Targeting Src in endometriosis-associated ovarian cancer

The SRC proto-oncogene is commonly overexpressed or activated during cancer development. Src family kinase inhibitors are approved for the treatment of certain leukemias, and are in clinical trials for the treatment of solid tumors. Src signaling is activated in endometriosis, a precursor of clear cell and endometrioid subtypes of epithelial ovarian cancers (OCs). We examined the expression of phosphorylated Src (Src-pY416) in 381 primary OC tissues. Thirty-six percent of OCs expressed Src-pY416. Src-pY416 expression was most common in endometriosis-associated OCs (EAOCs) (P=0.011), particularly in clear cell OCs where 58.5% of cases expressed Src-pY416. Src-pY416 expression was associated with shorter overall survival (log rank P=0.002). In vitro inhibition of Src signaling using 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2) resulted in reduced anchorage-independent and -dependent growth, and in three-dimensional cell culture models PP2 disrupted aggregate formation in Src-pY416-positive but not in Src-pY416-negative cell lines. These data suggest that targeting active Src signaling could be a novel therapeutic opportunity for EAOCs, and support the further pre-clinical investigation of Src family kinase inhibitors for treating OCs expressing Src-pY416

Characterization of an Aggregated Three-Dimensional Cell Culture Model by Multimodal Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) is an established analytical tool capable of defining and understanding complex tissues by determining the spatial distribution of biological molecules. Three-dimensional (3D) cell culture models mimic the pathophysiological environment of in vivo tumors and are rapidly emerging as a valuable research tool. Here, multimodal MSI techniques were employed to characterize a novel aggregated 3D lung adenocarcinoma model, developed by the group to mimic the in vivo tissue. Regions of tumor heterogeneity and the hypoxic microenvironment were observed based on the spatial distribution of a variety of endogenous molecules. Desorption electrospray ionization (DESI)-MSI defined regions of a hypoxic core and a proliferative outer layer from metabolite distribution. Targeted metabolites (e.g., lactate, glutamine, and citrate) were mapped to pathways of glycolysis and the TCA cycle demonstrating tumor metabolic behavior. The first application of imaging mass cytometry (IMC) with 3D cell culture enabled single-cell phenotyping at 1 μm spatial resolution. Protein markers of proliferation (Ki-67) and hypoxia (glucose transporter 1) defined metabolic signaling in the aggregoid model, which complemented the metabolite data. Laser ablation inductively coupled plasma (LA-ICP)-MSI analysis localized endogenous elements including magnesium and copper, further differentiating the hypoxia gradient and validating the protein expression. Obtaining a large amount of molecular information on a complementary nature enabled an in-depth understanding of the biological processes within the novel tumor model. Combining powerful imaging techniques to characterize the aggregated 3D culture highlighted a future methodology with potential applications in cancer research and drug development

Expression of BNIP3 in invasive breast cancer: correlations with the hypoxic response and clinicopathological features

<p>Abstract</p> <p>Background</p> <p>Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a pro-apoptotic member of the Bcl-2 family induced under hypoxia. Low or absent expression has recently been described in human tumors, including gastrointestinal tumors, resulting in poor prognosis. Little is known about BNIP3 expression in invasive breast cancer. The aim of the present study was to investigate the expression of BNIP3 in invasive breast cancer at the mRNA and protein level in correlation with the hypoxic response and clinicopathological features.</p> <p>Methods</p> <p>In 40 cases of invasive breast cancer, BNIP3 mRNA <it>in situ </it>hybridization was performed on frozen sections with a digoxigenin labeled anti-BNIP3 probe. Paraffin embedded sections of the same specimens were used to determine protein expression of BNIP3, Hypoxia Inducible Factor 1 alpha (HIF-1α) and its downstream targets Glucose Transporter 1 (Glut-1) and Carbonic Anhydrase (CAIX) by immunohistochemistry.</p> <p>Results</p> <p>BNIP3 mRNA was expressed in 16/40 (40%) of the cases and correlated with BNIP3 protein expression (p = 0.0218). Neither BNIP3 protein nor mRNA expression correlated with expression of HIF-1α expression or its downstream targets. Tumors which showed loss of expression of BNIP3 had significantly more often lymph node metastases (82% vs 39%, p = 0.010) and showed a higher mitotic activity index (p = 0.027). BNIP3 protein expression was often nuclear in normal breast, but cytoplasmic in tumor cells.</p> <p>Conclusion</p> <p>BNIP3 expression is lost in a significant portion of invasive breast cancers, which is correlated with poor prognostic features such as positive lymph node status and high proliferation, but not with the hypoxic response.</p

Aberrant DNA methylation associated with silencing BNIP3 gene expression in haematopoietic tumours

