Identification of a Potential Ovarian Cancer Stem Cell Gene Expression Profile from Advanced Stage Papillary Serous Ovarian Cancer

Identification of gene expression profiles of cancer stem cells may have significant implications in the understanding of tumor biology and for the design of novel treatments targeted toward these cells. Here we report a potential ovarian cancer stem cell gene expression profile from isolated side population of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Affymetrix U133 Plus 2.0 microarrays were used to interrogate the differentially expressed genes between side population (SP) and main population (MP), and the results were analyzed by paired T-test using BRB-ArrayTools. We identified 138 up-regulated and 302 down-regulated genes that were differentially expressed between all 10 SP/MP pairs. Microarray data was validated using qRT-PCR and17/19 (89.5%) genes showed robust correlations between microarray and qRT-PCR expression data. The Pathway Studio analysis identified several genes involved in cell survival, differentiation, proliferation, and apoptosis which are unique to SP cells and a mechanism for the activation of Notch signaling is identified. To validate these findings, we have identified and isolated SP cells enriched for cancer stem cells from human ovarian cancer cell lines. The SP populations were having a higher colony forming efficiency in comparison to its MP counterpart and also capable of sustained expansion and differentiation in to SP and MP phenotypes. 50,000 SP cells produced tumor in nude mice whereas the same number of MP cells failed to give any tumor at 8 weeks after injection. The SP cells demonstrated a dose dependent sensitivity to specific γ-secretase inhibitors implicating the role of Notch signaling pathway in SP cell survival. Further the generated SP gene list was found to be enriched in recurrent ovarian cancer tumors

Inhibition of the JAK2/STAT3 pathway in ovarian cancer results in the loss of cancer stem cell-like characteristics and a reduced tumor burden

Background Current treatment of ovarian cancer patients with chemotherapy leaves behind a residual tumor which results in recurrent ovarian cancer within a short time frame. We have previously demonstrated that a single short-term treatment of ovarian cancer cells with chemotherapy in vitro resulted in a cancer stem cell (CSC)-like enriched residual population which generated significantly greater tumor burden compared to the tumor burden generated by control untreated cells. In this report we looked at the mechanisms of the enrichment of CSC-like residual cells in response to paclitaxel treatment. Methods The mechanism of survival of paclitaxel-treated residual cells at a growth inhibitory concentration of 50% (GI50) was determined on isolated tumor cells from the ascites of recurrent ovarian cancer patients and HEY ovarian cancer cell line by in vitro assays and in a mouse xenograft model. Results Treatment of isolated tumor cells from the ascites of ovarian cancer patients and HEY ovarian cancer cell line with paclitaxel resulted in a CSC-like residual population which coincided with the activation of Janus activated kinase 2 (JAK2) and signal transducer and activation of transcription 3 (STAT3) pathway in paclitaxel surviving cells. Both paclitaxel-induced JAK2/STAT3 activation and CSC-like characteristics were inhibited by a low dose JAK2-specific small molecule inhibitor CYT387 (1 μM) in vitro. Subsequent, in vivo transplantation of paclitaxel and CYT387-treated HEY cells in mice resulted in a significantly reduced tumor burden compared to that seen with paclitaxel only-treated transplanted cells. In vitro analysis of tumor xenografts at protein and mRNA levels demonstrated a loss of CSC-like markers and CA125 expression in paclitaxel and CYT387-treated cell-derived xenografts, compared to paclitaxel only-treated cell-derived xenografts. These results were consistent with significantly reduced activation of JAK2 and STAT3 in paclitaxel and CYT387-treated cell-derived xenografts compared to paclitaxel only-treated cell derived xenografts. Conclusions This proof of principle study demonstrates that inhibition of the JAK2/STAT3 pathway by the addition of CYT387 suppresses the ‘stemness’ profile in chemotherapy-treated residual cells in vitro, which is replicated in vivo, leading to a reduced tumor burden. These findings have important implications for ovarian cancer patients who are treated with taxane and/or platinum-based therapies. Keywords: Ovarian carcinoma, Cancer stem cell, Metastasis, Ascites, Chemoresistance, Recurrence, JAK2/STAT3 pathwa

