O-GlcNAcylation regulates cancer metabolism and survival stress signaling via regulation of the HIF-1 pathway.

The hexosamine biosynthetic pathway elevates posttranslational addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) on intracellular proteins. Cancer cells elevate total O-GlcNAcylation by increasing O-GlcNAc transferase (OGT) and/or decreasing O-GlcNAcase (OGA) levels. Reducing O-GlcNAcylation inhibits oncogenesis. Here, we demonstrate that O-GlcNAcylation regulates glycolysis in cancer cells via hypoxia-inducible factor 1 (HIF-1α) and its transcriptional target GLUT1. Reducing O-GlcNAcylation increases α-ketoglutarate, HIF-1 hydroxylation, and interaction with von Hippel-Lindau protein (pVHL), resulting in HIF-1α degradation. Reducing O-GlcNAcylation in cancer cells results in activation of endoplasmic reticulum (ER) stress and cancer cell apoptosis mediated through C/EBP homologous protein (CHOP). HIF-1α and GLUT1 are critical for OGT-mediated regulation of metabolic stress, as overexpression of stable HIF-1 or GLUT1 rescues metabolic defects. Human breast cancers with high levels of HIF-1α contain elevated OGT, and lower OGA levels correlate independently with poor patient outcome. Thus, O-GlcNAcylation regulates cancer cell metabolic reprograming and survival stress signaling via regulation of HIF-1α.

ITGA6 is directly regulated by hypoxia-inducible factors and enriches for cancer stem cell activity and invasion in metastatic breast cancer models.

BACKGROUND: Hypoxia-inducible factors (HIFs) are well-established mediators of tumor growth, the epithelial to mesenchymal transition (EMT) and metastasis. In several types of solid tumors, including breast cancers, the HIFs play a critical role in maintaining cancer stem cell (CSC) activity. Thus, we hypothesized that HIFs may also regulate transcription of markers of breast CSC activity. One approach to enrich for breast cells with stem-like phenotypes is FACS sorting, in which sub-populations of live cells are gated based on the expression of cell surface antigens, including various integrin subunits. Integrin alpha 6 (ITGA6; CD49f) is routinely used in combination with other integrin subunits to enrich for breast stem cells by FACS. Integrins not only mediate interactions with the extracellular matrix (ECM), but also drive intracellular signaling events that communicate from the tumor microenvironment to inside of the tumor cell to alter phenotypes including migration and invasion. METHODS: We used two models of metastatic breast cancer (MBC), polyoma middle T (MMTV-PyMT) and MDA-MB-231 cells, to compare the expression of ITGA6 in wild type and knockout (KO) or knockdown cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays verified that ITGA6 is a direct HIF transcriptional target. We also used FACS sorting to enrich for CD49f (+) cells to compare tumorsphere formation, tumor initiating cell activity, invasion and HIF activity relative to CD49f(neg or low) cells. Knockdown of ITGA6 significantly reduced invasion, whereas re-expression of ITGA6 in the context of HIF knockdown partially rescued invasion. A search of public databases also revealed that ITGA6 expression is an independent prognostic factor of survival in breast cancer patients. RESULTS: We report that ITGA6 is a HIF-dependent target gene and that high ITGA6 expression enhances invasion and tumor-initiating cell activities in models of MBC. Moreover, cells that express high levels of ITGA6 are enriched for HIF-1α expression and the expression of HIF-dependent target genes. CONCLUSIONS: Our data suggest that HIF-dependent regulation of ITGA6 is one mechanism by which sorting for CD49f (+) cells enhances CSC and metastatic phenotypes in breast cancers. Our results are particularly relevant to basal-like breast cancers which express higher levels of the HIFα subunits, core HIF-dependent target genes and ITGA6 relative to other molecular subtypes.

The oncogene HER2/neu (ERBB2) requires the hypoxia-inducible factor HIF-1 for mammary tumor growth and anoikis resistance.

