Normal Ductal Cells Research Articles

Expression of growth hormone receptor in duct cells of pancreatic carcinoma-chronic pancreatitis and impact of growth hormone on PCNA of inoculated pancreatic tumor

目的 观察生长激素受体(GHR)在胰腺癌一慢性胰腺炎导管细胞的表达及生长激素(GH)对移植瘤增殖细胞核抗原(PCNA)的影响.方法 免疫组化法检测GHR在胰腺癌细胞(Cap1、Pane-1、Mia、Colo、SW-1990、Aspe、P3)、移植瘤及人胰腺癌切除标本的表达,并以人混合淋巴细胞、人正常胰腺及慢性胰腺炎导管细胞作对照;裸鼠成瘤后随机分为注射GH的实验组(GH组,按剂量及时间分为4 mg 2周、2 mg 2周、2 mg 3周3个亚组)及对照组(NS组),于最后一次注药后切除肿瘤,用免疫组化法观察GH对肿瘤细胞中PCNA表达的影响.结果 GHR在各胰腺癌细胞株上有不同程度的表达;在人胰腺癌及慢性胰腺炎切除标本,GHR在导管细胞虽呈阳性表达,但与人正常胰腺的强表达形成对照,阳性率低于后者(P＜0.05);胰腺癌细胞PCNA阳性率在组间无差别.结论 GHR在胰腺癌、慢性胰腺炎导管细胞有一定程度的表达,但表达强度弱于正常胰腺,GH不改变肿瘤细胞中PCNA的表达。

MYRICETIN INDUCES APOPTOSIS VIA CASPASE ACTIVATION AND INHIBITION OF PI-3 KINASE/AKT AND ERK PATHWAYS IN HUMAN PANCREATIC CELLS

Myricetin a polyphenolic flavonoid is present in fruits and vegetables. Studies have suggested that various flavonoids also induce cell death in cancer cells and may influence various intracellular signaling pathways, including Mitogen-Activated Protein Kinases (MAPKs) and PI-3 kinases. In this study, we evaluated the effect of myricetin on the viability and apoptosis in pancreatic cancer cells, and evaluated its effect on PI-3K signaling pathways, which leads to cell proliferation, migration, and apoptosis. Methods: Pancreatic cancer cells (MiaPaCa-2/PANC-1, S2013, and S2VP10) and normal pancreatic ductal cells were treated with myricetin 25-200 μM. Cell viability was evaluated by CCK-8. The parameters of apoptosis, Annexin V and Caspase 3/9 activation were measured by flow cytometry and luminescence-based caspase assay, respectively. The effect on the activation of Akt, MAPK, and p38 was measured by evaluating the level of the active phosphorilated form of these enzymes by western blotting. Results: Myricetin reduced the viability of pancreatic cancer cell lines in a dose-dependent and time-dependent fashion without affecting the viability of normal pancreatic ductal cells. Myricetin induces apoptosis in pancreatic cancer cells, as suggested by increased annexin V staining and activation of caspase 3/9. Myricetin reduced the levels of phosphorilated p-Akt, p-MAPK, and p-P38 as measured by western blot, which suggests their inactivation. Conclusion: Myricetin induces apoptotic cell death in pancreatic cancer cells without influencing the viability of normal ductal cells. It is a potential therapeutic candidate for treating pancreatic cancer.

15-Lipoxygenase-1 Production is Lost in Pancreatic Cancer and Overexpression of the Gene Inhibits Tumor Cell Growth

