Cell-Transforming Bystander Signals
Sponsor: DOE Office of Biological and Environmental Research Low Dose Radiation Research Program
Contact: Tom Weber, Lee Opresko, Diana Bigelow
Radiation-induced paracrine factors with the potential to influence carcinogenesis outcomes have been observed in a number of experimental model systems, however, the identity and molecular mechanism(s) of bystander signals remain unclear. The goal of our research is to test the hypothesis that annexin II and osteopontin are paracrine factors that significantly contribute to radiation carcinogenesis.
We are determining whether annexin II is a trigger for specific epigenetic events in radiation carcinogenesis. Using mass spectrometry and Western blot analysis we have identified annexin II and osteopontin as candidates for the transforming paracrine signal observed. We hypothesize that annexin II is a trigger that initiates the proteinase-dependent secretion of osteopontin, which in turn, is sufficient to induce cell transformation. We will quantify annexin II, target proteinase, and osteopontin levels (both total cellular and secreted forms) to facilitate interpretation of the bystander-specific transformation response within the context of these target proteins.
We will determine the role of annexin II/osteopontin in clonogenic survival and transformation response to low-dose radiation using specific small interfering RNA and neutralizing antibodies. A 23 kDa secreted form of annexin II has been detected that may be the product of regulated proteolysis. We will examine the role of regulated proteolysis in annexin II/osteopontin secretion and determine whether the annexin II fragment differentially modulates the activity of a relevant protein complex (tissue plasminogen activator/plasminogen/annexin II complex), relative to full-length annexin II.
We will test the hypothesis that secreted annexin II exhibits a number of discrete activities that integrate in carcinogenesis responses. These include prosurvival functions and activation of proteinases that in turn, induce the release of tumor promoters and tissue architectural remodeling.
Another aim of our research is to determine whether secreted annexin II remodels tissue architecture. Annexin II-dependent activation of proteinases is expected to result in the remodeling of tissue architecture that controls tumor progression. The role of annexin II in low dose radiation-induced remodeling of mammary epithelial acinar architecture will be determined using annexin II-specific siRNA and neutralizing antibody in a three-dimensional reconstituted basement membrane assay (matrigel).
We have recently demonstrated that irradiated cells secrete a soluble factor that induces the transformation of bystander cells. Annexin II was identified by mass spectrometry as an abundant paracrine signal in our model. Importantly, annexin II appears to play an important role in the etiology of oxidative stress-induced cancers and also functions as a prosurvival factor. Further, annexin II is linked to the activation of matrix metalloproteinases (MMPs) that in turn, regulate the release of tumor promoters from the plasma membrane. Osteopontin has been identified as a novel substrate for MMPs and consistent with the link between annexin II and MMP activation, we provide evidence that osteopontin secretion is specifically induced by low dose radiation in an MMP-dependent fashion. Because osteopontin treatment alone is sufficient to induce cell transformation, we hypothesize that the annexin II-osteopontin pathway is a likely candidate for the observed transforming bystander signal. This is important because annexin II and osteopontin are widely implicated in the etiology of human cancers.
These studies directly address a specific molecular mechanism for a radiation bystander effect that is highly relevant to tissue responses and a variety of human cancers. If our hypothesis is correct, the results of these studies will be of particular interest to a number of researchers within the DOE Low Dose Radiation Research Program and will have logical implications for high dose radiation responses of interest to NASA. Because both annexin II and osteopontin are implicated in prosurvival responses, the results of this work may have cross-cutting implications in the adaptive response.