Hypoxia-induced Cell Cycle Arrest Research Articles

FF-MAS prevents porcine ovarian granulosa cells from hypoxia-induced apoptosis via inhibiting STAT4 expression.

Follicular fluid meiosis-activating sterol (FF-MAS) is a small molecule compound found in follicular fluid, named for its ability to induce oocyte resumption of meiosis. Granulosa cells (GCs) within the follicle are typically located in a hypoxic environment under physiologic conditions due to limited vascular distribution. Previous research suggests that hypoxia-induced cell cycle arrest and apoptosis in GCs may be crucial triggering factors in porcine follicular atresia. However, the impact of FF-MAS on GCs within follicles has not been explored so far. In this study, we uncovered a novel role of FF-MAS in facilitating GC survival under hypoxic conditions by inhibiting STAT4 expression. We found that STAT4 expression was upregulated in porcine GCs exposed to 1% O2. Both gain and loss of function assays confirmed that STAT4 was required for cell apoptosis under hypoxia conditions, and that the GC apoptosis caused by hypoxia was markedly attenuated following FF-MAS treatment through inhibition of STAT4 expression. Correlation analysis in vivo revealed that GC apoptosis was associated with increased STAT4 expression, while the FF-MAS content in follicular fluid was negatively correlated with STAT4 mRNA levels and cell apoptosis. These findings elucidate a novel role of FF-MAS-mediated protection of GCs by inhibiting STAT4 expression under hypoxia, which might contribute to the mechanistic understanding of follicular development.

Abstract 3267: Understanding the role of dNTP pool on double-strand break and resistant to therapy in quiescent hypoxic glioblastoma

Abstract Glioblastoma (GBM) is the deadliest type of primary brain tumor. After surgical resection of GBM, chemotherapy and radiotherapy (RT) are used as treatment modalities to induce double-strand breaks (DSBs), the most cytotoxic and lethal form of DNA damage. However, an inadequate oxygen supply and the high metabolic demand in rapidly growing solid tumors like GBM result in severe hypoxic regions, contributing to altered cellular metabolism and resistance to these cancer therapies. These subpopulations of resistant and refractory hypoxic cancer cells are clinically significant as they lead to poor prognosis and adverse clinical outcomes in patients. Ionizing radiation (IR) and chemotherapeutics such as temozolomide induce DNA damage, such as base deletions, single-strand breaks (SSBs), and DSBs. Rapidly replicating cells are more sensitive to these therapeutic agents partly due to replication-induced conversion of SSB and other manageable damages to highly lethal DSB. A balanced deoxyribonucleoside triphosphate (dNTP) is crucial for DNA replication, repair, and maintenance of genomic stability. Cellular dNTP is mainly regulated by ribonucleotide reductase (RNR) and the SAM- and HD-domain-containing protein (SAMHD1). RNR boosts the cellular dNTP pool through the de novo synthesis, while SAMHD1 depletes the pool by hydrolyzing dNTPs. Also, previous studies have shown that hypoxic cancer cells have low dNTP levels due to oxygen dependency of its rate-limiting RNR enzyme, which could result in reduced DNA replication and cell cycle arrest. Here, we show the direct relationships between dNTP levels, DNA replication, and DSB induction in hypoxic GBM cancer cells, which is currently unknown. To bridge this knowledge gap, we exposed GBM cells to severe hypoxia and utilized cellular dNTP analysis to demonstrate that their dNTP pool can be elevated. Our results show that elevating the cellular dNTP pool relieved hypoxia-induced cell cycle arrest as determined by flow cytometry and increased their DNA replication. A single-cell microscopic analysis also revealed that increasing the dNTP pool significantly enhanced the induction of DSBs following exposure to IR and bleomycin. Furthermore, we detected an increased efficacy of DSB-inducing agents in hypoxic GBM, as demonstrated by apoptosis pathway activation. Our result suggests that increasing the intracellular dNTP pool in hypoxic GBM will stimulate DNA replication, increase genotoxin-induced DSB, and improve DSB-inducing therapeutic efficacy. In conclusion, this study provides a preclinical justification for using cellular dNTP modulation as a novel therapeutic strategy and an adjunct to classical DSB-inducing agents in hypoxic GBM. Citation Format: Edidiong R. Usoro, Dominique Monroe, Kristen Carver, Emily Steinbeck, Arilyn Williams, Shelby Aycox, Waaqo Daddacha. Understanding the role of dNTP pool on double-strand break and resistant to therapy in quiescent hypoxic glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3267.

