The hypoxic microenvironment is one of the major features of solid tumors, which regulates cell malignancy in multiple ways. As a response to hypoxia, a large number of target genes involved in cell growth, metabolism, metastasis and immunity are activated in cancer cells. Hypoxia-inducible factor 1 (HIF-1), as a heterodimeric DNA-binding complex, is comprised of a constitutively expressed HIF-1β subunit and an oxygen sensitive HIF-1α subunit, thus, adapts to decreased oxygen availability as a transcriptional factor. HIF-1 regulates many genes involved in tumorigenesis. Here, we focus on cancer cell metabolism and metastasis regulated by hypoxia.

Mechanism of PAH mixtures, using granulosa tumour cells, was investigated. Cells were exposed to a mixture of all 16 priority PAHs (M1) or a mixture of five PAHs not classified as human carcinogens (M2). The effect of siAHR, siAHRR and siNFKB2 on the expression of CYP1A1, CYP1B1, GSTM1, ERα, AR and cell proliferation was described. M1 decreased AhR and CYP1A1, while increased AhRR and ARNT expression. M2 also decreased AhR and CYP1A1 but had no effect on AhRR expression. siAHRR reversed the inhibitory effect of M1 on AhR and CYP1A1,while inhibitory effect of M2 was still observed. siNFKB2 reversed inhibitory effect of both mixtures on AhR and CYP1A1 expression and stimulatory effect of M1 on AhRR expression. siAHR reversed stimulatory effect of both mixtures on ERα expression. Stimulatory effect of M1 on cell proliferation was not observed in siAHR, was still observed in siESR1 cells. M2 had no effect on cell proliferation, however stimulatory effect was appeared in siAHR and siESR1cells. In conclusion: M1 by activation of AhRR and NFkB p52, but M2 only by activation of NFκB attenuated AhR signalling and ligand-induced CYP1A1 expression. Interaction between AhR and ER following M1 and M2 exposure is primarily initiated through AhR.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that consists of two subunits, the HIF-1α and HIF-1β (ARNT). Under hypoxic conditions, HIF-1 is an adaptive system that regulates the transcription of multiple genes associated with growth, angiogenesis, proliferation, glucose transport, metabolism, pH regulation and cell death. However, aberrant HIF-1 activation contributes to the pathophysiology of several human diseases such as cancer, ischemic cardiovascular disorders, and pulmonary and kidney diseases. A growing body of evidence indicates that curcumin, a natural bioactive compound of turmeric root, significantly targets both HIF-1 subunits, but is more potent against HIF-1α. In this review, we have summarized the knowledge about the pharmacological effects of curcumin on HIF-1 and the related molecular mechanisms that may be effective candidates for the development of multi-targeted therapy for several human diseases.

BACKGROUND/AIM: Predicting response to treatment in high-grade serous ovarian carcinoma (HGSOC) still remains a clinical challenge. The standard-of-care for first-line chemotherapy, based on a combination of carboplatin and paclitaxel, achieves a high response rate. However, the development of drug resistance is one of the major limitations to efficacy. Therefore, identification of biomarkers able to predict response to chemotherapy in patients with HGSOC is a critical step for prognosis and treatment of the disease. Several studies suggest that angiogenesis is an important process in the development of ovarian carcinoma and chemoresistance. The aim of this study was to identify a profile of angiogenesis-related genes as a biomarker for response to first-line chemotherapy in HGSOC.
MATERIALS AND METHODS: Formalin-fixed paraffin-embedded samples from 39 patients with HGSOC who underwent surgical cytoreduction and received a first-line chemotherapy with carboplatin and paclitaxel were included in this study. Expression levels of 82 angiogenesis-related genes were measured by quantitative real-time polymerase chain reaction using TaqMan low-density arrays.
RESULTS: Univariate analysis identified five genes [angiopoietin 1 (ANGPT1), aryl hydrocarbon receptor nuclear translocator (ARNT), CD34, epidermal growth factor (EGF) and matrix metallopeptidase 3 (MMP3)] as being statistically associated with response to treatment. Multivariable analysis by Lasso-penalized Cox regression generated a model with the combined expression of seven genes [angiotensinogen (AGT), CD34, EGF, erythropoietin receptor (EPOR), interleukin 8 (IL8), MMP3 and MMP7)]. The area under the receiver operating characteristics curve (0.679) and cross-validated Kaplan-Meier survival curves were used to estimate the accuracy of these predictors.
CONCLUSION: An angiogenesis-related gene expression profile useful for response prediction in HGSOC was identified, supporting the important role of angiogenesis in HGSOC.

