Gail E. Sonenshein

Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer.

Expression of nuclear factor-kappaB (NF-kappaB)/Rel transcription factors has recently been found to promote cell survival, inhibiting the induction of apoptosis. In most cells other than B lymphocytes, NF-kappaB/Rel is inactive, sequestered in the cytoplasm. For example, nuclear extracts from two human untransformed breast epithelial cell lines expressed only very low levels of NF-kappaB. Unexpectedly, nuclear extracts from two human breast tumor cell lines displayed significant levels of NF-kappaB/Rel. Direct inhibition of this NF-kappaB/ Rel activity in breast cancer cells induced apoptosis. High levels of NF-kappaB/Rel binding were also observed in carcinogen-induced primary rat mammary tumors, whereas only expectedly low levels were seen in normal rat mammary glands. Furthermore, multiple human breast cancer specimens contained significant levels of nuclear NF-kappaB/Rel subunits. Thus, aberrant nuclear expression of NF-kappaB/Rel is associated with breast cancer. Given the role of NF-kappaB/Rel factors in cell survival, this aberrant activity may play a role in tumor progression, and represents a possible therapeutic target in the treatment of these tumors.

Inhibition of NF-kappaB/Rel induces apoptosis of murine B cells.

Apoptosis of the WEHI 231 immature B cell lymphoma line following membrane interaction with an antibody against the surface IgM chains (anti‐IgM) is preceded by dramatic changes in Nuclear Factor‐kappaB (NF‐kappaB)/ Rel binding activities. An early transient increase in NF‐kappaB/Rel binding is followed by a significant decrease in intensity below basal levels. Here we have explored the role of these changes in Rel‐related factors in B cell apoptosis. Treatment of WEH1 231 cells with N‐tosyl‐L‐phenylalanine chloromethyl ketone (TPCK), a protease inhibitor which prevents degradation of the inhibitor of NF‐kappaB (IkappaB)‐alpha, or with low doses of pyrrolidinedithiocarbamate (PDTC) selectively inhibited NF‐kappaB/Rel factor binding and induced apoptosis. Bcl‐XL expression protected WEHI 231 cells from apoptosis induced by these agents. Microinjection of WEHI 231 cells with either IkappaB‐alpha‐GST protein or a c‐Rel affinity‐purified antibody induced apoptosis. Ectopic c‐Rel expression ablated apoptosis induced by TPCK or anti‐IgM. Treatment of BALENLM 17 and A20 B lymphoma cells or normal murine splenic B lymphocytes with either TPCK or PDTC also resulted in apoptosis. These findings indicate that the drop in NF‐kappaB/Rel binding following anti‐IgM treatment activates apoptosis of WEHI 231 cells; furthermore, they implicate the NF‐kappaB/Rel family in control of apoptosis of normal and transformed B cells.

Cell-cycle control of c-myc but not c-ras expression is lost following chemical transformation

Cellular oncogenes are DNA sequences implicated in the genesis of cancer, but their functions in the transformation process are not understood. Our experiments provide data linking expression of two well-studied proto-oncogenes, c-myc and c-rasKi, to current knowledge of proliferation control and its perturbation by differentiation and chemical transformation. Growth stimulation of quiescent cells by serum elevates expression of the myc proto-oncogene in Balb/c 3T3 (A31) cells. In two chemically transformed A31 derivatives (BPA31 and DA31), c-myc expression is constitutive. The levels of c-myc mRNA in quiescent and growing transformed cells are nearly the same, and are only slightly elevated compared to the level found in growing A31 cells. By contrast, c-rasKi expression is cell-cycle-dependent in BPA31 cells. The relative abundance of c-rasKi mRNA begins to increase in mid- to late G0/G1. During terminal differentiation of teratocarcinoma stem cells (F9) into nonproliferating endoderm, relative mRNA abundance is diminished more markedly for c-myc than for c-rasKi. These results demonstrate that expression of the myc and rasKi proto-oncogenes is dependent upon the cellular growth state, and that growth control exhibits growth-factor-dependent, cell-cycle-timed oncogene expression. In the case of the BPA31 cells, c-myc is not rearranged, amplified, or overexpressed. However, the oncogene has lost its cycle-dependent regulation in the chemically transformed cells.

