Oncogenic transformation and hypoxia both induce glut1 mRNA. We studied the interaction between the ras oncogene and hypoxia in up-regulating glut1 mRNA levels using Rat1 fibroblasts transformed with H-ras (Rat1-ras). Transformation with H-ras led to a substantial increase in glut1 mRNA levels under normoxic conditions and additively increased glut1 mRNA levels in concert with hypoxia. Using a luciferase reporter construct containing 6 kilobase pairs of the glut1 promoter, we showed that this effect was mediated at the transcriptional level. Promoter activity was much higher in Rat1-ras cells than in Rat1 cells and could be down-regulated by cotransfection with a dominant negative Ras construct (RasN17). A 480-base pair (bp) cobalt/hypoxia-responsive fragment of the promoter containing a HIF-1 binding site showed significantly higher activity in Rat1-ras cells than in Rat1 cells, suggesting that Ras might mediate its effect through HIF-1 even under normoxic conditions. Consistent with this, Rat1-ras cells displayed higher levels of HIF1-alpha protein under normoxic conditions. In addition, a promoter construct containing a 4-bp mutation in the HIF1 binding site showed lower activity in Rat1-ras cells than a construct with an intact HIF1 binding site. The activity of the latter construct but not the former could be down-regulated by RasN17, supporting the importance of the HIF1 binding site in regulation by Ras. The phosphatidylinositol 3-kinase inhibitor LY29004 down-regulated glut1 promoter activity and mRNA levels under normoxia and also decreased HIF1alpha protein levels in these cells. Collectively these results indicate that H-Ras up-regulates the glut1 promoter, at least in part, by increasing HIF-1alpha protein levels leading to transactivation of promoter through the HIF-1 binding site.

In the continuing search for effective treatments for cancer, the emerging model is the combination of traditional chemotherapy with anti-angiogenesis agents that inhibit blood vessel growth. However, the implementation of this strategy has faced two major obstacles. First, the long-term shutdown of tumour blood vessels by the anti-angiogenesis agent can prevent the tumour from receiving a therapeutic concentration of the chemotherapy agent. Second, inhibiting blood supply drives the intra-tumoural accumulation of hypoxia-inducible factor-1alpha (HIF1-alpha); overexpression of HIF1-alpha is correlated with increased tumour invasiveness and resistance to chemotherapy. Here we report the disease-driven engineering of a drug delivery system, a 'nanocell', which overcomes these barriers unique to solid tumours. The nanocell comprises a nuclear nanoparticle within an extranuclear pegylated-lipid envelope, and is preferentially taken up by the tumour. The nanocell enables a temporal release of two drugs: the outer envelope first releases an anti-angiogenesis agent, causing a vascular shutdown; the inner nanoparticle, which is trapped inside the tumour, then releases a chemotherapy agent. This focal release within a tumour results in improved therapeutic index with reduced toxicity. The technology can be extended to additional agents, so as to target multiple signalling pathways or distinct tumour compartments, enabling the model of an 'integrative' approach in cancer therapy.

Immune cells are often exposed to low oxygen tensions, which markedly affect cellular metabolism. We describe how activated T cells adapt to the changing energy supplies in hypoxic areas of inflamed tissues by using hypoxia-inducible factor 1 (HIF1) to switch to glycolysis as the main source of energy and by signalling through extracellular-adenosine receptors. This hypoxic regulation might alter the balance between T helper 1 cells and T helper 2 cells and might alter the activities of cells of the innate immune system, thereby qualitatively and quantitatively affecting immune responses. This regulatory mechanism should be taken into account in the design and interpretation of in vitro and in vivo studies of immune-cell effector functions.

