Roles of DKK3 in cellular adhesion, motility, and invasion through extracellular interaction with TGFBI.

Dickkopf-related protein (DKK) 3, a member of the DKK family, is a secreted glycoprotein that acts as a modulator of Wnt signaling in organogenesis and carcinogenesis. Recent studies have demonstrated that DKK3 has a variety of functions, suggesting that it plays roles not only in tumorigenesis, but also tumor neovascularization, prostatic acinus formation, cardiac vascular remodeling, renal and cardiac fibrosis, and immunological activity. The function of DKK3 is therefore of great interest, but details of the receptors and mechanisms involved have remained unclear. Here, we focused on the extracellular function of DKK3 as a secreted protein, and identified transforming growth factor beta induced protein ig-h3 (TGFBI) as a secreted protein interacting with DKK3. To investigate the function of these secreted proteins, we employed an in vitro cell model involving human hepatocellular carcinoma cells, human embryonic kidney cells, or non-neoplastic hepatocyte cells. We showed that DKK3 functions as an extracellular matrix-like molecule supporting adhesion, motility, and invasion, and that its interaction with TGFBI inhibits the functions of secreted DKK3 in cells expressing both proteins. These results suggest that this extracellular interaction between DKK3 and TGFBI modulates cell adhesion and motility through focal adhesion kinase signaling, and that this might serve as a potential therapeutic target in the context of cancer invasion and metastasis.

High expression of Ras-specific guanine nucleotide-releasing factor 2 (RasGRF2) in lung adenocarcinoma is associated with tumor invasion and poor prognosis.

The expression of Ras-specific guanine nucleotide-releasing factor 2 (RasGRF2) in lung adenocarcinomas was examined using immunohistochemistry in relation to clinicopathological characteristics and prognosis. In comparison to low expression, high expression of RasGRF2 was more closely associated with poor prognosis. Interestingly, expression of phosphorylated epithelial cell transforming 2 (pECT2), which - like RasGRF2 - is also a guanine-nucleotide exchange factor, was also associated with prognosis, and patients with high expression of both RasGRF2 and pECT2 had a much poorer outcome than those who were negative for both.

Mechanomics Biomarker for Cancer Cells Unidentifiable through Morphology and Elastic Modulus.

Cellular mechanical properties are potential cancer biomarkers used for objective cytology to replace the current subjective method relying on cytomorphology. However, heterogeneity among intra/intercellular mechanics and the interplay between cytoskeletal prestress and elastic modulus obscured the difference detectable between malignant and benign cells. In this work, we collected high density nanoscale prestress and elastic modulus data from a single cell by AFM indentation to generate a cellular mechanome. Such high dimensional mechanome data was used to train a malignancy classifier through machine learning. The classifier was tested on 340 single cells of various origins, malignancy, and degrees of similarity in morphology and elastic modulus. The classifier showed instrument-independent robustness and classification accuracy of 89% with an AUC-ROC value of 93%. A signal-to-noise ratio 8 times that of the human-cytologist-based morphological method was also demonstrated, in differentiating precancerous hyperplasia cells from normal cells derived from the same lung cancer patient.

Dickkopf-related protein 3 promotes cell adhesion and invasion during progression of lung adenocarcinoma.

Dickkopf-related protein 3 (DKK3) is one of the DKK family (DKK1-4), an evolutionally conserved group of secreted glycoproteins characterized by two distinct cysteine-rich domains. DKK3 is considered to be a tumor suppressor gene. However, it has been shown that 30-50% of various cancers are DKK3-positive, suggesting that DKK3 may have an additional function other than tumor suppression. In this study, we focused on lung adenocarcinoma, which is the major histological type of lung cancer. We analyzed the relationship between DKK3 expression and clinicopathological features by immunohistochemistry, using 200 lung adenocarcinoma specimens. We found that 40.5% and 59.5% of cases were DKK3-positive and -negative, respectively, and that positive cases had a greater tendency for progression than negative cases (P < 0.05). Furthermore, in vitro analyses demonstrated that DKK3 suppression affected cell adhesion in three DKK3-expressing lung adenocarcinoma cell lines and that DKK3-knockdown cells were less invasive in comparison to control cells. These results suggest that DKK3 plays a role in the progression of lung adenocarcinoma by promoting cell adhesion and invasion. DKK3 might be a new extracellular cancer therapeutic target, and it seems important to clarify molecular mechanisms underlying the DKK3 functions depending on cell context.

