Stable reintroduction of wild-type P53 (MTmp53ts) causes the induction of apoptosis and neuroendocrine-like differentiation in human ductal pancreatic carcinoma cells.

Pancreatic ductal adenocarcinoma is one of the major causes of cancer mortality in the industrialized world, having among the poorest prognosis of any malignancy. Mutations or alterations in the p53 tumor suppressor gene/protein are observed in 50-70% of these cancers, yet little information is available regarding the phenotypic effects of restoration of wild-type (wt) p53 function in pancreatic ductal carcinoma cells. The consequences of stable reintroduction of wt p53 on apoptosis and differentiation was examined in a poorly differentiated pancreatic carcinoma cell line (Panc-1), possessing only mutant (mt) p53 (codon 273 mutation). Cells were transfected with a temperature-sensitive mouse p53val135 (tsp53) vector under additional control of a genetically-modified metallothionein promoter. This tsp53 has a 'mt' phenotype at 37.5 degrees C, and a 'wt' phenotype at 32.5 degrees C and the presence of 100 microM ZnCl2. Stable expression of wt p53 caused upregulation of the p21/WAF1 gene, and G1 growth arrest as shown by flow cytometry and BrdU labeling. Additionally, apoptosis was induced 8-12 post-induction in the majority of the cells (60-70%), as demonstrated by morphological changes, in situ TdT labeling and internucleosomal laddering. However, a subpopulation (30%) of the transfectants survived this apoptotic fate. Unlike the epithelial parental Panc-1 cells, these cells exhibited the appearance of a neuroendocrine-like phenotype with extensive branch-like processes, and marked cytoplasmic and cytoskeletal immunostaining for tau-2, synaptophysin, and chromogranin A. These studies suggest that stable and regulated expression of wt p53 can have multiple phenotypic consequences (apoptosis and altered differentiation to a neuroendocrine-like phenotype) in poorly-differentiated pancreatic carcinoma cells.

The Trk tyrosine kinase inhibitor CEP-701 (KT-5555) exhibits significant antitumor efficacy in preclinical xenograft models of human pancreatic ductal adenocarcinoma.

The aggressive behavior and poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is associated with an increased expression of many growth factors and their cognate receptors. We have previously demonstrated the aberrant expression of the Trk receptors (Trks A, B, and C), enhanced tumor stromal expression of neurotrophins in primary PDAC specimens and human PDAC-derived cell lines, and a dose-dependent biological response of PDAC cells (in vitro invasiveness) to selective neurotrophins (Miknyoczki, S. J., et al., Int. J. Cancer, 81: 417-427, 1999). On the basis of these data, we have evaluated the therapeutic potential of inhibiting neurotrophin-Trk interactions using a selective and potent Trk tyrosine kinase inhibitor (CEP-701) in several preclinical models of human PDAC. CEP-701 is currently approved for clinical trials within the United States We demonstrate that CEP-701 administration at 10 mg/kg s.c. b.i.d. 5 days a week for 21-28 days inhibited tumor growth in a statistically significant manner in Panc-1, AsPc-1, BxPc-3, Colo 357, and MiaPaCa2 s.c. xenografts in athymic nude mice compared with vehicle-treated controls. Reductions in tumor growth volume of 50-70% relative to vehicle-treated controls were observed in xenografts responsive to CEP-701 administration. Significant reductions of in vivo PDAC tumor invasiveness were likewise observed in four of six CEP-701-treated rat tracheal xenografts implanted s.c. in athymic nude mice. The antitumor efficacy of CEP-701 was observed in the absence of pronounced morbidity or toxicity in vivo. Taken together, these data suggest that CEP-701 may be effective as a potential therapeutic agent in the treatment or management of PDAC.

Pan-trk inhibition decreases metastasis and enhances host survival in experimental models as a result of its selective induction of apoptosis of prostate cancer cells.