Clear-cell renal carcinoma is associated with inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. VHL is the substrate recognition subunit of an E3 ligase, known to target the alpha subunits of the HIF heterodimeric transcription factor for ubiquitin-mediated degradation under normoxic conditions. We demonstrate that competitive inhibition of the VHL substrate recognition site with a peptide derived from the oxygen degradation domain of HIF1alpha recapitulates the tumorigenic phenotype of VHL-deficient tumor cells. These studies prove that VHL substrate recognition is essential to the tumor suppressor function of VHL. We further demonstrate that normoxic stabilization of HIF1alpha alone, while capable of mimicking some aspects of VHL loss, is not sufficient to reproduce tumorigenesis, indicating that it is not the critical oncogenic substrate of VHL.

Constitutive expression of hypoxia-inducible factor (HIF) has been implicated in several proliferative disorders. Constitutive expression of HIF1 alpha and HIF2 alpha has been linked to a number of human cancers, especially renal cell carcinoma (RCC), in which HIF2 alpha expression is the more important contributor. Expression of HIF1 alpha is dependent on the mammalian target of rapamycin (mTOR) and is sensitive to rapamycin. In contrast, there have been no reports linking HIF2 alpha expression with mTOR. mTOR exists in two complexes, mTORC1 and mTORC2, which are differentially sensitive to rapamycin. We report here that although there are clear differences in the sensitivity of HIF1 alpha and HIF2 alpha to rapamycin, both HIF1 alpha and HIF2 alpha expression is dependent on mTOR. HIF1 alpha expression was dependent on both Raptor (a constituent of mTORC1) and Rictor (a constitutive of mTORC2). In contrast, HIF2 alpha was dependent only on the mTORC2 constituent Rictor. These data indicate that although HIF1 alpha is dependent on both mTORC1 and mTORC2, HIF2 alpha is dependent only on mTORC2. We also examined the dependence of HIF alpha expression on the mTORC2 substrate Akt, which exists as three different isoforms, Akt1, Akt2, and Akt3. Interestingly, the expression of HIF2 alpha was dependent on Akt2, whereas that of HIF1 alpha was dependent on Akt3. Because HIF2 alpha is apparently more critical in RCC, this study underscores the importance of targeting mTORC2 and perhaps Akt2 signaling in RCC and other proliferative disorders in which HIF2 alpha has been implicated.

To directly examine the role of vascular endothelial growth factor (VEGFA) in cartilage development, we conditionally knocked out Vegfa in chondrocytes, using the Col2a1 promoter to drive expression of Cre recombinase. Our study of Vegfa conditional knockout (CKO) mice provides new in-vivo evidence for two important functions of VEGFA in bone formation. First, VEGFA plays a significant role in both early and late stages of cartilage vascularization, since Vegfa CKO mice showed delayed invasion of blood vessels into primary ossification centers and delayed removal of terminal hypertrophic chondrocytes. Second, VEGFA is crucial for chondrocyte survival, since massive cell death was seen in joint and epiphyseal regions of Vegfa CKO endochondral bones. Chondrocytes in these regions were found to upregulate expression of Vegfa in wild-type mice at the time when massive cell death occurred in the Vegfa CKO mice. The expression of the VEGFA receptors Npr1 and Npr2 in epiphyseal chondrocytes and lack of blood vessel reduction in the vicinity of the cartilaginous elements in the Vegfa CKO mice raise the possibility that the observed cell death is the result of a direct involvement of VEGFA in chondrocyte survival. Interestingly, the extensive cell death seen in Vegfa CKO null bones had a striking similarity to the cell death phenotype observed when hypoxia-inducible factor 1 alpha (Hif1a) expression was abolished in developing cartilage. This similarity of cell death phenotypes and the deficient VEGFA production in Hif1a null epiphyseal chondrocytes demonstrate that HIF1 alpha and VEGFA are components of a key pathway to support chondrocyte survival during embryonic bone development.

