Constitutive Expression Of HIF-1α Research Articles

Abstract 120: HIF-1α regulation by Smurf2-mediated protein degradation in a VHL-independent manner

Abstract HIF-1α is the main mediator of hypoxic response in cells and contributes to angiogenesis, metastasis and therapy resistance in cancer. The canonical pathway of HIF-1α degradation in normoxia is through the Von Hippel-Lindau (VHL) E3 ubiquitin ligase. We previously found that knockdown of CDK4 decreased HIF-1α expression in a VHL-independent manner. To explore the mechanism of CDK4-induced HIF-1α stabilization, we performed a proteomic analysis on HIF-1α-interacting proteins with palbociclib treatment, a bioinformatic prediction of HIF-1α-targeting E3 ubiquitin ligases and identified SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) to be involved in a non-canonical HIF-1α regulation mechanism. Overexpression of Smurf2 enhanced the ubiquitination of HIF-1α and reduced expression of HIF-1α in HCT116 colorectal cancer cells. Smurf2 overexpression downregulated HIF-1α target gene SLC2A1 but not HIF1A mRNA level in hypoxia. Knockdown of SMURF2 increased HIF-1α expression in normoxia even when CDK4 was knocked-down. TCGA analysis showed correlation between high SMURF2 mRNA and better prognosis in kidney renal clear cell carcinoma (KIRC). In KIRC, VHL loss is a frequent mutational event that leads to high constitutive expression of HIF-1α and HIF-2α. Thus, we investigated a role for Smurf2 in HIF regulation in this cancer type. Overexpression of Smurf2 suppressed HIF-1α expression in RCC4 VHL-deficient clear cell renal cancer line in normoxia. Knockdown of SMURF2 increased HIF-1α expression in RCC4 cells. Treatment of RCC4 cells with Smurf2 inhibitor Heclin also increased HIF-1α expression in normoxia. HIF-2α expression was reduced upon Smurf2 overexpression in RCC4 cells under normoxia and in SW480 colorectal cancer cells under hypoxia. In summary, Smurf2 mediates VHL-independent HIF-1α degradation, which provides new insights for HIF-1α targeting in cancers especially with VHL deficiency, and targeting of Smurf2 to increase HIF activity may have functional consequences in non-neoplastic disorders. Citation Format: Shuai Zhao, Wafik S. El-Deiry. HIF-1α regulation by Smurf2-mediated protein degradation in a VHL-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 120.

Chetomin, targeting HIF-1α/p300 complex, exhibits antitumour activity in multiple myeloma

Background:Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. The constitutive expression of HIF-1α in MM suggests that inhibition of HIF-1α-mediated transcription represents an interesting target in MM.Methods:As p300 is a crucial co-activator of hypoxia-inducible transcription, disrupting the complex HIF-1α/p300 to target HIF activity appears to be an attractive strategy.Results:We reported that chetomin, an inhibitor of HIF-1α/p300 interaction, exhibits antitumour activity in human myeloma cell lines and primary MM cells from patients.Conclusions:Our data suggest that chetomin may be of clinical value in MM and especially for patients characterised by a high EP300/HIF-1α expression and a poor prognosis.

Intermittent Induction of HIF-1α Produces Lasting Effects on Malignant Progression Independent of Its Continued Expression

Dysregulation of hypoxia-inducible transcription factors HIF-1α and HIF-2α correlates with poor prognosis in human cancers; yet, divergent and sometimes opposing activities of these factors in cancer biology have been observed. Adding to this complexity is that HIF-1α apparently possesses tumor-suppressing activities, as indicated by the loss-of-function mutations or even homozygous deletion of HIF1A in certain human cancers. As a step towards understanding this complexity, we employed 8-week intermittent induction of a stable HIF-1α variant, HIF1α(PP), in various cancer cell lines and examined the effects on malignant progression in xenografts of immunocompromised mice in comparison to those of HIF2α(PP). Although 8-week treatment led to eventual loss of HIF1α(PP) expression, treated osteosarcoma U-2 OS cells acquired tumorigenicity in the subcutaneous tissue. Furthermore, the prior treatment resulted in widespread invasion of malignant glioma U-87 MG cells in the mouse brain and sustained growth of U-118 MG glioma cells. The lasting effects of HIF-1α on malignant progression are specific because neither HIF2α(PP) nor β-galactosidase yielded similar effects. By contrast, transient expression of HIF1α(PP) in U-87 MG cells or constitutive expression of HIF1α(PP) but not HIF2α(PP) in a patient-derived glioma sphere culture inhibited tumor growth and spread. Our results indicate that intermittent induction of HIF-1α produces lasting effects on malignant progression even at its own expense.