Cytoplasmic expression of epithelial cell transforming sequence 2 in lung adenocarcinoma and its implications for malignant progression.

Epithelial cell transforming sequence 2 (ECT2), a guanine nucleotide exchange factor, is predominantly localized in the nucleus of non-transformed cells and functions to regulate cytokinesis. ECT2 is also localized in the cytoplasm of cancer cells. Aberrant cytoplasmic expression of ECT2 is thought to drive tumor growth and invasion. In this study, we investigated the cytoplasmic expression of ECT2 and its prognostic and biological significance in lung adenocarcinoma. Western blotting of cellular fractions from the nucleus and cytoplasm was performed to determine the subcellular localization of ECT2 in lung adenocarcinoma cell lines. The cytoplasmic expression of ECT2 in 167 lung adenocarcinomas was evaluated by immunohistochemistry and its clinical significance was examined using Kaplan-Meier curves and Cox regression analysis. Scraping cytology specimens of 13 fresh lung adenocarcinomas were used to assess the subcellular localization of ECT2 and its phosphorylation at Thr790 (P-ECT2(T790)). We found that ECT2 was localized in both the nucleus and cytoplasm of lung adenocarcinoma cell lines and tumor tissues. Cytoplasmic expression of ECT2 was detected by immunohistochemistry in 83 (50%) of the lung adenocarcinomas, and was found to increase during cancer progression. It was expressed in 30 (29%) small adenocarcinomas ( ≤ 2 cm in diameter) and 53 (82%) advanced adenocarcinomas ( > 2 cm in diameter). Cytoplasmic positivity for ECT2 was associated with a poor outcome in terms of both disease-free and overall survival (both P < 0.001), and was an independent prognostic factor for overall survival (P = 0.025). Immunocytochemical staining for P-ECT2(T790) demonstrated cytoplasmic and membrane positivity in Calu-3 cells and scraping cytology specimens. Positive P-ECT2(T790) staining was correlated with cytoplasmic ECT2 expression in 6 of 13 scraped cytology specimens tested. In conclusion, our findings indicate that cytoplasmic ECT2 expression could promote the malignant progression of lung adenocarcinoma and may represent a potent therapeutic target for patients with lung adenocarcinoma.

Meridianin C inhibits the growth of YD-10B human tongue cancer cells through macropinocytosis and the down-regulation of Dickkopf-related protein-3.

Meridianin C is a marine natural product known for its anti-cancer activity. At present, the anti-tumour effects of meridianin C on oral squamous cell carcinoma are unknown. Here, we investigated the effect of meridianin C on the proliferation of four different human tongue cancer cells, YD-8, YD-10B, YD-38 and HSC-3. Among the cells tested, meridianin C most strongly reduced the growth of YD-10B cells; the most aggressive and tumorigenic of the cell lines tested. Strikingly, meridianin C induced a significant accumulation of macropinosomes in the YD-10B cells; confirmed by the microscopic and TEM analysis as well as the entry of FITC-dextran, which was sensitive to the macropinocytosis inhibitor amiloride. SEM data also revealed abundant long and thin membrane extensions that resemble lamellipodia on the surface of YD-10B cells treated with meridianin C, pointing out that meridianin C-induced macropinosomes was the result of macropinocytosis. In addition, meridianin C reduced cellular levels of Dickkopf-related protein-3 (DKK-3), a known negative regulator of macropinocytosis. A role for DKK-3 in regulating macropinocytosis in the YD-10B cells was confirmed by siRNA knockdown of endogenous DKK-3, which led to a partial accumulation of vacuoles and a reduction in cell proliferation, and by exogenous DKK-3 overexpression, which resulted in a considerable inhibition of the meridianin C-induced vacuole formation and decrease in cell survival. In summary, this is the first study reporting meridianin C has novel anti-proliferative effects via macropinocytosis in the highly tumorigenic YD-10B cell line and the effects are mediated in part through down-regulation of DKK-3.

Dickkopf 3 attenuates xanthine dehydrogenase expression to prevent oxidative stress-induced apoptosis.

