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Cancer biomarkers for the early detection of malignancies and selection of therapeutic strategies have been requested in the clinical field. Accurate and informative cancer biomarkers hold significant promise for improvements in the early detection of disease and in the selection of the most effective therapeutic strategies. Recently, significant progress in the comprehensive analysis of the human genome, epigenome, transcriptome, proteome and metabolome has led to revolutionary changes in the discovery of cancer biomarkers. The Human Proteome Organization has launched a global Human Proteome Project to map the entire human protein set. The Human Proteome Project research group has focused on three working proteomic pillars-mass spectrometry-based, antibody-based and knowledge-based proteomics-and each of these technologies is advancing rapidly. In this review, we introduce the proteomic platforms that are currently being used for cancer biomarker discovery, and describe examples of novel cancer biomarkers that were identified with each proteomic technology.

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., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objective: Emerging molecular targeting therapeutics have been incorporated into the management of advanced renal cell carcinoma; however, their efficacy remains limited. The aim of this study was to catalog potential therapeutic target molecules for renal cell carcinoma., Methods: We first selected genes up-regulated in clear cell renal cell carcinoma relative to surrounding normal kidney tissues in 10 patients (Study Cohort) using high-density exon arrays that detect all potential transcripts predicted in the human genome. The selected genes were subjected to independent validation in another set of 10 patients (Validation Cohort) using real-time reverse transcriptase polymerase chain reaction and functional screening using small interfering RNA in six clear cell renal cell carcinoma cell lines., Results: We identified 164 genes whose expression was significantly elevated in clear cell renal cell carcinoma (P< 0.0001 [Student's t-test] and at least a 3-fold change in transcription signal). We finally extracted 33 genes required for maintaining cell proliferation in at least two clear cell renal cell carcinoma cell lines. The 33 genes included 13 genes known to be associated with the development/progression of renal cell carcinoma, including CAIX and FLT-1, confirming the robustness of the current strategy., Conclusions: Through a combination of genome-wide expression and functional assays, we identified a set of genes with high potential as targets for drug development. This method is rapid and comprehensive and could be applied to the discovery of diagnostic biomarkers and therapeutic targets for cancers other than clear cell renal cell carcinoma.

The development of a new plasma biomarker for early detection would be necessary to improve the overall outcome of colorectal cancer. Here we report the identification and validation of the ninth component of complement (C9) as a novel plasma biomarker for colorectal cancer by cutting-edge proteomic technologies. Plasma proteins were enzymatically digested into a large array of peptides, and the relative quantity of a total of 94,803 peptide peaks was compared between 31 colorectal cancer patients and 59 age/sex-matched healthy controls using 2D image-converted analysis of liquid chromatography and mass spectrometry. The selected biomarker candidates were validated in 345 subjects (115 colorectal cancer patients and 230 age/sex-matched healthy controls) using high-density reverse-phase protein microarrays. Plasma levels of Apo AI and C9 in colorectal cancer patients significantly differed from healthy controls with P values of 7.94×10(-4) and 1.43×10(-12) (Student's t-test), respectively. In particular, C9 was elevated in patients with colorectal cancer, including those with stage-I and -II diseases (P=3.01×10(-3) and P=1.13×10(-5) , respectively). Although the significance of the present study must be validated in an independent clinical study, the increment of plasma C9 may be applicable to the early detection of colorectal cancer., (© 2010 Japanese Cancer Association.)

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.

Plasma proteome analysis requires sufficient power to compare numerous samples and detect changes in protein modification, because the protein content of human samples varies significantly among individuals, and many plasma proteins undergo changes in the bloodstream. A label-free proteomics platform developed in our laboratory, termed "Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL)," is capable of these tasks. Here, we describe successful detection of novel prolyl hydroxylation of alpha-fibrinogen using 2DICAL, based on comparison of plasma samples of 38 pancreatic cancer patients and 39 healthy subjects. Using a newly generated monoclonal antibody 11A5, we confirmed the increase in prolyl-hydroxylated alpha-fibrinogen plasma levels and identified prolyl 4-hydroxylase A1 as a key enzyme for the modification. Competitive enzyme-linked immunosorbent assay of 685 blood samples revealed dynamic changes in prolyl-hydroxylated alpha-fibrinogen plasma level depending on clinical status. Prolyl-hydroxylated alpha-fibrinogen is presumably controlled by multiple biological mechanisms, which remain to be clarified in future studies.

