H. Friess

Enhanced expression of transforming growth factor β isoforms in pancreatic cancer correlates with decreased survival

BACKGROUNDnTransforming growth factor beta s (TGF-beta s) constitute a family of bifunctional polypeptide growth factors that either inhibit or stimulate cell proliferation. Perturbations in TGF-beta expression and function may lead to loss of negative constraints on cell growth. In this study, we examined TGF-beta expression in human pancreatic cancer.nnnMETHODSnThe distribution of TGF-beta isoforms in 60 human pancreatic cancers was examined using immunohistochemical, Northern blot, and in situ hybridization techniques.nnnRESULTSnImmunohistochemical analysis showed the presence of TGF-beta 1 (47% of tumors), TGF-beta 2 (42% of tumors), and TGF-beta 3 (40% of tumors) in the cancer cells. The presence of TGF-beta 2 was associated with advanced tumor stage (P < 0.05). Furthermore, there was a significant correlation between the absence of TGF-beta s in the tumors and longer postoperative survival. Northern blot analysis indicated that, by comparison with the normal pancreas, pancreatic adenocarcinomas showed 11- (P < 0.001), 7- (P < 0.05), and 9-fold (P < 0.001) increases in the messenger RNA (mRNA) levels encoding TGF-beta 1, TGF-beta 2, and TGF-beta 3, respectively. By in situ hybridization, these mRNA moieties colocalized with their respective proteins in the cancer cells.nnnCONCLUSIONSnThese findings show that human pancreatic cancers show increased levels of TGF-beta isoforms and enhanced TGF-beta mRNA expression and suggest that the presence of TGF-beta s in pancreatic cancer cells may contribute to disease progression.

Overexpression of HER2/neu oncogene in human pancreatic carcinoma

The HER2/neu oncogene encodes a transmembrane protein that possesses intrinsic tyrosine kinase activity. Its overexpression has been associated with the malignant phenotype. In this study we examined HER2/neu expression in the normal and cancerous human pancreas. In the normal pancreas HER2/neu immunostaining was observed in acinar and ductal cells. HER2/neu immunoreactivity was expressed in 34 of 76 (45%) pancreatic carcinomas. There was a significant correlation between tumors with well-differentiated histology and HER2/neu expression. Northern blot analysis demonstrated HER2/neu mRNA expression in the normal pancreas and in situ hybridization confirmed its distribution in both acinar and ductal cells. In cancer tissues Northern blot analysis indicated that HER2/neu mRNA levels were elevated in 13 of 25 (52%) of the tumors in comparison with the normal tissues. In addition, in situ hybridization demonstrated a strong but heterogenous distribution of mRNA grains in these tumors. Southern blot analysis did not demonstrate HER2/neu gene amplification in any of the tumors. These data indicate that the HER2/neu protein is synthesized in the normal exocrine pancreas and is frequently overexpressed in well-differentiated adenocarcinomas of the pancreas as a result of increased HER2/neu mRNA levels.

Diagnosis of pancreatic cancer by 2[18F]-fluoro-2-deoxy-D-glucose positron emission tomography.

The detection of pancreatic cancer or the discrimination between pancreatic cancer and chronic pancreatitis remains an important diagnostic problem. The increased glucose metabolism in malignant tumours formed the basis for this investigation, which focused on the role of positron emission tomography (PET) with 2[18F]-fluoro-2-deoxy-D-glucose (FDG) in the detection of pancreatic cancer and its differentiation from chronic pancreatitis. Eighty patients admitted for elective pancreatic surgery received preoperatively 250-350 mBq FDG intravenously and emission scans were recorded 45 minutes later. Intense focal activity in the pancreatic region was taken at the time of scanning as showing the presence of pancreatic cancer. The presence of cancer was later confirmed by histological examination of the surgical specimens and histological findings were compared with the preoperative PET results. Forty one patients with pancreatic cancer (group I: n = 42) had a focally increased FDG uptake in the pancreatic region. Two patients with a periampullary carcinoma (group II: n = 6) failed to develop FDG accumulation. In 28 patients with chronic pancreatitis (group III: n = 32) no FDG accumulation occurred. Overall sensitivity and specificity of PET for malignancy (group I + II) were 94% (45 of 48) and 88% (28 of 32), respectively. The standard uptake value of the patients with pancreatic carcinoma was significantly higher than in patients with chronic pancreatitis (3.09 (2.18) v 0.87 (0.56); p < 0.001; median (interquartile range)). These findings show that FDG-PET represents a new and non-invasive diagnostic procedure for the diagnosis of pancreatic cancer and to differentiate pancreatic cancer from chronic pancreatitis. However, the diagnostic potential of this technique requires further evaluation.