Hypoxia is a key factor contributing to the progression of human neoplasias and to the development of resistance to chemotherapy. BNIP3 is a proapoptotic member of the Bcl-2 protein family involved in hypoxia-induced cell death. We evaluated the expression and methylation status of BNIP3 gene to better understand the role of epigenetic alteration of its expression in haematopoietic tumours. Methylation of the region around the BNIP3 transcription start site was detected in four acute lymphocytic leukaemia, one multiple myeloma and one Burkitt lymphoma cell lines, and was closely associated with silencing the gene. That expression of BNIP3 was restored by treatment with 5-aza2′-deoxycytidine (5-aza-dC), a methyltransferase inhibitor, which confirmed the gene to be epigenetically inactivated by methylation. Notably, re-expression of BNIP3 using 5-aza2-dC also restored hypoxia-mediated cell death in methylated cell lines. Acetylation of histone H3 in the 5′ region of the gene, which was assessed using chromatin immunoprecipitation assays, correlated directly with gene expression and inversely with DNA methylation. Among primary tumours, methylation of BNIP3 was detected in five of 34 (15%) acute lymphocytic leukaemias, six of 35 (17%) acute myelogenous leukaemias and three of 14 (21%) multiple myelomas. These results suggest that aberrant DNA methylation of the 5′ CpG island and histone deacetylation play key roles in silencing BNIP3 expression in haematopoietic tumours

RAD51C Germline Mutations in Breast and Ovarian Cancer Cases from High-Risk Families

BRCA1 and BRCA2 are the most well-known breast cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. One such gene, RAD51C, is essential for homologous recombination repair. Several likely pathogenic RAD51C mutations have been identified in BRCA1- and BRCA2-negative breast and ovarian cancer families. We performed complete sequencing of RAD51C in germline DNA of 286 female breast and/or ovarian cancer cases with a family history of breast and ovarian cancers, who had previously tested negative for mutations in BRCA1 and BRCA2. We screened 133 breast cancer cases, 119 ovarian cancer cases, and 34 with both breast and ovarian cancers. Fifteen DNA sequence variants were identified; including four intronic, one 5′ UTR, one promoter, three synonymous, and six non-synonymous variants. None were truncating. The in-silico SIFT and Polyphen programs were used to predict possible pathogenicity of the six non-synonomous variants based on sequence conservation. G153D and T287A were predicted to be likely pathogenic. Two additional variants, A126T and R214C alter amino acids in important domains of the protein such that they could be pathogenic. Two-hybrid screening and immunoblot analyses were performed to assess the functionality of these four non-synonomous variants in yeast. The RAD51C-G153D protein displayed no detectable interaction with either XRCC3 or RAD51B, and RAD51C-R214C displayed significantly decreased interaction with both XRCC3 and RAD51B (p<0.001). Immunoblots of RAD51C-Gal4 activation domain fusion peptides showed protein levels of RAD51C-G153D and RAD51C-R214C that were 50% and 60% of the wild-type, respectively. Based on these data, the RAD51C-G153D variant is likely to be pathogenic, while the RAD51C- R214C variant is hypomorphic of uncertain pathogenicity. These results provide further support that RAD51C is a rare breast and ovarian cancer susceptibility gene

Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor

<p>Abstract</p> <p>Background</p> <p>Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma.</p> <p>Methods</p> <p>HIF-1α and HIF-2α immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration.</p> <p>Results</p> <p>17/56 Ewing's tumours were HIF-1α-positive, 15 HIF-2α-positive and 10 positive for HIF-1α and HIF-2α. Expression of HIF-1α and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1α and HIF-2α in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2α in Ewing's. Downstream transcription was HIF-1α-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by ≥ 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration.</p> <p>Conclusions</p> <p>Co-localisation of HIF-1α and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in <it>in vivo </it>induction of HIF. <it>In vitro </it>data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.</p

Integrative Genomic Data Mining for Discovery of Potential Blood-Borne Biomarkers for Early Diagnosis of Cancer

Background: With the arrival of the postgenomic era, there is increasing interest in the discovery of biomarkers for the accurate diagnosis, prognosis, and early detection of cancer. Blood-borne cancer markers are favored by clinicians, because blood samples can be obtained and analyzed with relative ease. We have used a combined mining strategy based on an integrated cancer microarray platform, Oncomine, and the biomarker module of the Ingenuity Pathways Analysis (IPA) program to identify potential blood-based markers for six common human cancer types. Methodology/Principal Findings: In the Oncomine platform, the genes overexpressed in cancer tissues relative to their corresponding normal tissues were filtered by Gene Ontology keywords, with the extracellular environment stipulated and a corrected Q value (false discovery rate) cut-off implemented. The identified genes were imported to the IPA biomarker module to separate out those genes encoding putative secreted or cell-surface proteins as blood-borne (blood/serum/plasma) cancer markers. The filtered potential indicators were ranked and prioritized according to normalized absolute Student t values. The retrieval of numerous marker genes that are already clinically useful or under active investigation confirmed the effectiveness of our mining strategy. To identify the biomarkers that are unique for each cancer type, the upregulated marker genes that are in common between each two tumor types across the six human tumors were also analyzed by the IPA biomarker comparison function. Conclusion/Significance: The upregulated marker genes shared among the six cancer types may serve as a molecular tool to complement histopathologic examination, and the combination of the commonly upregulated and unique biomarkers may serve as differentiating markers for a specific cancer. This approach will be increasingly useful to discover diagnostic signatures as the mass of microarray data continues to grow in the ‘omics’ era

