RIP140 regulates transcription factor HES1 oscillatory expression and mitogenic activity in colon cancer cells.

The transcription factor receptor-interacting protein 140 (RIP140) regulates intestinal homeostasis and tumorigenesis through Wnt signaling. In this study, we investigated its effect on the Notch/HES1 signaling pathway. In colorectal cancer (CRC) cell lines, RIP140 positively regulated HES1 gene expression at the transcriptional level via a recombining binding protein suppressor of hairless (RBPJ)/neurogenic locus notch homolog protein 1 (NICD)-mediated mechanism. In support of these in vitro data, RIP140 and HES1 expression significantly correlated in mouse intestine and in a cohort of CRC samples, thus supporting the positive regulation of HES1 gene expression by RIP140. Interestingly, when the Notch pathway is fully activated, RIP140 exerted a strong inhibition of HES1 gene transcription controlled by the level of HES1 itself. Moreover, RIP140 directly interacts with HES1 and reversed its mitogenic activity in human CRC cells. In line with this observation, HES1 levels were associated with a better patient survival only when tumors expressed high levels of RIP140. Our data identify RIP140 as a key regulator of the Notch/HES1 signaling pathway, with a dual effect on HES1 gene expression at the transcriptional level and a strong impact on colon cancer cell proliferation.

Anti-cathepsin D immunotherapy triggers both innate and adaptive anti-tumour immunity in breast cancer.

BACKGROUND AND PURPOSE: Triple-negative breast cancer (TNBC) has poorer outcomes than other breast cancers (BC), including HER2+ BC. Cathepsin D (CathD) is a poor prognosis marker overproduced by BC cells, hypersecreted in the tumour microenvironment with tumour-promoting activity. Here, we characterized the immunomodulatory activity of the anti-CathD antibody F1 and its improved Fab-aglycosylated version (F1M1) in immunocompetent mouse models of TNBC (C57BL/6 mice harbouring E0771 cell grafts) and HER2-amplified BC (BALB/c mice harbouring TUBO cell grafts). EXPERIMENTAL APPROACH: CathD expression was evaluated by western blotting and immunofluorescence, and antibody binding to CathD by ELISA. Antibody anti-tumour efficacy was investigated in mouse models. Immune cell recruitment and activation were assessed by immunohistochemistry, immunophenotyping, and RT-qPCR. KEY RESULTS: F1 and F1M1 antibodies remodelled the tumour immune landscape. Both antibodies promoted innate antitumour immunity by preventing the recruitment of immunosuppressive M2-polarized tumour-associated macrophages (TAMs) and by activating natural killer cells in the tumour microenvironment of both models. This translated into a reduction of T-cell exhaustion markers in the tumour microenvironment that could be locally supported by enhanced activation of anti-tumour antigen-presenting cell (M1-polarized TAMs and cDC1 cells) functions. Both antibodies inhibited tumour growth in the highly-immunogenic E0771 model, but only marginally in the immune-excluded TUBO model, indicating that anti-CathD immunotherapy is more relevant for BC with a high immune cell infiltrate, as often observed in TNBC. CONCLUSION AND IMPLICATION: Anti-CathD antibody-based therapy triggers the anti-tumour innate and adaptive immunity in preclinical models of BC and is a promising immunotherapy for immunogenic TNBC.

RIP140 regulates POLK gene expression and the response to alkylating drugs in colon cancer cells.

Aim: The transcription factor RIP140 (receptor interacting protein of 140 kDa) is involved in intestinal tumorigenesis. It plays a role in the control of microsatellite instability (MSI), through the regulation of MSH2 and MSH6 gene expression. The aim of this study was to explore its effect on the expression of POLK, the gene encoding the specialized translesion synthesis (TLS) DNA polymerase κ known to perform accurate DNA synthesis at microsatellites. Methods: Different mouse models and engineered human colorectal cancer (CRC) cell lines were used to analyze by RT-qPCR, while Western blotting and luciferase assays were used to elucidate the role of RIP140 on POLK gene expression. Published DNA microarray datasets were reanalyzed. The in vitro sensitivity of CRC cells to methyl methane sulfonate and cisplatin was determined. Results: RIP140 positively regulates, at the transcriptional level, the expression of the POLK gene, and this effect involves, at least partly, the p53 tumor suppressor. In different cohorts of CRC biopsies (with or without MSI), a strong positive correlation was observed between RIP140 and POLK gene expression. In connection with its effect on POLK levels and the TLS function of this polymerase, the cellular response to methyl methane sulfonate was increased in cells lacking the Rip140 gene. Finally, the association of RIP140 expression with better overall survival of CRC patients was observed only when the corresponding tumors exhibited low levels of POLK, thus strengthening the functional link between the two genes in human CRC. Conclusion: The regulation of POLK gene expression by RIP140 could thus contribute to the maintenance of microsatellite stability, and more generally to the control of genome integrity.