Abstract 5132: Targeting hypoxia-mediated gene transcription with a novel polyamide drug designed to disrupt the HIF1α/β heterodimer binding overcomes resistance to hypoxia in myeloma cell lines

Abstract Despite recent advancements in therapy, multiple myeloma (MM) remains incurable. While the reasons for this are unclear, it is felt that the bone marrow (BM) microenvironment may confer protective advantages to MM. The BM is known to be hypoxic compared to other tissues, and while oxygen stress can kill tumor cells, it is also known that hypoxia promotes tumor progression, metastasis, angiogenesis, and resistance to chemo- and radiation therapy. This suggests that hypoxic conditions in the BM may contribute to MM resistance, and therefore targeting the hypoxia response may be of therapeutic benefit. The adaptive response to hypoxia is mediated by a constitutively expressed β-subunit (HIF-1β), which forms a heterodimer with the inducible α-subunit (HIF-1α), although HIF-1α is frequently found to be constitutively expressed in some MM cell lines. In order to target the hypoxic response in MM, we blocked HIF-mediated gene transcription using a novel class of DNA-binding pyrrole-imidazole (Py-Im) polyamide (PA) drugs that are designed to target and disrupt the HIF1α/β heterodimer from binding to its cognate DNA sequence. Using a hypoxia chamber, we cultured MM cell lines (OPM-2, 8226, U266, MM1.S) under normoxia (22% O2, 5% CO2) or hypoxia (0.1% O2, 5% CO2) for 72 hours and assayed for apoptosis. We found that 8226 and U266 cell lines were relatively resistant to hypoxia-mediated apoptosis (∼15-20% apoptosis), but that OPM-2 was much more sensitive (>50% apoptosis), whilst MM1.S was intermediate in sensitivity (∼25% apoptosis). Using immunoblots, we found that cell lines with constitutive expression of HIF-1α were resistance to hypoxia, suggesting that constitutive HIF activity predicts MM resistance to hypoxia. We then tested whether treatment with our novel PA drug could overcome resistance to hypoxia-induced apoptosis. After confirming the ability of the PA inhibitor to specifically block HIF-mediated transcription using MM cells with a HIF-response element-reporter construct, as well as inhibition of VEGF mRNA expression, we next assayed the effect of PA (0.1-10 μM) treatment in MM cells as described above. PA was only mildly cytotoxic to MM cells under normoxia, but we observed a significant synergistic induction of apoptosis under hypoxic conditions. These effects were further potentiated when PA treatment was combined with rapamycin (0.1-100 nM). Our data suggests that the HIF-mediated hypoxic response plays an important role in MM sensitivity to hypoxia, likely through the induction of specific survival genes. In conclusion, our preliminary data shows that targeting the hypoxic response in MM with a specific polyamide drug that targets the HIF-response, alone or in combination with mTOR inhibitors, may provide an effective and novel treatment in the hypoxic BM microenvironment. Citation Format: Veena S. Mysore, Nicholas G. Nickols, Jerzy Szablowski, Peter Dervan, Alan Lichtenstein, Patrick J. Frost. Targeting hypoxia-mediated gene transcription with a novel polyamide drug designed to disrupt the HIF1α/β heterodimer binding overcomes resistance to hypoxia in myeloma cell lines. [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 5132. doi:10.1158/1538-7445.AM2014-5132

Hypoxia-Inducible Factor-1α in Pulmonary Arterial Smooth Muscle Cells and Hypoxia-induced Pulmonary Hypertension

Hypoxia-Inducible Factor-1α in Pulmonary Arterial Smooth Muscle Cells and Hypoxia-induced Pulmonary Hypertension

Hypoxia inducible factor-1 alpha as a therapeutic target in multiple myeloma.