Pancreatic cancer patients have an abysmal prognosis because of late diagnosis and lack of therapeutic options. Pancreatic intraepithelial neoplasias (PaniNs), the precursor lesions, are a potential target for chemoprevention. Targeting eicosanoid pathways is an obvious choice because 5-lipoxygenase (5-LOX) has been suggested as a tumor promoter in pancreatic carcinogenesis. Here we provide evidence that 15-lipoxygenase-1 (15-LOX-1) expression and activity may exert antitumorigenic effects in pancreatic cancer. Reverse transcription- polymerase chain reaction (RTPCR) and Western blot analysis showed absence or very weak expression of 15-LOX-1 in all pancreatic cancer cell lines tested. 15-LOX-1 was strongly stained in normal ductal cells, tubular complexes, and centroacinar cells, but no staining was seen in islets, cancer cells, PanlN lesions, or in tumor cells in lymph node metastases, indicating that 15-LOX-1 expression is lost during tumor development in human pancreas. Overexpression of 15-LOX-1 in pancreatic tumor cells or treatment with its arachidonic acid-derived metabolite resulted in decreased cell growth. These findings provide evidence that loss of 15-LOX-1 may play an important role in pancreatic carcinogenesis, possibly as a tumor suppressor gene. Thus, induction of 15-LOX-1 expression may be an attractive option for the prevention and treatment of pancreatic cancer.

Over‐expression of S100A2 in pancreatic cancer correlates with progression and poor prognosis

Controversy exists regarding the clinical significance of S100A2 in the progression of tumours. In pancreatic cancer, little is known about the role of S100A2. The aim of this study was to clarify the clinical significance of S100A2 expression in pancreatic carcinogenesis. We microdissected invasive ductal carcinoma (IDC) cells from 22 lesions, pancreatic intraepithelial neoplasia (PanIN) cells from five lesions, intraductal papillary mucinous neoplasm (IPMN) cells from 38 lesions, pancreatitis-affected epithelial (PAE) cells from 16 lesions, and normal ductal cells from 18 normal pancreatic tissues. S100A2 expression in 14 pancreatic cancer cell lines, microdissected cells and formalin-fixed paraffin-embedded (FFPE) samples was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Microdissection analyses revealed that IDC cells expressed higher levels of S100A2 than did IPMN, PAE or normal cells (all comparisons, p < 0.007). Cell lines from metastatic sites expressed higher levels of S100A2 than those from primary sites. PanIN cells expressed higher levels of S100A2 than normal cells (p = 0.018). IDC cells associated with poorly differentiated adenocarcinoma expressed higher levels of S100A2 than did IDC cells without poorly differentiated adenocarcinoma (p = 0.006). Analyses of FFPE samples revealed that levels of S100A2 were higher in samples from patients who survived < 1000 days after surgery than in those from patients who survived > 1000 days (p = 0.043). Immunohistochemical analysis was consistent with qRT-PCR. S100A2 may be a marker of tumour progression or prognosis in pancreatic carcinogenesis and pancreatic cancer.

Prognostic Implications of Numb Immunoreactivity in Salivary Gland Carcinomas

The gene numb encodes for a protein (Numb) involved in cell fate decisions in Drosophila, with proposed endocytic and developmental functions in mammalians. The distribution pattern of Numb in human tissues however, has not been fully characterized. We set out to explore the immunohistochemical expression of Numb in normal and neoplastic (28 adenoid cystic and 34 mucoepidermoid carcinomas) salivary glands, and correlated the results with the clinico-pathologic features of the neoplasms. Intense Numb immunoreactivity was detected in normal ductal cells and in a subset of acinar cells. In salivary carcinomas, we detected diffuse and intense Numb immunostaining in 5 adenoid cystic and 8 mucoepidermoid carcinomas. By contrast, the majority of adenoid cystic and mucoepidermoid cancers showed only moderate (14 and 5 cases) or focal staining (9 and 21 cases), respectively. The corresponding expression of Numb mRNA was documented in normal parotid gland and adenoid cystic carcinoma. Numb immunoreactivity was inversely correlated with the histological grade and Ki-67 immunoreactivity of both adenoid cystic and mucoepidermoid carcinomas. In addition, while tumor grade, stage, Ki-67 and Numb immunoreactivity were associated with disease-free survival in univariate analysis, only Numb and Ki-67 immunoreactivities retained independent prognostic significance in multivariate analysis. These data suggest that loss of Numb is implicated in aberrant differentiation programs of salivary gland carcinomas and may serve as a prognostic indicator in patients treated for these neoplasms.

