BACKGROUND: Glioma, characterized by its undesirable prognosis and poor survival rate, is a serious threat to human health and lives. MicroRNA-9 (miR-9) is implicated in the regulation of multiple tumors, while the mechanisms underlying its aberrant expression and functional alterations in human glioma are still controversial.
METHODS: Expressions of miR-9 were measured in GEO database, patient specimens and glioma cell lines. Gain- and loss-of-function assays were applied to identify the effects of miR-9 on glioma cells and HUVECs in vitro and in vivo. Potential targets of miR-9 were predicted by bioinformatics and further verified via in vitro experiments. Transcriptional regulation of miR-9 by MYC and OCT4 was determined in glioma cells.
RESULTS: MiR-9 was frequently up-regulated in glioma specimens and cells, and could significantly enhance proliferation, migration and invasion of glioma cells. In addition, miR-9 could be secreted from glioma cells via exosomes and was then absorbed by vascular endothelial cells, leading to an increase in angiogenesis. COL18A1, THBS2, PTCH1 and PHD3 were verified as the direct targets of miR-9, which could elucidate the miR-9-induced malignant phenotypes in glioma cells. MYC and OCT4 were able to bind to the promoter region of miR-9 to trigger its transcription.
CONCLUSIONS: Our results highlight that miR-9 is pivotal for glioma pathogenesis and can be treated as a potential therapeutic target for glioma.

Most clear cell renal cell carcinomas (ccRCCs) have inactivation of the von Hippel-Lindau tumor suppressor protein (pVHL), resulting in the accumulation of hypoxia-inducible factor α-subunits (HIF-α) and their downstream targets. HIF-2α expression is particularly high in ccRCC and is associated with increased ccRCC growth and aggressiveness. In the canonical HIF signaling pathway, HIF-prolyl hydroxylase 3 (PHD3) suppresses HIF-2α protein by post-translational hydroxylation under sufficient oxygen availability. Here, using immunoblotting and immunofluorescence staining, qRT-PCR, and siRNA-mediated gene silencing, we show that unlike in the canonical pathway, PHD3 silencing in ccRCC cells leads to down-regulation of HIF-2α protein and mRNA. Depletion of other PHD family members had no effect on HIF-2α expression, and PHD3 knockdown in non-RCC cells resulted in the expected increase in HIF-2α protein expression. Accordingly, PHD3 knockdown decreased HIF-2α target gene expression in ccRCC cells and expression was restored upon forced HIF-2α expression. The effect of PHD3 depletion was pinpointed to

We have identified nine highly connected and differentially expressed gene subnetworks between aggressive primary tumors and metastatic lesions in endometrial carcinomas. We implemented a novel pipeline combining gene set and network approaches, which here allows integration of protein-protein interactions and gene expression data. The resulting subnetworks are significantly associated with disease progression across tumor stages from complex atypical hyperplasia, primary tumors to metastatic lesions. The nine subnetworks include genes related to metastasizing features such as epithelial-mesenchymal transition (EMT), hypoxia and cell proliferation. TCF4 and TWIST2 were found as central genes in the subnetwork related to EMT. Two of the identified subnetworks display statistically significant association to patient survival, which were further supported by an independent validation in the data from The Cancer Genome Atlas data collection. The first subnetwork contains genes related to cell proliferation and cell cycle, while the second contains genes involved in hypoxia such as HIF1A and EGLN3. Our findings provide a promising context to elucidate the biological mechanisms of metastasis, suggest potential prognostic markers and further identify therapeutic targets. The pipeline R source code is freely available, including permutation tests to assess statistical significance of the identified subnetworks.

Prostate cancer (PCA) is one of the most common cancer types in men, with cancer progression being linked to hypoxia and the induction of hypoxia-inducible factor (HIF).We investigated the expression of pyruvate kinase M2 (PKM2), its regulation by HIF isoforms 1α and 2α, and its role in HIF stabilization. We additionally examined cell survival in the prostate cancer cell lines PC3 and LNCaP under severe hypoxic (0.1% O2) and normoxic (20% O2) conditions. qRT-PCR showed higher up-regulation of PKM2 mRNA expression in LNCaP cells than in PC3 cells, while western blotting showed that PKM2 protein levels were up-regulated only in LNCaP cells. Inhibition of HIF-1α and HIF-2α by small interfering RNA (si-RNA) demonstrated HIF-1α dependent up-regulation of PKM2 at the mRNA and protein levels in LNCaP cells. PKM2 inhibition by si-RNA significantly decreased hypoxia-response element (HRE) activation in a gene reporter assay and down-regulated HIF-1α target vascular endothelial growth factor (VEGF) mRNA expression in PC3 cells, whereas HIF-1α protein levels were not significantly reduced. Additionally, PKM2 inhibition significantly reduced clonogenic survival in both cell lines in a colony formation assay. Prolyl hydroxylase 3 (PHD3) mRNA expression was up-regulated in both cell lines. It has been shown that PKM2 expression is regulated by HIF-1α and that PKM2 favors HIF-1α transactivation under mild (1% O2) but not severe (0.1% O2) hypoxic conditions, and some of our findings are consistent with these previous results. However, this mechanism was not fully observed in our studied cell lines, as PKM2 regulation and HIF-1α stabilization at the transactivation level occurred under severe hypoxic conditions. This discrepancy suggests that tumor tissue origin and cell type influence this model. Our findings expand the current knowledge of the mechanisms of PCA regulation, and would be important in developing novel therapeutic strategies.

