Conditional deletion of p53 and Rb in the renin-expressing compartment of the pancreas leads to a highly penetrant metastatic pancreatic neuroendocrine carcinoma.

Efforts to model human pancreatic neuroendocrine tumors (PanNETs) in animals have been moderately successful, with minimal evidence for glucagonomas or metastatic spread. The renin gene, although classically associated with expression in the kidney, is also expressed in many other extrarenal tissues including the pancreas. To induce tumorigenesis within rennin-specific tissues, floxed alleles of p53 and Rb were selectively abrogated using Cre-recombinase driven by the renin promoter. The primary neoplasm generated is a highly metastatic islet cell carcinoma of the pancreas. Lineage tracing identifies descendants of renin-expressing cells as pancreatic alpha cells despite a lack of active renin expression in the mature pancreas. Both primary and metastatic tumors express high levels of glucagon; furthermore, an increased level of glucagon is found in the serum, identifying the pancreatic cancer as a functional glucagonoma. This new model is highly penetrant and exhibits robust frequency of metastases to the lymph nodes and the liver, mimicking human disease, and provides a useful platform for better understanding pancreatic endocrine differentiation and development, as well as islet cell carcinogenesis. The use of fluorescent reporters for lineage tracing of the cells contributing to disease initiation and progression provides an unique opportunity to dissect the timeline of disease, examining mechanisms of the metastatic process, as well as recovering primary and metastatic cells for identifying cooperating mutations that are necessary for progression of disease.

Epidermal growth factor receptor-directed therapy in esophageal cancer.

Esophageal adenocarcinoma (EAC) is one of the fastest growing malignancies in the US. The long-term survival of patients with this cancer remains poor; only 25% of patients undergoing surgical excision are alive after 5 years. Multimodal programs that incorporate radiotherapy, chemotherapy and surgery for localized tumors may result in a modest survival advantage. However, significant strides in this disease can result from the inclusion of targeted therapies. The epidermal growth factor receptor (EGFR) family represents one such target and is receiving increasing attention due to the advent of specific inhibitors. Studies conducted by us and others have shown that the overexpression of EGFR family signaling intermediates is common in Barrett's esophagus and EAC. In the latter case, EGFR expression may have prognostic significance. EGFR inhibitors, including oral tyrosine kinase inhibitors and monoclonal antibodies, result in a synergistic antitumor effect with chemotherapeutic agents or with radiotherapy. Therefore, several ongoing studies include EGFR-directed therapy either alone or in combination with chemoradiotherapy for this disease. Our study of gefitinib, oxaliplatin and radiotherapy suggested that gefitinib can be safely incorporated into an oxaliplatin-based chemoradiation program for esophageal cancer, although the clinical activity of this combination is modest. Herein, we review the current literature on this subject.

EGF-dependent cell cycle progression is controlled by density-dependent regulation of Akt activation.

The normal human breast epithelial cell line, MCF10A, was used to investigate the mechanism by which high-density inhibits EGF-dependent cell cycle progression. EGF-dependent Akt activation was found to be transient in high-density cells and sustained in low-density cells. High-density cells also showed decreased EGF receptor (EGFR) autophosphorylation, decreased retinoblastoma protein phosphorylation, and increased p27 protein expression. Although EGFR activation was decreased in the high-density cells, the activation was sufficient to stimulate EGFR substrates comparable to low-density cells. EGF-dependent activation of the Erk1/2 pathway and the upstream activators of Akt (Gab1, erbB3, PI3 kinase, and PDK1) showed no density dependency. Antagonists of Akt activity provided further evidence that regulation of Akt activation is the critical signal transduction step controlling EGF-dependent cell cycle progression. Both adenovirus-mediated expression of dominant-negative Akt and inhibition of PI3 kinase-mediated Akt activation with LY294002 blocked cell cycle progression of low-density cells. In summary, we report the novel finding that high-density blocks EGF-dependent cell cycle progression by inhibiting EGF signaling at the level of EGF-dependent Akt activation rather than at the level of EGFR activation.

PTP LAR expression compared to prognostic indices in metastatic and non-metastatic breast cancer.

