Genomic and functional characterizations of phosphodiesterase subtype 4D in human cancers.

Discovery of cancer genes through interrogation of genomic dosage is one of the major approaches in cancer research. In this study, we report that phosphodiesterase subtype 4D (PDE4D) gene was homozygously deleted in 198 cases of 5,569 primary solid tumors (3.56%), with most being internal microdeletions. Unexpectedly, the microdeletions did not result in loss of their gene products. Screening PDE4D expression in 11 different types of primary tumor samples (n = 165) with immunohistochemistry staining revealed that its protein levels were up-regulated compared with corresponding nontransformed tissues. Importantly, depletion of endogenous PDE4D with three independent shRNAs caused apoptosis and growth inhibition in multiple types of cancer cells, including breast, lung, ovary, endometrium, gastric, and melanoma, which could be rescued by reexpression of PDE4D. We further showed that antitumor events triggered by PDE4D suppression were lineage-dependently associated with Bcl-2 interacting mediator of cell death (BIM) induction and microphthalmia-associated transcription factor (MITF) down-regulation. Furthermore, ectopic expression of the PDE4D short isoform, PDE4D2, enhanced the proliferation of cancer cells both in vitro and in vivo. Moreover, treatment of cancer cells with a unique specific PDE4D inhibitor, 26B, triggered massive cell death and growth retardation. Notably, these antineoplastic effects induced by either shRNAs or small molecule occurred preferentially in cancer cells but not in nonmalignant epithelial cells. These results suggest that although targeted by genomic homozygous microdeletions, PDE4D functions as a tumor-promoting factor and represents a unique targetable enzyme of cancer cells.

Evaluation of ovarian cancer remission markers HE4, MMP7 and Mesothelin by comparison to the established marker CA125.

OBJECTIVE: Evaluate and compare the effectiveness of CA125, HE4, Mesothelin and MMP7 marker levels to monitor ovarian cancer patients after surgery and chemotherapy. Evaluate the lead time of a rise of marker levels before recurrence. METHODS: The study consists of 23 patients with advanced stage ovarian/fallopian tube cancer. Blood was drawn after front line surgery and chemotherapy treatment and at 3 month intervals thereafter. One patient had chemoresistant disease, two patients remained in remission and 20 patients had recurring disease and were used for marker evaluation. RESULTS: In five patients HE4 was the only marker to elevate before recurrence with a lead time of up to 4½ months including one patient who did not have a CA125 response at all. In a further two patients, HE4 increased before CA125 did. In four of these seven patients, HE4 levels were consistently at or above threshold during remission when both CA125 and imaging results were negative. MMP7 elevated before recurrence in one patient who was negative for the other markers. Mesothelin elevated in two patients who were also positive for CA125 and HE4. CONCLUSIONS: HE4 can predict ovarian cancer recurrence earlier than CA125 and it can be elevated in patients that do not express CA125 at sufficient levels to make a clinical decision. MMP7 and Mesothelin have lower potential as markers for ovarian cancer recurrence to complement CA125. A failure of HE4 levels to normalize at completion of standard therapy may indicate a poor prognosis.

The HE4 (WFDC2) protein is a biomarker for ovarian carcinoma.

The WFDC2 (HE4) gene is amplified in ovarian carcinomas, whereas its expression in normal tissues, including ovary, is low. Although the function of the HE4 protein is unknown,it is a member of a family of stable 4-disulfide core proteins that are secreted at high levels. We therefore performed experiments to explore whether quantitation of HE4 protein levels in serum can be used as a biomarker for ovarian carcinoma. A fusion gene was constructed encoding the HE4 protein fused to a gene encoding the murine IgG2a Fc domain. Subsequently, protein produced in mammalian cells was purified by affinity chromatography and used to immunize mice to generate hybridomas specific for HE4. Hybridoma supernatants were screened for binding to a similar fusion protein that, instead, had a human immunoglobulin tail. Two hybridomas, 2H5 and 3D8, were selected that produce monoclonal antibodies to different HE4 epitopes, and a double determinant ("Sandwich") ELISA was constructed and shown to detect a signal at the 160-pg level. Blinded studies on sera from postmenopausal patients with ovarian carcinoma and controls indicate that the specificity and sensitivity of the HE4-based ELISA is equivalent to that of the CA125 assay. However, the HE4 assay may have an advantage over the CA125 assay in that it is less frequently positive in patients with nonmalignant disease.