BACKGROUND: Gastrointestinal stromal tumour (GIST) is the most common soft tissue sarcoma. The identification of the molecular mechanisms regulating GIST progression is vital for its treatment and prevention. Increasing reports have demonstrated that epigenetic alterations play critical roles in GIST development. However, the role of the histone demethylase KDM4D in GIST progression is poorly understood.
METHODS: In clinically matched GIST tissues, KDM4D protein levels were measured by Western blot and immunohistochemical (IHC) staining. KDM4D mRNA levels were examined by quantitative real-time PCR (qRT-PCR). Bioinformatics analysis was used to examine KDM4D expression. The biological effects of KDM4D were investigated in vitro using CCK-8, BrdU/PI, wound healing, colony formation, tube formation and Transwell assays and in vivo using a xenograft mice model. Luciferase assays were used to assess regulation of HIF1β gene promoter activity by KDM4D. ChIP assays were performed to assess KDM4D, H3K36me3 and H3K9me3 occupancy on the HIF1β gene promoter.
RESULTS: We observed a significant upregulation of KDM4D in GIST tissue compared with matched normal tissue and further explored the oncogenic function of KDM4D both in vitro and in vivo. Furthermore, we demonstrated that KDM4D directly interacted with the HIF1β gene promoter and regulated its activity, promoting tumour angiogenesis and GIST progression both in vitro and in vivo. Finally, we demonstrated that KDM4D transcriptionally activates HIF1β expression via H3K9me3 and H3K36me3 demethylation at the promoter region.
CONCLUSIONS: Our findings reveal the important roles of the KDM4D/HIF1β/VEGFA signalling pathway in GIST progression, and this pathway may act as a potential therapeutic target for GIST patients.

Oral squamous cell carcinoma has a high potential for locoregional invasion and nodal metastasis. Consequently, early detection of such malignancies is of immense importance. The melanoma inhibitory activity (MIA) gene family comprises MIA, MIA2, transport and Golgi organization protein 1 (TANGO), and otoraplin (OTOR). These members of the MIA gene family have a highly conserved Src homology 3 (SH3)-like structure. Although the molecules of this family share 34-45% amino acid homology and 47-59% cDNA sequence homology, those members, excluding OTOR, play different tumor-associated functions. MIA has a pivotal role in the progression and metastasis of melanoma; MIA2 and TANGO have been suggested to possess tumor-suppressive functions; and OTOR is uniquely expressed in cochlea of the inner ear. Therefore, the definite functions of the MIA gene family in cancer cells remain unclear. Since the members of the MIA gene family are secreted proteins, these molecules might be useful tumor markers that can be detected in the body fluids, including serum and saliva. In this review, we described the molecular biological functions of the MIA gene family in oral cancer.

BACKGROUND: Various endocrine signals oscillate over the 24-hour period and so does the responsiveness of target tissues. These daily oscillations do not occur solely in response to external stimuli but are also under the control of an intrinsic circadian clock.
DESIGN: We searched the PubMed database to identify studies describing the associations of clock genes with endocrine diseases.
RESULTS: Various human single nucleotide polymorphisms of brain and muscle ARNT-like 1 (BMAL1) and Circadian Locomotor Output Cycles Kaput (CLOCK) genes exhibited significant associations with type 2 diabetes mellitus. ARNTL2 gene expression and upregulation of BMAL1 and PER1 were associated with the development of type 1 diabetes mellitus. Thyroid hormones modulated PER2 expression in a tissue-specific way, whereas BMAL1 regulated the expression of type 2 iodothyronine deiodinase in specific tissues. Adrenal gland and adrenal adenoma expressed PER1, PER2, CRY2, CLOCK and BMAL1 genes. Adrenal sensitivity to adrenocorticotrophin was also affected by circadian oscillations. A significant correlation between the expression of propio-melanocorticotrophin and PER 2, as well as between prolactin and CLOCK, was found in corticotroph and lactosomatotroph cells, respectively, in the pituitary. Clock genes and especially BMAL1 showed an important role in fertility, whereas oestradiol and androgens exhibited tissue-specific effects on clock gene expression. Metabolic disorders were also associated with circadian dysregulation according to studies in shift workers.
CONCLUSIONS: Clock genes are associated with various endocrine disorders through complex mechanisms. However, data on humans are scarce. Moreover, clock genes exhibit a tissue-specific expression representing an additional level of regulation. Their specific role in endocrine disorders and their potential implications remain to be further clarified.

BACKGROUND: Carotid body tumor (CBT) is a form of head and neck paragangliomas (HNPGLs) arising at the bifurcation of carotid arteries. Paragangliomas are commonly associated with germline and somatic mutations involving at least one of more than thirty causative genes. However, the specific functionality of a number of these genes involved in the formation of paragangliomas has not yet been fully investigated.
METHODS: Exome library preparation was carried out using Nextera® Rapid Capture Exome Kit (Illumina, USA). Sequencing was performed on NextSeq 500 System (Illumina).
RESULTS: Exome analysis of 52 CBTs revealed potential driver mutations (PDMs) in 21 genes: ARNT, BAP1, BRAF, BRCA1, BRCA2, CDKN2A, CSDE1, FGFR3, IDH1, KIF1B, KMT2D, MEN1, RET, SDHA, SDHB, SDHC, SDHD, SETD2, TP53BP1, TP53BP2, and TP53I13. In many samples, more than one PDM was identified. There are also 41% of samples in which we did not identify any PDM; in these cases, the formation of CBT was probably caused by the cumulative effect of several not highly pathogenic mutations. Estimation of average mutation load demonstrated 6-8 mutations per megabase (Mb). Genes with the highest mutation rate were identified.
CONCLUSIONS: Exome analysis of 52 CBTs for the first time revealed the average mutation load for these tumors and also identified potential driver mutations as well as their frequencies and co-occurrence with the other PDMs.