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.

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, Welch's 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.

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.

Accumulation of the beta-catenin protein and transactivation of a certain set of T-cell factor (TCF)-4 target genes by accumulated beta-catenin have been considered crucial in colorectal carcinogenesis. In the present review, we summarize nuclear proteins that interact with, and regulate, the beta-catenin and TCF and lymphoid enhancer factor (LEF) transcriptional complexes. Our recent series of proteomic studies has also revealed that various classes of nuclear proteins participate in the beta-catenin-TCF-4 complex and modulate its transcriptional activity. Furthermore, the protein composition of the TCF-4-containing nuclear complex is not fixed, but is regulated dynamically by endogenous programs associated with intestinal epithelial cell differentiation and exogenous stimuli. Restoration of the loss-of-function mutation of the adenomatous polyposis coli (APC) gene in colorectal cancer cells does not seem to be a realistic approach with currently available medical technologies, and only signaling molecules downstream of the APC gene product can be considered as targets of pharmacological intervention. Nuclear proteins associated with the beta-catenin-TCF-4 complex may include feasible targets for molecular therapy against colorectal cancer. Recently, an inhibitor of the interaction between CREB-binding protein and beta-catenin was shown to efficiently shut down the transcriptional activity of TCF-4 and induce apoptosis of colorectal cancer cells. We also summarize current strategies in the development of drugs against Wnt signaling.

Aberrant transactivation of a certain set of target genes by the beta-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 beta-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 beta-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 mm(3) per female [P = 0.0008], 11.3 +/- 3.4 versus 0.6 +/- 0.2 mm(3) 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 beta-catenin-mediated regulation of proliferation and differentiation of intestinal epithelial cells.

Background & Aims: The Wnt signaling pathway is activated constitutively in the majority of colorectal cancers as a result of mutation in either the adenomatous polyposis coli or the CTNNB1 gene, and blockage of the pathway has been considered feasible as molecular therapy against colorectal cancer. DNA topoisomerase IIalpha (Topo IIalpha) is a component of the beta-catenin/T-cell factor-4 (TCF-4) nuclear complex. We examined the functional significance of Topo IIalpha in Wnt signaling., Methods: The physical and functional interaction between Topo IIalpha and the beta-catenin/TCF-4 nuclear complex was evaluated by immunoprecipitation, immunofluorescence microscopy, 2-hybrid assay, and luciferase reporter assay., Results: Amino acids 951-1301 of Topo IIalpha were necessary for binding to beta-catenin. Over expression of Topo IIalpha enhanced the TCF/lymphoid enhancer factor transcriptional activity in a dose-dependent manner, and knockdown of Topo IIalpha by RNA interference conversely attenuated the transcriptional activity. The Topo II inhibitors, merbarone and etoposide, suppressed the beta-catenin-mediated TCF/lymphoid enhancer factor transcriptional activity. The catalytic activity of Topo II was augmented by overexpression of beta-catenin as measured by the decatenation of kinetoplast DNA. Topo IIalpha was highly expressed and colocalized with beta-catenin in tumor cells of patients with familial adenomatous polyposis syndrome and patients with sporadic colorectal cancer., Conclusions: Topo IIalpha interacts with beta-catenin as a novel transcriptional co-activator. A new drug targeting the interaction of Topo IIalpha with beta-catenin as well as its catalytic activity might be more effective for suppressing aberrant Wnt signaling and proliferation of colorectal cancer cells than the current Topo II inhibitors.

