Functional Characterization Of OLA1, A Stress Response Protein And Potential Molecular Target For Anti-tumor Therapy

Cancer deaths are attributable not only to the primary tumor but also metastasis. Current therapies for late stage cancers are largely ineffective or only able to extend life slightly. Research on novel anti-cancer mechanisms effective for both primary and metastatic tumors is warranted for a significant improvement of cancer mortality. The overall goal of this project is to establish a novel anti-cancer mechanism by targeting Obg-like ATPase (OLA1), a newly identified stress response protein and putative regulator of protein S-glutathionylation. OLA1 is a ubiquitously expressed cytosolic protein, and belongs to Obg family of P-loop ATPases highly conserved from bacteria to human. However, besides our own publications, only a couple of references are available regarding its biological function. In 2009 our group first demonstrated that OLA1 is an endogenous regulator of cellular antioxidant responses and acts through a posttranscriptional mechanism. Our recent studies have shown that knockdown of OLA1 in cultured human cancer cells lead to impairment of cell migration and loss of invasiveness. In the present study we further confirmed that deficiency of OLA1 will also cause enhanced detachment-induced cell death (anoikis), another important aspect that could contribute to the inhibition of cancer metastasis. More importantly, this study has introduced an in vivo model, the Olal knockout mice, and by studying knockout mice and knockout mouse embryonic fibroblast (MEF) cells, we revealed some essential in vivo functions of OLA1. Putting together the in vivo data that present consistently the prenatal and postnatal growth retardation in knockout mice, and in vitro observation for the reduced growth rate in Ola1-/-primary MEF cells, it's now conclusive that Ola is implicated in cell growth, and inhibition of OLA1 results in growth suppression. The anti-growth effect of OLA1 inhibition is also confirmed in cancer cell, which was achieved through the stable shRNA transfection strategies. Thus far, at least 4 beneficial effects related to anti-cancer therapy:inhibition of growth, suppressed motility, reduced invasion, as well as the enhanced anoikis, are obtained by merely inhibition of a single gene (OLA1). It is strongly suggestive that OLA1 can be a novel target for anti-cancer and/or anti-metastasis treatment.In the present study, we have also explored the molecular mechanisms that may contribute to the above anti-cancer/anti-metastasis effects. By studying the Ola1 knockdown and knockout cells using a panel of molecular biology and biochemistry methods, we have demonstrated that OLA1 is an as-yet-unidentified regulator of protein S-glutathionylation. The reversible formation of protein mixed disulfides between cysteine residues and glutathione (S-glutathionylation) is an important posttranslational modification (PTM) mechanism that occurs during oxidative stress as well as under normal physiological conditions. It has been previously hypothesized that PTM by S-glutathionylation is an intrinsic mechanism for fine-tuning protein function and signal transduction, similar to the role of another PTM, notably the protein phosphorylation. However, unlike protein phosphorylation, protein glutathionylation is largely underexplored with regard to both basic mechanisms and its implications in human diseases, primarily due to lack of identification of any upstream mediators involved in this process. In this project we discovered that OLA1 is an inhibitory factor of global protein glutathionylation, and may function through modulating the levels of glutathionylation effector proteins including GSTπ(glutathione S-transferaseπ). We have confirmed the negative correlation of OLA1 content and the levels of S-glutathionylation in actin and many cellular proteins in multiple cell types including cancer cells and primary MEF cultures, at both baseline (unstressed) and oxidative stress conditions. Moreover,particular pathways that are potentially affected by this OLA 1-mediated glutathionylation alteration have also been discussed, for example, that on the Akt pathways leading to the inhibition of cell growth, and on actin and other cytoskeletal proteins leading to the impairment of cell migration and invasion.In summary, the results of this project may lead to the establishment of a new anti-cancer strategy based on targeting OLA1, an important posttranslational regulator of many cellular processes including protein S-glutathionylation, providing valuable new options to battle the fatal disease. This project also provides unique insights into the understanding of basic functions of OLA1, as well as the regulatory mechanisms in the entire S-glutathionylation pathway.