Our understanding of the pathways that regulate lymphocyte metabolism, as well as the effects of metabolism and its products on the immune response, is still limited. We report that a metabolic program controlled by the transcription factors hypoxia inducible factor-1α (HIF1-α) and aryl hydrocarbon receptor (AHR) supports the differentiation of type 1 regulatory T cell (Tr1) cells. HIF1-α controls the early metabolic reprograming of Tr1 cells. At later time points, AHR promotes HIF1-α degradation and takes control of Tr1 cell metabolism. Extracellular ATP (eATP) and hypoxia, linked to inflammation, trigger AHR inactivation by HIF1-α and inhibit Tr1 cell differentiation. Conversely, CD39 promotes Tr1 cell differentiation by depleting eATP. CD39 also contributes to Tr1 suppressive activity by generating adenosine in cooperation with CD73 expressed by responder T cells and antigen-presenting cells. These results suggest that HIF1-α and AHR integrate immunological, metabolic and environmental signals to regulate the immune response.

Our recent studies have mechanistically implicated a loss of stromal Cav-1 expression and HIF1-alpha-activation in driving the cancer-associated fibroblast phenotype, through the paracrine production of nutrients via autophagy and aerobic glycolysis. However, it remains unknown if HIF1a-activation is sufficient to confer the cancer-associated fibroblast phenotype. To test this hypothesis directly, we stably-expressed activated HIF1a in fibroblasts and then examined their ability to promote tumor growth using a xenograft model employing human breast cancer cells (MDA-MB-231). Fibroblasts harboring activated HIF1a showed a dramatic reduction in Cav-1 levels and a shift towards aerobic glycolysis, as evidenced by a loss of mitochondrial activity, and an increase in lactate production. Activated HIF1a also induced BNIP3 and BNIP3L expression, markers for the autophagic destruction of mitochondria. Most importantly, fibroblasts expressing activated HIF1a increased tumor mass by ∼2-fold and tumor volume by ∼3-fold, without a significant increase in tumor angiogenesis. In this context, HIF1a also induced an increase in the lymph node metastasis of cancer cells. Similar results were obtained by driving NFκB activation in fibroblasts, another inducer of autophagy. Thus, activated HIF1a is sufficient to functionally confer the cancer-associated fibroblast phenotype. It is also known that HIF1a expression is required for the induction of autophagy in cancer cells. As such, we next directly expressed activated HIF1a in MDA-MB-231 cells and assessed its effect on tumor growth via xenograft analysis. Surprisingly, activated HIF1a in cancer cells dramatically suppressed tumor growth, resulting in a 2-fold reduction in tumor mass and a 3-fold reduction in tumor volume. We conclude that HIF1a activation in different cell types can either promote or repress tumorigenesis. Based on these studies, we suggest that autophagy in cancer-associated fibroblasts promotes tumor growth via the paracrine production of recycled nutrients, which can directly "feed" cancer cells. Conversely, autophagy in cancer cells represses tumor growth via their "self-digestion". Thus, we should consider that the activities of various known oncogenes and tumor-suppressors may be compartment and cell-type specific, and are not necessarily an intrinsic property of the molecule itself. As such, other "classic" oncogenes and tumor suppressors will have to be re-evaluated to determine their compartment specific effects on tumor growth and metastasis. Lastly, our results provide direct experimental support for the recently proposed "Autophagic Tumor Stroma Model of Cancer".

The mammalian AP-endonuclease (APE1/Ref-1) plays a central role in the repair of oxidized and alkylated bases in mammalian genomes via the base excision repair (BER) pathway. However, APE1, unlike its E. coli prototype Xth, has two unique and apparently distinct transcriptional regulatory activities. APE1 functions as a redox effector factor (Ref-1) for several transcription factors including AP-1, HIF1-alpha, and p53. APE1 was also identified as a direct trans-acting factor for repressing human parathyroid hormone (PTH) and renin genes by binding to the negative calcium-response element (nCaRE) in their promoters. We have characterized APE1's post-translational modification, namely, acetylation which modulates its transcriptional regulatory function. Furthermore, stable interaction of APE1 with several other trans-acting factors including HIF-1alpha, STAT3, YB-1, HDAC1, and CBP/p300 and formation of distinct trans-acting complexes support APE1's direct regulatory function for diverse genes. Multiple functions of mammalian APE1, both in DNA repair and gene regulation, warrant extensive analysis of its own regulation and dissection of the mechanisms. In this review, we have discussed APE1's own regulation and its role as a transcriptional coactivator or corepressor by both redox-dependent and redox-independent (acetylation-mediated) mechanisms, and explore the potential utility of targeting these functions for enhancing drug sensitivity of cancer cells.