Quantitative Proteomics Analysis Reveals That Proteins Differentially Expressed in Chronic Pancreatitis Are Also Frequently Involved in Pancreatic Cancer

The effective treatment of pancreatic cancer relies on the diagnosis of the disease at an early stage, a difficult challenge. One major obstacle in the development of diagnostic biomarkers of early pancreatic cancer has been the dual expression of potential biomarkers in both chronic pancreatitis and cancer. To better understand the limitations of potential protein biomarkers, we used ICAT technology and tandem mass spectrometry-based proteomics to systematically study protein expression in chronic pancreatitis. Among the 116 differentially expressed proteins identified in chronic pancreatitis, most biological processes were responses to wounding and inflammation, a finding consistent with the underlining inflammation and tissue repair associated with chronic pancreatitis. Furthermore 40% of the differentially expressed proteins identified in chronic pancreatitis have been implicated previously in pancreatic cancer, suggesting some commonality in protein expression between these two diseases. Biological network analysis further identified c-MYC as a common prominent regulatory protein in pancreatic cancer and chronic pancreatitis. Lastly five proteins were selected for validation by Western blot and immunohistochemistry. Annexin A2 and insulin-like growth factor-binding protein 2 were overexpressed in cancer but not in chronic pancreatitis, making them promising biomarker candidates for pancreatic cancer. In addition, our study validated that cathepsin D, integrin beta1, and plasminogen were overexpressed in both pancreatic cancer and chronic pancreatitis. The positive involvement of these proteins in chronic pancreatitis and pancreatic cancer will potentially lower the specificity of these proteins as biomarker candidates for pancreatic cancer. Altogether our study provides some insights into the molecular events in chronic pancreatitis that may lead to diverse strategies for diagnosis and treatment of these diseases.

Transcription factor AP-2α represses both the mucin MUC4 expression and pancreatic cancer cell proliferation

MUC4 is a transmembrane mucin expressed in pancreatic ductal adenocarcinoma (DAC) in contrast to normal pancreas, and is an independent predictor of poor prognosis in patients with invasive DAC. Our aim was therefore to investigate the mechanisms that control MUC4 expression in pancreatic cancer cells. We focused our study on activator protein (AP)-2alpha transcription factor that acts as a tumour suppressor gene in several cancers. In a series of 18 human DAC, using immunohistochemistry, we confirmed that MUC4 was exclusively expressed in cancerous or preneoplastic lesions in 83% of the samples. On the contrary, AP-2 was mainly expressed by non-tumoural ductal cells (61%) or endocrine cells (67%). Moreover, MUC4 and AP-2 were never found co-expressed suggesting an inhibitory role of AP-2alpha in normal ductal cells. In CAPAN-1 and CAPAN-2 cells, transient AP-2alpha over-expression decreased both MUC4 mRNA and apomucin levels by 20-40% by a mechanism involving inhibition of MUC4 promoter. By chromatin immunoprecipitation and gel-shift assays, we demonstrated that this inhibition involved two AP-2 cis-elements located in the -475/-238 region of the promoter. CAPAN-1 clones, which stably over-expressed AP-2alpha, displayed a strong MUC4 down-regulation (-38 to -100%), a significant decrease of both cell proliferation and invasion concomitant to the up-regulation of p27 cyclin-dependent kinase inhibitor. In conclusion, our data provide evidence that AP-2alpha is an important in vivo negative regulator of MUC4 expression in human pancreatic tissue and that AP-2alpha may play a tumour-suppressive role in pancreatic DAC.

Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis

BackgroundInvasive ductal and lobular carcinomas (IDC and ILC) are the most common histological types of breast cancer. Clinical follow-up data and metastatic patterns suggest that the development and progression of these tumors are different. The aim of our study was to identify gene expression profiles of IDC and ILC in relation to normal breast epithelial cells.MethodsWe examined 30 samples (normal ductal and lobular cells from 10 patients, IDC cells from 5 patients, ILC cells from 5 patients) microdissected from cryosections of ten mastectomy specimens from postmenopausal patients. Fifty nanograms of total RNA were amplified and labeled by PCR and in vitro transcription. Samples were analysed upon Affymetrix U133 Plus 2.0 Arrays. The expression of seven differentially expressed genes (CDH1, EMP1, DDR1, DVL1, KRT5, KRT6, KRT17) was verified by immunohistochemistry on tissue microarrays. Expression of ASPN mRNA was validated by in situ hybridization on frozen sections, and CTHRC1, ASPN and COL3A1 were tested by PCR.ResultsUsing GCOS pairwise comparison algorithm and rank products we have identified 84 named genes common to ILC versus normal cell types, 74 named genes common to IDC versus normal cell types, 78 named genes differentially expressed between normal ductal and lobular cells, and 28 named genes between IDC and ILC. Genes distinguishing between IDC and ILC are involved in epithelial-mesenchymal transition, TGF-beta and Wnt signaling. These changes were present in both tumor types but appeared to be more prominent in ILC. Immunohistochemistry for several novel markers (EMP1, DVL1, DDR1) distinguished large sets of IDC from ILC.ConclusionIDC and ILC can be differentiated both at the gene and protein levels. In this study we report two candidate genes, asporin (ASPN) and collagen triple helix repeat containing 1 (CTHRC1) which might be significant in breast carcinogenesis. Besides E-cadherin, the proteins validated on tissue microarrays (EMP1, DVL1, DDR1) may represent novel immunohistochemical markers helpful in distinguishing between IDC and ILC. Further studies with larger sets of patients are needed to verify the gene expression profiles of various histological types of breast cancer in order to determine molecular subclassifications, prognosis and the optimum treatment strategies.

Immunohistochemical expression of the β‐catenin‐interacting protein B9L is associated with histological high nuclear grade and immunohistochemical ErbB2/HER‐2 expression in breast cancers

B9L/BCL9-2, a novel beta-catenin-interacting protein, plays an important role in colorectal carcinogenesis by translocating beta-catenin to the nucleus and enhancing beta-catenin-T-cell factor-mediated transcription. To elucidate the role of B9L in breast cancers, we studied B9L expression in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of the breast immunohistochemically and compared it to the immunohistochemical expression of known proteins involved in breast carcinogenesis. In breast tissues, B9L immunoreactivity was present exclusively in the nuclei of normal and neoplastic ductal cells. In DCIS, immunohistochemical B9L expression was significantly associated with the tumor nuclear grade, comedo necrosis and the expression of ErbB2/HER-2, c-myc and p53. In IDC, B9L expression was correlated with ErbB2/HER-2 expression and tumor nuclear grade only. In both DCIS and IDC, immunohistochemical B9L expression was not related to the expression of cytoplasmic beta-catenin. We demonstrated that nuclear B9L expression was closely associated with the high nuclear grade cancer phenotype and the expression of ErbB2/HER-2 in breast cancers.

TRIPTOLIDE INDUCES APOPTOSIS IN PANCREATIC CANCER CELLS VIA INHIBITION OF HEAT SHOCK PROTEIN 70 (HSP70)

Pancreatic cancer is the fourth leading cause of cancer deaths in the United States. Owing to the lack of success with current chemotherapy agents used in pancreatic cancer, we were interested in examining triptolide, a diterpenoid triepoxide component of a Chinese herb that has anti-tumor properties. We have recently demonstrated that an increased level of HSP70 in pancreatic cancer cells confers resistance to apoptosis and that inhibition of HSP70 induces apoptosis in these cells. Given this, we were interested in examining the effect of triptolide on HSP70 expression in pancreatic cancer cells. Therefore, the aims of this study were to examine the effect of triptolide on i) pancreatic cancer cells by assessing viability, apoptosis and HSP70 levels; and ii) pancreatic cancer growth in vivo. Methods: Pancreatic cancer cells (MiaPaCa-2/PANC-1) or normal pancreatic duct cells (PDC) were incubated with triptolide (100-200nM) or vehicle (control, C) for 24h. Cell viability and apoptosis were assessed. HSP70 levels were assessed by Western blot. Triptolide (0.2mg/kg) or vehicle was administered daily to nude mice 5 days after injection of MiaPaCa-2 into the pancreas. Results: Incubation of PANC-1 and MiaPaCa-2 cells with triptolide significantly reduced cell viability, but had no effect on normal duct cell viability (Triptolide 200nM - % of C: MiaPaCa-2 55±4, PANC-1 61±11, PDC 95±16). Triptolide induced apoptosis in the cancer cells as evidenced by a significant increase in Annexin V, caspase 3 (Table) and TUNEL stain. Triptolide also decreased HSP70 levels in both cell lines. Triptolide administered in vivo decreased pancreatic cancer growth (Tumor Volume cm3: Control 1.03±0.43 vs Triptolide 0.14±0.05, data expressed as Mean±SE).Table: No Caption Available.Conclusion: Triptolide causes pancreatic cancer cell death in vitro and in vivo by induction of apoptosis and its mechanism of action may be mediated via the inhibition of HSP70. Implication - Triptolide is a potential therapeutic agent to prevent the progression of pancreatic cancer via its inhibition of HSP70.

