Aprill Watanabe

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

Down-regulation of Yes Associated Protein 1 expression reduces cell proliferation and clonogenicity of pancreatic cancer cells.

Background The Hippo pathway regulates organ size by inhibiting cell proliferation and promoting cell apoptosis upon its activation. The Yes Associated Protein 1 (YAP1) is a nuclear effector of the Hippo pathway that promotes cell growth as a transcription co-activator. In human cancer, the YAP1 gene was reported as amplified and over-expressed in several tumor types. Methods Immunohistochemical staining of YAP1 protein was used to assess the expression of YAP1 in pancreatic tumor tissues. siRNA oligonucleotides were used to knockdown the expression of YAP1 and their effects on pancreatic cancer cells were investigated using cell proliferation, apoptosis, and anchorage-independent growth assays. The Wilcoxon signed-rank, Pearson correlation coefficient, Kendalls Tau, Spearmans Rho, and an independent two-sample t (two-tailed) test were used to determine the statistical significance of the data. Results Immunohistochemistry studies in pancreatic tumor tissues revealed YAP1 staining intensities were moderate to strong in the nucleus and cytoplasm of the tumor cells, whereas the adjacent normal epithelial showed negative to weak staining. In cultured cells, YAP1 expression and localization was modulated by cell density. YAP1 total protein expression increased in the nuclear fractions in BxPC-3 and PANC-1, while it declined in HPDE6 as cell density increased. Additionally, treatment of pancreatic cancer cell lines, BxPC-3 and PANC-1, with YAP1-targeting siRNA oligonucleotides significantly reduced their proliferation in vitro. Furthermore, treatment with YAP1 siRNA oligonucleotides diminished the anchorage-independent growth on soft agar of pancreatic cancer cells, suggesting a role of YAP1 in pancreatic cancer tumorigenesis. Conclusions YAP1 is overexpressed in pancreatic cancer tissues and potentially plays an important role in the clonogenicity and growth of pancreatic cancer cells.

Tissue microarrays: applications in genomic research.

The widespread application of tissue microarrays in cancer research and the clinical pathology laboratory demonstrates a versatile and portable technology. The rapid integration of tissue microarrays into biomarker discovery and validation processes reflects the forward thinking of researchers who have pioneered the high-density tissue microarray. The precise arrangement of hundreds of archival clinical tissue samples into a composite tissue microarray block is now a proven method for the efficient and standardized analysis of molecular markers. With applications in cancer research, tissue microarrays are a valuable tool in validating candidate markers discovered in highly sensitive genome-wide microarray experiments. With applications in clinical pathology, tissue microarrays are used widely in immunohistochemistry quality control and quality assurance. The timeline of a biomarker implicated in prostate neoplasia, which was identified by complementary DNA expression profiling, validated by tissue microarrays and is now used as a prognostic immunohistochemistry marker, is reviewed. The tissue microarray format provides opportunities for digital imaging acquisition, image processing and database integration. Advances in digital imaging help to alleviate previous bottlenecks in the research pipeline, permit computer image scoring and convey telepathology opportunities for remote image analysis. The tissue microarray industry now includes public and private sectors with varying degrees of research utility and offers a range of potential tissue microarray applications in basic research, prognostic oncology and drug discovery.

Hedgehog signaling pathway molecules and ALDH1A1 expression in early-stage non-small cell lung cancer

INTRODUCTION The Hedgehog Signaling Pathway (HHSP) has been implicated in the development of multiple cancers. HHSP activation may primarily be hedgehog ligand-dependent in non-small cell lung cancer (NSCLC); while a subset may be ligand-independent. In this study NSCLC primary tumors were used to identify correlations between multiple biomarkers thought to be involved in the HHSP and the clinical outcomes of patients with NSCLC. Identification of such correlations could be used to aid in NSCLC treatment and predicting patient prognosis. METHODS A tissue microarray representing 248 clinically annotated stage I-II NSCLC cases was stained using immunohistochemistry (IHC) and scored for HHSP proteins namely, SHH, PTCH1, SMO, GLI1, and GLI2; as well as, ALDH1A1, a putative cancer stem cell marker. Data was analyzed for correlation between IHC staining, EGFR and KRAS mutations, and clinical characteristics including relapse-free survival (RFS) and overall survival (OS). RESULTS In adenocarcinoma, there were significant correlations of IHC expression between SHH and downstream HHSP receptor SMO (p=0.017) and transcription factor GLI1 (p=0.001), while SMO correlated with GLI1 (p=0.007). In squamous cell carcinoma, SHH significantly correlated with GLI2 protein expression (p=0.003). After multiple testing correction, there was no significant correlation between any of the six markers and RFS or OS. CONCLUSIONS Key downstream components of the HHSP show correlation with sonic hedgehog ligand (SHH) expression, suggesting that ligand-dependent signaling is more prevalent in primary NSCLC tumors. Surprisingly, in early-stage NSCLC, there were no significant correlations between HHSP proteins or ALDH1A1 and RFS or OS.