Degree of histological differentiation is an important characteristic of cancers and may be associated with malignant potential. However, in squamous cell carcinomas, a key transcriptional factor regulating tumor differentiation is largely unknown. Chemoradiotherapy (CRT) is a standard treatment for locally advanced esophageal squamous cell carcinoma; however, the survival rate is still below 40%. From microarray data, single-minded 2 (SIM2) was overexpressed in the epithelial subtype. Here, we investigated the correlation between SIM2 expression and its clinical implication, and in vitro and in vivo functions of SIM2 in tumor differentiation and in CRT sensitivity. Although SIM2 was suppressed in cancerous tissues, SIM2-high ESCC showed a favorable prognosis in CRT. Transient SIM2 expression followed by 3D culture induced expression of differentiation markers and suppressed epithelial-mesenchymal transition- and basal-cell markers. Levels of PDPN-high tumor basal cells and of expression of genes for DNA repair and antioxidant enzymes were reduced in stable transfectants, and they showed high CDDP and H

Chromatin immunoprecipitation (ChIP) is a powerful method to determine whether a protein of interest binds to specific regulatory elements of the genome. Herein, we outline protocols optimized to detect binding of Hypoxia-Inducible Factor (HIF)-1α or HIF-2α to putative hypoxia response elements (HREs) within HIF target genes expressed in breast tumor epithelial cells.

Increasing epidemiological and animal experimental data provide substantial support for the role of aryl hydrocarbon receptor (AhR) in mammary tumorigenesis. The effects of AhR have been clearly demonstrated in rodent models of breast carcinogenesis and in several established human breast cancer cell lines following exposure to AhR ligands or AhR overexpression. However, relatively little is known about the role of AhR in human breast cancers. AhR has always been considered to be a regulator of toxic and carcinogenic responses to environmental contaminants such as TCDD (dioxin) and benzo[a]pyrene (BaP). The aim of this study was to identify the type of breast tumors (ERα-positive or ERα-negative) that express AHR and how AhR affects human tumorigenesis. The levels of AHR, AHR nuclear translocator (ARNT) and AHR repressor (AHRR) mRNA expression were analyzed in a cohort of 439 breast tumors, demonstrating a weak association between high AHR expression and age greater than fifty years and ERα-negative status, and HR-/ERBB2 breast cancer subtypes. AHRR mRNA expression was associated with metastasis-free survival, while AHR mRNA expression was not. Immunohistochemistry revealed the presence of AhR protein in both tumor cells (nucleus and/or cytoplasm) and the tumor microenvironment (including endothelial cells and lymphocytes). High AHR expression was correlated with high expression of several genes involved in signaling pathways related to inflammation (IL1B, IL6, TNF, IL8 and CXCR4), metabolism (IDO1 and TDO2 from the kynurenine pathway), invasion (MMP1, MMP2 and PLAU), and IGF signaling (IGF2R, IGF1R and TGFB1). Two well-known ligands for AHR (TCDD and BaP) induced mRNA expression of IL1B and IL6 in an ERα-negative breast tumor cell line. The breast cancer ER status likely influences AhR activity involved in these signaling pathways. The mechanisms involved in AhR activation and target gene expression in breast cancers are also discussed.

Aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor and involved in tumorigenesis of many cancers. However there are no reports on AHR in human meningioma. Therefore we examined the status of the AHR and its signalling molecules in human meningioma by using tumor biopsy samples and autopsy control meninges. We report the up regulation of AHR pathway genes like aryl hydrocarbon receptor nuclear translocator (ARNT), aldehyde dehydrogenase1family memberA3 (ALDH1A3), cytochrome P450, family1, subfamily A polypeptide1 (CYP1A1) and TCCD induced poly ADP ribose polymerase (TIPARP) gene expression in human meningioma. Further, AHR protein expression was found to be up regulated in all grades of human meningioma. We found that AHR localized in the nucleus for high grade anaplastic meningioma through immunohistochemical analysis. Since AHR signalling pathway was known to involve in inhibition of apoptosis in cancer cells, we evaluated the cyclophilin D levels which maintains mitochondrial permeability transition pore a critical event during apoptosis. We report that cyclophilin D levels were upregulated in all grades of human meningioma compared to control meninges. Finally we also evaluated c-Fos protein levels as its levels were regulated by AHR. Here we report that c-Fos protein levels were down regulated in all grades of human meningioma compared to control meninges. To sum-up we found that AHR signalling pathway components were upregulated, as the grade of the meningioma progresses from low to high grade, suggesting an important role of AHR signalling pathway in human meningioma.

