Kristin L.M. Boylan

Identification of Candidate Biomarkers in Ovarian Cancer Serum by Depletion of Highly Abundant Proteins and Differential In Gel Electrophoresis

Ovarian cancer is the fifth leading cause of cancer death for women in the US, yet survival rates are over 90% when it is diagnosed at an early stage, highlighting the need for biomarkers for early detection. To enhance the discovery of tumor‐specific proteins that could represent novel serum biomarkers for ovarian cancer, we depleted serum of highly abundant proteins which can mask the detection of proteins present in serum at low concentrations. Three commercial immunoaffinity columns were used in parallel to deplete the highly abundant proteins in serum from 60 patients with serous ovarian carcinoma and 60 non‐cancer controls. Medium and low abundance serum proteins from each serum pool were then evaluated by the quantitative proteomic technique of differential in‐gel electrophoresis. The number of protein spots that were elevated in ovarian cancer sera by at least twofold ranged from 36 to 248, depending upon the depletion and separation methods. From the 33 spots picked for MS analysis, nine different proteins were identified, including the novel candidate ovarian cancer biomarkers leucine‐rich α2 glycoprotein‐1 and ficolin 3. Western blotting validated the relative increases in serum protein levels for three of the proteins identified, demonstrating the utility of this approach for the identification of novel serum biomarkers for ovarian cancer.

Leucine-rich alpha-2-glycoprotein-1 is upregulated in sera and tumors of ovarian cancer patients

BackgroundNew biomarkers that replace or are used in conjunction with the current ovarian cancer diagnostic antigen, CA125, are needed for detection of ovarian cancer in the presurgical setting, as well as for detection of disease recurrence. We previously demonstrated the upregulation of leucine-rich alpha-2-glycoprotein-1 (LRG1) in the sera of ovarian cancer patients compared to healthy women using quantitative mass spectrometry.MethodsLRG1 was quantified by ELISA in serum from two relatively large cohorts of women with ovarian cancer and benign gynecological disease. The expression of LRG1 in ovarian cancer tissues and cell lines was examined by gene microarray, reverse-transcriptase polymerase chain reaction (RT-PCR), Western blot, immunocytochemistry and mass spectrometry.ResultsMean serum LRG1 was higher in 58 ovarian cancer patients than in 56 healthy women (89.33 ± 77.90 vs. 42.99 ± 9.88 ug/ml; p = 0.0008) and was highest among stage III/IV patients. In a separate set of 193 pre-surgical samples, LRG1 was higher in patients with serous or clear cell ovarian cancer (145.82 ± 65.99 ug/ml) compared to patients with benign gynecological diseases (82.53 ± 76.67 ug/ml, p < 0.0001). CA125 and LRG1 levels were moderately correlated (r = 0.47, p < 0.0001). LRG1 mRNA levels were higher in ovarian cancer tissues and cell lines compared to their normal counterparts when analyzed by gene microarray and RT-PCR. LRG1 protein was detected in ovarian cancer tissue samples and cell lines by immunocytochemistry and Western blotting. Multiple iosforms of LRG1 were observed by Western blot and were shown to represent different glycosylation states by digestion with glycosidase. LRG1 protein was also detected in the conditioned media of ovarian cancer cell culture by ELISA, Western blotting, and mass spectrometry.ConclusionsSerum LRG1 was significantly elevated in women with ovarian cancer compared to healthy women and women with benign gynecological disease, and was only moderately correlated with CA125. Ovarian cancer cells secrete LRG1 and may contribute directly to the elevated levels of LRG1 observed in the serum of ovarian cancer patients. Future studies will determine whether LRG1 may serve as a biomarker for presurgical diagnosis, disease recurrence, and/or as a target for therapy.

