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PDF file - 292K, Supplementary Figure 1: Structure of DPM and H&E-stained end stage tumor sections. Supplementary Figure 2: No gross changes in cellular proliferation, apoptosis or microvessel density are observed in end stage tumors in response to DPM therapy. Supplementary Figure 3: DPM treatment does not maintain expression of ER in late stage carcinomas. Supplementary Figure 4: DPM treatment does not reduce the expression of the PyMT transgene in tumors.

PDF file - 119K, Table 1A. Genes enriched as over-expressed in DPM treatment group Table 1B. Genes enriched as under-expressed in DPM treatment group Table 2A. Functional enrichment (based on GO Biological Process) of genes over-expressed in DPM treatment group using DAVID. Table 2B. Functional enrichment (based on GO Biological Process) of genes under-expressed in DPM treatment group using DAVID. Table 3. Cell growth, proliferation and migration related functions of those genes differentially-enriched in the DPM treatment group Table 4. Comparison of gene expression fold changes between microarray and qPCR. Table 5. Differentially expressed miRNAs in DPM treatment group

PDF file - 164K, Preclinical chemoprevention experimental design

Supplemental Figure 1: OGT expression in oncogene expressing MCF-10A cells. Supplemental Figure 2: Inhibition of PI3K and mTOR does not reduce OGT RNA levels in breast cancer cells. Supplemental Figure 3: OGA expression in breast cancer cells treated with inhibitors. Supplemental Figure 4: MEK/ERK pathway is not sufficient to regulate OGT and O-GlcNAcylation levels. Supplemental Figure 5: OGA expression is reduced in TSC2 -/- MEFs. Supplemental Figure 6: Myc does not regulate OGT RNA levels in cancer cells. Supplemental Figure 7: Proteasomal degradation regulates unfolded OGT. Supplemental Figure 8: MYC regulates OGT protein via transcriptional target HSP90. Supplemental Figure 9: MYC-driven breast cancers overexpress OGT.

The hexosamine biosynthetic pathway elevates posttranslational addition of O-linked β-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.

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. Cancer Prev Res; 6(5); 437–47. ©2013 AACR.

Dysregulated tumor cell metabolism is a hallmark of cancer progression and therapeutic resistance. In a screen for Hypoxia-Inducible Factor (HIF)-dependent genes regulating metabolism, we identified creatine kinase, brain isoform (CKB) as down-regulated in HIF-1 knockout mammary tumor cells. Creatine kinases (CKs) reversibly catalyze the transfer of a high-energy phosphoryl group from ATP to creatine, generating phosphocreatine in the forward reaction, and ATP in the reverse reaction. CKs are up-regulated in a variety of solid tumors, including ovarian, breast, colon, lung and brain. Knockdown of CKB in the polyoma middle T (PyMT) transgenic mouse model of metastatic breast cancer suppressed the production of intracellular ATP and invasion in vitro, and inhibited metastasis from the mammary gland to the lung in vivo. CK activity is known to be inhibited by cyclocreatine, a creatine kinase substrate that represses CK-dependent generation of ATP from phosphocreatine. When female FVB/Nj mice were injected with wild type PyMT cells in a tail vein assay and then treated with cCr (1g/kg/day, IP), lung metastasis was repressed to the same extent as Ckb gene knockdown. Moreover, when cCr therapy was administered 7 days after tail vein injection, cCr was effective in preventing the transition of lung micrometastases to macrometastases. To explore the role of CK activity in regulating cell proliferation, survival in suspension, cellular metabolism and invasion, we next created CKB loss- and gain-of-function models using human breast cancer cell lines, and compared phenotypes to cCr treatment. Whereas deletion of CKB had no effect on cell proliferation or survival in adherent conditions or in suspension, either deletion of CKB or cCr therapy potently reduced ATP levels and invasive potential in vitro. Preliminary data also indicate that pre-treatment of triple negative breast cancer cell lines with cCr sensitizes cells to doxorubicin. Together, these data suggest that inhibition of CK activity may be effective in treating stage IV breast cancer. We are currently testing whether cCr has anti-metastatic efficacy as a monotherapy, or in combination with conventional chemotherapies, using luciferase-labeled patient-derived xenograft (PDX) models. This work was sponsored by the NIH (CA138488), the Dept. of Defense (BC150640), the METAvivor foundation, and the West Cancer Center in Memphis, TN. Citation Format: Hilaire Barch, Danielle L. Brooks, Raya Krutilina, Luciana P. Schwab, Deanna Parke, Tiffany N. Seagroves. HIF-1-dependent regulation of creatine kinase metabolism promotes breast cancer invasion and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4511. doi:10.1158/1538-7445.AM2017-4511

To develop potent HIF-1α inhibitors for potential treatment of cancer.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.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.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.

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 β-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.

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 CD49fneg 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. Electronic supplementary material The online version of this article (doi:10.1186/s12943-016-0510-x) contains supplementary material, which is available to authorized users.

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.