OBJECTIVE

We sought to examine the presence of hypoxia in human carotid atherosclerosis and its association with hypoxia-inducible transcription factor (HIF) and intraplaque angiogenesis.

BACKGROUND

Atherosclerotic plaques develop intraplaque angiogenesis, which is a typical feature of hypoxic tissue and expression of HIF.

METHODS

To examine the presence of hypoxia in atherosclerotic plaques, the hypoxia marker pimonidazole was infused before carotid endarterectomy in 7 symptomatic patients. Also, the messenger ribonucleic acid (mRNA) and protein expression of HIF1 alpha, HIF2 alpha, HIF-responsive genes (vascular endothelial growth factor [VEGF], glucose transporter [GLUT]1, GLUT3, hexokinase [HK]1, and HK2), and microvessel density were determined in a larger series of nondiseased and atherosclerotic carotid arteries with microarray, quantitative reverse transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry.

RESULTS

Pimonidazole immunohistochemistry demonstrated the presence of hypoxia, especially within the macrophage-rich center of the lesions. Hypoxia correlated with the presence of a thrombus, angiogenesis, and expression of CD68, HIF, and VEGF. The mRNA and protein expression of HIF, its target genes, and microvessel density increased from early to stable lesions, but no changes were observed between stable and ruptured lesions.

CONCLUSIONS

This is the first study directly demonstrating hypoxia in advanced human atherosclerosis and its correlation with the presence of macrophages and the expression of HIF and VEGF. Also, the HIF pathway was associated with lesion progression and angiogenesis, suggesting its involvement in the response to hypoxia and the regulation of human intraplaque angiogenesis.

Fibroblasts are the most abundant "non-cancerous" cells in tumors. However, it remains largely unknown how these cancer-associated fibroblasts (CAFs) promote tumor growth and metastasis, driving chemotherapy resistance and poor clinical outcome. This review summarizes new findings on CAF signaling pathways and their emerging metabolic phenotypes that promote tumor growth. Although it is well established that altered cancer metabolism enhances tumor growth, little is known about the role of fibroblast metabolism in tumor growth. New studies reveal that metabolic coupling occurs between catabolic fibroblasts and anabolic cancer cells, in many types of human tumors, including breast, prostate, and head & neck cancers, as well as lymphomas. These catabolic phenotypes observed in CAFs are secondary to a ROS-induced metabolic stress response. Mechanistically, this occurs via HIF1-alpha and NFκB signaling, driving oxidative stress, autophagy, glycolysis and senescence in stromal fibroblasts. These catabolic CAFs then create a nutrient-rich microenvironment, to metabolically support tumor growth, via the local stromal generation of mitochondrial fuels (lactate, ketone bodies, fatty acids, glutamine, and other amino acids). New biomarkers of this catabolic CAF phenotype (such as caveolin-1 (Cav-1) and MCT4), which are reversible upon treatment with anti-oxidants, are strong predictors of poor clinical outcome in various types of human cancers. How cancer cells metabolically reprogram fibroblasts can also help us to understand the effects of cancer cells at an organismal level, explaining para-neoplastic phenomena, such as cancer cachexia. In conclusion, cancer should be viewed more as a systemic disease, that engages the host-organism in various forms of energy-transfer and metabolic co-operation, across a whole-body "ecosystem".

The basic helix-loop-helix-PAS (bHLH-PAS) protein ARNT is a dimeric partner of the Ah receptor (AHR) and hypoxia inducible factor 1 alpha(HIF1 alpha). These dimers mediate biological responses to xenobiotic exposure and low oxygen tension. The recent cloning of ARNT and HIF1(homologues (ARNT2 and HIF2 alpha) indicates that at least six distinct bHLH-PAS heterodimeric combinations can occur in response to a number of environmental stimuli. In an effort to understand the biological relevance of this combinatorial complexity, we characterized their relative expression at a number of developmental time points by parallel in situ hybridization of adjacent tissue sections. Our results reveal that in general there is limited redundancy in the expression of these six transcription factors and that each of these bHLH-PAS members displays a unique pattern of developmental expression emerging as early as embryonic day 9.5.

