Reiko Satow

Combined Functional Genome Survey of Therapeutic Targets for Hepatocellular Carcinoma

Purpose: The outcome of patients with advanced hepatocellular carcinoma (HCC) has remained unsatisfactory. Patients with HCC suffer from chronic hepatitis or liver cirrhosis, and their reserve liver function is often limited. Experimental Design: To develop new therapeutic agents that act specifically on HCC but interfere only minimally with residual liver function, we searched for genes that were upregulated in 20 cases of HCC [namely, discovery sets 1 (n = 10) and 2 (n = 10)] in comparison with corresponding nontumorous liver and a panel representing normal organs using high-density microarrays capable of detecting all exons in the human genome. Results: Eleven transcripts whose expression was significantly increased in HCC were subjected to siRNA-based secondary screening of genes required for HCC cell proliferation as well as quantitative reverse transcription-PCR analysis [validation sets 1 (n = 20) and 2 (n = 44)] and immunohistochemistry (n = 19). We finally extracted four genes, AKR1B10, HCAP-G, RRM2, and TPX2, as candidate therapeutic targets for HCC. siRNA-mediated knockdown of these candidate genes inhibited the proliferation of HCC cells and the growth of HCC xenografts transplanted into immunodeficient mice. Conclusions: The four genes we identified were highly expressed in HCC, and HCC cells are highly dependent on these genes for proliferation. Although many important genes must have been overlooked, the selected genes were biologically relevant. The combination of genome-wide expression and functional screening described here is a rapid and comprehensive approach that could be applied in the identification of therapeutic targets in any type of human malignancy. Clin Cancer Res; 16(9); 2518–28. ©2010 AACR.

Traf2- and Nck-Interacting Kinase Is Essential for Wnt Signaling and Colorectal Cancer Growth

T-cell factor-4 (TCF4) is a transcription factor essential for maintaining the undifferentiated status and self-renewal of intestinal epithelial cells. It has therefore been considered that constitutive activation of TCF4 by aberrant Wnt signaling is a major force driving colorectal carcinogenesis. We previously identified Traf2- and Nck-interacting kinase (TNIK) as one of the proteins that interact with TCF4 in colorectal cancer cells, but its functional significance has not been elucidated. Here, we report that TNIK is an activating kinase for TCF4 and essential for colorectal cancer growth. TNIK, but not its catalytically inactive mutant, phosphorylated the conserved serine 154 residue of TCF4. Small interfering RNA targeting TNIK inhibited the proliferation of colorectal cancer cells and the growth of tumors produced by injecting colorectal cancer cells s.c. into immunodeficient mice. The growth inhibition was abolished by restoring the catalytic domain of TNIK, thus confirming that its enzyme activity is essential for the maintenance of colorectal cancer growth. Several ATP-competing kinase inhibitors have been applied to cancer treatment and have shown significant activity. Our findings suggest TNIK as a feasible target for pharmacologic intervention to ablate aberrant Wnt signaling in colorectal cancer.

Reduced Argininosuccinate Synthetase Is a Predictive Biomarker for the Development of Pulmonary Metastasis in Patients with Osteosarcoma

Pulmonary metastasis is the most significant prognostic determinant for osteosarcoma, but methods for its prediction and treatment have not been established. Using oligonucleotide microarrays, we compared the global gene expression of biopsy samples between seven osteosarcoma patients who developed pulmonary metastasis within 4 years after neoadjuvant chemotherapy and curative resection, and 12 patients who did not relapse. We identified argininosuccinate synthetase (ASS) as a gene differentially expressed with the highest statistical significance (Welchs t test, P = 2.2 × 10−5). Immunohistochemical analysis of an independent cohort of 62 osteosarcoma cases confirmed that reduced expression of ASS protein was significantly correlated with the development of pulmonary metastasis after surgery (log-rank test, P < 0.05). Cox regression analysis revealed that ASS was the sole significant predictive factor (P = 0.039; hazard ratio, 0.319; 95% confidence interval, 0.108-0.945). ASS is one of the enzymes required for the production of a nonessential amino acid, arginine. We showed that osteosarcoma cells lacking ASS expression were auxotrophic for arginine and underwent G0-G1 arrest in arginine-free medium, suggesting that an arginine deprivation therapy could be effective in patients with osteosarcoma. Recently, phase I and II clinical trials in patients with melanoma and hepatocellular carcinoma have shown the safety and efficacy of plasma arginine depletion by stabilized arginine deiminase. Our data indicate that in patients with osteosarcoma, reduced expression of ASS is not only a novel predictive biomarker for the development of metastasis, but also a potential target for pharmacologic intervention. Mol Cancer Ther; 9(3); 535–44

