Doublecortin-Like Kinase 1 Is Elevated Serologically in Pancreatic Ductal Adenocarcinoma and Widely Expressed on Circulating Tumor Cells

Doublecortin-like kinase 1 (DCLK1) is a putative pancreatic stem cell marker and is upregulated in pancreatic cancer, colorectal cancer, and many other solid tumors. It marks tumor stem cells in mouse models of intestinal neoplasia. Here we sought to determine whether DCLK1 protein can be detected in the bloodstream and if its levels in archived serum samples could be quantitatively assessed in pancreatic cancer patients. DCLK1 specific ELISA, western blotting, and immunohistochemical analyses were used to determine expression levels in the serum and staining intensity in archived tumor tissues of pancreatic ductal adenocarcinoma (PDAC) patients and in pancreatic cancer mouse models. DCLK1 levels in the serum were elevated in early stages of PDAC (stages I and II) compared to healthy volunteers (normal controls). No differences were observed between stages III/IV and normal controls. In resected surgical tissues, DCLK1 expression intensity in the stromal cells was significantly higher than that observed in tumor epithelial cells. Circulating tumor cells were isolated from KPCY mice and approximately 52% of these cells were positive for Dclk1 staining. Dclk1 levels in the serum of KPC mice were also elevated. We have previously demonstrated that DCLK1 plays a potential role in regulating epithelial mesenchymal transition (EMT). Given the increasingly recognized role of EMT derived stem cells in cancer progression and metastasis, we hypothesize that DCLK1 may contribute to the metastatic process. Taken together, our results suggest that DCLK1 serum levels and DCLK1 positive circulating tumor cells should be further assessed for their potential diagnostic and prognostic significance.     

Characterization of proteins in human pancreatic cancer serum using differential gel electrophoresis and tandem mass spectrometry

The purpose of this study was to develop techniques for identifying cancer biomarkers in human serum using differential in-gel electrophoresis (DIGE), and characterizing the protein biomarkers using tandem mass spectrometry (MS/MS). A major problem in profiling protein expression by DIGE comes from the presence of high concentrations of a small number of proteins. Therefore, serum samples were first chromatographed using an immunoaffinity HPLC column (Agilent Technologies), to selectively remove albumin, immunoglobulins, transferrin, haptoglobin, and antitrypsin. Serum samples from three individuals with pancreatic cancer and three individuals without cancer were compared. Serum samples were processed using the immunoaffinity column. Differential protein analysis was performed using DIGE. A total of 56 protein spot-features were found to be significantly increased and 43 significantly decreased in cancer serum samples. These spot features were excised, trypsin digested, and analyzed by MALDI/TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems). We identified 24 unique proteins that were increased and 17 unique proteins that were decreased in cancer serum samples. Western blot analysis confirmed increased levels of several of these proteins in the pancreatic cancer serum samples. In an independent series of serum samples from 20 patients with pancreatic cancer and 14 controls, increased levels of apolipoprotein E, alpha-1-antichymotrypsin, and inter-alpha-trypsin inhibitor were found to be associated with pancreatic cancer. These results suggest that affinity column enrichment and 2-D DIGE can be used to identify numerous proteins differentially expressed in serum from individuals with pancreatic cancer.     

Expression and prognostic role of SKIP in human breast carcinoma

Ski-interacting protein (SKIP) is a nuclear hormone receptor-interacting cofactor, interactions with the proto-oncogene Ski, appears to modulate a number of signalling pathways involved in control of cell proliferation and differentiation, and may play a critical role in oncogenesis. In the present study, to investigate the potential roles of SKIP in breast cancer, expression patterns, interaction and the correlation with clinical/prognostic factors of SKIP and Ki-67 were examined among patients with breast cancer. Immunohistochemistry and Western blot analysis were performed for SKIP in 85 breast carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine their prognostic significance. We found that SKIP was over expressed in breast carcinoma as compared with the adjacent normal tissues. High expression of SKIP was positively associated with histological grade (P = 0.01) and Ki-67 (P = 0.004). Univariate analysis showed that SKIP expression was associated with a poor prognosis (P = 0.006). While in vitro, following release of breast cancer cell lines from serum starvation, the expression of SKIP was up-regulated, whereas p27 was down-regulated. In addition, we employed small interfering RNA (siRNA) technique to knock down SKIP expression and observed it effects on MDA-MB-231 cells growth. SKIP depletion by siRNA inhibited cell proliferation, blocked S phase and decreased cyclin A and cyclin B levels. On the basis of these results, we suggested that SKIP overexpression was involved in the pathogenesis of breast cancer, which might serve as a future target for breast cancer.     