Several prognostic indices in breast cancer, including c-erbB2, epithelial growth factor receptors (EGFR), estrogen and progesterone receptors are signal transduction molecules. Recently, expression of another signal transduction molecule, the protein tyrosine phosphatase LAR, has been suggested to be increased in breast cancer. The objective of the current investigation was to examine the relationship between LAR expression and prognostic parameters in breast cancer. LAR expression was associated with metastatic potential in the well-characterized 13762NF rat mammary adenocarcinoma clones. The metastatic MTLn3 and MTLn2 clones expressed sizable amounts of LAR. The essentially non-metastatic MTC clone had little LAR expression. C-erbB2 had highest expression in the highly metastatic MTLn3 clone, but c-erbB2 levels were sizeable in the weakly metastatic MTLn2 and non-metastatic MTC clone. EGFR expression had the strongest association with a clone's metastatic potential, being very high in MTLn3, weak in MTLn2, and undetectable in MTC. In human breast cancer specimens, LAR expression was strongly positive in 50% of metastatic cases but in only 21% of 'non-metastatic' cases. As with the 13762NF-derived clones, c-erbB2 expression was strongly positive independent of metastatic phenotype. However, 46% (6/13) of cases that were strongly positive for c-erbB2 were strongly positive for LAR. Only 17% (2/11) of negative or weakly c-erbB2 positive samples were strongly positive for LAR. All ER+ positive tumors (n = 15) were positive for LAR and 53% of these tumors were strongly positive for LAR. In ER negative cases, only 1 of 11 was strongly positive for LAR. While the current data indicate a strong association between ER and LAR expression in breast cancer tissue (p = 0.003), additional studies are warranted to further explore the relationship between LAR and prognostic indices of breast cancer progression.

A newly recognized entity: intraductal "oncocytic" papillary neoplasm of the pancreas.

Intraductal papillary-mucinous tumors of the pancreas are increasingly recognized, and their characteristic endoscopic and radiological features are well reported in the literature in recent years. Oncocytic features in these tumors are uncommon and unrecognized. Intraductal oncocytic papillary neoplasm is a distinct pancreatic tumor and is a recently recognized entity. We report a case of a 69-yr-old patient who presented with symptoms mimicking pancreatitis, resulting in delay in the diagnosis of her pancreatic tumor. She underwent a successful Whipple's procedure and subsequently has remained well. The resected specimen showed an intraductal oncocytic papillary-mucinous neoplasm. The entity is new and the literature information is inadequate at present to judge the biological behavior of this tumor. We discuss this recently recognized entity.

Congenital tuberculosis in a neonatal intensive care unit: case report, epidemiological investigation, and management of exposures.

Disseminated tuberculosis was diagnosed at the autopsy of a 65-day-old premature infant who died in a 52-bed neonatal intensive care unit (NICU). Both parents and one sibling had previously had positive tuberculin skin tests (TSTs); none had active pulmonary tuberculosis, but a second sibling had hilar adenopathy. Congenital transmission was confirmed by isolation of Mycobacterium tuberculosis from the mother's endometrium and the infant's lung tissue. Both strains were identical by DNA restriction fragment analysis. TSTs were performed on 14 neonates, 27 NICU visitors, 11 contacts of the family, and 260 health care workers. TST conversion occurred in two nurses (0.8%); both had normal chest radiographs and received isoniazid therapy. Exposed neonates had negative chest radiographs, had negative gastric aspirates for acid-fast bacilli, and received isoniazid preventive therapy. Diagnosis of congenital tuberculosis requires a high index of suspicion. Transmission of tuberculosis in the NICU setting is unusual but can occur.

Ligand and p185c-neu density govern receptor interactions and tyrosine kinase activation.

The neu protooncogene (also known as c-erbB2, NGL, and HER2) encodes a 185-kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity that resembles the receptor for epidermal growth factor. The p185 gene and protein were originally identified in the brain and are thought to play a critical role in neurogenesis. Aberrant c-erbB2 protein overexpression also occurs in several human adenocarcinomas. A ligand for p185, neu-activating factor (NAF), specifically binds to neu receptor and increases the p185c-neu tyrosine phosphorylation in vitro and in vivo in a dose-dependent manner. We now show that NAF specifically binds to purified p185 expressed in baculovirus. Direct binding analysis showed that NAF binds with high affinity (Kd = 1.3 nM). We have investigated changes in the structure and association state of baculovirus-produced neu holoreceptor that are induced by ligand binding. In this study, we used sucrose gradients to show that purified p185c-neu exists mainly in the monomeric form at low concentrations, whereas at higher concentrations p185c-neu exists as dimers or multimers. At low concentrations, but in the presence of ligand, p185c-neu sediments as a dimeric or multimeric form. Monomer-oligomer interconversion is absolutely ligand dependent at low receptor concentrations. The high molecular weight form of the receptor is enzymatically more active, as a consequence of ligand-driven activation of the receptor kinase. Oncogenic p185neu receptors sediment predominantly as high molecular weight forms and have constitutively active kinases.

Heterodimerization of epidermal growth factor receptor and wild-type or kinase-deficient Neu: a mechanism of interreceptor kinase activation and transphosphorylation.

