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1.  The biological kinship of hypoxia with CSC and EMT and their relationship with deregulated expression of miRNAs and tumor aggressiveness 
Biochimica et biophysica acta  2012;1826(2):272-296.
Hypoxia is one of the fundamental biological phenomena that are intricately associated with the development and aggressiveness of a variety of solid tumors. Hypoxia-inducible factors (HIF) function as a master transcription factor, which regulates hypoxia responsive genes and has been recognized to play critical roles in tumor invasion, metastasis, and chemo-radiation resistance, and contributes to increased cell proliferation, survival, angiogenesis and metastasis. Therefore, tumor hypoxia with deregulated expression of HIF and its biological consequence lead to poor prognosis of patients diagnosed with solid tumors, resulting in higher mortality, suggesting that understanding of the molecular relationship of hypoxia with other cellular features of tumor aggressiveness would be invaluable for developing newer targeted therapy for solid tumors. It has been well recognized that cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) phenotypic cells are associated with therapeutic resistance and contribute to aggressive tumor growth, invasion, metastasis and believed to be the cause of tumor recurrence. Interestingly, hypoxia and HIF signaling pathway are known to play an important role in the regulation and sustenance of CSCs and EMT phenotype. However, the molecular relationship between HIF signaling pathway with the biology of CSCs and EMT remains unclear although NF-κB, PI3K/Akt/mTOR, Notch, Wnt/β-catenin, and Hedgehog signaling pathways have been recognized as important regulators of CSCs and EMT. In this article, we will discuss the state of our knowledge on the role of HIF-hypoxia signaling pathway and its kinship with CSCs and EMT within the tumor microenvironment. We will also discuss the potential role of hypoxia-induced microRNAs (miRNAs) in tumor development and aggressiveness, and finally discuss the potential effects of nutraceuticals on the biology of CSCs and EMT in the context of tumor hypoxia.
doi:10.1016/j.bbcan.2012.04.008
PMCID: PMC3788359  PMID: 22579961
Hypoxia; HIF; CSC; EMT; miRNAs
2.  Differential expression of HIF-1α in CD44+CD24-/low breast ductal carcinomas 
Diagnostic Pathology  2011;6:73.
Background
Cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of tumor. Stem cells renewal and differentiation can be directly influenced by the oxygen levels of determined tissues, probably by the reduction of oxidative DNA damage in hypoxic regions, thus leading to a friendlier microenvironment, regarding to clonal expansion and for resistance to chemotherapeutic regimens. Furthermore, there have been strong data indicating a pivotal role of hypoxic niche in cancer stem cells development. There are evidence that hypoxia could drive the maintenance of CSC, via HIF-1α expression, but it still to be determined whether hypoxia markers are expressed in breast tumors presenting CD44+CD24-/low immunophenotype.
Methods
Immunohistochemical analysis of CD44+CD24-/low expression and its relationship with hypoxia markers and clinical outcome were evaluated in 253 samples of breast ductal carcinomas. Double-immunolabeling was performed using EnVision Doublestain System (Dako, Carpinteria, CA, USA). Slides were then scanned into high-resolution images using Aperio ScanScope XT and then, visualized in the software Image Scope (Aperio, Vista, CA, USA).
Results
In univariate analysis, CD44+CD24-/low expression showed association with death due to breast cancer (p = 0.035). Breast tumors expressing CD44+CD24-/low immunophenotype showed relationship with HIF-1α (p = 0.039) and negativity for HER-2 (p = 0.013).
Conclusion
Considering that there are strong evidences that the fraction of a tumour considered to be cancer stem cells is plastic depending upon microenvironmental signals, our findings provide further evidence that hypoxia might be related to the worse prognosis found in CD44+CD24-/low positive breast tumors.
doi:10.1186/1746-1596-6-73
PMCID: PMC3170242  PMID: 21824412
breast cancer; CD44; CD24; HIF-1α; hypoxia; immunohistochemistry; prognosis; stem cell
3.  Hypoxia-Induced Aggressiveness of Pancreatic Cancer Cells Is Due to Increased Expression of VEGF, IL-6 and miR-21, Which Can Be Attenuated by CDF Treatment 
PLoS ONE  2012;7(12):e50165.
Hypoxia is known to play critical roles in cell survival, angiogenesis, tumor invasion, and metastasis. Hypoxia mediated over-expression of hypoxia-inducible factor (HIF) has been shown to be associated with therapeutic resistance, and contributes to poor prognosis of cancer patients. Emerging evidence suggest that hypoxia and HIF pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cell (CSC) functions, and also maintains the vicious cycle of inflammation-all which lead to therapeutic resistance. However, the precise molecular mechanism(s) by which hypoxia/HIF drives these events are not fully understood. Here, we show, for the first time, that hypoxia leads to increased expression of VEGF, IL-6, and CSC signature genes Nanog, Oct4 and EZH2 consistent with increased cell migration/invasion and angiogenesis, and the formation of pancreatospheres, concomitant with increased expression of miR-21 and miR-210 in human pancreatic cancer (PC) cells. The treatment of PC cells with CDF, a novel synthetic compound inhibited the production of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 and miR-210 under hypoxia. CDF also led to decreased cell migration/invasion, angiogenesis, and formation of pancreatospheres under hypoxia. Moreover, CDF decreased gene expression of miR-21, miR-210, IL-6, HIF-1α, VEGF, and CSC signatures in vivo in a mouse orthotopic model of human PC. Collectively, these results suggest that the anti-tumor activity of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become a novel, and effective anti-tumor agent for PC therapy.
doi:10.1371/journal.pone.0050165
PMCID: PMC3521759  PMID: 23272057
4.  Hypoxia Induced MLL1 Regulates Glioma Stem Cell Tumorigenic Potential 
Cell Death and Differentiation  2011;19(3):428-439.
