Raphaëlle Romieu-Mourez

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.

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.

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.

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

NF-κB/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-κB 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-κB 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-κB elements, and in induction of endogenous c-Myc protein levels. A novel AhR/RelA-containing NF-κB 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-κB 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.

Inducible IκB Kinase/IκB Kinase ε Expression Is Induced by CK2 and Promotes Aberrant Nuclear Factor-κB Activation in Breast Cancer Cells

Aberrant activation of nuclear factor-κB (NF-κB) transcription factors has been implicated in the pathogenesis of breast cancer. We previously showed elevated activity of IκB kinase α (IKKα), IKKβ, and protein kinase CK2 in primary human breast cancer specimens and cultured cells. A novel inducible IKK protein termed IKK-i/IKKe has been characterized as a potential NF-κB activator. Here, we provide evidence that implicates IKK-i/IKKe in the pathogenesis of breast cancer. We show IKK-i/IKKe expression in primary human breast cancer specimens and carcinogen-induced mouse mammary tumors. Multiple breast cancer cell lines showed higher levels of IKK-i/IKKe and kinase activity compared with untransformed MCF-10F breast epithelial cells. Interestingly, IKK-i/IKKe expression correlated with CK2α expression in mammary glands and breast tumors derived from MMTV-CK2α transgenic mice. Ectopic CK2 expression in untransformed cells led to increased IKK-i/IKKe mRNA and protein levels. Inhibition of CK2α via the pharmacologic inhibitor apigenin or upon transfection of a CK2 kinase-inactive subunit reduced IKK-i/IKKe levels. Expression of a kinase-inactive IKK-i/IKKe mutant in breast cancer cells reduced NF-κB activity as judged by transfection assays of reporters driven either by NF-κB elements or the promoters of two NF-κB target genes, cyclin D1 and relB. Importantly, the kinase-inactive IKK-i/IKKe mutant reduced the endogenous levels of these genes as well as the ability of breast cancer cells to grow in soft agar or form invasive colonies in Matrigel. Thus, CK2 induces functional IKK-i/IKKe, which is an important mediator of the activation of NF-κB that plays a critical role in the pathogenesis of breast cancer. (Cancer Res 2005; 65(24): 11375-83)

Mouse Mammary Tumor Virus c-rel Transgenic Mice Develop Mammary Tumors

ABSTRACT Amplification, overexpression, or rearrangement of the c-rel gene, encoding the c-Rel NF-κB subunit, has been reported in solid and hematopoietic malignancies. For example, many primary human breast cancer tissue samples express high levels of nuclear c-Rel. While the Rev-T oncogene v-rel causes tumors in birds, the ability of c-Rel to transform in vivo has not been demonstrated. To directly test the role of c-Rel in breast tumorigenesis, mice were generated in which overexpression of mouse c-rel cDNA was driven by the hormone-responsive mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, and four founder lines identified. In the first cycle of pregnancy, the expression of transgenic c-rel mRNA was observed, and levels of c-Rel protein were increased in the mammary gland. Importantly, 31.6% of mice developed one or more mammary tumors at an average age of 19.9 months. Mammary tumors were of diverse histology and expressed increased levels of nuclear NF-κB. Analysis of the composition of NF-κB complexes in the tumors revealed aberrant nuclear expression of multiple subunits, including c-Rel, p50, p52, RelA, RelB, and the Bcl-3 protein, as observed previously in human primary breast cancers. Expression of the cancer-related NF-κB target genes cyclin D1, c-myc, and bcl-xl was significantly increased in grossly normal transgenic mammary glands starting the first cycle of pregnancy and increased further in mammary carcinomas compared to mammary glands from wild-type mice or virgin transgenic mice. In transient transfection analysis in untransformed breast epithelial cells, c-Rel-p52 or -p50 heterodimers either potently or modestly induced cyclin D1 promoter activity, respectively. Lastly, stable overexpression of c-Rel resulted in increased cyclin D1 and NF-κB p52 and p50 subunit protein levels. These results indicate for the first time that dysregulated expression of c-Rel, as observed in breast cancers, is capable of contributing to mammary tumorigenesis.