A Truncated NRIP1 Mutant Amplifies Microsatellite Instability of Colorectal Cancer by Regulating MSH2/MSH6 Expression, and Is a Prognostic Marker of Stage III Tumors.

Microsatellite instability (MSI) is related to the alteration of mismatch repair (MMR) genes and plays a key role in colorectal cancer (CRC) pathogenesis. We previously reported that the transcription factor Nuclear Receptor Interacting Protein 1 (NRIP1) is involved in sporadic intestinal tumorigenesis. The aim of this study was to decipher its role in MSI CRC. By using different mouse models and engineered cell lines, we demonstrated that NRIP1 increased MSH2 and MSH6 MMR gene transcription and mRNA/protein levels. In human CRC cells, NRIP1 expression was associated with decreased MSI and the hypermutator phenotype, and with resistance to chemotherapy drugs. Using a cohort of 194 CRC patients, we detected in 22% of the cases a MSI-induced frameshift mutation in the NRIP1 coding sequence. This genetic alteration generates a truncated protein with a dominant negative activity that increased human CRC cell proliferation and impaired the regulation of MSH2 and MSH6 gene expression. Moreover, the NRIP1 mutant correlated with a decreased overall survival of patients with advanced CRC, especially when MLH1-deficient. By decreasing the expression of MSH2 and MSH6 gene expression, the NRIP1 variant may amplify MLH1-dependent CRC progression and behave as a new prognostic marker of advanced MSI CRC.

RIP140 Represses Intestinal Paneth Cell Differentiation and Interplays with SOX9 Signaling in Colorectal Cancer.

RIP140 is a major transcriptional coregulator of gut homeostasis and tumorigenesis through the regulation of Wnt/APC signaling. Here, we investigated the effect of RIP140 on Paneth cell differentiation and its interplay with the transcription factor SOX9. Using loss of function mouse models, human colon cancer cells, and tumor microarray data sets we evaluated the role of RIP140 in SOX9 expression and activity using RT-qPCR, immunohistochemistry, luciferase reporter assays, and GST-pull down. We first evidence that RIP140 strongly represses the Paneth cell lineage in the intestinal epithelium cells by inhibiting Sox9 expression. We then demonstrate that RIP140 interacts with SOX9 and inhibits its transcriptional activity. Our results reveal that the Wnt signaling pathway exerts an opposite regulation on SOX9 and RIP140. Finally, the levels of expression of RIP140 and SOX9 exhibit a reverse response and prognosis value in human colorectal cancer biopsies. This work highlights an intimate transcriptional cross-talk between RIP140 and SOX9 in intestinal physiopathology.

Adsorption of proteins on TiO2 particles influences their aggregation and cell penetration.

TiO2 nanoparticles known as E171 are one controversial food additive due to its potential toxicity. In this work, the main hypothesis is that the proteins adsorbed on the TiO2 nanoparticles prevent their aggregation and favor the cell penetration. To do so, the TiO2 nanoparticles were coated with gelatin and ß-lactoglobulin to reach interfacial concentrations about 0.25 mg/mg and 0.32 mg/mg, respectively. The measurement of NP size showed that the protein coating improve the colloidal stability of TiO2 nanoparticles. The FTIR analysis suggests that the ß-lactoglobulin structure is modified after adsorption. The penetration of TiO2 penetration inside human intestinal epithelial cells was shown and quantify by using confocal Raman microscopy. The promoting role of the protein coating on the cell penetration was demonstrated for both the gelatin and ß-lactoglobulin. Finally, the results allow establishing a correlation between the ability of proteins to prevent NP aggregation and the cell penetration.

Increased expression of the HDAC9 gene is associated with antiestrogen resistance of breast cancers.