Normal stem cells reside in functional niches critical for self-renewal and maintenance. Neural and hematopoietic stem cell niches, in particular, are characterized by restricted availability of oxygen and the resulting regulation by hypoxia inducible factors (HIFs). Glioblastoma multiforme (GBM) is the most common malignant brain tumor and also contains high degrees of hypoxia. Heterogeneity within the neoplastic compartment has been well characterized in GBM and may be derived from genetic and epigenetic sources that co-evolve during malignant progression. Recent experimental evidence has supported the importance of hypoxia in cancer stem cell (CSC) niches. We hypothesized that HIFs require epigenetic modifying proteins in order to promote tumor malignancy in GBM. Here we demonstrate that in GBM the histone methyltransferase Mixed-Lineage Leukemia 1 (MLL1) is induced by hypoxia and enhances hypoxic responses. Loss of MLL1 reduces expression of HIF transcripts and HIF2α protein. Targeting MLL1 by RNA interference inhibited expression of HIF2α and target genes, including VEGF. CSCs expressed higher levels of MLL1 than matched nonstem tumor cells and depletion of MLL1 reduced CSC self-renewal, growth, and tumorigenicity. These studies have uncovered a novel mechanism mediating tumor hypoxic responses linking microenvironmental regulation of epigenetic modifying proteins to cellular heterogeneity and provide rationale for the design of more sophisticated clinical approaches targeting epigenetic regulation.
doi:10.1038/cdd.2011.109
PMCID: PMC3229666  PMID: 21836617
hypoxia; MLL1; cancer stem cell; HIF2α; epigenetics
5.  Basal/HER2 breast carcinomas 
Cell Cycle  2013;12(2):225-245.
High rates of inherent primary resistance to the humanized monoclonal antibody trastuzumab (Herceptin) are frequent among HER2 gene-amplified breast carcinomas in both metastatic and adjuvant settings. The clinical efficacy of trastuzumab is highly correlated with its ability to specifically and efficiently target HER2-driven populations of breast cancer stem cells (CSCs). Intriguingly, many of the possible mechanisms by which cancer cells escape trastuzumab involve many of the same biomarkers that have been implicated in the biology of CS-like tumor-initiating cells. In the traditional, one-way hierarchy of CSCs in which all cancer cells descend from special self-renewing CSCs, HER2-positive CSCs can occur solely by self-renewal. Therefore, by targeting CSC self-renewal and resistance, trastuzumab is expected to induce tumor shrinkage and further reduce breast cancer recurrence rates when used alongside traditional therapies. In a new, alternate model, more differentiated non-stem cancer cells can revert to trastuzumab-refractory, CS-like cells via the activation of intrinsic or microenvironmental paths-to-stemness, such as the epithelial-to-mesenchymal transition (EMT). Alternatively, stochastic transitions of trastuzumab-responsive CSCs might also give rise to non-CSC cellular states that lack major attributes of CSCs and, therefore, can remain “hidden” from trastuzumab activity. Here, we hypothesize that a better understanding of the CSC/non-CSC social structure within HER2-overexpressing breast carcinomas is critical for trastuzumab-based treatment decisions in the clinic. First, we decipher the biological significance of CSC features and the EMT on the molecular effects and efficacy of trastuzumab in HER2-positive breast cancer cells. Second, we reinterpret the genetic heterogeneity that differentiates trastuzumab-responders from non-responders in terms of CSC cellular states. Finally, we propose that novel predictive approaches aimed at better forecasting early tumor responses to trastuzumab should identify biological determinants that causally underlie the intrinsic flexibility of HER2-positive CSCs to “enter” into or “exit” from trastuzumab-sensitive states. An accurate integration of CSC cellular states and EMT-related biomarkers with the currently available breast cancer molecular taxonomy may significantly improve our ability to make a priori decisions about whether patients belonging to HER2 subtypes differentially enriched with a “mesenchymal transition signature” (e.g., luminal/HER2 vs. basal/HER2) would distinctly benefit from trastuzumab-based therapy ab initio.
doi:10.4161/cc.23274
PMCID: PMC3575452  PMID: 23255137
basal-like; cancer stem cells; EMT; HER2; trastuzumab; breast cancer; reprogramming
6.  The Hypoxic Microenvironment Maintains Glioblastoma Stem Cells and Promotes Reprogramming towards a Cancer Stem Cell Phenotype 
Cell cycle (Georgetown, Tex.)  2009;8(20):3274-3284.
Glioblastomas are highly lethal cancers that contain cellular hierarchies with self-renewing cancer stem cells that can propagate tumors in secondary transplant assays. The potential significance of cancer stem cells in cancer biology has been demonstrated by studies showing contributions to therapeutic resistance, angiogenesis, and tumor dispersal. We recently reported that physiologic oxygen levels differentially induce hypoxia inducible factor-2α (HIF2α) levels in cancer stem cells. HIF1α functioned in proliferation and survival of all cancer cells but also was activated in normal neural progenitors suggesting a potentially restricted therapeutic index while HIF2α was essential in only in cancer stem cells and was not expressed by normal neural progenitors demonstrating HIF2α is a cancer stem cell specific target. We now extend these studies to examine the role of hypoxia in regulating tumor cell plasticity. We find that hypoxia promotes the self-renewal capability of the stem and non-stem population as well as promoting a more stem-like phenotype in the non-stem population with increased neurosphere formation as well as upregulation of important stem cell factors, such as OCT4, NANOG, and c-MYC. The importance of HIF2α was further supported as forced expression of non-degradable HIF2α induced a cancer stem cell marker and augmented the tumorigenic potential of the non-stem population. This novel finding may indicate a specific role of HIF2α in promoting glioma tumorigenesis. The unexpected plasticity of the non-stem glioma population and the stem-like phenotype emphasizes the importance of developing therapeutic strategies targeting the microenvironmental influence on the tumor in addition to cancer stem cells.
PMCID: PMC2825672  PMID: 19770585
Cancer Stem Cell; Hypoxia; Hypoxia Inducible Factor; HIF2α; Glioblastoma
7.  Mutation of von Hippel–Lindau Tumour Suppressor and Human Cardiopulmonary Physiology 
PLoS Medicine  2006;3(7):e290.
Background
The von Hippel–Lindau tumour suppressor protein–hypoxia-inducible factor (VHL–HIF) pathway has attracted widespread medical interest as a transcriptional system controlling cellular responses to hypoxia, yet insights into its role in systemic human physiology remain limited. Chuvash polycythaemia has recently been defined as a new form of VHL-associated disease, distinct from the classical VHL-associated inherited cancer syndrome, in which germline homozygosity for a hypomorphic VHL allele causes a generalised abnormality in VHL–HIF signalling. Affected individuals thus provide a unique opportunity to explore the integrative physiology of this signalling pathway. This study investigated patients with Chuvash polycythaemia in order to analyse the role of the VHL–HIF pathway in systemic human cardiopulmonary physiology.
