Kruppel-like factor 8 regulates triple negative breast cancer stem cell-like activity

IntroductionBreast tumor development is regulated by a sub-population of breast cancer cells, termed cancer stem-like cells (CSC), which are capable of self-renewing and differentiating, and are involved in promoting breast cancer invasion, metastasis, drug resistance and relapse. CSCs are highly adaptable, capable of reprogramming their own metabolism and signaling activity in response to stimuli within the tumor microenvironment. Recently, the nutrient sensor O-GlcNAc transferase (OGT) and O-GlcNAcylation was shown to be enriched in CSC populations, where it promotes the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. This enrichment was associated with upregulation of the transcription factor Kruppel-like-factor 8 (KLF8) suggesting a potential role of KLF8 in regulating CSCs properties.MethodsTriple-negative breast cancer cells were genetically modified to generate KLF8 overexpressing or KLF8 knock-down cells. Cancer cells, control or with altered KLF8 expression were analyzed to assess mammosphere formation efficiency, CSCs frequency and expression of CSCs factors. Tumor growth in vivo of control or KLF8 knock-down cells was assessed by fat-pad injection of these cell in immunocompromised mice.ResultsHere, we show that KLF8 is required and sufficient for regulating CSC phenotypes and regulating transcription factors SOX2, NANOG, OCT4 and c-MYC. KLF8 levels are associated with chemoresistance in triple negative breast cancer patients and overexpression in breast cancer cells increased paclitaxel resistance. KLF8 and OGT co-regulate each other to form a feed-forward loop to promote CSCs phenotype and mammosphere formation of breast cancer cells.DiscussionThese results suggest a critical role of KLF8 and OGT in promoting CSCs and cancer progression, that may serve as potential targets for developing strategy to target CSCs specifically

On a Sugar High: Role of O-GlcNAcylation in Cancer

Recent advances in the understanding of the molecular mechanisms underlying cancer progression have led to the development of novel therapeutic targeting strategies. Aberrant glycosylation patterns and their implication in cancer have gained increasing attention as potential targets due to the critical role of glycosylation in regulating tumor-specific pathways that contribute to cancer cell survival, proliferation, and progression. A special type of glycosylation that has been gaining momentum in cancer research is the modification of nuclear, cytoplasmic, and mitochondrial proteins, termed O-GlcNAcylation. This protein modification is catalyzed by an enzyme called O-GlcNAc transferase (OGT), which uses the final product of the Hexosamine Biosynthetic Pathway (HBP) to connect altered nutrient availability to changes in cellular signaling that contribute to multiple aspects of tumor progression. Both O-GlcNAc and its enzyme OGT are highly elevated in cancer and fulfill the crucial role in regulating many hallmarks of cancer. In this review, we present and discuss the latest findings elucidating the involvement of OGT and O-GlcNAc in cancer

Nutrient sensor O-GlcNAc transferase controls cancer lipid metabolism via SREBP-1 regulation

Elevated O-GlcNAcylation is associated with disease states such as diabetes and cancer. O-GlcNAc transferase (OGT) is elevated in multiple cancers and inhibition of this enzyme genetically or pharmacologically inhibits oncogenesis. Here we show that O-GlcNAcylation modulates lipid metabolism in cancer cells. OGT regulates expression of the master lipid regulator the transcription factor sterol regulatory element binding protein 1 (SREBP-1) and its transcriptional targets both in cancer and lipogenic tissue. OGT regulates SREBP-1 protein expression via AMP-activated protein kinase (AMPK). SREBP-1 is critical for OGT-mediated regulation of cell survival and of lipid synthesis, as overexpression of SREBP-1 rescues lipogenic defects associated with OGT suppression, and tumor growth in vitro and in vivo. These results unravel a previously unidentified link between O-GlcNAcylation, lipid metabolism and the regulation of SREBP-1 in cancer and suggests a crucial role for O-GlcNAc signaling in transducing nutritional state to regulate lipid metabolism

Kruppel-Like Factor 8 Regulates Triple Negative Breast Cancer Stem Cell-Like Activity