The increasing importance of hypoxia-inducible factor-1α (HIF-1α) in tumorigenesis raises the possibility that agents which specifically inhibit this transcription factor, would provide significant therapeutic benefit. The constitutive expression of HIF-1α in about 35% of Multiple Myeloma (MM) patients suggests HIF-1α suppression might be part of a therapeutic strategy. Accordingly, we explored the effect of EZN-2968, a small 3rd generation antisense oligonucleotide against HIF-1α, in a panel of MM cell lines and primary patients samples. Here, we demonstrated that EZN-2968 is highly specific for HIF-1α mRNA and that exposure of MM cells to EZN-2968 resulted in an efficient and homogeneous loading of the cells showing a long lasting low HIF-1α protein level. In MM cells, HIF-1α suppression induced a permanent cell cycle arrest by prolonging S-phase through cyclin A modulation and in addition it induced a mild apoptotic cell death. Moreover, HIF-1α suppression caused a metabolic shift that leaded to increased production of ATP by oxidative phosphorylation (i.e. Warburg effect reversion), that was confirmed by the observed mitochondrial membrane potential decrease. These results show that HIF-1α is an important player in MM homeostasis and that its inhibition by small antisense oligonucleotides provides a rationale for novel therapeutic strategy to improving MM treatment.

Restricted Expression of miR-30c-2-3p and miR-30a-3p in Clear Cell Renal Cell Carcinomas Enhances HIF2α Activity

Inactivation of the von-Hippel Lindau (VHL) tumor suppressor gene occurs in 90% of human clear cell renal cell carcinomas (ccRCC) and leads to the stable expression of the hypoxia-inducible factors HIF1α and HIF2α. The constitutive expression of HIF1α in a majority of VHL-deficient tumors is counterintuitive, given that HIF1α functions as a tumor suppressor in ccRCC, whereas HIF2α clearly enhances tumor growth. We demonstrate here that miR-30c-2-3p and miR-30a-3p specifically bind and inhibit expression of HIF2A transcripts, and that the locus encoding miR-30c-2-3p and miR-30a-3p is selectively repressed in "H1H2" VHL-deficient tumors expressing both HIF1α and HIF2α proteins. Inhibiting miR-30a-3p expression increases HIF2α levels in H1H2 ccRCC cells and promotes cellular proliferation, angiogenesis, and xenograft tumor growth. Our results indicate that miR-30c-2-3p and miR-30a-3p repression enhances HIF2α expression and suggests a mechanism whereby the tumor-suppressive effects of constitutive HIF1α expression are attenuated in VHL-deficient H1H2 tumors.

Hypoxia‐Inducible Factor‐1 Is a Determinant of Lobular Structure and Oxygen Consumption in the Liver

Hypoxia-inducible factor is a hypoxia-responsive transcriptional factor that controls the expression of proteins contributing to homeostatic responses to hypoxia. Spatial heterogeneity of tissue oxygenation has been postulated as a determinant of structure and function of hepatic lobules, although its molecular mechanisms remain unknown. This study aimed to examine the role of HIF-1 expressed in hepatocytes in regulation of hepatic microcirculation. We have generated mice harboring a floxed HIF-1α allele, and employed the albumin-Cre transgenic line to inactivate the gene site-specifically in hepatocytes. Intravital observation of the hepatic microcirculation revealed extension of hepatic lobules in HIF-1α-deficient mice. Measurement of microvascular diameter, velocity, and local oxygen tension by laser-assisted phosphorimetry showed that the oxygen consumption in the lobules of HIF-1α-deficient mice was greater than that in those of control mice. Isolated hepatocytes from HIF-1α-deficient mice also stimulated oxygen consumptions with increased contents of mtDNA. Overexpression of HIF-1α decreased the expression of PGC-1α mRNA, whereas the knockdown of the HIF-1α gene increased it, suggesting that HIF-1 regulates cellular respiration through mitochondrial biogenesis. Our results suggest that constitutive expression of HIF-1α in hepatocytes acts as a determinant of hepatic lobular structure and oxygen consumption by changing mitochondrial contents.

Abstract 2935: Cyclin-dependent kinase 1 regualtes HIF-1α expression to promote tumorigenesis.