Methods and Findings
Twelve participants, three with Chuvash polycythaemia and nine controls, were studied at baseline and during hypoxia. Participants breathed through a mouthpiece, and pulmonary ventilation was measured while pulmonary vascular tone was assessed echocardiographically. Individuals with Chuvash polycythaemia were found to have striking abnormalities in respiratory and pulmonary vascular regulation. Basal ventilation and pulmonary vascular tone were elevated, and ventilatory, pulmonary vasoconstrictive, and heart rate responses to acute hypoxia were greatly increased.
Conclusions
The features observed in this small group of patients with Chuvash polycythaemia are highly characteristic of those associated with acclimatisation to the hypoxia of high altitude. More generally, the phenotype associated with Chuvash polycythaemia demonstrates that VHL plays a major role in the underlying calibration and homeostasis of the respiratory and cardiovascular systems, most likely through its central role in the regulation of HIF.
Editors' Summary
Background.
Human cells (like those of other multicellular animals) use oxygen to provide the energy needed for daily life. Having not enough oxygen is a problem, but having too much is also dangerous because it damages proteins, DNA, and other large molecules that keep cells functioning. Consequently, the physiological systems—including the heart, lungs, and circulation—work together to balance oxygen supply and demand throughout the body. When oxygen is limiting (a condition called hypoxia), as happens at high altitudes, the cellular oxygen supply is maintained by increasing the heart rate, increasing the speed and depth of breathing (hyperventilation), constricting the blood vessels in the lung (pulmonary vasoconstriction), and increasing the number of oxygen-carrying cells in the blood. All these physiological changes increase the amount of oxygen that can be absorbed from the air, but how they are regulated is poorly understood. By contrast, researchers know quite a bit about how individual cells respond to hypoxia. When oxygen is limited, a protein called hypoxia-inducible factor (or HIF) activates a number of target proteins that help the cell get enough oxygen (for example, proteins that stimulate the growth of new blood vessels). When there is plenty of oxygen, another protein, called von Hippel–Lindau tumor suppressor (abbreviated VHL), rapidly destroys HIF. Recently, researchers discovered that a genetic condition called Chuvash polycythaemia, characterised by the overproduction of red blood cells, is caused by a specific defect in VHL that reduces its ability to destroy HIF. As a result, the expression of certain HIF target proteins is increased even when oxygen levels are normal.
Why Was This Study Done?
Chuvash polycythaemia is very rare, and so far little is known about how this genetic abnormality affects the physiology and long-term health of patients. By studying heart and lung function in patients with Chuvash polycythaemia, the researchers involved in this study hoped to discover more about the health consequences of the condition and to find out whether the VHL–HIF system controls systemic responses to hypoxia as well as cellular responses.
What Did the Researchers Do and Find?
The researchers recruited and studied three patients with Chuvash polycythaemia, and, as controls for the comparison, several normal individuals and patients with an unrelated form of polycythaemia. They then measured how the lungs and hearts of these people reacted to mild hypoxia (similar to that experienced on commercial air flights) and moderate hypoxia (equiv alent to being on the top of an Alpine peak). They found that patients with Chuvash polycythaemia naturally breathe slightly quicker and deeper than normal individuals, and that their breathing rate increased dramatically and abnormally when oxygen was reduced. They also found that at normal oxygen levels the pulmonary blood vessels of these patients were more constricted than those of control individuals, and that they reacted more extremely to hypoxia. Similarly, the normal heart rate of the patients was slightly higher than that of the controls and increased much more in response to mild hypoxia.
What Do These Findings Mean?
The physiological differences measured by the researchers between Chuvash polycythaemia patients and control individuals are similar to the adaptations seen in people traveling to high altitudes where oxygen is limited. Thus, the VHL–HIF proteins may regulate the response to different oxygen concentrations both in individual cells and at the systemic level, although more physiological studies are needed to confirm this. Because the pulmonary blood vessels of patients with Chuvash polycythaemia are always abnormally constricted, and even more so when oxygen is limited, these people should avoid living at high altitude and should minimise air travel, suggest the researchers. The increased blood pressure in their lungs (pulmonary hypertension) could conceivably cause heart failure under such circumstances. Finally, this study has implications for the development of drugs directed at the VHL–HIF system. Agents are currently being designed to promote the development of new blood vessels after strokes or heart attacks by preventing the destruction of HIF, but based on the findings here such agents might have undesirable physiological affects. Conversely, HIF inhibitors (which act as anti-cancer reagents by increasing hypoxia in the centre of tumors and so inhibiting their growth) might be useful in the treatment of pulmonary hypertension.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030290.
• Online Mendelian Inheritance in Man page on Chuvash polycythaemia
• Information from the VHL Family Alliance on von Hippel–Lindau disease, including information on Chuvash polycythaemia
• Wikipedia page on polycythaemia and von Hippel–Lindau disease (note: Wikipedia is a free online encyclopaedia that anyone can edit)
Physiological study of patients with Chuvash polycythemia (caused by mutation of VHL) reveals characteristics similar to those associated with acclimatization to the hypoxia of high altitude.
doi:10.1371/journal.pmed.0030290
PMCID: PMC1479389  PMID: 16768548
8.  The Hypoxia-Associated Factor Switches Cells from HIF-1α– to HIF-2α–Dependent Signaling Promoting Stem Cell Characteristics, Aggressive Tumor Growth and Invasion 
Cancer Research  2011;71(11):4015-4027.
Most solid tumors and their metastases experience periods of low oxygen or hypoxia, which is of major clinical significance as it promotes both tumor progression and resistance to therapy. Critical mediators of the hypoxic response are the hypoxia-inducible factors HIF-1α and HIF-2α. The HIFs are nonredundant and regulate both overlapping and unique downstream target genes. Here, we describe a novel mechanism for the switch between HIF-1α– and HIF-2α–dependent transcription during tumor hypoxia caused by the hypoxia associated factor (HAF). HAF is overexpressed in a variety of tumors and its levels are decreased during acute hypoxia, but increased following prolonged hypoxia. We have previously identified HAF as an E3 ubiquitin ligase that binds and ubiquitinates HIF-1α by an oxygen and pVHL-independent mechanism, thus targeting HIF-1α for proteasomal degradation. Here, we show that HAF also binds to HIF-2α, but at a different site than HIF-1α, and increases HIF-2α transactivation without causing its degradation. HAF, thus, switches the hypoxic response of the cancer cell from HIF-1α–dependent to HIF-2α–dependent transcription and activates genes involved in invasion such as MMP9, PAI-1, and the stem cell factor OCT-3/4. The switch to HIF-2α–dependent gene expression caused by HAF also promotes an enriched tumor stem cell population, resulting in highly aggressive tumors in vivo. Thus, HAF, by causing a switch from a HIF-1α– to HIF-2α–dependent response to hypoxia, provides a mechanism for more aggressive growth of tumors under prolonged hypoxia.
doi:10.1158/0008-5472.CAN-10-4142
PMCID: PMC3268651  PMID: 21512133
9.  Hypoxia–Induced Cytotoxic Drug Resistance in Osteosarcoma Is Independent of HIF-1Alpha 
PLoS ONE  2013;8(6):e65304.