INTRODUCTION: Breast tumor development is regulated by a sub-population of breast cancer cells, termed cancer stem-like cells (CSC), which are capable of self-renewing and differentiating, and are involved in promoting breast cancer invasion, metastasis, drug resistance and relapse. CSCs are highly adaptable, capable of reprogramming their own metabolism and signaling activity in response to stimuli within the tumor microenvironment. Recently, the nutrient sensor O-GlcNAc transferase (OGT) and O-GlcNAcylation was shown to be enriched in CSC populations, where it promotes the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. This enrichment was associated with upregulation of the transcription factor Kruppel-like-factor 8 (KLF8) suggesting a potential role of KLF8 in regulating CSCs properties. METHODS: Triple-negative breast cancer cells were genetically modified to generate KLF8 overexpressing or KLF8 knock-down cells. Cancer cells, control or with altered KLF8 expression were analyzed to assess mammosphere formation efficiency, CSCs frequency and expression of CSCs factors. Tumor growth in vivo of control or KLF8 knock-down cells was assessed by fat-pad injection of these cell in immunocompromised mice. RESULTS: Here, we show that KLF8 is required and sufficient for regulating CSC phenotypes and regulating transcription factors SOX2, NANOG, OCT4 and c-MYC. KLF8 levels are associated with chemoresistance in triple negative breast cancer patients and overexpression in breast cancer cells increased paclitaxel resistance. KLF8 and OGT co-regulate each other to form a feed-forward loop to promote CSCs phenotype and mammosphere formation of breast cancer cells. DISCUSSION: These results suggest a critical role of KLF8 and OGT in promoting CSCs and cancer progression, that may serve as potential targets for developing strategy to target CSCs specifically

Control of FLIP L expression and TRAIL resistance by the extracellular signal-regulated kinase1/2 pathway in breast epithelial cells

Increased activation of the epidermal growth factor receptor (EGFR) is frequently observed in tumors, and inhibition of the signaling pathways originated in the EGFR normally renders tumor cells more sensitive to apoptotic stimuli. However, we show that inhibition of EGFR signaling in non-transformed breast epithelial cells by EGF deprivation or gefitinib, an inhibitor of EGFR tyrosine kinase, causes the upregulation of the long isoform of caspase-8 inhibitor FLICE-inhibitory protein (FLIP L) and makes these cells more resistant to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway plays a pivotal role in the regulation of FLIP L levels and sensitivity to TRAIL-induced apoptosis by EGF. Upregulation of FLIP L upon EGF deprivation correlates with a decrease in c-Myc levels and c-Myc knockdown by siRNA induces FLIP L expression. FLIP L upregulation and resistance to TRAIL in EGF-deprived cells are reversed following activation of an estrogen activatable form of c-Myc (c-Myc-ER). Finally, constitutive activation of the ERK1/2 pathway in HER2/ERBB2-transformed cells prevents EGF deprivation-induced FLIP L upregulation and TRAIL resistance. Collectively, our results suggest that a regulated ERK1/2 pathway is crucial to control FLIP L levels and sensitivity to TRAIL in non-transformed cells, and this mechanism may explain the increased sensitivity of tumor cells to TRAIL, in which the ERK1/2 pathway is frequently deregulated. © 2012 Macmillan Publishers Limited All rights reserved.This work was supported by grants from Ministerio de Ciencia e Innovación (SAF2009-07163), Red Temática de Investigación Cooperativa en Cáncer (RTICC: RD06/0020/0068), the European Community through the regional development funding program (FEDER) and Junta de Andalucía (P09-CVI-4497) to ALR. CP and RY were supported by contracts from Ministerio de Ciencia e Innovación and Junta de Andalucía, respectively.Peer Reviewe

Cellular FLIPL plays a survival role and regulates morphogenesis in breast epithelial cells

