provide the rationale to develop effective individualized treatment strategies to overcome disparities.

Methylation-associated silencing of SFRP1 with an 8p11-12 amplification inhibits canonical and non-canonical WNT pathways in breast cancers

Recently, we analysed the 8p11‐12 genomic region for copy number and gene expression changes in a panel of human breast cancer cell lines and primary specimens. We found that SFRP1 (Secreted frizzled related protein 1) is frequently under expressed even in breast tumours with copy number increases in this genomic region. SFRP1 encodes a WNT signalling antagonist, and plays a role in the development of multiple solid tumour types. In this study, we analysed methylation‐associated silencing of the SFRP1 gene in breast cancer cells with the 8p11‐12 amplicon, and investigated the tumour suppressor properties of SFRP1 in breast cancer cells. SFRP1 expression was markedly reduced in both the breast cancer cell lines and primary tumour specimens relative to normal primary human mammary epithelial cells even when SFRP1 is amplified. Suppression of SFRP1 expression in breast cancer cells with an SFRP1 gene amplification is associated with SFRP1 promoter methylation. Furthermore, restoration of SFRP1 expression suppressed the growth of breast cancer cells in monolayer, and inhibited anchorage independent growth. We also examined the relationship between the silencing of SFRP1 gene and WNT signalling in breast cancer. Ectopic SFRP1 expression in breast cancer cells suppressed both canonical and non‐canonical WNT signalling pathways, and SFRP1 expression was negatively associated with the expression of a subset of WNT responsive genes including RET and MSX2. Thus, down‐regulation of SFRP1 can be triggered by epigenetic and/or genetic events and may contribute to the tumourigenesis of human breast cancer through both canonical and non‐canonical WNT signalling pathways.

Fibroblast Growth Factor Receptor 1 Amplification in Non-Small Cell Lung Cancer by Quantitative Real-Time PCR

Introduction Amplification of the fibroblast growth factor receptor 1 (FGFR1) gene has been described in tumors of non-small-cell lung cancer (NSCLC) patients. Prior reports showed conflicting rates of amplification frequency and clinical relevance. Materials and Methods We developed a reliable real-time quantitative PCR assay to assess the frequency of FGFR1 amplification and assessed the optimal cutoff level of amplification for clinical application. Results In a training cohort of 203 NSCLCs, we established that a 3.5-fold amplification optimally divided patients into groups with different survival rates with a clear threshold level. Those with FGFR1 amplification levels above 3.5-fold had an inferior survival. These data were confirmed in a validation cohort of 142 NSCLC. After adjusting for age, sex, performance status, stage, and histology, patients with FGFR1 amplification levels above 3.5 fold had a hazard ratio of 2.91 (95% CI- 1.14, 7.41; pvalue-0.025) for death in the validation cohort. The rates of FGFR1 amplification using the cutoff level of 3.5 were 5.1% in squamous cell and 4.1% in adenocarcinomas. There was a non-significant trend towards higher amplifications rates in heavy smokers (> 15 pack-years of cigarette consumption) as compared to light smokers. Discussion Our data suggest that a 3.5-fold amplification of FGFR1 is of clinical importance in NSCLC. Our cutpoint analysis showed a clear threshold effect for the impact of FGFR1 amplification on patients’ survival, which can be used as an initial guide for patient selection in trials assessing efficacy of novel FGFR inhibitors.

Oncogene activation induces metabolic transformation resulting in insulin-independence in human breast cancer cells

Normal breast epithelial cells require insulin and EGF for growth in serum-free media. We previously demonstrated that over expression of breast cancer oncogenes transforms MCF10A cells to an insulin-independent phenotype. Additionally, most breast cancer cell lines are insulin-independent for growth. In this study, we investigated the mechanism by which oncogene over expression transforms MCF10A cells to an insulin-independent phenotype. Analysis of the effects of various concentrations of insulin and/or IGF-I on proliferation of MCF10A cells demonstrated that some of the effects of insulin were independent from those of IGF-I, suggesting that oncogene over expression drives a true insulin-independent proliferative phenotype. To test this hypothesis, we examined metabolic functions of insulin signaling in insulin-dependent and insulin-independent cells. HER2 over expression in MCF10A cells resulted in glucose uptake in the absence of insulin at a rate equal to insulin-induced glucose uptake in non-transduced cells. We found that a diverse set of oncogenes induced the same result. To gain insight into how HER2 oncogene signaling affected increased insulin-independent glucose uptake we compared HER2-regulated gene expression signatures in MCF10A and HER2 over expressing MCF10A cells by differential analysis of time series gene expression data from cells treated with a HER2 inhibitor. This analysis identified genes specifically regulated by the HER2 oncogene, including VAMP8 and PHGDH, which have known functions in glucose uptake and processing of glycolytic intermediates, respectively. Moreover, these genes specifically implicated in HER2 oncogene-driven transformation are commonly altered in human breast cancer cells. These results highlight the diversity of oncogene effects on cell regulatory pathways and the importance of oncogene-driven metabolic transformation in breast cancer.