Regulation of Wnt Signaling by the Nuclear Pore Complex

BACKGROUND & AIMS The function of beta-catenin as a transcriptional coactivator of T-cell factor-4 (TCF-4) is crucial for colorectal carcinogenesis. However, beta-catenin has no nuclear localization signal, and the mechanisms by which beta-catenin is imported into the nucleus and forms a complex with the TCF-4 nuclear protein are poorly understood. METHODS Proteins of 2 colorectal cancer cell lines, HCT-116 and DLD1, were immunoprecipitated with anti-TCF-4 antibody and analyzed directly by nanoflow liquid chromatography and mass spectrometry. The functional significance of nuclear pore complex (NPC) proteins in Wnt signaling was evaluated by in vitro and in vivo sumoylation, luciferase reporter, and colony formation assays. RESULTS TCF-4 interacted with a large variety of NPC proteins including ras-related nuclear protein (Ran), Ran binding protein-2 (RanBP2), and Ran GTPase-activating protein-1 (RanGAP1). The NPC protein RanBP2 functioned as the small ubiquitin-related modifier (SUMO) E3 ligase of TCF-4, and sumoylation of TCF-4 enhanced the interaction between TCF-4 and beta-catenin. The overexpression of NPC proteins increased the nuclear import of the TCF-4 and beta-catenin proteins and enhanced the transcriptional activity. NPC proteins increased the growth of colorectal cancer cells, whereas sentrin-specific protease-2 inhibited it. CONCLUSIONS Through a comprehensive proteomics approach, we revealed that NPC functions as a novel regulator of Wnt signaling and is possibly involved in colorectal carcinogenesis. A new drug targeting the interactions of TCF-4 with NPC proteins as well as their sumoylation activity might be effective for suppressing aberrant Wnt signaling and the proliferation of colorectal cancer cells.

Quantitative proteomics using formalin-fixed paraffin-embedded tissues of oral squamous cell carcinoma

Clinical proteomics using a large archive of formalin‐fixed paraffin‐embedded (FFPE) tissue blocks has long been a challenge. Recently, a method for extracting proteins from FFPE tissue in the form of tryptic peptides was developed. Here we report the application of a highly sensitive mass spectrometry (MS)‐based quantitative proteome method to a small amount of samples obtained by laser microdissection from FFPE tissues. Cancerous and adjacent normal epithelia were microdissected from FFPE tissue blocks of 10 squamous cell carcinomas of the tongue. Proteins were extracted in the form of tryptic peptides and analyzed by 2‐dimensional image‐converted analysis of liquid chromatography and mass spectrometry (2DICAL), a label‐free quantitative proteomics method developed in our laboratory. From a total of 25 018 peaks we selected 72 mass peaks whose expression differed significantly between cancer and normal tissues (P < 0.001, paired t‐test). The expression of transglutaminase 3 (TGM3) was significantly down‐regulated in cancer and correlated with loss of histological differentiation. Hypermethylation of TGM3 gene CpG islands was observed in 12 oral squamous cell carcinoma (OSCC) cell lines with reduced TGM3 expression. These results suggest that epigenetic silencing of TGM3 plays certain roles in the process of oral carcinogenesis. The method for quantitative proteomic analysis of FFPE tissue described here offers new opportunities to identify disease‐specific biomarkers and therapeutic targets using widely available archival samples with corresponding detailed pathological and clinical records. (Cancer Sci 2009; 100: 1605–1611)

Large-scale quantitative clinical proteomics by label-free liquid chromatography and mass spectrometry

We previously reported the development of an integrated proteome platform, namely 2‐Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL), for quantitative comparison of large peptide datasets generated by nano‐flow liquid chromatography (LC) and mass spectrometry (MS). The key technology of 2DICAL was the precise adjustment of the retention time of LC by dynamic programming. In order to apply 2DICAL to clinical studies that require comparison of a large number of patient samples we further refined the calculation algorithm and increased the accuracy and speed of the peptide peak alignment using a greedy algorithm, which had been used for fast DNA sequence alignment. The peptide peaks of each sample with the same m/z were extracted every 1 m/z and displayed with along the horizontal axis. Here we report a precise comparison of more than 150 000 typtic peptide ion peaks derived from 70 serum samples (40 patients with uterine endometrial cancer and 30 controls). The levels of 49 MS peaks were found to differ significantly between cancer patients and controls (P < 0.01, Welchs t‐test and interquartile range [IQR] of >40), and the differential expression and identification of selected three proteins was validated by immunoblotting. 2DICAL was is highly advantageous for large‐scale clinical proteomics because of its simple procedure, high throughput, and quantification accuracy. (Cancer Sci 2009; 100: 514–519)

Functional genome screen for therapeutic targets of osteosarcoma

Osteosarcoma (OS) is the most frequent primary malignant bone tumor of children and young adults. Although the introduction of combined neoadjuvant chemotherapy has markedly improved survival, the outcome of OS patients with distant metastasis and/or poor response to chemotherapy is still unsatisfactory. Therefore there is a need to develop new therapeutic agents that suppress OS cell proliferation with higher efficacy. The protein kinases are a family of genes that play critical roles in various signaling pathways. Some cancer cells show addiction to constitutive activation of certain signaling pathways for proliferation and survival. To identify new drug targets for OS, we screened a panel of small interfering RNAs (siRNAs) that target 691 genes encoding human protein kinases and related proteins. We found that different constructs of siRNA specifically targeting polo‐like 1 kinase (PLK1) significantly caused mitotic cell cycle arrest and subsequent apoptotic cell death in a variety of OS cell lines. siRNA targeting PLK1 also suppressed the growth of OS xenografts established in immunodeficient mice. Recently, phase I clinical trials of PLK1 chemical inhibitors have been reported. Our results indicate that PLK1 is a promising molecular target for pharmacologic intervention in OS. (Cancer Sci 2009; 100: 2268–2274)