BACKGROUND A hypoxic microenvironment is associated with resistance to tyrosine kinase inhibitors (TKIs) and a poor prognosis in chronic myeloid leukemia (CML). The E3 ubiquitin ligase Siah2 plays a vital role in the regulation of hypoxia response, as well as in leukemogenesis. However, the role of Siah2 in CML resistance is unclear, and it is unknown whether vitaminK3 (a Siah2 inhibitor) can improve the chemo-sensitivity of CML cells in a hypoxic microenvironment. MATERIAL AND METHODS The expression of Siah2 was detected in CML patients (CML-CP and CML-BC), K562 cells, and K562-imatinib-resistant cells (K562-R cells). We measured the expression of PHD3, HIF-1α, and VEGF in both cell lines under normoxia and hypoxic conditions, and the degree of leukemic sensitivity to imatinib and VitaminK3 were evaluated. RESULTS Siah2 was overexpressed in CML-BC patients (n=9) as compared to CML-CP patients (n=13). Similarly, K562-imatinib-resistant cells (K562-R cells) showed a significantly higher expression of Siah2 as compared to K562 cells in a hypoxic microenvironment. Compared to normoxia, under hypoxic conditions, both cell lines had lower PHD3, higher HIF-1α, and higher VEGF expression. Additionally, Vitamin K3 (an inhibitor of Siah2) reversed these changes and promoted a higher degree of leukemic sensitivity to imatinib. CONCLUSIONS Our findings indicate that the Siah2-PHD3- HIF-1α-VEGF axis is an important hypoxic signaling pathway in a leukemic microenvironment. An inhibitor of Siah2, combined with TKIs, might be a promising therapy for relapsing and refractory CML patients.

Neuroblastoma (NB) is the most common solid tumor during infancy and the first cause of death among the preschool age diseases. The availability of several NB genomic profiles improves the prognostic ability, but the outcome prediction for this pathology remains imperfect. We previously produced a novel prognostic gene signature based on the response of NB cells to hypoxia, a condition of tumor microenvironment strictly connected with cancer aggressiveness. Here we attempted to further define the expression of hypoxia-modulated specific genes, looking at their protein level in NB specimens, considering in particular the hypoxia inducible factor-1α (HIF-1α), the mitochondrial pyruvate dehydrogenase kinase 1 (PDK1), and the HIF-prolyl hydroxylase domain 3 (PHD3). The evaluation of expression was performed by Western blot and immunocytochemistry on NB cell lines and by immunohistochemistry on tumor specimens. Stimulation of both HIF-1α and PDK1 and inhibition of PHD3 expression were observed in NB cell lines cultured under prolonged hypoxic conditions as well as in most of the tumors with poor outcome. Our results indicate that the immunohistochemistry analysis of the protein expression of PDK1, PHD3, and HIF-1α defines the hypoxic status of NB tumors and can be used as a simple and relevant tool to stratify high-risk patients.

Prolyl hydroxylase 3 (PHD3) is widely accepted as a tumor suppressor; however, the expression of PHD3 in various cancer types remains controversial. The present study aimed to investigate the association between PHD3 expression and the clinicopathological features of gastric cancer using reverse transcription‑quantitative polymerase chain reaction and immunohistochemistry. The effects of PHD3 in gastric cancer cell lines were assessed using western blot analysis and transwell migration assays. The present results revealed that PHD3 expression was increased in adjacent non‑cancerous tissue compared with in gastric cancer tissue, and PHD3 overexpression was correlated with the presence of well‑differentiated cancer cells, early cancer stage classification and the absence of lymph node metastasis. In vitro experiments demonstrated that PHD3 may act as a negative regulator of hypoxia‑inducible factor‑1α and vascular endothelial growth factor, both of which participate in tumor angiogenesis. In conclusion, the present results suggested that PHD3 may act as a tumor suppressor in gastric cancer. Therefore, the targeted regulation of PHD3 may have potential as a novel therapeutic approach for the treatment of patients with gastric cancer.

BACKGROUND: Solid tumours are less oxygenated than normal tissues. Consequently, cancer cells acquire to be adapted to a hypoxic environment. The poor oxygenation of solid tumours is also a major indicator of an adverse cancer prognosis and leads to resistance to conventional anticancer treatments. We previously showed the involvement of Che-1/AATF (Che-1) in cancer cell survival under stress conditions. Herein we hypothesized that Che-1 plays a role in the response of cancer cells to hypoxia.
METHODS: The human colon adenocarcinoma HCT116 and HT29 cell lines undepleted or depleted for Che-1 expression by siRNA, were treated under normoxic and hypoxic conditions to perform studies regarding the role of this protein in metabolic adaptation and cell proliferation. Che-1 expression was detected using western blot assays; cell metabolism was assessed by NMR spectroscopy and functional assays. Additional molecular studies were performed by RNA seq, qRT-PCR and ChIP analyses.
RESULTS: Here we report that Che-1 expression is required for the adaptation of cells to hypoxia, playing an important role in metabolic modulation. Indeed, Che-1 depletion impacted on HIF-1α stabilization, thus downregulating the expression of several genes involved in the response to hypoxia and affecting glucose metabolism.
CONCLUSIONS: We show that Che-1 a novel player in the regulation of HIF-1α in response to hypoxia. Notably, we found that Che-1 is required for SIAH-2 expression, a member of E3 ubiquitin ligase family that is involved in the degradation of the hydroxylase PHD3, the master regulator of HIF-1α stability.