Protein kinase CK2 in mammary gland tumorigenesis

Protein kinase CK2 is a ubiquitous and evolutionarily conserved serine/threonine kinase that is upregulated in many human cancers and can serve as an oncogene in lymphocytes. Recently, we have demonstrated that CK2 potentiates Wnt/β-catenin signaling in mammary epithelial cells. To determine whether CK2 overexpression contributes to mammary tumorigenesis, we have performed comparative studies of human and rat breast cancer specimens and we have engineered transgenic mice with dysregulated expression of CK2α in the mammary gland. We find that CK2 is highly expressed in human breast tumor specimens and in carcinogen-induced rat mammary tumors. Overexpression of CK2α in the mammary gland of transgenic mice, under control of the MMTV-LTR, causes hyperplasia and dysplasia of the female mammary gland. Thirty per cent of the female MMTV-CK2α transgenic mice develop mammary adenocarcinomas at a median of 23 months of age, often associated with Wnt pathway activation, as evidenced by upregulation of β-catenin protein. NF-κB activation and upregulation of c-Myc also occur frequently. Thus, in mice, rats, and humans, dysregulated expression of CK2 is associated with and is capable of contributing to mammary tumorigenesis. Targeted inhibition of CK2 could be useful in the treatment of breast cancer.

NF-κB and Epithelial to Mesenchymal Transition of Cancer

During progression of an in situ to an invasive cancer, epithelial cells lose expression of proteins that promote cell–cell contact, and acquire mesenchymal markers, which promote cell migration and invasion. These events bear extensive similarities to the process of epithelial to mesenchymal transition (EMT), which has been recognized for several decades as critical feature of embryogenesis. The NF‐κB family of transcription factors plays pivotal roles in both promoting and maintaining an invasive phenotype. After briefly describing the NF‐κB family and its role in cancer, in this review we will first describe studies elucidating the functions of NF‐κB in transcription of master regulator genes that repress an epithelial phenotype. In the second half, we discuss the roles of NF‐κB in control of mesenchymal genes critical for promoting and maintaining an invasive phenotype. Overall, NF‐κB is identified as a key target in prevention and in the treatment of invasive carcinomas. J. Cell. Biochem. 104: 733–744, 2008.

Her-2/neu overexpression induces NF-κB via a PI3-kinase/Akt pathway involving calpain-mediated degradation of IκB-α that can be inhibited by the tumor suppressor PTEN

The Nuclear Factor (NF)-κB family of transcription factors controls expression of genes which promote cell growth, survival, and neoplastic transformation. Recently we demonstrated aberrant constitutive activation of NF-κB in primary human and rat breast cancer specimens and in cell lines. Overexpression of the epidermal growth factor receptor (EGFR) family member Her-2/neu, seen in approximately 30% of breast cancers, is associated with poor prognosis. Previously, Her-2/neu has been shown to signal via a phosphatidylinositol 3 (PI3)-kinase to Akt/protein kinase B (PKB) pathway. Since this signaling pathway was recently shown to activate NF-κB, here we have tested the hypothesis that Her-2/neu can activate NF-κB in breast cancer. Overexpression of Her-2/neu and EGFR-4 in Ba/F3 cells led to constitutive PI3- and Akt kinase activities, and induction of classical NF-κB (p50/p65). Similarly, a tumor cell line and tumors derived from MMTV-Her-2/neu transgenic mice displayed elevated levels of classical NF-κB. Engagement of Her-2/neu receptor downregulated the level of NF-κB. NF-κB binding and activity in the cultured cells was reduced upon inhibition of the PI3- to Akt kinase signaling pathway via ectopic expression of kinase inactive mutants, incubation with wortmannin, or expression of the tumor suppressor phosphatase PTEN. Inhibitors of calpain, but not the proteasome, blocked IκB-α degradation. Inhibition of Akt did not affect IKK activity. These results indicate that Her-2/neu activates NF-κB via a PI3- to Akt kinase signaling pathway that can be inhibited via the tumor suppressor PTEN, and is mediated by calpain rather than the IκB kinase complex.

Notch1 augments NF-κB activity by facilitating its nuclear retention

Notch1 specifically upregulates expression of the cytokine interferon‐γ in peripheral T cells through activation of NF‐κB. However, how Notch mediates NF‐κB activation remains unclear. Here, we examined the temporal relationship between Notch signaling and NF‐κB induction during T‐cell activation. NF‐κB activation occurs within minutes of T‐cell receptor (TCR) engagement and this activation is sustained for at least 48 h following TCR signaling. We used γ‐secretase inhibitor (GSI) to prevent the cleavage and subsequent activation of Notch family members. We demonstrate that GSI blocked the later, sustained NF‐κB activation, but did not affect the initial activation of NF‐κB. Using biochemical approaches, as well as confocal microscopy, we show that the intracellular domain of Notch1 (N1IC) directly interacts with NF‐κB and competes with IκBα, leading to retention of NF‐κB in the nucleus. Additionally, we show that N1IC can directly regulate IFN‐γ expression through complexes formed on the IFN‐γ promoter. Taken together, these data suggest that there are two ‘waves’ of NF‐κB activation: an initial, Notch‐independent phase, and a later, sustained activation of NF‐κB, which is Notch dependent.