Dickkopf (DKK) 3 is a DKK glycoprotein family member that controls cell fate during embryogenesis and exerts opposing effects on survival in a cell type-dependent manner; however, the mechanisms governing its pro-apoptosis versus pro-survival functions remain unclear. Here, we investigated DKK3 function in Li21 hepatoma cells and tPH5CH immortalized hepatocytes. DKK3 knockdown by siRNA resulted in reactive oxygen species accumulation and subsequent apoptosis, which were abrogated by administration of the antioxidant N-acetyl-cysteine. Moreover, forced DKK3 over-expression induced resistance to hydrogen peroxide (H2 O2 )-induced apoptosis. Expression analysis by cDNA microarray showed that xanthine dehydrogenase (XDH) expression was significantly lower in Li21 and tPH5CHDKK3-over-expressing cells in response to H2 O2 treatment when compared to that in their respective mock-transfected controls, whereas a marked increase was observed in H2 O2 -treated DKK3 knockdown cells. Thus, these data suggest that DKK3 promotes cell survival during oxidative stress by suppressing the expression of the superoxide-producing enzyme XDH.

miR-3941: A novel microRNA that controls IGBP1 expression and is associated with malignant progression of lung adenocarcinoma.

Immunoglobulin (CD79a) binding protein 1 (IGBP1) is universally overexpressed in lung adenocarcinoma and exerts an anti-apoptotic effect by binding to PP2Ac. However, the molecular mechanism of IGBP1 overexpression is still unclear. In the present study, we used a microRNA (miRNA) array and TargetScan Human software to detect IGBP1-related miRNAs that regulate IGBP1 expression. The miRNA array analysis revealed more than 100 miRNAs that are dysregulated in early invasive adenocarcinoma. On the other hand, in silico analysis using TargetScan Human revealed 79 miRNAs that are associated with IGBP1 protein expression. Among the miRNAs selected by miRNA array analysis, six (miR-34b, miR-138, miR-374a, miR-374b, miR-1909, miR-3941) were also included among those selected by TargetScan analysis. Real-time reverse transcription PCR (real-time RT-PCR) showed that the six microRNAs were downregulated in invasive adenocarcinoma (IGBP1+) relative to adjacent normal lung tissue (IGBP1-). Among these microRNAs, only miR-34b and miR-3941 depressed luciferase activity by targeting 3'UTR-IGBP1 in the luciferase vector. We transfected miR-34b and miR-3941 into lung adenocarcinoma cell lines (A549, PC-9), and both of them suppressed IGBP1 expression and cell proliferation. Moreover, the transfected miR-34b and miR-3941 induced apoptosis of a lung adenocarcinoma cell line, similarly to the effect of siIGBP1 RNA. As well as miR-34b, we found that miR-3941 targeted IGBP1 specifically and was able to exclusively downregulate IGBP1 expression. These findings indicate that suppression of miR-3941 has an important role in the progression of lung adenocarcinoma at an early stage.

DNMT3a expression pattern and its prognostic value in lung adenocarcinoma.

OBJECTIVES: DNA methyltransferases (DNMTs) are an important part of the methylation pathway that is highly correlated with the pathophysiology of cancers. Several studies have reported overexpression of DNMTs in human lung cancer, but none have compared the expression pattern to pathological features. In this study, we clarified the association of DNMT3a expression pattern with pathological features and prognosis of lung adenocarcinoma. MATERIALS AND METHODS: 135 cases of surgically resected lung adenocarcinoma specimens were used for DNMT3a immunohistochemistry (IHC). IHC score was determined by counting the number of positive nuclei. The ROC curve was drawn to determine the best cut-off point of the score; this was set at 57.5. Western blot also implemented and confirmed the specificity of the antibody. Correlations between expression pattern and clinicopathological features and prognosis were analyzed using chi-squared method and Cox proportional hazards model respectively. RESULT: Seventy-nine of the 135 cases (58.5%) showed strong positive reactivity to anti-DNMT3a. In terms of histological subtypes, among invasive lung adenocarcinomas 41 out of 53 lepidic adenocarcinomas (77%) were strongly positive, while among the other histological subtypes only 23 out of 66 cases (34.8%) showed a positive reaction. Among non-invasive lung adenocarcinomas 15 out of 16 cases (93.8%) were strongly positive. The level of DNMT3a expression was associated with patient outcome, and patients with weak expression of DNMT3a had a poorer outcome than those with strong expression. Multivariate analysis also indicated that DNMT3a is an independent prognostic marker in lung adenocarcinoma. CONCLUSION: Our results indicate that DNMT3a expression in lung adenocarcinoma is associated with the histologically non-invasive type and lepidic subtype, and a favorable prognosis. We also showed that DNMT3a expression is an independent prognostic marker in lung adenocarcinoma. Since lack of DNMT3a is thought to facilitate tumor progression, DNMT3a might be clinically applicable as an indicator of favorable prognosis.