Lactate in breast cancer cells is associated with evasion of hypoxia-induced cell cycle arrest and adverse patient outcome.

Tumor hypoxia is a common microenvironmental factor in breast cancers, resulting in stabilization of Hypoxia-Inducible Factor 1 (HIF-1), the master regulator of hypoxic response in cells. Metabolic adaptation by HIF-1 results in inhibition of citric acid cycle, causing accumulation of lactate in large concentrations in hypoxic cancers. Lactate can therefore serve as a secondary microenvironmental factor influencing cellular response to hypoxia. Presence of lactate can alter the hypoxic response of breast cancers in many ways, sometimes in opposite manners. Lactate stabilizes HIF-1 in oxidative condition, as well as destabilizes HIF-1 in hypoxia, increases cellular acidification, and mitigates HIF-1-driven inhibition of cellular respiration. We therefore tested the effect of lactate in MDA-MB-231 under hypoxia, finding that lactate can activate pathways associated with DNA replication, and cell cycling, as well as tissue morphogenesis associated with invasive processes. Using a bioengineered nano-patterned stromal invasion assay, we also confirmed that high lactate and induced HIF-1α gene overexpression can synergistically promote MDA-MB-231 dissemination and stromal trespass. Furthermore, using The Cancer Genome Atlas, we also surprisingly found that lactate in hypoxia promotes gene expression signatures prognosticating low survival in breast cancer patients. Our work documents that lactate accumulation contributes to increased heterogeneity in breast cancer gene expression promoting cancer growth and reducing patient survival.

Abstract B020: Understanding the role of dNTP pool on double-strand break and resistance to therapy in quiescent hypoxic glioblastoma

Abstract Glioblastoma (GBM) is the deadliest type of primary brain tumor. After surgical resection, chemotherapy, and radiotherapy (RT) are used as treatment modalities to induce double-strand breaks (DSBs), the most cytotoxic and lethal form of DNA damage. However, an inadequate oxygen supply and the high metabolic demand in rapidly growing solid tumors like GBM result in severe hypoxic regions contributing to altered cellular metabolism and resistance to these cancer therapies. These subpopulations of resistant and refractory hypoxic cancer cells are clinically significant as they lead to poor prognosis and adverse clinical outcomes in patients. Ionizing radiation (IR) and chemotherapeutics such as temozolomide induce DNA damage, such as base deletions, single-strand breaks (SSBs), and DSBs. Rapidly replicating cells are more sensitive to these therapeutic agents partly due to replication-induced conversion of SSB and other manageable damages to highly lethal DSB. A balanced deoxyribonucleoside triphosphate (dNTP) is crucial for DNA replication, repair, and maintenance of genomic stability. Cellular dNTP is mainly regulated by ribonucleotide reductase (RNR) and the SAM- and HD-domain-containing protein (SAMHD1). RNR boosts the cellular dNTP pool through the de novo synthesis while SAMHD1 depletes the pool by hydrolyzing dNTPs. Previous studies have shown that hypoxic cancer cells have low dNTP levels due to oxygen dependency of its rate limiting RNR enzyme, which could result in reduced DNA replication and cell cycle arrest. Here, we show the direct relationships between dNTP levels, DNA replication, and DSB induction in hypoxic GBM cancer cells, which is currently unknown. To bridge this knowledge gap, we exposed GBM cells to severe hypoxia and utilized cellular dNTP analysis to demonstrate that their dNTP pool can be elevated. Our result shows that elevating cellular dNTP pool relieved hypoxia-induced cell cycle arrest as determined by flow cytometry and increased their DNA replication. A single cell microscopic analysis also revealed that increasing the dNTP pool significantly enhanced the induction of DSBs following exposure to IR and bleomycin. Furthermore, we detected an increased efficacy of DSB-inducing agents in hypoxic GBM as demonstrated by apoptosis pathway activation. Our result suggest that increasing the intracellular dNTP pool in hypoxic GBM will stimulate DNA replication, increase genotoxin-induced DSB, and improve DSB-inducing therapeutics' efficacy. This study provides a preclinical justification for using cellular dNTP modulation as a novel therapeutic strategy and an adjunct to classical DSB-inducing agents in hypoxic GBM. Citation Format: Edidiong R. Usoro, Dominique Monroe, Kristen Carver, Maya Jenkins, Emily Steinbeck, Arilyn Williams, Shelby Aycox, Waaqo Daddacha. Understanding the role of dNTP pool on double-strand break and resistance to therapy in quiescent hypoxic glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B020.

Protective effects of agrimonolide on hypoxia-induced H9c2 cell injury by maintaining mitochondrial homeostasis.