During the progression of prostate cancer, molecular changes occur resulting in the autocrine production of a series of neurotrophins by the malignant cells. This is coupled with expression of high-affinity cognate receptors for these ligands, termed trk receptors, by these cancer cells. The binding of the neurotrophins to their trk receptors activates the receptor's latent tyrosine kinase activity inducing a series of signal transduction pathways within these prostate cancer cells. These molecular changes result in the acquisition by prostate cancer cells of a restricted requirement for these trk signaling pathways for optimal survival. CEP-701 is an indolocarbazole compound specifically designed as a potent inhibitor (IC(50), 4 nM) of the tyrosine kinase activity of the trk receptors required for initiation of these survival pathways. In the present studies, the consequences of CEP-701 inhibition of these trk signaling survival pathways were tested in vivo using both rat (R3327 AT 6.3 and H) and human (TSU-pr1 and CWR-22Rv1) prostatic cancer models. These in vivo studies demonstrated that treatment with CEP-701 inhibits the growth of both rodent and human prostate cancers, without being toxic to the normal tissue including the host prostate. Because of this selective effect, CEP-701 inhibits metastasis and growth of both primary and metastatic sites of prostate cancer. Based upon this profile, long-term survival studies were performed using the slow-growing Dunning H rat prostate cancer model. For these latter studies, the dosing regimen was 10 mg CEP-701/kg/dose twice a day via gavage 5 days a week. This regimen maintains CEP-701 tumor tissue concentrations of 25-50 nM. Such chronic dosing increased (P < 0.001) the median survival of rats bearing the slow growing H prostate cancers from 408 days (395-432 days, 95% confidence interval) for the vehicle group (n = 18) to 566 days (497-598 days, 95% confidence interval) for the CEP-701-treated group (n = 24).

Role of neurotrophin-trk interactions in oncology: the anti-tumor efficacy of potent and selective trk tyrosine kinase inhibitors in pre-clinical tumor models.

The increasing knowledge of the pathogenic mechanisms involved in cancer cell growth has enabled the implementation of mechanism based drug design approaches rather than the more conventional nonspecific approaches. Drugs that interfere with cellular signal transduction pathways are currently a major focus in development of newer therapeutic technologies in oncology. The therapeutic utility of potent and selective tyrosine kinase inhibitors in oncologic applications has become widely recognized for several years, however targeting neurotrophin receptors as a molecular target driven approach has only recently been realized. This review presents the hypothesis of the neurotrophin-trk receptors as a viable molecular target for medicinal chemical intervention in tumor biology, followed by an overview of the pre-clinical studies which culminated in the advancement of the first potent trk tyrosine kinase inhibitor CEP-2563 (KT-8391), and the orally active K-252a analog, CEP-701 (KT-5555) into clinical evaluation.

The novel Trk receptor tyrosine kinase inhibitor CEP-701 (KT-5555) exhibits antitumor efficacy against human pancreatic carcinoma (Panc1) xenograft growth and in vivo invasiveness.

The survival rate for patients with pancreatic ductal adenocarcinoma (PDAC) is among the poorest for all cancers. The factors that contribute to this poor prognosis are lack of effective early detection, high rate of metastases and a generally refractory response to available treatment modalities. The most commonly used treatment methods--chemotherapy and radiation therapy--are mainly used for symptom palliation, with surgery being the only "curative" treatment option. The use of combinations of treatment modalities is the only therapy available to patients with locally advanced disease or that which is surgically unresectable. These options are still not sufficient to increase patient survival time significantly. The aggressive behavior and poor prognosis of this cancer is associated with an increased expression of many growth factors and their cognate receptors. We have demonstrated previously the aberrant expression of the Trk receptors (Trks A, B, and C) in PDAC specimens and human PDAC-derived cell lines and a biphasic, dose-dependent response of specific neurotrophic agents on the in vitro invasiveness of PDAC cells. Based on these data we have evaluated the therapeutic potential of inhibiting neurotrophin-Trk interactions using a selective Trk tyrosine kinase inhibitor (CEP-701) on subcutaneous (s.c.) and tracheal xenografts derived from the poorly differentiated PDAC cell line, Panc1. We demonstrate that CEP-701 administration at 10 mg/kg s.c. BID for 21 days inhibited tumor growth of the Panc1 s.c. xenografts in a statistically-significant manner (p < 0.01) compared to vehicle controls, in the absence of morbidity and mortality. A T/C value of 25% was observed for CEP-701-treated s.c. xenografts. In addition, CEP-701 administration inhibited tumor cell invasion in the s.c. tracheal xenograft model of in vivo invasiveness. Taken together, these data suggest that further studies are warranted to evaluate CEP-701 as a potential therapeutic agent in the treatment of PDAC.

Chemopreventive activity of Oltipraz against N-nitrosobis(2-oxopropyl)amine (BOP)-induced ductal pancreatic carcinoma development and effects on survival of Syrian golden hamsters.