Abstract Hypoxia-inducible factor 1 (HIF-1) is a major mediator of tumor physiology, and its activation is correlated with tumor progression, metastasis, and therapeutic resistance. HIF-1 is activated in a broad range of solid tumors due to intra-tumoral hypoxia or genetic alterations that enhance its expression or inhibit its degradation. As a result, decreasing HIF-1α expression represents an attractive strategy to sensitize hypoxic tumors to standard or targeted therapies. Here, we show that cyclin dependent kinase 1 (CDK1) regulates the expression of HIF-1α, independent of its known regulators, such as VHL, p53, and prolyl hydroxolases. Inhibition of CDK1 enhances the proteasomal degradation of HIF-1α, reducing its half-life and steady state levels. In vitro kinase assays reveal that CDK1 directly phosphorylates HIF-1α at a previously unidentified regulatory site, Ser668, to regulate its basal levels and rate of turnover. Mutation of HIF-1α Ser668 to an alanine (S668A) significantly decreased the half-life and steady state levels of HIF-1α, where as mutation to a glutamic acid (S668E) enhanced HIF-1α expression and stability. Importantly, constitutive phosphorylation of HIF-1α at Ser668 was sufficient to stabilize HIF-1α under normoxic conditions, resulting in the transcription of HIF-1 target genes and increased tumor cell invasion in vitro. Furthermore, in vivo xenograft tumor models of HCT116 and RKO colorectal cancer cell lines revealed that stable expression of a phospho-mimetic of HIF-1α this site (S668E) significantly increased tumor proliferation and angiogenesis, resulting in dramatically enhanced tumor volume compared to cells expressing wild-type HIF-1α. Taken together, these findings provide new insights into the regulation of HIF-1α expression and suggest a novel mechanism that may promote HIF-1 deregulation in tumorigenesis. Our results demonstrating CDK-dependent HIF-1α stabilization raise the question of whether the expression of HIF-1α is controlled in a cell cycle-dependent manner during tumorigenesis, even prior to the evolution of significant hypoxia associated with bulky tumor growth in vivo. This finding has previously unrecognized implications for a critical connection between CDK activity and tumor growth, beyond the cell cycle engine. HIF-1α is frequently highly expressed in tumors, independent of hypoxia or its known regulators. The identification of Ser668 phosphorylation as an important regulator of HIF-1α provides a novel mechanism to explain the constitutive expression of HIF-1α in some tumors, most of which also have increased CDK1 and CDK4 activity. It has been well established that HIF-1 inhibition sensitizes solid tumors to radiation, standard chemotherapies and targeted agents. Therefore, inhibition CDK1/4 represents a novel strategy for the treatment of cancer cells with constitutively active HIF-1. Citation Format: Noel A. Warfel, Nathan G. Dolloff, Wafik S. El-Deiry. Cyclin-dependent kinase 1 regualtes HIF-1α expression to promote tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2935. doi:10.1158/1538-7445.AM2013-2935

Abstract 4735: Epigenetic modification of the PI3K/Akt/HIF1α pathway in lymphoma cells by HDAC inhibition