Cardiomyocyte death caused by hypoxia is one of the main causes of myocardial infarction or heart failure, and mitochondria play an important role in this process. Agrimonolide (AM) is a monomeric component extracted from Agrimonia pilosa L. and has antioxidant, antitumor, and anti-inflammatory effects. This study aimed to investigate the role and mechanism of AM in improving hypoxia-induced H9c2 cell damage. The results showed that low AM concentrations promote H9c2 cell proliferation and increase cellular ATP content. Transcriptome sequencing showed that AM induces differential expression of genes in H9c2 cells. Gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that these genes were concentrated in mitochondrial function. Subsequent experiments confirmed that AM regulates hypoxia-induced cell cycle arrest. AM inhibited the rate of apoptosis by regulating the expression of apoptosis-related proteins, reducing the level of cleaved Caspase 3 and Bax, and increasing the level of Bcl2, thereby protecting H9c2 cells from hypoxia-induced apoptosis. AM restored the mitochondrial membrane potential, inhibited the generation of ROS, maintained the normal shape of the mitochondria, improved the level of the mitochondrial functional proteins OPA1, MFN1, MFN2, Tom20, and increased the level of ATP. In conclusion, AM protects H9c2 cells from hypoxia-induced cell damage.

The RNA-Binding Protein RBM3 Promotes Neural Stem Cell (NSC) Proliferation Under Hypoxia.

Neural stem cells (NSCs) reside physiologically in a hypoxic niche to maintain self-renewal and multipotency. Whereas mild hypoxia is known to promote NSC proliferation, severe hypoxia in pathological conditions exerts the reverse effect. The multi-functional RNA-binding protein RBM3 is abundant in NSCs and can be regulated by hypoxic exposure. Although RBM3 has been shown to accelerate cell growth in many cell types, whether and how it affects NSC proliferation in hypoxic environment remains largely unknown. In this study, we tested how RBM3 regulates cell proliferation under hypoxia in C17.2 mouse NSC cell line and in primary mouse NSCs from both the forebrain of postnatal day 0 (P0) mice and the subgranular zone (SGZ) of adult mice. Our results demonstrated that RBM3 expression was highly sensitive to hypoxia, and NSCs were arrested in G0/G1 phase by 5, 2.5, and 1% O2 treatment. When we overexpressed RBM3, hypoxia-induced cell cycle arrest in G0/G1 phase was relieved and more cell transit into S phase was observed. Furthermore, cell viability under hypoxia was also increased by RBM3. In contrast, in RBM3-depleted primary NSCs, less BrdU-incorporated cells were detected, indicating exacerbated cell cycle arrest in G1 to S phase transition. Instead, overexpressed RBM3 significantly increased proliferation ratio in primary NSCs. Our findings indicate RBM3 as a potential target to maintain the proliferation capacity of NSCs under hypoxia, which can be important in NSC-based therapies of acute brain injury and chronic neurodegenerative diseases.

MicroRNA-493 targets STMN-1 and promotes hypoxia-induced epithelial cell cycle arrest in G2/M and renal fibrosis.

Hypoxia plays an important role in the development of renal fibrosis. G2/M arrest in renal tubular cells is an important pathway in the development of chronic kidney disease. It is unknown whether hypoxia leads to renal fibrosis via the regulation of G2/M arrest in renal tubular epithelial cells. For the first time, to our knowledge, we showed that hypoxia induces G2/M arrest in renal tubular cells leading to renal fibrosis, and microRNAs are involved in this regulation. We compared microRNA expression between hypoxia and normoxia in HK2 cells and found microRNA (miR)-493 to be highly expressed at 24 and 48 h after hypoxia. The overexpression of miR-493 reduced the expression of the cell cycle regulator, Stathmin (STMN)-1, and increased the percentage of G2/M phase cells and profibrotic factors in HK2 cells. Targeting STMN-1 with short hairpin RNA produced an effect similar to that of miR-493 overexpression. On contrast, the miR-493 inhibitor reversed these effects in vitro. Consistent with these results, miR-493 sponge adeno-associated virus reduced the expression of profibrotic factors and increased STMN-1 in vivo. In summary, these results suggest that the miR-493-STMN-1 pathway contributes to hypoxia-induced tubular epithelial cell G2/M arrest and renal fibrosis. Abrogating G2/M arrest and blocking the miR-493-STMN-1 pathway will provide further insight for the development of antifibrosis therapy.-Liu, T., Liu, L., Liu, M., Du, R., Dang, Y., Bai, M., Zhang, L., Ma, F., Yang, X., Ning, X., Sun, S. MicroRNA-493 targets STMN-1 and promotes hypoxia-induced epithelial cell cycle arrest In G2/M and renal fibrosis.