The synthetic dithiolethione Oltipraz has marked cancer chemopreventive and phase II enzyme inducing activity in various animal carcinogenesis models, but has not been examined in any animal models of ductal pancreatic cancer relevant to the human disease. The chemopreventive potential of Oltipraz on pancreatic tumor incidence and multiplicity was examined in the N-nitrosobis(2-oxopropyl)-amine (BOP)-induced ductal pancreatic adenocarcinoma model in Syrian hamsters. Animals were maintained on control semipurified diets or semipurified diets containing 300 and 600 mg/kg Oltipraz beginning 2 weeks prior to BOP initiation and throughout the 26 week study. Oltipraz at 300 mg/kg had no effect on the incidence or multiplicity of preneoplastic, neoplastic or metastatic lesions, while at 600 mg/kg dietary Oltipraz the incidence of pancreatic adenocarcinomas was reduced significantly (P < or = 0.05) compared to BOP-treated controls. Dietary Oltipraz at both doses had a significant influence on reducing mortality and morbidity in tumor-bearing animals with metastatic disease. At 26 weeks, total hepatic glutathione-S transferase (GST) activity and GST mu activity were elevated significantly in Oltipraz-treated animals, while total pancreatic GST activity was reduced, albeit not significantly. Serum lipase activity, a marker for pancreatic damage, exhibited a progressive decline in BOP-treated animals administered Oltipraz compared to BOP-treated controls at 12 weeks of the study; by week 26, lipase activity was comparable in all groups and reduced compared to activity at week 12. Positive nuclear immunostaining for the p53 tumor suppressor protein, a hallmark of human pancreatic cancer and a transient response to DNA damage, was observed in only a small percentage of BOP-induced pancreatic lesions and was not influenced Oltipraz administration. Further chemoprevention and pharmacologic studies of Oltipraz in relevant animal models of ductal pancreatic cancer could provide a foundation for future studies in human populations at potential risk for pancreatic cancer.

Neurotrophin-Trk receptor interactions in neoplasia: a possible role in interstitial and perineural invasion in ductal pancreatic cancer.

Autocrine and paracrine influences of growth factors play a critical role in the regulation of the growth, survival, differentiation, and invasion potential of tumor cells. These influences on the neoplastic phenotype are particularly important in those cancers in which tumor-stroma interactions constitute important components of tumor development, as exemplified by prostatic, breast, and pancreatic carcinomas. The neurotrophins and their corresponding Trk and p75(NGFR) receptor subtypes are families of growth factors and receptors that have received relatively little attention with respect to neoplasia. This review attempts to summarize their biochemical properties, their role in neuronal and non-neuronal systems, and their involvement in the development of a variety of cancers, particularly those in which perineural invasion and/or metastasis to the CNS are a part of the pathophysiological presentation. In this regard, we have focused on our studies of neurotrophin-Trk/p75(NGFR) expression and interactions in pancreatic ductal carcinoma (PDAC) and their potential role in the perineural invasive phenotype characteristic of this cancer.

Neurotrophins and Trk receptors in human pancreatic ductal adenocarcinoma: expression patterns and effects on in vitro invasive behavior.

The aggressive and highly metastatic behavior observed in pancreatic ductal adenocarcinoma (PDAC) may be due to autocrine and/or paracrine interactions (tumor/stromal) involving altered expression of peptide growth factors and their corresponding receptors. The neurotrophin (NT) growth factor family and their cognate receptors have been demonstrated to play a role in the invasiveness, chemotactic behavior and tumor cell survival of both neuronal and non-neuronal cancers. We hypothesized that aberrant expression of the NTs and/or the Trk receptors may contribute to the malignant phenotype of PDAC, specifically tumor cell invasiveness, through autocrine and/or paracrine interactions. In this study, we examined the expression of NTs, Trks and p75NGFR by immunohistochemical and in situ hybridization analyses in both normal (n=14) and neoplastic pancreas (n=47) and PDAC-derived cell lines (n=6). Further, we evaluated the effects of various NTs on the in vitro invasive and chemotactic behavior on 6 human PDAC-derived cell lines in a modified Boyden chamber assay. Brain-derived nerve growth factor (BDNF), NT-3, NT-4/5 and Trks A, B and C exhibited diffuse cytoplasmic and membranous immunostaining patterns in both the ducts and the acini of the exocrine pancreas and the islets of the endocrine pancreas of both normal and PDAC specimens. NT expression was primarily within the stromal compartment of the tumor, while Trk expression was weak or absent. We observed a 68%, 64% and 66% increase in the expression of Trks A, B and C, respectively, in the ductal elements of the PDAC samples examined compared with the normal adjacent tissue. Invasiveness of 4 of 6 PDAC cell lines was significantly inhibited (p<0.05) when the cells were incubated with 100 ng/ml NT. However, when select cell lines were incubated with lower concentrations of NT-3 and BDNF (0, 1, 5, 25 and 50 ng/ml), invasiveness was significantly stimulated (p<0.05) through the Matrigel matrix. Collectively, our data suggest the possibility that paracrine and/or autocrine NT-Trk interactions may influence the phenotype (possibly the invasive behavior) of PDAC.