While much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition.

The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1-3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel-Lindau protein (VHL)-elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.

Seven-in-absentia homolog (SIAH) proteins are evolutionary conserved RING type E3 ubiquitin ligases responsible for the degradation of key molecules regulating DNA damage response, hypoxic adaptation, apoptosis, angiogenesis, and cell proliferation. Many studies suggest a tumorigenic role for SIAH2. In breast cancer patients SIAH2 expression levels correlate with cancer aggressiveness and overall patient survival. In addition, SIAH inhibition reduced metastasis in melanoma. The role of SIAH1 in breast cancer is still ambiguous; both tumorigenic and tumor suppressive functions have been reported. Other studies categorized SIAH ligases as either pro- or antimigratory, while the significance for metastasis is largely unknown. Here, we re-evaluated the effects of SIAH1 and SIAH2 depletion in breast cancer cell lines, focusing on migration and invasion. We successfully knocked down SIAH1 and SIAH2 in several breast cancer cell lines. In luminal type MCF7 cells, this led to stabilization of the SIAH substrate Prolyl Hydroxylase Domain protein 3 (PHD3) and reduced Hypoxia-Inducible Factor 1α (HIF1α) protein levels. Both the knockdown of SIAH1 or SIAH2 led to increased apoptosis and reduced proliferation, with comparable effects. These results point to a tumor promoting role for SIAH1 in breast cancer similar to SIAH2. In addition, depletion of SIAH1 or SIAH2 also led to decreased cell migration and invasion in breast cancer cells. SIAH knockdown also controlled microtubule dynamics by markedly decreasing the protein levels of stathmin, most likely via p27(Kip1). Collectively, these results suggest that both SIAH ligases promote a migratory cancer cell phenotype and could contribute to metastasis in breast cancer.

BACKGROUND: Hypoxia can halt cell cycle progression of several cell types at the G1/S interface. The arrest needs to be overcome by cancer cells. We have previously shown that the hypoxia-inducible cellular oxygen sensor PHD3/EGLN3 enhances hypoxic cell cycle entry at the G1/S boundary.
METHODS: We used PHD3 knockdown by siRNA and shRNA in HeLa and 786-0 renal cancer cells. Flow cytometry with cell synchronization was used to study cell growth at different cell cycle phases. Total and phosphospecific antibodies together with cycloheximide chase were used to study p27/CDKN1B expression and fractionations for subcellular protein localization.
RESULTS: Here we show that PHD3 enhances cell cycle by decreasing the expression of the CDK inhibitor p27/CDKN1B. PHD3 reduction led to increased p27 expression under hypoxia or VHL mutation. p27 was both required and sufficient for the PHD3 knockdown induced cell cycle block. PHD3 knockdown did not affect p27 transcription and the effect was HIF-independent. In contrast, PHD3 depletion increased the p27 half-life from G0 to S-phase. PHD3 depletion led to an increase in p27 phosphorylation at serine 10 without affecting threonine phosphorylation. Intact serine 10 was required for normal hypoxic and PHD3-mediated degradation of p27.
CONCLUSIONS: The data demonstrates that PHD3 can drive cell cycle entry at the G1/S transition through decreasing the half-life of p27 that occurs by attenuating p27S10 phosphorylation.

UNLABELLED: Renal cell carcinoma (RCC) is the most common cancer arising from the kidney in adults, with clear cell RCC (ccRCC) representing the majority of all RCCs. Expression of a human HIF1α triple-mutant (P402A, P564A, and N803A) construct in the proximal tubule cells of C57BL/6 mice [TRAnsgenic model of Cancer of the Kidney (TRACK); ref. 1] mimics the histologic changes found in early stage human ccRCC. To better understand the genomic landscape, a high-throughput sequence analysis was performed with cDNA libraries (RNAseq) derived from TRACK transgenic positive (TG(+)) kidney cortex along with human ccRCC transcripts from the Oncomine and The Cancer Genome Atlas databases. Importantly, the expression profiles of TRACK TG(+) kidneys show significant similarities with those observed in human ccRCC, including increased expression of genes involved in glycolysis and the tricarboxylic acid cycle. Some of the transcripts overexpressed in both the TRACK mouse model and human ccRCC include ANKRD37, CA9, EGLN3, HK2, NDUFA4L2, and SLC16A3. These data suggest that constitutive activation of HIF1α in kidney proximal tubule cells transcriptionally reprograms the regulation of metabolic pathways in the kidney and that HIF1α is a major contributor to the altered metabolism observed in human ccRCC.
IMPLICATIONS: TRACK (GGT-HIF1αM3) kidney mRNA profiles show similarities to human ccRCC transcriptome and phenotypes associated with the Warburg effect.