ERBB2, a receptor tyrosine kinase amplified in breast cancer, is a well established regulator of tumor growth in vivo and anoikis resistance leading to disruption of architecture in three-dimensional mammary epithelial acinar structures in vitro. ERBB2 promotes anoikis resistance by maintaining signaling pathways and by rescuing metabolic defects and thus inhibiting accumulation of deleterious reactive oxygen species. Recent evidence suggests that hypoxia, via hypoxia-inducible factors (HIFs), can inhibit anoikis; thus, we hypothesized that HIF-1 may play a role in ERBB2-mediated anoikis resistance and oncogenesis. Indeed, tumors isolated from MMTV-Neu mice contain elevated HIF-1α levels and tumor cells created from MMTV-Neu mice harboring deletion of Hif1α alleles reduced primary tumor growth in vivo. ERBB2 overexpressing cancer cells stabilize HIF under normoxic conditions and require HIF-1 for ERBB2-mediated anchorage-independence, three-dimensional culture growth and anoikis resistance. HIF-1 reduction in ERBB2 cells was associated with induction of the pro-anoikis protein BIM and decreased ERK and AKT signaling during cell detachment. ERBB2-mediated inhibition of metabolic defects, including decreased reactive oxygen species generation in suspension, required HIF-1 expression that was critical for ERBB2-mediated oncogenesis. Gene expression profiling of hypoxic three-dimensional acinar structures identified a number of genes elevated in response to hypoxia that are known ERBB2 targets, suggesting that hypoxic conditions and ERBB2 overexpression share both phenotypic and genetic components via HIF-1 regulation. Thus, our data demonstrate that ERBB2 requires HIF-1 for tumor growth and suggest that HIF is a major downstream regulator of ERBB2 that protects cells from anoikis and metabolic stress caused by decreased matrix adhesion.

Anti-matrix metalloproteinase-9 DNAzyme decreases tumor growth in the MMTV-PyMT mouse model of breast cancer.

INTRODUCTION: Despite continued improvements in diagnosis, surgical techniques, and chemotherapy, breast cancer patients are still overcome by cancer metastasis. Tumor cell proliferation, invasion and metastasis are mediated, at least in part, through degradation of basement membrane by neutral matrix metalloproteinases (MMP) produced by tumor and stromal cells. Evidence suggests that MMP-9 plays a significant role in breast tumor cell invasion and metastasis. DNAzymes or catalytic oligonucleotides are new classes of gene targeting molecules that bind and cleave a specific mRNA, resulting in decreased protein expression. METHODS: The application of anti-MMP-9 DNAzyme (AM9D) for the treatment of primary and metastatic breast cancer was evaluated in vitro and in vivo using MDA-MB-231 cells and the MMTV-PyMT transgenic breast cancer mouse model. Spontaneously developed mammary tumors in MMTV-PyMT transgenic mice were treated intratumorally with naked AM9D, once a week for 4 weeks. The stability of DNAzyme was determined in vitro and in vivo using fluorescently labeled DNAzyme. RESULTS: AM9D specifically inhibited expression of MMP-9 in MDA-MB-231 cells resulting in reduced invasive property of these cells by 43%. Weekly intratumoral treatment of spontaneously developed mammary tumors in MMTV-PyMT transgenic mice was sufficient to significantly reduce the rate of tumor growth and final tumor load in a dose dependent and statistically significant manner (P < 0.05). This decrease in tumor growth was correlated with decreased MMP-9 protein production within the treated tumor tissues. Tumors treated with AM9D were also less vascularized and contained more apoptotic cells compared to control and untreated tumors. CONCLUSIONS: These results show that targeting and down regulation of MMP-9 by AM9D could prove useful as a therapy against breast carcinoma tumor growth and invasion.

Hypoxia-inducible factor 1α promotes primary tumor growth and tumor-initiating cell activity in breast cancer.