The incidence of endometrial cancer is predicted to increase in developed countries. Because of the relatively high incidence of complications and low diagnostic sensitivity associated with endometrial tissue sampling, there is an urgent need for the development of a safe and non-invasive diagnostic method. The proteomic spectrum of albumin-associated peptides was obtained from a total of 125 serum samples (92 from endometrial cancer patients and 33 from controls) by matrix-assisted laser desorption/ionization hybrid quadrupole time-of-flight mass spectrometry, and the candidate markers were selected by the Mann-Whitney U-test and receiver operator characteristics analysis. We selected three mass peaks at 4769, 6254 and 11 792 m/z from a total of 507 peaks as distinguishing cancer patients from controls (P < 0.00001 and area under curve of over 0.8). When the cut-off points were defined as the averages of the values in the controls + 2 SD, the combination of the three peptides detected endometrial cancer with a sensitivity of 65.2% (60/92). Even stage I early endometrial cancers were detected with a sensitivity of 60.3% (38/63). Unfortunately, the three peptides were also detected in 44.6% (33/74) of myoma uteri patients, indicating that they are not specific to endometrial cancer. Although a large-scale study is necessary to confirm the clinical significance of the peptide biomarkers identified in this study, direct profiling of serum-albumin-bound peptides by high-resolution mass spectrometry was proven to have potential as a means of identifying biomarkers for a variety of diseases.

Background and Aims: beta-Catenin is the downstream effector of the Wnt signaling pathway and is involved in the process of colorectal carcinogenesis. However, it is still uncertain whether beta-catenin exerts its oncogenic function solely by coactivating the target genes of T-cell factor-4 (TCF4). We previously reported that the beta-catenin/TCF4 complex contains several classes of RNA-binding proteins and regulates the premessenger RNA splicing reaction, but the identity of the exact effector molecule downstream of the beta-catenin/TCF4 complex has not been established., Methods: Using isotope-coded affinity tagging and mass spectrometry, we examined more than 4000 peptides derived from colorectal cancer cells and identified that splicing factor-1 (SF1) was one of the proteins whose expression is regulated by the beta-catenin/TCF4 complex., Results: The expression of SF1 was found to be correlated with the differentiation status of intestinal epithelial cells and inversely correlated with tumorigenesis. Immunoprecipitation and immunofluorescence microscopy revealed that SF1 was a complex, and beta-catenin-evoked gene transactivation and cell proliferation were negatively regulated by SF1 complementary DNA transfection. SF1 was essential for the induction of alternative splicing by the beta-catenin/TCF4 complex, and SF1 complementary DNA transfection induced known cancer-related splice variants, such as Wnt-induced secreted protein-1v and fibroblast growth factor receptor-3-ATII., Conclusions: The beta-catenin/TCF4 complex regulates the level of SF1 protein expression, and, conversely, SF1 interacts with the complex and regulates its gene transactivation and premessenger RNA splicing activities. Identification of the interaction may shed light on a novel aspect of the Wnt signaling pathway.

Formation of the T-cell factor-4 (TCF-4) and beta-catenin nuclear complex is considered crucial to embryonic development and colorectal carcinogenesis. We previously reported that poly(ADP-ribose) polymerase-1 (PARP-1) interacts with the TCF-4 and beta-catenin complex and enhances its transcriptional activity. However, its biological significance remains unexplained. Using immunoprecipitation and mass spectrometry, we found that two Ku proteins, Ku70 and Ku80, were also associated with the complex. Knockdown of Ku70 by RNA interference increased the amount of beta-catenin associated with TCF-4 and enhanced the transcriptional activity. PARP-1 competed with Ku70 for binding to TCF-4. Treatment with bleomycin, a DNA-damaging alkylating agent, induced polyADP-ribosylation of PARP-1 protein and inhibited its interaction with TCF-4. Bleomycin conversely increased the amounts of Ku70 coimmunoprecipitated with TCF-4 and removed beta-catenin from TCF-4. We propose a working model in which the transcriptional activity of TCF-4 is regulated by the relative amount of Ku70, PARP-1, and beta-catenin proteins binding to TCF-4. Identification of the functional interaction of Ku70 as well as PARP-1 with the TCF-4 and beta-catenin transcriptional complex may provide insights into a novel linkage between DNA damage recognition/repair and Wnt signaling.