Solid tumours are exposed to microenvironmental factors such as hypoxia that normally inhibit cell growth. However, tumour cells are capable of counteracting these signals through mechanisms that are largely unknown. Here we show that the prolyl hydroxylase PHD3 restrains tumour growth in response to microenvironmental cues through the control of EGFR. PHD3 silencing in human gliomas or genetic deletion in a murine high-grade astrocytoma model markedly promotes tumour growth and the ability of tumours to continue growing under unfavourable conditions. The growth-suppressive function of PHD3 is independent of the established PHD3 targets HIF and NF-κB and its hydroxylase activity. Instead, loss of PHD3 results in hyperphosphorylation of epidermal growth factor receptor (EGFR). Importantly, epigenetic/genetic silencing of PHD3 preferentially occurs in gliomas without EGFR amplification. Our findings reveal that PHD3 inactivation provides an alternative route of EGFR activation through which tumour cells sustain proliferative signalling even under conditions of limited oxygen availability.

Tumours exploit their hypoxic microenvironment to induce a more aggressive phenotype, while curtailing the growth-inhibitory effects of hypoxia through mechanisms that are poorly understood. The prolyl hydroxylase PHD3 is regulated by hypoxia and plays an important role in tumour progression. Here we identify PHD3 as a central regulator of epidermal growth factor receptor (EGFR) activity through the control of EGFR internalization to restrain tumour growth. PHD3 controls EGFR activity by acting as a scaffolding protein that associates with the endocytic adaptor Eps15 and promotes the internalization of EGFR. In consequence, loss of PHD3 in tumour cells suppresses EGFR internalization and hyperactivates EGFR signalling to enhance cell proliferation and survival. Our findings reveal that PHD3 inactivation provides a novel route of EGFR activation to sustain proliferative signalling in the hypoxic microenvironment.

Tumor microenvironment favors the escape from immunosurveillance by promoting immunosuppression and blunting pro-inflammatory responses. Since most tumor-associated macrophages (TAM) exhibit an M2-like tumor cell growth promoting polarization, we have studied the role of 2G-03NN24 carbosilane dendrimer in M2 macrophage polarization to evaluate the potential application of dendrimers in tumor immunotherapy. We found that the 2G-03NN24 dendrimer decreases LPS-induced IL-10 production from in vitro generated monocyte-derived M2 macrophages, and also switches their gene expression profile towards the acquisition of M1 polarization markers (INHBA, SERPINE1, FLT1, EGLN3 and ALDH1A2) and the loss of M2 polarization-associated markers (EMR1, IGF1, FOLR2 and SLC40A1). Furthermore, 2G-03NN24 dendrimer decreases STAT3 activation. Our results indicate that the 2G-03NN24 dendrimer can be a useful tool for antitumor therapy by virtue of its potential ability to limit the M2-like polarization of TAM.

The extensive lipid accumulation occurring in clear-cell renal cell carcinoma (ccRCC) results in a clear-cell cytoplasm. Hypoxia-inducible factor α (HIF-α) is constitutively expressed in many ccRCC and transcriptionally regulates >100 genes. In a recent breakthrough study, HIF-1α induced ccRCC in transgenic mice. On the basis of these findings, we developed a hypothesis that accounted for HIF-α generation of the clear-cell phenotype. The aim of the present study was to use immunohistochemical staining methods in tissue microarray to determine the extent to which the clear-cell phenotype coincided with HIF-α expression in primary and metastatic ccRCC. In addition, we studied whether the prolyl-hydroxylases (PHD2,3) play a role in promoting the elevated expression of HIF-α in tumor cells. The clear-cell phenotype was observed in all primary and metastatic cases of ccRCC examined. A total of 168 renal cell carcinomas were evaluated by immunohistochemical methods; 141 of the 168 (84%) tumors expressed HIF-α (HIF-1α and/or HIF-2α). In contrast, HIF-α was expressed in only 1 of the 23 (4%) non-ccRCCs. These data supported the hypothesis that in the majority of the tumors HIF-α expression overlapped with the clear-cell phenotype and was indicative of an HIF-α-mediated lipid accumulation. In a smaller percentage of ccRCC cases (16%), HIF-α was not detected in the tumor cells and suggested that lipid accumulation by HIF-α-lipid-independent process. PHD3 was undetectable in both primary and metastatic ccRCC cases. We concluded that the undetectable PHD3 could contribute to the higher HIF-α expression in ccRCC.