Survival rates from childhood cancer have improved dramatically in the last 40 years, such that over 80% of children are now cured. However in certain subgroups, including metastatic osteosarcoma, survival has remained stubbornly poor, despite dose intensive multi-agent chemotherapy regimens, and new therapeutic approaches are needed. Hypoxia is common in adult solid tumours and is associated with treatment resistance and poorer outcome. Hypoxia induces chemotherapy resistance in paediatric tumours including neuroblastoma, rhabdomyosarcoma and Ewing’s sarcoma, in vitro, and this drug resistance is dependent on the oxygen-regulated transcription factor hypoxia inducible factor-1 (HIF-1). In this study the effects of hypoxia on the response of the osteosarcoma cell lines 791T, HOS and U2OS to the clinically relevant cytotoxics cisplatin, doxorubicin and etoposide were evaluated. Significant hypoxia-induced resistance to all three agents was seen in all three cell lines and hypoxia significantly reduced drug-induced apoptosis. Hypoxia also attenuated drug-induced activation of p53 in the p53 wild-type U2OS osteosarcoma cells. Drug resistance was not induced by HIF-1α stabilisation in normoxia by cobalt chloride nor reversed by the suppression of HIF-1α in hypoxia by shRNAi, siRNA, dominant negative HIF or inhibition with the small molecule NSC-134754, strongly suggesting that hypoxia-induced drug resistance in osteosarcoma cells is independent of HIF-1α. Inhibition of the phosphoinositide 3-kinase (PI3K) pathway using the inhibitor PI-103 did not reverse hypoxia-induced drug resistance, suggesting the hypoxic activation of Akt in osteosarcoma cells does not play a significant role in hypoxia-induced drug resistance. Targeting hypoxia is an exciting prospect to improve current anti-cancer therapy and combat drug resistance. Significant hypoxia-induced drug resistance in osteosarcoma cells highlights the potential importance of hypoxia as a target to reverse drug resistance in paediatric osteosarcoma. The novel finding of HIF-1α independent drug resistance suggests however other hypoxia related targets may be more relevant in paediatric osteosarcoma.
doi:10.1371/journal.pone.0065304
PMCID: PMC3681794  PMID: 23785417
10.  HSP90 inhibitor 17-AAG selectively eradicates lymphoma stem cells 
Cancer research  2012;72(17):4551-4561.
Cancer stem cells (CSCs, also called tumor initiating cells) comprise tumor cell subpopulations that preserve the properties of quiescence, self-renewal and differentiation of normal stem cells. CSCs are also therapeutically important because of their key contributions to drug resistance. The hypoxia inducible transcription factor HIF1α is critical for CSC maintenance in mouse lymphoma. Here we show that low concentrations of the HSP90 inhibitor 17-AAG eliminate lymphoma CSCs in vitro and in vivo by disrupting the transcriptional function of HIF1α, a client protein of HSP90. 17-AAG preferentially induced apoptosis and eliminated the colony formation capacity of mouse lymphoma CSCs and human acute myeloid leukemia (AML) CSCs. However, low concentrations of 17-AAG failed to eliminate highly proliferative lymphoma and AML cells (non-CSCs), in which the AKT-GSK3 signaling pathway is constitutively active. The heat shock transcription factor HSF1 is highly expressed in non-CSCs but it was weakly expressed in lymphoma CSCs. However, siRNA-mediated attenuation of HSF1 abrogated the colony formation ability of both lymphoma and AML CSCs. This study supports the use of 17-AAG as a CSC targeting agent, and it also shows that HSF1 is an important target for elimination of both CSCs and non-CSCs in cancer.
doi:10.1158/0008-5472.CAN-11-3600
PMCID: PMC3443561  PMID: 22751135
11.  Hypoxia Induced Aggressiveness of Prostate Cancer Cells Is Linked with Deregulated Expression of VEGF, IL-6 and miRNAs That Are Attenuated by CDF 
PLoS ONE  2012;7(8):e43726.
Tumor hypoxia with deregulated expression of hypoxia inducing factor (HIF) and its biological consequence leads to poor prognosis of patients diagnosed with solid tumors, resulting in higher mortality, suggesting that understanding of the molecular relationship of hypoxia with other cellular features of tumor aggressiveness would be invaluable for developing newer targeted therapy for solid tumors. Emerging evidence also suggest that hypoxia and HIF signaling pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cell (CSC) functions, and also maintains the vicious cycle of inflammation, all of which contribute to radiation therapy and chemotherapy resistance. However, the detailed mechanisms by which hypoxia/HIF drive these events are not fully understood. Here, we have shown that hypoxia leads to increased expression of VEGF, IL-6, and CSC marker genes such as Nanog, Oct4 and EZH2, and also increased the expression of miR-21, an oncogenic miRNA, in prostate cancer (PCa) cells (PC-3 and LNCaP). The treatment of PCa cells with CDF, a novel Curcumin-derived synthetic analogue previously showed anti-tumor activity in vivo, inhibited the productions of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 under hypoxic condition. Moreover, CDF treatment of PCa cells led to decreased cell migration under hypoxic condition. Taken together, these results suggest that the anti-tumor effect of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become useful for cancer therapy.
doi:10.1371/journal.pone.0043726
PMCID: PMC3428287  PMID: 22952749
12.  Imaging and Targeting of the Hypoxia-inducible Factor 1-active Microenvironment 
Journal of Toxicologic Pathology  2009;22(2):93-100.