Abstract 1031: Fumarate hydratase deficiency redirects glucose metabolism of hypoxic cancer cells into the pentose phosphate pathway

Abstract Hypoxia is a common feature of all solid cancers and strongly correlated with poor prognosis. As an adaptive response to hypoxia, cancer cells reprogram their metabolism by increasing glycolysis and reductive carboxylation at the expense of mitochondrial respiration, a phenomenon orchestrated by the transcription factor hypoxia inducible factor (HIF) -1. Mutations in the gene encoding for the mitochondrial enzyme fumarate hydratase (FH), found in the hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome, lead to a similar phenotype despite the presence of O2, a phenomenon due to normoxic stabilization of HIF-1 (pseudohypoxia). Here, we report for the first time that FH loss-of-function (LOF) redirects glucose metabolism into the pentose phosphate pathway (PPP) in non-RCC cells subjected to severe hypoxia (O2< .02%). We show that this metabolic shift favors the buildup of biosynthetic precursors supporting hypoxic cell growth and proliferation. HCT-116 (colon), HeLa (cervix) and H460 (lung) adenocarcinoma cells were transfected with lentiviral vectors encoding for shRNAs targeting FH. Immunoblot analysis showed that FH LOF did not induce pseudohypoxia in these cells. In contrast, HLRCC-derived UOK262 cells showed accumulation of HIF-1 under normoxia which was reversed upon FH re-introduction. A comprehensive analysis utilizing a RT-qPCR array to profile the mRNA expression of 84 HIF-1 target genes, further confirmed that FH LOF did not result in a pseudohypoxic phenotype in HCT-116 cells. An unbiased analysis of 250 metabolites detected by liquid chromatography-tandem mass spectrometry followed by quantitative enrichment analysis, identified glycolysis and the PPP among the most enriched metabolic pathways in hypoxic FH knockdown cells (P< 5×10-8). Since the PPP provides precursors for synthesis of nucleic acids, we analyzed the effect of FH LOF on cell cycle progression and found an inhibition of hypoxia-induced cell cycle arrest in HCT-116 and HeLa cells. Our study reveals novel insights into the effect of FH loss-of-function in cancer cells and indicates a stark contrast between the pseudohypoxic phenotype described in kidney cancer cell lines obtained from HLRCC patients (i.e, UOK-262) and a HIF- independent mechanism of metabolic rerouting in colon, lung and cervix cancer cell lines. Our data show that FH LOF promotes an anabolic phenotype in hypoxic cancer cells that could be exploited to enhance the therapeutic response targeting this resistant subset of cancer cells. Citation Format: Luana Schito, Sergio Rey, Judy Pawling, James W. Dennis, Bradly G. Wouters, Marianne Koritzinsky. Fumarate hydratase deficiency redirects glucose metabolism of hypoxic cancer cells into the pentose phosphate pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1031.

Involvement of Cold Inducible RNA-Binding Protein in Severe Hypoxia-Induced Growth Arrest of Neural Stem Cells In Vitro

Neonatal hypoxia is the leading cause of brain damage with birth complications. Many studies have reported proliferation-promoting effect of mild hypoxia on neural stem cells (NSCs). However, how severe hypoxia influences the behavior of NSCs has been poorly explored. In the present study, we investigated the effects of 5, 3, and 1 % oxygen exposure on NSCs in vitro. MTT, neurosphere assay, and 5-ethynyl-2′-deoxyuridine (EdU) incorporation revealed a quick growth arrest of C17.2 cells and primary NSCs induced by 1 % oxygen exposure. Cell cycle analysis showed that this hypoxia exposure caused a significant increase of cells in G0/G1 phase and decrease of cells in S phase that is associated with decrease of Cyclin D1. Interestingly, the expression of cold inducible RNA-binding protein (CIRBP), a cold responsive gene reacting to multiple cellular stresses, was decreased in parallel with the 1 % oxygen-induced proliferation inhibition. Forced expression of CIRBP under hypoxia could restore the proliferation of NSCs, as showed by EdU incorporation and cell cycle analysis. Furthermore, the expression of Cyclin D1 under hypoxia was also restored by CIRBP overexpression. Taken together, these data suggested a growth-suppressing effect of severe hypoxia on NSCs and, for the first time, revealed a novel role of CIRBP in hypoxia-induced cell cycle arrest, suggesting that modulating CIRBP may be utilized for preventing hypoxia-induced neonatal brain injury.

