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    The Role of Toll-Like Receptors in Breast Cancer

    Scientific Abstract:
    The Role of Toll-Like Receptors in Breast Cancer (Scientific Abstract) Background: The transcription factor NF-kB is a central regulator of the inflammatory gene program in addition to its role in activating cell survival, proliferation and cell differentiation genes. Toll-like receptors (TLRs) are related to the IL-1 receptor and detect molecular patterns on bacterial, viral proteins and products and initiate the innate host defense system largely through activation of the NF-kB signaling pathway. Inappropriate activation and/or chronic activation of NF-kB are commonly observed in many tumor types and are an essential component of the cellular transformation and metastatic program. NF-kB plays a role in normal breast development and is chronically activated in a majority of breast cancer (BC) tumors. We have discovered that various subsets of the TLRs are upregulated in expression in a majority of breast cancer tumors but not in normal breast tissue and the expression are apparent in the mammary epithelium. Furthermore, a number of breast cancer cell lines, MDA-MB-468 and MDA-MB-231 BC cell lines in particular, produce soluble factors that are capable of potently activating NF-kB via a subset of the TLRs. Objective/Hypothesis: We have observed that breast cancer cells produce factors that activate TLR receptors and we hypothesize that these factors (T-Fs) are recognized by TLRs on BC tumor cells present on a subset of BCs in an autocrine fashion to activate NF-kB. It is also likely that surrounding normal tissue also produces TLR-recognizable ligands (T-Fs) in response to tumor cell instruction and lead to NF-kB activation in BC tumor cells in a paracrine fashion. We also believe that since a majority of BC tumors express a varied subset of TLRs that there is clinical significance to the pattern of TLR expression in BC. To test these hypotheses, we propose the following Specific Aims. Specific Aim I. Identify and molecularly clone autocrine or paracrine released TLR ligands from individual BC cell lines or BC tumor explants. Factors present in the conditioned culture supernatants from established BC cell lines or from tumor cell explants grown in culture will be detected using the various TLR stably transfected cell lines and purified using standard biochemical techniques and sequenced by microelectrospray ionization-ion trap mass spectrometry and identified from database searching. We will make antibodies and nucleic acid probes to detect its expression in normal and BC tissue samples and BC cell lines. Specific Aim II. Examine TLR and T-F expression patterns on tissue microarrays (TMAs) and correlating expression patterns using TLR- and T-F-specific antibodies and also with in situ hybridization using TLRs and T-F gene-specific riboprobes with known clinical outcomes available from our institutional database of over 600 samples that also have frozen tissue available for additional studies. Clinical outcomes to be examined include correlation with metastasis (organ-specific or not), hormone dependent/independent status, responsiveness/non-responsiveness to common treatment modalities. Specific Aim III. Will examine effects on cell survival and proliferation by expressing dominant-negative alleles or silencing RNAs (siRNA) plasmids of the various TLRs and T-Fs in MDA-MB-468 and MDA-MB-231 BC cell lines and challenging them with commonly used chemotherapeutic modalities. Study Design: BC cell lines produce factors that are recognized by a subset of TLRs and lead to NF-kB DNA binding activity in exposed cells expressing the specific TLR. We will biochemically fractionate the conditioned growth media from a number of T-F producing BC cell lines and media derived from fresh BC tumor explants and assay NF-kB stimulation by transient expression assay of an NF-kB-driven reporter gene expression in various TLR stably expressing cell lines and in control cells. Proteins in enriched fractions will be sequenced by microelectrospray ionization-ion trap mass spectrometry, molecularly cloned and assayed. Antibodies will be prepared against the cloned T-F expressed proteins using phage display single chain antibody techniques currently used in our laboratory and used along with TLR and T-F-specific riboprobes to examine the localization and abundance of the proteins and mRNAs in BC cell lines and in BC tumor tissues using TMAs. We will construct a large BC TMA with assistance of our molecular pathology department and a pathologist will score it. Clinical outcomes to be examined include correlation with metastasis (organ-specific or not), hormone dependent/independent status, and responsiveness/non-responsiveness to common treatment modalities. Expression of dominant-negative alleles or siRNAs of the various TLRs by recombinant lentiviruses in BC cell lines will be examined for their ability to induce BC cell death or inhibit cellular proliferation in response to no treatment or treatment with chemotherapeutic agents. Potential Outcomes and Benefits of the Research: We anticipate that the identification and detection of novel TLR ligands produced by BC tumor cells and TLR expression patterns in BC tumor tissue will segregate and reflect clinically relevant outcomes and will serve as not only a outcome-specific prognosticative readout but will identify a new class of BC drug targets and ultimately aid in combating breast cancer.

