Integrin-linked kinase (ILK) in pulmonary fibrosis

Pulmonary fibrosis is a common feature of a large group of lung diseases. The molecular mechanisms underlying pulmonary fibrosis and the key macromolecules involved are not fully understood yet. In an effort to better understand aspects of pulmonary fibrosis, the established bleomycin injection model in mice was used and the focus of the present study was on integrin-linked kinase (ILK) expression. ILK is an intracellular protein involved in the regulation of integrin-mediated processes. In fibrosis, ILK has been examined in the kidney and in the liver where it mediates epithelial to mesenchymal transition (EMT) and hepatic stellate cell activation, respectively. However, information on ILK’s involvement in lung fibrosis is missing. In order to examine ILK’s role in pulmonary fibrosis, we used both an in vivo and an in vitro approach. In vivo, the bleomycin model was used in order to examine ILK’s expression and localization in the fibrotic lung. In vitro, transforming growth factor-β1 was used to induce fibrotic characteristics and EMT in alveolar epithelial cells. ILK’s role in alveolar EMT was studied by siRNA. Our results demonstrate that in the animal model used, ILK exhibits a decrease in expression at early stages of the fibrotic process and that a specific subset of fibroblasts is expressing ILK. The in vitro experiments suggested that ILK is not directly involved in E-cadherin downregulation and initiation of EMT (as is the case in renal fibrosis) but is involved in upregulation of vimentin. These results suggest that ILK is involved in lung fibrosis in a tissue-specific manner and raise the possibility to use it as a specific therapeutic target for lung fibrosis in the future.     

Prognostic significance of immunohistochemical RhoA expression on survival in pancreatic ductal adenocarcinoma: a high-throughput analysis

Among all human carcinomas, pancreatic cancer has one of the worst survival rates. Most patients will die of this cancer shortly after diagnosis, and currently, surgery is the only potential cure. Ductal adenocarcinoma is the most common histologic type. The search for prognostic parameters has progressed from mere physical or histomorphological tumor properties to molecular parameters. These, in turn, might point toward new therapeutic strategies. The K-ras oncogene is known to play a role in early stages of ductal adenocarcinoma carcinogenesis, and ras homologues are differentially expressed in cancerous versus normal ductal cells. RhoA belongs to a family of ras homologues comprising RhoA, RhoB, and RhoC. It is a guanosine triphosphatase associated with the cytoskeleton that seems to be involved in epithelial mesenchymal transition, a process of dedifferentiation. Immunohistologic RhoA expression was studied in a tissue microarray of 94 pancreatic ductal adenocarcinomas and correlated with clinicopathologic parameters and follow-up. RhoA protein expression, measured as labeling intensity or evaluated as percentage of reactive tumor cells, correlated with overall survival. A multivariate analysis demonstrated that RhoA protein expression is independent from other known prognostic parameters such as tumor size or grade. Moreover, a score combining RhoA expression with tumor size and grade resulted in a highly significant increase in the prognostic value for the overall survival of patients with pancreatic ductal adenocarcinoma.     

Analysis of the E-cadherin repressor Snail in primary human cancers

Epithelial-mesenchymal transition (EMT), a normal developmental process, is known to play a crucial role in tumor progression. Molecules involved in this process, such as the E-cadherin repressor Snail, facilitate migration and invasion of carcinoma cells. A growing number of studies addressing the expression of Snail in clinical samples have been reported and are discussed in this review. A total of 2,112 cases from 9 different tumor types were evaluated. So far, a clear picture has emerged only in some cancer types analyzed with regard to overexpression of Snail and clinical-pathological parameters. Currently, it seems that Snail may play a role in hormone-dependent carcinomas but may be of minor importance in gastrointestinal cancers for tumor dedifferentiation and the maintenance of the invasive phenotype. It should be kept in mind, however, that the threshold for Snail activity does not have to be the same in every tumor type analyzed. The recent introduction of well-characterized novel monoclonal antibodies reacting with the short-lived nuclear Snail protein may help to establish a potential clinical usefulness for this master molecule of EMT, at least for certain types of cancer.     