INTRODUCTION: Overexpression of the oxygen-responsive transcription factor hypoxia-inducible factor 1α (HIF-1α) correlates with poor prognosis in breast cancer patients. The mouse mammary tumor virus polyoma virus middle T (MMTV-PyMT) mouse is a widely utilized preclinical mouse model that resembles human luminal breast cancer and is highly metastatic. Prior studies in which the PyMT model was used demonstrated that HIF-1α is essential to promoting carcinoma onset and lung metastasis, although no differences in primary tumor end point size were observed. Using a refined model system, we investigated whether HIF-1α is directly implicated in the regulation of tumor-initiating cells (TICs) in breast cancer. METHODS: Mammary tumor epithelial cells were created from MMTV-PyMT mice harboring conditional alleles of Hif1a, followed by transduction ex vivo with either adenovirus ß-galactosidase or adenovirus Cre to generate wild-type (WT) and HIF-1α-null (KO) cells, respectively. The impact of HIF-1α deletion on tumor-initiating potential was investigated using tumorsphere assays, limiting dilution transplantation and gene expression analysis. RESULTS: Efficient deletion of HIF-1α reduced primary tumor growth and suppressed lung metastases, prolonging survival. Loss of HIF-1α led to reduced expression of markers of the basal lineage (K5/K14) in cells and tumors and of multiple genes involved in the epithelial-to-mesenchymal transition. HIF-1α also enhanced tumorsphere formation at normoxia and hypoxia. Decreased expression of several genes in the Notch pathway as well as Vegf and Prominin-1 (CD133)was observed in response to Hif1a deletion. Immunohistochemistry confirmed that CD133 expression was reduced in KO cells and in tumorspheres. Tumorsphere formation was enhanced in CD133hi versus CD133neg cells sorted from PyMT tumors. Limiting dilution transplantation of WT and KO tumor cells into immunocompetent recipients revealed > 30-fold enrichment of TICs in WT cells. CONCLUSION: These results demonstrate that HIF-1α plays a key role in promoting primary mammary tumor growth and metastasis, in part through regulation of TICs. HIF-1α regulates expression of several members of the Notch pathway, CD133 and markers of the basal lineage in mammary tumors. Our results suggest that CD133, which has not been profiled extensively in breast cancer, may be a useful marker of TICs in the PyMT mouse model. These data reveal for the first time that HIF-1α directly regulates breast TIC activity in vivo.

VHL deletion impairs mammary alveologenesis but is not sufficient for mammary tumorigenesis.

Overexpression of hypoxia inducible factor-1 (HIF-1)alpha, which is common in most solid tumors, correlates with poor prognosis and high metastatic risk in breast cancer patients. Because HIF-1alpha protein stability is tightly controlled by the tumor suppressor von Hippel-Lindau (VHL), deletion of VHL results in constitutive HIF-1alpha expression. To determine whether VHL plays a role in normal mammary gland development, and if HIF-1alpha overexpression is sufficient to initiate breast cancer, Vhl was conditionally deleted in the mammary epithelium using the Cre/loxP system. During first pregnancy, loss of Vhl resulted in decreased mammary epithelial cell proliferation and impaired alveolar differentiation; despite these phenotypes, lactation was sufficient to support pup growth. In contrast, in multiparous dams, Vhl(-/-) mammary glands exhibited a progressive loss of alveolar epithelium, culminating in lactation failure. Deletion of Vhl in the epithelium also impacted the mammary stroma, as there was increased microvessel density accompanied by hemorrhage and increased immune cell infiltration. However, deletion of Vhl was not sufficient to induce mammary tumorigenesis in dams bred continuously for up to 24 months of age. Moreover, co-deletion of Hif1a could not rescue the Vhl(-/-)-dependent phenotype as dams were unable to successfully lactate during the first lactation. These results suggest that additional VHL-regulated genes besides HIF1A function to maintain the proliferative and regenerative potential of the breast epithelium.

HIF-Dependent CKB Expression Promotes Breast Cancer Metastasis, Whereas Cyclocreatine Therapy Impairs Cellular Invasion and Improves Chemotherapy Efficacy.

The oxygen-responsive hypoxia inducible factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple-negative breast cancers (TNBCs) and is correlated with poor patient survival. Inhibiting the HIF transcription factors with small molecules is challenging; therefore, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss- and gain-of-function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and lung metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, the formation of invadopodia and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth.

Titanium particles and surface-bound LPS activate different pathways in IC-21 macrophages.