Pancreatic cancer is the fifth leading cause of cancer-related mortality in Japan. Early detection in pancreatic cancer is one of the most feasible strategies to improve outcome. We compared plasma proteome between pancreatic cancer patients and healthy controls using surface-enhanced laser desorption/ionization coupled with hybrid quadrupole time-of-flight mass spectrometry. Proteomic spectra were generated from a total of 245 plasma samples obtained from two institutes. A discriminating proteomic pattern was built from training cohort using machine learning algorithm and was applied two validation cohorts. This set discriminating cancer patients in the first validation cohort with a sensitivity of 90.9% and a specificity of 91.9%, and was further validated in an independent cohort at a second institution. When combined with CA19-9, 100% tumor of pancreatic cancers, including early stage tumors, were detected. In this report, we describe a possible detection of early pancreatic cancer using novel proteomic technique.

Background & Aims: beta-Catenin is a downstream effector of the Wnt signaling pathway and is believed to exert its oncogenic function by activating T-cell factor (TCF)/lymphoid enhancer factor (LEF) family transcriptional factors. However, it is still uncertain whether the diverse effects of beta-catenin are caused solely by aberrant gene transactivation. In this study, we used a proteomics approach to obtain further insight into the functional properties of nuclear beta-catenin., Methods: The protein assembly of a native beta-catenin-containing complex in nuclear extracts from a colorectal cancer cell line, DLD-1, was identified using immunoprecipitation and mass spectrometry., Results: beta-Catenin physically interacted with fusion (FUS)/translocated in liposarcoma (TLS) and various RNA-binding proteins. The expression of FUS/TLS was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. The transient transfection of FUS suppressed beta-catenin-evoked gene transactivation of TCF/LEF, and beta-catenin transfection affected the splicing pattern of the E1A minigene and induced a novel splicing variant of estrogen receptor (ER)-beta exerting a dominant-negative activity., Conclusions: Human cancer expresses a large variety of alternatively spliced messenger RNA (mRNA), but the precise molecular mechanisms responsible for cancer-related alternative splicing are largely unknown. In this study, we demonstrated the interaction of beta-catenin with FUS/TLS and other RNA-binding proteins involved in the regulation of pre-mRNA splicing. Certain mRNA splicing abbreviations seen in human cancers may be induced by the activation of the Wnt signaling pathway.

The E-cadherin/catenin system acts as an invasion suppressor of epithelial malignancies. This invasion suppressive activity seems be mediated not only by the cell adhesive activity of E-cadherin but by other undetermined signaling pathways elicited by beta-catenin. In fact, cancer cells that have infiltrated the stroma reduce the expression of E-cadherin and accumulate beta-catenin. We attempted to identify the alternative partner proteins that make complexes with beta-catenin in the absence of E-cadherin. An approximately 100-kDa protein was constantly coimmunoprecipitated with beta-catenin from SW480 colorectal cancer cells, which lack the expression of E-cadherin, and was identified as actinin-4 by mass spectrometry. Transfection of E-cadherin cDNA suppressed the association between beta-catenin and actinin-4. Inhibition of E-cadherin by RNA interference transferred the beta-catenin and actinin-4 proteins into the membrane protrusions of DLD-1 cells. Immunofluorescence histochemistry of clinical colorectal cancer specimens showed that the beta-catenin and actinin-4 proteins were colocalized in colorectal cancer cells infiltrating the stroma. We reported previously that overexpression of actinin-4 induces cell motility and specifically promotes lymph node metastasis by colorectal cancer. The association between beta-catenin and actinin-4 and its regulation by E-cadherin may represent a novel molecular link connecting cell adhesion and motility. Shutting down the signals mediating this association may be worth considering as a therapeutic approach to cancer invasion and metastasis.