We have shown that members of the erbB family undergo homodimer and heterodimer formation. The rat p185c-neu and the epidermal growth factor receptor (EGFR) can associate into an active heterodimeric tyrosine kinase. Overexpression of these two receptors also results in a transformed phenotype. We now show that mutant Neu proteins resulting from a point mutation at the ATP-binding site (N757) or cytoplasmic domain deletions (N691stop) are still able to undergo EGF-induced heterodimerization with EGFR. Analysis of heterodimer formation between EGFR and truncated Neu proteins revealed that heterodimerization is preferred over homodimerization of EGFR. N757 can be transphosphorylated by associated EGFR upon EGF stimulation. However, the heterodimer composed of EGFR and N691stop is kinase inactive. These results provided evidence that the Neu ectodomain is sufficient to associate with EGFR physically, and the cytoplasmic domain interaction is required for heterodimeric kinase activation, indicating that Neu/c-erbB2 is not just a simple substrate for EGFR but a transactivator as well.

A structural and kinetic comparison of proto-oncogenic and oncogenic neu holo-receptors expressed in insect cells.

The proto-oncogenic and oncogenic forms of the rat neu receptors were expressed in the baculovirus system to characterize their structural and enzymatic differences. The epitopes of their extracellular domains, their molecular weights, and kinase activities were similar to rat neu receptors expressed in fibroblasts. The receptors were partially purified using a phospho-agarose column and were analyzed to compare kinetic parameters using ATP as a substrate. The oncogenic form of the receptor showed a significant increase in Vmax (56%) over the proto-oncogenic form. Structural analysis of these proteins using sucrose gradients showed the oncogenic receptors to have a 62.6% increase in aggregated receptors when compared to the proto-oncogenic receptors. These studies are the first to link enzymatic activation and the physical form of the receptor using isolated receptor species.

Expression, purification, and characterization of Bacneu. A soluble protein tyrosine kinase domain encoded by the neu-oncogene.

To further characterize the structure and regulation of the tyrosine kinase encoded by the rodent neu oncogene, its cytoplasmic tyrosine kinase domain has been expressed as a soluble protein, called Bacneu, in Sf9 insect cells, using the baculovirus expression system. Expression of Bacneu was detected by immunoblotting with anti p185neu antisera and in vitro autophosphorylation analysis as early as 24 h postinfection. Maximal expression was observed at 48 h postinfection. The soluble kinase was purified to near homogeneity by sequential chromatography on DEAE-Sepharose, phosphocellulose, poly-L-lysine, and Sephacryl 300, yielding 0.55 mg Bacneu per L of Sf9 cells (4% yield). The kinase is more active in the presence of Mn2+ compared to Mg2+ ions. The specific activity of the kinase using poly(Glu4Tyr1) as a substrate is 179 nmol/min/mg. Maximal incorporation of 1.4 mol of phosphate per mol of enzyme by autophosphorylation was found to increase the activity of the enzyme 1.5- to twofold. These results indicate that the Bacneu kinase is activated by phosphorylation. Therefore, it will be a useful reagent for characterizing the effects that phosphorylation by other cellular kinases and dephosphorylation by phosphatases have on its activity.

Anti-receptor antibodies reverse the phenotype of cells transformed by two interacting proto-oncogene encoded receptor proteins.

The neu oncogene product, p185neu, is a tyrosine kinase receptor with structural similarity to the epidermal growth factor (EGF) receptor. We have recently described that coexpression of EGF receptors and high levels of normal p185c-neu lead to transformation of rodent fibroblasts. Anti-EGF receptor and anti-p185neu monoclonal antibodies inhibited tumorigenic growth of these transformants implanted into nude mice. These monoclonal antibodies also suppressed focus formation of the cells transformed by the synergistic action of these receptor proteins in vitro. However, EGF enhanced focus formation and stimulated cell growth when added to cells transfected just with the EGF receptor encoding cDNA. These data suggest that receptor specific effectors may have potentially useful applications in cancer therapy for neoplasms which demonstrate increased receptor densities. In addition the data suggest novel differences in the actions of tyrosine kinases when acting alone or in concert with other receptors.

Synergistic interaction of p185c-neu and the EGF receptor leads to transformation of rodent fibroblasts.

The protein product of the rodent neu oncogene, p185neu, is a tyrosine kinase with structural similarity to the epidermal growth factor receptor (EGFR). Transfection and subsequent overexpression of the human p185c-erbB-2 protein transforms NIH 3T3 cells in vitro. However, NIH 3T3 cells are not transformed by overexpressed rodent p185c-neu. NIH 3T3 transfectants overexpressing EGF receptors are not transformed unless incompletely transformed. Several groups have recently demonstrated EGF-induced, EGFR-mediated phosphorylation of p185c-neu. During efforts to characterize the interaction of p185c-neu with EGFR further, we created cell lines that simultaneously overexpress both p185c-neu and EGFR and observed that these cells become transformed. These observations demonstrate that two distinct, overexpressed tyrosine kinases can act synergistically to transform NIH 3T3 cells, thus identifying a novel mechanism that can lead to transformation.