Lysophosphatidic acid is a bioactive mediator in ovarian cancer.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that exhibits pleiotrophic biological activities, ranging from rapid morphological changes to long-term cellular effects such as induction of gene expression and stimulation of cell proliferation and survival on a wide spectrum of cell types. LPA binds and activates distinct members of the Edg/LP subfamily of G protein-coupled receptors that link to multiple G proteins including Gi, Gq and G12/13 to elicit cellular responses. LPA plays a critical role as a general growth, survival and pro-angiogenic factor, in the regulation of physiological and pathophysiological processes in vivo and in vitro. Our previous work indicates that abnormalities in LPA metabolism and function in ovarian cancer patients may contribute to the initiation and progression of the disease. Thus, LPA could be a potential target for cancer therapy. This review summarizes evidence that implicates LPA in the pathophysiology of human ovarian cancer and likely other types of human malignancies.

Evaluating Serum Markers for Hormone Receptor-Negative Breast Cancer.

INTRODUCTION: Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide. Death rates have been declining, largely as a result of early detection through mammography and improved treatment, but mammographic screening is controversial because of over-diagnosis of breast disease that might not require treatment, and under-diagnosis of cancer in women with dense breasts. Breast cancer screening could be improved by pairing mammography with a tumor circulating marker, of which there are currently none. Given genomic similarities between the basal breast cancer subtype and serous ovarian cancer, and given our success in identifying circulating markers for ovarian cancer, we investigated the performance in hormone receptor-negative breast cancer detection of both previously identified ovarian serum markers and circulating markers associated with transcripts that were differentially expressed in breast cancer tissue compared to healthy breast tissue from reduction mammaplasties. METHODS: We evaluated a total of 15 analytes (13 proteins, 1 miRNA, 1 autoantibody) in sera drawn at or before breast cancer surgery from 43 breast cancer cases (28 triple-negative-TN-and 15 hormone receptor-negative-HRN-/ HER2-positive) and 87 matched controls. RESULTS: In the analysis of our whole cohort of breast cancer cases, autoantibodies to TP53 performed significantly better than the other selected 14 analytes showing 25.6% and 34.9% sensitivity at 95% and 90% specificity respectively with AUC: 0.7 (p<0.001). The subset of 28 TN cancers showed very similar results. We observed no correlation between anti-TP53 and the 14 other markers; however, anti-TP53 expression correlated with Body-Mass-Index. It did not correlate with tumor size, positive lymph nodes, tumor stage, the presence of metastases or recurrence. CONCLUSION: None of the 13 serum proteins nor miRNA 135b identified women with HRN or TN breast cancer. TP53 autoantibodies identified women with HRN breast cancer and may have potential for early detection, confirming earlier reports. TP53 autoantibodies are long lasting in serum but may be affected by storage duration. Autoantibodies to TP53 might correlate with Body-Mass-Index.

Breast cancer genomics: normal tissue and cancer markers.

Mammography is a powerful screening tool for early detection of breast cancer, but it has limitations in terms of both specificity and sensitivity. Imaging tools such as MRI that complement mammography are too costly to serve as first-line screens. Recently, progress has been made on blood markers, particularly microRNAs and proteins. There are new methods for protein marker discovery directly in blood, but they are limited in the number of patients that can be examined. An alternative is to discover markers as transcripts in tissues, followed by development of blood protein tests for those that perform best. To identify genes that are overexpressed in malignancy it is paramount to include normal control tissues from healthy individuals. Here we report the identification of potential breast cancer markers, including some that are overexpressed in aggressive disease.

