Aimed at discovering diagnostic biomarkers and targets of molecular therapy, comprehensive protein and gene expression profiling of cancer cell lines, cancer tissues, and sera/plasma of cancer patients has been undertaken at the Chemotherapy Division and Cancer Proteomics Project.

b-Catenin is the most downstream effector in the Wnt signaling pathway, and evidence exists to suggest its involvement in the process of early colorectal carcinogenesis. b-Catenin is believed to exert its oncogenic action by activating TCF4, a member of the TCF/lymphoid enhancer factor (LEF) family of transcriptional factors. Several studies (including ours) have used cDNA/ oligonucleotide microarrays in an attempt to identify the immediate transcriptional targets of TCF4, but the extensive protein network of b-catenin-mediated intestinal carcinogenesis seems to be complicated and has not yet been fully elucidated.

Nano (nL/minute)-level flow rate HPLC (high-performance liquid chromatography) chromatography and mass spectrometry (MS)-based proteomics has recently attracted considerable attention because of its capacity for comprehensive protein identification and quantification. More than 4000 peptides derived from colorectal cancer cells were examined by LC-MS, and 87 proteins whose expression was significantly up- or downregulated by induction of dominant-negative TCF4 were identified. A zinc finger protein SF1/ZNF162/ZFM1 was one of the proteins negatively regulated by b-catenin ⁽³⁵⁾.

The expression of SF1 was correlated with the differentiation status of intestinal epithelial cells and inversely correlated with tumorigenesis. Furthermore, SF1 was found to be a novel component of the TCF4/b-catenin complex. SF1 cDNA transfection markedly inhibited the transcriptional activity of the TCF4/b-catenin complex and suppressed b-catenin-evoked colony formation by HEK293 cells. Consistent with this, Sf1^+/- mice exhibited greater susceptibility to colon tumorigenesis induced by azoxymethane than wild-type (Sf1^+/+) mice, prompting us to conclude that SF1 is a negative regulator of the oncogenic activity of the b-catenin/TCF4 complex ⁽³⁶⁾.

The b-catenin/TCF4 complex physically interacts with poly (ADP-ribose) polymerase-1 (PARP-1) and Ku70, and the transcriptional activity of TCF4 is regulated competitively by the relative amount of Ku70 and PARP-1 proteins binding to TCF4. In response to DNA damage, PARP-1 polyADP-ribosylates its own auto- modification domain. This modification inhibits the interaction between PARP-1 and TCF4, and the dissociation of PARP-1 from TCF4 allows Ku70 to interact with TCF4. The recruitment of Ku70 into TCF4 inhibits the interaction between b-catenin and TCF4 and, thereby, the transcriptional activity of TCF4 ⁽³⁷⁾.

Topoisomerase IIa (Topo IIa) was found to be another functional component of the b-catenin/TCF4 complex. Overexpression of Topo IIa enhanced the TCF/LEF transcriptional activity, and conversely, knockdown of Topo IIa by RNA interference attenuated the transcriptional activity. The Topo II inhibitors, merbarone and etoposide, suppressed the b-catenin-mediated TCF/LEF transcriptional activity. Topo II is a known target of drugs that are currently being widely used for cancer chemotherapy. A new drug targeting the interaction of Topo IIa with b-catenin as well as its catalytic activity might be more effective for suppressing aberrant Wnt signaling and proliferation of colorectal cancer cells than the currently used Topo II inhibitors ⁽³⁸⁾.

Actinin-4 was identified as an actin-binding protein associated with cancer invasion and metastasis, and significant clinicopathological implications of actinin-4 have been demonstrated in various human malignancies, including breast, lung, colorectal, and ovarian cancers ⁽³⁹⁾. However, the level of actinin-4 expression was found to be significantly lower in prostate cancer cells than in non-cancerous basal cells, and restoration of actinin-4 expression inhibited proliferation of prostate cancer cells. MS-based proteomic analysis revealed that actinin-4 forms native complexes with several endocytosis-related proteins. The decreased expression of actinin-4 protein in prostate cancer cells may cause aberrations in the intracellular trafficking of various cell surface molecules and contribute to carcinogenesis ⁽⁴⁰⁾.

A large variety of low-molecular-weight protein fragments are known to be produced as a consequence of the proteolytic processes that occur in the microenvironment of cancer tissues. These protein fragments are released into the blood circulation, where they become bound to high-abundance proteins, such as serum albumin. Serum-albumin-associated peptide fragments may accumulate over time in the body during the course of chronic diseases, such as cancer. Detection and quantification of such peptides could be applicable to cancer diagnosis. Using high-resolution MS ⁽⁴¹⁾ the amounts of 3 albumin-associated peptides in the blood were identified to be significantly different between endometrial cancer patients and controls ⁽⁴²⁾.

Natural killer T (NKT) cells have a strong antitumor activity and easily expand in vitro in the presence of a-galactosylceramide. Therefore, in-vitro-expanded NKT cells are considered to be promising effector cells for cancer immunotherapy. The efficiency of in-vitro expansion of NKT cells is determined by the ratio of production of interleukin-4 and IFN-g by the NKT cells. The effective expansion of NKT cells requires T helper type 2-biased culture conditions ⁽⁴³⁾.

The antitumor activities and modes of actions of new compounds isolated from antibiotics were examined to find new agents targeting new molecular compounds in tumor cells. Pironetin, isolated from Streptomyces sp., with the unique characteristic of binding covalently to a-tublin was effective not only against human tumor cell lines resistant to microtubule (b-tublin)-targeted drugs, but also against multidrug-resistant cells showing mdr1 gene expression ⁽⁴⁴⁾.