Human solid tumors contain hypoxic regions that have considerably lower oxygen tension than normal tissues. They are refractory to radiotherapy and anticancer chemotherapy. Although more than half a century has passed since it was suggested that tumour hypoxia correlates with poor treatment outcomes and contributes to recurrence of cancer, no fundamental solution to this problem has been found. Hypoxia-inducible factor-1(HIF-1) is the main transcription factor that regulates the cellular response to hypoxia. It induces various genes, whose function is strongly associated with malignant alteration of the entire tumour. The cellular changes induced by HIF-1 are extremely important therapeutic targets of cancer therapy, particularly in therapy against refractory cancers. Therefore, targeting strategies to overcome the HIF-1-active microenvironment are important for cancer therapy. To Target HIF-1-active/ hypoxic tumor cells, we developed a fusion protein drug, PTD-ODD-Procaspase-3 that selectively induces cell death in HIF-1-active/hypoxic cells. The drug consists of the following three functional domains: the protein transduction domain (PTD), which efficiently delivers the fusion protein to hypoxic tumor cells, the ODD domain, which has a VHL-mediated protein destruction motif of human HIF-1α protein and confers hypoxia-dependent stabilization to the fusion proteins, and the human procaspase-3 proenzyme responsible for the cytocidal activity of the protein drug. In vivo imaging systems capable of monitoring HIF-1 activity in transplanted human cancer cells in mice are useful in evaluating the efficiency of these drugs and in study of HIF-1-active tumor cells.
doi:10.1293/tox.22.93
PMCID: PMC3246054  PMID: 22271982
hypoxia-inducible factor 1 (HIF-1); tumour hypoxia; hypoxia responsive element (HRE); protein transduction domain (PTD); bioluminescence; in vivo imaging
13.  Identification of a stem-like cell population by exposing metastatic breast cancer cell lines to repetitive cycles of hypoxia and reoxygenation 
Introduction
The irregular vasculature of solid tumors creates hypoxic regions, which are characterized by cyclic periods of hypoxia and reoxygenation. Accumulated evidence suggests that chronic and repetitive exposure to hypoxia and reoxygenation seem to provide an advantage to tumor growth. Although the development of hypoxia tolerance in tumors predicts poor prognosis, mechanisms contributing to hypoxia tolerance remain to be elucidated. Recent studies have described a subpopulation of cancer stem cells (CSC) within tumors, which have stem-like properties such as self-renewal and the ability to differentiate into multiple cell types. The cancer stem cell theory suggests CSCs persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Since hypoxia is considered to be one of the critical niche factors to promote invasive growth of tumors, we hypothesize that repetitive cycles of hypoxia/reoxygenation also play a role in the enrichment of breast CSCs.
Methods
Two metastatic human breast cancer cell lines (MDA-MB 231 and BCM2) were used to optimize the conditions of hypoxia and reoxygenation cycles. The percentage of CSCs in the cycling hypoxia selected subpopulation was analyzed based on the CD44, CD24, ESA, and E-cadherin expression by three-color flow cytometry. Colony formation assays were used to assess the ability of this subpopulation to self-renew. Limiting dilution assays were performed to evaluate the tumor-initiating and metastatic ability of this subpopulation. Induction of EMT was examined by the expression of EMT-associated markers and EMT-associated microRNAs.
Results
Using an optimized hypoxia and reoxygenation regimen, we identified a novel cycling hypoxia-selected subpopulation from human breast cancer cell lines and demonstrated that a stem-like breast cancer cell subpopulation could be expanded through repetitive hypoxia/reoxygenation cycles without genetic manipulation. We also found that cells derived from this novel subpopulation form colonies readily, are highly tumorigenic in immune-deficient mice, and exhibit both stem-like and EMT phenotypes.
Conclusions
These results provide the validity to the newly developed hypoxia/reoxygenation culture system for examining the regulation of CSCs in breast cancer cell lines by niche factors in the tumor microenvironment and developing differential targeting strategies to eradicate breast CSCs.
doi:10.1186/bcr2773
PMCID: PMC3046435  PMID: 21067584
14.  Suppression of apoptosis inhibitor c-FLIP selectively eliminates breast cancer stem cell activity in response to the anti-cancer agent, TRAIL 
Introduction
It is postulated that breast cancer stem cells (bCSCs) mediate disease recurrence and drive formation of distant metastases - the principal cause of mortality in breast cancer patients. Therapeutic targeting of bCSCs, however, is hampered by their heterogeneity and resistance to existing therapeutics. In order to identify strategies to selectively remove bCSCs from breast cancers, irrespective of their clinical subtype, we sought an apoptosis mechanism that would target bCSCs yet would not kill normal cells. Suppression of the apoptosis inhibitor cellular FLICE-Like Inhibitory Protein (c-FLIP) partially sensitizes breast cancer cells to the anti-cancer agent Tumour Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL). Here we demonstrate in breast cancer cell lines that bCSCs are exquisitely sensitive to the de-repression of this pro-apoptotic pathway, resulting in a dramatic reduction in experimental metastases and the loss of bCSC self-renewal.
Methods
Suppression c-FLIP was performed by siRNA (FLIPi) in four breast cancer cell lines and by conditional gene-knockout in murine mammary glands. Sensitivity of these cells to TRAIL was determined by complementary cell apoptosis assays, including a novel heterotypic cell assay, while tumour-initiating potential of cancer stem cell subpopulations was determined by mammosphere cultures, aldefluor assay and in vivo transplantation.
Results
Genetic suppression of c-FLIP resulted in the partial sensitization of TRAIL-resistant cancer lines to the pro-apoptotic effects of TRAIL, irrespective of their cellular phenotype, yet normal mammary epithelial cells remained refractory to killing. While 10% to 30% of the cancer cell populations remained viable after TRAIL/FLIPi treatment, subsequent mammosphere and aldefluor assays demonstrated that this pro-apoptotic stimulus selectively targeted the functional bCSC pool, eliminating stem cell renewal. This culminated in an 80% reduction in primary tumours and a 98% reduction in metastases following transplantation. The recurrence of residual tumour initiating capacity was consistent with the observation that post-treated adherent cultures re-acquired bCSC-like properties in vitro. Importantly however this recurrent bCSC activity was attenuated following repeated TRAIL/FLIPi treatment.
Conclusions
We describe an apoptotic mechanism that selectively and repeatedly removes bCSC activity from breast cancer cell lines and suggest that a combined TRAIL/FLIPi therapy could prevent metastatic disease progression in a broad range of breast cancer subtypes.
doi:10.1186/bcr2945
PMCID: PMC3262200  PMID: 21914219
15.  Hypoxia Potentiates Glioma-Mediated Immunosuppression 
PLoS ONE  2011;6(1):e16195.