Purpose: Surface enhanced laser desorption/ionization mass spectrometry can generate robust information from a small amount of clinical samples such as serum and plasma. In this study we identified novel diagnostic biomarkers of renal cell carcinoma (RCC) by large-scale serum protein profiling using surface enhanced laser desorption/ionization mass spectrometry., Materials and Methods: Proteomic spectra were generated by a time of flight mass spectrometer from a set of training samples (21 patients with RCC and 24 healthy volunteers) and another set of validation samples (19 patients with RCC, 20 healthy volunteers and 5 patients with pyelonephritis). Information on the peaks (intensity and m/z) was extracted from the mass spectra using newly developed algorithms, and the Mann-Whitney's U test and linear support vector machine were used to identify the peaks distinguishing RCC samples from the controls., Results: Two peaks with molecular masses of 4,151 and 8,968 m/z were selected as significantly more prominent in RCC samples (p <0.01) among the 3,539 peaks in the range of 3,000 to 30,000 m/z obtained from the training samples. Simultaneous recognition of these 2 biomarkers was shown to have a sensitivity of 89.5% for the diagnosis of RCC and an overall specificity of 80.0% (95% [19 of 20] of healthy volunteers and 20% [1 of 5] of patients with pyelonephritis) in the blinded validation samples, and to allow detection of RCC in stage I (UICC) in 88.9% (16 of 18) of the cases., Conclusions: We identified 2 serum biomarkers potentially useful for the early diagnosis of RCC. This finding warrants a further large-scale multi-institutional analysis for clinical evaluation of the diagnostic significance of these biomarkers.

Background & Aims: T-cell factor (TCF)-4 regulates a certain set of genes related to growth and differentiation of intestinal epithelial cells. Aberrant transactivation of these TCF-4-regulated genes by beta-catenin protein plays a crucial role in early intestinal carcinogenesis, and the transcriptional machinery of the TCF-4/beta-catenin complex is likely to contain targets for molecular therapy. We explored the molecular composition of the TCF-4/beta-catenin transcriptional complex by means of proteomics., Methods & Results: A protein of approximately 112 kilodaltons was consistently coimmunoprecipitated with FLAG-tagged TCF-4 transiently expressed in HEK293 cells, and the protein was identified by mass spectrometry as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 physically interacted with TCF-4 and augmented the transcriptional activity of the beta-catenin/TCF-4 complex. Knockdown of PARP-1 by RNA interference significantly suppressed both transcriptional activity and proliferation by colorectal cancer cells. Auto-polyADP-ribosylation of the PARP-1 protein induced by DNA damage inhibited the functional interaction of PARP-1 with TCF-4. PARP-1 was overexpressed in the intestinal adenomas of patients with familial adenomatous polyposis and multiple intestinal polyposis mice. The expression of PARP-1 was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells., Conclusions: In this study, we identified PARP-1 as a novel coactivator of the beta-catenin/TCF-4 complex. Although PARP-1 has been believed to play a protective role against carcinogenesis, these expression patterns and functional properties of PARP-1 were highly suggestive of its participation in early colorectal carcinogenesis.

