Breast Cancer and the NFκB Transcription Factor Family
Breast cancer is the most common non-skin cancer among women in the Western world, and is the second most frequent cause of death due to cancer among women in the United States. Over the past 50 years, there has been a profound increase in the incidence of breast cancer worldwide, in both industrialized and developing countries. The life-time risk of developing breast cancer for American women has increased from 1 in 20 in 1960, to 1 in 8 today. Changes in genetic predispositions cannot explain the rising incidence of disease over a relatively short time frame. Studies have shown that sporadic breast cancer incidence varies with environmental influences, including exposure to carcinogens, as well as diet. For example, populations that migrate from nations with a low incidence of breast cancer to those with a high incidence manifest a high incidence of this disease within 1 or 2 generations.
Our laboratory focuses on the NF-κB family of dimeric transcription factors, which is composed of five members (c-Rel, RelA, RelB, p50, and p52). While many signals can activate NF-κB factors, they were believed to be expressed constitutively only in B cells. However, we discovered that most human and rodent breast cancers aberrantly express NF-κB factors constitutively. Using a mouse model in which c-Rel expression was driven in the mammary gland, we showed that NF-κB can play a causal role in mammary tumorigenesis (Figure 1). NF-κB is now known to regulate genes that promote cancer cell proliferation, survival and a more mesenchymal phenotype.
Figure 1. In vivo demonstration of a causal role of the aberrant NF-κB c-Rel activation in mammary tumorigenesis. Representative histopathologies of mammary tumors that developed in MMTV-c-rel transgenic mice after multiple cycles of pregnancy and regression. A) Adenocarcinoma; B) Pulmonary metastasis in a mouse with mammary adenocarcinomas; C) Adenosquamous carcinoma showing areas with extracellular squamous differentiation (arrow); D) Squamous cell carcinoma; E) Spindle cell carcinoma; F) Immunohistochemistry for cytokeratin 8 expression in the spindle cell carcinoma shown in 2E; Note the staining of many of the spindle cells and staining of luminal epithelium in the glands (arrow).
Our current research is focused on the three NF-κB subunits c-Rel, RelB and p65, which contain transactivation domains. We are also elucidating the roles of several upstream kinases in NF-κB activation including IKKε/i, which we first demonstrated is expressed in breast cancer and promotes a more malignant phenotype via activation of the NF-κB RelB subunit . The gene encoding the IKK/i kinase is amplified in many breast cancers. We are also studying downstream mediators of NF-κB, which represent potential novel therapeutic targets. One mediator that we have identified is called the B lymphocyte-induced maturation protein 1 (Blimp1), see discussion below. Lastly, we are studying dietary compounds and environmental factors that can revert/prevent epithelial to mesenchymal (EMT) conversion, causing the cell to return to or maintain a more normal phenotype. In particular, studies are focused on elucidating the mechanism of action of green tea polyphenols (Figure 2) and 1,25-Dihydroxyvitamin D3.
Figure 2. Green tea and its polyphenol EGCG prevent or reverse invasive phenotype of mammary cancer cells induced by the carcinogen DMBA. (Top) Representative results of E-cadherin staining in paraffin-embedded sections of histologically normal glands from Sprague-Dawley rats given vehicle sesame oil and water to drink, and DCIS in mammary glands of rats treated with 15 mg/kg DMBA and given either water or green tea as drinking fluid. (Bottom) rel-3983V (untreated) or rel-3983D DMBA-transformed cells in the presence of either carrier DMSO or 60 μg/ml EGCG were grown in Matrigel. After 5 days, the colonies were photographed.
Carcinogenesis and Breast Cancer Signaling
Several years ago, we proposed that exposure to environmental carcinogens can alter the epithelial phenotype of normal mammary cells by activating signaling cascades that convert them to a more mesenchymal (migratory or invasive) phenotype. These pathways include those regulated by the aromatic hydrocarbon receptor (AhR), protein kinase CK2, Wnt and NF-κB. Our lab is elucidating the mechanisms by which NF-κB expression is activated by environmental carcinogens and how changes in signaling promote cancer cell survival and more invasive phenotype (Figure 3). In particular, we collaborate with Dr David H Sherr (Boston University School of Public Health) and Dr David C Seldin (Hematology/Oncology, Boston University School of Medicine), respectively, to study the cross-talk of NF-κB with the AhR and its repressor (AhRR) and CK2 and Wnt signaling - to elucidate signaling changes that are induced by carcinogen exposure that promotes this transformation process.
Figure 3. Scheme of the cross-talk of signal transduction pathways induced by environmental carcinogens that promote a more invasive phenotype of breast cancer cells.
Lysyl Oxidase Propeptide Inhibits Ras Signaling and Transformation
The lysyl oxidase (LOX) gene was identified as the ras recision gene (rrg), with ability to suppress Ha-Ras-induced transformed phenotype. The LOX gene suppressed Ras-mediated activation of NF-κB factors in NIH 3T3 cells via the Raf/MEK and PI3K/Akt pathways. LOX is synthesized as a 50 kDa pro-enzyme, secreted into the extracellular environment where it is processed to a functional 32 kDa enzyme and an 18 kDa propeptide (LOX-PP). With our collaborators Philip Trackman and Kathrin H. Kirsch (Boston University), we determined that the LOX-PP, and not the LOX enzyme, inhibits ras-dependent transformation of NIH 3T3, and these observations were extended to breast cancers driven by Her-2/neu, and to lung and pancreatic cancers, which are driven by Ras signaling. Experiments are continuing to elucidate the mechanism of action of LOX-PP using yeast two-hybrid, co-immunoprecipitation and microarray studies to identify interacting proteins and affected pathways. Furthermore, we recently demonstrated that a non-synonymous single nucleotide polymorphism (SNP), which maps to a highly conserved region within the DNA encoding LOX-PP, dramatically impairs its ability to revert invasive phenotype in vitro and in a mouse xenograft model. Collaborative work with Drs L Rosenberg and J Palmer of the Boston University Slone Epidemiology has suggested that the SNP is associated with increased risk of ERα negative breast cancer in African-American women. Studies on the role of the SNP in breast, lung and pancreatic cancers are ongoing.
BLIMP1 Promotes a More Migratory Phenotype in Lung and Breast Cancer Cells
The Zn finger protein Blimp1, which was originally identified as a silencer of IFN-β gene transcription, is a repressor of transcription. Blimp1 functions as a master regulator of development of antibody secreting B lymphocytes and of T cell homeostasis. Blimp1 also plays key roles in specification of primordial germ cells and promotes their ability to migrate. Although the exact mechanism by which repression occurs is not fully understood, Blimp1 possesses DNA-binding activity and can recruit histone deacetylases, histone methyltransferases and the co-repressor Groucho. We recently discovered that Blimp1 is expressed in breast cancer cells and functions as a potent repressor of ERα synthesis. Furthermore, we showed that Blimp1 promotes the migratory phenotype of breast cancer cells. Currently, the lab is elucidating the mechanism of Blimp1 activation and the downstream targets of Blimp1 that are mediating the transformed phenotype of breast and lung cancer cells.