MicroRNAs serve a crucial role in the regulation of malignant biological behavior of Ewing's sarcoma (ES). Abnormal expression of miR-107 has been reported in a cohort of cancers, while its exact function in ES remains unclear. Hence, we explored the expression of miR-107 in ES cells and detected its effects on the malignant phenotype of ES cells. Firstly, we perceived the under-expression of miR-107 in human ES cells contrast with the human mesenchymal stem cells. Over-expression of miR-107 restrained cell proliferation and tube formation, arrested cell cycle progression, and facilitated cell apoptosis in SK-ES-1 and RD-ES cell lines. Furthermore, hypoxia inducible factor-1β (HIF-1β) was assumed as a target gene of miR-107. We confirmed the target role of HIF-1β in ES cells. Finally, restoring the expression of HIF-1β could partly abolish miR-107-mediated tumor suppression in ES cells. In conclusion, our results advised that miR-107 suppressed the malignant biological ability of ES cells through targeting HIF-1β.

INTRODUCTION: TRIP11 is a multifunctional protein localizing either to Golgi apparatus, acting as a golgin, or in the nucleus, acting as coactivator of transcription mediated by thyroid hormone receptor (THR) and hypoxia induced factor (HIF). Triiodothyronine (T3) regulates nuclear localization of TRIP11 by inducing its phosphorylation. The exact mechanism of this regulation unknown. The expressions of THR and HIF are disturbed in various cancers, including renal cell cancer (RCC). In this study we aimed to analyze: 1) the mechanism of T3-dependent subcellular localization of TRIP11; 2) the significance of TRIP11 and T3 signaling pathway in RCC progression.
MATERIAL AND METHODS: TRIP11 subcellular localization was analyzed using immunocytochemistry in RCC-derived cell line treated with T3, T3-agarose and PI3K inhibitor, wortmannin. The expressions of TRIP11 and genes involved in T3 signaling and hypoxia were investigated using qPRC in 36 pairs of RCC tumor-control samples, followed by validation/survival analysis in an independent cohort of >450 renal cancer patients.
RESULTS: Wortmannin disrupted T3-dependent nuclear transport of TRIP11. T3-agarose did not change TRIP11 localization, precluding extracellular T3-mediated mechanism. The expressions of TRIP11, HIF-1β, THRA, THRB, FURIN, VEGFA, and GLUT1 were disturbed in renal cancer. Expressions of TRIP11 and HIF-1β correlated with tumor grades. Decreased expressions of TRIP11, THRA, and THRB correlated with poor survival of RCC patients.
CONCLUSIONS: 1) T3 induces nuclear TRIP11 localization via PI3K-dependent mechanism; 2) disturbed expression of T3 signaling pathway genes correlates with RCC progression. The specific mechanisms by which altered T3 signaling may contribute to RCC progression require further investigation.

Protein-coding mutations in clear cell renal cell carcinoma (ccRCC) have been extensively characterized, frequently involving inactivation of the von Hippel-Lindau (

BACKGROUND: A substantial fraction of familial colorectal cancer (CRC) and polyposis heritability remains unexplained. This study aimed to identify predisposing loci in patients with these disorders.
METHODS: Homozygosity mapping was performed using 222 563 SNPs in 302 index patients with various colorectal neoplasms and 3367 controls. Linkage analysis, exome and whole-genome sequencing were performed in a family affected by microsatellite stable CRCs. Candidate variants were genotyped in 10 554 cases and 21 480 controls. Gene expression was assessed at the mRNA and protein level.
RESULTS: Homozygosity mapping revealed a disease-associated region at 1q32.3 which was part of the linkage region 1q32.2-42.2 identified in the CRC family. This includes a region previously associated with risk of CRC. Sequencing identified the p.Asp1432Glu variant in the MIA3 gene (known as TANGO1 or TANGO) and 472 additional rare, shared variants within the linkage region. In both cases and controls the population frequency was 0.02% for this MIA3 variant. The MIA3 mutant allele showed predominant mRNA expression in normal, cancer and precancerous tissues. Furthermore, immunohistochemistry revealed increased expression of MIA3 in adenomatous tissues.
CONCLUSIONS: Taken together, our two independent strategies associate genetic variations in chromosome 1q loci and predisposition to familial CRC and polyps, which warrants further investigation.

