Pharmacologic downregulation of c-FLIP(L) restores juxtacrine death receptor-mediated apoptosis in cancer cells in a peritoneal carcinomatosis model.

Metastasis occurs when circulating cancer cells implant in normal secondary tissues. Paradoxically, many cancer cells express death receptors while many normal tissues express the cognate death receptor ligands, suggesting that cancer cells possess mechanisms to inhibit death receptor signaling. Pharmacological restoration of juxtacrine-mediated death receptor signaling could prevent cancer cells from implanting in normal tissues such as the peritoneum. The results showed that BAY 11-7085 significantly inhibited peritoneal carcinomatosis in mice following the introduction of colon and pancreatic cancer cell lines into the intra-abdominal cavity. Treatment with BAY 11-7085 restored juxtacrine death receptor signaling during the adhesion of the cancer cells to mesothelial cells, which line the peritoneum. BAY 11-7085 rapidly inhibited c-FLIP(L) expression in colon and pancreatic cancer cell lines during adhesion to mesothelial cells. Pancreatic cancer cells sorted for high c-FLIP(L) expression formed peritoneal implants much more readily than cells with low c-FLIP(L) expression, and RNAi inhibition of c-FLIP(L) in colon cancer cells dramatically reduced peritoneal implantation. This is a novel demonstration that the restoration of death receptor-mediated apoptotic signaling in cancer cells through the pharmacological inhibition of c-FLIP(L) can inhibit tumor implantation in a clinically relevant model of peritoneal carcinomatosis, a fatal disease. Pharmacological inhibitors of FLIP hold promise as a way to curtail cancer cell colonization of secondary tissues.

Conditional deletion of beta1 integrins in the intestinal epithelium causes a loss of Hedgehog expression, intestinal hyperplasia, and early postnatal lethality.

Conditional deletion of beta1 integrins in the intestinal epithelium, unlike in epidermal and mammary epithelia, of mice does not result in decreased cell adhesion and proliferation, but instead causes a profound increase in epithelial proliferation with dysplasia and polypoid structures. The increased epithelial proliferation inhibited epithelial differentiation that caused severe malnutrition and early postnatal lethality. The striking similarities between beta1 integrin-deleted mice and neonatal mice with defective Hedgehog signaling led to the discovery that Hedgehog expression was markedly reduced in the former mice. beta1 integrins were found to drive the expression of Hedgehogs in intestinal epithelial cells in an HNF-3beta (Foxa2)-dependent fashion. The expression of Tcf-4, a transcription factor known to be required for intestinal epithelial stem cell proliferation, was increased and mislocalized in the intestinal epithelia of the beta1 integrin-deleted mice and in newborn mice treated with the Hedgehog signaling inhibitor cyclopamine. This study shows that beta1 integrins are key regulators of proliferation and homeostasis in the intestine and achieve this not through anchorage-dependent effects but by generating Hh expression and signaling.

The marine alkaloid naamidine A promotes caspase-dependent apoptosis in tumor cells.

Apoptosis is important for normal development and removal of damaged cells. Evasion of apoptosis by cancer cells is one of the key characteristics of many tumor types. Thus, discovering agents that promote apoptosis in tumor cells could have great therapeutic value. Marine natural products have demonstrated great potential as anticancer agents, and the proapoptotic activity of some of these products is emerging as a potentially useful property for cancer treatments. Using a tumor xenograft assay in rodents, we previously found that the marine alkaloid naamidine A is a potent antitumor agent. In this study, we further characterize the mechanism of action of naamidine A. In cultured tumor cells, we find that naamidine A induces cell death, which is accompanied with annexin V staining, disruption of the mitochondrial membrane potential, and cleavage and activation of caspases 3, 8, and 9, all of which are hallmarks of apoptosis. Furthermore, naamidine A-induced cell death is caspase dependent. We also find that under conditions where naamidine A inhibits tumor xenograft growth, it induces activation of caspase 3, suggesting that apoptosis is part of its antitumorigenic activity in vivo. Apoptosis is not dependent on extracellular signal-regulated kinase 1/2, previously characterized molecular targets of naamidine A, nor does it require functional p53. Our studies support the continued study of naamidine A and its target(s) for the potential development of better clinical treatments for cancer.

Effects of trastuzumab on epidermal growth factor receptor-dependent and -independent human colon cancer cells.

Little information is available as to the potential role of HER-2 as a therapeutic target in colon cancers, which express much fewer HER-2 receptors than breast cancer cells. Treatment of certain human colon cancer cell lines with the HER-2 inhibitory antibody mAb 4D5 demonstrated a role for HER-2 in mediating proliferation, apoptosis and tumorigenicity. However, only the cell lines that were dependent on autocrine EGFR-mediated cell proliferation were susceptible to the antiproliferative and antitumorigenic effects of HER-2 inhibition. The relative levels of HER-2, EGFR, HER-3 and HER-4 were not predictive of responsiveness to mAb 4D5. Treatment with HER-2 antibodies caused a decrease in HER-2 protein levels in all of the colon cancer cell lines and also significantly decreased EGFR levels but only in the EGFR-dependent cell lines. Treatment with mAb 4D5 caused the rapid ubiquitination and ligand-dependent downregulation of the EGFR in an EGFR-dependent colon cancer cell line. Treatment of athymic mice engrafted with EGFR-dependent colon cancer cells with mAb 4D5 caused tumor regression and a decrease in EGFR tyrosine phosphorylation in the tumor cells. EGFR-independent colon cancer cell xenografts were resistant to mAb 4D5 therapy. Combined inhibition of HER-2 and EGFR caused large areas of necrosis in EGFR-dependent colon cancer xenografts, suggesting a benefit of combined HER-2 and EGFR inhibitor therapy. Predicting clinical responsiveness of human colon cancer cells to anti-HER-2 and anti-EGFR therapy may require demonstration of EGFR tyrosine kinase dependency of the cells.