Application of laser capture microdissection combined with two-dimensional electrophoresis for the discovery of differentially regulated proteins in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal of all the common malignancies and markers for early detection or targets for treatment of this disease are urgently required. The disease is characterised by a strong stromal response, with cancer cells usually representing a relatively small proportion of the cells in the tumor mass. We therefore performed laser capture microdissection (LCM) to enrich for both normal and malignant pancreatic ductal epithelial cells. Proteins extracted from these cells were then separated by two-dimensional gel electrophoresis (2-DE). The limited amounts of protein in the LCM procured samples necessitated the detection of 2-DE resolved proteins by silver staining. Consequently, loading equivalent amounts of protein onto gels was essential. However, we found that conventional means of measuring total protein in the samples were not sufficiently accurate. We therefore adopted a strategy in which the samples were first separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis, stained with silver stain and subjected to densitometry. Evaluation of the staining intensity was then used to normalise the samples. We found that the protein profiles from undissected normal pancreas and LCM-acquired non-malignant ductal epithelial cells from the same tissue block were different, underpinning the value of LCM in our analysis. The comparisons of protein profiles from nonmalignant and malignant ductal epithelial cells revealed nine protein spots that were consistently differentially regulated. Five of these proteins showed increased expression in tumor cells while four showed diminished expression in these cells. One of the proteins displaying enhanced expression in tumor cells was identified as the calcium-binding protein, S100A6. To determine the incidence of S100A6 overexpression in pancreatic cancer, we carried out immunohistochemical analysis on sections from a pancreas cancer tissue array containing 174 duplicate normal and malignant pancreatic tissue samples, from 46 pancreas cancer patients. Normal pancreatic ductal epithelia were either devoid of detectable S100A6 or showed weak expression only. Moderately or poorly differentiated tumors, by contrast, showed a higher incidence and a higher level of S100A6 expression. These observations indicate that the combination of LCM with 2-DE provides an effective strategy to discover proteins that are differentially expressed in PDAC.     

MHCI链相关分子A／B在胰腺癌组织中的表达及临床意义

目的：探讨MHCI链相关分子A／B（MICA／B）在胰腺癌（PDA）组织中的表达及其与胰腺癌发生发展的相关性。方法：应用免疫组化检测MICA／B在胰腺癌中的表达,分析MICA／B与胰腺癌临床病理学特征的相关性。结果：MICA／B在PDA组织中的阳性表达率为89．58％（43／48）,而慢性胰腺炎和正常胰腺组织中不表达或微弱表达,癌组织与慢性胰腺炎及正常胰腺组差异显著（X2=36．069,p〈0．001）。MICA／B在胰腺癌组织中的表达水平与肿瘤的临床分期和病理分级有密切的关系（X2=8．398,10．000;P=0．015,0．003）,在早期、分化较好的胰腺癌组织中MICA／B表达水平较高,而在分化较差的胰腺癌组织中表达水平较低,随着胰腺癌进展,MICA／B从肿瘤细胞表面脱落到癌细胞周围的间质组织中,晚期胰腺癌患者MICA／B的表达水平较低甚至不表达。Spearman等级相关分析显示MICA／B表达程度与分化程度正相关（r=0．331）。结论：MICA／B作为诱导蛋白通过MICA／B—NKG2D介导的免疫反应可能在早期清除胰腺癌细胞中起重要作用。但随着肿瘤的进展,MICAfB从肿瘤细胞表面脱落,释放到肿瘤间质组织进而引起肿瘤免疫逃避。因而我们推测,MICA／B-NKG2D介导的免疫监视障碍可能促进了胰腺癌的进展。     

Methylation status and protein expression of RASSF1A in breast cancer patients

Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.     

Search for the tumor-related proteins of transition cell carcinoma in Taiwan by proteomic analysis

To better understand the carcinogenesis of bladder cancer in Taiwan, we utilized the proteomic approach to search for potential biomarkers of transitional cell carcinoma (TCC). Analysis by 2-DE and MS/MS indicated that seven proteins are down-regulated and three proteins up-regulated in grade III samples as compared with those of grade II. Of these deregulated proteins, fatty acid binding proteins, annexin V, heat-shock protein 27, and lactate dehydrogenase have been shown to be associated with bladder cancer. Our studies also found altered expression of a group of proteins that have not been documented previously in bladder cancer, including annexin I, 15-hydroxyprostaglandin dehydrogenase, galectin-1, lysophospholipase and mitochondrial short-chain enoyl-coenzyme A hydratase 1 precursor. These results illustrate a pattern of differential protein expression between low- and high-grade tumors and it may be utilized as the molecular fingerprinting of a subset of bladder cancers. In addition, the present study provides a valuable resource in the study of pathological mechanisms in cancers of urothelial origin. The immunohistochemical staining of grade II and III TCC samples with antiserum to annexin I protein was utilized to confirm that the annexin I protein is up-regulated in grade III TCC.     

