Research Grants Awarded
Synergistic Cooperation Of Snail And Beta-Catenin In The Initiation Of Breast Cancer Metastasis
Investigator Initiated Research
Background: Approximately 90% of breast cancer deaths are caused by metastasis. A major challenge in breast cancer research is to identify the extrinsic signals and intrinsic factors that initiate breast cancer metastasis. The increased motility and invasiveness of metastatic tumor cells are reminiscent of the events at the epithelial-mesenchymal transition (EMT), which is characteristic of embryonic development, tissue remodeling, and wound healing. A hallmark of EMT is the loss of E-cadherin. Loss of E-cadherin is often correlated with tumor grade and metastasis because it results in the disruption of cell-cell adhesion and increases nuclear translocation of beta-catenin, a key player in the Wnt/beta-catenin pathway that is implicated in stem cell renewal, proliferation, and anti-apoptosis. On one hand, beta-catenin is an essential component of adherent junctions, where it provides the link between E-cadherin and alpha-catenin and modulates cell-cell adhesion and cell migration. On the other hand, beta-catenin also functions as a transcription cofactor to enhance cell proliferation and protect apoptosis. Aberrant activation of the Wnt/beta-catenin pathway has been associated with more than 50% of breast carcinomas. Strikingly, only nuclear beta-catenin is found in cancer cells at the invasive front (the tumor?host interface), and its presence correlates with an aggressive tumor phenotype and poor survival. It remains unclear, however, what signaling mechanisms induce EMT and initiate beta-catenin nuclear translocation at the invasive front. Interestingly, under normal physiological conditions, EMT takes place at the edge of injury during wound healing. Similarly, this process occurs at the invasive front of metastatic cancers. These observations suggest that EMT is triggered by an extrinsic signal that emanates from an inflammatory microenvironment. Consistent with our hypothesis, we recently found that the invasiveness of breast cancer cells was greatly enhanced by inflammatory cytokines. We showed that inflammatory cytokines significantly induced the protein stabilization of Snail, a transcription suppressor of E-cadherin and a critical inducer of EMT in breast cancer cells. In addition, we found that Snail is required for the synergistic activation of beta-catenin by inflammatory cytokines and the Wnt ligand. Thus, the interactions between the inflammatory microenvironment, Snail/EMT, and beta-catenin emerge as a major culprit in the initiation of breast cancer metastasis at the invasive front.
Hypothesis/Objective: The central hypothesis of the proposed research is that breast cancer metastasis is initiated and propelled by the synergistic cooperation between the intrinsic activation of the Wnt/beta-catenin pathway and the extrinsic inflammatory microenvironment (i.e., macrophage infiltration) at the invasive front of breast cancer via the activation of the Snail/EMT program. We propose a two-step event for the initiation of breast cancer metastasis. In the first step, inappropriate activation of the beta-catenin pathway in selected tumor cells initiates the formation of early breast adenomas. At this stage, tumor cells are attached to each other in an epithelial context, and only low quantities of beta-catenin can be detected in the nucleus. In the second step, an inflammatory microenvironment is generated at the tumor-host boundary as the tumor develops. The inflammatory cytokines induce stabilization of Snail and trigger EMT in breast cancer cells at the invasive front. However, because inflammatory cytokines also elicit cytotoxic effects that are detrimental to the cells, the downregulation of E-cadherin mediated by Snail/EMT initiates more nuclear translocation of beta-catenin and thus, provides the potential for cancer cells to counteract this cytotoxic effect by becoming invincible during dissemination and invasion. The objective of this proposal is to dissect the signal interplay among inflammation, Snail/EMT, and beta-catenin and, by discovery of the aberrant regulations, to identify molecules that may serve as therapeutic targets for preventing breast cancer metastasis.
Specific Aims: To test our central hypothesis and achieve the objective of this proposal, we prepare three aims: (1) to delineate the mechanism of Snail stabilization and EMT induction by inflammation; (2) to analyze the synergistic effect of Snail and beta-catenin on cell migration and invasion; and (3) to define the roles of Snail and beta-catenin in breast cancer metastasis in vivo and in tumor samples.
