Nuclear factor-kappaB activation: a molecular therapeutic target for estrogen receptor-negative and epidermal growth factor receptor family receptor-positive human breast cancer.

Nuclear factor-kappaB (NF-kappaB), a transcription factor with pleotropic effects, is a downstream mediator of growth signaling in estrogen receptor (ER)-negative and erbB family particularly erbB2 (HER-2/neu) receptor-positive cancer. We previously reported activation of NF-kappaB in ER-negative breast cancer cells and breast tumor specimens, but the consequence of inhibiting NF-kappaB activation in this subclass of breast cancer has not been shown. In this study, we investigated the role of NF-kappaB activation by studying the tumorigenic potential of cells expressing genetically manipulated, inducible, dominant-negative inhibitory kappaB kinase (IKK) beta in xenograft tumor model. Conditional inhibition of NF-kappaB activation by the inducible expression of dominant-negative IKKbeta simultaneously blocked cell proliferation, reinstated apoptosis, and dramatically blocked xenograft tumor formation. Secondly, the humanized anti-erbB2 antibody trastuzumab (Herceptin) and the specific IKK inhibitor NF-kappaB essential modifier-binding domain peptide both blocked NF-kappaB activation and cell proliferation and reinstated apoptosis in two ER-negative and erbB2-positive human breast cancer cell lines that are used as representative model systems. Combinations of these two target-specific inhibitors synergistically blocked cell proliferation at concentrations that were singly ineffective. Inhibition of NF-kappaB activation with two other low molecular weight compounds, PS1145 and PS341, which inhibited IKK activity and proteasome-mediated phosphorylated inhibitory kappaB protein degradation, respectively, blocked erbB2-mediated cell growth and reversed antiapoptotic machinery. These results implicate NF-kappaB activation in the tumorigenesis and progression of ER-negative breast cancer. It is postulated that this transcription factor and its activation cascade offer therapeutic targets for erbB2-positive and ER-negative breast cancer.

Crossroads of estrogen receptor and NF-kappaB signaling.

Cellular homeostasis in higher organisms is maintained by balancing cell growth, differentiation, and death. Two important systems that transmit extracellular signals into the machinery of the cell nucleus are the signaling pathways that activate nuclear factor kappaB (NF-kappaB) and estrogen receptor (ER). These two transcription factors induce expression of genes that control cell fates, including proliferation and cell death (apoptosis). However, ER has anti-inflammatory effects, whereas activated NF-kappaB initiates and maintains cellular inflammatory responses. Recent investigations elucidated a nonclassical and nongenomic effect of ER: inhibition of NF-kappaB activation and the inflammatory response. In breast cancer, antiestrogen therapy might cause reactivation of NF-kappaB, potentially rerouting a proliferative signal to breast cancer cells and contributing to hormone resistance. Thus, ER ligands that selectively block NF-kappaB activation could provide specific potential therapy for hormone-resistant ER-positive breast cancers.

Apoptosis caused by chemotherapeutic inhibition of nuclear factor-kappaB activation.

Both the protein kinase C (alpha/beta) inhibitor Go6976 and expression of dominant-negative nuclear factor (NF)-kappaB inhibitor kinase mutants: (a) blocked the growth and caused regression of a mammary tumor insyngeneic mice; (b) inhibited epidermal growth factor (EGF)-induced activation, nuclear translocation, and DNA-binding activity of NF-kappaB; and (c) caused apoptosis of EGF-stimulated cultured mammary tumor cells. cDNA microarray analysis revealed that these treatments reversed the expression changes of a subset of genes altered by EGF treatment. These included: up-regulation of proapoptotic genes of the tumor necrosis factor (TNF) pathway, death-associated protein (DAP) kinase, p53, and p21/Waf1; and down-regulation of inhibitors of apoptosis: inhibitor of apoptosis(IAP)-1 and X-IAP, TNF receptor-associated factor (TRAF)-2, and factors OX40 and 4-1BB. These results and our previous studies suggest the practicality of a target-directed chemotherapy for EGF-responsive breast cancers, by blocking NF-kappaB activation and thereby reinstating apoptosis.

RelA-Induced Interferon Response Negatively Regulates Proliferation.