Aberrant transactivation of a certain set of target genes by the beta-catenin and T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor complexes has been implicated in the process of intestinal epithelial cells entering early colorectal carcinogenesis. A rat intestinal epithelial cell line IEC6 became elongated, extended protrusions at cell periphery, and increased stress fibers and focal contacts upon the induction of beta-catenin protein stabilized by deletion of the N-terminal glycogen synthase kinase-3beta (GSKbeta) phosphorylation sites (beta-catenin DeltaN89). We used the GeneChiptrade mark oligonucleotide microarray system to examine approximately 24 000 genes and identified 13 genes whose expression was altered during the course of this morphological transformation. Those genes included known negative regulators of the Wnt signaling pathway, Sfrp4 and Axin2; extracellular matrix and related molecule, Hxb and Crtl1; cell adhesion and cytoskeletal proteins, Podxl, Igaf4, and Itab6; and molecules involved in the insulin and insulin-like growth factor (IGF) signaling pathways, Enpp1, Igfbp2, and Sgk. We report the finding that insulin-like growth factor-binding protein-2 (IGFBP2) is a direct target gene of the beta-catenin and TCF/LEF complexes. The IGFBP2 protein interacts with integrins. Disruption of the multigene network system regulating cell adhesion and cytoskeleton may be crucial in the initiation of colorectal carcinogenesis.

Background & Aims: Enhanced motility of cancer cells by remodeling of the actin cytoskeleton seems crucial in the process of cancer invasion and metastasis. We previously identified an actin-binding protein, actinin-4, as a new biomarker of cancer invasion and an indicator of prognosis for patients with breast cancer. However, its involvement in the mechanisms of cancer invasion and metastasis remains undetermined. The current study tested the role of actinin-4 in the motility and metastatic potential of colorectal cancer cells., Methods & Results: Quantitative immunofluorescence histochemistry showed that the expression level of the actinin-4 protein was increased in 73.1% (19/26) of the cases of colorectal cancer over the corresponding normal intestinal epithelium. The increased expression of actinin-4 was most significant in dedifferentiated cancer cells at the invasive front. A colorectal cancer cell clone capable of inducing actinin-4 using the tetracycline-regulatory system (designated DLD1 Tet-off ACTN-4) was established. Upon the induction of actinin-4, DLD1 Tet-off ACTN-4 cells spread filopodia and significantly increased their motility ( P = .00027); actinin-4 protein was concentrated at the leading edges of these actin-rich podia. When injected into the mesocecum of severe combined immunodeficient mice, DLD1 Tet-off ACTN4 cells, but not the control cells, metastasized into regional mesenteric lymph nodes, resembling the behavior of clinical cancers. The expression of actinin-4 in focally dedifferentiated cancer cells at the invasive front was significantly correlated with the frequency of lymph node metastasis of colorectal cancer ( P = .038)., Conclusions: Actinin-4 actively increases cell motility and promotes lymph node metastasis of colorectal cancer.

Tumor-associated alternative RNA splicing has gained considerable attention. We identified a novel alternative splice variant RNA of actinin-4 in human small cell lung cancer (SCLC). Expression of the splice variant was highly specific to SCLC cell lines (10/10), biopsies (3/3), and testis. The variant encoded a peptide with a three amino-acid change in exon 8, where the germline missense mutation takes place in familial focal segmental glomerulosclerosis (FSGS). The variant protein showed high affinity to filamentous actin polymers and was not localized with cortical actin. Alternatively spliced actinin-4 may be a new diagnostic marker of SCLC and a candidate target for selective therapy.

Aberrant accumulation of beta-catenin protein because of mutation of either the beta-catenin or adenomatous polyposis coli gene plays an essential role in the development of colorectal carcinoma. We established previously a stable clone of the rat small intestinal epithelial cell line IEC6, which is capable of inducing stabilized beta-catenin protein lacking NH(2)-terminal glycogen synthase kinase-3beta phosphorylation site under a strict control of the tetracycline-regulatory system. This clone, IEC6-TetOFF-beta-catenin DeltaN89, shows in vitro polypoid growth on the removal of doxycycline and seems to be an appropriate model for analyzing the molecular mechanisms of early intestinal carcinogenesis. Of >2000 protein spots displayed by newly developed two-dimensional difference gel electrophoresis, 22 were found to be up- or down-regulated on the induction of stabilized beta-catenin. The majority of these proteins fell into two categories: (a) redox-status regulatory proteins and (b) cytoskeleton-associated proteins. Representatively, a key redox-status regulatory protein, manganese superoxide dismutase, up-regulated in IEC6 cells expressing stabilized beta-catenin protein, was overexpressed in adenoma and adenocarcinoma cells of familial adenomatous polyposis patients in parallel with the accumulation of beta-catenin. These results suggest that aberrant accumulation of beta-catenin might contribute to colorectal carcinogenesis by affecting redox status in the mitochondria of intestinal epithelial cells.