Traf2- and Nck-interacting Kinase Is Essential for Canonical Wnt Signaling in Xenopus Axis Formation

Wnt signaling pathways play important roles in various stages of developmental events and several aspects of adult homeostasis. Aberrant activation of Wnt signaling has also been associated with several types of cancer. We have recently identified Traf2- and Nck-interacting kinase (TNIK) as a novel activator of Wnt signaling through a comprehensive proteomic approach in human colorectal cancer cell lines. TNIK is an activating kinase for T-cell factor-4 (TCF4) and essential for the β-catenin-TCF4 transactivation and colorectal cancer growth. Here, we report the essential role of TNIK in Wnt signaling during Xenopus development. We found that Xenopus TNIK (XTNIK) was expressed maternally and that the functional knockdown of XTNIK by catalytically inactive XTNIK (K54R) or antisense morpholino oligonucleotides resulted in significant malformations with a complete loss of head and axis structures. XTNIK enhanced β-catenin-induced axis duplication and the expression of β-catenin-TCF target genes, whereas knockdown of XTNIK inhibited it. XTNIK was recruited to the promoter region of β-catenin-TCF target genes in a β-catenin-dependent manner. These results demonstrate that XTNIK is an essential factor for the transcriptional activity of the β-catenin-TCF complex and dorsal axis determination in Xenopus embryos.

Increased susceptibility of Sf1+/– mice to azoxymethane‐induced colon tumorigenesis

Aberrant transactivation of a certain set of target genes by the β‐catenin and T‐cell factor‐4 nuclear complex has been considered crucial for the initiation of colorectal carcinogenesis. We previously identified splicing factor‐1 (SF1) as a novel component of the β‐catenin and T‐cell factor‐4 complex, and showed that the overexpression of SF1 inhibited the gene transactivational activity of the complex and markedly suppressed β‐catenin‐evoked colony formation by human embryonic kidney 293 cells. However, the involvement of SF1 in the process of carcinogenesis in vivo remains unclear. In the present study, we established SF1‐knockout mice using the gene trapping method. Homozygous mice (Sf1−/–) died during embryonic development before embryonic day (E)8.5, whereas heterozygous (Sf1+/–) mice were born alive and developed normally. Azoxymethane (AOM) was given at a dose of 10 mg/kg body weight once a week for 6 weeks to 7‐week‐old Sf1+/– and Sf1+/+ mice. At 23 weeks after the start of AOM the average number (5.5 ± 0.6 versus 2.2 ± 0.2 in females [P = 0.003, Mann–Whitney U‐test], 3.7 ± 0.2 versus 1.7 ± 0.7 in males [P = 0.014]) and volume of colon tumors per mouse (8.7 ± 1.6 versus 2.2 ± 0.5 mm3 per female [P = 0.0008], 11.3 ± 3.4 versus 0.6 ± 0.2 mm3 per male [P = 0.001]) were significantly higher in Sf1+/– than in Sf1+/+ mice. The increased susceptibility of Sf1+/– mice to AOM‐induced colon tumorigenesis indicates the crucial involvement of SF1 in the β‐catenin‐mediated regulation of proliferation and differentiation of intestinal epithelial cells. (Cancer Sci 2007; 98: 1862–1867)

β-Catenin Inhibits Promyelocytic Leukemia Protein Tumor Suppressor Function in Colorectal Cancer Cells

BACKGROUND & AIMS Loss of promyelocytic leukemia protein (PML) nuclear body (NB) formation has been reported in colorectal and other solid tumors. However, genetic alteration of PML is rarely observed in these tumors; the exact mechanisms that mediate loss of PML function are not known. METHODS We previously used a comprehensive shotgun mass spectrometry approach to identify PML as 1 of 70 proteins that coimmunoprecipitate with anti-T-cell factor 4 in DLD-1 and HCT116 colorectal cancer cell lines; we investigated the effects of altered β-catenin expression on PML function in these cells. RESULTS β-catenin specifically interacted with the product of PML transcript variant IV (PML-IV) through the armadillo repeat domain of β-catenin. Overexpression of β-catenin in colorectal cancer cells disrupted the subcellular compartmentalization of PML-IV, whereas knockdown of β-catenin restored formation of PML-NB. Modification of PML by the small ubiquitin-related modifier (SUMO) is required for proper assembly of PML-NB. β-catenin inhibited Ran-binding protein 2-mediated SUMOylation of PML-IV. CONCLUSIONS β-catenin interacts with PML isoform IV and disrupts PML-IV function and PML-NB formation by inhibiting Ran-binding protein 2-mediated SUMO modification of PML-IV. These findings indicate the involvement of a posttranslational mechanism in disruption of PML-NB organization in cancer cells and provide more information about the oncogenic functions of β-catenin.