Expression of the imprinted H19 gene is remarkably elevated in a large number of human cancers. Recently, we reported that H19 RNA is up-regulated in hypoxic stress and furthermore, it possesses oncogenic properties. However, the underlying mechanism(s) of these phenomena remain(s) unknown. Here we demonstrate a tight correlation between H19 RNA elevation by hypoxia and the status of the p53 tumor suppressor. Wild type p53 (p53(wt)) prevents the induction of H19 upon hypoxia, and upon its reconstitution in p53(null) cells. The last case is accompanied by a decrease in cell viability. The p53 effect is nuclear and seems independent of its tetramerization. Furthermore, using knockdown and over-expression approaches we identified HIF1-alpha as a critical factor that is responsible for H19 induction upon hypoxia. Knocking down HIF1-alpha abolishes H19 RNA induction, while its over-expression significantly enhances the H19 elevation in p53(null) hypoxic cells. In p53(wt) hypoxic cells simultaneous suppression of p53 and over-expression of HIF1-alpha are needed to induce H19 significantly, while each treatment separately resulting in a mild induction, indicating that the molecular mechanism of p53 suppression effect on H19 may at least in part involve interfering with HIF1-alpha activity. In vivo a significant increase in H19 expression occurred in tumors derived from p53(null) cells but not in p53(wt) cells. Taken together, our results indicate that a functional link exists between p53, HIF1-alpha and H19 that determines H19 elevation in hypoxic cancer cells. We suggest that this linkage plays a role in tumor development.

The insulin/TOR pathway is a conserved regulator of cell and organism growth in metazoans. Over the last several years, an array of signaling inputs to this pathway has been defined. However the growth-regulatory outputs are less clear. Drosophila has proven to be a powerful genetic model system in which to study insulin/TOR signaling. This review highlights recent studies in Drosophila that have identified essential outputs and key effectors of the pathway. These include the regulation of ribosome synthesis, mRNA translation, autophagy and endocytosis, through downstream effectors such as Myc, FOXO, HIF1-alpha, TIF-IA, 4EBP and Atg1. This network of outputs and effectors can regulate cell and organismal metabolism, and is essential for the control of tissue growth, responses to starvation and stress, and aging. The mechanisms identified in Drosophila likely operate in most metazoans, and are relevent to our understanding of diseases caused by aberrent insulin/TOR signaling such as cancer, diabetes and obesity.

Epithelial-to-mesenchymal transition (EMT) induced by chronic hypoxia is one of the critical causes of renal fibrosis. Twist, a basic helix-loop-helix transcription factor, is believed to be important in promoting EMT. We found that the expression of Twist was increased in human tubule cell lines (HK-2 and HKC) grown under hypoxic conditions. This was accompanied by reduced expression of the epithelial markers E-cadherin and ZO-1 and enhanced expression of the mesenchymal markers vimentin and alpha-smooth muscle actin. When Twist was overexpressed in these cells it induced a mesenchymal phenotype, whereas its knockdown by short interfering RNA (siRNA) effectively reversed hypoxia-induced EMT. We showed that transfection with siRNA to hypoxia-inducible factor-1alpha (HIF-1alpha), another basic helix-loop-helix transcription factor, reduced Twist expression. Twist promoters contain HIF1-alpha-binding sites and transfection of reporter constructs using the promoter showed increased transcription in cells subjected to hypoxia. Electrophoretic mobility shift and chromatin immunoprecipitation assays identified the presence of a functional HIF-1alpha-binding site within the proximal Twist gene promoter. In an in vivo assay using the rat remnant kidney we found that both Twist and HIF-1alpha were overexpressed in tubular epithelial cells showing EMT. These studies suggest that HIF-1alpha induces Twist expression in hypoxic tubular cells and that this plays a role in EMT during renal fibrogenesis.