Aberrant transactivation of a certain set of target genes by the beta-catenin and T-cell factor/lymphoid enhancer factor complex has been considered crucial for the initiation of intestinal tumorigenesis. The human multidrug resistance (MDR)1 (ABCB1) gene contains multiple beta-catenin-T-cell factor4-binding elements in its promoter and is one of the immediate targets of the complex. In the current study, we have further substantiated the biological involvement of MDR1 in intestinal tumorigenesis based on the following evidence: (a) aberrant induction of the Mdr1a (Abcb1a) gene product, P-glycoprotein, associated with nuclear accumulation of the beta-catenin protein, was observed even in nascent microscopic adenomas of Min mice; (b) Mdr1-deficient Min (Apc(Min/+)Mdr1a/b(-/-)) mice developed significantly fewer intestinal polyps than did Apc(Min/+)Mdr1a/b(+/+) mice; and (c) Inhibitors of P-glycoprotein, verapamil, and cyclosporin A had a suppressive effect on the in vitro polypoid growth of IEC6 expressing stabilized (DeltaN89) beta-catenin protein. Inhibitors of P-glycoprotein may be included in a novel class of chemopreventive agents against colorectal carcinogenesis.

Background: Several studies have shown the prognostic value of desmoplasia for lung adenocarcinomas. The authors evaluated the density and extent of desmoplasia by modifying the scar grade, as well as the prognostic impact on patient survival., Methods: Modified scar grade was defined as follows: Grade 1, no desmoplasia; Grade 2, sparse desmoplastic reaction; Grade 3, dense desmoplastic reaction with diameter of 10 mm or less; Grade 4, dense desmoplastic reaction with diameter exceeding 10 mm. In addition, the prognostic impact of conventional histologic factors and modified scar grade was analyzed in 239 cases of small peripheral lung adenocarcinoma (maximum dimension,

Purpose: Chromosome 3p is deleted frequently in various types of human cancers, including lung cancer. Recently, the RASSF1A gene was isolated from the 3p21.3 region homozygously deleted in lung and breast cancer cell lines, and it was shown to be inactivated by hypermethylation of the promoter region in lung cancers. In this study, we investigated the pathogenetic and clinicopathological significances of RASSF1A methylation in the development and/or progression of lung adenocarcinoma., Experimental Design: Association of RASSF1A methylation with clinicopathological features, allelic imbalance at 3p21.3, p53 mutations, and K-ras mutations was examined in 110 stage I lung adenocarcinomas., Results: Thirty-five of 110 (32%) tumors showed RASSF1A methylation. RASSF1A methylation was dominantly detected in tumors with vascular invasion (P = 0.0242) or pleural involvement (P = 0.0305), and was observed more frequently in poorly differentiated tumors than in well (P = 0.0005) or moderately (P = 0.0835) differentiated tumors. Furthermore, RASSF1A methylation correlated with adverse survival by univariate analysis (P = 0.0368; log-rank test) as well as multivariate analysis (P = 0.032,; risk ratio 2.357; 95% confidence interval, 1.075-5.169). The correlation between RASSF1A methylation and allelic imbalance at 3p21.3 was significant (P = 0.0005), whereas the correlation between RASSF1A methylation and p53 mutation was borderline (P = 0.0842). However, there was no correlation or inverse correlation between RASSF1A methylation and K-ras mutation (P = 0.2193)., Conclusions: These results indicated that epigenetic inactivation of RASSF1A plays an important role in the progression of lung adenocarcinoma, and that RASSF1A hypermethylation appears to be a useful molecular marker for the prognosis of patients with stage I lung adenocarcinoma.