Glioblastoma multiforme (GBM) is a lethal cancer that exerts potent immune suppression. Hypoxia is a predominant feature of GBM, but it is unclear to the degree in which tumor hypoxia contributes to this tumor-mediated immunosuppression. Utilizing GBM associated cancer stem cells (gCSCs) as a treatment resistant population that has been shown to inhibit both innate and adaptive immune responses, we compared immunosuppressive properties under both normoxic and hypoxic conditions. Functional immunosuppression was characterized based on production of immunosuppressive cytokines and chemokines, the inhibition of T cell proliferation and effector responses, induction of FoxP3+ regulatory T cells, effect on macrophage phagocytosis, and skewing to the immunosuppressive M2 phenotype. We found that hypoxia potentiated the gCSC-mediated inhibition of T cell proliferation and activation and especially the induction of FoxP3+T cells, and further inhibited macrophage phagocytosis compared to normoxia condition. These immunosuppressive hypoxic effects were mediated by signal transducer and activator of transcription 3 (STAT3) and its transcriptionally regulated products such as hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). Inhibitors of STAT3 and HIF-1α down modulated the gCSCs' hypoxia-induced immunosuppressive effects. Thus, hypoxia further enhances GBM-mediated immunosuppression, which can be reversed with therapeutic inhibition of STAT3 and HIF-1α and also helps to reconcile the disparate findings that immune therapeutic approaches can be used successfully in model systems but have yet to achieve generalized successful responses in the vast majority of GBM patients by demonstrating the importance of the tumor hypoxic environment.
doi:10.1371/journal.pone.0016195
PMCID: PMC3024401  PMID: 21283755
16.  Hypoxia Increases Gefitinib-Resistant Lung Cancer Stem Cells through the Activation of Insulin-Like Growth Factor 1 Receptor 
PLoS ONE  2014;9(1):e86459.
Accumulating evidence indicates that a small population of cancer stem cells (CSCs) is involved in intrinsic resistance to cancer treatment. The hypoxic microenvironment is an important stem cell niche that promotes the persistence of CSCs in tumors. Our aim here was to elucidate the role of hypoxia and CSCs in the resistance to gefitinib in non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9 and HCC827, which express the EGFR exon 19 deletion mutations, were exposed to high concentration of gefitinib under normoxic or hypoxic conditions. Seven days after gefitinib exposure, a small fraction of viable cells were detected, and these were referred to as “gefitinib-resistant persisters” (GRPs). CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1–all genes involved in stemness–were highly expressed in GRPs in PC9 and HCC827 cells, and PC9 GRPs exhibited a high potential for tumorigenicity in vivo. The expression of insulin-like growth factor 1 (IGF1) was also upregulated and IGF1 receptor (IGF1R) was activated on GRPs. Importantly, hypoxic exposure significantly increased sphere formation, reflecting the self-renewal capability, and the population of CD133- and Oct4-positive GRPs. Additionally, hypoxia upregulated IGF1 expression through hypoxia-inducible factor 1α (HIF1α), and markedly promoted the activation of IGF1R on GRPs. Knockdown of IGF1 expression significantly reduced phosphorylated IGF1R-expressing GRPs under hypoxic conditions. Finally, inhibition of HIF1α or IGF1R by specific inhibitors significantly decreased the population of CD133- and Oct4-positive GRPs, which were increased by hypoxia in PC9 and HCC827 cells. Collectively, these findings suggest that hypoxia increased the population of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC by activating IGF1R. Targeting the IGF1R pathway may be a promising strategy for overcoming gefitinib resistance in EGFR mutation-positive NSCLC induced by lung CSCs and microenvironment factors such as tumor hypoxia.
doi:10.1371/journal.pone.0086459
PMCID: PMC3904884  PMID: 24489728
17.  Knock down of HIF-1α in glioma cells reduces migration in vitro and invasion in vivo and impairs their ability to form tumor spheres 
Molecular Cancer  2010;9:133.
Background
Glioblastoma (GBM) is the most common and malignant primary intracranial human neoplasm. GBMs are characterized by the presence of extensive areas of necrosis and hypoxia. Hypoxia and its master regulator, hypoxia inducible factor 1 (HIF-1) play a key role in glioma invasion.
Results
To further elucidate the functional role of HIF-1α in glioma cell migration in vitro and in invasion in vivo, we used a shRNA approach to knock down HIF-1α expression complemented with genome-wide expression profiling, performed in both normoxic and hypoxic conditions. Our data show that knock down of HIF-1α in glioma cells significantly impairs their migration in vitro as well as their ability to invade into the brain parenchyma in vivo. Next, we assessed the role that HIF-1α plays in maintaining the characteristics of cancer stem cells (CSCs). By using the tumor sphere forming assay, we demonstrate that HIF-1α plays a role in the survival and self-renewal potential of CSCs. Finally, expression profiling experiments in glioma cells provided detailed insight into a broad range of specific biological pathways and processes downstream of HIF-1α. We discuss the role of these processes in the migratory and invasive properties, as well as the stem cell biology of glioblastomas
Conclusions
Our data show that knock down of HIF-1α in human and murine glioma cells impairs their migration in vitro and their invasion in vivo. In addition, our data suggest that HIF-1α plays a role in the survival and self-renewal potential of CSCs and identify genes that might further elucidate the role of HIF-1α in tumor migration, invasion and stem cell biology.
doi:10.1186/1476-4598-9-133
PMCID: PMC2896954  PMID: 20515450
18.  Bcl-2 Regulates HIF-1α Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90 
PLoS ONE  2010;5(7):e11772.
Background
Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.
Methodology/Principal Findings
By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1α protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1α protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1α protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1α stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1α protein. We also showed that bcl-2, HIF-1α and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1α protein during hypoxia, and in particular the isoform HSP90β is the main player in this phenomenon.
Conclusions/Significance
We identified the stabilization of HIF-1α protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the β isoform of molecular chaperone HSP90.
doi:10.1371/journal.pone.0011772
PMCID: PMC2910721  PMID: 20668552
19.  In vitro hypoxia-conditioned colon cancer cell lines derived from HCT116 and HT29 exhibit altered apoptosis susceptibility and a more angiogenic profile in vivo 
British Journal of Cancer  2005;93(12):1356-1363.