Targeting CD133 in an in vivo ovarian cancer model reduces ovarian cancer progression

OBJECTIVES While most women with ovarian cancer will achieve complete remission after treatment, the majority will relapse within two years, highlighting the need for novel therapies. Cancer stem cells (CSC) have been identified in ovarian cancer and most other carcinomas as a small population of cells that can self-renew. CSC are more chemoresistant and radio-resistant than the bulk tumor cells; it is likely that CSC are responsible for relapse, the major problem in cancer treatment. CD133 has emerged as one of the most promising markers for CSC in ovarian cancer. The hypothesis driving this study is that despite their low numbers in ovarian cancer tumors, CSC can be eradicated using CD133 targeted therapy and tumor growth can be inhibited. METHODS Ovarian cancer cell lines were evaluated using flow cytometry for expression of CD133. In vitro viability studies with an anti-CD133 targeted toxin were performed on one of the cell lines, NIH:OVCAR5. The drug was tested in vivo using a stably transfected luciferase-expressing NIH:OVCAR5 subline in nude mice, so that tumor growth could be monitored by digital imaging in real time. RESULTS Ovarian cancer cell lines showed 5.6% to 16.0% CD133 expression. dCD133KDEL inhibited the in vitro growth of NIH:OVCAR5 cells. Despite low numbers of CD133-expressing cells in the tumor population, intraperitoneal drug therapy caused a selective decrease in tumor progression in intraperitoneal NIH:OVCAR5-luc tumors. CONCLUSIONS Directly targeting CSC that are a major cause of drug resistant tumor relapse with an anti-CD133 targeted toxin shows promise for ovarian cancer therapy.

A Transgenic Mouse Model of Plasma Cell Malignancy Shows Phenotypic, Cytogenetic, and Gene Expression Heterogeneity Similar to Human Multiple Myeloma

Multiple myeloma is an incurable plasma cell malignancy for which existing animal models are limited. We have previously shown that the targeted expression of the transgenes c-Myc and Bcl-X(L) in murine plasma cells produces malignancy that displays features of human myeloma, such as localization of tumor cells to the bone marrow and lytic bone lesions. We have isolated and characterized in vitro cultures and adoptive transfers of tumors from Bcl-xl/Myc transgenic mice. Tumors have a plasmablastic morphology and variable expression of CD138, CD45, CD38, and CD19. Spectral karyotyping analysis of metaphase chromosomes from primary tumor cell cultures shows that the Bcl-xl/Myc tumors contain a variety of chromosomal abnormalities, including trisomies, translocations, and deletions. The most frequently aberrant chromosomes are 12 and 16. Three sites for recurring translocations were also identified on chromosomes 4D, 12F, and 16C. Gene expression profiling was used to identify differences in gene expression between tumor cells and normal plasma cells (NPC) and to cluster the tumors into two groups (tumor groups C and D), with distinct gene expression profiles. Four hundred and ninety-five genes were significantly different between both tumor groups and NPCs, whereas 124 genes were uniquely different from NPCs in tumor group C and 204 genes were uniquely different from NPCs in tumor group D. Similar to human myeloma, the cyclin D genes are differentially dysregulated in the mouse tumor groups. These data suggest the Bcl-xl/Myc tumors are similar to a subset of plasmablastic human myelomas and provide insight into the specific genes and pathways underlying the human disease.

Quantitative proteomic analysis by iTRAQ ® for the identification of candidate biomarkers in ovarian cancer serum

BackgroundOvarian cancer is the most lethal gynecologic malignancy, with the majority of cases diagnosed at an advanced stage when treatments are less successful. Novel serum protein markers are needed to detect ovarian cancer in its earliest stage; when detected early, survival rates are over 90%. The identification of new serum biomarkers is hindered by the presence of a small number of highly abundant proteins that comprise approximately 95% of serum total protein. In this study, we used pooled serum depleted of the most highly abundant proteins to reduce the dynamic range of proteins, and thereby enhance the identification of serum biomarkers using the quantitative proteomic method iTRAQ®.ResultsMedium and low abundance proteins from 6 serum pools of 10 patients each from women with serous ovarian carcinoma, and 6 non-cancer control pools were labeled with isobaric tags using iTRAQ® to determine the relative abundance of serum proteins identified by MS. A total of 220 unique proteins were identified and fourteen proteins were elevated in ovarian cancer compared to control serum pools, including several novel candidate ovarian cancer biomarkers: extracellular matrix protein-1, leucine-rich alpha-2 glycoprotein-1, lipopolysaccharide binding protein-1, and proteoglycan-4. Western immunoblotting validated the relative increases in serum protein levels for several of the proteins identified.ConclusionsThis study provides the first analysis of immunodepleted serum in combination with iTRAQ® to measure relative protein expression in ovarian cancer patients for the pursuit of serum biomarkers. Several candidate biomarkers were identified which warrant further development.

