Antiangiogenic therapy has shown promise in the treatment of patients with renal cell carcinoma (RCC). Two classes of antiangiogenic drugs, the anti-vascular endothelial growth factor antibody bevacizumab and the tyrosine kinase inhibitors sorafenib, sunitinib and pazopanib, have shown efficacy in patients with RCC and are approved by the US Food and Drug Administration for treatment of this cancer. In practice, the clinical benefit of antiangiogenic drugs in RCC has been heterogeneous, and in patients who do respond, benefits are modest and/or short-lived. To improve efficacy, combination targeted therapy has been attempted, but with either very limited additional efficacy or nontolerable toxicities. Recent advances in the molecular understanding of tumor angiogenesis and mechanism of resistance, along with the rapid development of targeted drug discovery, have made it possible to further explore novel combination therapy for RCC.

Antiangiogenic therapy has shown promise in the treatment of patients with hepatocellular carcinoma (HCC). Bevacizumab, sorafenib, and sunitinib showed efficacy in patients with HCC; and sorafenib is approved by the FDA for treatment of this cancer. In practice, the clinical benefit of these agents has been heterogeneous; and in patients who do respond, the benefit is modest and/or short-lived. Recent advances in the molecular understanding of tumor angiogenesis along with the rapid development of targeted drug discovery have made it possible to explore novel combination therapy for HCC. We review the clinical trial results, discuss possible molecular mechanisms of resistance, and suggest novel combinations with antiangiogenic therapy.

Hypoxia-inducible transcription factors (HIF) protect cells against oxygen deprivation, and HIF stabilization before ischemia mitigates tissue injury. Because ischemic acute kidney injury (AKI) often involves the thick ascending limb (TAL), modulation of HIF in this segment may be protective. Here, we generated mice with targeted TAL deletion of the von Hippel-Lindau protein (Vhl), which mediates HIF degradation under normoxia, using Tamm-Horsfall protein (Thp)-driven Cre expression. These mice showed strong expression of HIF-1α in TALs but no changes in kidney morphology or function under control conditions. Deficiency of Vhl in the TAL markedly attenuated proximal tubular injury and preserved TAL function following ischemia-reperfusion, which may be partially a result of enhanced expression of glycolytic enzymes and lactate metabolism. These results highlight the importance of the thick ascending limb in the pathogenesis of AKI and suggest that pharmacologically targeting the HIF system may have potential to prevent and mitigate AKI.

The relationship between hypoxic stress, autophagy, and specific cell-mediated cytotoxicity remains unknown. This study shows that hypoxia-induced resistance of lung tumor to cytolytic T lymphocyte (CTL)-mediated lysis is associated with autophagy induction in target cells. In turn, this correlates with STAT3 phosphorylation on tyrosine 705 residue (pSTAT3) and HIF-1α accumulation. Inhibition of autophagy by siRNA targeting of either beclin1 or Atg5 resulted in impairment of pSTAT3 and restoration of hypoxic tumor cell susceptibility to CTL-mediated lysis. Furthermore, inhibition of pSTAT3 in hypoxic Atg5 or beclin1-targeted tumor cells was found to be associated with the inhibition Src kinase (pSrc). Autophagy-induced pSTAT3 and pSrc regulation seemed to involve the ubiquitin proteasome system and p62/SQSTM1. In vivo experiments using B16-F10 melanoma tumor cells indicated that depletion of beclin1 resulted in an inhibition of B16-F10 tumor growth and increased tumor apoptosis. Moreover, in vivo inhibition of autophagy by hydroxychloroquine in B16-F10 tumor-bearing mice and mice vaccinated with tyrosinase-related protein-2 peptide dramatically increased tumor growth inhibition. Collectively, this study establishes a novel functional link between hypoxia-induced autophagy and the regulation of antigen-specific T-cell lysis and points to a major role of autophagy in the control of in vivo tumor growth.