Bone morphogenetic protein 2 exerts diverse effects on cell growth in vitro and is expressed in human pancreatic cancer in vivo

BACKGROUND & AIMSnBone morphogenetic proteins (BMPs) belong to the transforming growth factor beta superfamily of signaling molecules. We characterized the expression of BMP-2 and its receptors in human pancreatic tissues and pancreatic cancer cell lines and examined the effects of BMP-2 on mitogenesis.nnnMETHODSnExpression of BMP-2 and its receptors was determined by Northern blot analysis using specific complementary DNA probes. Distribution of BMP-2 in pancreatic cancers was examined by immunohistochemistry and in situ hybridization. Effects of BMP-2 on mitogenesis were assessed by monitoring cell proliferation and activation of mitogen-activated protein kinase (MAPK).nnnRESULTSnCompared with the normal pancreas, pancreatic cancers showed a 12.5-fold (P < 0.01), 2-fold (P < 0.01), and 8-fold (P < 0.01) increase of BMP-2, BMP receptor (R)-IA, and BMPR-II messenger RNA levels, respectively. By immunohistochemistry and in situ hybridization, BMP-2 was expressed in the cancer cells within the tumor mass. There was a significant correlation between the presence of BMP-2 immunostaining in the tumors and shorter postoperative survival. Pancreatic cancer cell lines expressed variable levels of messenger RNA encoding BMP-2 and its receptors. BMP-2 stimulated the growth of two pancreatic cancer cell lines (ASPC-1 and CAPAN-1). This mitogenic effect was associated with MAPK activation and blocked by the MAPK inhibitor PD98059 in CAPAN-1 but not in ASPC-1 cells. In both cell lines, expression of wild-type Smad4 abolished the BMP-2-mediated growth stimulation. BMP-2 inhibited the growth of COLO-357 cells, an effect that was blocked by expressing a dominant negative Smad4. BMP-2 had no effect in three cell lines that underexpressed either the BMP receptors or Smad1.nnnCONCLUSIONSnThese findings indicate that BMP-2 has the capacity to act as a mitogen when Smad4 is mutated and suggest that it might play a role in the pathobiology of human pancreatic cancer.

p53 mutations are common in pancreatic cancer and are absent in chronic pancreatitis.

Pancreatic expression of the p53 tumor suppressor gene was studied in pancreatic adenocarcinomas and chronic pancreatitis. By immunohistochemistry, 16 of 34 (47%) cancers and none of the 24 chronic pancreatitis samples revealed nuclear staining. Sequence analysis indicated that 8 of 24 (33%) cancers were mutated for the p53 gene. Point substitutions occurred at codons 35, 105, 133, 213, 213, 258, and 299. A three base-pair in-frame insertion was identified between codons 261 and 262. None of 8 chronic pancreatitis samples exhibited p53 gene mutations. These data support a role for p53 gene alterations in human pancreatic cancer, and suggest that loss of its regulatory functions may constitute one of the differences between pancreatic cancer and chronic pancreatitis.

