Deficiency in tumor suppressor p53 is required for doxorubicin induced transcriptional upregulation of NF-kB target genes in human breast cancer
ABSTRACT: NF-kB has been linked to doxorubicin-based chemotherapy resistance in breast cancer patients. NF-kB nuclear translocation and DNA binding in doxorubicin treated-breast cancer cells have been extensively examined, however its functional consequences in terms the spectrum of NF-kB -dependent genes expressed and, thus, the impact on tumour cell behaviour are unclear. We hypothesized that NF-kB gene expression profile induced by doxorubicin might be different among breast cancer cells and tumors. Doxorubicin treatment in the p53-mutated MDA-MB-231 cells resulted in NF-kB driven-gene transcription demonstrated by gene expression microarrays. Selected genes (ICAM-1, CXCL1, IL8) related with invasion, metastasis and chemoresistance expression were confirmed by RT-PCR in a subset of additional doxorubicin-treated cells and fresh primary human breast tumors. In both systems, p53-deficient background correlated with the activation of these NF-kB targeted genes. Overexpression of p53WT in the mutant p53 MDA-MB-231 cells impaired NF-kB driven transcription induced by doxorubicin. Moreover, tumors with a p53 deficient background and nuclear NF-kB /p65 expression correlated with reduced disease free-survival. This study supports that tumor molecular profiles for doxorubicin driven NF-kB-response are likely to exist. A link between p53 deficiency and the presence of active transcriptionally NF-kB could favour an aggressive behaviour and might have implications for doxorubicin-based chemotherapy in breast tumors exhibiting aberrant p53 activity 12 samples were analyzed: controls (n=3); Doxorubicin treated (n=3); MLN120B treated (n=3); MLN120B + Doxorubicin treated (n=3)
Project description:NF-?B has been linked to doxorubicin resistance in breast cancer patients. NF-?B nuclear translocation and DNA binding in doxorubicin treated-breast cancer cells have been extensively examined; however its functional relevance at transcriptional level on NF-?B-dependent genes and the biological consequences are unclear. We studied NF-?B-dependent gene expression induced by doxorubicin in breast cancer cells and fresh human cancer specimens with different genetic backgrounds focusing on their p53 status. NF-?B-dependent signature of doxorubicin was identified by gene expression microarrays in breast cancer cells treated with doxorubicin and the IKK?-inhibitor MLN120B, and confirmed ex vivo in human cancer samples. The association with p53 was functionally validated. Finally, NF-?B activation and p53 status was determined in a cohort of breast cancer patients treated with adjuvant doxorubicin-based chemotherapy. Doxorubicin treatment in the p53-mutated MDA-MB-231 cells resulted in NF-?B driven-gene transcription signature. Modulation of genes related with invasion, metastasis and chemoresistance (ICAM-1, CXCL1, TNFAIP3, IL8) were confirmed in additional doxorubicin-treated cell lines and fresh primary human breast tumors. In both systems, p53-deficient background correlated with the activation of the NF-?B-dependent signature. Furthermore, restoration of p53WT in the mutant p53 MDA-MB-231 cells impaired NF-?B driven transcription induced by doxorubicin. Moreover, a p53 deficient background and nuclear NF-?B/p65 in breast cancer patients correlated with reduced disease free-survival. This study supports that p53 deficiency is necessary for a doxorubicin driven NF-?B-response that limits doxorubicin cytotoxicity in breast cancer and is linked to an aggressive clinical behavior.
Project description:Metformin was shown to sensitize multidrug resistant breast cancer cells; however, the mechanisms involved in this capacity need to be clarified. We investigated oxidative stress and inflammatory-related pathways during the induction of doxorubicin resistance in MCF-7 and MDA-MB-231 human breast cancer cells (DOX-res group), and evaluated metformin-induced cellular responses that resulted in the prevention of doxorubicin resistance (Met-DOX group). Microarray analysis demonstrated that DOX-res changed the expression of genes involved in oxidative stress (OS) and the TGF- β1 pathway. The DOX-res group presented increased thiols and reduced lipoperoxidation, increased levels of nitric oxide, nuclear NF-kB and Nrf2, and reduced nuclear p53 labelling. Analysis of the TGF-β1 signaling pathway by RT-PCR array showed that DOX-res developed adaptive responses, such as resistance against apoptosis and OS. Metformin treatment modified gene expression related to OS and the IFN-α signaling pathway. The Met-DOX group was more sensitive to DOX-induced OS, presented lower levels of nitric oxide, nuclear NF-kB and Nrf2, and increased nuclear p53. Analysis of the IFN-α signaling pathway showed that Met-DOX presented more sensitivity to apoptosis and OS. Our findings indicate that metformin is a promising tool in the prevention of chemoresistance in patients with breast cancer submitted to doxorubicin-based treatments.