The integration of chemistry and molecular biology with imaging is providing some of the most exciting opportunities in the treatment of cancer. The field of theranostic imaging, where diagnosis is combined with therapy, is particularly suitable for a disease as complex as cancer, especially now that genomic and proteomic profiling can provide an extensive 'fingerprint' of each tumor. Using this information, theranostic agents can be shaped for personalized treatment to target specific compartments, such as the tumor microenvironment (TME), whilst minimizing damage to normal tissue. These theranostic agents can also be used to target multiple pathways or networks by incorporating multiple small interfering RNAs (siRNAs) within a single agent. A decade ago genetic alterations were the primary focus in cancer research. Now it is apparent that the tumor physiological microenvironment, interactions between cancer cells and stromal cells, such as endothelial cells, fibroblasts and macrophages, the extracellular matrix (ECM), and a host of secreted factors and cytokines, influence progression to metastatic disease, aggressiveness and the response of the disease to treatment. In this review, we outline some of the characteristics of the TME, describe the theranostic agents currently available to target the TME and discuss the unique opportunities the TME provides for the design of novel theranostic agents for cancer therapy.

The cytotoxic marine red algal metabolite thyrsiferol (1) was found to inhibit hypoxia-induced hypoxia-inducible factor-1 (HIF-1) activation in T47D human breast tumor cells (66% inhibition at 3 μM). Compound 1 also suppressed hypoxic induction of HIF-1 target genes (VEGF, GLUT-1) at the mRNA level, and displayed tumor cell line-selective time-dependent inhibition of cell viability/proliferation. Mechanistic studies revealed that 1 selectively suppressed mitochondrial respiration at Complex I (IC(50) 3 μM). Thyrsiferol represents a prototypical, structurally unique electron transport chain inhibitor. The apparent rotenone-like activity may contribute to the observed cytotoxicity of 1 and play an important role in Laurencia chemical defense.

Endothelial cells (ECs) are the primary sensors of variations in blood oxygen concentrations. They use the hypoxia-sensitive stabilization of the hypoxia-inducible factor-1α (HIF-1α) transcription factor to engage specific transcriptional programs in response to oxygen changes. The regulation of HIF-1α expression is well documented at the protein level, but much less is known about the control of its mRNA stability. Using small interfering RNA knockdown experiments, reporter gene analyses, ribonucleoprotein immunoprecipitations, and mRNA half-life determinations, we report a new regulatory mechanism of HIF-1α expression in ECs. We demonstrate that 1) sustained hypoxia progressively decreases HIF-1α mRNA while HIF-1α protein levels rapidly peak after 3 h and then slowly decay; 2) silencing the mRNA-destabilizing protein tristetraprolin (TTP) in ECs reverses hypoxia-induced down-regulation of HIF-1α mRNA; 3) the decrease in the half-life of Luciferase-HIF-1α-3'UTR reporter transcript that is observed after prolonged hypoxia is mediated by TTP; 4) TTP binds specifically to HIF-1α 3'UTR; and 5) the most distal AU-rich elements present in HIF-1α 3'UTR (composed of two hexamers) are sufficient for TTP-mediated repression. Finally, we bring evidence that silencing TTP expression enhances hypoxia-induced increase in HIF-1α protein levels with a concomitant increase in the levels of the carbonic anhydrase enzyme CA IX, thus suggesting that TTP physiologically controls the expression of a panel of HIF-1α target genes. Altogether, these data reveal a new role for TTP in the control of gene expression during the response of endothelial cell to hypoxia.

MicroRNAs (MiRNAs) are short, non-coding RNA that regulate a variety of cellular functions by suppressing target protein expression. We hypothesized that a set of microRNA regulate tumor responses to hypoxia by inhibiting components of the hypoxia signaling pathway. We found that miR-22 expression in human colon cancer is lower than in normal colon tissue. We also found that miR-22 controls hypoxia inducible factor 1α (HIF-1α) expression in the HCT116 colon cancer cell line. Over-expression of miR-22 inhibits HIF-1α expression, repressing vascular endothelial growth factor (VEGF) production during hypoxia. Conversely, knockdown of endogenous miR-22 enhances hypoxia induced expression of HIF-1α and VEGF. The conditioned media from cells over-expressing miR-22 contain less VEGF protein than control cells, and also induce less endothelial cell growth and invasion, suggesting miR-22 in adjacent cells influences endothelial cell function. Taken together, our data suggest that miR-22 might have an anti-angiogenic effect in colon cancer.