Estrogens play a pivotal role in breast cancer etiology, and endocrine therapy remains the main first line treatment for estrogen receptor-alpha (ERα)-positive breast cancer. ER are transcription factors whose activity is finely regulated by various regulatory complexes, including histone deacetylases (HDACs). Here, we investigated the role of HDAC9 in ERα signaling and response to antiestrogens in breast cancer cells. Various Michigan Cancer Foundation-7 (MCF7) breast cancer cell lines that overexpress class IIa HDAC9 or that are resistant to the partial antiestrogen 4-hydroxy-tamoxifen (OHTam) were used to study phenotypic changes in response to ER ligands by using transcriptomic and gene set enrichment analyses. Kaplan-Meier survival analyses were performed using public transcriptomic datasets from human breast cancer biopsies. In MCF7 breast cancer cells, HDAC9 decreased ERα mRNA and protein expression and inhibited its transcriptional activity. Conversely, HDAC9 mRNA was strongly overexpressed in OHTam-resistant MCF7 cells and in ERα-negative breast tumor cell lines. Moreover, HDAC9-overexpressing cells were less sensitive to OHTam antiproliferative effects compared with parental MCF7 cells. Several genes (including MUC1, SMC3 and S100P) were similarly deregulated in OHTam-resistant and in HDAC9-overexpressing MCF7 cells. Finally, HDAC9 expression was positively associated with genes upregulated in endocrine therapy-resistant breast cancers and high HDAC9 levels were associated with worse prognosis in patients treated with OHTam. These results demonstrate the complex interactions of class IIa HDAC9 with ERα signaling in breast cancer cells and its effect on the response to hormone therapy.

Expression and role of nuclear receptor coregulators in colorectal cancer.

Colorectal cancer (CRC) is one of the most common human cancers and the cause of about 700000 deaths per year worldwide. Deregulation of the WNT/ß-catenin pathway is a key event in CRC initiation. This pathway interacts with other nuclear signaling pathways, including members of the nuclear receptor superfamily and their transcription coregulators. In this review, we provide an overview of the literature dealing with the main coactivators (NCoA-1 to 3, NCoA-6, PGC1-α, p300, CREBBP and MED1) and corepressors (N-CoR1 and 2, NRIP1 and MTA1) of nuclear receptors and summarize their links with the WNT/ß-catenin signaling cascade, their expression in CRC and their role in intestinal physiopathology.

The IL-17B-IL-17 receptor B pathway promotes resistance to paclitaxel in breast tumors through activation of the ERK1/2 pathway.

Interleukin 17B (IL-17B) is a pro-inflammatory cytokine that belongs to the IL-17 cytokines family and binds to IL-17 receptor B (IL-17RB). Here we found that high expression of IL-17B and IL-17RB is associated with poor prognosis in patients with breast cancer and that IL-17B expression upregulation is specifically associated with poorer survival in patients with basal-like breast cancer. We thus focused on IL-17B role in breast cancer by using luminal and triple negative (TN)/basal-like tumor cell lines. We found that IL-17B induces resistance to conventional chemotherapeutic agents. In vivo, IL-17B induced resistance to paclitaxel and treatment with an anti-IL-17RB neutralizing antibody completely restored breast tumor chemosensitivity, leading to tumor shrinkage. We next focused on the signaling pathways activated in human breast cancer cell lines upon incubation with IL-17B. We observed that IL-17B induces ERK1/2 pathway activation, leading to upregulation of anti-apoptotic proteins of the BCL-2 family. IL-17B-induced chemoresistance was completely abolished by incubation with PD98059, an inhibitor of the MAPK/ERK pathway, indicating that the ERK pathway plays a crucial role. Altogether our results emphasize the role of the IL-17B/IL-17RB signaling pathway in breast tumors and identify IL-17B and its receptor as attractive therapeutic targets for potentiating breast cancer chemotherapy.

Histone deacetylase 9 regulates breast cancer cell proliferation and the response to histone deacetylase inhibitors.

Histone lysine acetylation is an epigenetic mark regulated by histone acetyltransferases and histone deacetylases (HDAC) which plays an important role in tumorigenesis. In this study, we observed a strong overexpression of class IIa HDAC9, at the mRNA and protein levels, in the most aggressive human breast cancer cell lines (i.e. in basal breast cancer cells vs luminal ones or in malignant vs begnin MCF10A breast epithelial cell lines). HDAC9 overexpression was associated with higher rates of gene transcription and increased epigenetic marks on the HDAC9 promoter. Ectopic expression of HDAC9 in MCF7 luminal breast cancer cells led to an increase in cell proliferation and to a decrease in apoptosis. These effects were associated with a deregulated expression of several genes controlled by HDAC inhibitors such as CDKN1A, BAX and TNFRSF10A. Inversely, knock-down of HDAC9 expression in MDA-MB436 basal breast cancer cells reduced cell proliferation. Moreover, high HDAC9 expression decreased the efficacy of HDAC inhibitors to reduce cell proliferation and to regulate CDKN1A gene expression. Interestingly, the gene encoding the transcription factor SOX9 was identified by a global transcriptomic approach as an HDAC9 target gene. In stably transfected MCF7 cells, SOX9 silencing significantly decreased HDAC9 mitogenic activity. Finally, in a large panel of breast cancer biopsies, HDAC9 expression was significantly increased in tumors of the basal subtype, correlated with SOX9 expression and associated with poor prognosis. Altogether, these results indicate that HDAC9 is a key factor involved in mammary carcinogenesis and in the response to HDAC inhibitors.