Epidemiological studies have shown a link between problems with offspring of couples living in a contaminated environment in comparison to those who live in an uncontaminated environment. We measured the concentrations of 16 priority polycyclic aromatic hydrocarbons (PAHs) in maternal and cord blood. To explore the mechanism of the effects of PAH mixtures on nonluteinized granulosa cells (HGrC1) and granulosa tumor cells (COV434), as well as cell proliferation and apoptosis, we investigated the effect of PAH mixtures on the expression of the aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT) and aryl hydrocarbon receptor repressor (AHRR) genes, as well as the expression and activity of target genes cytochrome P450 1A1 (CYP1A1) and catechol-O-methyltransferase (COMT). The cells were exposed to mixture 1 (M1), composed of all 16 priority PAHs, and mixture 2 (M2), composed of five PAHs which are not classified as human carcinogens, and which are observed in the highest amounts both in maternal and cord blood. All 16 priority PAHs were bioavailable in maternal and cord plasma, suggesting that perinatal exposure should be considered. In HGrC1 cells, M1 increased AHR and ARNT, but decreased AHRR expression, in parallel with increased CYP1A1 and COMT expression and activity. M2 decreased AHR and AHRR, and increased ARNT, with no effect on CYP1A1 expression and activity; however, it did increase COMT expression and activity. In tumor cells, M1 lowered AHR and up-regulated AHRR and ARNT expression, consequently decreasing CYP1A1 expression and COMT activity. M2 up-regulated AHR and ARNT, down-regulated AHRR, and had no effect on CYP1A1 and COMT expression, but decreased COMT activity. We hypothesise that, dependent on composition, mixtures of PAHs activate the AHR differently through varying transcription responses: in HGrC1, a canonical AHR mechanism of M1, with activation of CYP1A1 important for detoxication, while in COV434, a noncanonical AHR mechanism, probably by activation the nuclear factor NFkB.

BACKGROUND: Two key issues in prostate cancer (PCa) that demand attention currently are the need for a more precise and minimally invasive screening test owing to the inaccuracy of prostate-specific antigen and differential diagnosis to distinguish advanced vs. indolent cancers. This continues to pose a tremendous challenge in diagnosis and prognosis of PCa and could potentially lead to overdiagnosis and overtreatment complications. Copy number variations (CNVs) in the human genome have been linked to various carcinomas including PCa. Detection of these variants may improve clinical treatment as well as an understanding of the pathobiology underlying this complex disease.
METHODS: To this end, we undertook a pilot genome-wide CNV analysis approach in 36 subjects (18 patients with high-grade PCa and 18 controls that were matched by age and ethnicity) in search of more accurate biomarkers that could potentially explain susceptibility toward high-grade PCa. We conducted this study using the array comparative genomic hybridization technique. Array results were validated in 92 independent samples (46 high-grade PCa, 23 benign prostatic hyperplasia, and 23 healthy controls) using polymerase chain reaction-based copy number counting method.
RESULTS: A total of 314 CNV regions were found to be unique to PCa subjects in this cohort (P<0.05). A log
CONCLUSION: In conclusion, we postulated that the CNVs identified in this study could provide an insight into the development of advanced PCa.

Recent studies have shown that disruption of the circadian rhythm was one of the endogenous factors contributing to tumorigenesis of various human malignancies, including colorectal cancer (CRC). However, the roles of circadian genes in the development of CRC are still unexplored. In this study, we investigated the expression pattern and the underlying mechanism of human Clock gene (hClock) in CRC progression. Multiple methods such as qRT-PCR, immunohistochemistry, and western blotting were performed to evaluate the expression pattern of the gene hClock, as well as to observe the changes of angiogenesis-related proteins and EMT-related proteins. Transwell cell migration assays and an animal tumor metastasis model were used to examine the impact of hClock on the metastatic ability of CRC cells in vitro and in vivo. Our results showed that the expression level of hClock significantly increased in human CRC tissues, which strongly associated with late TNM stage and positive lymph node metastasis. Moreover, a higher level of hClock expression was found in CRC cell lines with a higher metastatic potential. Furthermore, ectopic expression of hClock promoted the migration of SW480 CRC cells, while knockdown of hClock inhibited the tumor metastasis of SW620 CRC cells, and targeting hClock by shRNA effectively suppressed the metastatic ability of SW620 CRC cells in nude mice. Finally, we found that overexpression of hClock enhanced the expression of angiogenesis-related genes such as HIF-1α, ARNT and VEGF, and promoted epithelial-mesenchymal (-like) transition (EMT) in CRC cells, both of which are considered to be critical for tumor progression. These findings suggest that upregulation of the circadian gene hClock plays an important role in metastasis of colorectal cancer.