DIFFERENTIAL EXPRESSIONS OF MRNA IN TUMOR PROGRESSION OF PANCREATIC DUCTAL CARCINOMA AND INTRADUCTAL PAPILLARY MUCINOUS NEOPLASM

Background and Aim: Recent microarray analyses suggested that various novel genes were up-regulated in pancreatic ductal carcinoma and intraductal papillary mucinous neoplasm (IPMN). In the present study, we quantitatively measured the mRNA expression of such genes in microdissected cells of normal ducts, PanINs, IPMNs, and invasive pancreatic cancer (IDC) by real time RT-PCR. Furthermore, we evaluated mRNA expression in pancreatic juice samples from patients with chronic pancreatitis, IPMN or IDC. Method: We designed gene-specific primers of MUC1, MUC5AC, S100P, S100A6, S100A11, Twist, and house keeping genes of beta-actin and 18SrRNA. We used one-step real time RT-PCR with gene-specific priming, short amplicons, and normalization to house keeping genes to achieve reliable quantification of mRNA from moderately to extensively fragmented samples of microdissected cells or pancreatic juice. After histological evaluation on H&E stained sections, normal duct cells, PanIN cells (PanIN 1B or PanIN 2), IPMN cells (IPM adenoma or IPM borderline), and IDC cells were isolated selectively from frozen sections with a laser microdissection and pressure catapulting system. Result: All six genes showed significantly higher expressions in IDC cells than in normal duct. Stepwise up-regulations were observed in MUC5AC, MUC1, S100A6 and Twist in relation to the tumor progression of pancreatic ductal carcinoma, from normal duct to PanINs and IDC. Levels of S100P mRNA in PanINs were comparable to those of IDC, and that of S100A11 was significantly higher in PanINs than in IDC. Meanwhile, MUC5AC, S100P and S100A11 showed significantly higher expression in IPMN than in IDC. Twist expression was significantly lower in IPMN than in IDC. Expression of mRNA in pancreatic juice was similar to that in microdissected cells, and Twist was most useful to differentiate pancreatic cancer from IPMN. Conclusion: Quantitative measurement of mRNA levels of these genes was successfully achieved in microdissected target cells from pancreatic tissues and differential expressions were observed in pancreatic cancer and IPMN. These genes may become promising diagnostic markers and therapeutic targets for pancreatic cancer.

Stromal and Epithelial Caveolin-1 Both Confer a Protective Effect Against Mammary Hyperplasia and Tumorigenesis: Caveolin-1 Antagonizes Cyclin D1 Function in Mammary Epithelial Cells