Abstract Hypoxia-inducible factors (HIFs) strongly influence many cellular process such as erythropoiesis, angiogenesis, energy metabolism, pH regulation, cell migration and tumor invasion. Under normoxic conditions, HIF-1α possesses a very short half-life and is rapidly degraded by the ubiquitin proteasome pathway and remains inactive. In hypoxia, HIF-1α is stabilized and forms a complex with HIF-1α that allows HIF-1 to function as a transcription factor. HIF-1α is activated only during hypoxia under normal physiologic conditions. By contrast, HIF-1α is frequently activated in cancer cells, including under normoxic conditions by oncogene products or impaired activity of tumor suppressor genes. Recent studies have shown that the expression of HIF1α is increased in a variety of human tumors a significant fraction of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma patients, implicating a potential role of dysregulated HIF activation. Constitutive expression of HIF-1α enhances vascularization, increases glucose metabolism, and induces the expression of anti-apoptotic proteins in cancer tissues, thus making it an attractive target for cancer therapy. We hypothesized that epigenetic modification of lymphoma cells by histone deacetylase (HDAC) inhibition may modify HIF-1α expression and lead to cell death. We therefore studied the role of the pan HDACi PCI-24781 (PCI) in HIF1α mediated cell death. Western blot analysis of cells treated with 0.125μM to 0.5μM of PCI show significant down regulation of HIF1α protein in all NHL cell lines. To confirm that down regulation of HIF1α is an important factor in cell apoptosis induced by PCI, we incubated cells with the small molecule inhibitor of HIF1α, PX-478 in combination with PCI. Combination of PCI and PX-478 at concentrations of 0.5μM and 10μM respectively, showed an increase in apoptosis as compared to either agent alone in all the DLBCL cell lines. Moreover, knocking down HIF1α in SUDHL4 and OCI-LY3 cell lines using shRNA followed by treatment with PCI also showed the similar effect. We further show that down regulation of HIF1α by PCI is mediated via the PI3K/Akt pathway and inhibition of the PI3K/Akt pathway by LY294002 inhibits HIF1α protein expression. Furthermore pre-incubation of cells with LY inhibits PCI-induced apoptosis. By contrast, constitutive activation of AKT using Myr-Akt-CA shows an increase in HIF1α protein and partial attenuation of PCI-induced apoptosis.We conclude that the activation of the PI3K/Akt/HIF-1α pathway plays a critical role in mediating hypoxia-induced drug resistance leading to unfavorable treatment outcome in lymphoma. Our study suggests a mechanism by which lymphoma cells become resistant to HDAC inhibitors. 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 4735. doi:1538-7445.AM2012-4735

Abstract 3093: Gene regulation induced by constitutive expression of HIF-1α in transgenic mice

Abstract Hypoxia-inducible factor-1α (HIF-1α) has a central role on regulation of various genes under hypoxic condition linked to regulation of angiogenesis, metabolism and tumor development. Expression of HIF-1α was reported in many cancers associated with tumor progression, invasion and metastasis. We reported that constitutive expression of HIF-1α in transgenic mice induced tumors in lymphoid, lung and breast tissues in last this meeting. The incidence of tumors in transgenic mice was up to 90% until 18 months after birth. To clarify the molecular mechanism of lymphomagenesis in the transgenic mice, lymphocyte subsets, gene expression and mitogenic responsiveness on lymphoid tissues were analyzed. Although population of T and B cell subset in spleen, maturation pattern of T cells in thymus, or cell growth rate after treatment with various mitogens were not changed by HIF-1α overexpression, the lymphocytes from transgernic mice showed longer survival than those from wild mice. The lymphocytes also showed resistance for genotoxic stimuli like topoisomerase inhibitors. We next compared gene expression profile on lymphocyte from transgenic mice and wild mice by cDNA microarray and RT-PCR. The expression of over a hundred of genes including HIF-1α itself was altered on T and B cells in transgenic mice. Among them the gene associated with cell survival and cell transformation-related genes were further analyzed by validation study. In this paper, we present the results concerning the role of HIF-1α in lymphoma occurrence, and will discuss the contribution of the transcription factor in human cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3093. doi:10.1158/1538-7445.AM2011-3093

Abstract 3512: Regulation of hypoxia inducible factor alpha in clear cell renal cell carcinoma