Increased expression of erbB3 in colorectal cancer is associated with concomitant increase in the level of erbB2

ErbB3 is a transmembrane signaling molecule that shares close structural homology with epidermal growth factor receptor (EGFR), erbB2, and erbB4. They have all been implicated in cell transformation and tumor pathogenesis, but very little is known about the role of erbB3 in normal colon and colorectal cancer. Therefore, in the current study, we determined whether erbB3 is found in normal human colon and whether its expression is altered in colorectal cancer. Because of some evidence that erbB3 might interact with erbB2 and EGFR, respectively, by heterodimerization, we also included erbB2 and EGFR analysis with special regard to coexpression. The study was performed on 35 patients operated on for colorectal carcinoma. The normal human colon showed weak erbB3 and erbB2 immunostaining, predominantly in surface epithelial cells. EGFR immunoreactivity in normal colon varied from weak to strong. In contrast, in 31 of 35 (89%) and in 29 of 35 (83%) colonic cancers, moderate to strong immunoreactivity for erbB3 and erbB2, respectively, was present in most epithelial cancer cells. A concomitant erbB3 and erbB2 immunostaining advantage could be found in 77% of cancerous tissues in comparison with the normal colon. No difference in EGFR immunostaining was evident between normal colon and cancer. Northern blot analysis showed an increase in erbB3 and erbB2 mRNA levels in 64% of cancers in comparison with normal colon samples. By densitometry, 2.3-fold and a 1.5-fold significant increases in erbB3 and erbB2 mRNA levels, respectively, were calculated in the cancerous tissues. Eighty-five percent of cancers with erbB3 mRNA overexpression showed an increase in erbB2 mRNA. Southern blot analysis did not indicate any gene amplification or rearrangement responsible for erbB2 or erbB3 overexpression. EGFR, however, was decreased in cancer on mRNA level. These findings indicate that erbB2 and erbB3, but not EGFR, may contribute to tumor growth and disease progression in colon cancer. The correlation between increased erbB2 and erbB3 expression in both Northern blots and immunohistochemical analysis suggests a co-overexpression of erbB2 and erbB3 and might support the hypothesis that these two growth factor receptors act together by heterodimer formation.

Chronic pancreatitis is associated with increased concentrations of epidermal growth factor receptor, transforming growth factor alpha, and phospholipase C gamma.

The epidermal growth factor (EGF) receptor is a transmembrane protein that binds EGF and transforming growth factor alpha (TGF alpha), and that stimulates phospholipase C gamma 1 (PLC gamma 1) activity. In this study the role of the EGF receptor in chronic pancreatitis was studied. By immunohistochemistry, the EGF receptor, TGF alpha, and PLC gamma 1 were found to be expressed at high concentrations in pancreatic ductal and acinar cells from chronic pancreatitis patients. Northern blot analysis showed that, by comparison with normal controls, 19 of 27 chronic pancreatitis tissues exhibited a 5.7-fold increase in EGF receptor mRNA concentrations, and 20 of 27 chronic pancreatitis tissues exhibited a sixfold increase in TGF alpha mRNA concentrations. In situ hybridisation confirmed that overexpression occurred in ductal and acinar cells, and showed that both mRNA moieties colocalised with their respective proteins. These findings suggest that TGF alpha may act through autocrine and paracrine mechanisms to excessively activate the overexpressed EGF receptor in the two major cell types of the exocrine pancreas, thereby contributing to the pathobiology of this disorder.

Hyperlipaemia intensifies the course of acute oedematous and acute necrotising pancreatitis in the rat.

