Date of Award

5-1-2011

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Watabe, Kounosuke

Abstract

Metastasis suppressors regulate multiple steps during the process of dissemination of tumor cells from primary sites to distant organs, while they do not affect the growth of the primary tumor. Previously, we identified NDRG1 (N-myc downstream regulated gene 1) as a tumor metastasis suppressor gene and found that it is negatively involved in metastatic progression of prostate and breast cancers. To elucidate the molecular mechanism of NDRG1 function, we used the yeast two-hybrid system to identify proteins interacting with NDRG1. In the first part of this project, we demonstrate that NDRG1, interacts with the Wnt receptor, LRP6, followed by blocking of the Wnt signaling, and therefore, orchestrates a cellular network that impairs the metastatic progression of tumor cells in vitro and in animal model. We also found that restoring NDRG1 expression by a small molecule compound significantly suppressed the capability of otherwise highly metastatic tumor cells to thrive in circulation and distant organs in animal models. In addition, our analysis of clinical cohorts data indicate that Wnt+/NDRG-/LRP+ signature has a strong predictable value for recurrence-free survival of cancer patients. Collectively, we have identified NDRG1 as a negative master regulator of Wnt signaling during the metastatic progression, and therefore revealed a novel control mechanism of Wnt signaling in tumor progression. Previously, we identified the metastasis promoting transcription factor, ATF3, as a downstream target of NDRG1. Further analysis revealed that the KAI1 promoter contained a consensus binding motif of ATF3, suggesting a possibility that NDRG1 suppresses metastasis through inhibition of ATF3 expression followed by activation of KAI1 gene. In the second part of this project, we examine a possible link between two metastasis suppressor genes, NDRG1 and KAI1, through ATF3. We demonstrated that ectopic expression of NDRG1 was able to augment endogenous KAI1gene expression in prostate cancer cell lines, while silencing NDRG1 accompanied with significant decrease in KAI1 expression in vitro and in vivo. In addition, our results of ChIP analysis indicate that ATF3 indeed bound to the promoter of KAI1 gene. Importantly, our promoter-based analysis revealed that ATF3 modulated KAI1 transcription through cooperation with other endogenous transcription factor as co-activator (ATF3-JunB) or co-repressor (ATF3-NFêB). Moreover, loss of KAI1 expression significantly abrogated NDRG1-mediated metastatic suppression in vitro as well as in a spontaneous metastasis animal model, indicating that KA11 is a functional down-stream target of NDRG1 pathway. Our result of immunohistochemical analysis showed that loss of NDRG1 and KAI1 occurs in parallel as prostate cancer progresses. We also found that a combined expression status of these two genes serves as a strong independent prognostic marker to predict metastasis-free survival of prostate cancer patients. Taken together, our result revealed a novel regulatory network of two metastasis suppressor genes, NDRG1 and KAI1, which together concerted metastasis-suppressive activities through intrinsic transcriptional cascade.

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