Strong evidences support the inhibitory activity of cellular FLICE-inhibitory protein (FLIP) in the apoptotic signalling by death receptors in tumor cells. However, little is known about the role of FLIP in the regulation of apoptosis in non-transformed cells. In this report, we demonstrate that FLIPL plays an important role as a survival protein in non-transformed breast epithelial cells. Silencing of FLIPL by siRNA methodology enhances TRAIL-R2 expression and activates a caspase-dependent cell death process in breast epithelial cells. This cell death requires the expression of TRAIL, TRAIL-R2, FADD and procaspase-8 proteins. A mitochondria-operated apoptotic pathway is partially required for FLIPL siRNA-induced apoptosis. Interestingly, FLIPL silencing markedly abrogates formation of acinus-like structures in a three-dimensional basement membrane culture model (3D) of the human mammary MCF-10A cell line through a caspase-8 dependent process. Furthermore, over-expression of FLIPL in MCF-10A cells delayed lumen formation in 3D cultures. Our results highlight the central role of FLIP in maintaining breast epithelial cell viability and suggest that the mechanisms regulating FLIP levels should be finely controlled to prevent unwanted cell demise.This work was supported by grants from Ministerio de Educación y Ciencia (SAF2006-00633 and SAF2009-07163), Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD06/0020/0068) and Junta de Andalucía (CTS-211 and CVI-4497) to AL-R. RY and CP were supported by contracts from Instituto de Salud Carlos III and Junta de Andalucía, respectively.Peer Reviewe

Control of FLIP(L) expression and TRAIL resistance by the extracellular signal-regulated kinase1/2 pathway in breast epithelial cells

Póster presentado en 8th European Workshops on Cell Death: Death à la carte, celebrado en Monêtier-les-Bains, Serre Chevalier Valley (France), del 3 al 8 de junio de 2012Increased activation of the epidermal growth factor receptor (EGFR) is frequently observed in tumors, and inhibition of the signaling pathways originated in the EGFR normally renders tumor cells more sensitive to apoptotic stimuli. However, we show that inhibition of EGFR signaling in non-transformed breast epithelial cells by EGF deprivation or gefitinib, an inhibitor of EGFR tyrosine kinase, causes the upregulation of the long isoform of caspase-8 inhibitor FLICE-inhibitory protein (FLIP(L)) and makes these cells more resistant to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway plays a pivotal role in the regulation of FLIP(L) levels and sensitivity to TRAIL-induced apoptosis by EGF. Upregulation of FLIP(L) upon EGF deprivation correlates with a decrease in c-Myc levels and c-Myc knockdown by siRNA induces FLIP(L) expression. FLIP(L) upregulation and resistance to TRAIL in EGF-deprived cells are reversed following activation of an estrogen activatable form of c-Myc (c-Myc-ER). Finally, constitutive activation of the ERK1/2 pathway in HER2/ERBB2-transformed cells prevents EGF deprivation-induced FLIP(L) upregulation and TRAIL resistance. Collectively, our results suggest that a regulated ERK1/2 pathway is crucial to control FLIP(L) levels and sensitivity to TRAIL in non-transformed cells, and this mechanism may explain the increased sensitivity of tumor cells to TRAIL, in which the ERK1/2 pathway is frequently deregulated.Peer Reviewe

O-GlcNAcylation regulates breast cancer metastasis via SIRT1 modulation of FOXM1 pathway

Tumors utilize aerobic glycolysis to support growth and invasion. However, the molecular mechanisms that link metabolism with invasion are not well understood. The nutrient sensor O-linked-β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) modifies intracellular proteins with N-acetylglucosamine. Cancers display elevated O-GlcNAcylation and suppression of O-GlcNAcylation inhibits cancer invasion and metastasis. Here, we show that the regulation of cancer invasion by OGT is dependent on the NAD+-dependent deacetylase SIRT1. Reducing O-GlcNAcylation elevates SIRT1 levels and activity in an AMPK-dependent manner. Reduced O-GlcNAcylation in cancer cells leads to SIRT1-mediated proteasomal degradation of oncogenic transcription factor FOXM1 in a MEK/ERK-dependent manner. SIRT1 is critical for OGT-mediated regulation of FOXM1 ubiquitination and reducing SIRT1 activity reverses OGT-mediated regulation of FOXM1. Moreover, we show that SIRT1 levels are required for OGT-mediated regulation of invasion and metastasis in breast cancer cells. Thus, O-GlcNAcylation is a central component linking metabolism to invasion and metastasis via a SIRT1/ /FOXM1 axis