Aberrant DNA methylation at CpG islands has been implicated as a critical player in colorectal cancer (CRC). However, its biological role and clinical significance in carcinogenesis have not been clearly clarified in Chinese CRC patients. In order to examine the methylation status of cancer-related genes in CRC progression, 184 tumor tissues were collected from Chinese patients diagnosed with CRC during 2008-2011. Promoter methylation was assessed by combined bisulphite-restriction analysis, methylation-specific PCR, and bisulphite sequencing PCR . The relationship between the gene promoter methylation status and clinicopathological factors/CRC mortality was examined by using the chi-square test/Cox-proportional hazards models. Promoter hypermethylation of MLH1, p16, SFRP2, PHD3, KLOTHO, and IGFBP7 was observed in 1.6, 10.9, 97.3, 44.0, 59.8, and 88.6 % of CRC samples, respectively. KLOTHO promoter methylation reduced with age (P = 0.018) whereas p16 promoter methylation increased with age (P = 0.044) and was more frequent among males (P = 0.017). Tumor tissues (73.9 %) had concurrent methylation of two or more genes, with the most frequent combination as KLOTHO and IGFBP7 (53.8 %). Concurrent methylation of KLOTHO and IGFBP7 occurred more frequently among patients less than 70 years old (P = 0.035) and those with poor differentiation (P = 0.024). CRC-specific mortality was not associated with promoter methylation and clinicopathological features except for age (P = 0.038; risk ratio (RR), 1.96; 95 % confidence interval (CI), 1.04-3.70) and TNM stage (P = 0.034; RR, 3.47; 95 % CI, 1.10-10.92). Methylation frequencies of MLH1, p16, PHD3, KLOTHO, and IGFBP7 in CRC tissues were significantly higher than that in the paired normal tissues, while promoter hypermethylation of SFRP2 was widespread in normal tissues. In conclusion, we suggest that methylation of some genes (MLH1, PHD3, KLOTHO, p16, and IGFBP7) is important in CRC progression whereas SFRP2 methylation is unlikely to contribute to CRC development in Chinese patients. Besides, by identifying the characteristics of concordant methylation, we confirm the multifactorial nature of tumor progression.

BACKGROUND: Aberrant DNA methylation is often associated with cancers. Thus, screening genes with cancer-associated aberrant DNA methylation is a useful method to identify candidate cancer-causing genes. Aberrant DNA methylation is also genotype dependent. Thus, the selection of genes with genotype-specific aberrant DNA methylation in cancers is potentially important for tailor-made medicine. The selected genes are important candidate drug targets.
RESULTS: The recently proposed principal component analysis based selection of genes with aberrant DNA methylation was applied to genotype and DNA methylation patterns in squamous cell carcinoma measured using single nucleotide polymorphism (SNP) arrays. SNPs that are frequently found in cancers are usually highly methylated, and the genes that were selected using this method were reported previously to be related to cancers. Thus, genes with genotype-specific DNA methylation patterns will be good therapeutic candidates. The tertiary structures of the proteins encoded by the selected genes were successfully inferred using two profile-based protein structure servers, FAMS and Phyre2. Candidate drugs for three of these proteins, tyrosine kinase receptor (ALK), EGLN3 protein, and NUAK family SNF1-like kinase 1 (NUAK1), were identified by ChooseLD.
CONCLUSIONS: We detected genes with genotype-specific DNA methylation in squamous cell carcinoma that are candidate drug targets. Using in silico drug discovery, we successfully identified several candidate drugs for the ALK, EGLN3 and NUAK1 genes that displayed genotype-specific DNA methylation.

PURPOSE: We previously found that hypoxia-inducible factor (HIF) prolyl hydroxylase-3 (PHD3) was frequently overexpressed in renal cell carcinomas (RCCs), unlike in normal tissues, and therefore, we studied the mechanism and role of PHD3 expression in RCC.
METHODS: The von Hippel-Lindau (VHL)-gene-mutant RCC cell lines SMKT-R2 and SMKT-R3 and wild-type VHL cell lines Caki-1 and ACHN were used. Associations of the expression of PHD3 with HIF-α proteins and signal transduction pathways were evaluated under normoxic conditions. The effect of PHD3 on cell proliferation was also examined by small interference RNA and cDNA transfection. Moreover, the prognostic impact of PHD3 expression in clear cell RCC (CCRCC) was evaluated using primary cancer tissues.
RESULTS: In SMKT-R2 and SMKT-R3, HIF-α proteins were expressed and PHD3 was highly expressed. On the other hand, ACHN had low expression of HIF-α proteins and PHD3. However, Caki-1 had high expression of PHD3 even though there was no distinct expression of HIF-α proteins. PHD3 expression was inhibited by blockade of Akt and mammalian target of rapamycin (mTOR), but not by HIF-1α and HIF-2α double knockdown. In addition, PHD3 knockdown resulted in the promotion of cell proliferation in SMKT-R2, SMKT-R3 and Caki-1. On the other hand, forced expression of PHD3 reduced cell proliferation in ACHN. In immunohistochemistry, PHD3 expression was a significant factor for better recurrence-free survival in patients with CCRCC.
CONCLUSIONS: PHD3 expression can be induced by the phosphatidylinositol-3 kinase/Akt/mTOR pathway in RCC independently of HIF proteins. Furthermore, PHD3 has an antiproliferative function independent of HIF protein status in RCC, indicating a novel expression mechanism and function of PHD3.