Comparison of breast cancer to healthy control tissue discovers novel markers with potential for prognosis and early detection.

This study was initiated to identify biomarkers with potential value for the early detection of poor-outcome breast cancer. Two sets of well-characterized tissues were utilized: one from breast cancer patients with favorable vs. poor outcome and the other from healthy women undergoing reduction mammaplasty. Over 46 differentially expressed genes were identified from a large list of potential targets by a) mining publicly available expression data (identifying 134 genes for quantitative PCR) and b) utilizing a commercial PCR array. Three genes show elevated expression in cancers with poor outcome and low expression in all other tissues, warranting further investigation as potential blood markers for early detection of cancers with poor outcome. Twelve genes showed lower expression in cancers with poor outcome than in cancers with favorable outcome but no differential expression between aggressive cancers and most healthy controls. These genes are more likely to be useful as prognostic tissue markers than as serum markers for early detection of aggressive disease. As a secondary finding was that, when histologically normal breast tissue was removed from a distant site in a breast with cancer, 7 of 38 specimens displayed a cancer-like expression profile, while the remaining 31 were genetically similar to the reduction mammaplasty control group. This finding suggests that some regions of ipsilateral histologically 'normal' breast tissue are predisposed to becoming malignant and that normal-appearing tissue with malignant signature might warrant treatment to prevent new primary tumors.

Activation of the MEK-S6 pathway in high-grade ovarian cancers.

The primary objective of this study is to show the activation and analyze the regulation of the MEK- S6 kinase pathway in high-grade ovarian cancer. Phospho-ERK (pERK), a direct substrate of MEK and 2 phosphorylation sites on the ribosomal protein, S6, Ser235/236, and Ser240/244, which are both targeted by the MEK and PI3-kinase/AKT pathways, were analyzed in 13 cell lines, 28 primary cancers and 8 cases of cancer cells from ascites. In primary cancers, ERK and S6 phosphorylation was measured by immunohistochemistry (IHC). pERK, pS6, pAKT, and p4EBP1 were also measured by Western blotting (WB). The regulation of S6 phosphorylation by the MEK and PI3-kinase pathways was determined in ovarian cancer cell lines. We observed frequent pERK expression in primary ovarian cancers (100% by WB, 75% by IHC) but not in ovarian cancer cells from ascites (25% of cases by WB). The activation of the AKT pathway, measured by pAKT expression occurred in 7 cases of primary ovarian cancer by WB, but in none of the ascites samples. In ovarian cancer cell lines, the MEK pathway had a greater effect on S6 phosphorylation in cells without hyperactive AKT signaling. Our data suggest that MEK is a potential drug target in high-grade ovarian cancer, however, cancer cells with hyperactive AKT and cancer cells in ascites may be less responsive to MEK inhibition. The phosphorylation of S6 as a specific biomarker for either MEK or PI3-kinase pathway activation should be used with caution.

Proteomic analysis of ovarian cancer cells reveals dynamic processes of protein secretion and shedding of extra-cellular domains.

BACKGROUND: Elucidation of the repertoire of secreted and cell surface proteins of tumor cells is relevant to molecular diagnostics, tumor imaging and targeted therapies. We have characterized the cell surface proteome and the proteins released into the extra-cellular milieu of three ovarian cancer cell lines, CaOV3, OVCAR3 and ES2 and of ovarian tumor cells enriched from ascites fluid. METHODOLOGY AND FINDINGS: To differentiate proteins released into the media from protein constituents of media utilized for culture, cells were grown in the presence of [(13)C]-labeled lysine. A biotinylation-based approach was used to capture cell surface associated proteins. Our general experimental strategy consisted of fractionation of proteins from individual compartments followed by proteolytic digestion and LC-MS/MS analysis. In total, some 6,400 proteins were identified with high confidence across all specimens and fractions. CONCLUSIONS AND SIGNIFICANCE: Protein profiles of the cell lines had substantial similarity to the profiles of human ovarian cancer cells from ascites fluid and included protein markers known to be associated with ovarian cancer. Proteomic analysis indicated extensive shedding from extra-cellular domains of proteins expressed on the cell surface, and remarkably high secretion rates for some proteins (nanograms per million cells per hour). Cell surface and secreted proteins identified by in-depth proteomic profiling of ovarian cancer cells may provide new targets for diagnosis and therapy.