Hypoxia-inducible factor-1α (HIF-1α) is a widely studied protein with significant biomedical impact. Care is needed to stabilize HIF-1α protein during sample preparation for Western blot analysis due to its rapid degradation in the presence of oxygen. Enzyme inhibitor cocktails can be complex and expensive. We present a protease inhibitor-free buffer, containing cobalt chloride, which is effective at stabilizing HIF-1α, while being inexpensive, straightforward, and convenient, and has potential for widespread application.

Kirsten Ras (K-Ras) mutations have been implicated as a key predictive marker of resistance to therapies targeting the epidermal growth factor receptor (EGFR). To determine whether Harvey Ras (H-Ras) mutations also can confer resistance to EGFR-targeted therapy, we expressed a constitutively active H-Ras (Ras G12V) in A431 human vulvar squamous carcinoma cells. Compared with corresponding control cells, A431-Ras cells exhibited marked resistance to the EGFR inhibitors cetuximab and gefitinib, reducing inhibition of Akt and Erk phosphorylation, inhibition of HIF-1α expression and transcriptional activity, and antitumor effects in vitro and in vivo. Our data indicate that constitutively active H-Ras can also confer resistance to anti-EGFR therapy in cancer cells.

Most cases of breast cancer (BrCa) mortality are due to vascular metastasis. BrCa cells must intravasate through endothelial cells (ECs) to enter a blood vessel in the primary tumor and then adhere to ECs and extravasate at the metastatic site. In this study we demonstrate that inhibition of hypoxia-inducible factor (HIF) activity in BrCa cells by RNA interference or digoxin treatment inhibits primary tumor growth and also inhibits the metastasis of BrCa cells to the lungs by blocking the expression of angiopoietin-like 4 (ANGPTL4) and L1 cell adhesion molecule (L1CAM). ANGPTL4 is a secreted factor that inhibits EC-EC interaction, whereas L1CAM increases the adherence of BrCa cells to ECs. Interference with HIF, ANGPTL4 or L1CAM expression inhibits vascular metastasis of BrCa cells to the lungs.

The maintenance of oxygen homeostasis is critical for survival, and the master regulator of this process in metazoan species is hypoxia-inducible factor 1 (HIF-1), which controls both O(2) delivery and utilization. Under conditions of reduced O(2) availability, HIF-1 activates the transcription of genes, whose protein products mediate a switch from oxidative to glycolytic metabolism. HIF-1 is activated in cancer cells as a result of intratumoral hypoxia and/or genetic alterations. In cancer cells, metabolism is reprogrammed to favor glycolysis even under aerobic conditions. Pyruvate kinase M2 (PKM2) has been implicated in cancer growth and metabolism, although the mechanism by which it exerts these effects is unclear. Recent studies indicate that PKM2 interacts with HIF-1α physically and functionally to stimulate the binding of HIF-1 at target genes, the recruitment of coactivators, histone acetylation, and gene transcription. Interaction with HIF-1α is facilitated by hydroxylation of PKM2 at proline-403 and -408 by PHD3. Knockdown of PHD3 decreases glucose transporter 1, lactate dehydrogenase A, and pyruvate dehydrogenase kinase 1 expression; decreases glucose uptake and lactate production; and increases O(2) consumption. The effect of PKM2/PHD3 is not limited to genes encoding metabolic enzymes because VEGF is similarly regulated. These results provide a mechanism by which PKM2 promotes metabolic reprogramming and suggest that it plays a broader role in cancer progression than has previously been appreciated.

Adaptation of cancer cells to their microenvironment is an important driving force in the clonal selection that leads to invasive and metastatic disease. O2 concentrations are markedly reduced in many human cancers compared with normal tissue, and a major mechanism mediating adaptive responses to reduced O2 availability (hypoxia) is the regulation of transcription by hypoxia-inducible factor 1 (HIF-1). This review summarizes the current state of knowledge regarding the molecular mechanisms by which HIF-1 contributes to cancer progression, focusing on (1) clinical data associating increased HIF-1 levels with patient mortality;(2) preclinical data linking HIF-1 activity with tumor growth;(3) molecular data linking specific HIF-1 target gene products to critical aspects of cancer biology and (4) pharmacological data showing anticancer effects of HIF-1 inhibitors in mouse models of human cancer.