Hypoxia is an important selective force in the clonal evolution of tumours. Through HIF-1 and other transcription factors combined with tumour-specific genetic alterations, hypoxia is a dominant factor in the angiogenic phenotype. Cellular adaptation to hypoxia is an important requirement of tumour progression independent of angiogenesis. The adaptive changes, insofar as they alter hypoxia-induced apoptosis, are likely to determine responsiveness to antiangiogenic strategies. To investigate this adaptation of tumour cells to hypoxia, we recreated in vitro the in vivo situation of chronic intermittent exposure to low-oxygen levels. The colon carcinoma cell lines HT29 and HCT116 were subjected to 40 episodes of sublethal hypoxia (4 h) three times a week. The resulting two hypoxia-conditioned cell lines have been maintained in culture for more than 2 years. In both cell lines changes in doubling times occurred: in HT29 an increase, and in HCT116 a decrease. Cell survival in response to hypoxia and to DNA damage differed strikingly in the two cell lines. The HT29 hypoxia-conditioned cells were more resistant than the parental line to a 24 h hypoxic challenge, while those from HCT116 surprisingly were more sensitive. Sensitivity to cisplatin in vitro was also significantly different for the hypoxia-conditioned compared with the parental lines, suggesting a change in pathways leading to apoptosis following DNA damage signaling. The growth of both conditioned cell lines in vivo as xenografts in immunodeficient (SCID) mice was more rapid than their parental lines, and was accompanied in each by evidence of enhanced vascular proliferation as a consequence of the hypoxia-conditioning. Thus the changes in apoptotic susceptibility were independent of altered angiogenesis. The derivation of these lines provides a model for events within hypoxic regions of colon cancers, and for the acquisition of resistance and sensitivity characteristics that may have therapeutic implications for the use of antiangiogenesis drugs.
doi:10.1038/sj.bjc.6602864
PMCID: PMC2361533  PMID: 16333244
hypoxia; angiogenesis; colon cancer; cisplatin; apoptosis
20.  Cobalt stimulates HIF-1-dependent but inhibits HIF-2-dependent gene expression in liver cancer cells 
The international journal of biochemistry & cell biology  2013;45(11):10.1016/j.biocel.2013.07.025.
Hypoxia-inducible factors (HIFs) are transcriptional regulators that mediate the cellular response to low oxygen. Although HIF-1 is usually considered as the principal mediator of hypoxic adaptation, several tissues and different cell types express both HIF-1 and HIF-2 isoforms under hypoxia or when treated with hypoxia mimetic chemicals such as cobalt. However, the similarities or differences between HIF-1 and HIF-2, in terms of their tissue- and inducer-specific activation and function, are not adequately characterized. To address this issue, we investigated the effects of true hypoxia and hypoxia mimetics on HIF-1 and HIF-2 induction and specific gene transcriptional activity in two hepatic cancer cell lines, Huh7 and HepG2. Both hypoxia and cobalt caused rapid induction of both HIF-1α and HIF-2α proteins. Hypoxia induced erythropoietin (EPO) expression and secretion in a HIF-2-dependent way. Surprisingly, however, EPO expression was not induced when cells were treated with cobalt. In agreement, both HIF-1- and HIF-2-dependent promoters (of PGK and SOD2 genes, respectively) were activated by hypoxia while cobalt only activated the HIF-1-dependent PGK promoter. Unlike cobalt, other hypoxia mimetics such as DFO and DMOG activated both types of promoters. Furthermore, cobalt impaired the hypoxic stimulation of HIF-2, but not HIF-1, activity and cobalt-induced HIF-2α interacted poorly with USF-2, a HIF-2-specific co-activator. These data show that, despite similar induction of HIF-1α and HIF-2α protein expression, HIF-1 and HIF-2 specific gene activating functions respond differently to different stimuli and suggest the operation of oxygen-independent and gene- or tissue-specific regulatory mechanisms involving additional transcription factors or co-activators.
doi:10.1016/j.biocel.2013.07.025
PMCID: PMC3855297  PMID: 23958427
HIF-2α; EPO; SOD2; Hypoxia; Cobalt; USF2
21.  Induction of Cancer Stem Cell Properties in Colon Cancer Cells by Defined Factors 
PLoS ONE  2014;9(7):e101735.
Cancer stem cells (CSCs) are considered to be responsible for the dismal prognosis of cancer patients. However, little is known about the molecular mechanisms underlying the acquisition and maintenance of CSC properties in cancer cells because of their rarity in clinical samples. We herein induced CSC properties in cancer cells using defined factors. We retrovirally introduced a set of defined factors (OCT3/4, SOX2 and KLF4) into human colon cancer cells, followed by culture with conventional serum-containing medium, not human embryonic stem cell medium. We then evaluated the CSC properties in the cells. The colon cancer cells transduced with the three factors showed significantly enhanced CSC properties in terms of the marker gene expression, sphere formation, chemoresistance and tumorigenicity. We designated the cells with CSC properties induced by the factors, a subset of the transduced cells, as induced CSCs (iCSCs). Moreover, we established a novel technology to isolate and collect the iCSCs based on the differences in the degree of the dye-effluxing activity enhancement. The xenografts derived from our iCSCs were not teratomas. Notably, in contrast to the tumors from the parental cancer cells, the iCSC-based tumors mimicked actual human colon cancer tissues in terms of their immunohistological findings, which showed colonic lineage differentiation. In addition, we confirmed that the phenotypes of our iCSCs were reproducible in serial transplantation experiments. By introducing defined factors, we generated iCSCs with lineage specificity directly from cancer cells, not via an induced pluripotent stem cell state. The novel method enables us to obtain abundant materials of CSCs that not only have enhanced tumorigenicity, but also the ability to differentiate to recapitulate a specific type of cancer tissues. Our method can be of great value to fully understand CSCs and develop new therapies targeting CSCs.
doi:10.1371/journal.pone.0101735
PMCID: PMC4090165  PMID: 25006808
22.  c-kit-Positive Cardiac Stem Cells Nested in Hypoxic Niches are Activated by Stem Cell Factor Reversing the Aging Myopathy 
Circulation research  2013;114(1):41-55.
Rationale
Hypoxia favors stem cell quiescence, while normoxia is required for their activation; but whether cardiac stem cell (CSC) function is regulated by the hypoxic/normoxic state of the cell is currently unknown.
Objective
A balance between hypoxic and normoxic CSCs may be present in the young heart, although this homeostatic control may be disrupted with aging. Defects in tissue oxygenation occur in the old myocardium, and this phenomenon may expand the pool of hypoxic CSCs, which are no longer involved in myocyte renewal.
Methods and Results
Here we show that the senescent heart is characterized by an increased number of quiescent CSCs with intact telomeres that cannot reenter the cell cycle and form a differentiated progeny. Conversely, myocyte replacement is controlled only by frequently dividing CSCs with shortened telomeres; these CSCs generate a myocyte population that is chronologically young but phenotypically old. Telomere dysfunction dictates their actual age and mechanical behavior. However, the residual subset of quiescent young CSCs can be stimulated in situ by stem cell factor reversing the aging myopathy.