Gene cloning of immunogenic antigens overexpressed in pancreatic cancer

The serological analysis of recombinant cDNA expression libraries (SEREX) by utilizing a library derived from a human pancreatic adenocarcinoma cell line and IgG antibodies from an allogeneic patient serum led to the identification of 18 genes: 13 of these were known genes, and 5 were unknown genes. In Northern and RT-PCR analyses, we found that the expression of mRNA of 14 genes was elevated in pancreatic cancer cell lines compared with the levels in normal pancreatic tissues. In addition, the expression of mRNA of hsp105 in colon cancer was greater than that in normal colon tissue. Immunohistochemical analysis using anti-hsp105 antibody revealed that an increased expression of hsp105 is a characteristic feature of pancreatic ductal and colon adenocarcinoma. Furthermore, hsp105 immunoreactivity in some cases of gastric, esophageal, and hepatocellular carcinoma was much stronger than that in normal corresponding tissues. These molecules identified may provide good diagnostic markers for cancer cells.     

The novel mTORC1/2 dual inhibitor INK-128 suppresses survival and proliferation of primary and transformed human pancreatic cancer cells

Pancreatic cancer has one of worst prognosis among all human malignancies around the world, the development of novel and more efficient anti-cancer agents against this disease is urgent. In the current study, we tested the potential effect of INK-128, a novel mammalian target of rapamycin (mTOR) complex 1 and 2 (mTORC1/2) dual inhibitor, against pancreatic cancer cells in vitro. Our results demonstrated that INK-128 concentration- and time-dependently inhibited the survival and growth of pancreatic cancer cells (both primary cells and transformed cells). INK-128 induced pancreatic cancer cell apoptosis and necrosis simultaneously. Further, INK-128 dramatically inhibited phosphorylation of 4E-binding protein 1 (4E-BP1), ribosomal S6 kinase 1 (S6K1) and Akt at Ser 473 in pancreatic cancer cells. Meanwhile, it downregulated cyclin D1 expression and caused cell cycle arrest. Finally, we found that a low concentration of INK-128 significantly increased the sensitivity of pancreatic cancer cells to gemcitabine. Together, our in vitro results suggest that INK-128 might be further investigated as a novel anti-cancer agent or chemo-adjuvant for pancreatic cancer treatment.     

Mitomycin C modulates DNA-double strand break repair genes in cervical carcinoma cells

In a previous study, we elucidated the apoptotic mechanism mediated via Fas/FasL-dependent pathway in mitomycin C-treated cervical carcinoma cells. In this study, 2-D and MALDI-TOF analyses were performed in order to search mitomycin C-induced modulators in cervical carcinoma cells. Some protein spots down- or up-regulated by mitomycin C were separately selected from the 2-D gels. Twenty protein spots were identified from the 2-D gels. Among the 20 spots, 11 spots were down-regulated, whereas 9 spots were up-regulated in SiHa/pRSV-luc cells by mitomycin C. Three spots have not been identified in the database. Ku70-binding protein (KUB3), MHC class I antigen, MHC class I chain-related protein A or multi-PDZ domain protein 1, MAGUK P55 subfamily member 3 or lamda/iota protein kinase C-interacting protein, and GL014 or Sad1/unc-84 protein-like 1 were suppressed by mitomycin C treatment. Heat shock 60 kDa protein 1 (chaperonin), similar to heat shock protein 90 kDa protein alpha or ninein centrosomal protein isoform C, NADP-dependent malic enzyme, mitochondrial precursor, GRB10 adaptor protein, glycogenin-interacting protein 1, cystathionine gamma-lyase, G₂/mitotic-specific cyclin B2 or heat shock 90 kDa protein 1 alpha, peptidyl-prolyl cis–trans isomerase B, and PARP-2 (fragment) were induced by mitomycin C. KUB3, Brca1, and E6 gene expressions were down-regulated by mitomycin C in HPV-positive cervical cancer cells, SiHa/pRSV-luc and SiHa. In these studies, we suggest that MMC down-regulated the expression levels of the upstream molecules of DNA-double strand break repair system, non-homologous end joining or homologous recombination, resulting in the suppression of cervical cancer cell growth.     