Claudin 4 is differentially expressed between ovarian cancer subtypes and plays a role in spheroid formation

Claudin 4 is a cellular adhesion molecule that is frequently overexpressed in ovarian cancer and other epithelial cancers. In this study, we sought to determine whether the expression of claudin 4 is associated with outcome in ovarian cancer patients and may be involved in tumor progression. We examined claudin 4 expression in ovarian cancer tissues and cell lines, as well as by immunohistochemical staining of tissue microarrays (TMAs; n = 500), spheroids present in patients’ ascites, and spheroids formed in vitro. Claudin 4 was expressed in nearly 70% of the ovarian cancer tissues examined and was differentially expressed across ovarian cancer subtypes, with the lowest expression in clear cell subtype. No association was found between claudin 4 expression and disease-specific survival in any subtype. Claudin 4 expression was also observed in multicellular spheroids obtained from patients’ ascites. Using an in vitro spheroid formation assay, we found that NIH:OVCAR5 cells treated with shRNA against claudin 4 required a longer time to form compact spheroids compared to control NIH:OVCAR5 cells that expressed high levels of claudin 4. The inability of the NIH:OVCAR5 cells treated with claudin 4 shRNA to form compact spheroids was verified by FITC-dextran exclusion. These results demonstrate a role for claudin 4 and tight junctions in spheroid formation and integrity.

S100A1 Expression in Ovarian and Endometrial Endometrioid Carcinomas Is a Prognostic Indicator of Relapse-Free Survival

We sought to investigate the expression levels of S100A1 in ovarian cancer cell lines and tissues to correlate S100A1 with subtype, stage, grade, and relapse-free survival. S100A1 messenger RNA and protein were up-regulated in ovarian cancer cell lines and tumors compared with normal ovarian cell lines and tissues by gene microarray analysis, reverse transcriptase-polymerase chain reaction, quantitative reverse transcriptase-polymerase chain reaction, and Western immunoblotting. In the study, 63.7% of serous, 21.2% of clear cell, 11.2% of endometrioid, and 3% of mucinous ovarian (1/31) cancers were S100A1+ by immunohistochemical staining of tissue microarrays (n = 500). S100A1 expression increased with increasing Silverberg grade but not stage in serous tumors. Endometrial tissue microarrays (n = 127) were 9.4% S100A1+; no correlation with stage or grade and S100A1 was found. In the endometrioid subtype of ovarian and endometrial cancers, relapse-free survival was decreased for patients with S100A1+ tumors. These data suggest that S100A1 is a marker for poor prognosis of endometrioid subtypes of cancer.

Motility Screen Identifies Drosophila IGF-II mRNA-Binding Protein—Zipcode-Binding Protein Acting in Oogenesis and Synaptogenesis