Repression of transcription of the p27 Kip1 cyclin-dependent kinase inhibitor gene by c-Myc

Upon engagement of the B Cell Receptor (BCR) of WEHI 231 immature B cells, a drop in c-Myc expression is followed by activation of the cyclin-dependent kinase inhibitor (CKI) p27Kip1, which induces growth arrest and apoptosis. Here, we report inverse patterns of p27 and c-Myc protein expression follow BCR engagement. We present evidence demonstrating, for the first time, that the p27Kip1 gene is a target of transcriptional repression by c-Myc. Specifically, the changes in p27 protein levels correlated with changes in p27 mRNA levels, and gene transcription. Induction of p27 promoter activity followed BCR engagement of WEHI 231 cells, and this induction could be repressed upon co-transfection of a c-Myc expression vector. Inhibition of the TATA-less p27 promoter by c-Myc was also observed in Jurkat T cells, vascular smooth muscle, and Hs578T breast cancer cells, extending the observation beyond immune cells. Consistent with a putative Inr element CCAGACC (where +1 is underlined) at the start site of transcription in the p27 promoter, deletion of Myc homology box II reduced the extent of repression. Furthermore, enhanced repression was observed upon transfection of the c-Myc ‘super-repressor’, with mutation of Phe115 to Leu. The sequences mediating transcriptional activity and c-Myc repression were mapped to bp −20 to +20 of the p27 gene. Finally, binding of Max was shown to facilitate c-Myc binding and repression of p27 promoter activity. Overall, these studies identify the p27 CKI gene as a new target whereby c-Myc can control cell proliferation, survival and neoplastic transformation.

TGFβ1 Inhibits NF-κB/Rel Activity Inducing Apoptosis of B Cells: Transcriptional Activation of IκBα

Abstract TGFβ1 treatment of B cell lymphomas decreases c- myc gene expression and induces apoptosis. Since we have demonstrated NF-κB/Rel factors play a key role in transcriptional control of c- myc , we explored the effects of TGFβ1 on WEHI 231 immature B cells. A reduction in NF-κB/Rel activity followed TGFβ1 treatment. In WEHI 231 and CH33 cells, we observed an increase in IκBα, a specific NF-κB/Rel inhibitor, due to transcriptional induction. Engagement of surface CD40 or ectopic c-Rel led to maintenance of NF-κB/Rel and c-Myc expression and protection of WEHI 231 cells from TGFβ1-mediated apoptosis. Ectopic c-Myc expression overrode apoptosis induced by TGFβ1. Thus, downmodulation of NF-κB/Rel reduces c-Myc expression, which leads to apoptosis in these immature B cell models of clonal deletion. The inhibition of NF-κB/Rel activity represents a novel TGFβ signaling mechanism.

Differential regulation of the c-myc oncogene promoter by the NF-kappa B rel family of transcription factors.

The murine c-myc gene contains two elements responsive to the rel-oncogene-related family of NF-kappa B factors. Previously we have shown that factor binding to these two NF-kappa B elements mediates induction of transcription of the c-myc promoter upon interleukin-1 treatment of human dermal fibroblasts and human T-cell leukemia virus type I tax gene expression in T cells (D. J. Kessler, M. P. Duyao, D. B. Spicer, and G. E. Sonenshein, J. Exp. Med. 176:787-792, 1992; M. P. Duyao, D. J. Kessler, D. B. Spicer, C. Bartholomew, J. L. Cleveland, M. Siekevitz, and G. E. Sonenshein, J. Biol. Chem. 267:16288-16291, 1992). To begin to delineate the specific roles of the individual members of the NF-kappa B family, here we have tested their effects on activation of a c-myc promoter/exon 1-CAT construct in NIH 3T3 cells. Classical NF-kappa B (p65/p50) was a potent transcriptional activator of the c-myc promoter. Cotransfection with either p65 alone or p65 in combination with p50 mediated significant induction. In contrast, expression of either v-rel or chicken c-rel failed to transactivate, while murine c-rel induced c-myc promoter activity only slightly. Furthermore, induction by classical NF-kappa B was inhibited by coexpression of either v-rel or chicken c-rel. Thus, individual members of the rel family have differential effects of the c-myc promoter, which can modulate overall transcriptional activity and allow for precise regulation of this oncogene under diverse physiologic conditions.