OBJECTIVES: The purpose of this study was to identify featured miRNAs of hepatocellular carcinoma (HCC) by comparing normal and cancer cell line samples and find potential utility as biomarkers for early diagnosis and treatment of HCC.
MATERIALS AND METHODS: We downloaded the gene expression profile GSE41077 from Gene Expression Omnibus database which included 6 HCC cell lines samples and 2 controls. Differentially expressed miRNAs were identified by multtest package in R language after the data normalization. The selected differentially expressed miRNAs were further analyzed using bioinformatics methods. Target genes of these miRNAs were predicted using miRTarBase and miRecords databases. STRING software was used to construct the interaction network of target genes. Finally, we made module analysis by using Cytoscape software and its plugins--MCODE and BiNGO.
RESULTS: A total of 40 differentially expressed miRNAs were identified and the remarkably down-regulated miRNA was hsa-miR-122 which included 29 high confident target genes. The interaction network of target genes was constructed among 629 interaction pairs. Four functional modules in the network were obtained, from which EGLN3, ALDOA, NCAM1 and AACS were the high confident target genes, respectively. Genes in the modules most related to biological functions of signal transmission, regulation of macromolecule metabolic process.
CONCLUSIONS: Low level of expression of hsa-miR-122 in HCC cell line is consistent with the existed previous studies. It is not only confirm the importance role of such miRNA in HCC cells, but also provide important help in identifying specific biomarker of HCC cells.

The histone lysine demethylase KDM5B regulates gene transcription and cell differentiation and is implicated in carcinogenesis. It contains multiple conserved chromatin-associated domains, including three PHD fingers of unknown function. Here, we show that the first and third, but not the second, PHD fingers of KDM5B possess histone binding activities. The PHD1 finger is highly specific for unmodified histone H3 (H3K4me0), whereas the PHD3 finger shows preference for the trimethylated histone mark H3K4me3. RNA-seq analysis indicates that KDM5B functions as a transcriptional repressor for genes involved in inflammatory responses, cell proliferation, adhesion, and migration. Biochemical analysis reveals that KDM5B associates with components of the nucleosome remodeling and deacetylase (NuRD) complex and may cooperate with the histone deacetylase 1 (HDAC1) in gene repression. KDM5B is downregulated in triple-negative breast cancer relative to estrogen-receptor-positive breast cancer. Overexpression of KDM5B in the MDA-MB 231 breast cancer cells suppresses cell migration and invasion, and the PHD1-H3K4me0 interaction is essential for inhibiting migration. These findings highlight tumor-suppressive functions of KDM5B in triple-negative breast cancer cells and suggest a multivalent mechanism for KDM5B-mediated transcriptional regulation.

Prolyl-4-hydroxylation by the intracellular prolyl-4-hydroxylase enzymes (PHD1-3) serves as a master regulator of environmental oxygen sensing. The activity of these enzymes is tightly tied to tumorigenesis, as they regulate cell metabolism and angiogenesis through their control of hypoxia-inducible factor (HIF) stability. PHD3 specifically, is gaining attention for its broad function and rapidly accumulating array of non-HIF target proteins. Data from several recent studies suggest a role for PHD3 in the regulation of cell morphology and cell migration. In this study, we aimed to investigate this role by closely examining the relationship between PHD3 expression and epithelial-to-mesenchymal transition (EMT); a transcriptional program that plays a major role in controlling cell morphology and migratory capacity. Using human pancreatic ductal adenocarcinoma (PDA) cell lines and Madin-Darby Canine Kidney (MDCK) cells, we examined the correlation between several markers of EMT and PHD3 expression. We demonstrated that loss of PHD3 expression in PDA cell lines is highly correlated with a mesenchymal-like morphology and an increase in cell migratory capacity. We also found that induction of EMT in MDCK cells resulted in the specific downregulation of PHD3, whereas the expression of the other HIF-PHD enzymes was not affected. The results of this study clearly support a model by which the basal expression and hypoxic induction of PHD3 is suppressed by the EMT transcriptional program. This may be a novel mechanism by which migratory or metastasizing cells alter signaling through specific pathways that are sensitive to regulation by O2. The identification of downstream pathways that are affected by the suppression of PHD3 expression during EMT may provide important insight into the crosstalk between O2 and the migratory and metastatic potential of tumor cells.

In this paper, we provide a comprehensive summary of available clear cell renal cell carcinoma (ccRCC) microarray data in the form of meta-analysis of genes differentially regulated in tumors as compared to healthy tissue, using effect size to measure the strength of a relationship between the disease and gene expression. We identified 725 differentially regulated genes, with a number of interesting targets, such as TMEM213, SMIM5, or ATPases: ATP6V0A4 and ATP6V1G3, of which limited or no information is available in terms of their function in ccRCC pathology. Downregulated genes tended to represent pathways related to tissue remodeling, blood clotting, vasodilation, and energy metabolism, while upregulated genes were classified into pathways generally deregulated in cancers: immune system response, inflammatory response, angiogenesis, and apoptosis. One hundred fifteen deregulated genes were included in network analysis, with EGLN3, AP-2, NR3C1, HIF1A, and EPAS1 (gene encoding HIF2-α) as points of functional convergence, but, interestingly, 610 genes failed to join previously identified molecular networks. Furthermore, we validated the expression of 14 top deregulated genes in independent sample set of 32 ccRCC tumors by qPCR and tested if it could serve as a marker of disease progression. We found a correlation of high fucosyltransferase 11 (FUT11) expression with non-symptomatic course of the disease, which suggests that FUT11's expression might be potentially used as a biomarker of disease progression.