Study Design: In Aim 1, we will delineate (i) how the inflammatory cytokine induces protein stabilization of Snail; and (ii) whether activation of the NF-kappaB pathway is required for this process; and (iii) whether the stabilization of Snail requires the suppression of its phosphorylation and ubiquitination. In Aim 2, we will examine (i) whether knockdown of Snail expression inhibits cell migration/invasion mediated by inflammation; and (ii) whether knockdown of beta-catenin expression induces apoptosis mediated by inflammation; and (iii) whether the inflammation-mediated regulation of Snail or beta-catenin is influenced by 3D culture. In Aim 3, we will determine (i) whether knockdown of Snail or beta-catenin expression blocks breast cancer metastasis in vivo; and (ii) whether Snail expression correlates with nuclear beta-catenin and macrophage infiltration in breast cancer specimens.
Breast cancer is the most common cancer in women. The clinical symptoms and outcome of breast cancer depend largely on whether it is confined to the breast or has spread to adjacent or distant parts of the body. Despite more than 3 decades of research, approximately 90% of breast cancer deaths are caused by local invasion and distant metastasis of tumor cells, and the average time to live after documentation of metastasis is approximately 2 years. Thus, novel treatment strategies based on the biology of how breast cancer metastasizes are urgently needed to combat this life-threatening disease.
The process of metastasis involves multiple steps. At the initial step, tumor cells at the tumor-host boundary detach from the primary tumor and start to invade the adjacent tissue. This early event is crucial for the entire metastatic process. The major challenge in breast cancer research is to identify the factors within the cell and the signals outside the cell that initiate this early event. The increased motility and invasiveness of tumor cells at the initial step of metastasis are similar to the occurrence of the epithelial-mesenchymal transition (EMT) which is required for embryonic development, tissue remodeling, and wound healing. In EMT, cells change from ?static? to ?motile? in appearance and start to migrate, this is because the cell-cell adhesion molecule E-cadherin is lost, and tumor cells break away from one other and start to migrate and invade the surrounding tissue. At the same time the tumor cells lose E-cadherin, they activate a survival mechanism called the beta-catenin pathway to protect them from death during the odyssey of invasion and metastasis. The signal that triggers the EMT process and the activation of the beta-catenin pathway in breast cancer remains a mystery. Interestingly, under normal physiological condition, EMT takes place at the edge of the site of injury during wound healing. Similarly, in many tumors, EMT occurs at the invasive front (the tumor?host boundary) of metastatic cancers. As cancer has been called as ?a wound that never heals,? these observations suggest that EMT is triggered by inflammation that occurs at the wound or at the tumor-host boundary of cancer. Consistent with our hypothesis, we recently found that the amount of Snail, a protein that acts as a master switch at the nucleus to control EMT and the mobility of the cell, is dramatically increased by inflammation. When too much Snail protein is generated, as occurs in many cases of metastatic breast cancer, it suppresses the expression of E-cadherin and induces the activation of the ?survival? beta-catenin pathway. In fact, high levels of Snail have been linked to metastasis, tumor cell survival, and recurrence of breast cancer and predict a poor clinical outcome. Based on our novel findings, we propose that an inflammatory microenvironment at the tumor-host boundary induces the production of Snail in the tumor cells, which results in the suppression of E-cadherin and the induction of EMT, and consequently leads to the activation of beta-catenin pathway. We hypothesize that Snail and beta-catenin work cooperatively to initiate breast cancer metastasis. We will test this hypothesis by determining (1) how Snail stabilization and EMT are induced by inflammation; (2) the synergistic effect of Snail and beta-catenin on cell migration and invasion; and (3) the role of Snail and beta-catenin in breast cancer metastasis in vivo.
We will accomplish our goal by utilizing contemporary methods such as, co-culture experiment with macrophages (inflammatory cells), siRNA techniques, 3D cultures, animal models, and analyses of human breast cancer specimens in our study. In addition, we have designed a 3D macrophage co-culture experiment that is built to mimic the invasive front of breast cancer in vivo. By using multiple and complementary approaches, we will carefully examine the dialogue or signaling between breast cancer cells and inflammatory cells (macrophages). Furthermore, we will validate our finding by using breast cancer metastasis model in mice that have human breast tumors growing in their mammary glands. We will induce an inflammation in these mice and examine the signaling of Snail and beta-catenin during the development of breast cancer metastasis. Finally, we will compare and evaluate the inflammation status and the expression of these molecules in 120 tumor samples from patient with breast cancer. We believe that our systematic study will not only enhance our knowledge of how breast cancer metastasis develops but also lead to the development of new treatment strategies that will be tested in clinical trials for the benefit of patients with metastatic breast cancer.