Tumor-associated inflammation can induce various molecules expressed from the tumors themselves or surrounding cells to create a microenvironment that potentially promotes cancer development. Inflammation, particularly chronic inflammation, is often linked to cancer development, even though its evolutionary role should impair nonself objects including tumors. The inflammation amplifier, a hyperinducer of chemokines in nonimmune cells, is the principal machinery for inflammation and is activated by the simultaneous stimulation of NF-κB and STAT3. We have redefined inflammation as local activation of the inflammation amplifier, which causes an accumulation of various immune cells followed by dysregulation of local homeostasis. Genes related to the inflammation amplifier have been genetically associated with various human inflammatory diseases. Here, we describe how cancer-associated genes, including interleukin (IL)-6, Ptgs2, ErbB1, Gas1, Serpine1, cMyc, and Vegf-α, are strongly enriched in genes related to the amplifier. The inflammation amplifier is activated by the stimulation of cytokines, such as TNF-α, IL-17, and IL-6, resulting in the subsequent expression of various target genes for chemokines and tumor-related genes like BCL2L11, CPNE7, FAS, HIF1-α, IL-1RAP, and SOD2. Thus, we conclude that inflammation does indeed associate with the development of cancer. The identified genes associated with the inflammation amplifier may thus make potential therapeutic targets of cancers.

Melanomas are very aggressive neoplasms with notorious resistance to therapeutics. It was recently proposed that the remarkable phenotypic plasticity of melanoma cells allows for the rapid development of both resistance to chemotherapeutic drugs and invasive properties. Indeed, the capacity of melanoma cells to form distant metastases is the main cause of mortality in melanoma patients. Therefore, the identification of the mechanism controlling melanoma phenotype is of paramount importance. In the present report, we show that deletion of microphthalmia-associated transcription factor (MITF), the master gene in melanocyte differentiation, is sufficient to increase the metastatic potential of mouse and human melanoma cells. MITF silencing also increases fibronectin and Snail, two mesenchymal markers that might explain the increased invasiveness in vitro and in vivo. Furthermore, ablation of this population by Forskolin-induced differentiation or MITF-forced expression significantly decreases tumour and metastasis formation, suggesting that eradication of low-MITF cells might improve melanoma treatment. Moreover, we demonstrate that a hypoxic microenvironment decreases MITF expression through an indirect, hypoxia-inducible factor 1 (HIF1)α-dependant transcriptional mechanism, and increases the tumourigenic and metastatic properties of melanoma cells. We identified Bhlhb2, a new factor in melanoma biology, as the mediator of hypoxia/HIF1α inhibitory effect on MITF expression. Our results reveal a hypoxia-HIF1α-BHLHB2-MITF cascade controlling the phenotypic plasticity in melanoma cells and favouring metastasis development. Targeting this pathway might be helpful in the design of new anti-melanoma therapies.

Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases.

OBJECTIVE

We have previously shown the prognostic significance of BCL2 expression in the activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) patients treated with cyclophosphamide-Adriamycin-vincristine-prednisone (CHOP) or CHOP-like therapy. However, after the inclusion of rituximab (R) in the CHOP regimen, several conflicting observations about the prognostic value of BCL2 expression have been reported.

METHODS

We evaluated the R-CHOP cohort of 221 DLBCL cases with gene expression profiling data. BCL2 protein (n = 169), mRNA (n = 221) expression, and t(14;18) (n = 144) were correlated with clinical outcome. The CHOP cohort (n = 181) was used for comparative analysis.