Convergence of Hypoxia and TGFβ Pathways on Cell Cycle Regulation in Human Hematopoietic Stem/Progenitor Cells

Although it has been shown that HIF1 and 2 fulfill essential roles within the hematopoietic system and in the regulation of HSC fate, little is currently known about the specific mechanisms that are involved. We identified transcriptome changes induced by hypoxia, constitutively active HIF1(P402/564) and HIF2(P405/531) in human cord blood CD34+ cells. Thus, we were able to identify common hypoxia-HIF1-HIF2 gene signatures, but we also identified specific target genes that were exclusively regulated by HIF1, HIF2 or hypoxia. Geneset enrichment analysis (GSEA) revealed that, besides known pathways associated with “hypoxia-induced signaling”, also significant enrichment for the Transforming Growth Factor beta (TGFβ) pathway was observed within the hypoxia/HIF1/HIF2 transcriptomes. One of the most significantly upregulated genes in both gene sets was the cyclin dependent kinase inhibitor CDKN1C (p57kip2). Combined hypoxia treatment or HIF overexpression together with TGFβ stimulation resulted in enhanced expression of CDKN1C and enhanced cell cycle arrest within the CD34+/CD38− stem cell compartment. Interestingly, we observed that CD34+ cells cultured under hypoxic conditions secreted high levels of latent TGFβ, suggesting an auto- or paracrine role of TGFβ in the regulation of quiescence of these cells. However, knockdown of SMAD4 could not rescue the hypoxia induced cell cycle arrest, arguing against direct effects of hypoxia-induced secreted TGFβ. Finally, the Gα-coupled receptor GTPase RGS1 was identified as a HIF-dependent hypoxia target that dampens SDF1-induced migration and signal transduction in human CD34+ stem/progenitor cells.

Severe hypoxia induces complete antifolate resistance in carcinoma cells due to cell cycle arrest

Antifolates have a crucial role in the treatment of various cancers by inhibiting key enzymes in purine and thymidylate biosynthesis. However, the frequent emergence of inherent and acquired antifolate resistance in solid tumors calls for the development of novel therapeutic strategies to overcome this chemoresistance. The core of solid tumors is highly hypoxic due to poor blood circulation, and this hypoxia is considered to be a major contributor to drug resistance. However, the cytotoxic activity of antifolates under hypoxia is poorly characterized. Here we show that under severe hypoxia, gene expression of ubiquitously expressed key enzymes and transporters in folate metabolism and nucleoside homeostasis is downregulated. We further demonstrate that carcinoma cells become completely refractory, even at sub-millimolar concentrations, to all hydrophilic and lipophilic antifolates tested. Moreover, tumor cells retained sensitivity to the proteasome inhibitor bortezomib and the topoisomerase II inhibitor doxorubicin, which are independent of cell cycle. We provide evidence that this antifolate resistance, associated with repression of folate metabolism, is a result of the inability of antifolates to induce DNA damage under hypoxia, and is attributable to a hypoxia-induced cell cycle arrest, rather than a general anti-apoptotic mechanism. Our findings suggest that solid tumors harboring a hypoxic core of cell cycle-arrested cells may display antifolate resistance while retaining sensitivity to the chemotherapeutics bortezomib and doxorubicin. This study bears important implications for the molecular basis underlying antifolate resistance under hypoxia and its rational overcoming in solid tumors.