Distribution of somatostatin in pancreatic ductal adenocarcinoma remodels the normal pattern of the protein during foetal pancreatic development: an immunohistochemical analysis

AIM: To determine the immunoreactivity of somatostatin during the development of the human fetal pancreas and pancreatic ductal adenocarcinoma, given that, somatostatin-positive cells were demonstrated either into its embryonic anlage or into pancreatic cancer. METHODS: Tissue sections from 15 pancreatic fetal specimens, and an equal number of ductal adenocarcinoma specimens were assessed. RESULTS: The density of positive cells in the primitive exocrine ductal epithelium and endocrine epithelium was significantly different from the relevant density in the neoplastic pancreatic tissue of mixed (ductal-endocrine) and pure ductal type (P1=0.021 P2=0.001, P3<0.0001, P4=0.003 respectively). The above values were estimated from the 8th to 10th week. There was no significant difference in the density of positive cells in the mantle zone of the islets from the 13th to the 24th week, and the neoplastic tissue of mixed (P5=0.16) and pure ductal type (P6=0.65). CONCLUSION: The immunostaining for somatostatin identifies a subgroup of pancreatic ductal adenocarcinomas with a neuroendocrine component, (initially considered as pure ductal tumors), and mixed ductal and neuroendocrine tumors. This pattern of expression in neoplasms recapitulates the normal pattern during the embryonal development of the organ, raising the question of therapeutic efficacy of somatostatin and analogues as monotherapy in pancreatic cancer management.     

Distribution of somatostatin in pancreatic ductal adenocarcinoma remodels the normal pattern of the protein during foetal pancreatic development: an immunohistochemical analysis

Abstract Aim: To determine the immunoreactivity of somatostatin during the development of the human fetal pancreas and pancreatic ductal adenocarcinoma, given that, somatostatin-positive cells were demonstrated either into its embryonic anlage or into pancreatic cancer. Methods: Tissue sections from 15 pancreatic fetal specimens, and an equal number of ductal adenocarcinoma specimens were assessed. Results: The density of positive cells in the primitive exocrine ductal epithelium and endocrine epithelium was significantly different from the relevant density in the neoplastic pancreatic tissue of mixed (ductal-endocrine) and pure ductal type (P1=0.021 P2=0.001, P3<0.0001, P4=0.003 respectively). The above values were estimated from the 8th to 10th week. There was no significant difference in the density of positive cells in the mantle zone of the islets from the 13th to the 24th week, and the neoplastic tissue of mixed (P5=0.16) and pure ductal type (P6=0.65). Conclusion: The immunostaining for somatostatin identifies a subgroup of pancreatic ductal adenocarcinomas with a neuroendocrine component, (initially considered as pure ductal tumors), and mixed ductal and neuroendocrine tumors. This pattern of expression in neoplasms recapitulates the normal pattern during the embryonal development of the organ, raising the question of therapeutic efficacy of somatostatin and analogues as monotherapy in pancreatic cancer management.     

Cyclooxygenase 2 expression in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia: an immunohistochemical analysis with automated cellular imaging

We immunohistochemically examined material from 36 pancreata (adenocarcinomas, 30 lesions; pancreatic intraepithelial neoplasia [PanIN], 65; normal pancreatic ducts, 30) for cyclooxygenase 2 (COX-2) with an automated platform. We analyzed 7 to 10 discrete foci and generated an average percentage of positive cells and average staining intensity for each lesion. These 2 values were then multiplied to create an overall "HistoScore" for each lesion. COX-2 demonstrated considerable heterogeneity of expression between and within cases. The overall average percentage of positive cells in adenocarcinomas was 47.3%; in PanINs, 36.3%; and in normal ducts, 19.2%. COX-2 was expressed in more than 20% of cells in 23 adenocarcinomas (77%), 42 PanINs (65%), and 12 normal ducts (40%). The overall average HistoScore for adenocarcinomas was 6.1; for PanINs, 5.4; and for normal ducts, 3.5. Significant differences in COX-2 expression were demonstrable in adenocarcinomas vs normal ducts, PanINs vs normal ducts, and PanIN 2/3 vs PanIN 1a/1b. In general, the pattern of COX-2 expression increased from normal to PanIN to adenocarcinoma. The up-regulation of COX-2 in a subset of noninvasive precursor lesions makes it a potential target for chemoprevention with selective COX-2 inhibitors.     