Here, we investigate the role of caveolin-1 (Cav-1) in breast cancer onset and progression, with a focus on epithelial-stromal interactions, ie, the tumor microenvironment. Cav-1 is highly expressed in adipocytes and is abundant in mammary fat pads (stroma), but it remains unknown whether loss of Cav-1 within mammary stromal cells affects the differentiated state of mammary epithelia via paracrine signaling. To address this issue, we characterized the development of the mammary ductal system in Cav-1-/- mice and performed a series of mammary transplant studies, using both wild-type and Cav-1-/- mammary fat pads. Cav-1-/- mammary epithelia were hyperproliferative in vivo, with dramatic increases in terminal end bud area and mammary ductal thickness as well as increases in bromodeoxyuridine incorporation, extracellular signal-regulated kinase-1/2 hyperactivation, and up-regulation of STAT5a and cyclin D1. Consistent with these findings, loss of Cav-1 dramatically exacerbated mammary lobulo-alveolar hyperplasia in cyclin D1 Tg mice, whereas overexpression of Cav-1 caused reversion of this phenotype. Most importantly, Cav-1-/- mammary stromal cells (fat pads) promoted the growth of both normal mammary ductal epithelia and mammary tumor cells. Thus, Cav-1 expression in both epithelial and stromal cells provides a protective effect against mammary hyperplasia as well as mammary tumorigenesis.

RNA Interference Suppression of MUC1 Reduces the Growth Rate and Metastatic Phenotype of Human Pancreatic Cancer Cells

MUC1 is a highly glycosylated, type I transmembrane protein expressed by normal ductal epithelial cells of the pancreas, breast, lung, and gastrointestinal tract, and overexpressed in many cases of adenocarcinoma. We down-regulated MUC1 expression by RNA interference and investigated the effects on malignant and metastatic potential of a human pancreatic cancer cell line, S2-013. MUC1-suppressed clones, S2-013.MTII.C1 and S2-013.MTII.C2, were established by targeting a sequence 3,151 bp from the initiation codon and characterized in vitro for proliferation, invasion, and adhesion. We evaluated the effects of MUC1 suppression in vivo on tumor growth and metastatic properties following implantation into the cecum or pancreas of athymic mice. MUC1-suppressed clones showed significantly decreased proliferation in vitro and in vivo. Global gene expression was evaluated by oligonucleotide microarray analysis. Surprisingly, genes predicted to increase doubling times (cyclin B1 and cyclin D3) were overexpressed in MUC1-suppressed clones. There were alterations in expression of several genes that may affect the malignant properties of pancreatic cancer. Adhesion of MUC1-suppressed cells in vitro to type IV collagen and fibronectin was slightly increased, and adhesion was slightly decreased to type I collagen and laminin. Results of implantation to cecum and pancreas showed significant reduction of metastasis to lymph nodes, lung, or peritoneal sites compared with S2-013.gfp-neo control cells. These results support the hypothesis that MUC1 contributes significantly to growth and metastasis, and that down-regulation of MUC1 protein expression decreases the metastatic potential of pancreatic adenocarcinoma.

Angiogenesis-associated protein annexin II in breast cancer: Selective expression in invasive breast cancer and contribution to tumor invasion and progression

Many advanced human tumors including breast cancer overproduce plasmin that is known to promote angiogenesis and metastasis. The mechanism of this effect is poorly understood. Here we report that annexin II, an endothelial co-receptor for tPA (tissue-type plasminogen activator) and plasminogen, was undetectable in normal and hyperplastic ductal epithelial cells and ductal complexes. By contrast, it was consistently expressed in invasive breast cancer and ductal carcinoma in situ (DCIS) indicating its involvement in breast cancer. Using the well established invasive/metastatic MDA-MB231 cell line and the noninvasive/nonmetastatic MCF-7 human breast cancer cell line, we investigated the mechanism by which annexin II regulates breast cancer progression and metastasis. Western and Northern blot analyses demonstrate selective expression of annexin II in MDA-MB231 cells but not in poorly invasive MCF-7 cells suggesting its participation in invasive breast cancer. Since annexin II is a receptor for plasminogen, we tested whether MDA-MB231 cells are capable of producing plasmin in vitro. MDA-MB231 cell membranes induced plasmin generation in a time-dependent manner while those from MCF-7 cells failed to convert plasminogen to plasmin. The generated plasmin is capable of degrading ECM consequently facilitating cell invasion and migration, biological functions required for angiogenesis and metastasis. Plasmin generation and its dependent invasion and migration can be blocked by a monoclonal antibody to annexin II or angiostatin, potent inhibitors of angiogenesis, breast cancer, and metastasis. Our findings indicate that annexin II-dependent localized plasmin generation by human breast cancer cells could contribute to angiogenesis and metastasis. These results suggest that annexin II may be an attractive target for new anti-angiogenic and anti-breast cancer therapies.

