Nathalie Jacobs

NF-κB transcription factor induces drug resistance through MDR1 expression in cancer cells

The ubiquitous NF-κB transcription factor has been reported to inhibit apoptosis and to induce drug resistance in cancer cells. Drug resistance is the major reason for cancer therapy failure and neoplastic cells often develop multiple mechanisms of drug resistance during tumor progression. We observed that NF-κB or P-glycoprotein inhibition in the HCT15 colon cancer cells led to increased apoptotic cell death in response to daunomycin treatment. Interestingly, NF-κB inhibition through transfection of a plasmid coding for a mutated IκB-α inhibitor increased daunomycin cell uptake. Indeed, the inhibition of NF-κB reduced mdr1 mRNA and P-glycoprotein expression in HCT15 cells. We identified a consensus NF-κB binding site in the first intron of the human mdr1 gene and demonstrated that NF-κB complexes could bind with this intronic site. Moreover, NF-κB transactivates an mdr1 promoter luciferase construct. Our data thus demonstrate a role for NF-κB in the regulation of the mdr1 gene expression in cancer cells and in drug resistance.

Regulation of vimentin by SIP1 in human epithelial breast tumor cells

The expression of Smad interacting protein-1 (SIP1; ZEB2) and the de novo expression of vimentin are frequently involved in epithelial-to-mesenchymal transitions (EMTs) under both normal and pathological conditions. In the present study, we investigated the potential role of SIP1 in the regulation of vimentin during the EMT associated with breast tumor cell migration and invasion. Examining several breast tumor cell lines displaying various degrees of invasiveness, we found SIP1 and vimentin expression only in invasive cell lines. Also, using a model of cell migration with human mammary MCF10A cells, we showed that SIP1 is induced specifically in vimentin-positive migratory cells. Furthermore, transfection of SIP1 cDNA in MCF10A cells increased their vimentin expression both at the mRNA and protein levels and enhanced their migratory abilities in Boyden Chamber assays. Inversely, inhibition of SIP1 expression by RNAi strategies in BT-549 cells and MCF10A cells decreased vimentin expression. We also showed that SIP1 transfection did not activate the TOP-FLASH reporter system, suggesting that the β-catenin/TCF pathway is not implicated in the regulation of vimentin by SIP1. Our results therefore implicate SIP1 in the regulation of vimentin observed in the EMT associated with breast tumor cell migration, a pathway that may contribute to the metastatic progression of breast cancer.

Regulation of NF-kappaB activity by I kappaB-related proteins in adenocarcinoma cells.

Constitutive NF-κB activity varies widely among cancer cell lines. In this report, we studied the expression and the role of different IκB inhibitors in adenocarcinoma cell lines. High constitutive NF-κB activity and low IκB-α expression was found in a number of these cell lines. Moreover, some of these cells showed a high p100 expression, responsible for the cytoplasmic sequestration of most of p65 complexes. Treatment of these cells with TNF-α or other NF-κB activating agents induced only weakly nuclear NF-κB activity without significant p100 processing and led to a very weak transcription of NF-κB-dependent reporter gene. Induction of NF-κB activity can be restored by expression of the Tax protein or by treatment with antisense p100 oligonucleotides. In MCF7 A/Z cells stably transfected with a p100 expression vector, p65 complexes were sequestered in the cytoplasm by p100. These cells showed a reduced nuclear NF-κB induction and NF-κB-dependent gene transcription following TNF-α stimulation. As a consequence of a competition between IκB-α and p100, cells expressing high levels of p100 respond poorly to NF-κB activating stimuli as TNF-α.