Somatic mutations or loss of expression of tumor suppressor VHL happen in the vast majority of clear cell Renal Cell Carcinoma, and it's causal for kidney cancer development. Without VHL, constitutively active transcription factor HIF is strongly oncogenic and is essential for tumor growth. However, the contribution of individual HIF-responsive genes to tumor growth is not well understood. In this study we examined the contribution of important HIF-responsive genes such as VEGF, CCND1, ANGPTL4, EGLN3, ENO2, GLUT1 and IGFBP3 to tumor growth in a xenograft model using immune-compromised nude mice. We found that the suppression of VEGF or CCND1 impaired tumor growth, suggesting that they are tumor-promoting genes. We further discovered that the lack of ANGPTL4, EGLN3 or ENO2 expression did not change tumor growth. Surprisingly, depletion of GLUT1 or IGFBP3 significantly increased tumor growth, suggesting that they have tumor-inhibitory functions. Depletion of IGFBP3 did not lead to obvious activation of IGFIR. Unexpectedly, the depletion of IGFIR protein led to significant increase of IGFBP3 at both the protein and mRNA levels. Concomitantly, the tumor growth was greatly impaired, suggesting that IGFBP3 might suppress tumor growth in an IGFIR-independent manner. In summary, although the overall transcriptional activity of HIF is strongly tumor-promoting, the expression of each individual HIF-responsive gene could either enhance, reduce or do nothing to the kidney cancer tumor growth.

BACKGROUND: Colorectal cancer (CRC) is one of the most common and comprehensively studied malignancies. Hypoxic conditions during formation of CRC may support the development of more aggressive cancers. Hypoxia inducible factor (HIF), a major player in cancerous tissue adaptation to hypoxia, is negatively regulated by the family of prolyl hydroxylase enzymes (PHD1, PHD2, PHD3) and asparaginyl hydroxylase, called factor inhibiting HIF (FIH).
METHODS: PHD1, PHD2, PHD3 and FIH gene expression was evaluated using quantitative RT-PCR and western blotting in primary colonic adenocarcinoma and adjacent histopathologically unchanged colonic mucosa from patients who underwent radical surgical resection of the colon (n=90), and the same methods were used for assessment of PHD3 gene expression in HCT116 and DLD-1 CRC cell lines. DNA methylation levels of the CpG island in the promoter regulatory region of PHD1, PHD2, PHD3 and FIH were assessed using bisulfite DNA sequencing and high resolution melting analysis (HRM) for patients and HRM analysis for CRC cell lines.
RESULTS: We found significantly lower levels of PHD1, PHD2 and PHD3 transcripts (p=0.00026; p<0.00001; p<0.00001) and proteins (p=0.004164; p=0.0071; p<0.00001) in primary cancerous than in histopathologically unchanged tissues. Despite this, we did not observe statistically significant differences in FIH transcript levels between cancerous and histopathologically unchanged colorectal tissue, but we found a significantly increased level of FIH protein in CRC (p=0.0169). The reduced PHD3 expression was correlated with significantly increased DNA methylation in the CpG island of the PHD3 promoter regulatory region (p<0.0001). We did not observe DNA methylation in the CpG island of the PHD1, PHD2 or FIH promoter in cancerous and histopathologically unchanged colorectal tissue. We also showed that 5-Aza-2'-deoxycytidine induced DNA demethylation leading to increased PHD3 transcript and protein level in HCT116 cells.
CONCLUSION: We demonstrated that reduced PHD3 expression in cancerous tissue was accompanied by methylation of the CpG rich region located within the first exon and intron of the PHD3 gene. The diminished expression of PHD1 and PHD2 and elevated level of FIH protein in cancerous tissue compared to histopathologically unchanged colonic mucosa was not associated with DNA methylation within the CpG islands of the PHD1, PHD2 and FIH genes.

We have previously reported on the inhibition of HIF-1α (hypoxia-inducible factor α)-regulated pathways by HEXIM1 [HMBA (hexamethylene-bis-acetamide)-inducible protein 1]. Disruption of HEXIM1 activity in a knock-in mouse model expressing a mutant HEXIM1 protein resulted in increased susceptibility to the development of mammary tumours, partly by up-regulation of VEGF (vascular endothelial growth factor) expression, HIF-1α expression and aberrant vascularization. We now report on the mechanistic basis for HEXIM1 regulation of HIF-1α. We observed direct interaction between HIF-1α and HEXIM1, and HEXIM1 up-regulated hydroxylation of HIF-1α, resulting in the induction of the interaction of HIF-1α with pVHL (von Hippel-Lindau protein) and ubiquitination of HIF-1α. The up-regulation of hydroxylation involves HEXIM1-mediated induction of PHD3 (prolyl hydroxylase 3) expression and interaction of PHD3 with HIF-1α. Acetylation of HIF-1α has been proposed to result in increased interaction of HIF-1α with pVHL and induced pVHL-mediated ubiquitination, which leads to the proteasomal degradation of HIF-1α. HEXIM1 also attenuated the interaction of HIF-1α with HDAC1 (histone deacetylase 1), resulting in acetylation of HIF-1α. The consequence of HEXIM1 down-regulation of HIF-1α protein expression is attenuated expression of HIF-1α target genes in addition to VEGF and inhibition of HIF-1α-regulated cell invasion.