Both oncogenic and tumor-suppressor activities are attributed to the Nuclear Factor kappa B (NF-kB) pathway. Moreover, NF-kB may positively or negatively regulate proliferation. The molecular determinants of these opposing roles of NF-kB are unclear. Using primary human mammary epithelial cells (HMEC) as a model, we show that increased RelA levels and consequent increase in basal transcriptional activity of RelA induces IRF1, a target gene. Induced IRF1 upregulates STAT1 and IRF7, and in consort, these factors induce the expression of interferon response genes. Activation of the interferon pathway down-regulates CDK4 and up-regulates p27 resulting in Rb hypo-phosphorylation and cell cycle arrest. Stimulation of HMEC with IFN-γ elicits similar phenotypic and molecular changes suggesting that basal activity of RelA and IFN-γ converge on IRF1 to regulate proliferation. The anti-proliferative RelA-IRF1-CDK4 signaling axis is retained in ER+/HER2- breast tumors analyzed by The Cancer Genome Atlas (TCGA). Using immuno-histochemical analysis of breast tumors, we confirm the negative correlation between RelA levels and proliferation rate in ER+/HER2- breast tumors. These findings attribute an anti-proliferative tumor-suppressor role to basal RelA activity. Inactivation of Rb, down-regulation of RelA or IRF1, or upregulation of CDK4 or IRF2 rescues the RelA-IRF1-CDK4 induced proliferation arrest in HMEC and are points of disruption in aggressive tumors. Activity of the RelA-IRF1-CDK4 axis may explain favorable response to CDK4/6 inhibition observed in patients with ER+ Rb competent tumors.

Linkage between EGFR family receptors and nuclear factor kappaB (NF-kappaB) signaling in breast cancer.

In the United States there are 225,000 new cases of invasive breast cancer annually, and at least 50,000 women are diagnosed with ductal carcinoma in situ (DCIS). Breast cancer is a collection of disorders of mammary epithelial cells with distinct pathological characteristics and diverse clinical manifestations. Breast cancers are divided broadly into four classes by the level of the biomarkers HER2 (erbB2/neu) and the estrogen receptor (ER). Histologic grade is also an important modifier of breast cancer taxonomy and behavior. Broadly speaking, breast cancer can be divided into those that are HER2-positive, containing cancers that are both ER-positive and negative, cancers that are ER-positive and divided into high-grade and low-grade tumors, and the remaining but important class of cancers that are both ER-negative and HER2-negative. These last cancers are called basal-like and were first recognized as a distinct group by gene expression arrays. Nuclear factor kappaB (NF-kappaB) is family of multifunctional transcription factors that when activated generate pleotrophic changes in target cells. Elevated levels of active NF-kappaB are detected in many human diseases including breast cancers. High-level active NF-kappaB is detected in specific subclasses of breast cancers briefly described above, predominantly in ER-negative and epidermal growth factor family receptor (EGFR) overexpressing breast cancers (predominantly HER2 amplified cancers). This article is focused on the role of NF-kappaB activation initiated by the EGFR family receptors in subclasses of breast cancer. The combined influence of EGFR family receptors and NF-kappaB signaling on the transformation of ER-negative human mammary epithelial cell is illustrated.

NF-kappa B activation in human breast cancer specimens and its role in cell proliferation and apoptosis.

Lack of molecular targets in estrogen receptor-negative (ER-negative) breast cancer is a major therapeutic hurdle. We studied NF-kappa B activation in human breast tumors and in carcinoma cell lines. Activated NF-kappa B was detected predominantly in ER-negative vs. ER-positive breast tumors and mostly in ER-negative and ErbB2-positive tumors (86%). These in vivo results demonstrate association of activated NF-kappa B with a subgroup of human breast tumors and are consistent with previously reported in vitro observations using similar classes of human breast cancer cell lines. Finding such an association suggested functional and biological significance. Immunofluorescence demonstrated increased nuclear p65, a component of the active NF-kappa B complex, in cytokeratin 19 (CK19)-positive epithelial cells of ER-negative/ErbB2-positive tumor samples. In contrast, nuclear NF-kappa B was detected mostly in stroma of ER-negative and ErbB2-negative tumors, suggesting a role of activated NF-kappa B in intercellular signaling between epithelial and stromal cells in this type of breast cancers. To elucidate roles of activated NF-kappa B, we used an ER-negative and ErbB2-positive human breast tumor cell line (SKBr3). The polypeptide heregulin beta1 stimulated, and herceptin, the anti-ErbB2 antibody, inhibited, NF-kappa B activation in SKBr3 cells. The NF-kappa B essential modulator (NEMO)-binding domain (NBD) peptide, an established selective inhibitor of I kappa B-kinase (IKK), blocked heregulin-mediated activation of NF-kappa B and cell proliferation, and simultaneously induced apoptosis only in proliferating and not resting cells. These results substantiate the hypothesis that certain breast cancer cells rely on NF-kappa B for aberrant cell proliferation and simultaneously avoid apoptosis, thus implicating activated NF-kappa B as a therapeutic target for distinctive subclasses of ER-negative breast cancers.