RESULTS

BCL2 protein expression has significant impact on overall survival (OS) and event-free survival (EFS) in DLBCL (OS, P = 0.009; EFS, P = 0.001) and GCB-DLBCL (OS, P = 0.03; EFS, P = 0.002) but not in ABC-DLBCL in the R-CHOP cohort. The survival differences for EFS in GCB-DLBCL were still observed in multivariate analysis. At the mRNA level, this correlation was observed in EFS in DLBCL (P = 0.006), but only a trend was observed in GCB-DLBCL (P = 0.09). The t(14;18) was detected in 34% of GCB-DLBCL but was not associated with significant differences in survival. Gene enrichment analysis identified significant enrichment of the DLBCL "stromal-1" signatures and hypoxia-inducible factor 1 (HIF1-α) signature in BCL2(-)GCB-DLBCL, whereas T(FH) cell signatures were enriched in BCL2(+)GCB-DLBCL.

CONCLUSIONS

The prognostic significance of BCL2 has changed after inclusion of rituximab in the treatment protocol and is observed in the GCB-DLBCL rather than the ABC-DLBCL. Although rituximab has benefited patients in both DLBCL subgroups, the BCL2(+)GCB-DLBCL seems to receive less benefit from this treatment and may require other novel therapeutic intervention.

Lysophosphatidic acid (LPA) is elevated in ascites of ovarian cancer patients and stimulates growth and other activities of ovarian cancer cells in vitro. Tissue hypoxia is a critical factor for tumor aggressiveness and metastasis in cancers. We tested whether the ascites of ovarian cancer is hypoxic and whether hypoxia influences the effects of LPA on ovarian cancer cells. We found that ovarian ascitic fluids were hypoxic in vivo. Enhanced cellular responsiveness to LPA, including migration and/or invasion of ovarian cancer cells, was observed under hypoxic conditions. This enhancement could be completely blocked by geldanamycin or a small interfering RNA targeting hypoxia-inducible factor 1 alpha (HIF1 alpha). LPA-induced cell migration required cytosolic phospholipase A(2) (cPLA(2)) and LPA stimulates cPLA(2) phosphorylation in a HIF1 alpha-dependent manner under hypoxia conditions. Furthermore, we show for the first time that exogenous LPA enhances tumor metastasis in an orthotopic ovarian cancer model and HIF alpha expression in tumors. 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (an inhibitor of the heat shock protein 90) effectively blocked LPA-induced tumor metastasis in vivo. Together, our data indicate that hypoxic conditions are likely to be pathologically important for ovarian cancer development. HIF1 alpha plays a critical role in enhancing and/or sensitizing the role of LPA on cell migration and invasion under hypoxic conditions, where cPLA(2) is required for LPA-induced cell migration.

Synergy between Toll-like receptor (TLR) and adenosine A2A receptor (A2AR) signaling switches macrophages from production of inflammatory cytokines such as tumor necrosis factor-alpha to production of the angiogenic growth factor vascular endothelial growth factor (VEGF). We show in this study that this switch critically requires signaling through MyD88, IRAK4, and TRAF6. Macrophages from mice lacking MyD88 (MyD88(-/-)) or IRAK4 (IRAK4(-/-)) lacked responsiveness to TLR agonists and did not respond to A2AR agonists by expressing VEGF. Suppression of TRAF6 expression with siRNA in RAW264.7 macrophages also blocked their response to TLR and A2AR agonists. Excisional skin wounds in MyD88(-/-) mice healed at a markedly slower rate than wounds in wild-type MyD88(+/+) mice, showing delayed contraction, decreased and delayed granulation tissue formation, and reduced new blood vessel density. Although macrophages accumulated to higher levels in MyD88(-/-) wounds than in controls, expression of VEGF and HIF1-alpha mRNAs was elevated in MyD88(+/+) wounds. CGS21680, an A2AR agonist, promoted repair in MyD88(+/+) wounds and stimulated angiogenesis but had no significant effect on healing of MyD88(-/-) wounds. These results suggest that the synergistic interaction between TLR and A(2A)R signaling observed in vitro that switches macrophages from an inflammatory to an angiogenic phenotype also plays a role in wound healing in vivo.