It is still unknown if wear-debris particles themselves induce osteolysis or if they serve a functional role as receptors for ligands that incite an inflammatory response that ultimately leads to bone resorption. In this study, commercially pure titanium particles (cpTi) were subjected to a serial combination of different cleaning methods to remove Lipopolysaccharide (LPS) or were incubated in LPS solutions of known concentrations. Then, the response of the macrophage cell line IC-21 to the cleaned particles, LPS-bound Ti particles, and soluble LPS was examined. It was found that cleaned particles up to 1000 particles per cell did not stimulate macrophages to release Tumor necrosis factor-alpha (TNF-alpha) or Interleukin 6 (IL-6), but they significantly increased the release of Prostaglandin E(2) (PGE(2)) when the particle concentration was higher than 500 particles per cell. At one particle per cell, Ti particles bound with LPS stimulated the release of IL-6 and TNF-alpha by macrophages. The level of released cytokines was dependent on, and correlated with, the amount of LPS present on the particles. The macrophages were more sensitive to soluble LPS than to particle-bound LPS, and the simultaneous addition of cleaned Ti particles did not have additional effects on the effects of soluble LPS. This study shows evidence that, cpTi particles and LPS have distinct mechanisms of action on the IC-21 macrophages, but that both contribute to the development of an inflammatory response.

mTOR/MYC Axis Regulates O-GlcNAc Transferase Expression and O-GlcNAcylation in Breast Cancer.

UNLABELLED: Cancers exhibit altered metabolism characterized by increased glucose and glutamine uptake. The hexosamine biosynthetic pathway (HBP) uses glucose and glutamine, and directly contributes to O-linked-ß-N-acetylglucosamine (O-GlcNAc) modifications on intracellular proteins. Multiple tumor types contain elevated total O-GlcNAcylation, in part, by increasing O-GlcNAc transferase (OGT) levels, the enzyme that catalyzes this modification. Although cancer cells require OGT for oncogenesis, it is not clear how tumor cells regulate OGT expression and O-GlcNAcylation. Here, it is shown that the PI3K-mTOR-MYC signaling pathway is required for elevation of OGT and O-GlcNAcylation in breast cancer cells. Treatment with PI3K and mTOR inhibitors reduced OGT protein expression and decreased levels of overall O-GlcNAcylation. In addition, both AKT and mTOR activation is sufficient to elevate OGT/O-GlcNAcylation. Downstream of mTOR, the oncogenic transcription factor c-MYC is required and sufficient for increased OGT protein expression in an RNA-independent manner and c-MYC regulation of OGT mechanistically requires the expression of c-MYC transcriptional target HSP90A. Finally, mammary tumor epithelial cells derived from MMTV-c-myc transgenic mice contain elevated OGT and O-GlcNAcylation and OGT inhibition in this model induces apoptosis. Thus, OGT and O-GlcNAcylation levels are elevated via activation of an mTOR/MYC cascade. IMPLICATIONS: Evidence indicates OGT as a therapeutic target in c-MYC-amplified cancers.

Benzimidazole analogs as potent hypoxia inducible factor inhibitors: synthesis, biological evaluation, and profiling drug-like properties.

AIM: To develop potent HIF-1α inhibitors for potential treatment of cancer. MATERIALS AND METHODS: Chemical synthesis, HIF-luciferase assay, cytotoxic assay, platelet aggregation assay, western blot analysis, quantitative real-time PCR, aqueous solubility, protein binding, metabolic stability, and metabolic pathways. RESULTS: Thirteen novel benzimidazole analogs were synthesized. Compounds 3a and 3k showed the highest anti-HIF-1α activity. They are significantly more effective than YC-1 in the suppression of HIF-1α protein expression based on western blot assay. They show comparable potency in inhibition of cancer cell migration. They are less potent in the inhibition of platelet aggregation. 3k had the most favorable drug-like properties, including long half-life in human liver microsomes, medium protein binding level and reasonable aqueous solubility. CONCLUSION: The potent anti-HIF-1α activity and favorable drug-like properties of compound 3k suggest that it may hold great potential as an adjuvant therapy for cancer treatment through repression of HIF-1α protein expression.