The aryl hydrocarbon receptor (AhR) is a potential clinical target for cancer and autoimmune dysfunction. Identifying selective AhR modulators that produce desirable clinical outcomes represents an opportunity for developing new anti-cancer agents. Repurposing clinically-used drugs with established safety profiles that activate the AhR represents a good starting place to pursue this goal. In this study, we characterized the AhR-dependent effects of SU5416 (Semaxanib) following its identification in a small-molecule library screen. SU5416 potently activated AhR-dependent reporter genes, induced AhR nuclear localization, facilitated AhR-DNA binding, and increased, expression of its endogenous target genes. SU5416 significantly inhibited proliferation of Hepa1 hepatoma cells in an AhR-dependent manner, but did not induce apoptosis. SU5416 also inhibited the growth of human HepG2 liver cancer cells. The effects of SU5416 correlated with an increased G1 population and increased expression of cell cycle inhibitor p21cip1/waf1 at both the mRNA and protein level. Increased expression of p21cip1/waf1 by SU5416 required expression of both AhR and Arnt. In addition, evidence for long-term activation of the AhR in vivo by a single dose of SU5416 was identified by analyzing published microarray data. Our results provide support for continued investigation of the AhR as therapeutic for cancers such as hepatocellular carcinoma. In addition, our findings raise the possibility that some of the previously observed anti-proliferative effects of SU5416 may be due to activation of the AhR.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is known as a mediator of toxic responses. Recently, it was shown that the AhR has dual functions. Besides being a transcription factor, it also possesses an intrinsic E3 ubiquitin ligase function that targets, e.g., the steroid receptors for proteasomal degradation. The aim of this study was to identify the molecular switch that determines whether the AhR acts as a transcription factor or an E3 ubiquitin ligase. To do this, we used the breast cancer cell line MCF7, which expresses a functional estrogen receptor alpha (ERα) signaling pathway. Our data suggest that aryl hydrocarbon receptor nuclear translocator (ARNT) plays an important role in the modulation of the dual functions of the AhR. ARNT knockdown dramatically impaired the transcriptional activation properties of the ligand-activated AhR but did not affect its E3 ubiquitin ligase function. The availability of ARNT itself is modulated by another basic helix-loop-helix (bHLH)-Per-ARNT-SIM (PAS) protein, the repressor of AhR function (AhRR). MCF7 cells overexpressing the AhRR showed lower ERα protein levels, reduced responsiveness to estradiol, and reduced growth rates. Importantly, when these cells were used to produce estrogen-dependent xenograft tumors in SCID mice, we also observed lower ERα protein levels and a reduced tumor mass, implying a tumor-suppressive-like function of the AhR in MCF7 xenograft tumors.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica dahurica is a commonly used traditional Chinese medicine to treat migraine headache, toothache and cancer. Imperatorin is an active natural furocoumarin component originating from Angelica dahurica and has been shown to exhibit multiple bioeffector functions, including anti-cancer activity. However, the mechanism by which imperatorin inhibits tumor growth is not fully understood.
AIM OF THE STUDY: The aim of this study was to investigate the effectiveness of imperatorin as a treatment of cancer and to identify the underlying mechanisms of its anticancer activity.
MATERIALS AND METHODS: HCT116, HeLa, and Hep3B cells were used in this study. Major assays were promoter-reporter gene assay, MTT, western blot analysis, immunofluorescence assay, reverse transcription-PCR (RT-PCR), flow cytometric analysis, clonogenic assay, EdU labeling and immunofluorescence, xenografted assay, and VEGF ELISA.
RESULTS: We here demonstrated the effect of imperatorin on hypoxia-inducible factor-1 (HIF-1) activation. Imperatorin showed a potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently, whereas it did not affect the expressions of HIF-1β and topoisomerase-I (Topo-I). Further analysis revealed that imperatorin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), eIF4E binding protein-1 (4E-BP1), eukaryotic initiation factor 4E (eIF4E), extracellular signal-regulated kinase-1/2 (ERK1/2), SAPK/JNK and p38 were significantly suppressed by imperatorin. Furthermore, imperatorin prevented hypoxia-induced expression of HIF-1 target genes and flow cytometric analysis indicated that imperatorin induced G1 phase arrest in human colon cancer cell (HCT116). We found that imperatorin administration inhibits tumor growth and blocks tumor angiogenesis in a xenograft tumor model.
CONCLUSIONS: These results show that imperatorin inhibited HIF-1α protein synthesis by downregulating the mTOR/p70S6K/4E-BP1 and MAPK pathways. These conclusions suggest that imperatorin is an effective inhibitor of HIF-1 and provide new perspectives into the mechanism of its anticancer activity.

