Studies show that glycoprotein-130 may hold the key to bladder cancer treatment. Inhibition of this IL-6 cytokine receptor can reduce bladder cancer cell proliferation and tumor volume in mice, as shown in a study performed by Dr. Darryl Martin, an Associate Research Scientist working with Dr. Robert Weiss at Yale University in the Department of Urology.
In the search for bladder cancer targets, the Weiss lab found that the cytokine IL-6 was overexpressed in several bladder cancer cell lines. Unfortunately, inhibition of IL-6 expression did not alter cancer cell proliferation–but the scientists were not discouraged. The IL-6 receptor is located at a central point between 3 important pathways involved in cancer regulation: JAK/STAT, PI3K/AKT and ERK/MAPK. Perhaps greater success would be found by studying the IL-6 receptor. Glycoprotein-130, or GP-130, is a critical component of the IL-6 receptor, important for activation of downstream targets stimulated by IL-6 binding.
To begin, Martin evaluated GP-130 expression in human bladder cancer specimens by immunohistochemistry and found that levels of GP-130 protein were correlated to tumor grade. He also looked at GP-130 levels in cell lines derived from human bladder cancers. Cell lines from high grade tumors showed elevated GP-130, while a cell line from a low grade papillary tumor showed less protein as seen by Western blot.
Martin then began experiments knocking out GP-130 in vitro. siRNA for GP-130 caused a 60% decrease in cell viability and 50% reduced migration in the scratch assay. Crystal violet staining of cells showed a reduced number of cells. This showed promise for GP-130 as a target for bladder cancer treatment.
To explore the mechanism by which GP-130 regulates cell proliferation and viability, the effects of GP-130 knockdown on the PI3/STAT pathway were investigated. Western blots showed reduction of AKT by 20% and a 50% decrease in mTOR, suggesting that GP-130 indeed utilizes this pathway, but that others may be involved, as well. ERK1/2 and other pathways are currently being explored.
In his final set of experiments, Martin evaluated the effectiveness of GP-130 inhibition in vivo. Heterotransplant tumors were formed by subcutaneous injection of UMUC3 bladder cancer cells in nude mice. Upon tumor formation, GP-130 siRNA encapsulated in chitosan-coated nanoparticles was injected into tumors. Martin and Weiss developed this delivery system in collaboration with Drs. W. Mark Saltzman and Jill Steinbach in the Department of Biomedical Engineering at Yale in an attempt to enhance the amount of siRNA delivered to the cancer site. By delivering siRNA within nanoparticles, its stability is increased, and lower doses are required. One issue with cancer treatments instilled in the bladder is that the drugs do not always penetrate the urothelial wall. The coating of chitosan, a mucoadhesive polysaccharide, allows greater penetration of this bladder permeability barrier.
When the tumors were observed following treatment, tumor volume was significantly decreased in the siRNA-treated mice compared to the control mice. In addition, levels of the GP-130 protein were reduced, as well as those of the CK20 protein, a marker correlated with advanced tumor stage. The success of this experiment was a critical step for the advancement o f GP-130 as a bladder cancer target, as tests in animals are required before clinical trials may begin.
Future experiments will include injection of cancer cells into the bladder to create a model more similar to bladder cancer in humans. This would also allow Martin to evaluate the effectiveness of the chitosan nanoparticle to penetrate the urothelial wall, inhibit GP-130 expression, and tumor growth.