BACKGROUND: Previous studies suggest that expression of hypoxia markers may be associated with response to antiangiogenic drugs. Thus, we aimed to identify predictors of sunitinib outcome in clear-cell renal cell carcinoma (ccRCC).
PATIENTS AND METHODS: The expression of eight key proteins related to hypoxia (CAIX, HIF1A, HIF2A, VEGFA, VEGFR1, VEGFR2, VEGFR3 and PDGFRB) and P-glycoprotein were assessed by immunohistochemistry in 67 primary ccRCC samples from prospectively recruited patients treated with first-line sunitinib. The proteins expression, VHL inactivation and EGLN3 mRNA content were compared with the patients' response to sunitinib.
RESULTS: High expression of HIF2A and PDGFRB was associated with better sunitinib RECIST objective response (P = 0.024 and P = 0.026; respectively) and increased VEGFR3 expression was associated with longer progression-free survival (P = 0.012). VEGFR3 overexpression showed a negative correlation with VEGFR3 polymorphism rs307826 (P = 0.002), a sunitinib resistance predictor. With respect to overall survival (OS), high VEGFA was associated with short (P = 0.009) and HIF2A with long (P = 0.048) survival times. High EGLN3 mRNA content was associated with shorter OS (P = 0.023).
CONCLUSIONS: We found an association between several proteins involved in hypoxia and sunitinib efficacy. In addition, low VEGFR3 expression was associated with worse outcome and with VEGFR3 rs307826 variant allele, reinforcing VEGFR3 as a marker of sunitinib resistance.

Our previous studies revealed that gambogic acid (GA), the major active ingredient of gamboge, possessed antiangiogenic activities. In this study, we further explored the mechanism of inhibition effects of GA in tumor angiogenesis. The results of luciferase, RT-PCR, and ELISA assays indicated that GA significantly decreased transcription activation, mRNA expression, and secretion of VEGF in hypoxia. We detected that GA had no effect on mRNA level of HIF-1α which targets VEGF gene, but the increase of HIF-1α protein expression in hypoxia was repressed by GA, which can be reversed by proteasomal inhibitor MG132 and siRNA of VHL. But GA exhibited no effect on expression of VHL both in normoxia and hypoxia. HIF prolyl hydroxylases (PHD enzymes) act as oxygen sensors regulating HIF, and hence angiogenesis. Our results showed that GA potentially enhanced level of PHD2, the most important HIF hydroxylase, and showed no effect on PHD1 and PHD3. Transient transfection of siRNA of PHD2 could eliminate GA-induced VEGF secretion increase. Growth of HepG2 xenografts in BALB/cA nude mice was inhibited by GA and angiogenesis was repressed significantly in tumor xenografts by immunohistochemical staining of CD-31, a vascular endothelial marker, accompanied with decrease of HIF-1α and increase of PHD2 expression in tissue extracts. This work provides the demonstration that GA shows anti-angiogenic effects via inhibiting PHD2-VHL-HIF-1α pathway.

STAT5 fulfills essential roles in hematopoietic stem cell (HSC) self-renewal and chronic myeloid leukemia (CML), a prototypical stem cell malignancy. However, the specific contributions of the two related genes STAT5A and STAT5B have not been determined. In this study, we used a RNAi-based strategy to establish participation of these genes to CML disease and persistence following targeted therapy. We showed that STAT5A/STAT5B double-knockdown triggers CML cell apoptosis and suppresses both normal and CML HSC long-term clonogenic potential. STAT5A and STAT5B exhibited similar prosurvival activity, but STAT5A attenuation alone was ineffective at impairing growth of normal and CML CD34(+) cells isolated at diagnosis. In contrast, STAT5A attenuation was sufficient to enhance basal oxidative stress and DNA damage of normal CD34(+) and CML cells. Furthermore, it weakened the ability to manage exogenous oxidative stress, increased p53 (TRP53)/CHK-2 (CHEK2) stress pathway activation, and enhanced prolyl hydroxylase domain (PHD)-3 (EGLN3) mRNA expression. Only STAT5A and its transactivation domain-deficient mutant STAT5AΔ749 specifically rescued these activities. STAT5A attenuation was also active at inhibiting growth of CML CD34(+) cells from patients with acquired resistance to imatinib. Our findings show that STAT5A has a selective role in contributing to stress resistance through unconventional mechanisms, offering new opportunities to eradicate the most primitive and tyrosine kinase inhibitor-resistant CML cells with an additional potential to eradicate persistent stem cell populations.