Quantitative Analysis of Human Telomerase Reverse Transcriptase in Pancreatic Cancer

Although telomerase activity is a promising diagnostic marker, clinical introduction of this marker for cancer diagnosis is still problematic due to the lack of means of evaluating sample quality. Human telomerase reverse transcriptase (hTERT), one of the subunits of telomerase, is also a promising diagnostic marker. In the present study, we did large-scale analysis of 88 pancreatic juice samples to determine the feasibility of quantitative analysis of hTERT mRNA for diagnosis of pancreatic cancer. We found significant differences in hTERT expression among carcinoma-derived, intraductal papillary mucinous neoplasm (IPMN)-derived, and chronic pancreatitis-derived juice samples. Results showed that quantitative analyses of hTERT mRNAs are more useful in discriminating carcinoma from IPMN than from chronic pancreatitis. When the specificity was set at 100%, the sensitivity for differentiation between carcinoma and IPMN was 43.5%, whereas the sensitivity of cytologic examination was 22.0%. There were significant differences in hTERT expression among carcinoma cells, IPMN cells, and normal ductal cells isolated from pancreatic tissues by microdissection. Lymphocytes and hyperplastic epithelial cells isolated from tissues with the histologic appearance of pancreatitis showed various expression levels of hTERT. Our results suggest that quantitative analysis of hTERT mRNA in pancreatic juice is advantageous over cytologic analysis for differentiation between carcinoma and IPMN but probably not for differentiation between carcinoma and chronic pancreatitis.

Of Cilia and Cysts: Modeling Pancreatic Polycystic Disease

Of Cilia and Cysts: Modeling Pancreatic Polycystic Disease

Overexpression of c-met in the early stage of pancreatic carcinogenesis; altered expression is not sufficient for progression from chronic pancreatitis to pancreatic cancer

To investigate c-met expression during early pancreatic carcinogenesis. We used 46 bulk tissues and 36 micro-dissected samples, including normal pancreas, chronic pancreatitis, and pancreatic cancer, for quantitative real-time reverse transcription-polymerase chain reaction. In bulk tissue analyses, pancreatic cancer tissues expressed significantly higher levels of c-met than did chronic pancreatitis and normal pancreas tissues. c-met levels did not differ between chronic pancreatitis and normal pancreas tissues. In microdissection-based analyses, c-met was expressed at higher levels in microdissected pancreatic cancer cells and pancreatitis-affected epithelial cells than in normal ductal epithelial cells (both, P < 0.01). Interestingly, pancreatitis-affected epithelial cells expressed levels of c-met similar to those of pancreatic cancer cells. Overexpression of c-met occurs during the early stage of pancreatic carcinogenesis, and a single alteration of c-met expression is not sufficient for progression of chronic pancreatitis-affected epithelial cells to pancreatic cancer cells.

Changes in Cytoskeletal Protein Composition Indicative of an Epithelial-Mesenchymal Transition in Human Micrometastatic and Primary Breast Carcinoma Cells