BACKGROUND: Current angiogenic therapies for cancers and cardiovascular diseases have not yet achieved expected benefits, which reflects the need for improved understanding of angiogenesis. In this study, we focused on solving the problem of whether tissues have different angiogenic potentials (APs) in physiological conditions and how angiogenesis is regulated in various disease conditions.
METHODS: In healthy and diseased human and mouse tissues, we profiled the expression of 163 angiogenic genes, including transcription regulators (TRs), growth factors and receptors (GF/Rs), cytokines and chemokines (C/Cs), and proteases and inhibitors (P/Is). TRs were categorized as inflammatory, homeostatic, and endothelial cell-specific TRs, and C/Cs were categorized as pro-angiogenic, anti-angiogenic, and bi-functional C/Cs.
RESULTS: We made the following findings: (1) the human heart, muscle, eye, pancreas, and lymph node are among the tissues with the highest APs; (2) tissues with high APs have more active angiogenic pathways and angiogenic C/C responses; (3) inflammatory TRs dominate regulation of all angiogenic C/Cs; homeostatic TRs regulate all to a lower extent, while endothelial cell-specific TRs mainly regulate pro-angiogenic and bi-functional C/Cs; (4) tissue AP is positively correlated with the expression of oxygen sensors PHD2 and HIF1B, VEGF pathway gene VEGFB, and stem cell gene SOX2; (5) cancers of the digestive system tend to have increased angiogenesis dominated by endothelial cell-specific pro-angiogenic pathways, while lung cancer and prostate cancer have significantly decreased angiogenesis; and (6) endothelial cell-specific pro-angiogenic pathways are significantly increased in thrombus-derived leukocytes in patients with acute coronary artery disease.
CONCLUSIONS: Our results demonstrate that thrombus-derived leukocytes express more endothelial cell-specific angiogenic markers to directly promote angiogenesis after myocardial infarction and that certain solid tumors may be more sensitive to anti-angiogenic therapies than others.

The loss of the Von Hippel-Lindau tumor suppressor (VHL) is a key oncogenic event in the vast majority of patients with clear cell renal cell carcinoma (ccRCC). With the loss of the VHL protein (pVHL) function, the hypoxia inducible factor α (HIF-α) accumulates inside the tumor cell and dimerizes with HIF-β. The HIF-α/HIF-β complex transcriptionally activates hundreds of genes promoting the adaptation to hypoxia that is implicated in tumor development. There is growing evidence showing that HIF-2α subunit has a central role in ccRCC over HIF-1α. Thus, efforts have been made to specifically target this pathway. PT2385 and PT2399 are first-in-class, orally available, small molecule inhibitors of HIF-2 that selectively disrupt the heterodimerization of HIF-2α with HIF-1β. Preclinical and clinical data indicate that these new molecules are effective in blocking cancer cell growth, proliferation, and tumor angiogenesis characteristic in ccRCC. Treatment with HIF-2α specific antagonists, either alone or in combination with immunotherapy or other antiangiogenic agents have the potential to transform the therapeutic landscape in this tumor in the future. Herein, we summarize the molecular background behind the use of HIF-2α inhibitors in ccRCC and give an overview of the development of new agents in this setting.

INTRODUCTION: Gene-expression signatures for prognosis have been reported in localized renal cell carcinoma (RCC). The aim of this study was to test the predictive power of two different signatures, ClearCode34, a 34-gene signature model [Eur Urol 2014;66:77-84], and an 8-gene signature model [Eur Urol 2015;67:17-20], in the setting of systemic therapy for metastatic disease.
MATERIALS AND METHODS: Metastatic RCC (mRCC) patients from five institutions who were part of TCGA were identified and clinical data were retrieved. We trained and implemented each gene model as described by the original study. The latter was demonstrated by faithful regeneration of a figure and results from the original study. mRCC patients were dichotomized to good or poor prognostic risk groups using each gene model. Cox proportional hazard regression and concordance index (C-Index) analysis were used to investigate an association between each prognostic risk model and overall survival (OS) from first-line therapy.
RESULTS: Overall, 54 patients were included in the final analysis. The primary endpoint was OS. Applying the ClearCode34 model, median survival for the low-risk-ccA (
CONCLUSION: The ClearCode34 but not the 8-gene molecular model improved the prognostic predictive power of the IMDC model in this cohort of 54 patients with metastatic clear cell RCC.

Hepatocellular carcinoma (HCC) is the major threat to human health, and disruption of circadian clock genes is implicated in hepatocarcinogenesis. This study examined the dysregulation of metallothioneins and circadian genes in achieved human HCC (n = 24), peri-HCC tissues (n = 24) as compared with normal human livers (n = 36). Total RNA was extracted and reverse transcribed. Real-time RT-qPCR was performed to determine the expression of genes of interest. The results demonstrated the downregulation of metallothionein-1 (MT-1), MT-2, and metal transcription factor-1 (MFT-1) in human HCC as compared with Peri-HCC and normal tissues. MTs are a biomarker for HCC and have typical circadian rhythms; the expression of major circadian clock genes was also determined. HCC produced a dramatic decrease in the expression of core clock genes, circadian locomotor output cycles kaput (Clock) and brain and muscle Arnt-like protein 1 (Bmal1), and decreased the expression of the clock feedback control genes, Periods (Per1, Per2) and Cryptochromes (Cry1, Cry2). On the other hand, the expression of clock target genes nuclear orphan receptor factor protein (Nr1d1) and D-box-binding protein (Dbp) was upregulated as compared with Peri-HCC and normal livers. Peri-HCC also had mild alterations in these gene expressions. In summary, the present study clearly demonstrated the dysregulation of MTs and circadian clock genes in human HCC, which could provide the information of targeting MT and circadian clock in HCC management.