Stratifin accelerates progression of lung adenocarcinoma at an early stage.

BACKGROUNDS: Adenocarcinoma in situ (AIS) of the lung has an extremely favorable prognosis. However, early but invasive adenocarcinoma (eIA) sometimes has a fatal outcome. We had previously compared the expression profiles of AIS with those of eIA showing lymph node metastasis or a fatal outcome, and found that stratifin (SFN, 14-3-3 sigma) was a differentially expressed gene related to cell proliferation. Here, we performed an in vivo study to clarify the role of SFN in initiation and progression of lung adenocarcinoma. FINDINGS: Suppression of SFN expression in A549 (a human lung adenocarcinoma cell line) by siSFN significantly reduced cell proliferation activity and the S-phase subpopulation. In vivo, tumor development or metastasis to the lung was reduced in shSFN-transfected A549 cells. Moreover, we generated SFN-transgenic mice (Tg-SPC-SFN(+/-)) showing lung-specific expression of human SFN under the control of a tissue-specific enhancer, the SPC promoter. We found that Tg-SPC-SFN(+/-) mice developed lung tumors at a significantly higher rate than control mice after administration of chemical carcinogen, NNK. Interestingly, several Tg-SPC-SFN(+/-) mice developed tumors without NNK. These tumor cells showed high hSFN expression. CONCLUSION: These results suggest that SFN facilitates lung tumor development and progression. SFN appears to be a novel oncogene with potential as a therapeutic target.

Moesin overexpression is a unique biomarker of adenomyosis.

Adenomyosis is characterized by extension of endometrial glands and stromal cells into the myometrium. Here we proved that 'moesin' is a unique biomarker of adenomyosis. We selected two cases of adenomyosis that had been surgically resected and fixed with formalin. Proteins were extracted from the infiltrating adenomyosis lesions and normal endometrium by tissue microdissection. The extracted proteins were examined using a LC-MS/MS system and the expression profiles of each region were compared. Two hundred and sixty proteins were detected, among which 73 were expressed more in adenomyosis than in normal endometrium. Among these proteins, we focused on overexpression of moesin in adenomyosis. Expression of moesin estimated semiquantitatively using an immunohistochemistry score was higher in adenomyosis than in normal endometrium. In particular, moesin was significanly overexpressed in stromal cells of adenomyosis than in those of normal endometrium. Relative to normal endometrium, moesin was also overexpressed at the RNA level in 9 of 14 cases of adenomyosis and at the protein level in all 14 cases. We also detected activated (phosphorylated) moesin in adenomyosis lesions. The present findings suggest that moesin is characteristically overexpressed and activated in adenomyosis, and that moesin activation may be related to extension of adenomyosis in the myometrium.

PRKAR1A overexpression is associated with increased ECPKA autoantibody in liver fluke-associated cholangiocarcinoma: application for assessment of the risk group.