The emerging role of the transcriptional coregulator RIP140 in solid tumors.

RIP140 is a transcriptional coregulator (also known as NRIP1) which plays very important physiological roles by finely tuning the activity of a large number of transcription factors. Noticeably, the RIP140 gene has been shown to be involved in the regulation of energy expenditure, in mammary gland development and intestinal homeostasis as well as in behavior and cognition. RIP140 is also involved in the regulation of various oncogenic signaling pathways and participates in the development and progression of solid tumors. This short review aims to summarize the role of this transcription factor on nuclear estrogen receptors, E2F and Wnt signaling pathways based on recent observations focusing on breast, ovary, liver and colon tumors.

Expression and role of RIP140/NRIP1 in chronic lymphocytic leukemia.

RIP140 is a transcriptional coregulator, (also known as NRIP1), which finely tunes the activity of various transcription factors and plays very important physiological roles. Noticeably, the RIP140 gene has been implicated in the control of energy expenditure, behavior, cognition, mammary gland development and intestinal homeostasis. RIP140 is also involved in the regulation of various oncogenic signaling pathways and participates in the development and progression of solid tumors. During the past years, several papers have reported evidences linking RIP140 to hematologic malignancies. Among them, two recent studies with correlative data suggested that gene expression signatures including RIP140 can predict survival in chronic lymphocytic leukemia (CLL). This review aims to summarize the literature dealing with the expression of RIP140 in CLL and to explore the potential impact of this factor on transcription pathways which play key roles in this pathology.

RIP140 increases APC expression and controls intestinal homeostasis and tumorigenesis.

Deregulation of the Wnt/APC/ß-catenin signaling pathway is an important consequence of tumor suppressor APC dysfunction. Genetic and molecular data have established that disruption of this pathway contributes to the development of colorectal cancer. Here, we demonstrate that the transcriptional coregulator RIP140 regulates intestinal homeostasis and tumorigenesis. Using Rip140-null mice and mice overexpressing human RIP140, we found that RIP140 inhibited intestinal epithelial cell proliferation and apoptosis. Interestingly, following whole-body irradiation, mice lacking RIP140 exhibited improved regenerative capacity in the intestine, while mice overexpressing RIP140 displayed reduced recovery. Enhanced RIP140 expression strongly repressed human colon cancer cell proliferation in vitro and after grafting onto nude mice. Moreover, in murine tissues and human cancer cells, RIP140 stimulated APC transcription and inhibited ß-catenin activation and target gene expression. Finally, RIP140 mRNA and RIP140 protein levels were decreased in human colon cancers compared with those in normal mucosal tissue, and low levels of RIP140 expression in adenocarcinomas from patients correlated with poor prognosis. Together, these results support a tumor suppressor role for RIP140 in colon cancer.

Regulation of intestinal homeostasis and tumorigenesis by the transcriptional coregulator RIP140.

Colon cancer frequently results from mutations that constitutively activate the Wnt signaling pathway, a major target being the tumor suppressor gene adenomatous polyposis coli (APC). We recently identified the transcription factor RIP140 as a new inducer of APC gene transcription that inhibits colon cancer cell growth and impedes the Wnt signaling pathway by reducing ß-catenin activation.

Efficient new constructs against triple negative breast cancer cells: synthesis and preliminary biological study of ferrocifen-SAHA hybrids and related species.

Chemotherapeutic agents combining several active groups within a single molecule can modulate multiple cellular pathways and, thus, exhibit higher efficacy than single-target drugs. In this study, six new hybrid compounds combining tamoxifen (TAM) or ferrocifen (FcTAM) structural motifs with suberoylanilide hydroxamic acid (SAHA) were synthesised and evaluated. Antiproliferative activity was first explored in cancer cell lines. Combining FcTAM and SAHA structural motifs to form the unprecedented FcTAM­SAHA hybrid molecule led to an increased cytotoxicity (IC50 = 0.7 µM) in triple-negative MDA-MB-231 breast cancer cells when compared to FcTAM or SAHA alone (IC50 = 2.6 µM and 3.6 µM, respectively), while the organic hybrid analogue TAM­SAHA was far less cytotoxic (IC50 = 8.6 µM). In hormone-dependent MCF-7 breast cancer cells, FcTAM­SAHA was more active (IC50 = 2.0 µM) than FcTAM (IC50 = 4.4 µM) and TAM­SAHA (IC50 > 10 µM), but less toxic than SAHA (IC50 = 1.0 µM). Surprisingly, FcTAM­PSA, an N1-phenylsuberamide derivative, also possessed strong antiproliferative activity (IC50 = 0.5 µM and 1.8 µM in MDA-MB-231 and MCF-7 cells, respectively). Subsequent biochemical studies indicate that estrogen receptor alpha (ERα) and histone deacetylases (HDAC) are not the main targets of the hybrid compounds for their antiproliferative effect. Interestingly, both organometallic compounds were able to induce p21waf1/cip1 gene expression in MCF-7 breast cancer cells in accordance with their antiproliferative activity.