Abstract Clear cell renal cell carcinoma (CCRC) is the most common kidney cancer with high incidence of hypoxia-inducible factor 1 and 2 alpha (HIF-α) expression as a consequence of the nonfunctional Von Hippel-Lindau (VHL). The purpose of the study is: a) to investigate the expression levels and relationship between HIF-α and prolyl hydroxylases (PHDs), the negative regulators of HIF-α in tissue microarray (TMA) of human surgical specimens. b) to demonstrate whether methylselenenic acid (MSA) would degrade HIF-α, independent of VHL. c) to determine whether inhibition of HIF-α would enhance cytotoxicity of docetaxel.The results of immunohistochemical evaluation revealed that 92% (81 out of 88) combined incidence of both HIF-1α and HIF-2α with low levels of PHD2 (35%, 31 out of 88) and completely deficient with PHD3, the enzymes responsible for the hydroxylation of HIF-α and subsequent degradation in proteasome. In contrast, we found high incidence of PHD2 positive cases with low HIF-α in both head and neck (86% PHD2 with 38% HIF-α, n=210) and colorectal (90% PHD2 with 26% HIF-α, n=64) indicate high incidence of PHD2 is associated with low incidence of HIF-α. The expression of PHD3 was found in both head & neck (21%) and colorectal (50%) cancer specimens which have low HIF-α incidence compared to CCRC. These data indicate that there is an inverse relationship between HIF-α and PHD2 in CCRC clinical samples in addition to VHL deficiency. In agreement with the surgical specimens, CCRC cells RC2 and 786- O express PHD2 protein but not PHD3 with constitutive expression of HIF-1α in RC2 and HIF-2α in 786 O. We have demonstrated the degradation of both HIF-1α and HIF-2α by MSA that leads to the significant reduction of HIF-α transcriptionally regulated gene VEGF in RC2 and 786-O. MSA exhibits unique property of degrading both HIF-1α and HIF-2α that has not been reported earlier by any other agent. Proteasome inhibitor MG132 reversed the HIF-1α degradation by MSA indicates HIF-1α degradation is proteasome dependent and VHL independent since VHL is nonfunctional in CCRC. Based on these findings we hypothesize that the degradation of HIF-α by MSA is VHL independent. We found RC2 cells with constitutive HIF-1α are 5 fold resistant to docetaxel compared to RC2-VHL cells with no detectable HIF-1α due to overexpression of VHL. As we expected, the combination of MSA enhanced the docetaxel efficacy only in RC2 cells, but not in RC2-VHL shows that the effect of MSA is through HIF-1α. These findings demonstrated the therapeutic potential of selenium for the treatment of CCRC. Thus, the unique molecular properties of CCRC being VHL deficient, express high constitutive HIF-1α and HIF-2α protein with low PHD2 and deficient in PHD3 could provide the rational for the development and evaluation of selenium in combination with the clinically approved drugs for kidney cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3512. doi:10.1158/1538-7445.AM2011-3512

Expression of Hypoxia-Inducible Factor (HIF) Is An Independent Favorable Prognostic Factor in Diffuse Large B-Cell Lymphoma (DLBCL) Treated with R-CHOP

HIF is a transcription factor that regulates gene expression in response to decreases in cellular oxygenation (hypoxia). Genes activated by HIF are involved in glycolysis, glucose transport, angiogenesis, cell proliferation, cell migration, cell metabolism, and cell survival. Many of these genes confer protection against the consequences of oxygen deprivation while others enhance resistance to chemotherapy or radiotherapy. Clinically, evidence of elevated HIF protein correlates with poor prognosis in lung, breast, colorectal, brain, pancreatic, ovarian, renal, and bladder cancers. Our preliminary tissue micro array (TMA) data suggested that HIF is frequently stabilized in lymphoma (BJH 2008; 141:676), especially among patients with diffuse large B-cell lymphoma (DLBCL). We also found that constitutive expression of HIF-1α and/or HIF-2α is stabilized in a set of established lymphoma cell lines, underscoring the potential impact of HIF in lymphoma. To further investigate the importance of HIF, we examined TMAs for HIF-1α expression from 153 patients with DLBCL treated with CHOP or rituximab-CHOP (R-CHOP) at the BCCA between 1999–2002. Treatment was before (pre-rituximab) and after (post-rituximab) institution of a policy recommending the combination of rituximab with CHOP for all patients with newly diagnosed advanced-stage DLBCL (March 2001). Tissue sections were stained with monoclonal antibody to HIF-1α and expression (nuclear) was scored by computer (Automated Cellular Imaging System) and validated by pathology review. HIF was dichotomized as either negative (neg; no HIF staining) or positive (pos; score of 1 or greater). Of the 153 patients, 78 were consecutively treated with R-CHOP, while 75 had received CHOP. We found HIF pos expression in 63% (49/78) of patients treated with R-CHOP and 59% (44/75) treated with CHOP (p=NS). Further, based on the Hans algorithm, HIF was expressed in 62% of germinal center (GC) patients and 59% of non-GC patients (P=NS). In the R-CHOP group (see Figure 1A and 1B), the 5-year progression-free survival (PFS) was 45% for HIF neg patients vs 62% for HIF pos (log rank p=0.0187); while the 5-year overall survival (OS) was 48% for HIF neg vs 76% for HIF pos (log rank p=0.025).In patients treated with CHOP, there was no difference in outcome between HIF pos and HIF neg subsets. In multivariate analysis in the R-CHOP treated group, controlling for the International Prognostic Index (IPI), HIF pos expression remained a significant independent predictor for OS (p=0.03), while the IPI was of borderline significance (p=0.051). Comparison with other biomarkers showed that HIF did not correlate with expression of bcl-2, CD10, MUM-1, or FOXP1; while a high correlation was detected with bcl-6 and HIF (among HIF pos R-CHOP patients, 94% were bcl-6 pos and 6% were bcl-6 neg; p=0.004). We conclude that expression of HIF-1α is an important and heretofore undiscovered independent favorable prognostic factor for PFS and OS in DLBCL patients treated with R-CHOP, but not in patients treated with CHOP. These data suggest there may be an important biological interaction between CD20 monoclonal antibody-based therapy and HIF or downstream genes regulated by HIF. Further study of this observation is warranted. [Display omitted]