The notion that microvascular abnormalities contribute to deleterious changes in the Alzheimer's disease (AD) brain is supported by work from our laboratory and others demonstrating biochemical and functional alterations of the microcirculation in AD. The objective of this study is to determine whether levels of neurotoxic (thrombin) and inflammatory (interleukin 8 (IL-8), integrins alphaVbeta3 and alphaVbeta5) proteins are altered in microvessels isolated from AD patients compared to levels in vessels obtained from non-demented age-matched controls. We also evaluate in AD and control microvessels expression of the transcription factor hypoxia-inducible factor 1-alpha(HIF1-alpha), which regulates pro-inflammatory gene expression, and the regulation of HIF1-alpha expression by thrombin in cultured brain endothelial cells. Our results indicate that in AD there are high levels of expression of the neurotoxic protease thrombin and the inflammation-associated proteins IL-8 and alphaVbeta3 and alphaVbeta5 integrins. HIF1-alpha is higher in AD microvessels compared to control and thrombin treatment of cultured brain endothelial cells results in increased expression of HIF1-alpha. These data suggest that in AD the cerebral microcirculation is a source of neurotoxic and inflammatory mediators and as such contributory to pathologic processes ongoing in the AD brain.

BACKGROUND

Pim-1 is a proto-oncogene involved in cell survival, differentiation and proliferation in several hematologic and epithelial malignancies. Clinically, absence of Pim-1 expression correlates with poor prognosis in prostate cancer. In the present study, the expression of Pim-1 is analyzed in pancreatic cancer and correlated to clinicopathological parameters.

RESULTS

Compared to benign, inflammatory and pre-malignant conditions (i.e., the normal pancreas, chronic pancreatitis and benign intraductal papillary mucinous neoplasm), expression of Pim-1 mRNA and protein increased significantly in pancreatic malignancies. Absence of Pim-1 immunopositivity in cancer cells strongly correlated with a poor prognosis (median survival 13.8 vs. 23.4 months, p = 0.0016). In vitro, rapidly dividing (high versus low serum concentrations) and hypoxic cells displayed higher Pim-1 mRNA and protein levels.

METHODS

Pim-1 mRNA and protein was evaluated with quantitative real-time RT-PCR, immunofluorescence and immunocytochemistry analyses. Ex vivo expression analysis using semi-quantitative immunohistochemistry was performed using human pancreatic tissues of the normal pancreas (n = 10), chronic pancreatitis (n = 30), pancreatic ductal adenocarcinoma (n = 59) and other pancreatic tumors (n = 42). In consecutive sections HIF1-alpha was used as a marker of hypoxia. Survival of patients (n = 35) was compared using the Kaplan-Meier method and a log-rank test. In vitro analyses were performed using cultured pancreatic cancer cell lines (n = 8) and primary human pancreatic stellate cells.

CONCLUSIONS

Hypoxia is a novel inducer of Pim-1 expression. Compared to non-malignant tissues Pim-1 significantly increases in pancreatic cancer. However, the presence of Pim-1 in cancer cells has a positive prognostic impact.

Cancer cells employ both conventional oxidative metabolism and glycolytic anaerobic metabolism. However, their proliferation is marked by a shift towards increasing glycolytic metabolism even in the presence of O2 (Warburg effect). HIF1, a major hypoxia induced transcription factor, promotes a dissociation between glycolysis and the tricarboxylic acid cycle, a process limiting the efficient production of ATP and citrate which otherwise would arrest glycolysis. The Warburg effect also favors an intracellular alkaline pH which is a driving force in many aspects of cancer cell proliferation (enhancement of glycolysis and cell cycle progression) and of cancer aggressiveness (resistance to various processes including hypoxia, apoptosis, cytotoxic drugs and immune response). This metabolism leads to epigenetic and genetic alterations with the occurrence of multiple new cell phenotypes which enhance cancer cell growth and aggressiveness. In depth understanding of these metabolic changes in cancer cells may lead to the development of novel therapeutic strategies, which when combined with existing cancer treatments, might improve their effectiveness and/or overcome chemoresistance.