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Simple Summary Targeting dysregulated cellular metabolism is a promising avenue to treat metastatic disease. The aim of our study was to identify genes downstream of the hypoxia-inducible factor (HIF)-1 transcription factor that are amenable to therapeutic intervention to treat metastatic breast cancer (MBC). We identified creatine kinase, brain isoform (CKB) as an HIF-dependent gene that strongly promotes invasion and metastasis in estrogen-receptor (ER)-negative breast cancer models. Deletion of Ckb also repressed glycolysis and mitochondrial respiration, leading to a reduction in intracellular ATP. Either the deletion of Ckb or inhibition of creatine kinase (CK) activity using the creatine analog cyclocreatine (cCr) repressed cell invasion, the formation of invadopodia and lung metastasis. In addition, when paired with paclitaxel or doxorubicin, cCr enhanced growth inhibition in an additive or synergistic manner. cCr may be an effective anti-metastatic agent in ER-negative, HIF-1α-positive breast cancers, targeting both cellular metabolism and motility, particularly when paired with conventional cytotoxic agents. Abstract 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.

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.

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-β-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. Mol Cancer Res; 13(5); 923–33. ©2015 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

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.

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.

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.

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

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.