Project description:We present a new model of ESR1 network regulation based on analysis of Doxorubicin, Estradiol, and TNF? combination treatment in MCF-7. We used Doxorubicin as a therapeutic agent, TNF? as marker and mediator of an inflammatory microenvironment and 17?-Estradiol (E2) as an agonist of Estrogen Receptors, known predisposing factor for hormone-driven breast cancer, whose pharmacological inhibition reduces the risk of breast cancer recurrence. Based on the results of transcriptomics analysis, we found 71 differentially expressed genes that are specific for the combination treatment with Doxorubicin?+?Estradiol?+?TNF? in comparison with single or double treatments. The responsiveness to the triple treatment was examined for seven genes by qPCR, of which six were validated, and then extended to four additional cell lines differing for p53 and/or ER status. The results of differential regulation enrichment analysis highlight the role of the ESR1 network that included 36 of 71 specific differentially expressed genes. We propose that the combined activation of p53 and NF-kB transcription factors significantly influences ligand-dependent, ER-driven transcriptional responses, also of the ESR1 gene itself. These results provide a model of coordinated interaction of TFs to explain the Doxorubicin, E2 and TNF? induced repression mechanisms.
Project description:The tandem duplicator phenotype (TDP) is a genome-wide instability configuration primarily observed in breast, ovarian, and endometrial carcinomas. Here, we stratify TDP tumors by classifying their tandem duplications (TDs) into three span intervals, with modal values of 11 kb, 231 kb, and 1.7 Mb, respectively. TDPs with ?11 kb TDs feature loss of TP53 and BRCA1. TDPs with ?231 kb and ?1.7 Mb TDs associate with CCNE1 pathway activation and CDK12 disruptions, respectively. We demonstrate that p53 and BRCA1 conjoint abrogation drives TDP induction by generating short-span TDP mammary tumors in genetically modified mice lacking them. Lastly, we show how TDs in TDP tumors disrupt heterogeneous combinations of tumor suppressors and chromatin topologically associating domains while duplicating oncogenes and super-enhancers.
Project description:Galectin-7 was initially described as a marker of epithelial differentiation expressed in the stratified epithelium of various tissues. Like other members of the galectin family, its expression level is often significantly altered in cancer cells. In breast cancer, its expression is significantly augmented in aggressive molecular subtypes, most notably in estrogen receptor-negative tumors and in cell lines with a basal-like phenotype. Studies using experimental mouse models have further shown high expression of galectin-7 was sufficient to increase the metastatic behavior of poorly metastatic breast cancer cells, rendering them more resistant to apoptosis. This expression pattern in breast cancer cells is unexpected because galectin-7 was originally identified as a p53-induced gene. To address this paradox, we have examined the molecular mechanisms regulating galectin-7 in breast cancer cells. Our results showed that transfection of breast cancer cells with expression vectors encoding mutant p53 was sufficient to induce galectin-7 at both mRNA and protein levels. Doxorubicin treatment of breast cancer cells harboring a mutant p53 also induced galectin-7. This induction was specific since knockdown of endogenous mutant p53 inhibited doxorubicin-induced galectin-7 expression. The p53-induced galectin-7 expression in breast cancer cells correlated with increased NF-?B activity and was inhibited by NF-?B inhibitors, indicating that the ability of mutant p53 to induce galectin-7 was dependent on NF-?B activity. The implication of NF-?B was further supported by data showing that NF-?B bound to the endogenous galectin-7 promoter and that TNF?-induced galectin-7 expression was abolished by NF-?B inhibitors. Taken together, our data provide an explanation to the observed high galectin-7 expression levels in cancer cells and suggest that galectin-7 could be part of a common pathway used by mutant p53 to promote cancer progression.