Cytokine expression in squamous intraepithelial lesions of the uterine cervix: implications for the generation of local immunosuppression

We have addressed the notion that the progression of cancer of the uterine cervix is associated with a preferential constraint on the development of a type 1 cellular mediated response, which is necessary to efficiently eliminate (pre)neoplastic cells. Based on the importance of cytokines in the regulation of an appropriate immune response, we have evaluated the expression of IL‐12p40, IL‐10 and transforming growth factor‐beta 1 (TGF‐β1). Using reverse transcriptase‐polymerase chain reaction (RT‐PCR), the expression of these three cytokines was evaluated in both low‐grade (LG) and high‐grade (HG) cervical squamous intraepithelial lesions (SIL) and in normal exocervix and transformation zone biopsies. Our results show that the average level of IL‐12 increases within both the LG and HG SIL, compared with both control groups. Interestingly, the percentage of HG SIL expressing IL‐12p40 was lower compared with LG SIL. In contrast, the expression of IL‐10 increased in parallel with the severity of the lesion to a maximal level in HG SIL. Using immunohistochemistry, we ascertained the presence of IL‐12 protein in SIL and IL‐10 protein in the transformation zone and SIL biopsies. Both IL‐12‐ and IL‐10‐producing cells were localized in the stroma, not within the SIL. Furthermore, in this study we also observed that the region of the cervix the most sensitive to lesion development, the transformation zone, was associated with higher average levels of the immunosuppressive cytokines IL‐10 and TGF‐β1.

The cross-talk between dendritic and regulatory T cells: good or evil?

Immune responses against pathogens require fine regulation to avoid excessive inflammation, which could be harmful to the host. Moreover, the immune system must be tolerant to nonpathogenic antigens to prevent allergy, autoimmunity, and transplant rejection. There is accumulating evidence that interactions between dendritic cells (DC) and regulatory T (Treg) cells play a crucial role in the balance between immune response and tolerance. Communications between these cells are complex, bidirectional, and mediated by soluble or cell surface molecules. The maturation status of DC, which may be influenced by different microenvironmental factors, is considered as an important checkpoint for the induction of peripheral tolerance through modifications of the activation status of T cells. Moreover, several lines of experimental evidence suggest that different subsets or the functional status of DC are also involved in the promotion of Treg cell differentiation. A better knowledge of the regulatory mechanisms of the immune response induced or inhibited by DC via their interactions with Treg cells could be relevant for the development of new, immunotherapeutic approaches.

Innate lymphocyte and dendritic cell cross-talk: a key factor in the regulation of the immune response

Dendritic cells (DC) are specialized in the presentation of antigens and the initiation of specific immune responses. They have been involved recently in supporting innate immunity by interacting with various innate lymphocytes, such as natural killer (NK), NK T or T cell receptor (TCR)‐γδ cells. The functional links between innate lymphocytes and DC have been investigated widely and different studies demonstrated that reciprocal activations follow on from NK/DC interactions. The cross‐talk between innate cells and DC which leads to innate lymphocyte activation and DC maturation was found to be multi‐directional, involving not only cell–cell contacts but also soluble factors. The final outcome of these cellular interactions may have a dramatic impact on the quality and strength of the down‐stream immune responses, mainly in the context of early responses to tumour cells and infectious agents. Interestingly, DC, NK and TCR‐γδ cells also share similar functions, such as antigen uptake and presentation, as well as cytotoxic and tumoricidal activity. In addition, NK and NK T cells have the ability to kill DC. This review will focus upon the different aspects of the cross‐talk between DC and innate lymphocytes and its key role in all the steps of the immune response. These cellular interactions may be particularly critical in situations where immune surveillance requires efficient early innate responses.

Natural killer cells: role in local tumor growth and metastasis

Historically, the name of natural killer (NK) cells came from their natural ability to kill tumor cells in vitro. From the 1970s to date, accumulating data highlighted the importance of NK cells in host immune response against cancer and in therapy-induced antitumor response. The recognition and the lysis of tumor cells by NK cells are regulated by a complex balance of inhibitory and activating signals. This review summarizes NK cell mechanisms to kill cancer cells, their role in host immune responses against tumor growth or metastasis, and their implications in antitumor immunotherapies via cytokines, antibodies, or in combination with other therapies. The regulatory role of NK cells in autoimmunity is also discussed.