Long-term treatment with the pure anti-estrogen fulvestrant durably remodels estrogen signaling in BG-1 ovarian cancer cells.

Most ovarian cancers are estrogen-positive and hormonal treatments using anti-estrogens or aromatase inhibitors are under investigation for treating the tumors that are resistant to conventional therapies. In this study, the long-term effects of two anti-estrogens, namely 4-hydroxytamoxifen and fulvestrant (or ICI182,780), were investigated in ERα-positive BG1 epithelial ovarian cancer cells. To this aim, cells were grown in the presence of anti-estrogen concentrations that were sufficient to saturate the estrogen receptors, but were neither cytotoxic nor cytostatic as indicated by the absence of inhibition of cell proliferation. In these conditions and despite the lack of cytostatic effect of the drugs, long-term treatment (3 months) with the pure anti-estrogen fulvestrant induced a specific, reproducible and irreversible inhibition of ERα expression. This inhibition was accompanied by loss of estrogen-induced cell proliferation and gene expression as indicated by the analysis of several estrogen-responsive genes. ERα down-regulation was not linked to deregulated expression of transcription factors which drive ERα transcription and did not involve DNA methylation or histone deacetylation. Altogether, these results demonstrate that non-cytotoxic concentrations of pure anti-estrogens affect estrogen signaling and might be relevant for the treatment for ovarian cancers.

The RIP140 gene is a transcriptional target of E2F1.

RIP140 is a transcriptional coregulator involved in energy homeostasis and ovulation which is controlled at the transcriptional level by several nuclear receptors. We demonstrate here that RIP140 is a novel target gene of the E2F1 transcription factor. Bioinformatics analysis, gel shift assay, and chromatin immunoprecipitation demonstrate that the RIP140 promoter contains bona fide E2F response elements. In transiently transfected MCF-7 breast cancer cells, the RIP140 promoter is transactivated by overexpression of E2F1/DP1. Interestingly, RIP140 mRNA is finely regulated during cell cycle progression (5-fold increase at the G1/S and G2/M transitions). The positive regulation by E2F1 requires sequences located in the proximal region of the promoter (-73/+167), involves Sp1 transcription factors, and undergoes a negative feedback control by RIP140. Finally, we show that E2F1 participates in the induction of RIP140 expression during adipocyte differentiation. Altogether, this work identifies the RIP140 gene as a new transcriptional target of E2F1 which may explain some of the effect of E2F1 in both cancer and metabolic diseases.

Inhibitory feature of the proprotein convertases prosegments.

The proprotein convertases (PCs) are serine proteases involved in various physiological processes and their overactivity or inactivity has been linked to different disorders. PCs are responsible for the proteolytic processing of various polypeptide precursors. Here, we discuss the effect of their N-terminal prosegments on various PC substrates processing and functions.

Opposing function of the proprotein convertases furin and PACE4 on breast cancer cells' malignant phenotypes: role of tissue inhibitors of metalloproteinase-1.

Proteolytic cleavage of various cancer-related substrates by the proprotein convertases (PC) was reported to be important in the processes of neoplasia. These enzymes are inhibited by their naturally occurring inhibitors, the prosegments (ppPC), and by the engineered general PC inhibitor, the serpin variant alpha1-PDX. In the present study, we sought to compare the effect of these PC inhibitors on malignant phenotypes of breast cancer cells. Overexpression in a stable manner of alpha1-PDX and the prosegment ppPACE4 in MDA-MB-231 breast cancer cells resulted in increased matrix metalloproteinase (MMP)-9 (but not MMP-2) activity and a reduced secretion of tissue inhibitor of metalloproteinase 1 (TIMP-1). This was associated with significant enhancement in cell motility, migration, and invasion of collagen in vitro. In contrast, ppFurin expression in these cells decreased MMP-9 activity and diminished these biological functions, but had no significant effect on TIMP-1 secretion. Taken together, these data showed the specific and opposing roles of Furin and PACE4 in the regulation of MMP-9/TIMP-1-mediated cell motility and invasion.