Chemoprevention activity of dipyridamole in the MMTV-PyMT transgenic mouse model of breast cancer.

Dipyridamole (DPM) is widely used to prevent strokes and vascular thrombosis. Combination therapy of DPM and antimetabolites has shown synergistic anticancer activity. This study investigated the chemopreventive effects of DPM in the mouse mammary tumor virus promoter-driven polyoma middle T oncoprotein metastatic breast cancer model. We also investigated the effects of DPM on gene and miRNA expression. Chemopreventive activity was assessed by comparing the time to onset of palpable lesions, primary tumor growth kinetics, and the number of lung metastases in transgenic mice treated with DPM or vehicle. Gene expression and miRNA expression profiles of mammary tumor tissues were then analyzed using the Affymetrix GeneChip or miRNA 2.0 arrays. Real-time quantitative PCR was used to confirm changes in gene expression. Treatment with DPM beginning at the age of 4 weeks delayed the onset of palpable lesions, delayed tumor progression, and suppressed lung metastasis. Microarray gene expression analysis identified 253 genes differentially expressed between DPM-treated and control mammary tumors. miRNA expression analysis revealed that 53 miRNAs were altered by DPM treatment. The results indicate that DPM has chemoprevention activity against breast cancer tumorigenesis and metastasis in mice. The array analyses provide insights into potential mechanisms of DPM's chemopreventive effects, involving upregulation of several genes and miRNAs known to suppress cancer growth and/or metastasis and downregulation of genes known to promote cancer. Some of these genes have not been previously studied in breast cancer and may serve as novel molecular targets for breast cancer chemoprevention.

Macrophage response to high number of titanium particles is cytotoxic and COX-2 mediated and it is not affected by the particle's endotoxin content or the cleaning treatment.

Periprosthetic osteolysis is a progressive deterioration of bone around prostheses resulting primarily from the presence of wear debris. Particulate material, number, and their interactions with environmental factors play important roles in macrophage activation around implants. We have previously shown that macrophages cultured in the presence of high numbers of cleaned titanium (Ti) particles released significant amounts of PGE2 that is potentially detrimental for bone. Cleaning of particles has become routine in most studies of macrophage/particle interactions as contaminating endotoxin elicits a macrophage cytokine response and since numerous studies have suggested that endotoxins may be present on implant materials. However, the strenuous cleaning procedure itself represents a possible source of other contaminants (such as material by-products) that may be relevant to the prostanoid response of macrophages. To analyze this hypothesis, the macrophage response to high numbers of cleaned Ti particles was compared to that of unclean particles and to particles that were subjected to a short version of the cleaning procedure. It was found that neither the high amount of endotoxin on the unclean particles nor the duration of the cleaning procedure had an effect on the release of PGE2 and the decrease in cell viability in response to high number of Ti particles. Evidence of a possible relationship between these two variables is presented.

Titanium particles that have undergone phagocytosis by macrophages lose the ability to activate other macrophages.

Titanium particles derived from the wear of the orthopaedic implant surfaces can activate macrophages to secrete cytokines and stimulate osteoclastic bone resorption, causing osteolysis around orthopaedic implants. However, what happens to the titanium particles after being phagocytosed by macrophages is not known. We prepared titanium particles (as received, clean, and LPS-coated), and exposed them to macrophages in culture. Free particles were washed away after 24 h and the intracellular particles were kept in culture for additional 48 h until being harvested by lysing the cells. Particles that had been cell treated or noncell treated were examined by scanning electronic microscopy to analyze the shape, size, and concentration of the particles. The cell treated and noncell treated particles were exposed to macrophages in culture with a particle to cell ratio of 300:1. After 18 h, the levels of TNF-alpha in culture medium and the viability of the cells were examined. Clean particles did not stimulate TNF-alpha secretion by macrophages, while LPS-coated particles dramatically increased that response. Phagocytosis by macrophages did not change the shape and size of the particles, but depleted the ability of the particles to stimulate TNF-alpha secretion by macrophages. This indicates that macrophages are capable of rendering titanium particles inactive without degrading the particles, possibly by altering the surface chemistry of the particles.