Inverse modulation of IL-10 and IL-12 in the blood of women with preneoplastic lesions of the uterine cervix

It has been postulated that an impaired immune response may contribute to the progression of human papillomavirus (HPV)‐associated preneoplastic lesions. Based on this hypothesis, we evaluated the cytokine production in the blood of patients with squamous intraepithelial lesions (SIL) of the uterine cervix. The levels of type‐1 (interferon‐gamma (IFN‐γ) and IL‐12) and type‐2 (IL‐4 and IL‐10) cytokines were measured in whole blood culture supernatants of patients with low‐ and high‐grade SIL and control women. There was no difference in IL‐4 and IFN‐γ levels between patients with SIL and the control group. In contrast, the ratio of IL‐12/IL‐10 levels was significantly lower in patients with SIL compared with the control group. A lower IL‐12/IL‐10 ratio in women with SIL was also observed when peripheral blood mononuclear cell (PBMC) culture supernatants and plasma samples were analysed. In patients, neither the lower expression of the CD3ε chain nor the higher frequency of HLA‐DRB1*1501 expression could be correlated with abnormal cytokine production. These results suggest that a part of the cytokine network, namely IL‐10 and IL‐12, is perturbed in patients with SIL. A better knowledge of the role of these cytokines in regulating the growth of HPV‐associated SIL might have practical implications for the development of vaccines or immunomodulatory strategies in the treatment of cervical cancers.

Downregulation of ICAM-1 and VCAM-1 expression in endothelial cells treated by photodynamic therapy

Photodynamic therapy (PDT) is a treatment for cancer and several noncancerous proliferating cell diseases that depends on the uptake of a photosensitizing compound followed by selective irradiation with visible light. In the presence of oxygen, irradiation leads to the production of reactive oxygen species (ROS). A large production of ROS induces the death of cancer cells by apoptosis or necrosis. A small ROS production can activate various cellular pathways. Here, we show that PDT by pyropheophorbide-a methyl ester (PPME) induces the activation of nuclear factor kappa B (NF-κB) in HMEC-1 cells. NF-κB is active since it binds to the NF-κB sites of both ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) promoters and induces the transcription of several NF-κB target genes such as those of IL-6, ICAM-1, VCAM-1. In contrast, expression of ICAM-1 and VCAM-1 at the protein level was not observed, although we measured an IL-6 secretion. Using specific chemical inhibitors, we showed that the lack of ICAM-1 and VCAM-1 expression is the consequence of their degradation by lysosomal proteases. The proteasome and calpain pathways were not involved. All these observations were consistent with the fact that no adhesion of granulocytes was observed in these conditions.

Caspase-8-Dependent HER-2 Cleavage in Response to Tumor Necrosis Factor {alpha} Stimulation Is Counteracted by Nuclear Factor {kappa}B through c-FLIP-L Expression

The oncoprotein HER-2/neu is a prosurvival factor, and its overexpression has been correlated with poor prognosis in patients with breast cancer. We report that HER-2 is a new substrate for caspase-8 and that tumor necrosis factor alpha (TNF-alpha) stimulation leads to an early caspase-8-dependent HER-2 cleavage in MCF7 A/Z breast adenocarcinoma cells defective for nuclear factor kappaB (NFkappaB) activation. We show that the antiapoptotic transcription factor NFkappaB counteracts this cleavage through induction of the caspase-8 inhibitor c-FLIP. Our results also demonstrate that this HER-2 cleavage contributes to the TNF-alpha-induced apoptosis pathway because ectopic expression of an uncleavable HER-2 protects NFkappaB-defective cells against TNF-alpha-mediated cell death. Therefore, we propose an original model in which NFkappaB exerts a new antiapoptotic function by counteracting TNF-alpha-triggered cleavage of the HER-2 survival factor.