Cholangiocarcinoma (CCA) associated with Opisthorchis viverrini (Ov) chronic infection is the most frequent primary liver cancer in Thailand, and current approaches to early diagnosis and curative treatments are largely disappointing. We hypothesize a role for protein kinase A (PKA) in Ov-induced CCA. First, we studied the PKA isozyme switching in the liver from the hamster CCA model using quantitative (q) PCR, in situ hybridization, and immunohistochemical and western blot analysis. Second, the presence of extracellular PKA (ECPKA) in CCA cell lines and their conditioned media was demonstrated by western blot and PKA activity assay. Third, we determined the association between PRKAR1A expression and serum ECPKA autoantibody in patients with CCA by ELISA. We demonstrated that an increased PRKAR1A expression is restricted to the biliary cells starting from week 1, with remarkable up-regulation when CCA has completely developed by week 24. The switching of the PKA regulatory subunit isoforms from PRKAR2B/PKAII to PRKAR1A/PKAI is significantly associated with cholangiocyte proliferation. Further, we observed that human CCA cell lines express PRKAR1A but not PRKAR2B and excrete ECPKA. Finally, ECPKA autoantibodies are detected in serum of patients with CCA, adenocarcinoma, and Ov infection with periductal fibrosis, but not from Ov-infected subjects without periductal fibrosis lesion and healthy controls. We conclude that PKA isozyme switching and the PRKAR1A/PKAI pathway might contribute to the induction of cholangiocyte transformation and proliferation in Ov-induced CCA. Overexpression of PRKAR1A leads to secretion of ECPKA which is associated with serum autoantibody that may constitute a biomarker for human CCA genesis.

Abnormality of the hepatocyte growth factor/MET pathway in pulmonary adenocarcinogenesis.

BACKGROUND: Signaling mediated by hepatocyte growth factor (HGF)/MET promotes multiple biological activities, including cell proliferation, motility, invasion, angiogenesis, and morphogenesis. Overexpression of HGF and MET and an increase of the MET gene copy number have recently been found in various cancers that had a poor outcome. Here we investigated the copy number of the MET gene and expression of MET and HGF in small pulmonary adenocarcinomas. METHODS: Tumor tissues were obtained from 106 pulmonary small adenocarcinomas 2 cm or less in diameter. MET gene copy number, and the expression of MET and HGF, were analyzed using fluorescence in situ hybridization (FISH) and immunohistochemistry, respectively. RESULTS: MET FISH-positive signals were observed in 11 (10.4%) of 106 cases. One case (0.9%) showed gene amplification and 10 (9.4%) exhibited high polysomy. High immunoreactivity for MET and HGF in tumor cells was found in 30 (28.3%) and 19 cases (17.9%), respectively. HGF was also expressed in stromal cells in 32 cases (30.2%). No cases of non-invasive adenocarcinoma (adenocarcinoma in situ, localized bronchioloalveolar carcinoma) showed MET FISH-positive signals or high expression of HGF in the tumor cells. Expression of both MET and stromal HGF was stronger in invasive than in non-invasive adenocarcinoma. MET FISH-positive signals and high immunoreactivity for MET and HGF in tumor cells were associated with factors indicative of poor prognosis such as pleural invasion, vascular invasion, lymphatic permeation, lymph node metastasis, and nuclear grading. Univariate and multivariate analyses that included these factors showed that all statuses except for MET and HGF immunoreactivity were significantly associated with an increased risk of death. However, multivariate analysis revealed no independent factors related to poor prognosis. CONCLUSION: Our results suggest that abnormality of the HGF/MET pathway occurs during the course of progression from non-invasive to invasive pulmonary adenocarcinoma. An increased MET gene copy number is indicative of a poor outcome in patients with small pulmonary adenocarcinomas.

IGFBP-1 is expressed specifically in ovarian clear cell adenocarcinoma.

AIMS: To investigate the specific expression of insulin-like growth factor binding protein-1 (IGFBP-1) in ovarian clear cell adenocarcinoma (CCA). METHODS AND RESULTS: Immunohistochemistry and in situ hybridization for IGFBP-1 were performed in normal endometrium, placenta, and 100 surgically resected cases of ovarian cancer including 31 CCAs and 69 non-CCAs. Immunohistochemistry for hepatocyte nuclear factor-1 (HNF-1ß) was also examined in all cases. Specific expression of IGFBP-1 was confirmed in secretory endometrium, decidua of placenta and Arias-Stella glands of miscarriage material. Among ovarian cancers, almost all cases of CCA showed expression of both IGFBP-1 protein and mRNA, but non-CCA hardly expressed IGFBP-1. There was a significant difference between CCA and non-CCA in the expression of IGFBP-1 protein and mRNA. No correlation was found between the rate of IGFBP-1 expression and pathological T and N factors of the tumour-node-metastasis (TNM) classification. All CCA cases except for one exhibited expression of HNF-1ß protein, whereas only 15.9% of non-CCAs did so. CONCLUSION: The expression of IGFBP-1 in CCA is more specific than that of HNF-1ß. IGFBP-1 shows expression by decidual endometrium and Arias-Stella glands, and CCA also exhibits characteristic expression. These results indicate that IGFBP-1 is a immunohistochemical marker for CCA.