Convergence Of Hypoxia and TGFβ Pathways On Cell Cycle Regulation In Human Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cells reside within specialized hypoxic niches in the bone marrow where they are kept in a relative quiescent state. One of the key pathways activated under low oxygen conditions is the Hypoxia Induced Factor (HIF) pathway. HIF1 and HIF2 act as oxygen sensors that are degraded by the Von Hippel Lindau (VHL) tumor suppressor protein under normoxic conditions but not when oxygen levels are low, resulting in stabilization of the HIF proteins, translocation to the nucleus and initiation of target gene transcription.Although it has been shown that HIF1 and 2 fulfill essential roles in the regulation of HSC fate, little is known about the mechanisms that are involved. Here, we set out to investigate the effects of hypoxia, HIF1 and HIF2 on gene transcription in human hematopoietic stem/progenitor cells. Cord blood (CB) CD34+ cells were cultured under low oxygen conditions (2%), or were lentivirally transduced with constitutively active HIF1(P402/564) and HIF2(P405/531) constructs under normoxic conditions and after 24 hrs transcriptome changes were analyzed by Illumina BeadArray analysis. This provided the possibility to identify common hypoxia-HIF1-HIF2 gene signatures, but also the identification of specific target genes that were exclusively regulated by HIF1, HIF2 or hypoxia. Geneset enrichment analysis (GSEA) using Gene Ontology genesets revealed that overexpression of HIF1 and -2 resulted in significant enrichment for known pathways like “hypoxia induced signaling”, but unexpectedly also for the Transforming Growth Factor beta (TGFβ) pathway. GSEA using a published dataset of TGFβ stimulated CB CD34+ cells indeed confirmed a high correlation between hypoxia target genes and TGFβ induced genes. Two of the most significantly upregulated genes in both gene sets were the cyclin dependent kinase inhibitor CDKN1C (p57kip2) and Regulator of G-protein signaling (RGS)1.q-RT-PCR analysis demonstrated enhanced expression of CDKN1C by hypoxia treatment or HIF overexpression under normoxia in combination with TGFβ stimulation. Although it was demonstrated that CD34+cells cultured under hypoxic conditions secreted high levels of latent TGFβ, no rescue of the hypoxia induced cell cycle arrest was demonstrated by knockdown of SMAD4, arguing against direct effects of hypoxia-induced secreted TGFβ on cell cycle quiescence.RGS1 is a member of the RGS family, involved in the negative regulation of G-protein coupled receptor signaling. Overexpression studies under normoxic conditions in CB CD34+cells demonstrated a decrease of SDF1-mediated migration. Furthermore, overexpression of RGS1 attenuated SDF1 and GM-CSF-induced ERK phosphorylation whereas the GM-CSF-induced STAT5 tyrosine phosphorylation was unaffected. These findings indicate that RGS1 can interfere with specific signaling pathways involved in the regulation of cell proliferation and migration.Analysis of the CDKN1C as well as the RGS1 promoters revealed binding sites for both HIF and SMAD2/3/4 in the proximal part, suggesting that both pathways can indeed converge on the regulation of these important proteins that control cell cycle progression and the response to stimulatory cytokines in human stem/progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Hypoxia and cell cycle regulation of the von Hippel–Lindau tumor suppressor

Inactivation of von Hippel-Lindau tumor suppressor protein (pVHL) is associated with von Hippel-Lindau disease, an inherited cancer syndrome, as well as the majority of patients with sporadic clear cell renal carcinoma (RCC). While the involvement of pVHL in oxygen sensing through targeting HIFα subunits to ubiquitin-dependent proteolysis has been well documented, less is known about pVHL regulation under both normoxic and hypoxic conditions. We found that pVHL levels decreased in hypoxia and that hypoxia-induced cell cycle arrest is associated with pVHL expression in RCC cells. pVHL levels fluctuate during the cell cycle, paralleling cyclin B1 levels, with decreased levels in mitosis and G1. pVHL contains consensus Destruction box sequences, and pVHL associates with Cdh1, an activator of the anaphase promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. We show that pVHL has a decreased half-life in G1, Cdh1 downregulation results in increased pVHL expression, while Cdh1 overexpression results in decreased pVHL expression. Taken together these results suggest that pVHL is a novel substrate of APC/CCdh1. Destruction box-independent pVHL degradation was also detected, indicating that other ubiquitin ligases are also activated for pVHL degradation.

Abstract A65: MicroRNA miR-210 modulates cellular response to hypoxia through the MYC antagonist MNT

Abstract Intratumoral hypoxia is well known as a hallmark of most solid tumors. Here, we studied expression level of about 200 microRNAs in a panel of cancer cell lines from different tissues, and identified a microRNA miR210, its expression was prominently up-regulated by hypoxia. Over expression of miR210 in cancer cell lines partially reversed the hypoxic gene expression signature and allowed cells to escape from hypoxia-induced cell-cycle arrest. We then identified MNT, a known MYC antagonist, as a key miR-210 target. miR-210 directly down-regulated MNT transcript and protein via recognition sites in the transcript 3′ UTR. Consistent with a role for MNT as a miR-210 target, RNA interference-mediated knockdown of MNT phenocopied miR-210 over expression cell cycle effect. Furthermore, loss of MYC abolished the ability of miR-210 to override hypoxia-induced cell-cycle arrest. Thus, miR-210 could function as a modulator in cancer cells in response to hypoxic conditions through Myc network. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A65.

