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Lab of Collaborative Research (Masuda Group)
Genomic and Proteomic Approaches to Colorectal Carcinogenesis
β-Catenin is the downstream effector of the Wnt signaling pathway and is involved in the process of colorectal carcinogenesis. T-cell factor-4 (TCF4) regulates a certain set of genes related to growth and differentiation of intestinal epithelial cells, and aberrant transactivation of these TCF4-regulated genes by β-catenin protein plays a crucial role in early intestinal carcinogenesis.
Suppression of β-catenin-evoked gene transactivation of colorectal cancer cells by dominant-negative TCF4 switches off genes involved in cell proliferation and switches on genes involved in cell differentiation (1). Induction of dominant-negative TCF4 has been reported to restore the epithelial cell polarity of a colorectal cancer cell line and convert the cell line into a single layer of columnar epithelium, indicating that colorectal cancer cells still require accumulation of β-catenin protein, thereby transactivating the target genes of TCF4, to maintain cell proliferation, depolarization, and dedifferentiation (2).
By using global gene (GeneChip oligonucleotide microarray) and protein (2D-DIGE and isotope-coded affinity tagging and mass spectrometry) expression analyses we succeeded in identifying several molecules whose expression is regulated by the β-catenin/TCF4 complex (3,4,9). The human multidrug resistance-1 (MDR1) (ABCB1) gene contains multiple TCF/LEF-binding elements in its promoter and is one of the immediate targets of the β-catenin/TCF4 complex (1).
Biological involvement of MDR1 in intestinal tumorigenesis has been verified in a mouse model. Mdr1-deficient Min (ApcMin/+Mdr1a/b-/-) mice developed significantly fewer intestinal polyps than did control (ApcMin/+Mdr1a/b+/+) mice (5). SF1 negatively regulates negatively -catenin-evoked gene transactivation and cell proliferation (9), and consistent with this Sf1+/- mice exhibited greater susceptibility to colon tumorigenesis induced by azoxymethane than wild-type (Sf1+/+) mice (10).
In our series of proteomic studies we identified fusion/translocated in liposarcoma (FUS/TLS), poly(ADP-ribose) polymerase-1 (PARP-1), Ku70, Ku80, DNA topoisomerase IIα (Topo IIα), and splicing factor-1 (SF1) as putative components of the β-catenin and TCF4 nuclear complex (6,7,8,9,12). Two of these component proteins, Topo IIα(11) and PARP-1(6,8), are enhancers of the β-catenin and TCF/LEF transcriptional complexes, and SF1 (9, 10), FUS/TLS (7), and Ku70 (8) are suppressors. Topo II is a known target of drugs that are currently being widely used for cancer chemotherapy. We have demonstrated that Topo IIα a functional component of the β-catenin and TCF4 complex (11) and a potential drug target. Although there is a correlation between nuclear localization of ƒÀ-catenin and neoplastic phonotypes of colorectal cancer, the mechanisms underlying nuclear import of β-catenin, which lacks a nuclear localization signal, remain poorly understood. We have found that TCF-4 interacts with nuclear pore complex (NPC) including SUMO E3 ligase, Ran BP2, that sumoylates TCF4(PDF:122KB). This sumoylated TCF-4 reinforces the interaction between TCF-4 and ƒÀ-catenin and thereafter accelerates nuclear transport of ƒÀ-catenin/TCF-4 complex (12). We therefore believe that RanBP2 becomes a therapeutic target for colorectal cancer.
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Identification of Druggable Molecular Targets in Hepatocellular Carcinoma
Unresectable hepatocellular carcinoma (HCC) is an aggressive disease and its management remains a challenge. Since the approval in 2009, the multikinase inhibitor sorafenib has been the only available therapeutic for advanced HCC with modest results. Predictive biomarkers of sorafenib efficacy in HCC have been extensively investigated worldwide, which led only to potential candidates. We also identified p-RPS6 S235/236 as a potential predictor for sorafenib efficacy(PDF156KB) (1). Despite little progress in developing a molecularly targeted therapy for advanced HCC over the last decade, both regorafenib and lenvatinib are expected to be approved in the near future for treating advanced HCC in Japan. Treatment options however are still limited for advanced HCC. In an effort to develop new therapeutic agents that act specifically on HCC, but interfere minimally with residual liver function, we searched for genes upregulated in HCC compared with background non-tumorous liver tissue in 84 paired clinical specimens using microarrays and real-time PCR followed by siRNA-based screening of genes required for HCC cell proliferation. As a result, we identified potential molecular targets including aurora kinase A and its interacting protein TPX2 (2).
- Masuda et al., Mol Cell Proteomics 6:1429-38, 2014.
- Satow et al., Clin Cancer Res. 16(9):2518-28, 2010.
- Development of a Reverse-Phase Protein Array Platform (RPPA) The reverse-phase protein array (RPPA) is an antibody-based proteomic technology ideally suited for profiling both protein expression and post-translational modifications including phosphorylation (1,2) (PDF: 338KB). Its throughput, sensitivity, cost effectiveness and ability to handle miniscule amounts of samples have accelerated the recent evolution of RPPA technology in basic, preclinical and clinical research fields. Possible utilization of the RPPA platform for refining precision medicine includes patient stratification; discovery of therapeutic targets and predictive biomarkers; elucidating the mechanism of drug resistance; and monitoring patient response to treatment. We have generated our in-house RPPA platform in 2010 (3). Drs. Tesshi Yamada, Satoshi Nishizuka and Mari Masuda of our laboratory are members of the Global RPPA Workshop launched in 2011 and have been sharing RPPA-related knowledge and technologies with other laboratories worldwide. In a continued effort to improve and update our in-house RPPA platform, we have been pushing for RPPA application in clinical laboratory testing.
- Development of a Biomarker to Identify Patients with Lung Adenocarcinomas Who Could Benefit from Postsurgical Adjuvant Therapy
Actinin-4 was identified in our laboratory as a novel actin-binding protein associated with increased cell motility and cancer invasion (4). Expression of Actinin-4 has also been found to be significantly correlated with the outcomes of colorectal (PDF:27KB) (25), ovarian (6) and pancreatic cancer patients (7). In addition, gene amplification of ACTN4 has been observed in ovarian (8) and pancreatic cancers (7). We have found an increase in the DNA copy number for ACTN4 appears to predict poor prognosis in patients with high-grade ovarian carcinomas (PDF:23KB) (58). By conducting fluorescence in situ hybridization (FISH) analysis in 543 patients, who underwent surgical resections of lung carcinomas at the NCC Hospital in Tokyo and the NCC Hospital East in Kashiwa, Chiba, we found amplification of the ACTN4 gene could be a useful biomarker to identify stage I lung adenocarcinoma patients likely to benefit from postsurgical adjuvant therapy (9). We are currently planning to conduct a prospective randomized study to determine possible stratification of stage I lung adenocarcinoma patients on the basis of their ACTN4 gene copy number. FISH-based molecular diagnostics generally require highly experienced pathologists for precise diagnosis and involve a relatively long turn-around time. In an effort to circumvent these drawbacks, we are currently developing in collaboration with Sysmex (Kobe, Japan) a novel technology that would provide accurate and rapid molecular testing in the near future.
- Masuda et al., Expert Rev Proteomics, 2017.
- Masuda et al., Biochim Biophys Acta 6:651-7, 2015.
- Masuda et al., Mol Cell Proteomics 6:1429-38, 2014.
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- Noro et al., Ann Oncol 10:2594-600, 2013