RelB/p52 NF-κB Complexes Rescue an Early Delay in Mammary Gland Development in Transgenic Mice with Targeted Superrepressor IκB-α Expression and Promote Carcinogenesis of the Mammary Gland

ABSTRACT Classical NF-κB (p65/p50) transcription factors display dynamic induction in the mammary gland during pregnancy. To further elucidate the role of NF-κB factors in breast development, we generated a transgenic mouse expressing the IκB-α S32/36A superrepressor (SR) protein under control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. A transient delay in mammary ductal branching was observed in MMTV-SR-IκB-α mice early during pregnancy at day 5.5 (d5.5) and d7.5; however, development recovered by mid- to late pregnancy (d14.5). Recovery correlated with induction of nuclear cyclin D1 and RelB/p52 NF-κB complexes. RelB/p52 complexes induced cyclin D1 and c-myc promoter activities and failed in electrophoretic mobility shift assay to interact with IκB-α-glutathione S-transferase, indicating that their weak interaction with IκB-α can account for the observed recovery of mammary gland development. Activation of IKKα and NF-κB-inducing kinase was detected by d5.5, implicating the alternative NF-κB signaling pathway in RelB/p52 induction. Constitutively active IKKα induced p52, RelB, and cyclin D1 in untransformed mammary epithelial cells. Moreover, mouse mammary tumors induced by 7,12-dimethylbenz(a)anthracene treatment displayed increased RelB/p52 activity. Inhibition of RelB in breast cancer cells repressed cyclin D1 and c-Myc levels and growth in soft agar. These results implicate RelB/p52 complexes in mammary gland development and carcinogenesis.

Distinct Roles for IFN Regulatory Factor (IRF)-3 and IRF-7 in the Activation of Antitumor Properties of Human Macrophages

When properly activated, macrophages can be tumoricidal, thus making them attractive additions to standard cancer therapies. To this end, tolerance and activity of human autologous IFN-gamma-activated macrophages, produced in large scale for clinical use (MAK cells), have been assessed in pilot trials in cancer patients. In the present study, we tested the hypothesis that activation of IFN regulatory factor (IRF)-3 and IRF-7, with subsequent type I IFN production, may be involved in the acquisition of new antitumor functions by macrophages. Adenoviral vectors were generated for the delivery of constitutively active forms of IRF-3 (Ad-IRF-3) or IRF-7 (Ad-IRF-7) into primary human macrophages. Cell death was observed in Ad-IRF-3-transduced macrophages, whereas Ad-IRF-7-transduced macrophages produced type I IFNs and displayed increased expression of genes encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand, interleukin (IL)-12, IL-15, and CD80, persisting for at least 96 hours. Expression of iNOS, TNF-alpha, FasL, IL-1, and IL-6 genes was unaltered by Ad-IRF-7 transduction. Interestingly, Ad-IRF-3 or Ad-IRF-7 transduction negatively regulated the transcription of protumorigenic genes encoding vascular endothelial growth factor and matrix metalloproteinase-2. Furthermore, Ad-IRF-7-transduced macrophages exerted a cytostatic activity on different cancer cell lines, including SK-BR-3, MCF-7, and COLO-205; the latter cells were shown previously to be insensitive to MAK cells. In conclusion, transduction of active forms of IRF-3 or IRF-7 differentially modulate the apoptotic and antitumor properties of primary macrophages, with active IRF-7 leading to the acquisition of novel antitumor effector functions.

Mesenchymal Stromal Cells Expressing ErbB-2/neu Elicit Protective Anti–Breast Tumor Immunity In vivo, which is Paradoxically Suppressed by IFN-γ and Tumor Necrosis Factor-α Priming

It is unknown whether mesenchymal stromal cells (MSC) can regulate immune responses targeting tumor autoantigens of low immunogenicity. We tested here whether immunization with MSC could break immune tolerance towards the ErbB-2/HER-2/neu tumor antigen and the effects of priming with IFN-γ and tumor necrosis factor-α (TNF-α) on this process. BALB/c- and C57BL/6-derived MSC were lentivirally transduced to express a kinase-inactive rat neu mutant (MSC/Neu). Immunization of BALB/c mice with nontreated or IFN-γ-primed allogeneic or syngeneic MSC/Neu induced similar levels of anti-neu antibody titers; however, only syngeneic MSC/Neu induced protective neu-specific CD8(+) T cell responses. Compared to immunization with nontreated or IFN-γ-primed syngeneic MSC/Neu, the number of circulating neu-specific CD8(+) T cells and titers of anti-neu antibodies were observed to be decreased after immunizations with IFN-γ- plus TNF-α-primed MSC/Neu. In addition, syngeneic MSC/Neu seemed more efficient than IFN-γ-primed MSC/Neu at inducing a protective therapeutic antitumor immune response resulting in the regression of transplanted neu-expressing mammary tumor cells. In vitro antigen-presenting cell assays performed with paraformaldehyde-fixed or live MSC showed that priming with IFN-γ plus TNF-α, compared to priming with IFN-γ alone, increased antigen presentation as well as the production of immunosuppressive factors. These data suggest that whereas MSC could effectively serve as antigen-presenting cells to induce immune responses aimed at tumor autoantigens, these functions are critically regulated by IFN-γ and TNF-α.