MicroRNA miR-210 modulates cellular response to hypoxia through the MYC antagonist MNT

The hypoxia-inducible factor (HIF) pathway is essential for cell survival under low oxygen and plays an important role in tumor cell homeostasis. We investigated the function of miR-210, the most prominent microRNA up-regulated by hypoxia and a direct transcriptional target of HIFs. miR-210 expression was elevated in multiple cancer types and correlated with metastasis of breast and melanoma tumors. miR-210 overexpression in cancer cell lines bypassed hypoxia-induced cell-cycle arrest and partially reversed the hypoxic gene expression signature. We identified MNT, a known MYC antagonist, as a miR-210 target. MNT mRNA contains multiple miR-210 binding sites in the 3' UTR and its knockdown phenocopied miR-210 overexpression. Furthermore, loss of MYC abolished miR-210-mediated override of hypoxia-induced cell-cycle arrest. Comparison of miR-210 and MYC overexpression with MNT knockdown signatures also indicated that miR-210 triggered a "MYC-like" transcriptional response. Thus, miR-210 influences the hypoxia response in tumor cells through targeting a key transcriptional repressor of the MYC-MAX network.

Erbb2 Suppresses DNA Damage-Induced Checkpoint Activation and UV-Induced Mouse Skin Tumorigenesis

The Erbb2 receptor is activated by UV irradiation, the primary cause of non-melanoma skin cancer. We hypothesized that Erbb2 activation contributes to UV-induced skin tumorigenesis by suppressing cell cycle arrest. Consistent with this hypothesis, inhibition of Erbb2 in v-ras(Ha) transgenic mice before UV exposure resulted in both 56% fewer skin tumors and tumors that were 70% smaller. Inhibition of the UV-induced activation of Erbb2 also resulted in milder epidermal hyperplasia, S-phase accumulation, and decreased levels of the cell cycle regulator Cdc25a, suggesting altered cell cycle regulation on inhibition of Erbb2. Further investigation using inhibition or genetic deletion of Erbb2 in vitro revealed reduced Cdc25a levels and increased S-phase arrest in UV-irradiated cells lacking Erbb2 activity. Ectopic expression of Cdc25a prevented UV-induced S-phase arrest in keratinocytes lacking Erbb2 activity, demonstrating that maintenance of Cdc25a by Erbb2 suppresses cell cycle arrest. Examination of checkpoint pathway activation upstream of Cdc25a revealed Erbb2 activation did not alter Ataxia Telangiectasia and Rad3-related/Ataxia Telangiectasia Mutated activity but increased inhibitory phosphorylation of Chk1-Ser(280). Since Akt phosphorylates Chk1-Ser(280), the effect of Erbb2 on phosphatidyl inositol-3-kinase (PI3K)/Akt signaling during UV-induced cell cycle arrest was determined. Erbb2 ablation reduced the UV-induced activation of PI3K while inhibition of PI3K/Akt increased UV-induced S-phase arrest. Thus, UV-induced Erbb2 activation increases skin tumorigenesis through inhibitory phosphorylation of Chk1, Cdc25a maintenance, and suppression of S-phase arrest via a PI3K/Akt-dependent mechanism.

SWI/SNF Regulates the Cellular Response to Hypoxia

Hypoxia induces a variety of cellular responses such as cell cycle arrest, apoptosis, and autophagy. Most of these responses are mediated by the hypoxia-inducible factor-1alpha. To induce target genes, hypoxia-inducible factor-1alpha requires a chromatin environment conducive to allow binding to specific sequences. Here, we have studied the role of the chromatin-remodeling complex SWI/SNF in the cellular response to hypoxia. We find that SWI/SNF is required for several of the cellular responses induced by hypoxia. Surprisingly, hypoxia-inducible factor-1alpha is a direct target of the SWI/SNF chromatin-remodeling complex. SWI/SNF components are found associated with the hypoxia-inducible factor-1alpha promoter and modulation of SWI/SNF levels results in pronounced changes in hypoxia-inducible factor-1alpha expression and its ability to transactivate target genes. Furthermore, impairment of SWI/SNF function renders cells resistant to hypoxia-induced cell cycle arrest. These results reveal a previously uncharacterized dependence of hypoxia signaling on the SWI/SNF complex and demonstrate a new level of control over the hypoxia-inducible factor-1alpha system.

