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P53-Dependent Dysregulation of IL-6/raft/STAT3 Signaling in Breast Cancer
1) Interleukin-6 (IL-6) has an established role in the dyshesion, scattering and epithelioid to mesenchymal transition of ductal breast cancer cells. IL-6 signals to the interior of normal and cancerous cells via the activation of the Janus Kinase /Signal transducer and activator of transcription (JAK/STAT) pathway. Activated STAT3 levels are elevated in breast carcinoma tissue as well as breast cancer cell lines. We have previously shown that gp130, the signal transducing receptor chain for IL-6, STAT3 and the integral lipid raft protein caveolin-1 (cav-1) localize to plasma membrane lipid raft microdomains and that functional IL-6/STAT3 signaling is largely initiated at the level of such cav-1 enriched lipid raft microdomains. Furthermore, we have demonstrated a dynamic interaction of cav-1 with STAT3 at the level of the plasma membrane rafts and downstream scaffolding complexes in numerous cell lines as well as rat lung tissue. While the mechanism of raft-localization of several signaling molecules (for e.g. EGF receptor, eNOS) is well known, the mechanisms by which STAT3 and gp130 are targeted to raft microdomains, including their direct or indirect interactions with cav-1 remain poorly understood.
2) Previously, our lab has reported the link between the proliferative IL-6/STAT3 signaling and mutations in the tumor suppressor protein p53 through a novel observation (wild type p53-dependent "STAT3 masking"). Expression of temperature-sensitive (ts) p53-Val135 mutant (p53-Val135 has a mutant conformation at 37°C and a wt-like conformation at 32.5°C) in human hepatoma Hep3B cells results in a marked inhibition in IL-6/STAT3 signaling at 32.5°C (the wt-p53 phenotype) but not at 37°C (the mutant p53 phenotype). This p53-mediated “STAT3 masking” is dependent upon p53-mediated transcriptional regulation of an unidentified protein. STAT3 masking has now been independently confirmed by other investigators including in breast cancer cell lines but its mechanism remains unknown. p53 is now known to upregulate the expression of cav-1. Our preliminary data shows that overexpression of cav-1 in Hep3B cells as well as other cell lines leads to an inhibition of IL-6/STAT3 signaling. Re-expression of cav-1 has been shown to inhibit growth of breast cancer cells. Is cav-1, itself a putative tumor suppressor, the mediator of p53-dependent STAT3 masking ?
Specific Aims: Specific Aim 1 of this study proposes to investigate the underlying mechanisms that target STAT3 and gp130 to cav-1-enriched lipid rafts and the dysregulation of such pre-assembled raft structures in breast cancer. This study will perform mutational and truncational analyses to dissect out the domains within STAT3 and gp130 that are responsible for their localization to rafts. We will also determine if the interactions of gp130 and STAT3 with cav-1 are direct or indirect and identify adapter protein partners if it is indirect.
Specific Aim 2 of this project links the IL-6/raft/STAT3 signaling to mutations in p53 and its regulation of this pathway. Specifically, we propose to investigate whether cav-1 is the mediator of p53-dependent inhibition of IL-6/STAT3 signaling and how this mechanism is modulated in breast cancer cell lines.
The proposed studies of such tumor suppressor mechanisms are of broad significance in that IL-6/raft/STAT3 signaling has an established role in breast cancer and its metastasis but very little is known about the mechanism of localization of STAT3 and gp130 to the lipid rafts. The mechanism by which the tumor suppressor p53 mediates an inhibition of IL-6/STAT3 signaling is a potential target for therapeutic intervention in breast cancer. Establishing the role of cav-1, itself a putative tumor suppressor, in mediating this inhibition gives us the first insight into the regulation of IL-6/STAT3 signaling by p53 in breast cancer.
Cells in the body have a normal cycle of growth, division and rest phase, governed by an interplay of several proteins. Cancer is defined as the uncontrolled and unchecked growth and multiplication of cells or a tissue due to a loss of normal body checks and controls. A variety of factors such as harmful chemicals, radiation and even viruses can result in a loss of this normal regulation and cause the cell to divide in an unlimited manner and the resulting mass of cells is detected as a tumor or cancer. Most of these events also cause damage to the DNA of the cell, the molecules that are the "information storage" mechanism of the cell. Normally, a protein called p53 is activated upon DNA damage and it leads the cell to commit suicide instead of multiplying in an uncontrolled manner. Thus, p53 is a "tumor suppressor". In breast cancer, however, it is frequently found that this tumor suppressor itself is damaged.
Cytokines are a class proteins that circulate in the body and help the body in its defense mechanism against microbes and also against the body's own cells that function abnormally. A balance between those cytokines that signal for cell growth and those that inhibit cell growth governs the normal cell cycle of growth, division and rest. Interleukin-6 (IL-6) is one such cytokine that gives proliferation signals to the cells by sequentially activating two other proteins inside the cell - gp130 and STAT3. Previously investigators have found that in patients with breast cancer, especially the type that metastasizes (scientific word for cancer that spreads), levels of IL-6 and activated STAT3 are highly increased. Thus it is essential to understand how in normal cells gp130 and STAT3 are activated by IL-6, as if we are able to disable this activation mechanism by development of newer drugs, we will very likely be able to check cell growth and division in breast cancer and its further spread inside the body. One such mechanism that we have found is by a protein called cav-1, which binds to gp130 and STAT3 and normally serves to keep them inactive. Other investigators have reported the loss of cav-1 in breast cancer, which would then lead to an unchecked activation of gp130 and STAT3. The first aim of this project is to investigate the exact mechanism by which cav-1 keeps gp130 and STAT3 in check and how this regulation is lost in breast cancer.
Our lab has previously shown that in liver cancer cells, the tumor suppressor p53 also functions to inactivate the STAT3 protein. This is an exciting discovery as it provides an important mechanism to control proliferation in breast cancer. The second aim of our project is to investigate whether p53 can inactivate STAT3 in breast cancer also. The final twist to the tale comes by way of the fact that p53 increases cav-1 inside cells, which is understandable as both p53 and cav-1 serve to control proliferative signals inside the cells. As part of our second aim we will also investigate whether inactivation of STAT3 by p53 is via cav-1.
The studies are of tremendous significance as they will investigate a new "tumor suppressor mechanism" in breast cancer. There is a strong body of evidence to believe that this suppression is very likely on account of the mechanism we propose. Eventually, this mechanism will help other investigators to then develop new drugs to treat breast cancer and control its spread. It will also serve to develop vaccines against breast cancer that will reduce world-wide mortality from breast cancer.