Functional role of miR-10b in tamoxifen resistance of ER-positive breast cancer cells through down-regulation of HDAC4

BackgroundFor breast cancer patients diagnosed with estrogen receptor (ER)-positive tumors, treatment with tamoxifen is the gold standard. A significant number of patients, however, develop resistance to tamoxifen, and management of such tamoxifen-resistant patients is a major clinical challenge. With an eye to identify novel targets for the treatment of tamoxifen-resistant tumors, we observed that tamoxifen-resistant cells derived from ER-positive MCF-7 cells (MCF7TR) exhibit an increased expression of microRNA-10b (miR-10b). A role of miR-10b in drug-resistance of breast cancer cells has never been investigated, although its is very well known to influence invasion and metastasis.MethodsTo dileneate a role of miR-10b in tamoxifen-resistance, we over-expressed miR-10b in MCF-7 cells and down-regulated its levels in MCF7TR cells. The mechanistic role of HDAC4 in miR-10b-mediated tamoxifen resistance was studied using HDAC4 cDNA and HDAC4-specific siRNA in appropriate models.ResultsOver-expression of miR-10b in ER-positive MCF-7 and T47D cells led to increased resistance to tamoxifen and an attenuation of tamoxifen-mediated inhibition of migration, whereas down-regulation of miR-10b in MCF7TR cells resulted in increased sensitivity to tamoxifen. Luciferase assays identified HDAC4 as a direct target of miR-10b. In MCF7TR cells, we observed down-regulation of HDAC4 by miR-10b. HDAC4-specific siRNA-mediated inactivation of HDAC4 in MCF-7 cells led to acquisition of tamoxifen resistance, and, moreover, reduction of HDAC4 in MCF7TR cells by HDAC4-specific siRNA transfection resulted in further enhancement of tamoxifen-resistance.ConclusionsWe propose miR-10b-HDAC4 nexus as one of the molecular mechanism of tamoxifen resistance which can potentially be expolited as a novel targeted therapeutic approach for the clinical management of tamoxifen-resistant breast cancers.

Amyloid-beta neuroprotection mediated by a targeted antioxidant

Amyloid-beta (Aβ)-induced neurotoxicity is a major contributor to the pathologies associated with Alzheimers disease (AD). The formation of reactive oxygen species (ROS), an early response induced by the peptide and oligomeric derivatives of Aβ, plays a significant role in effecting cellular pathogenesis. Here we employ particularly toxic forms of Aβ with cultured primary cortical/hippocampal neurons to elicit ROS and drive cellular dysfunction. To prevent and even reverse such effects, we utilized a cell-penetrating, peroxisome-targeted, protein biologic – called CAT-SKL. We show the recombinant enzyme enters neurons, reverses Aβ-induced oxidative stress, and increases cell viability. Dramatic restorative effects on damaged neuronal processes were also observed. In addition, we used DNA microarrays to determine Aβs effects on gene expression in neurons, as well as the ability of CAT-SKL to modify such Aβ-induced expression profiles. Our results suggest that CAT-SKL, a targeted antioxidant, may represent a new therapeutic approach for treatment of disorders, like Alzheimers disease, that are driven through oxidative stress. Preclinical testing is ongoing.

Racial Diversity of Actionable Mutations in Non–Small Cell Lung Cancer

Introduction: Lung cancer is the leading cause of cancer-related deaths in the US. The reasons for higher incidence and poorer survival rates among black compared with white lung cancer patients have not been defined. We hypothesized that differential incidence of somatic cancer gene mutations may be a contributing factor. Previous genomic studies of non–small cell lung cancer (NSCLC) have not adequately represented black patients. Methods: A matrix-assisted laser desorption/ionization and time-of-flight mass spectrometry approach was used to analyze tumor DNA for 214 coding mutations in 26 cancer genes previously identified in NSCLC. The samples included NSCLC from 335 white patients and 137 black patients. For 299 of these, normal matched DNA was available and analyzed. Results: Epidermal growth factor receptor (EGFR) exon 19 deletions were only detected in women cases, with increased odds for black women compared with white women (odds ratio = 3.914, 95% confidence interval: 1.014–15.099, p = 0.048). Beyond race, variations in mutation frequencies were seen by histology. DDR2 alterations, previously described as somatic mutations, were identified as constitutional variants. Conclusions: This study is among the largest comparing somatic mutations in black and white patients. The results point to the molecular diversity of NSCLC and raise new questions as to the importance of inherited alleles. Genomic tumor testing will benefit both populations, although the mutation spectrum appears to vary by sex, race, and histology.