Identification of Novel Therapeutic Targets in Microdissected Clear Cell Ovarian Cancers

Clear cell ovarian cancer is an epithelial ovarian cancer histotype that is less responsive to chemotherapy and carries poorer prognosis than serous and endometrioid histotypes. Despite this, patients with these tumors are treated in a similar fashion as all other ovarian cancers. Previous genomic analysis has suggested that clear cell cancers represent a unique tumor subtype. Here we generated the first whole genomic expression profiling using epithelial component of clear cell ovarian cancers and normal ovarian surface specimens isolated by laser capture microdissection. All the arrays were analyzed using BRB ArrayTools and PathwayStudio software to identify the signaling pathways. Identified pathways validated using serous, clear cell cancer cell lines and RNAi technology. In vivo validations carried out using an orthotopic mouse model and liposomal encapsulated siRNA. Patient-derived clear cell and serous ovarian tumors were grafted under the renal capsule of NOD-SCID mice to evaluate the therapeutic potential of the identified pathway. We identified major activated pathways in clear cells involving in hypoxic cell growth, angiogenesis, and glucose metabolism not seen in other histotypes. Knockdown of key genes in these pathways sensitized clear cell ovarian cancer cell lines to hypoxia/glucose deprivation. In vivo experiments using patient derived tumors demonstrate that clear cell tumors are exquisitely sensitive to antiangiogenesis therapy (i.e. sunitinib) compared with serous tumors. We generated a histotype specific, gene signature associated with clear cell ovarian cancer which identifies important activated pathways critical for their clinicopathologic characteristics. These results provide a rational basis for a radically different treatment for ovarian clear cell patients

Expression profiling of SP/MP cells from ascites specimens.

<p>(A) Heatmap showing the expression pattern of 438 probesets that discriminated SP cells from MP cells in ovarian cancer patients. Vertical columns represent individual samples; horizontal rows represent individual genes. The red and blue color indicates up and down-regulation respectively. (B) Validation of microarray data using qRT-PCR: 19 randomly selected genes were used to validate the microarray data. To calculate the relative expression for each gene, the 2^−ΔΔCT method was used averaging the CT values for the three housekeeping genes (Cyclophilin A, GUSB, GAPDH) for a single reference gene value. (C) Representative plots for correlation analysis between the microarray and qRT-PCR data: The 2^−ΔCT values (qRT-PCR) were plotted against signal intensity values (Microarray). The correlation analysis was performed for each gene by Pearson's and Spearman's method using GraphPad Prism version 4.00.</p

Identification and validation of side population cells from ovarian cancer cell lines.

<p>(A) Identification of SP cells in established human ovarian cancer cell lines. SKOV3 and A224 cell lines were labeled with Hoechst 33342 dye and analyzed by flow cytometry. The SP cells, which disappeared in the presence of Verapamil (a multidrug transporter inhibitor), are outlined and shown as the percentage of the total cell population. Similar results were obtained for three independent measurements. (B) Validation of randomly selected genes from the SP gene list on the SP and MP cells isolated from the SKOV3 cell lines. (C, D) Colony forming efficiency assay: Colony forming efficiency of sorted SP and MP cells from SKOV3 and A224 cell lines. For the analysis of colony forming efficiency (CFE), SP and MP cells were sorted and plated in equal numbers in tissue culture six well plates and grown for 14 days. The cells were then fixed with cold methanol and stained with 0.5% crystal violet solution to count the number of colonies by microscopy. The experiments were carried out in triplicates. (E, F) Passage Number: Colony forming efficiency of sorted SP and MP cells from SKOV3 and A224 cell lines were evaluated as a function of passage number. Cells were fixed with cold methanol and stained with 0.5% crystal violet.</p

Validation of SP cells from cell lines and the effect of GSI-IX inhibitor on SP cells colony forming efficiency.