The ductal phenotypic expression of the E-cadherin/catenin complex in tubulolobular carcinoma of the breast: an immunohistochemical and clinicopathologic study.

Tubulolobular carcinoma is a type of mammary carcinoma that displays an admixture of invasive tubules and lobular-like cells. Previous reports have shown it to share clinical similarities to lobular carcinoma, whereas more recent studies have shown it to be E-cadherin positive. The aim of the current study was to further explore the immunophenotype of tubulolobular carcinoma, and to document its natural behavior. Nineteen cases of tubulolobular carcinoma and 10 cases each of tubular and lobular carcinoma were retrieved for comparison analysis. Immunohistochemistry was performed with antibodies against estrogen receptor, progesterone receptor, HER2/neu, 34betaE12, E-cadherin, and the catenins. Twenty-five percent of patients with tubulolobular carcinoma presented with greater than stage I disease, compared to 0 and 60% of patients with tubular and lobular carcinoma, respectively. Two patients with tubulolobular carcinoma had tumor recurrence, one of whom also developed metastasis. The majority of all carcinomas were estrogen and progesterone receptor positive. E-cadherin displayed membranous staining in all tubular and tubulolobular carcinomas, and was negative in all lobular carcinomas. Half of each carcinoma subtype displayed granular cytoplasmic 34betaE12 immunoreactivity. alpha-Catenin exhibited partial or complete membranous staining in all tubulolobular and tubular carcinomas, and was negative in all lobular carcinomas. beta-Catenin displayed membranous staining in tubulolobular and tubular carcinomas, whereas all lobular carcinomas had coarse cytoplasmic immunoreactivity. p120 and gamma-catenin displayed membranous staining in 100% of tubulolobular and tubular carcinomas and cytoplasmic staining in 100% of lobular carcinomas. Tubulolobular carcinoma of the breast is thus a distinct type of mammary carcinoma that displays both tubular and lobular patterns histologically but displays the membranous E-cadherin/catenin complex characteristic of the ductal immunophenotype. Tubulolobular carcinoma appears to be more aggressive than tubular carcinoma, as 16% of patients had lymph node metastases, although all were alive at a mean follow-up of 40 months.     

胰腺导管腺癌中CD74的表达及临床意义

目的 检测CD74蛋白在胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)组织中的表达并探讨其临床意义.方法 收集59例PDAC和23例慢性胰腺炎(chronic pancreatitis,CP)的存档石蜡包埋标本.HE染色、镜下观察神经纤维周围淋巴细胞病灶.免疫组化检测CD74的表达,统计分析其与PDAC临床病理参数的关系.结果 PDAC神经纤维周围淋巴细胞病灶发生率为67.8%.CD74在肿瘤细胞中阳性表达率为61%(36/59),明显高于CP导管上皮(13%,3/23,P=0.000).CD74表达与神经纤维淋巴细胞病灶密切相关(P=0.009),与其它临床病理参数无关.CD74阳性的PDAC患者术后生存时间明显短于CD74阴性患者(P=0.006 9).CD74在肿瘤细胞中的表达(P=0.009;rs=2.590)及Pt(P=0.022;rs=1.482)为影响PDAC患者预后的独立危险因素.结论 CD74在胰腺导管腺癌组织中高表达与神经纤维周围淋巴细胞病灶及肿瘤预后相关,可能成为PDAC预后相关的分子靶标.     