Project description:Breast cancer is the most common cancer and the second leading cause of cancer death in industrialized countries. Systemic treatment of breast cancer is effective at the beginning of therapy. However, after a variable period of time, progression occurs due to therapy resistance. Artesunate, clinically used as anti-malarial agent, has recently revealed remarkable anti-tumor activity offering a role as novel candidate for cancer chemotherapy. We analyzed the anti-tumor effects of artesunate in metastasizing breast carcinoma in vitro and in vivo. Unlike as expected, artesunate induced resistance in highly metastatic human breast cancer cells MDA-MB-231. Likewise acquired resistance led to abolishment of apoptosis and cytotoxicity in pre-treated MDA-MB-231 cells. In contrast, artesunate was more cytotoxic towards the less tumorigenic MDA-MB-468 cells without showing resistance. Unraveling the underlying molecular mechanisms, we found that resistance was induced due to activation of the tumor progression related transcription factors NF?B and AP-1. Thereby transcription, expression and activity of the matrix-degrading enzyme MMP-1, whose function is correlated with increased invasion and metastasis, was up-regulated upon acquisition of resistance. Additionally, activation of the apoptosis-related factor NF?B lead to increased expression of ant-apoptotic bcl2 and reduced expression of pro-apoptotic bax. Application of artesunate in vivo in a model of xenografted breast cancer showed, that tumors growth was not efficiently abolished as compared to the control drug doxorubicin. Taken together our in vitro and in vivo results correlate well showing for the first time that artesunate induces resistance in highly metastatic breast tumors.
Project description:Reovirus is a ubiquitous RNA virus that exploits aberrant signaling pathways for its replication. The oncolytic potential of reovirus against numerous cancers under pre-clinical/clinical conditions has been documented by us and others. Despite its proven clinical activity, the underlying mechanisms of reovirus oncolysis is still not well elucidated. If reovirus therapy is to be optimized for cancer, including breast cancer patients, it is imperative to understand the mechanisms of reovirus oncolysis, especially in treatment of resistant tumour.In the present study global gene expression profiling was utilized as a preliminary roadmap to tease-out pivotal molecules involved in reovirus induced apoptosis in breast cancer. Reovirus treated HTB133 and MCF7 breast cancer cells revealed transcriptional alteration of a defined subset of apoptotic genes and members of the nuclear factor-kappa B (NF-kB) family and p53 upregulated modulator of apoptosis (PUMA) were prominent. Since NF-kB can paradoxically suppress or promote apoptosis in cancer, the significance of NF-kB in reovirus oncolysis of breast cancer was investigated. Real time PCR analysis indicated a 2.9-4.3 fold increase in NF-kB p65 message levels following reovirus infection of MCF7 and HTB133, respectively. Nuclear translocation of NF-kB p65 protein was also dramatically augmented post reovirus treatment and correlated with enhanced DNA binding. Pharmacologic inhibition of NF-kB lead to oncolytic protection and significant down regulation of PUMA message levels. PUMA down regulation using siRNA suppressed reovirus oncolysis via significantly repressed apoptosis in p53 mutant HTB133 cells.This study demonstrates for the first time that a prominent pathway of reovirus oncolysis of breast cancer is mediated through NF-kB and that PUMA upregulation is dependent on NF-kB activation. These findings represent potential therapeutic indicators of reovirus treatment in future clinical trials.
Project description:The aim of this work was to understand whether the nature of breast cancer cells could modify the nature of the dialog of mesenchymal stem cells (MSCs) with cancer cells. By treating MSCs with the conditioned medium of metastatic Estrogen-receptor (ER)-negative MDA-MB-231, or non-metastatic ER-positive MCF-7 breast cancer cells, we observed that a number of chemokines were produced at higher levels by MSCs treated with MDA-MB-231 conditioned medium (CM). MDA-MB-231 cells were able to induce NF-?B signaling in MSC cells. This was shown by the use of a NF-kB chemical inhibitor or an I?B dominant negative mutant, nuclear translocation of p65 and induction of NF-?B signature. Our results suggest that MDA-MB-231 cells exert their effects on MSCs through the secretion of IL-1?, that activates MSCs and induces the same chemokines as the MDA-MB-231CM. In addition, inhibition of IL-1? secretion in the MDA-MB-231 cells reduces the induced production of a panel of chemokines by MSCs, as well the motility of MDA-MB-231 cells. Our data suggest that aggressive breast cancer cells secrete IL-1?, which increases the production of chemokines by MSCs.