<p>(A) Repopulation Assay: The SKOV3 and A224 cells were stained with Hoechst 33342 dye and sorted for SP and MP populations. The cells were cultured for 8 days for repopulation, and then reanalyzed by flow cytometry. This demonstrated the enrichment of SP cells (22.5% and 10.4% for SKOV3 and A224 resp.) with a capacity to regenerate to MP cells. (B, C, D) qRT-PCR analysis for stem cell marker genes and transporter genes (p<0.01). To calculate the relative expression for each gene, the 2^−ΔΔCT method was used averaging the CT values for the three housekeeping genes (Cyclophilin A, GUSB, GAPDH) for a single reference gene value. (E, F) Inhibitory effect of GSI-IX on Colony forming efficiency of sorted SP and MP cells from SKOV3. Cells were sorted to SP and MP populations and treated with 10 and 20 µg of GSI-IX. The inhibitor carrier DMSO is used as a control.</p

Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues.

Archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a readily available but largely untapped resource for gene expression profiling-based biomarker discovery. Several technologies have been proposed to cope with the bias from RNA cross-linking and degradation associated with archival specimens to generate data comparable with RNA from fresh-frozen materials. Direct comparison studies of these RNA expression platforms remain rare. We compared two commercially available platforms for RNA expression profiling of archival FFPE specimens from clinical studies of prostate and ovarian cancer: the Affymetrix Human Gene 1.0ST Array following whole-transcriptome amplification using the NuGen WT-Ovation FFPE System V2, and the NanoString nCounter without amplification. For each assay, we profiled 7 prostate and 11 ovarian cancer specimens, with a block age of 4 to 21 years. Both platforms produced gene expression profiles with high sensitivity and reproducibility through technical repeats from FFPE materials. Sensitivity and reproducibility remained high across block age within each cohort. A strong concordance was shown for the transcript expression values for genes detected by both platforms. We showed the biological validity of specific gene signatures generated by both platforms for both cohorts. Our study supports the feasibility of gene expression profiling and large-scale signature validation on archival prostate and ovarian tumor specimens using commercial platforms. These approaches have the potential to aid precision medicine with biomarker discovery and validation

The percentage of the SP cells identified in ascites samples.

<p>n.d. (not determined).</p

Identification of putative signaling events contributing to ovarian cancer SP cell survival, self-renewal and tumor maintenance.

<p>(A) Gene ontology analysis of microarray data (P value<0.01): The pie diagram shows the biological functions of the differentially expressed genes among SP and MP cells. The gene signature was enriched for genes in gene ontology biological processes of apoptosis, cell cycle, cell proliferation, transport, signal transduction, transcription, translation, protein modification, metabolism and proteolysis. Other represents genes involved in defense response, vasculogenesis, blood coagulation, visual perception, ontogenesis, cell matrix adhesion, and initiation of primordial ovarian follicle growth. (B) Graphical representation of the literature derived facts about the biological pathways involved in SP cells. Pathway Studio 6.0 software was used to identify the activated pathways in SP cells. Solid symbols representing the genes (EGFR, TNFRSF6, BCL2L11, FOXO3A, RUNX1) as down regulated in SP cells, the open symbols represents the upregulated genes (EPHB4, EPHB2, PAWR, AKT1) and gray symbols are the genes whose expression did not change significantly between SP and MP cells. (C) The Notch signaling pathway: The figure shows the schematic of Notch signal transduction elements. The overexpressed proteins in SP cells (FPTG, ST3GAL6 and ADAM19) are shown in blue color. Post-translational modification of precursor Notch-protein includes cleavage by proteases and glycosylations in the trans-Golgi. Adherence of Notch extracellular domain (ECN) with Notch intracellular domain (ICN) results in mature Notch heterodimers and they are transferred to the cell membrane. Receptor interaction with the ligands of the DSL family (Delta, Serrate, Lag3) on neighboring cells is modulated by glycosylations of ECN. Successful interaction of extracellular ligand regions with EGF-like repeats of ECN lead to the proteolytic cleavages of Notch transmembrane domain by two sequential steps by ADAM proteases and presenilins with γ–secretase activity. The released Notch intracellular domain is translocated to the nuclease where it interacts with CSL1, replacing CSL repressors and forming a transcription complex with Mastermind-like factors and transcriptional coactivators. This transcriptional complex activates downstream target genes and may account for cancer stem cell survival, self-renewal and tumor maintenance in ovary.</p