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.

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.

Putative role of HIF transcriptional activity in melanocytes and melanoma biology.

Hypoxia-inducible factor-1α (HIF-1α) is a highly oxygen sensitive bHLH protein that is part of the heterodimeric HIF-1 transcription factor. Under hypoxic stress, HIF-1 activity is induced to control expression of multiple downstream target genes, including vascular endothelial growth factor (VEGF). The normal epidermis exists in a constant mild hypoxic microenvironment and constitutively expresses HIF-1α and HIF-2α. Expression of HIF-1α and/or HIF-2α has been suggested to correlate with the increased malignant potential of melanocytes, therefore, failures of melanoma therapies may be partially linked to high HIF activity. Notably, melanomas that have the V600E BRAF mutation exhibit increased HIF-1α expression. We have utilized a bioinformatics approach to identify putative hypoxia response elements (HREs) in a set of genes known to participate in the process of melanogenesis (includingTRPM1, SLC45A2, HRAS, C-KIT, PMEL and CRH). While some of the mechanistic links between these genes and the HIF pathway have been previously explored, others await further investigation. Although agents targeting HIF activity have been proposed as novel treatment modalities for melanoma, there are currently no clinical trials in progress to test their efficacy in melanoma.

Breast tumor kinase (Brk/PTK6) is a mediator of hypoxia-associated breast cancer progression.

Basal-type triple-negative breast cancers (TNBC) are aggressive and difficult to treat relative to luminal-type breast cancers. TNBC often express abundant Met receptors and are enriched for transcriptional targets regulated by hypoxia-inducible factor-1α (HIF-1α), which independently predict cancer relapse and increased risk of metastasis. Brk/PTK6 is a critical downstream effector of Met signaling and is required for hepatocyte growth factor (HGF)-induced cell migration. Herein, we examined the regulation of Brk by HIFs in TNBC in vitro and in vivo. Brk mRNA and protein levels are upregulated strongly in vitro by hypoxia, low glucose, and reactive oxygen species. In HIF-silenced cells, Brk expression relied upon both HIF-1α and HIF-2α, which we found to regulate BRK transcription directly. HIF-1α/2α silencing in MDA-MB-231 cells diminished xenograft growth and Brk reexpression reversed this effect. These findings were pursued in vivo by crossing WAP-Brk (FVB) transgenic mice into the MET(Mut) knockin (FVB) model. In this setting, Brk expression augmented MET(Mut)-induced mammary tumor formation and metastasis. Unexpectedly, tumors arising in either MET(Mut) or WAP-Brk × MET(Mut) mice expressed abundant levels of Sik, the mouse homolog of Brk, which conferred increased tumor formation and decreased survival. Taken together, our results identify HIF-1α/2α as novel regulators of Brk expression and suggest that Brk is a key mediator of hypoxia-induced breast cancer progression. Targeting Brk expression or activity may provide an effective means to block the progression of aggressive breast cancers.

Prognostic significance of growth kinetics in newly diagnosed glioblastomas revealed by combining serial imaging with a novel biomathematical model.

Glioblastomas are the most aggressive primary brain tumors, characterized by their rapid proliferation and diffuse infiltration of the brain tissue. Survival patterns in patients with glioblastoma have been associated with a number of clinicopathologic factors including age and neurologic status, yet a significant quantitative link to in vivo growth kinetics of each glioma has remained elusive. Exploiting a recently developed tool for quantifying glioma net proliferation and invasion rates in individual patients using routinely available magnetic resonance images (MRI), we propose to link these patient-specific kinetic rates of biological aggressiveness to prognostic significance. Using our biologically based mathematical model for glioma growth and invasion, examination of serial pretreatment MRIs of 32 glioblastoma patients allowed quantification of these rates for each patient's tumor. Survival analyses revealed that even when controlling for standard clinical parameters (e.g., age and Karnofsky performance status), these model-defined parameters quantifying biological aggressiveness (net proliferation and invasion rates) were significantly associated with prognosis. One hypothesis generated was that the ratio of the actual survival time after whatever therapies were used to the duration of survival predicted (by the model) without any therapy would provide a therapeutic response index (TRI) of the overall effectiveness of the therapies. The TRI may provide important information, not otherwise available, about the effectiveness of the treatments in individual patients. To our knowledge, this is the first report indicating that dynamic insight from routinely obtained pretreatment imaging may be quantitatively useful in characterizing the survival of individual patients with glioblastoma. Such a hybrid tool bridging mathematical modeling and clinical imaging may allow for stratifying patients for clinical studies relative to their pretreatment biological aggressiveness.