Investigation of oncogene amplification or deletion,and oncoprotein expression in papillary thyroid cancer

AIM: Assessment of occurrence and possible prognostic significance of c-myc and Ha-ras amplification, p53 deletion and overexpression of cyclin D1, p53 and p21 in papillary thyroid cancer. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tumor tissue from 24 patients were investigated. Dot-blot DNA hybridization was used to detect oncogene amplification or deletion. The expression of oncoproteins was determined by immunohistochemical method. RESULTS: In our samples neither Ha-ras amplification nor p53 deletion were found. Low c-myc amplification (mean: 2.55) occured in 4 cases (17%). p53 protein was detected in 16 samples (66.6%), with p21 expression (chi(2)=7.02, p<0.01) in 6 cases (25%). The p53 expression did not influence the tumor fenotype. Cyclin D1 overexpression was found in 12 cases (50%), it was often associated with p21 expression (chi2=10.1, p<0.001) and in inverse relation to the tumor lymphocytic infiltration (chi(2)=5.35, p<0.05). Increased expression of estrogen receptor was shown in 4 cyclin D1 positive samples (17%). CONCLUSIONS: The p53 detected in our study is likely not to be mutant protein in all cases because its presence was associated with p21 expression that the mutant protein cannot induce and also it did not mean more aggressive tumor phenotype. The connection of cyclin D1 overexpression with the lymphocytic infiltration of the tumor suggests that the increased expression of cyclin D1 means poor prognosis. The coexpression of cyclin D1 and p21 raises the modulative character of the p21 protein, thought to be a tumor suppressor originally, but we find a CDK-independent, estrogen receptor mediated effect of cyclin D1 more likely, which has been described in breast cancer and is also proved by the coexpression of cyclin D1 and estrogen receptor detected here.     

Heat shock protein 27 as a prognostic and predictive biomarker in pancreatic ductal adenocarcinoma

A role of heat shock protein 27 (HSP27) as a potential biomarker has been reported in various tumour entities, but comprehensive studies in pancreatic cancer are lacking. Applying tissue microarray (TMA) analysis, we correlated HSP27 protein expression status with clinicopathologic parameters in pancreatic ductal adenocarcinoma specimens from 86 patients. Complementary, we established HSP27 overexpression and RNA-interference models to assess the impact of HSP27 on chemo- and radiosensitivity directly in pancreatic cancer cells. In the TMA study, HSP27 expression was found in 49% of tumour samples. Applying univariate analyses, a significant correlation was found between HSP27 expression and survival. In the multivariate Cox-regression model, HSP27 expression emerged as an independent prognostic factor. HSP27 expression also correlated inversely with nuclear p53 accumulation, indicating either protein interactions between HSP27 and p53 or TP53 mutation-dependent HSP27-regulation in pancreatic cancer. In the sensitivity studies, HSP27 overexpression rendered HSP27 low-expressing PL5 pancreatic cancer cells more susceptible towards treatment with gemcitabine. Vice versa, HSP27 protein depletion in HSP27 high-expressing AsPC-1 cells caused increased gemcitabine resistance. Importantly, HSP27 expression was inducible in pancreatic cancer cell lines as well as primary cells. Taken together, our study suggests a role for HSP27 as a prognostic and predictive marker in pancreatic cancer. Assessment of HSP27 expression could thus facilitate the identification of specific patient subpopulations that might benefit from individualized treatment options. Additional studies need to clarify whether modulation of HSP27 expression could represent an attractive concept to support the incorporation of hyperthermia in clinical treatment protocols for pancreatic cancer.     

Diagnostic significance of serum HMGB1 in colorectal carcinomas

High mobility group box 1 protein (HMGB1), a nuclear protein, can be translocated to the cytoplasm and secreted in colon cancer cells. However, the diagnostic significance of HMGB1 has not been evaluated in colorectal carcinomas. For this purpose, we have screened the expression and secretion of HMGB1 in 10 colon cancer cell lines and 1 control cell line and found that HMGB1 was detected in the culture medium. To evaluate the diagnostic value of HMGB1, we performed an enzyme-linked immunosorbent assay to measure HMGB1 levels and compared them to carcinoembryonic antigen (CEA) levels in the serum samples of 219 colorectal carcinoma patients and 75 healthy control subjects. We found that the serum HMGB1 level was increased by 1.5-fold in patients with colorectal carcinoma compared to those in healthy controls. When HMGB1 and CEA levels were compared, HMGB1 had similar efficacy as CEA regarding cancer detection (the sensitivity was 20.1% for HMGB1 vs. 25.6% for CEA, and the specificity was 96% for HMGB1 vs. 90.7% for CEA). Moreover, the diagnostic accuracy of HMGB1 for stage I cancer was significantly higher than that of CEA (sensitivity: 41.2% vs. 5.9%; specificity: 96% vs. 90.7). When we combined HMGB1 and CEA, the overall diagnostic sensitivity was higher than that of CEA alone (42% vs. 25.6%), and the diagnostic sensitivity for stage I was also elevated (47% vs. 5.9%). However, the prognosis of patients was not related with serum HMGB1 concentrations. Our findings indicate that serum HMGB1 levels are increased in a subset of colorectal carcinomas, suggesting their potential utility as a supportive diagnostic marker for colorectal carcinomas.     