SAG (sensitive to apoptosis gene) or ROC2/RBX2 is the second family member of ROC1/RBX1, a component of SCF (Skp1, Cullin, F-box protein) and VCB (von Hippel-Lindau (VHL), Cullin and Elongin B/C) E3 ubiquitin ligases. SAG protected cells from hypoxia-induced apoptosis when overexpressed. We report here that SAG was subjected to hypoxia induction at the levels of mRNA and protein. Hypoxia induction of SAG was largely HIF-1alpha dependent. A consensus HIF-1-binding site, GCGTG was identified in the first intron of the SAG gene. In response to hypoxia, HIF-1 bound to this site and transactivated SAG expression. SAG transactivation required both the intact binding site in cis and HIF-1alpha in trans. On the other hand, like its family member, ROC1, SAG promoted VHL-mediated HIF-1alpha ubiquitination and degradation, which was significantly inhibited upon small interfering RNA silencing of SAG or ROC1. Furthermore, the endogenous HIF-1alpha at both basal and hypoxia-induced levels was significantly increased upon SAG silencing. Finally, SAG forms in vivo complex with Cul-5 and VHL under hypoxia condition. These results suggest an HIF-1-SAG feedback loop in response to hypoxia, as follows: hypoxia induces HIF-1 to transactivate SAG. Induced SAG then promotes HIF-1alpha ubiquitination and degradation. This feedback loop may serve as a cellular defensive mechanism to reduce potential cytotoxic effects of prolonged HIF-1 activation under hypoxia.

This review is divided into three parts:(a) The primary site of oxygen sensing is the carotid body which instantaneously respond to hypoxia without involving new protein synthesis, and is historically known as the first oxygen sensor and is therefore placed in the first section (Lahiri, Roy, Baby and Hoshi). The carotid body senses oxygen in acute hypoxia, and produces appropriate responses such as increases in breathing, replenishing oxygen from air. How this oxygen is sensed at a relatively high level (arterial PO2 approximately 50 Torr) which would not be perceptible by other cells in the body, is a mystery. This response is seen in afferent nerves which are connected synaptically to type I or glomus cells of the carotid body. The major effect of oxygen sensing is the increase in cytosolic calcium, ultimately by influx from extracellular calcium whose concentration is 2 x 10(4) times greater. There are several contesting hypotheses for this response: one, the mitochondrial hypothesis which states that the electron transport from the substrate to oxygen through the respiratory chain is retarded as the oxygen pressure falls, and the mitochondrial membrane is depolarized leading to the calcium release from the complex of mitochondria-endoplasmic reticulum. This is followed by influx of calcium. Also, the inhibitors of the respiratory chain result in mitochondrial depolarization and calcium release. The other hypothesis (membrane model) states that K(+) channels are suppressed by hypoxia which depolarizes the membrane leading to calcium influx and cytosolic calcium increase. Evidence supports both the hypotheses. Hypoxia also inhibits prolyl hydroxylases which are present in all the cells. This inhibition results in membrane K(+) current suppression which is followed by cell depolarization. The theme of this section covers first what and where the oxygen sensors are; second, what are the effectors; third, what couples oxygen sensors and the effectors.(b) All oxygen consuming cells have a built-in mechanism, the transcription factor HIF-1, the discovery of which has led to the delineation of oxygen-regulated gene expression. This response to chronic hypoxia needs new protein synthesis, and the proteins of these genes mediate the adaptive physiological responses. HIF-1alpha, which is a part of HIF-1, has come to be known as master regulator for oxygen homeostasis, and is precisely regulated by the cellular oxygen concentration. Thus, the HIF-1 encompasses the chronic responses (gene expression in all cells of the body). The molecular biology of oxygen sensing is reviewed in this section (Semenza).(c) Once oxygen is sensed and Ca(2+) is released, the neurotransmittesr will be elaborated from the glomus cells of the carotid body. Currently it is believed that hypoxia facilitates release of one or more excitatory transmitters from glomus cells, which by depolarizing the nearby afferent terminals, leads to increases in the sensory discharge. The transmitters expressed in the carotid body can be classified into two major categories: conventional and unconventional. The conventional neurotransmitters include those stored in synaptic vesicles and mediate their action via activation of specific membrane bound receptors often coupled to G-proteins. Unconventional neurotransmitters are those that are not stored in synaptic vesicles, but spontaneously generated by enzymatic reactions and exert their biological responses either by interacting with cytosolic enzymes or by direct modifications of proteins. The gas molecules such as NO and CO belong to this latter category of neurotransmitters and have unique functions. Co-localization and co-release of neurotransmitters have also been described. Often interactions between excitatory and inhibitory messenger molecules also occur. Carotid body contains all kinds of transmitters, and an interplay between them must occur. But very little has come to be known as yet. Glimpses of these interactions are evident in the discussion in the last section (Prabhakar).