The bone marrow is a frequent and clinically important homing site for early disseminated breast cancer cells. Here, we aimed to profile the protein expression of these cells using unique cell line models and to evaluate the prognostic relevance of candidate gene expression for breast cancer patients. To identify expression patterns characteristic for micrometastatic cells, three different cell lines (BC-K1, BC-P1, and BC-S1) established by SV40 immortalization of cancer cells isolated from the bone marrow of patients with breast cancer were compared with MCF-7 breast cancer and SV40 immortalized normal breast ductal cells (MTSV-1.7) using two-dimensional gel electrophoresis followed by MALDI-ToF analysis. The prognostic significance and clinicopathologic associations of selected differentially expressed proteins were evaluated using high-density breast cancer tissue microarrays. In contrast to MCF-7 and MTSV1-7 reference cell lines, all micrometastatic cancer cell lines displayed loss of epithelial cytokeratins (CK8, CK18, and CK19) and ectopic expression of vimentin commonly present in mesenchymal cells. Immunohistochemical analysis of 2,517 samples of breast cancer further showed that loss of cytokeratin and ectopic vimentin expression were significantly associated with a higher tumor grade, high mitotic index, and negative estrogen/progesterone-receptor status. Although in univariate analyses significantly related to clinical outcome, none of the cytokeratins analyzed were independently associated with either overall or cancer-specific survival. Micrometastatic cancer cells exhibit marked changes in the expression pattern of cytoskeletal proteins indicative of an epithelial-mesenchymal transition. This phenotypical change could already be detected in primary tumors and is associated with the aggressive behavior of breast cancer cells in vivo.

Expression of glial cell line–derived neurotrophic factor family members and their receptors in pancreatic cancers

The glial cell line-derived neurotrophic factor (GDNF) is a member of neurotrophic polypeptide family, which promotes survival and rescue of various neural cells in the central and peripheral nerve systems. We previously reported that GDNF promotes tumor cell invasion in pancreatic cancer cell lines. The purpose of this study was to investigate GDNF family expression and the status of related receptors in actual cancer tissues, and assess correlations with clinicopathologic behavior. Immunohistochemical assessment of GDNF, neurturin, persephin, artemin, GDNF family receptor alpha-1 and alpha-2, and RET was performed for 51 cases of surgically resected pancreatic cancer. In all intrapancreatic nerves, GDNF and artermin were expressed strongly. In pancreatic cancer tissues. The expression of RET was stronger than that seen in normal ductal cells and was significantly related to the survival rate after resection (P = .026) and lymphatic invasion (P = .014). Intrapancreatic neural invasion was significantly related to the expression of GDNF (P = .047). We conclude that the expression of RET in pancreatic cancer tissues may be a useful prognostic marker and GDNF may play an important role in neural invasion.

Overexpression of EphA2 gene in invasive human breast cancer and its association with hormone receptor status

9583 Background: EphA2 is a receptor tyrosine kinase for ephrin A-1 that has been implicated in a broad spectrum of functions, including angiogenesis, cell migration, and cancer development. Expression of EphA2 is regulated by p53. This study is aimed to determine if expression of EphA2 is associated with development of invasive human breast cancer and breast cancer histologic prognostic characteristics. Methods: Paraffin blocks of 134 invasive human breast cancer cases and fifteen normal human breast tissues were studied with immunohistochemistry staining using two polyclonal antibodies against EphA2 protein. The expression patterns were determined as overexpression (expression in more than 50% of cells) or low expression (expression in less than 10% of cells). Histologic data of these breast cancer cases were retrieved from computerized patient medical records. Results: In normal breast tissues, the expression of EphA2 protein was found to be low and to be predominantly restricted to the cytoplasm of discontinuous satellite myoepithelial cells and scattered epithelial cells. Normal breast ductal epithelial cells showed low EphA2 expression in less than 5% of cells. However, 110 out of 134 (82%) human invasive breast cancer cases showed overexpression of EphA2 protein. Three EphA2 protein expression patterns were identified in these breast cancer cases: predominantly nuclear expression, diffuse nuclear and cytoplasmic expression, and predominantly membrane expression. The expression of EphA2 is at least 10 fold higher in invasive human breast cancer than normal breast tissue. Interestingly, 90% of ER (estrogen receptor)-positive cases were found to overexpress EphA2, while only 60% of ER-negative cases overexpress EphA2 (p<0.001). In another word, 10% of ER-positive cases showed low expression of EphA2, compared to 40% of ER-negative cases. PR status, HER2/NEU overexpression, nodal status, and grade were not associated with pattern or intensity of EphA2 expression. Conclusions: Receptor tyrosine kinase EphA2 is highly overexpressed in more than 80% cases of invasive human breast cancer. Absence of overexpression of EphA2 was associated with negative ER status, and hence, poorer prognosis. No significant financial relationships to disclose.