Tropical peat surface oscillations are a function of peat condition at North Selangor peat swamp forest, Malaysia

Tropical peatland condition across southeast Asia is deteriorating as a result of conversion to agriculture and urban zones. Conversion begins by lowering the water table, which leads to peat decomposition, subsidence and increased risk of large-scale forest fires. Associated changes in mechanical peat properties impact the magnitude and timing of changes in peatland surface motion, making them a potential proxy for peatland condition. However, such a relationship is yet to be observed in a tropical peatland setting. This study aimed to establish whether patterns of tropical peatland surface motion were a function of peat condition at North Selangor Peat Swamp Forest in Selangor, Malaysia. Results showed that subsidence was greatest at fire-affected scrubland sites, whilst the lowest mean water table levels were found at smallholder oil palm sites. Peat condition and magnitude of tropical peat surface oscillation were significantly different between peat condition classes, whilst peat condition differed with depth. More degraded tropical peats with high bulk density throughout the peat profile due to high surface loading and low mean water table levels showed greater surface oscillation magnitudes. The dominant peat surface oscillation mechanisms present at all sites were compression and shrinkage from changes in water table level. Mean water table level and subsidence rate were related to surface oscillation magnitude. However further work towards measuring surface and within-water table range bulk densities and surface loading is required to better understand the controls on surface oscillation magnitudes

Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype

BACKGROUND: Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasis-associated genes. Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression. The aim of this study was to examine the as yet unknown relationship between hypoxia and CTCs. METHODS: We generated human MDA-MB-231 orthotopic xenografts and, using a new technology, isolated viable human CTCs from murine blood. The CTCs and parental MDA-MB-231 cells were incubated at 21 and 0.2% (hypoxia) oxygen, respectively. Colony formation was assayed and levels of hypoxia- and anoxia-inducible factors were measured. Xenografts generated from CTCs and parental cells were compared. RESULTS: MDA-MB-231 xenografts used to generate CTCs were hypoxic, expressing hypoxia factors: hypoxia-inducible factor1 alpha (HIF1alpha) and glucose transporter protein type 1 (GLUT1), and anoxia-induced factors: activating transcription factor 3 and 4 (ATF3 and ATF4). Parental MDA-MB-231 cells induced ATF3 in hypoxia, whereas CTCs expressed it constitutively. Asparagine synthetase (ASNS) expression was also higher in CTCs. Hypoxia induced ATF4 and the HIF1alpha target gene apelin in CTCs, but not in parental cells. Hypoxia induced lower levels of carbonic anhydrase IX (CAIX), GLUT1 and BCL2/adenovirus E1B 19-KD protein-interacting protein 3 (BNIP3) proteins in CTCs than in parental cells, supporting an altered hypoxia response. In chronic hypoxia, CTCs demonstrated greater colony formation than parental cells. Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts. CONCLUSION: CTCs show an altered hypoxia response and an enhanced aggressive phenotype in vitro and in vivo

Expression of hypoxia-inducible factor-1α and cell cycle proteins in invasive breast cancer are estrogen receptor related

BACKGROUND: The transcription factor hypoxia-inducible factor-1 (HIF-1) is a key regulator of the cellular response to hypoxia. Previous studies showed that concentrations of its subunit HIF-1α, as a surrogate for HIF-1 activity, are increased during breast carcinogenesis and can independently predict prognosis in breast cancer. During carcinogenesis, the cell cycle is progressively deregulated, and proliferation rate is a strong prognostic factor in breast cancer. In this study we undertook a detailed evaluation of the relationships between HIF-1α and cell cycle-associated proteins. METHODS: In a representative estrogen receptor (ER) group of 150 breast cancers, the expression of HIF-1α, vascular endothelial growth factor, the ER, HER-2/neu, Ki-67, cyclin A, cyclin D(1), p21, p53, and Bcl-2 was investigated by immunohistochemistry. RESULTS: High concentrations (5% or more) of HIF-1α were associated with increased proliferation as shown by positive correlations with Ki-67 (P < 0.001) and the late S–G2-phase protein cyclin A (P < 0.001), but not with the G1-phase protein cyclin D(1). High HIF-1α concentrations were also strongly associated with p53 positivity (P < 0.001) and loss of Bcl-2 expression (P = 0.013). No association was found between p21 and HIF-1α (P = 0.105) in the whole group of patients. However, the subgroup of ER-positive cancers was characterized by a strong positive association between HIF-1α and p21 (P = 0.023), and HIF-1α lacked any relation with proliferation. CONCLUSION: HIF-1α overexpression is associated with increased proliferation, which might explain the adverse prognostic impact of increased concentrations of HIF-1α in invasive breast cancer. In ER-positive tumors, HIF-1α is associated with p21 but not against proliferation. This shows the importance of further functional analysis to unravel the role of HIF-1 in late cell cycle progression, and the link between HIF-1, p21, and ER