BACKGROUND--Serum triglyceride concentrations higher than 10 to 20 mmol/l are probably a risk factor for developing acute pancreatitis in humans. AIMS--To therefore analyse the influence of hyperlipaemia on the course of acute oedematous and acute necrotising pancreatitis in rats. SUBJECTS--Male Wistar rats were used in all experiments. METHODS--Six different groups of animals were used: two groups without pancreatitis (controls), two with acute oedematous pancreatitis, and two with acute necrotising pancreatitis. One group from each pair was treated with Triton WR 1339, which induces endogenous hyperlipaemia. Blood samples were taken from all subjects to measure triglyceride, cholesterol, amylase, and lipase. Pancreatic tissue samples were taken and the degree of pancreatic damage was judged microscopically. RESULTS--In the control groups no significant changes occurred, either in serum enzyme activities or in histology. The hyperlipaemic subgroup of animals with acute oedematous pancreatitis developed significantly higher (p < 0.001) serum amylase activities and a greater degree of histological damage (p < 0.01) than the animals of the non-hyperlipaemic acute oedematous pancreatitis group. In the animals with necrotising pancreatitis, serum lipase activity and the histological degree of pancreatic damage were significantly higher in the hyperlipaemic animals than in the non-hyperlipaemic animals. CONCLUSION--This study shows that hyperlipaemia intensifies the course of acute oedematous and acute necrotising pancreatitis in rats.

Osteopontin influences the invasiveness of pancreatic cancer cells and is increased in neoplastic and inflammatory conditions

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with an overall 5-year survival rate of less then 5%. Invasive tumour growth and early metastasis are two important reasons for this dismal prognosis. Osteopontin (OPN) is a secretory protein with a variety of functions, for example in cell adhesion and migration, inflammatory reaction and apoptosis. In this study the functional role of OPN in human pancreatic cancer and its potential use as a disease marker were analyzed. By real time quantitative PCR, there was a 2.2-fold and 1.6-fold increase of OPN mRNA in pancreatic cancers (n=23) and chronic pancreatitis samples (n=22), respectively, compared to normal pancreatic tissues (n=20). Immunohistochemical analysis demonstrated OPN staining in 60% of the primary pancreatic tumours and in 72% of the lymph node and liver metastases. ELISA analysis of serum samples obtained from pancreatic cancer patients (n=70), chronic pancreatitis patients (n=12), and healthy donors (n=20) showed a 1.6-fold increase in OPN serum levels in patients with tumours and a 1.9–fold increase in patients with chronic pancreatitis. Recombinant human OPN significantly increased the invasiveness of pancreatic cancer cells, without having any impact on cell proliferation. In addition, down-regulation of OPN by specific siRNA molecules decreased pancreatic cancer cell invasion. In conclusion, OPN serum levels in pancreatic cancer and chronic pancreatitis patients are not significantly different, thereby restricting its role as a prognostic or follow-up marker. Our results do suggest, however, that blockade of OPN might be useful as a therapeutic approach to inhibit invasion and metastasis of pancreatic cancer cells.

AZGP1 is a tumor suppressor in pancreatic cancer inducing mesenchymal-to-epithelial transdifferentiation by inhibiting TGF-β-mediated ERK signaling

Epithelial-to-mesenchymal transdifferentiation (EMT) mediated by transforming growth factor-β (TGF-β) signaling leads to aggressive cancer progression. In this study, we identified zinc-α2-glycoprotein (AZGP1, ZAG) as a tumor suppressor in pancreatic ductal adenocarcinoma whose expression is lost due to histone deacetylation. In vitro, ZAG silencing strikingly increased invasiveness of pancreatic cancer cells accompanied by the induction of a mesenchymal phenotype. Expression analysis of a set of EMT markers showed an increase in the expression of mesenchymal markers (vimentin (VIM) and integrin-α5) and a concomitant reduction in the expression of epithelial markers (cadherin 1 (CDH1), desmoplakin and keratin-19). Blockade of endogenous TGF-β signaling inhibited these morphological changes and the downregulation of CDH1, as elicited by ZAG silencing. In a ZAG-negative cell line, human recombinant ZAG (rZAG) specifically inhibited exogenous TGF-β-mediated tumor cell invasion and VIM expression. Furthermore, rZAG blocked TGF-β-mediated ERK2 phosphorylation. PCR array analysis revealed that ZAG-induced epithelial transdifferentiation was accompanied by a series of concerted cellular events including a shift in the energy metabolism and prosurvival signals. Thus, epigenetically regulated ZAG is a novel tumor suppressor essential for maintaining an epithelial phenotype.