Clinicopathologic correlation of beclin-1 expression in pancreatic ductal adenocarcinoma

Beclin-1 has emerged as an autophagy gene in a variety of human carcinomas. The aim of this study was to evaluate beclin-1 expression and to determine its prognostic significance in patients with pancreatic ductal adenocarcinoma (PDA).We performed immunohistochemical staining for beclin-1 in 63 cases of PDA. We investigated whether beclin-1 expression correlated with clinicopathologic characteristics and patient outcomes.Beclin-1 expression was absent in normal pancreatic islet cells, acinar cells, and ductal epithelial cells. In contrast, in PDA, beclin-1 showed positive immunoreactivity in 14 of 63 (22.2%) PDA cases, while the remaining 49 (77.8%) PDA cases exhibited negative beclin-1 expression. We found significant associations between increased beclin-1 expression and the absence of lymphatic invasion (P = 0.032) and low rate of distant metastasis (P = 0.001). Univariate analysis of survival showed that the median distant metastasis-free survival of beclin-1-negative PDA patients (10 months) was significantly shorter than that of beclin-1-positive PDA patients (21 months; P = 0.031).Our results suggest that increased beclin-1 expression plays a role in the inhibition of PDA progression.     

Integrin-linked kinase (ILK) is required for polarizing the epiblast,cell adhesion,and controlling actin accumulation

Integrin-mediated cell-matrix interactions are essential for development, tissue homeostasis, and repair. Upon ligand binding, integrins are recruited into focal adhesions (FAs). Integrin-linked kinase (ILK) is an FA component that interacts with the cytoplasmic domains of integrins, recruits adaptor proteins that link integrins to the actin cytoskeleton, and phosphorylates the serine/threonine kinases PKB/Akt and GSK-3beta. Here we show that mice lacking ILK expression die at the peri-implantation stage because they fail to polarize their epiblast and to cavitate. The impaired epiblast polarization is associated with abnormal F-actin accumulation at sites of integrin attachments to the basement membrane (BM) zone. Likewise, ILK-deficient fibroblasts showed abnormal F-actin aggregates associated with impaired cell spreading and delayed formation of stress fibers and FAs. Finally, ILK-deficient fibroblasts have diminished proliferation rates. However, insulin or PDGF treatment did not impair phosphorylation of PKB/Akt and GSK-3beta, indicating that the proliferation defect is not due to absent or reduced ILK-mediated phosphorylation of these substrates in vivo. Furthermore, expression of a mutant ILK lacking kinase activity and/or paxillin binding in ILK-deficient fibroblasts can rescue cell spreading, F-actin organization, FA formation, and proliferation. Altogether these data show that mammalian ILK modulates actin rearrangements at integrin-adhesion sites.     

Over-expression of integrin-linked kinase correlates with aberrant expression of Snail, E-cadherin and N-cadherin in oral squamous cell carcinoma: implications in tumor progression and metastasis

Integrin-linked kinase (ILK), an intracellular protein with serine/threonine protein kinase activities, plays a key role in integrin mediated cell-excellular matrix interactions, regulating cell proliferation, apoptosis, differentiation, and migration. ILK has been implicated in the development and progression in several malignancies. However, the role of ILK and ILK-mediated epithelial?Cmensenchymal transition (EMT) in the progression of oral squamous cell carcinoma (OSCC) has not been well understood. Here, by immunohistochemistry, we studied the expression of ILK, Snail, E-cadherin and N-cadherin in 98 primary OSCC specimens and analyzed their correlations with clinicopathologic profiles and clinical outcome. We also investigated the expression of ILK in 42 corresponding lymph node metastases. Positive expression of ILK protein was detected in 87.8?% of the primary tumors and 100?% of metastatic lesions. Increased ILK expression was correlated strongly with enhanced tumor invasion, higher tumor grade, advanced clinical stage, positive lymph node status and increased risk of recurrence. Higher ILK expression was also observed in lymph node metastases in comparison with the corresponding primary tumor. Moreover, up-regulation of Snail and N-cadherin and down-regulation of E-cadherin correlated significantly with both ILK over-expression and tumor invasion. Patients with higher ILK expression exhibited shorter disease-free survival while those with absent E-cadherin expression exhibited shorter overall and disease-free survival. Taken together, our results suggest that ILK may have an important role in progression and metastasis of OSCC, possibly through EMT involving up-regulation of Snail and consequent aberrant expression of E-cadherin and N-cadherin. ILK should be considered as a critical prognostic indicator for patients with OSCC.     

