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Regulation Of Breast Cancer Progression Through The Transcriptional Repressor Hbp1
BACKGROUND: Constitutive activation of Wnt signaling is a feature of many invasive breast cancers; therefore, understanding the biochemical deregulation of this pathway will provide important insights into disease progression. Our published results demonstrate that the transcriptional repressor, HBP1, is able to suppress Wnt signaling by physical means. Our recent data suggests an alternative mechanism by which HBP1 potentially regulates Wnt signaling through SFRP1, a natural Wnt antagonist. This was based on data demonstrating that knockdown of HBP1 results in a downregulation of SFRP1. Low levels of SFRP1 are frequently found in breast cancer as well, contributing to the observed high Wnt signaling associated with this disease. The primary mechanism for the decrease in SFRP1 and the heightened Wnt signaling is epigenetic, involving DNA and histone methylation on histone 3 lysine 27 (H3K27). EZH2 is a histone methyltransferase that triggers H3K27 trimethylation, which itself has been linked to subsequent DNA methylation and silencing of tumor suppressor genes. Overexpression of EZH2 is also linked to invasive breast cancer. Intriguingly, our results demonstrate that HBP1 overexpression results in a downregulation of EZH2. Furthermore, knockdown of HBP1 results in an upregulation of EZH2. The goal of this proposal is to understand how HBP1 regulates Wnt signaling through SFRP1 and EZH2 in order to determine any clinical implications for invasive breast cancer.
OBJECTIVE/HYPOTHESIS: This proposal aims to understand the functional relationship between three proteins that are clinically linked to invasive breast cancer: HBP1, SFRP1 and EZH2. Our working hypothesis is that HBP1 regulates the expression of EZH2, which in turn, epigenetically regulates SFRP1 expression, through trimethylation of H3K27. Furthermore, we hypothesize that the relative expression of these three proteins dictates the onset of tumorigenesis and acquisition of invasiveness, two salient characteristics of invasive breast cancer. Lastly, we hypothesize that the molecular signature of these three proteins will define a clinically relevant network for determining invasive breast cancer prognosis and degree of relapse. Our specific aims are:
(1) To understand HBP1 and epigenetic mechanisms for regulating Wnt signaling
(2) To determine the roles of HBP1 and SFRP1 in models of invasive breast cancer
(3) To develop a mechanism-based multi-gene signature for invasive breast cancer.
STUDY DESIGN: In Aim 1, genetic (RNAi and retroviral transduction) and pharmacological approaches will be used to test a possible HBP1-EZH2-SFRP1 mechanism. Specifically, chromatin immunoprecipitation (ChIP) will determine whether trimethylated H3K27 occupies the SFRP1 promoter. In Aim 2, human mammary epithelial cells, orthotopic xenograft models and three-dimensional mammary models will be used to address the role of these proteins in salient characteristics of invasive breast cancer: increased tumorigenic proliferation and the acquisition of an invasive morphology. In Aim 3, clinical analyses of breast cancer specimens will be used to develop a mechanism-based signature to sub-classify invasive breast cancer for prognosis. qPCR and targeted database analysis will be used to link HBP1, SFRP1, and EZH2 expression with disease progression.
IMPACT AND CANCER RELEVANCE: This proposal brings together diverse experimental observations on Wnt signaling to investigate a potentially new epigenetics-based mechanism of regulating the oncogenic nature of Wnt signaling, a pathway often activated in invasive breast cancers. The investigation of a molecular network of three proteins that are clinically linked to invasive breast cancer may fill a gap in knowledge on the mechanisms that determine invasiveness and recurrence. The identification of new targets and mechanisms provide opportunities to improve current epigenetics-based therapies, which are limited by toxicity. Our hope is that information gained from this proposal will be useful in developing mechanism-based prognostic and therapeutic strategies for treating invasive breast cancer and preventing recurrence.
Breast cancer affects 1 in 8 women in the US over her lifetime. While early detection has greatly improved patient outcome, recurrence to metastatic disease represents a major obstacle to improved survival and a tragic outcome for invasive breast cancer. Invasive breast cancer is a frequent subtype that is particularly prone to recurrence and distant metastases. The two defining characteristics of initial invasive breast cancer are 1) increased tumorigenic proliferation and 2) local invasiveness. Understanding the clinically important mechanisms that underlie invasive breast cancer is central to designing better therapeutic strategies that definitively treat breast cancer to prevent recurrence to metastatic disease.
This proposal aims to understand how three genes (HBP1, SFRP1 and EZH2) in the context of a major oncogenic pathway (Wnt signaling) influence the onset and progression of invasive breast cancer. A unique aspect of our molecular investigations is that each of the principle players (HBP1, SFRP1 and EZH2) are clinically linked to invasive breast cancer, however, have not been investigated together in a potentially important clinically-relevant network. We believe these three proteins are involved in a cascade of molecular events that influences the onset of the two characteristics of invasive breast cancer (increased proliferation and increased invasiveness). We will use the molecular findings in order to better predict relapse and prognosis of patients. Understanding the mechanisms that determine the acquisition of invasiveness and recurrence are central to improving patient outcome.
Our goals include understanding the functional network of these three proteins and their impact on determining these two features of invasive breast cancer. This new knowledge should provide insights into new molecular and prognostic targets for invasive breast cancer and for preventing recurrence. By understanding the mechanisms that underlie invasive breast cancer, we hope to develop new cell-based and animal models that recapitulate aspects of invasive breast cancer and which can then be used to discover new, mechanism-based therapeutics. This proposal outlines studies that will go from bench-to-bedside-to-bench, using results from basic research to better predict clinical outcomes. Thus, our pre-clinical studies may provide insights and models for designing new clinical strategies to treat invasive breast cancers and prevent recurrence. The hope is that the clinically relevant results will be used to greatly improve the prognosis and treatment for breast cancer patients by providing the tools and knowledge for new and aggressive clinical strategies for definitive cure.
This training proposal is designed not only to provide new insights into breast cancer biology, but also to train a future breast cancer researcher that will make the next wave of discoveries. This training requires expertise in both basic science and clinical aspects of breast cancer with the theoretical knowledge of an oncology fellow. This multidisciplinary training is required to investigate the clinically important questions in breast cancer. This training will continue the mission of improving patient outcome and eventually eradicate breast cancer.