Conclusions
Our findings support the notion that strategies targeting CSC activation and growth interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of animal studies to human beings, our data strongly suggests that a pool of functionally-competent CSCs persists in the senescent heart and this stem cell compartment can promote myocyte regeneration effectively, correcting partly the aging myopathy.
doi:10.1161/CIRCRESAHA.114.302500
PMCID: PMC3959163  PMID: 24170267
Cardiac stem cells; hypoxia; myocardial aging; stem cell factor
23.  BMP2 sensitizes glioblastoma stem-like cells to Temozolomide by affecting HIF-1α stability and MGMT expression 
Cell Death & Disease  2012;3(10):e412-.
Glioblastoma multiforme (GBM) is the most common brain tumour, characterized by a central and partially necrotic (i.e., hypoxic) core enriched in cancer stem cells (CSCs). We previously showed that the most hypoxic and immature (i.e., CSCs) GBM cells were resistant to Temozolomide (TMZ) in vitro, owing to a particularly high expression of O6-methylguanine-DNA-methyltransferase (MGMT), the most important factor associated to therapy resistance in GBM. Bone morphogenetic proteins (BMPs), and in particular BMP2, are known to promote differentiation and growth inhibition in GBM cells. For this reason, we investigated whether a BMP2-based treatment would increase TMZ response in hypoxic drug-resistant GBM-derived cells. Here we show that BMP2 induced strong differentiation of GBM stem-like cells and subsequent addition of TMZ caused dramatic increase of apoptosis. Importantly, we correlated these effects to a BMP2-induced downregulation of both hypoxia-inducible factor-1α (HIF-1α) and MGMT. We report here a novel mechanism involving the HIF-1α-dependent regulation of MGMT, highlighting the existence of a HIF-1α/MGMT axis supporting GBM resistance to therapy. As confirmed from this evidence, over-stabilization of HIF-1α in TMZ-sensitive GBM cells abolished their responsiveness to it. In conclusion, we describe a HIF-1α-dependent regulation of MGMT and suggest that BMP2, by down-modulating the HIF-1α/MGMT axis, should increase GBM responsiveness to chemotherapy, thus opening the way to the development of future strategies for GBM treatment.
doi:10.1038/cddis.2012.153
PMCID: PMC3481140  PMID: 23076220
Glioblastoma; BMP2; temozolomide; hypoxia; HIF-1α; MGMT
24.  Hypoxia-resistant profile implies vulnerability of cancer stem cells to physiological agents, which suggests new therapeutic targets 
Cell Cycle  2013;13(2):268-278.
We have previously shown that peculiar metabolic features of cell adaptation and survival in hypoxia imply growth restriction points that are typical of embryonic stem cells and disappear with differentiation. Here we provide evidence that such restrictions can be exploited as specific antiblastic targets by physiological factors such as pyruvate, tetrahydrofolate, and glutamine. These metabolites act as powerful cytotoxic agents on cancer stem cells (CSCs) when supplied at doses that perturb the biochemical network, sustaining the resumption of aerobic growth after the hypoxic dormant state. Experiments were performed in vivo and in vitro using CSCs obtained from various anaplastic tumors: human melanoma, leukemia, and rat hepatoma cells. Pretreatment of melanoma CSCs with pyruvate significantly reduces their self-renewal in vitro and tumorigenicity in vivo. The metabolic network underlying the cytotoxic effect of the physiological factors was thoroughly defined, principally using AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem stage. This network, based on a tight integration of aerobic glycolysis, cellular redox state, and folate metabolism, is centered on the cellular NADP/NADPH ratio that controls the redox pathway of folate utilization in purine synthesis. On the whole, this study indicates that pyruvate, FH4, and glutamine display anticancer activity, because CSCs are committed to survive and maintain their stemness in hypoxia. When CSC need to differentiate and proliferate, they shift from anaerobic to aerobic status, and the few mitochondria available makes them susceptible to the injury of the above physiological factors. This vulnerability might be exploited for novel therapeutic treatments.
doi:10.4161/cc.27031
PMCID: PMC3906243  PMID: 24200964
Krebs cycle substrates; cancer stem cells; cellular redox state; folate metabolism; hypoxia
25.  Tight Control of Hypoxia-inducible Factor-α Transient Dynamics Is Essential for Cell Survival in Hypoxia 
The Journal of Biological Chemistry  2014;289(9):5549-5564.
Background: Hypoxia inducible factor-α (HIF-α) is the main transcription factor activated in low oxygen conditions.
Results: Single cell imaging reveals pulses in nuclear levels of HIF-α.
Conclusion: The transient nature of the HIF-α nuclear accumulation is required to avoid cell death.
Significance: The duration of HIF-α response depends on cellular oxygenation, and can encode information and dictate cell fate.
Intracellular signaling involving hypoxia-inducible factor (HIF) controls the adaptive responses to hypoxia. There is a growing body of evidence demonstrating that intracellular signals encode temporal information. Thus, the dynamics of protein levels, as well as protein quantity and/or localization, impacts on cell fate. We hypothesized that such temporal encoding has a role in HIF signaling and cell fate decisions triggered by hypoxic conditions. Using live cell imaging in a controlled oxygen environment, we observed transient 3-h pulses of HIF-1α and -2α expression under continuous hypoxia. We postulated that the well described prolyl hydroxylase (PHD) oxygen sensors and HIF negative feedback regulators could be the origin of the pulsatile HIF dynamics. We used iterative mathematical modeling and experimental analysis to scrutinize which parameter of the PHD feedback could control HIF timing and we probed for the functional redundancy between the three main PHD proteins. We identified PHD2 as the main PHD responsible for HIF peak duration. We then demonstrated that this has important consequences, because the transient nature of the HIF pulse prevents cell death by avoiding transcription of p53-dependent pro-apoptotic genes. We have further shown the importance of considering HIF dynamics for coupling mathematical models by using a described HIF-p53 mathematical model. Our results indicate that the tight control of HIF transient dynamics has important functional consequences on the cross-talk with key signaling pathways controlling cell survival, which is likely to impact on HIF targeting strategies for hypoxia-associated diseases such as tumor progression and ischemia.
doi:10.1074/jbc.M113.500405
PMCID: PMC3937633  PMID: 24394419
Cell Death; Hypoxia; Hypoxia-inducible Factor; Imaging; Mathematical Modeling; Negative Feedback Loop; p53; Prolyl Hydroxylase

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