Application of two-dimensional gel-based mass spectrometry to functionally dissect resistance to targeted cancer therapy

Purpose: The majority of gastric cancers are diagnosed at advanced stages, characterized by robust therapy resistance. The oncoprotein hypoxia-inducible factor 1 (HIF-1) is associated with therapy resistance, partly via activation of the DNA damage response. We have noted a robust ability of gastric cancer cells to functionally compensate the loss of HIF-1 in vitro. The purpose of this study was to identify molecular pathways that underlie this compensation.

Experimental design: We performed 2DE to compare the nuclear proteome of wild-type and HIF-1-deficient gastric cancer cells. Differently expressed protein spots were identified via MS). After bioinformatic evaluation, functional validation of selected identified pathways was performed.

Results: 2DE displayed a total of 2523 protein spots, from which 87 were identified as regulated by HIF-1. Seventy of the identified spots were different proteins and 17 were isoforms. Bioinformatic analyses revealed that a significant amount of the identified proteins were related to cellular survival pathways. Specifically, members of the proteasome pathway were found upregulated upon loss of HIF-1. Combined inhibition of HIF-1 and the proteasome inflicted significant DNA damage, supporting the hypothesis that the proteasome is of functional importance to compensate the loss of HIF-1.

Conclusions and clinical relevance: Our data show robust and functional changes of the nuclear proteome upon inactivation of the HIF-1 oncoprotein in gastric cancer cells. We propose that 2DE-MS represents a useful tool to functionally dissect resistance mechanisms to targeted therapy and to identify novel targets for antiproliferative combination therapy.