Cyclin D1 expression in ductal carcinoma in situ, atypical ductal hyperplasia and usual ductal hyperplasia: an immunohistochemical study

The cell cycle regulatory gene, Cyclin D1, plays a critical role in the growth and progression of several types of human cancer, including breast cancer. Immunohistochemical study of Cyclin D1 expression has been extensively reported in invasive ductal carcinoma (IDC). In contrast, there have been few reports concerning Cyclin D1 expression in ductal carcinoma in situ (DCIS) and their positive rates are variable. The differences in the reported frequency may be largely due to the differences in antibodies used, immunohistochemical methods and the positive cut-off point. However, we speculated that the strictness of diagnosis of DCIS might be somewhat responsible for these differences in frequency. Therefore, we selected cases of DCIS by carefully eliminating cases of predominantly intraductal carcinoma (PIC). Moreover, to clarify whether Cyclin D1 expression is involved in multistep carcinogenesis or the progression of human breast cancer, we immunohistochemically investigated Cyclin D1 expression in 57 DCIS, 10 atypical ductal hyperplasia (ADH), 70 usual ductal hyperplasia (UDH), 44 PIC and 92 IDC. Cyclin D1 expression was detected in 41 DCIS cases (72%), 22 PIC cases (50%) and 40 IDC cases (43%). No expression of Cyclin D1 was observed in either ADH or UDH. There were no significant correlations between Cyclin D1 expression and histological grade or estrogen receptor expression in DCIS. These results suggest that Cyclin D1 expression may play an important role in the early stages of carcinogenesis, and that immunohistochemical detection of Cyclin D1 expression may be helpful in differentiating low-grade DCIS from ADH.     

Yes-associated protein (YAP65) in relation to Smad7 expression in human pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by multiple alterations in the TGF-beta signaling pathway. Yes-associated protein (YAP65) interacts with Smad7 thereby influencing TGF-beta signaling. In the present study, the expression of YAP65 in PDAC was analyzed in order to elucidate the potential role of this molecule in the pathogenesis of pancreatic cancer. YAP65 mRNA expression levels in human pancreatic tissue samples and cell lines were analyzed by Northern blotting and quantitative RT-PCR. Immunohistochemistry was carried out to localize and quantify YAP65 expression in relation to Smad7 expression and Smad4 mutations. The effects of TGF-beta1 on Smad7 and YAP65 mRNA expression were analyzed by quantitative RT-PCR. Enhanced expression of YAP65 mRNA was identified by Northern blotting and quantitative RT-PCR in PDAC in comparison to the normal pancreas (2.5-fold increase) and to chronic pancreatitis (1.3-fold increase). In the normal pancreas, YAP65 was absent in acinar cells, large ducts and islet cells, but exhibited moderate to strong immunoreactivity in centroacinar cells and ductules. Tubular complexes in CP and CP-like lesions in PDAC also exhibited strong staining. In contrast, weak to moderate YAP65 immunoreactivity was present in the cancer cells. There was no correlation between YAP65 immunostaining and Smad7 staining or Smad4 mutations in the cancer samples. TGF-beta1 strongly induced Smad7 mRNA in Colo-357 and in Panc-1 cells, but only slightly induced YAP65 mRNA in Colo-357 cells. In conclusion, YAP65 is expressed mainly in centroacinar and small ductal cells in the normal pancreas. In PDAC, YAP65 is present in tubular complexes and to a lesser extent in cancer cells. Together with the known function of YAP65 in different growth and differentiation regulating pathways, it is suggested that this gene plays a role in the normal and diseased pancreas.