Abstract
Pancreatic cancer is a deadly disease and is the fourth leading cause of cancer deaths in the United States. It is characterized by poor prognosis, metastasis at the time of diagnosis and lack of response to conventional therapies. Surgical resection is the only effective treatment for this disease. However, at the time of diagnosis only 5% of patients are candidates for surgical resection and the 5 year survival rate after treatment in less than 2%.|Retinoids are differentiating agents, that have the potential to inhibit the proliferative capacity of cancer cells as well as induce morphological and biochemical changes. They can induce cancer cells to redifferentiate. Therefore, retinoids offer an attractive alternative to chemotherapeutic regimens in the treatment of cancer. An advantage in the use of retinoids as therapeutic agents is that they can target cancer cells, but have minimal effects on normal cells. They, can also inhibit cancer progression regardless of the p53 status.|The most successful therapeutic use of retinoids has been in changing the clinical course of acute promyelocytic leukemia (APL), from a highly lethal disease to complete remission in >90% of cases after treatment. Promising results have also been obtained with 9-cis- retinoic acid in combination with tamoxifen in Phase I trials in breast cancer. Retinoic acid has also been successful in the treatment of several other malignancies.|Clinical trials with 13-cis retinoic acid in combination with interferon-a in patients with pancreatic cancer have not been very successful. However, the limited effectiveness of retinoids in these patients was expected, since they were terminally ill cancer patients.|There have been conflicting reports regarding the efficacy of retinoids to induce anti-cancer effects in pancreatic cancer cells, which appear to be mainly due to differences in treatment conditions. Conditions for retinoic acid treatment were optimized in our laboratory. Under optimized conditions, all the pancreatic cancer cell lines tested were very sensitive to the growth inhibitory effects of all the three isoforms of retinoic acid (9-cis, 13-cis and all-trans retinoic acid), regardless of their p53 and differentiation status. However, the molecular pathways involved in mediating these growth inhibitory effects in pancreatic cancer cells have not been clearly elucidated.|In order to truly understand the potential of retinoids as anti-cancer agents, it is imperative to study the molecular pathways involved in mediating these effects in pancreatic cancer cells. The first step was to study the effect of retinoids in inducing cell cycle-related changes in these cells. These include changes in the cell cycle, as well as in cellular concentrations of cyclins and their associated cyclin-dependent kinase (CDK) sub-units and CDK inhibitors (CDKI) such as p21, p27 and p16. Previously published studies have shown minimal effects of natural retinoids in inducing such changes in pancreatic cancer cell lines.|In the present studies with optimized conditions, flow cytometry data indicated that retinoic acid induced cell cycle arrest at the G0/G1 and G2/M phases. The cyclin/CDKs associated G0/G1 and G2/M phases, such as cyclin D, but not CDK4, cyclin E /CDk2 and cyclin B/CDC2 were decreased by retinoic acid. This is consistent with studies that show that retinoic acid induces apoptosis, since an apoptotic cell population (sub G0/G1 peak) was seen in treated cells. Therefore, these results clearly demonstrate that retinoic acid triggered cell cycle related changes that are coupled with apoptosis in pancreatic cancer cells.|In one cell line levels of the CDKI, p21Waf1 protein were decreased, while those of p27Kip1 were unchanged in response to retinoic acid. The p21Waf1 mRNA expression levels were transiently increased at early time points (6-18hours), but were unchanged at later time points. In another cell line, p21Waf1 mRNA expression increased with time and concentration, while p21Waf1 protein levels were decreased. The decrease in the p21 protein levels could be blocked by proteosome inhibitors, indicating that retinoic acid was triggering the proteosomal degradation of p21 protein. This reflects a pro-survival mechanism of cancer cells in response to the therapeutic agent. While immunoprecipitation studies revealed that the degradation of p21 was ubiquitin-independent, an increase in expression of a novel E3 ligase p53RFP was also seen. Therefore, it can be concluded that (1) p21 can be subject to degradation by several systems, which are independently predominant under particular physiological conditions or stages of the cell cycle, (2) stoichiometrically, an increase of unbound p21, due to the decrease in cyclin/CDK levels, could be targeted directly to the proteosome.|Retinoic acid stimulated an marked reduction in the density and function of EGFR-mediated autocrine pathway. This is the major growth-sustaining pathway in pancreatic cancer cells. The down-regulation of the EGFR pathway was coupled to increased secretion of TGF-p (active and latent forms), a potent antiproliferative factor in epithelial cells. Further studies revealed that retinoic acid induced the secretion and activation of TGF-P2 in pancreatic cancer cells, regardless of their differentiation status. Retinoic acid also increased the bioactivity of TGF-p, as measured by the inhibition of proliferation of mink lung epithelial cells. Specificity of these cells to the presence of TGF-p was confirmed by a pan-neutralizing TGF-p antibody in the conditioned medium of retinoid treated cells. Pancreatic cancer cells were very sensitive to the growth inhibitory effects of TGF-P2. Cells with a homozygously deleted DPC-4 gene were also very sensitive to the growth inhibitory effects of TGF-p. These cells have been previously shown to be moderately sensitive to TGF-p. Immunoneutralization of TGF-p by pan-neutralizing antibodies partially, but significantly, prevented the growth-inhibitory effects of retinoic acid. Overall it can be concluded that other pathways in addition to the TGF-p signaling pathway may be involved in mediating the anti-proliferative effects of retinoic acid.|All of the current findings indicate that retinoic acid is a multi-potent differentiating agent with multiple effects on various cellular pathways. Its effects are mediated through multiple independent pathways. These studies indicate that retinoids are agents which are likely to be valuable in the treatment of pancreatic adenocarcinoma, which is at present the deadliest of cancers.