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-α) or Interleukin 6 (IL-6), but they significantly increased the release of Prostaglandin E2 (PGE2) 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-α 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. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006

Introduction: In response to hypoxic stress, tumors must adapt to the hostile microenvironment by regulating angiogenesis, cellular metabolism, cell survival and motility. The alpha subunits of the Hypoxia-Inducible Factors (HIF-1α and HIF-2α) are oxygen-responsive, master regulators of the hypoxic response. Over-expression of HIF-1α independently correlates with decreased survival of breast cancer patients, and a hypoxic core gene signature is enriched in triple negative breast cancers (TNBC) and HER2+ cancers relative to luminal tumors (ER+). In a variety of solid tumors, HIF-1α or HIF-2α also directly promotes tumor initiating cell (TIC) activity. Methods: Mammary tumor epithelial cells (MTECs) were established from late-stage tumors of MMTV-PyMT+; Hif1α floxed mice (which model ER-negative, luminal-like tumors), and exposed to either Adenovirus-β-gal or -Cre to create wild-type (WT) and knock-out (KO) cells, respectively. MDA-MB-231 cells (a model of TNBC) were generated in which either HIF1A or HIF2A or both genes (DKD) were knocked down (KD) via shRNA. Results: When PyMT+ MTECs were transplanted into female recipients, HIF-1 deletion reduced primary tumor growth by 60%, lung metastasis by 90% and TIC activity by >35-fold (Schwab et al. 2012). The expression of cell surface markers routinely used to enrich for TICs was assayed in PyMT+ and MDA-MB-231 cells. ITGA6 mRNA and CD49f protein expression were significantly decreased in response to HIF deletion at both normoxia and hypoxia. ChIP assays showed that either HIF-1 or HIF-2 bind to at least two HREs identified within the ITGA6 promoter. When WT PyMT tumors were sorted to enrich for CD49fhi cells, limiting dilution transplantation confirmed that TIC activity was increased relative to CD49low cells. Finally, data mining of breast cancer patient repositories showed that ITGA6 mRNA expression is independently and inversely correlated with overall and recurrence free survival. Conclusions: CD49f is a direct transcriptional target of HIF-1/HIF-2. Because CD49f is known to enrich for breast TICs, we suggest that HIF-dependent regulation of ITGA6 expression contributes at least partially to HIFs promotion of TIC activity in ER-negative breast cancers. Citation Format: Danielle L. Peacock, Luciana P. Schwab, Tiffany N. Seagroves. ITGA6 (CD49F) is directly regulated by hypoxia-inducible factors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3885. doi:10.1158/1538-7445.AM2014-3885

Introduction: Hypoxia is a hallmark of most solid tumors. Under hypoxic stress tumor cells adapt by regulating survival, metabolism and angiogenesis. The heterodimeric Hypoxia-Inducible Factor (HIF)-1 transcription factor is a master regulator of this response, and is comprised of HIF-1α, the oxygen-regulated subunit, and aryl hydrocarbon nuclear receptor translocator (ARNT), which is constitutively expressed. HIF-1α protein is over-expressed in ∼30% of primary breast tumors and ∼70% of metastases. Over-expression of HIF-1α is independently correlated with poor prognosis and decreased survival in breast cancer patients. In agreement with these observations, conditional deletion of Hif1a represses tumor initiation and lung metastasis in the MMTV-polyoma virus middle T (MMTV-PyMT) model of breast cancer. Methods: Primary mammary tumor epithelial cells (MTECs) were established from late stage carcinomas originating in PyMT+; Hif1a floxed mice maintained on an inbred background (FVB/Nj). MTECs were exposed ex vivo to Adenovirus-β-gal or -Cre to create wild type (WT) and knockout (KO) cells, respectively; cells were then re-introduced into the mammary fat pad of FVB/Nj recipients. HIF-1α deletion reduced primary tumor growth by ∼60% and lung macrometastases by ∼90%. To identify HIF-dependent target genes, microarray profiling was performed. Several genes were differentially expressed between both WT and KO cells cultured at normoxia (21% O2) or hypoxia (0.5% O2) and end-stage WT and KO tumors. Of particular interest, creatine kinase brain isoform (Ckb) mRNA levels were reduced >100-fold in KO cells and >2-fold in KO end-stage tumors; down-regulation at the protein level was confirmed by western blotting. Two independent shRNA constructs (shc59 and shc61) were generated to stably knockdown Ckb in WT cells to assay for effects on cell proliferation and metastasis. Results: In 2-D culture, no significant difference in cell growth was observed in either knockdown line versus the WT cells. In contrast, when one million cells were introduced into the circulation by tail vein injection, 100% of mice injected with WT cells developed macrometastases, whereas no macromets were derived from shc61 cells and only 20% of mice injected with shc59 cells developed macromets. Four putative hypoxic response elements (HREs) have been identified in the Ckb promoter; therefore, ChIP experiments are in progress to determine if Ckb is a direct HIF-1 target. Additional experiments will test for a role of Ckb in regulating primary tumor growth, cell migration or invasion, and, if cyclocreatine, a CKB inhibitor, is effective in reducing primary tumor growth and/or metastasis. Conclusions: Further characterization of the function of Ckb, and other genes downstream of HIF-1α that drive metastasis, may identify pathways amenable to therapeutic intervention to treat patients with metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5312. doi:1538-7445.AM2012-5312