Selecting differentially expressed genes from microarray experiments.

High throughput technologies, such as gene expression arrays and protein mass spectrometry, allow one to simultaneously evaluate thousands of potential biomarkers that could distinguish different tissue types. Of particular interest here is distinguishing between cancerous and normal organ tissues. We consider statistical methods to rank genes (or proteins) in regards to differential expression between tissues. Various statistical measures are considered, and we argue that two measures related to the Receiver Operating Characteristic Curve are particularly suitable for this purpose. We also propose that sampling variability in the gene rankings be quantified, and suggest using the "selection probability function," the probability distribution of rankings for each gene. This is estimated via the bootstrap. A real dataset, derived from gene expression arrays of 23 normal and 30 ovarian cancer tissues, is analyzed. Simulation studies are also used to assess the relative performance of different statistical gene ranking measures and our quantification of sampling variability. Our approach leads naturally to a procedure for sample-size calculations, appropriate for exploratory studies that seek to identify differentially expressed genes.

Serologic analysis of ovarian tumor antigens reveals a bias toward antigens encoded on 17q.

We utilized SEREX immunoscreening to identify a set of novel tumor antigens that are associated with human serous ovarian cancer and may prove useful for the early detection and treatment of this disease. Extensive screening with a panel of sera from 25 late-stage ovarian cancer patients against 3 independent cDNA libraries identified a set of 9 antigens that were immunogenic in more than 1 patient and not in a panel of 20-45 normal female serum donors. These antigens include p53, NY-ESO-1, UBQLN1, HOXB6, TOP2A, putative helicase-RUVBL (RUVBL), HMBA-inducible (HEXIM1), DDX5 and HDCMA. Ten of 25 ovarian cancer patients (40%) expressed serum IgG to at least 1 of these antigens, while 14% (4/25) had antibodies to 2 or more antigens. Unexpectedly, 4 antigens identified in this screen, DDX5, HEXIM1, TOP2A and HOXB6, are encoded within a region of 17q that also includes the genes for HER2/neu, Homeobox-B7 and BRCA1. Real-time RT-PCR analysis showed that mRNA for HER2/neu and 3 SEREX-defined antigens, TOP2A, HOXB6 and DDX5, was more abundant in ovarian tumors than most normal tissues, including normal and benign ovarian tissues, suggesting that elevated expression of genes encoded within this region of chromosome 17 is a common event in ovarian tumors. Thus, these abnormal expression patterns combined with the endogenous immune response suggests that these antigens represent potential targets for immunotherapy.

Differential gene expression profiling of adult murine hematopoietic stem cells.

Hematopoietic stem cells (HSCs) have self-renewal capacity and multilineage developmental potentials. The molecular mechanisms that control the self-renewal of HSCs are still largely unknown. Here, a systematic approach using bioinformatics and array hybridization techniques to analyze gene expression profiles in HSCs is described. To enrich mRNAs predominantly expressed in uncommitted cell lineages, 54 000 cDNA clones generated from a highly enriched population of HSCs and a mixed population of stem and early multipotent progenitor (MPP) cells were arrayed on nylon membranes (macroarray or high-density array), and subtracted with cDNA probes derived from mature lineage cells including spleen, thymus, and bone marrow. Five thousand cDNA clones with very low hybridization signals were selected for sequencing and further analysis using microarrays on glass slides. Two populations of cells, HSCs and MPP cells, were compared for differential gene expression using microarray analysis. HSCs have the ability to self-renew, while MPP cells have lost the capacity for self-renewal. A large number of genes that were differentially expressed by enriched populations of HSCs and MPP cells were identified. These included transcription factors, signaling molecules, and previously unknown genes.