INDUCTION OF APOPTOSIS IN CANCER CELLS
Siva Kumar Kolluri, Ph.D.
Summary: To effectively treat cancer, we want to protect normal cells and selectively kill cancer cells. A protein called Bcl-2 regulates apoptosis or cell death. An abundance of Bcl-2 produced in cancer cells prevents apoptosis. Prevention of apoptosis promotes cancer cell growth. Another protein called Nur77 can interact with Bcl-2 and convert Bcl-2 from an anti-apoptotic to a pro-apoptotic protein in cancer cells. We discovered that small fragments of Nur77 (peptides, or groups of amino acids that make proteins) could also interact with Bcl-2, leading to the death of cancer cells.
Damaged cells in the body are eliminated through a process involving cell suicide known as apoptosis. Since apoptosis normally eliminates cells with increased malignant potential, such as those with damaged DNA or aberrant cell cycling, inhibition of apoptosis is highly important to tumor initiation and progression. All cells contain both pro-death and pro-survival proteins that regulate the process of cell apoptosis. These pro-survival proteins are abundantly expressed in cancer cells, which help them to thrive and resist elimination upon treatment with anti-cancer drugs. Bcl-2—an acronym for B-cell lymphoma 2—is one such survival protein that is overexpressed in a variety of cancers, including leukemia and lung, liver, gastric, ovarian, prostate, and breast cancers.
Members of the Bcl-2 family of proteins modulate apoptosis in different cell types in response to various stimuli. Some of these members, such as Bcl-2 and Bcl-XL, act to inhibit apoptosis (anti-apoptotic), while others, such as Bax, Bak, Bid, and Bad, promote apoptosis (pro-apoptotic). All members of the Bcl-2 family possess at least one of the four structural domains called Bcl-2 homology (BH) domains. Domains are discrete, stable components of protein structure that combine in different ways to make different proteins. Proteins that contain only one BH domain (BH3) are proapoptotic. Interestingly, anti-apoptotic Bcl-2 members contain this BH3 domain along with three other BH domains (1, 2 and 4). To prevent apoptosis, anti-apoptotic members bind pro-apoptotic members containing only the BH3 domain, and while this occurs in normal cells, a significant upregulation of Bcl-2 in cancer cells disrupts the normal balance, allowing cancerous cells to survive.
Nur77 is a protein that belongs to the superfamily of nuclear receptors that controls gene expression in cells. Nur77 has the capacity to not only regulate gene expression but also to induce apoptosis. Importantly, the apoptotic effect of Nur77 family members is clinically relevant, since the expression of the Nur77 subfamily member Nor-1 improves survival in diffuse, large B-cell lymphoma patients treated with chemotherapeutic drugs. Gene expression profiles have also revealed that downregulation of Nur77 expression is associated with metastasis of primary solid tumors.
We had previously demonstrated a new paradigm in cancer cell apoptosis, whereby Nur77 moves from the cell's nucleus to its mitochondria, where it interacts with Bcl-2, converting this normally anti-apoptotic protein into a pro-apoptotic protein that induces cell death. While trying to understand this unexpected phenomenon, we discovered that Nur77 causes an alteration in the Bcl-2 structural conformation that exposes its hidden pro-apoptotic BH3 domain.
In collaboration with Drs. Xiao-kun Zhang, Arnold Sattherthwait, John Reed, and other colleagues at the Sanford-Burnham Medical Research Institute, we discovered that even short protein fragments or peptides from Nur77ís Bcl-2 binding region are able to induce the same conformation changes in Bcl-2 that Nur77 induces. We found that the Nur77-derived short peptides bound to Bcl-2 and exposed its BH3 domain, which allowed for Bcl-2 to activate two pro-apoptotic proteins, Bax and Bak, to initiate apoptosis. Importantly, the altered Bcl-2 also neutralized the antiapoptotic activity of other Bcl-2 family members. We then found that these peptides induced apoptosis in cancer cells in vitro and suppressed the growth of human cancer cells explanted in mice. To further confirm this relationship, we established that the pro-apoptotic activity of the Nur77 peptides was dependent on Bcl-2 expression.
A majority of solid tumors are protected from apoptosis by overexpression of Bcl-2. Accordingly, there have been efforts in the last few years to identify compounds that neutralize Bcl-2's anti-apoptotic functions to make cancer cells more susceptible to chemotherapeutic drugs or compounds. This has led to the development of approaches that either decrease Bcl-2 protein expression or neutralize the survival functions of Bcl-2 with small molecules. However, neutralizing Bcl-2's normal anti-apoptotic/pro-apoptotic function alone may not be sufficient, since other anti-apoptotic proteins must also be inhibited for the therapeutics to be effective. Importantly, our studies have established that Nur77 peptides or small molecule mimetics not only inhibit Bcl-2 survival function but also neutralize other Bcl-2 survival proteins by altering Bcl-2 conformation. This will likely have therapeutic value in treating a variety of cancers.
Pilot project funding from LPI helped us to conduct the experiments that reveal the mechanism of cell death induced by the converted Bcl-2. Furthermore, the LPI pilot project award helped us to establish assays to potentially identify additional compounds that convert Bcl-2 from a cell protector to a cell killer. This research helped to secure additional funding from the Breast Cancer Research Program of the Department of Defense.
Last updated November 2010