Quiescin Sulfhydryl Oxidase 1 Promotes Invasion of Pancreatic Tumor Cells Mediated by Matrix Metalloproteinases

Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. We previously mapped a peptide in plasma from pancreatic ductal adenocarcinoma (PDA) patients back to an overexpressed QSOX1 parent protein. In addition to overexpression in pancreatic cancer cell lines, 29 of 37 patients diagnosed with PDA expressed QSOX1 protein in tumor cells, but QSOX1 was not detected in normal adjacent tissues or in a transformed, but nontumorigenic cell line. To begin to evaluate the advantage QSOX1 might provide to tumors, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in two pancreatic cancer cell lines. Growth, cell cycle, apoptosis, invasion, and matrix metalloproteinase (MMP) activity were evaluated. QSOX1 shRNA suppressed both short and long isoforms of the protein, showing a significant effect on cell growth, cell cycle, and apoptosis. However, QSOX1 shRNA dramatically inhibited the abilities of BxPC-3 and Panc-1 pancreatic tumor cells to invade through Matrigel in a modified Boyden chamber assay. Mechanistically, gelatin zymography indicated that QSOX1 plays an important role in activation of MMP-2 and MMP-9. Taken together, our results suggest that the mechanism of QSOX1-mediated tumor cell invasion is by activation of MMP-2 and MMP-9. Mol Cancer Res; 9(12); 1621–31. ©2011 AACR.

Negative Regulation of NF-κB by the ING4 Tumor Suppressor in Breast Cancer

Nuclear Factor kappa B (NF-κB) is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4) tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227). Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65), an activated form of NF-κB (p = 0.018). Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in breast cancer.

Tissue Microarray Applications in Drug Discovery for Pancreatic Cancer

The rising incidence of pancreatic cancer combined with limited responses to chemotherapy highlights the need for improved molecular characterization in this highly lethal cancer. The discovery and validation of novel biomarkers are urgently needed to improve early detection and thereby improve survival outcomes. The introduction and widespread application of tissue microarrays (TMAs) over the past decade provide a valuable tool for validation of abundance and cellular localization of expressed proteins and RNA transcript levels contextually within the complex tissue morphology of pancreatic cancer. The TMA format offers a standardized platform to optimize and validate newly identified biomarkers and drug targets in pancreatic cancer research to patient tumor samples to provide critical links in advancing cancer research findings to the clinical setting.

Abstract 2135: TGFβRI inhibition results in reduced collagen expression in pancreatic ductal adenocarcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic ductal adenocarcinoma (PDAC) is characterized by a robust stromal component termed the desmoplastic reaction. Transforming growth factor β (TGFβ) has been implicated in the activation of the cancer-associated fibroblast (CAF) that leads to pancreatic desmoplasia and the increased deposition of extracellular matrix proteins. Desmoplasia and its effects are thought to contribute to poor drug efficacy in PDAC treatment. We therefore hypothesized that TGFβ receptor (TGFβR) inhibition may serve to improve the activity of chemotherapeutics in PDAC by reducing the effects of desmoplasia. Using the TGFβRI inhibitors, LY2157299 and SB431542, we assessed the effects of TGFβR inhibition on cell proliferation and collagen production in both mono- and co-cultured pancreatic tumor cells and CAFs. We found that although the TGFβRI inhibitors had moderate effect on the proliferation of PDAC cells and co-cultured CAF cells, they significantly inhibited collagen 1 expression in the co-culture of MIA PaCa-2 and SU86.86 cells with CAFs (2.3 and 6.0-fold at the mRNA level, respectively). Similar qualitative reductions were also observed in immunofluorescence microscopy and immunoblots probing for collagen 1 protein levels. We also sought to determine if TGFβR inhibition would result in a reduction in collagen expression in the more biologically relevant in vivo model for PDAC, the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mouse. KPC mice were treated orally, b.i.d., with 75mg/kg LY2157299 and by i.p., with gemcitabine (q3dx4) at 80mg/kg. Tumors in KPC mice treated with the combination of LY2157299 and gemcitabine showed a marked reduction in overall stromal content when compared to vehicle-treated mice. Our results suggest that TGFβR inhibition may be an effective means of ameliorating the effects of desmoplasia in PDAC, which may in turn improve the efficacy of current PDAC treatment regimens. Citation Format: Clifford J. Whatcott, Sabrina N. Dumas, Aprill Watanabe, Janine LoBello, Daniel D. Von Hoff, Haiyong Han. TGFβRI inhibition results in reduced collagen expression in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2135. doi:10.1158/1538-7445.AM2013-2135