Project description:The receptor activator of nuclear factor-kB (NF-kB) (RANK)/receptor activator of NF-kB ligand (RANKL) axis emerges as a key regulator of breast cancer initiation, progression and metastasis. RANK receptor is a tumor necrosis superfamily member, which upon ligand binding transduces a variety of survival, proliferation, differentiation and migration signals. The majority of these intracellular cues merge through the NF-kB transcription machinery.TNFRSF11A (RANK) variants were identified and cloned in mammalian expression vectors. Their expression was analyzed using real time PCR on RNA from normal tissue, cell lines and breast cancer specimens. Western blot analysis and immunofluoresence stainings were used to study expression and localization of protein isoforms in a panel of breast cancer cell lines and in transfected 293T cells. Luciferase assays were employed to assess the contribution of each isoform alone or in combinations on NF-kB activation. Isoform effect on cell survival after doxorubicin treatment was analyzed through MTT assay. Wound healing and transwell assays were employed to evaluate the effect of TNFRSF11A isoforms on migration of MDA-MB-231 and 293T cells.We report the identification of three novel TNFRSF11A (RANK) variants, named TNFRSF11A_?9, TNFRSF11A_?8,9 and TNFRSF11A_?7,8,9 which result from the alternative splicing of exons 7 to 9. Interestingly, variant TNFRSF11A_?7,8,9 was found to be upregulated in breast cancer cells lines and its expression inversely correlated with tumor grade and proliferation index. TNFRSF11A_?7,8,9 encodes a 40-45 kDa protein, we named RANK-c, which lacks the transmembrane domain and most of the intracellular part of the wild type receptor. Furthermore, we showed that RANK-c could act as a dominant negative regulator of RANK-dependent NF-kB activation, affecting cell survival after apoptosis induction. In addition, RANK-c suppresses cell migration and represses the tumorigenic properties of invasive breast carcinoma cells.In this study, we provide evidence of a complex regulatory network of RANK receptor splice variants with a role in breast cancer. We identify that the RANK-c isoform is expressed in breast cancer samples and its expression reversely correlates with histological grade. Finally, isoform RANK-c seems to have the capacity to regulate signaling through wild type RANK and moreover to inhibit cell motility and migration of breast cancer cells.
Project description:RAS mutations or its activation by upstream receptor tyrosine kinases are frequently associated with poor response of carcinomas to chemotherapy. The 18 kDa propeptide domain of lysyl oxidase (LOX-PP) released from the secreted precursor protein (Pro-LOX) has been shown to inhibit RAS signaling and the transformed phenotype of breast, pancreatic, lung, and prostate cancer cells in culture, and formation of tumors by Her-2/neu-driven breast cancer cells in a mouse xenograft model. Here, we tested the effects of LOX-PP on MIA PaCa-2 pancreatic cancer cells, driven by mutant RAS. In MIA PaCa-2 cells in culture, LOX-PP attenuated the ERK and AKT activities and decreased the levels of the NF-?B p65 and RelB subunits and cyclin D1, which are activated by RAS signaling. In mouse xenograft growth, LOX-PP reduced growth of tumors by these pancreatic cancer cells, and the nuclear levels of the p65 NF-?B subunit and cyclin D1 proteins. While biological agents attenuate tumor growth when used alone, often they have additive or synergistic effects when used in combination with chemotherapeutic agents. Thus, we next tested the hypotheses that LOX-PP sensitizes pancreatic and breast cancer cells to the chemotherapeutic agent doxorubicin. Purified LOX-PP enhanced the cytotoxic effects of doxorubicin in pancreatic and breast cancer cells, as judged by ATP production, Cell Death ELISA assays, caspase 3 activation, PARP cleavage, and Annexin V staining. Thus, LOX-PP potentiates the cytotoxicity of doxorubicin on breast and pancreatic cancer cells, warranting additional studies with a broader spectrum of current cancer treatment modalities.