Identification of Prognostic Biomarkers by Combined mRNA and miRNA Expression Microarray Analysis in Pancreatic Cancer

Pancreatic cancer is the fourth leading cause for cancer-related death, and early diagnosis is one key to improve the survival rate of this disease. Molecular biomarkers are an important method for diagnostic use in pancreatic cancer. We used data from three mRNA microarray datasets and a microRNA dataset (GSE16515, GSE15471, GSE28735, and GSE41372) to identify potential key genes. Differentially expressed genes (DEGs) and microRNAs (DEMs) were identified. Functional, pathway enrichment, and protein-protein interaction analyses were performed on common DEGs across all datasets. The target genes of the DEMs were identified. DEMs targets that were also DEGs were further scrutinized using overall survival analysis. A total of 236 DEGs and 21 DEMs were identified. There were a total of four DEGs (ECT2, NR5A2, NRP2, and TGFBI), which were also predicted target genes of DEMs. Overall survival analysis showed that high expression levels of three of these genes (ECT2, NRP2, and TGFBI) were associated with poor overall survival for pancreatic cancer patients. The basic expression of DEGs in pancreas stood lower level in various organ tissues. The expression of ECT2 and NRP2 was higher in different pancreatic cancer cell lines than normal pancreas cell line. Knockout of ECT2 by Crispr Cas9 gene editing system decreased proliferation and migration ability in pancreatic cancer cell line MiaPaCa2. In conclusion, we think that data mining method can do well in biomarker screening, and ECT2 and NRP2 can play as potential biomarker or therapy target by Crispr Cas9 in pancreatic cancer.     

Cyclin D1 Serves as a Poor Prognostic Biomarker in Stage I Gastric Cancer

TNM stage still serves as the best prognostic marker in gastric cancer (GC). The next step is to find prognostic biomarkers that detect subgroups with different prognoses in the same TNM stage. In this study, the expression levels of epidermal growth factor receptor (EGFR) and cyclin D1 were assessed in 96 tissue samples, including non-tumorous tissue, adenoma, and carcinoma. Then, the prognostic impact of EGFR and cyclin D1 was retrospectively investigated in 316 patients who underwent R0 resection for GC. EGFR positivity increased as gastric tissue became malignant, and cyclin D1 positivity was increased in all the tumorous tissues. However, there was no survival difference caused by the EGFR positivity, while the cyclin D1-postive group had worse overall survival (OS) than the cyclin D1-negative group in stage I GC (10-year survival rate (10-YSR): 62.8% vs. 86.5%, p = 0.010). In subgroup analyses for the propensity score-matched (PSM) cohort, there were also significant differences in the OS according to the cyclin D1 positivity in stage I GC but not in stage II and III GC. Upon multivariate analysis, cyclin D1 positivity was an independent prognostic factor in stage I GC. In conclusion, cyclin D1 may be a useful biomarker for predicting prognosis in stage I GC.     

Increased DNase I activity in diabetes might be associated with injury of pancreas

DNase I is an endonuclease responsible to destruction of chromatin during apoptosis. However, its role in diabetes is still unclear. With blood samples from our previous study related to type 2 diabetes, we examined the DNase I activity in the serum of these patients and the role of DNase I in the injury of pancreas was further investigated in rats and INS-1 cells. Serum and pancreatic tissues from human and rats were used for the study. Insulin resistance and diabetes were induced by high fat diet and STZ injection, respectively. DNase I activity was determined by radial enzyme-diffusion method. Expressions of DNase I and caspase-3 in pancreas were determined in rat pancreatic tissues and INS-1 cells. Apoptosis of INS-1 cells was determined by both TUNEL assay and Flow Cytometry. There was a significant elevation of DNase I activity in serum of patients with type 2 diabetes and rats with STZ injection. Moreover, increase in DNase I expression was observed in the pancreas of diabetic person and rats. Furthermore, high glucose induced both DNase I and caspase-3 expression and at the same time increased apoptosis rate of INS-1 cells. In conclusion, elevated DNase I in diabetes may be related to pancreatic injury and could be one of the causes that induce diabetes.