Michael S. Kobrin

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.

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.

Attenuated ALK5 receptor expression in human pancreatic cancer: Correlation with resistance to growth inhibition

Transforming growth factor‐β (TGF‐β) receptors constitute a family of transmembrane proteins that bind TGF‐β ligands. In this study we assessed the growth responsiveness to TGF‐β1 in pancreatic cancer cell lines and characterized the levels of expression of TGF‐β receptors in these cell lines and in human pancreatic cancer tissues. COLO 357 cells were most sensitive to the growth inhibitory actions of TGF‐β1, PANC‐1 cells exhibited moderate sensitivity, Hs766T cells exhibited slight sensitivity and MIA PaCa‐2 and T3M4 cells were resistant to TGF‐β1. Only COLO 357 cells expressed high levels of ALK5, the major type I TGF‐β receptor (TβRI). Hs766T and PANC‐1 cells expressed high levels of SKRI, another TβRI subtype. Only MIA PaCa‐2 cells did not exhibit the type II TGF‐β receptor (Tβ‐RII) transcript, whereas type III TGF‐β receptor (Tβ‐RIII) mRNA levels were elevated in this cell line and in HS766T cells. All the cell lines expressed TGF‐β1, but TGF‐β2 and TGF‐β3 mRNA levels were variable. ALK5 and SKRI mRNA levels were 6.8‐ and 9‐fold greater in the pancreatic tumors in comparison with the corresponding levels in the normal pancreas. However, in the cancer cells, ALK5 immunoreactivity was faint, whereas TβRII immunoreactivity was focal and intense. Conversely, in ductal cells adjacent to cancer cells ALK5 immunoreactivity was strong, whereas TβRII immunoreactivity was weak. Since ALK5 heterodimerization with TβRII is crucial for TGF‐β‐mediated signaling, our findings suggest that low levels of ALK5 in pancreatic cancer cells within a tumor may protect against growth inhibition.

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.

Transforming growth factor-α in normal and neoplastic human endocrine tissues

Abstract Transforming growth factor-α (TGF-α) is a polypeptide growth factor that binds to the epidermal growth factor receptor and is thought to stimulate cell proliferation. It has been believed to play a role in tumor initiation by inducing the reversible transformed phenotype; overexpression of TGF-α may be important for tumor progression via autocrine stimulation and oncogene overexpression. Expression of TGF-α and the epidermal growth factor receptor has been documented in several nontumorous tissues and in a variety of tumors. This study used immunohistochemistry to localize TGF-α expression in normal and neoplastic endocrine tissues. Transforming growth factor-α was found in nontumorous hypothalamus, pituitary, thyroid, parathyroid, adrenal cortex and medulla, and pancreatic islets. Immunoreactivity was detected in most benign and malignant tumors of these tissues, as well as in endocrine neoplasms of the lung and gastrointestinal tract. Hypothalamic gangliocytomas, pheochromocytomas, and adrenal cortical carcinomas showed consistently greater immunoreactivity than their normal counterpart, but there was no correlation between degree of reactivity and tumor grade, stage, or hormone content. These results suggest that in endocrine tissues, TGF-α is unlikely to prove useful as a tumor marker but that the growth factor may play a role in both normal physiology and tumorigenesis.

Expression of a truncated EGF receptor is associated with inhibition of pancreatic cancer cell growth and enhanced sensitivity to cisplatinum

Human pancreatic cancers over‐express the epidermal growth factor receptor (EGF‐R) and all 5 known ligands of the EGF family, including EGF, transforming growth factor‐alpha (TGF‐α), amphiregulin, betacellulin and heparin‐binding EGF‐like growth factor (HB‐EGF). The aim of the present study was to confirm the presence of EGF‐R‐dependent autocrine loops in a human pancreatic cancer cell line and to explore the possibility that interrupting EGF‐R activation by introducing a truncated receptor abrogates pancreatic cancer cell growth. The anchorage‐independent growth of PANC‐1 human pancreatic cancer cells, previously shown to express TGF‐α, was inhibited by specific anti TGF‐α antibodies. PANC‐ 1 cells were then either transfected with an expression plasmid encoding a kinase‐deficient EGF‐R cDNA (HER653) or infected with the same EGF‐R cDNA using a retroviral vector. Multiple transfected and infected clones co‐expressed the truncated EGF‐R and endogenous EGF‐R as revealed by Northern blot analysis and immunoblots. In these clones, there was a marked attenuation in EGF‐ and TGF‐α‐mediated EGF‐R tyrosine phosphorylation and c‐tos induction. There was also a significant decrease in colony formation in soft agar by comparison with control cells and a significant increase in the effect of the growth‐inhibitory effect of the alkylating agent cisplatinum in these clones. Our observations indicate that dominant negative inhibition of EGF‐R may have therapeutic potential in pancreatic cancer.