The localization of specific mRNAs can establish local protein gradients that generate and control the development of cellular asymmetries. While all evidence underscores the importance of the cytoskeleton in the transport and localization of RNAs, we have limited knowledge of how these events are regulated. Using a visual screen for motile proteins in a collection of GFP protein trap lines, we identified the Drosophila IGF-II mRNA-binding protein (Imp), an ortholog of Xenopus Vg1 RNA binding protein and chicken zipcode-binding protein. In Drosophila, Imp is part of a large, RNase-sensitive complex that is enriched in two polarized cell types, the developing oocyte and the neuron. Using time-lapse confocal microscopy, we establish that both dynein and kinesin contribute to the transport of GFP-Imp particles, and that regulation of transport in egg chambers appears to differ from that in neurons. In Drosophila, loss-of-function Imp mutations are zygotic lethal, and mutants die late as pharate adults. Imp has a function in Drosophila oogenesis that is not essential, as well as functions that are essential during embryogenesis and later development. Germline clones of Imp mutations do not block maternal mRNA localization or oocyte development, but overexpression of a specific Imp isoform disrupts dorsal/ventral polarity. We report here that loss-of-function Imp mutations, as well as Imp overexpression, can alter synaptic terminal growth. Our data show that Imp is transported to the neuromuscular junction, where it may modulate the translation of mRNA targets. In oocytes, where Imp function is not essential, we implicate a specific Imp domain in the establishment of dorsoventral polarity.

IL-15 super-agonist (ALT-803) enhances natural killer (NK) cell function against ovarian cancer

OBJECTIVE Natural killer (NK) cells represent a powerful immunotherapeutic target as they lyse tumors directly, do not require differentiation, and can elicit potent inflammatory responses. The objective of these studies was to use an IL-15 super-agonist complex, ALT-803 (Altor BioScience Corporation), to enhance the function of both normal and ovarian cancer patient derived NK cells by increasing cytotoxicity and cytokine production. METHODS NK cell function from normal donor peripheral blood mononuclear cells (PBMCs) and ovarian cancer patient ascites was assessed using flow cytometry and chromium release assays ±ALT-803 stimulation. To evaluate the ability of ALT-803 to enhance NK cell function in vivo against ovarian cancer, we used a MA148-luc ovarian cancer NOD scid gamma (NSG) xenogeneic mouse model with transferred human NK cells. RESULTS ALT-803 potently enhanced functionality of NK cells against all ovarian cancer cell lines with significant increases seen in CD107a, IFNγ and TNFα expression depending on target cell line. Function was also rescued in NK cells derived from ovarian cancer patient ascites. Finally, only animals treated with intraperitoneal ALT-803 displayed an NK dependent significant decrease in tumor. CONCLUSIONS ALT-803 enhances NK cell cytotoxicity against ovarian cancer in vitro and in vivo and is able to rescue functionality of NK cells derived from ovarian cancer patient ascites. These findings suggest that ALT-803 has the potential to enhance NK cell-based immunotherapeutic approaches for the treatment of ovarian cancer.

Mathematical Prognostic Biomarker Models for Treatment Response and Survival in Epithelial Ovarian Cancer

Following initial standard chemotherapy (platinum/taxol), more than 75% of those patients with advanced stage epithelial ovarian cancer (EOC) experience a recurrence. There are currently no accurate prognostic tests that, at the time of the diagnosis/surgery, can identify those patients with advanced stage EOC who will respond to chemotherapy. Using a novel mathematical theory, we have developed three prognostic biomarker models (complex mathematical functions) that—based on a global gene expression analysis of tumor tissue collected during surgery and prior to the commencement of chemotherapy—can identify with a high accuracy those patients with advanced stage EOC who will respond to the standard chemotherapy [long-term survivors (>7 yrs)] and those who will not do so [short-term survivors (<3 yrs)]. Our three prognostic biomarker models were developed with 34 subjects and validated with 20 unknown (new and different) subjects. Both the overall biomarker model sensitivity and specificity ranged from 95.83% to 100.00%. The 12 most significant genes identified, which are also the input variables to the three mathematical functions, constitute three distinct gene networks with the following functions: 1) production of cytoskeletal components, 2) cell proliferation, and 3) cell energy production. The first gene network is directly associated with the mechanism of action of anti-tubulin chemotherapeutic agents, such as taxanes and epothilones. This could have a significant impact in the discovery of new, more effective pharmacological treatments that may significantly extend the survival of patients with advanced stage EOC.