Constitutive Expression of HIF-1α and HIF-2α in Bone Marrow Stromal Cells Differentially Promotes Their Proangiogenic Properties

Bone marrow stromal cells (BMSCs) contain progenitors capable of participating in postnatal angiogenesis. Hypoxia-inducible factors (HIFs) mediate endothelial activation by driving the expression of multiple angiogenic factors. We explored the potential of HIF-1alpha and HIF-2alpha modification in BMSCs, as a tool to improve cell-based angiogenic therapy. BMSCs were retrovirally transduced to express stable forms of HIF-1alpha and HIF-2alpha. HIF-1alpha and, to a greater extent, HIF-2alpha overexpression promoted differentiation of BMSCs to the endothelial lineage, evident by CD31 and Tie-2 expression and improved adhesive properties. Whereas chemotaxis toward stromal-derived factor 1 was higher in both HIF-alpha-expressing BMSCs, enhanced migration toward vascular endothelial growth factor was found only following overexpression of HIF-2alpha, supported by a robust expression of its receptor, Flk-1. HIF-alpha expression was associated with upregulation of angiogenic proteins and improved tube formation. Cytokine arrays of endothelial cells stimulated by medium collected from HIF-alpha-expressing BMSCs revealed further angiogenic activation and improved adhesive capacity. Eventually, delivery of HIF-2alpha-transduced BMSCs induced a more robust angiogenic response, compared with sham-transduced or HIF-1alpha-transduced BMSCs in the corneal micropocket angiogenesis model. Our results support the use of HIF-alpha genes, particularly HIF-2alpha, to augment the efficacy of future cell-based therapy. Disclosure of potential conflicts of interest is found at the end of this article.

Hypoxia-inducible Factor-1-dependent Overexpression of Myeloid Cell Factor-1 Protects Hypoxic Cells against tert-Butyl Hydroperoxide-induced Apoptosis

Increased levels of Mcl-1 (myeloid cell factor-1) have been reported in several cancers, suggesting an important role played by Mcl-1 in cancer cell survival. Mcl-1 is an anti-apoptotic protein shown to delay or block apoptosis. In this work, using semiquantitative immunofluorescence, real-time PCR, and RNase protection assay, an increase in Mcl-1 expression was detected in hepatoma HepG2 cells incubated under hypoxia or in the presence of cobalt chloride. Through analysis of the Mcl-1 promoter sequence, a putative HIF-1 (hypoxiainducible factor-1) binding site was identified. A Mcl-1 promoter fragment containing this hypoxia-responsive element was able to bind HIF-1 in vitro. It also induced hypoxia-dependent transcription of a luciferase reporter gene, which was suppressed by anti-HIF-1alpha short interfering RNA. Finally, overexpression of Mcl-1 protected HepG2 cells against apoptosis induced by tert-butyl hydroperoxide as shown by inhibition of caspase-3 activation and DNA fragmentation. All these data suggest a potential anti-apoptotic role of HIF-1 that could protect cells against apoptosis under hypoxia by overexpression of the Mcl-1 protein.