Detection of cellular signal transduction pathways in platinum-resistant ovarian cancer

Ovarian cancer (OC) has the highest mortality among gynecologic cancers. Up to 75% of OC patients develop recurrent OC due to increasing resistance to platinum-based chemotherapy. Despite many attempts to find predictive gene profiles, the molecular mechanisms of resistance development still remain unknown. Here we report on a project to perform pathway analysis in fresh frozen OC tissues using the novel DIGI-West approach developed by the NMI, Reutlingen, Germany. DIGI-West uses the principles of the Western blot (protein separation by SDS-PAGE; protein immobilization on a solid support; detection by specific antibodies) and combines it with a multiplexed bead array as a readout system. The system thereby generates hundreds of bead-based Western blot equivalents from a few micrograms of protein, providing information on the expression and modification of hundreds of proteins.

We checked a patient collective of 24 women having undergone surgery resecting OC tissue. 12 patients were classified as 'resistant' if relapsing half a year after the beginning of a platinum-based chemotherapy or within one year after surgery and subsequent chemotherapy. The other 12 patients were stated as 'sensitive'. Fresh frozen tumor samples were cut with a cryotome into 10 µm slices, and HE-stained. The samples were then assessed by a pathologist, affirming a tumor cell content of 60 – 90%. Afterwards, specimen were lysed and analyzed by DIGI-West.

We performed comprehensive signal transduction analyses of clinical OC specimen. 280 of 466 specific antibodies were applied using lysates of a 5mm3 tissue equivalent from every patient to analyze the activation state of different signaling cascades. This was followed by a t-test with consecutive hierarchical clustering analysis, revealing signaling mechanisms triggering proliferation. Pronounced differences were detected upon activation of a growth promoting signaling pathway, a change of expression for a central epigenetic regulator as well as for regulators of the cell cycle.

The DIGI-West method enables highly parallel analysis of minute tissue samples on protein level allowing comprehensive characterization of the activation status of signaling pathways. Candidate proteins may be functionally involved in platinum resistance development, presumably by promoting cell division and preventing cell cycle check point signaling mechanisms.