Induction of IRF-3 and IRF-7 phosphorylation following activation of the RIG-I pathway.

The induction of type I interferon (IFN) and the development of the innate antiviral response are mediated by the activation of interferon regulatory factor (IRF)-3 and IRF-7 under the control of the non-canonical kinases TBK-1 and IKKepsilon. The initial sensing of infection by RNA viruses is mediated by the cytoplasmic, retinoic acid inducible gene I (RIG-I), via a Toll-like receptor (TLR) independent signaling pathway. In the present study, we identify key residues involved in IRF-3 and IRF-7 phosphorylation using TAP-tag purification of TBK-1 and IKKepsilon proteins. Based on the identification of an extended sequence motif--SxSxxxS--common to both IRF-3 and IRF-7, an IRF-7 pSer477/479 phosphospecific antibody was generated. Virus infection, TBK-1/IKKepsilon expression or co-expression of different signaling adaptors such as RIG-I, MAVS and TRIF, all stimulated pSer477/479 phosphorylation. Furthermore, the newly identified adaptor of the RIG-I pathway (MAVS/IPS-1/VISA/Cardif) was able to induce IRF and NF-kappaB dependent promoter activity as efficiently as the constitutively active form of RIG-I (DeltaRIG-I). Co-expression of the NS3/4A protease activity of hepatitis C virus however blocked MAVS-mediated gene activation in a dose dependent manner. These studies link RIG-I sensing of viral RNA to downstream kinase signaling and phosphorylation of IRF-3 and IRF-7 via the MAVS/IPS/VISA/Cardif adaptor.

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/beta-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 CK2alpha 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 CK2alpha 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-CK2alpha transgenic mice develop mammary adenocarcinomas at a median of 23 months of age, often associated with Wnt pathway activation, as evidenced by upregulation of beta-catenin protein. NF-kappaB 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.

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

The Nuclear Factor (NF)-kappaB family of transcription factors controls expression of genes which promote cell growth, survival, and neoplastic transformation. Recently we demonstrated aberrant constitutive activation of NF-kappaB 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-kappaB, here we have tested the hypothesis that Her-2/neu can activate NF-kappaB 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-kappaB (p50/p65). Similarly, a tumor cell line and tumors derived from MMTV-Her-2/neu transgenic mice displayed elevated levels of classical NF-kappaB. Engagement of Her-2/neu receptor downregulated the level of NF-kappaB. NF-kappaB 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 IkappaB-alpha degradation. Inhibition of Akt did not affect IKK activity. These results indicate that Her-2/neu activates NF-kappaB 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 IkappaB kinase complex.

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) p27(Kip1), 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 p27(Kip1) 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.

Roles of IKK kinases and protein kinase CK2 in activation of nuclear factor-kappaB in breast cancer.

Nuclear factor-kappaB (NF-kappaB)/Rel transcription factors regulate genes that control cell proliferation, survival, and transformation. In normal breast epithelial cells, NF-kappaB/Rel proteins are mainly sequestered in the cytoplasm bound to one of the specific inhibitory IkappaB proteins, whereas in breast cancers they are activated aberrantly. Human breast tumor cell lines, carcinogen-transformed mammary epithelial cells, and the majority of primary human or rodent breast tumor tissue samples express constitutively high levels of nuclear NF-kappaB/REL: To begin to understand the mechanism of this aberrant NF-kappaB/Rel expression, in this study we measured the activity of the major kinases implicated in regulation of IkappaB stability, namely IKKalpha, IKKbeta, and protein kinase, CK2 (formerly casein kinase II). Hs578T, D3-1, and BP-1 breast cancer cell lines displayed higher levels of IKKalpha, IKKbeta, and CK2 activity than untransformed MCF-10F mammary epithelial cells. Inhibition of IKK activity upon expression of dominant negative kinases or of CK2 activity by treatment with selective inhibitors decreased NF-kappaB/Rel activity in breast cancer cells. Inactivation of the IkappaB kinase complex in Hs578T cells via expression of a dominant negative IKKgamma/NF-kappaB essential modulator/IKK-associated protein 1 reduced soft agar colony growth. Thus, the aberrant expression of CK2 or IKK kinases promotes increased nuclear levels of NF-kappaB/Rel and transformation of breast cancer cells. Furthermore, primary human breast cancer specimens that displayed aberrant constitutive expression of NF-kappaB/Rel were found to exhibit increased CK2 and/or IKK kinase activity. These observations suggest these kinases play a similar role in an intracellular signaling pathway that leads to the elevated NF-kappaB/Rel levels seen in primary human mammary tumors and, therefore, represent potential therapeutic targets in the treatment of patients with breast cancer.