A Subgroup of Patients with Chronic Pancreatitis Overexpress the c-erb B-2 Protooncogene

ObjectiveChronic pancreatitis (CP) is a chronic condition associated with pancreatic fibrosis. A small subgroup of patients with CP develop enlargement of the head of the pancreas (EHP). This study examined some of the mechanisms that may lead to the development of EHP. Summary Background DataThe c-erb B-2 protooncogene encodes a 185-kDa transmembrane growth factor receptor (p185) that regulates cell growth and differentiation. MethodsThe authors analyzed c-erb B-2 expression in samples obtained from the head of the pancreas from 26 patients with CP (5 women, 21 men) using immunohistochemical and molecular techniques. A diagnosis of CP with EHP was made when the vertical pancreatic head diameter was greater than 4 cm (14 patients), as determined by contrast-enhanced computed axial tomography scan. Pancreatic tissues from 15 healthy organ donors served as control subjects. ResultsIn all patients without EHP and in the healthy control subjects, p185 immunoreactivity was present at low levels. In contrast, strong p185 immunoreactivity was observed in acinar and ductal cells in all patients with EHP. By in situ hybridization, c-erb B-2 messenger ribonucleic acid (mRNA) grains were expressed at high levels in patients with CP with EHP In both ductal and acinar cells. Northern blot analysis demonstrated a 4.5-fold increase (p < 0.001) in c-erb B-2 mRNA levels in patients with EHP compared with patients without EHP and healthy control subjects. Southern blot analysis did not reveal c-erb B-2 gene amplification or rearrangement. ConclusionsThese findings indicate the c-erb B-2 is not overexpressed in most patients with CP. However, its overexpression in patients with CP with EHP suggest that c-erb B-2 may contribute to the pathophysiologic processes that lead to pancreatic head enlargement.

Cytotoxic effects of TGF-α-Pseudomonas exotoxin A fusion protein in human pancreatic carcinoma cells

The epidermal growth factor (EGF) receptor is overexpressed in human pancreatic cancers and cultured cell lines. TP40 is a chimeric protein composed of transforming growth factor-α (TGF-α) linked to a modified Pseudomonas exotoxin A (PE40) that exerts growth inhibitory effects on cells bearing a high number of EGF receptors. Therefore, we compared the effect of TP40 on the growth of Chinese hamster ovary (CHO) cells expressing varying levels of the EGF receptor and on the growth of two human pancreatic cancer cell lines. The growth of CHO cells devoid of endogenous EGF receptors was minimally altered by high concentrations of TP40, even following a 72-h incubation period. In contrast, in CHO cells expressing ∼95,000 and 438,000 EGF receptors per cell, one-half maximal growth inhibition occurred at 5 and 3 ng/ml TP40, respectively. Following a 72-h incubation in T3M4 and COLO 357 human pancreatic cancer cells, one-half maximal growth inhibition occurred at 0.2 and 0.4 ng/ml TP40, respectively. This effect was significantly greater than that of native Pseudomonas exotoxin A. These findings indicate that human pancreatic cancer cells are markedly sensitive to the growth inhibitory effects of TP40 and raise the possibility that TP40 may have a therapeutic role in this disorder.

Single-strand conformation polymorphism analysis of the epidermal growth factor receptor at codon 497.

A variant human epidermal growth factor (EGF) receptor (HER) with a transition (G to A) at codon 497, resulting in a substitution of a lysine for an arginine, was recently demonstrated in several human pancreatic cancer cell lines. In the present study, we compared the frequency of expression of wild-type (HER497R) and variant (HER497K) EGF receptors in normal and malignant pancreatic tissue samples using single-strand conformation polymorphism analysis. Both receptors were expressed in normal (42%) and malignant (46%) tissues. Three samples (two normal and one cancer) expressed only HER497K. The cancer sample that was homozygous for HER497K exhibited strong EGF receptor immunore-activity. The high frequency of HER497K expression suggests that it is due to a relatively common polymorphism in the HER coding region. Its presence in some of the cancer samples suggests that, like the wild-type receptor, HER497K also has a role in pancreatic cancer.