    Lay Abstract:
    The Role of Toll-Like Receptors in Breast Cancer (Lay Abstract) The development of breast cancer occurs in the mammary epithelial cells. The initial event in tumorogenesis involves the loss or gain of functions of a variety of genes that ultimately lead to an event called cell transformation. Normally when this occurs the cell is programmed to undergo a programmed cell death. If this process is blocked the cell will not die and will continue to proliferate and grow. A family of proteins, known as NF-kB, bind to specific DNA sequences and activate a specific subset of genes. These genes are involved mainly in the inflammatory response but also a number of genes activated by NF-kB are involved in cell to cell attachment, cell to cell communication, cell survival and cell growth, cell differentiation and development and tissue remodeling. Activation of NF-kB is required for cells to undergo transformation and bypass the cellular death program. NF-kB activity also plays an important role in normal breast development and its activity is tightly controlled. In a majority of breast cancer tumors and cell lines derived from those tumors, NF-kB is chronically active. Inhibition of NF-kB in these cells diminishes their potential to metastasize in animal models of breast cancer and also sensitizes tumor cells to cell death induced by exposure to a variety of clinically used chemotherapeutic agents. Therefore, the ability to inhibit NF-kB activation in breast cancer cells would be expected to provide a beneficial effect in combating breast cancer. Chronic activation of NF-kB in breast cancer occurs in response to hormone growth factors in some tumor cells but additional activation pathways have not been elucidated or are only partially known. We have discovered an unknown NF-kB activation pathway in some breast cancer cells. This pathway involves the production of a factor by some breast cancer cells that is recognized by receptor protein that is a member of a large family of receptors called the Toll-like receptors (TLRs). TLRs normally recognize bacterial, fungal and viral pathogenic products and activate the NF-kB pathway leading to expression of genes involved in innate host response to deal immediately with pathogens and later play an essential role in allowing the host to mount an adaptive immunity (antibody) response. It is likely that in the breast cancer cells that produce these factors that the factor feeds back and activates NF-kB in those cells via recognition by the TLRs. We have examined the expression of all of the TLR family members in a number of established breast cancer cell lines and in a number of breast cancer tissues and normal breast tissue and found that various subsets of the TLRs are expressed in the breast cancer cell lines and breast cancer tissue but nearly absent in normal breast tissue. We believe that differential expression of the TLRs in breast cancer is clinically important. We propose the following specific aims to examine this possibility. In Specific Aim I we will purify and identify the factors released by the breast cancer cell lines or tumor tissues grown in tissue culture dishes and molecularly clone the gene for this (these) factors and develop reagents that can detect their presence at the mRNA level (riboprobes) or at the protein level (with antibodies). We will combine these tools with our ability to examine a large number of breast cancer clinical samples that has a database of clinical outcomes and parameters using a tissue microarray. Tissue microarrays allow up to a hundred or so clinical samples to be investigated at one time on one microscope slide. We will investigate TLR expression profiles and factor expression profiles in the breast cancer tumor and normal breast tissue samples using our available antibodies against the factor(s) and individual TLRs. A pathologist will read the results and we will compare and correlate the patterns of expression with known clinical outcomes available from our institutional database of over 600 samples that also have frozen tissue available for additional studies. Clinical outcomes to be examined include correlation with metastasis (organ-specific or not), hormone dependent/independent status, responsiveness/non-responsiveness to common treatment modalities. To examine the biological importance of responsiveness to the factors produced by the breast cancer cells we will express defective versions of either the factor or the various TLR genes in the breast cancer cell and assay the cells ability to grow and or to undergo the cell death program in response to treatment with various chemotherapeutically relevant drugs. We believe that blocking the activation pathway utilized by the factor will sensitize the tumor cell to undergo cell death. We anticipate that the identification and detection of novel TLR ligands produced by breast cancer tumor cells and TLR expression patterns in tumor tissue will segregate and reflect clinically relevant outcomes and will serve as not only a outcome-specific prognosticative readout but will identify a new class of drug targets and ultimately aid in combating breast cancer.