Binding of lactoferrin to IGBP1 triggers apoptosis in a lung adenocarcinoma cell line.

BACKGROUND: Lactoferrin (Lf), an iron-binding protein present in mammalian secretions, plays important roles in cancer prevention by inducing apoptosis. MATERIALS AND METHODS: PC-14 lung adenocarcinoma cells were exposed to bovine Lf (bLf) protein and the expression of caspase-3 and apoptosis protease-activating factor-1 (APAF-1) was assessed. To investigate the molecular mechanism of apoptosis induced by bLf, a major Lf-binding protein was screened using a protein microarray with bLf protein as the probe. Protein interaction was demonstrated by co-immunoprecipitation, immunofluorescence and phosphatase activity assay. RESULTS: Lf directly suppressed the proliferation of the PC-14 cells by triggering their apoptosis. Lf was shown to bind specifically with a 36-kDa protein, immunoglobulin (CD79A)-binding protein 1 (IGBP1). The binding complex interacted with the catalytic subunit of protein phosphatase 2A (PP2A), thus reducing the phosphatase activity of PP2A and triggering apoptosis. CONCLUSION: Lf binds IGBP1 and promotes the acceleration of cellular apoptosis.

High expression of stratifin is a universal abnormality during the course of malignant progression of early-stage lung adenocarcinoma.

Adenocarcinoma in situ (AIS) of the lung has an extremely favorable prognosis, with a 5-year survival rate of 100%. However, early invasive adenocarcinoma (EIA) often has a fatal outcome. In this study, we compared the expression profiles of AIS with those of EIA showing lymph node metastasis or a fatal outcome, and screened the differentially expressed genes by cDNA microarray. From the genes selected, we focused on Stratifin (SFN, 14-3-3 σ), which showed significantly higher expression in EIA than in AIS. Immunohistochemistry for SFN revealed that more than 95% of EIAs were immunopositive for SFN, in comparison to only 13% of AISs (p <0.05). Interestingly, positivity was detected not only in the invasive region but also in the in situ spreading component of EIA. Functionally, SFN facilitates the cell proliferation capacity of lung adenocarcinoma. These results indicate that SFN overexpression is a universal abnormality during the stepwise progression from in situ to invasive adenocarcinoma of the lung.

APAF-1 is related to an undifferentiated state in the testicular germ cell tumor pathway.

Apoptotic protease activating factor-1 (APAF-1) is a key regulator gene of apoptosis, located downstream from p53. Loss of APAF-1 expression is associated with chemorefractory malignant melanoma and neuronal cell differentiation. In order to make clear the function of APAF-1 in the carcinogenesis of germ cell tumors, we evaluated the expression levels of APAF-1 and several apoptosis and differentiation markers by immunohistochemistry in formalin-fixed paraffin-embedded samples from 43 cases of testicular germ cell tumor (TGCT) and six specimens of normal testis tissue. Expression of cleaved caspase-3, Oct-3/4, and Ki-67 were also examined by immunohistochemistry to evaluate apoptotic reactivity, tumor differentiation, and proliferation activity, respectively. APAF-1 was downregulated in two TGCT cell lines by siRNA transfection, and subsequent expression of the Ki-67 and Oct-3/4 genes and differentiation markers of three embryonic germ layers including keratin16 (KRT16) for ectoderm, vimentin (VIM) for mesoderm and GATA4 for endoderm were then tested. No significant relationship was found between APAF-1 expression and apoptotic activity in TGCTs. Expression of APAF-1, Oct-3/4, and Ki-67 was significantly higher in seminomas than in non-seminomas. In TGCTs, higher APAF-1 expression was correlated with higher proliferation (high Ki-67) and a lower degree of differentiation (high Oct-3/4). Interestingly, the expression of APAF-1 gradually decreased in accordance with tumor differentiation (seminoma and embryonal carcinoma > teratoma). Downregulation of APAF-1 in TGCT cell lines resulted in a decrease of Ki-67 and Oct-3/4 and an increase of VIM and KRT16 gene expression. These data show that higher expression of APAF-1 is related to an undifferentiated state in the TGCT pathway.