Tissue Microarray Applications: A Valuable Clinical and Research Resource

Abstract Widespread applications of tissue microarrays (TMAs) in both cancer research and clinical pathology demonstrate a versatile and portable technology. The rapid integration of TMA technology into the biomarker discovery and validation process reflects the forward thinking of researchers who pioneered the high-density TMA. The precise arrangement of hundreds of archival clinical tissue samples into a composite paraffin block is now a proven method for efficient and standardized analyses of molecular markers. With applications in cancer research, TMAs are a valuable tool in viiidating candidate markers identified in highly sensitive genome-wide microarray experiments. With applications in clinical pathology, TMAs are used widely in immunohistochemistry for quality control and quality assurance. TMAs can demonstrate antibody thresholds on a single slide, which aid in optimization where high and low signal intensities can be seen. This type of application lends itself to clinical pathology and immunohistochemistry panels run daily to improve biomarker identification and characterization. The TMA industry now includes public and private resources with varying degrees of research utility. In this article, we will explore several potential TMA applications in basic research, prognostic oncology, and drug discovery. (The J Histotechnol 28:223, 2005) Submitted September 6, 2005; accepted with revisions December 6, 2005

Abstract 5158: The H3K9 demethyltransferase gene JMJD1C has tumor suppressor functions in multiple myeloma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Increasing evidence points towards the importance of epigenetic changes in the pathogenesis of multiple myeloma (MM). The biochemical modifications that govern epigenetics are DNA methylation, and post-translational modifications of histone proteins. Histone methylation is catalyzed by histone methyltransferases (HMT) and histone demethylases (HDMT). The purpose of our study was to interrogate genomics data to identify HMT or HDMT genes that are altered in MM. We mined copy number, gene expression and whole genome sequencing data generated as part of the Multiple Myeloma Research Consortium Genomics Initiative. Collectively, the datasets converged on numerous alterations involving histone methylation of lysine 9 (H3K9). One of these genes, the HDMT JMJD1C, was downregulated in approximately 25% of MM samples examined and was associated with worst clinical outcome. We validated these data by quantitative RT-PCR and by immunohistochemistry (IHC) on a tissue microarray (TMA) consisting of over 60 MM samples. Next we demonstrated a negative correlation between JMJD1C expression and H3K9 methylation by IHC and western blot in 6/10 HMCL and in over 60% of clinical samples examined on the TMA. To study the direct effects of JMJD1C on H3K9 methylation and determine the functional significance, we generated a knockout isogenic cell line pair in KMS11 MM cells using zinc finger nuclease technology. JMJD1C depletion resulted in an increase in H3K9 mono- and dimethylation as demonstrated by western blot and immunofluorescence, confirming its association to H3K9 methylation. H3K9 trimethylation was unaffected by JMJD1C depletion. Differential gene expression analysis demonstrated defects in cell cycle and G2/M transition. Most notably, NEK2, Cyclin B, CDC20, PLK1 and TTK were among genes upregulated in response to JMJD1C depletion. Accordingly, we assessed cell growth using CellTiter-Glo® and demonstrated increased cell growth in JMJD1C-depleted cells. We also demonstrated a more pronounced G2/M peak by FACS. DNA methylation analysis using the HumanMethylation 450K BeadChip showed global hypomethylation in response to JMJD1C loss, a phenotype indicative of myeloma progression. Results to date validate the H3K9 demethylase activity of JMJD1C, and suggest a tumor suppressive function, which may be lost in MM. Data demonstrate a novel biological and molecular understanding of JMJD1C, pointing us to therapeutic vulnerabilities in MM. Studies are ongoing to further characterize the genomic, epigenomic and functional significance of JMJD1C in MM. Citation Format: Danielle DiPerna, Gerald C. Gooden, Brooke Hjelm, Sara Nasser, Janine LoBello, Aprill Watanabe, Bodour Salhia. The H3K9 demethyltransferase gene JMJD1C has tumor suppressor functions in multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5158. doi:10.1158/1538-7445.AM2014-5158