The hypoxia-inducible factor 1 (HIF-1) plays a critical role in cellular responses to hypoxia. The aim of the present study was to evaluate which genes are induced by hypoxia, and whether this induction is mediated by HIF-1, by expression microarray analysis of wt and HIF-1alpha null mouse fibroblasts. Forty-five genes were up-regulated by hypoxia and 40 (89%) of these were regulated by HIF-1. Of the 114 genes down-regulated by hypoxia, 19 (17%) were HIF-1-dependent. All glycolytic enzymes were strongly up-regulated by hypoxia in a HIF-1-dependent manner. Genes already known to be related to hypoxia, such as glucose transporter 1, BNIP3, and hypoxia-induced gene 1, were induced. In addition, multiple new HIF-1-regulated genes were identified, including genes involved in metabolism (adenylate kinase 4, galactokinase), apoptosis (galectin-3 and gelsolin), and invasion (RhoA). Genes down-regulated by hypoxia were involved in cytoskeleton maintenance (Rho kinase), mRNA processing (heterogeneous nuclear ribonucleoprotein H1 and splicing factor), and DNA repair (REV3). Furthermore, seven cDNAs from genes with unknown function or expressed sequence tags (ESTs) were up-regulated and 27 such cDNAs were down-regulated. In conclusion, hypoxia causes down- rather than up-regulation of gene expression and HIF-1 seems to play a major role in the regulation of hypoxia-induced genes.

Volatile anesthetics modulate a variety of physiological and pathophysiological responses including hypoxic responses. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates cellular and systemic homeostatic responses to reduced O(2) availability in mammals, including erythropoiesis, angiogenesis, and glycolysis. We demonstrate for the first time that the volatile anesthetic halothane blocks HIF-1 activity and downstream target gene expressions induced by hypoxia in the human hepatoma-derived cell line, Hep3B. Halothane reversibly blocks hypoxia-induced HIF-1alpha protein accumulation and transcriptional activity at clinically relevant doses.

Otto Warburg's classic treatise on the reprogramming of tumour metabolism from oxidative to glycolytic metabolism was published in London in 1930. Although the Warburg effect is one of the most universal characteristics of solid tumours, the molecular basis for this phenomenon has only recently been elucidated by studies indicating that increased expression of genes encoding glucose transporters and glycolytic enzymes in tumour cells is mediated by the transcription factors c-MYC and HIF-1. Whereas c-myc is a direct target for oncogenic mutations, expression of hypoxia-inducible factor 1 (HIF-1) is indirectly up-regulated via gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes that result increased HIF-1alpha protein expression and/or increased HIF-1 transcriptional activity in a cell-type-specific manner. As a result of genetic alterations and intratumoral hypoxia, HIF-1alpha is overexpressed in the majority of common human cancers relative to the surrounding normal tissue. In human breast cancer and brain tumours, HIF-1alpha overexpression is strongly correlated with tumour grade and vascularity.