Expression of the GA733 gene family and its relationship to prognosis in pulmonary adenocarcinoma.

The GA733 gene family is composed of GA733-1 (TROP2) and GA733-2 (Ep-CAM), whose expression has been examined in various carcinomas and reported to be significantly associated with prognosis. The aim of this study was to investigate the expression of GA733 gene family members and to compare their prognostic significance in pulmonary adenocarcinoma. One hundred thirty paraffin-embedded specimens of small-sized pulmonary adenocarcinoma, less than 2 cm in diameter, were categorized using the classification of small-sized pulmonary adenocarcinoma devised by Noguchi et al. (Cancer 75:2844-2852, 1995) and examined immunohistochemically using a murine monoclonal antibody against Ep-CAM and a goat polyclonal antibody against TROP2. The patient survival rate was calculated using the Kaplan-Meier method. Ep-CAM and TROP2 were similarly expressed in many small-sized pulmonary adenocarcinomas. The expression of Ep-CAM was significantly related to a favorable outcome (p = 0.0185), whereas TROP2 tended to be expressed in cases with an unfavorable outcome (p = 0.0564), and was significantly associated with an unfavorable outcome in nonlepidic-type adenocarcinomas (p = 0.0125). Multivariate analysis showed that TROP2 overexpression and lymph node metastasis were independent prognostic markers. Although the two GA733 proteins share structural similarity, they appear to have opposite biological significances in small-sized adenocarcinomas. As the expression of TROP2 was detected in more poorly differentiated tumors, the protein may have oncogenic activity.

Increased cytoplasmic S100A6 expression is associated with pulmonary adenocarcinoma progression.

S100A6 is a calcium-binding protein implicated in many cellular processes and frequently upregulated in cancer. Recently it was reported that S100A6 is one of the genes having higher expression in adenocarcinoma mixed subtype with a bronchioloalveolar carcinoma (BAC) component than in pure BAC. To clarify the association of S100A6 expression with stepwise progression of lung adenocarcinoma, S100A6 protein expression was examined on immunohistochemistry in 92 formalin-fixed and paraffin-embedded lung adenocarcinomas. Both the nucleus and cytoplasm of the tumor cells were stained, and the nuclear and cytoplasmic expression of S100A6 was assessed individually. In addition, six frozen surgical specimens were selected, and the expression of S100A6 was confirmed on western blotting. As a result, although it was not possible to detect any significant correlation between nuclear S100A6 immunoreactivity and tumor progression, advanced adenocarcinoma had significantly higher cytoplasmic S100A6 expression than non-invasive lesions or normal lung tissue (P < 0.05). Moreover, the BAC component tended to have weaker staining than any of the other components. These findings indicate that S100A6 may be associated with the stepwise progression and/or invasion of lung adenocarcinoma, especially BAC-type adenocarcinoma. The present results suggest the utility of S100A6 immunohistochemistry as a marker for estimation of malignancy in adenocarcinoma with a BAC component.

Overexpression of Dickkopf 3 in hepatoblastomas and hepatocellular carcinomas.

Dickkopf 3 (Dkk3) is a protein expressed at a very early stage of hepatogenesis. In this study, we examined whether Dkk3 was related to a premature or dedifferentiated nature in hepatoblastomas (HBLs) and hepatocellular carcinomas (HCCs). It was demonstrated that Dkk3 was overexpressed in HBLs and HCCs and that its expression was more frequent in the former than in the latter, being consistent with the fact that most HBLs show an embryonal or fetal hepatic histology, whereas there was no distinct relationship between Dkk3 expression and clinical data or histology. All of the HBLs expressed Dkk3, alpha-fetoprotein (AFP), or both proteins, suggesting that, similar to AFP, Dkk3 is another potentially useful biomarker detecting a wide range of HBLs. Furthermore, Dkk3 and AFP were expressed reciprocally in the tumors. These results suggest that Dkk3 may be related to the premature or dedifferentiated nature of HBLs and HCCs, whereas AFP may be related to a more differentiated nature. Thus, assessment of Dkk3 and AFP may be useful in the diagnosis of hepatic tumors.