Reactive oxygen species‐mediated cyclin D1 degradation mediates tumor growth retardation in hypoxia, independently of p21cip1 and hypoxia‐inducible factor

Cell growth arrest is an adaptation process for tumor survival in hypoxic environments. As proliferation is a very complicated and dynamic process, hypoxic growth arrest is not considered to be simply determined by a few molecules. Recently, several research groups have demonstrated that hypoxia-inducible factor (HIF)-1alpha plays a crucial role in hypoxia-induced cell-cycle arrest by inhibiting c-Myc and subsequently inducing p21(cip1) expression. However, we found that hypoxic growth arrest could occur even in p21-null cancer cells, and addressed the p21-independent process of cell-cycle arrest. We show that cyclin D1 was downregulated in various cancer cell lines under hypoxic conditions, which was independent of p21 and HIF-1 and -2alpha expression. It was also found that cyclin D1 was destabilized by the ubiquitin-proteasome system and this degradation process was highly activated by hypoxia. Moreover, antioxidants prevented the hypoxic degradation of cyclin D1 and hydrogen peroxide destabilized cyclin D1 in normoxia. Finally, we demonstrated that ectopic expression of cyclin D1 rescued hypoxic growth arrest in both p21+/+ and p21-/- HCT116 cells. Given the results, we here propose that reactive oxygen species-mediated cyclin D1 degradation contributes to tumor growth retardation in hypoxic environments.

Hypoxia‐inducible factor 1α is essential for hypoxic p27 induction in endometrioid endometrial carcinoma

Hypoxia-inducible factor 1alpha (HIF-1alpha) plays an essential role in the adaptive response of cells to hypoxia. The cyclin-dependent kinase inhibitor p27(Kip1) is highly expressed in the normal endometrium but is lost during endometrial carcinogenesis. However, in high-grade cancers, p27 re-expression is observed. We analysed the role of HIF-1alpha in hypoxia-induced expression of p27 in vitro and in vivo in endometrial cancer. Paraffin-embedded specimens from endometrioid endometrial carcinoma (n = 39) were stained immunohistochemically for HIF-1alpha, p27, and Ki67. HEC1B, an endometrial carcinoma cell line, was cultured under normoxic or hypoxic conditions in the presence or absence of transiently expressed short hairpin RNAs targeting HIF-1alpha. Protein expression of p27 and HIF-1alpha was assessed by western blotting. Immunohistochemical staining revealed perinecrotic HIF-1alpha expression in 67% of the cases and p27 staining centrally in the tumour islands, mostly around necrosis, in 46% of the cases. In 50% of the tumours with perinecrotic HIF-1alpha expression, p27 and HIF-1alpha perinecrotic/central co-localization was observed. In these tumour sections, hypoxia-associated p27 expression showed less proliferation around necrosis. Analysis of cultured endometrial carcinoma cells demonstrated that p27 protein expression is induced by hypoxia. This induction was abrogated by transient knockdown of HIF-1alpha using RNAi. Furthermore, hypoxia induced cell cycle arrest in HEC1B cells. We conclude that, in endometrioid endometrial carcinoma, p27 re-expression by hypoxia is HIF-1alpha-dependent and leads to cell cycle arrest. This may contribute to the survival of cancer cells in hypoxic parts of the tumour.

Hypoxia-regulated p53 and its effect on radiosensitivity in cancer cells

Purpose: To determine the response of tumor suppressor p53 to hypoxia in different tumor cell lines and the involvement of p53 activity regulation in the effect of hypoxia on tumor cell sensitivity to radiation and hyperthermia.Materials and methods: Three tumor cell lines with functional p53 were treated with chronic or cyclic hypoxia followed by radiation or hyperthermia to investigate p53 activity and cell survival. Flow cytometry was used to investigate the effect of hypoxia-induced cell cycle arrest on radiosensitivity in KHT-C (mouse fibrosarcoma) cells. Transient transfection was performed to determine the role of altered p53 activity in KHT-C and SCC VII (mouse squamous-cell carcinoma) radiosensitivity.Results: Aerobic radiosensitivity was decreased in KHT-C and SCC VII cells after in vitro chronic or cyclic hypoxia pretreatment, but in HT1080 cells, it was slightly increased after chronic hypoxia, and was unchanged after acute hypoxia pretreatment. Decreased radiosensitivity in hypoxia-pretreated KHT-C and SCC VII cells was unlikely due to hypoxia-induced cell cycle arrest, but rather seemed to be associated with increased expression of Mdm2 (mouse double minute-2) and decreased p53. Furthermore, hypoxia pretreatment inhibited the activation of p53 by radiation. Similar results were observed in hyperthermia treated KHT-C cells. Finally, decreased radiosensitivity was observed in both KHT-C and SCC VII cells transiently transfected with Mdm2 or anti-sense p53 cDNA.Conclusion: We demonstrated that hypoxia may decrease tumor cell radiosensitivity through the suppression of p53 activity in some tumor cell lines. These results suggested the response of p53 to hypoxia can be cell type specific and contribute to radiosensitivity of hypoxic cells.