Protein kinase CK2: signaling and tumorigenesis in the mammary gland.

Breast cancer is a major cause of cancer death in women, and the genetic abnormalities leading to the common sporadic forms of the disease are still under active investigation. CK2 has been reported to be upregulated in human breast cancer, which these studies confirm; CK2 is also upregulated in rat carcinogen-induced breast tumors. Transgenic mice overexpressing CK2alpha in the mammary gland develop mammary hyperplasia, dysplasia, and eventually adenocarcinomas, demonstrating that dysregulated expression of CK2 can contribute to transformation of the mammary epithelium. These mammary tumors have evidence of activation of the Wnt and NFkappaB pathways and upregulation of c-Myc. CK2 is capable of phosphorylating the key signaling molecule in the Wnt pathway, the transcriptional cofactor beta-catenin, and regulating its turnover. CK2 is known to phosphorylate IkappaB and thereby regulate basal NFkappaB levels; in the mammary cell lines and tumors, CK2 activity correlates with NFkappaB levels and inhibition of CK2 downregulates NFkappaB. Thus, CK2 may promote breast cancer through dysregulation of key pathways of transcriptional control in the mammary epithelium, and inhibition of CK2 has a potential role in the treatment of breast and other cancers.

The RelA NF-kappaB subunit and the aryl hydrocarbon receptor (AhR) cooperate to transactivate the c-myc promoter in mammary cells.

NF-kappaB/Rel transcription factors regulate many genes involved in control of cellular proliferation, neoplastic transformation, and apoptosis, including the c-myc oncogene. Recently, we have observed that levels of NF-kappaB and aryl hydrocarbon receptor (AhR), which mediates malignant transformation by environmental carcinogens, are highly elevated and appear constitutively active in breast cancer cells. Rel factors have been found to functionally interact with other transcription factors. Here we demonstrate a physical and functional association between the RelA subunit of NF-kappaB and AhR resulting in the activation of c-myc gene transcription in breast cancer cells. RelA and AhR proteins were co-immunoprecipitated from cytoplasmic and nuclear extracts of non-malignant MCF-10F breast epithelial and malignant Hs578T breast cancer cells. In transient co-transfection, RelA and AhR gene products demonstrated cooperation in transactivation of the c-myc promoter, which was dependent on the NF-kappaB elements, and in induction of endogenous c-Myc protein levels. A novel AhR/RelA-containing NF-kappaB element binding complex was identified by electrophoretic mobility shift analysis of nuclear extracts from RelA and AhR co-transfected Hs578T cells. Thus, the RelA and AhR proteins functionally cooperate to bind to NF-kappaB elements and induce c-myc gene expression. These findings suggest a novel signaling mechanism whereby the Ah receptor can stimulate proliferation and tumorigenesis of mammary cells.

Activation of NF-kappaB/Rel occurs early during neoplastic transformation of mammary cells.

NF-kappaB/Rel is a family of transcription factors which are expressed in all cells; however, in most non-B cells, they are sequestered in the cytoplasm in inactive complexes with specific inhibitory proteins, termed IkappaBs. We have recently shown that NF-kappaB/Rel factors are aberrantly activated in human breast cancer and rodent mammary tumors, and function to promote tumor cell survival and proliferation. Here, we have examined the time-course of induction of NF-kappaB/Rel factors upon carcinogen treatment of female Sprague-Dawley (S-D) rats in vivo and in human mammary epithelial cells (HMECs) in culture. We observed that NF-kappaB/Rel activation is an early event, occurring prior to malignant transformation. In S-D rats, increased NF-kappaB/Rel binding was detected in nuclear extracts of mammary glands from 40% of animals 3 weeks post-treatment with 15 mg/kg 7,12-dimethylbenz[a]anthracene (DMBA); this is prior to formation of tumors which normally begin to be detected after 7-9 weeks. In non-tumorigenic MCF-10F cells, in vitro malignant transformation upon treatment with either DMBA or benzo[a]pyrene (B[a]P) resulted in a 4- to 12-fold increase in activity of classical NF-kappaB (p65/p50). NF-kappaB induction was corrrelated with a decrease in the stability of the NF-kappaB-specific inhibitory protein IkappaB-alpha. Ectopic expression of the transactivating p65 subunit of NF-kappaB in MCF-10F cells induced the c-myc oncogene promoter, which is driven by two NF-kappaB elements, and endogenous c-Myc levels. Furthermore, reduction mammoplasty-derived HMECs, immortalized following B[a]P exposure, showed dysregulated induction of classical NF-kappaB prior to malignant transformation. Together these findings suggest that activation of NF-kappaB plays an early, critical role in the carcinogen-driven transformation of mammary glands.