Cell metabolism can be reprogrammed by tissue hypoxia leading to cell transformation and glioblastoma multiforme (GBM) progression. In response to hypoxia, GBM cells are able to express a transcription factor called hypoxia inducible factor-1 (HIF-1). HIF-1 belongs to a family of heterodimeric proteins that includes HIF-1α and HIF-1β subunits. HIF-1α has been reported to play a pivotal role in GBM development and progression. In the present review, we discuss the role of HIF-1α in glucose uptake, cancer proliferation, cell mobility and chemoresistance in GBM. Evidence from previous studies indicates that HIF-1α regulates angiogenesis, metabolic and transcriptional signaling pathways. Examples of such are the EGFR, PI3K/Akt and MAPK/ERK pathways. It affects cell migration and invasion by regulating glucose metabolism and growth in GBM cells. The present review focuses on the strategies through which to target HIF-1α and the related downstream genes highlighting their regulatory roles in angiogenesis, apoptosis, migration and glucose metabolism for the development of future GBM therapeutics. Combined treatment with inhibitors of HIF-1α and glycolysis may enhance antitumor effects in clinical settings.

Colorectal cancer (CRC) is a complex disease with still unsatisfactory prognosis even in western societies, although substantial progress has been made in pre-screening programs, surgical techniques and targeted therapy options. Mediator of motility-1 (Memo-1) was previously recognized as an important effector of cell migration downstream of receptor tyrosine kinase signaling in breast cancer. This study identified Memo-1 as frequently overexpressed in CRC and established a close link between extracellular HER2 activation, AhR/ARNT transcriptional activity and Memo-1 expression. Dissection of the hMemo-1 gene promoter using reporter assays and chromatin IP techniques revealed recruitment of Aryl hydrocarbon receptor (AhR)/Aryl hydrocarbon receptor nuclear-translocator (ARNT) complex, which positively influenced Memo-1 expression in cancer cells. We found that Memo-1 depletion negatively influenced the cellular actin network and that its expression is required for HER2-mediated cell migration and invasion. Moreover, analyses of Memo-1 expression in primary CRC revealed correlation with clinical parameters that point to Memo-1 as a new prognostic factor of aggressive disease in CRC patients. Altogether, these observations demonstrate that Memo-1 is an important downstream regulator of HER2-driven CRC cell migration and invasion through connecting extracellular signals from membrane to the cytoskeletal actin network.

BACKGROUND: Pomolic acid (PA), an active triterpenoid from Euscaphis japonica, inhibits the proliferation of a variety of cancer cells, but the molecular mechanisms of the anti-angiogenic potential of PA have not been fully elucidated in breast cancer cells.
HYPOTHESIS/PURPOSE: We investigated the molecular mechanisms underlying the anti-angiogenic effect of PA in epidermal growth factor (EGF)-responsive human breast cancer cells, MCF-7 and MDA-MB-231, and human umbilical vascular endothelial cells (HUVEC).
STUDY DESIGN/METHODS: Effects of PA on EGF-induced HIF1α/VEGF expression in MCF-7, MDA-MB-231 and HUVEC were assayed. As to the mechanisms, EGF-mediated MAPKs, PI3K/Akt, and mTOR signaling pathway were performed. Wound healing and invasion assay, tube formation assay, immunoblot assay, real-time PCR, luciferase gene assay, electrophoretic mobility shift assay and immunofluorescence staining were used for assessment.
RESULTS: PA significantly and selectively suppressed EGF-induced HIF1α/VEGF expression, whereas it did not affect the expression of HIF1β in MCF-7 and MDA-MB-231. Furthermore, PA inhibited EGF-induced angiogenesis in vitro and downregulated HIF1α/VEGF expression in HUVEC. Mechanistically, we found that the inhibitory effects of PA on HIF1α/VEGF expression are associated with inhibition of HIF1α/VEGF expression through an EGF-dependent mechanism. In addition, PA suppressed the EGF-induced phosphorylation of p38-MAPK and mTOR.
CONCLUSION: PA suppresses EGF-induced HIF1α protein translation by inhibiting the p38-MAPK and mTOR kinase signaling pathways and plays a novel anti-angiogenic role.

BACKGROUND: Despite the remarkable progress to fight against breast cancer, metastasis remains the dominant cause of treatment failure and recurrence. Therefore, control of invasiveness potential of breast cancer cells is crucial. Accumulating evidences suggest Aryl hydrocarbon receptor (Ahr), a helix-loop-helix transcription factor, as a promising target to control migration and invasion in breast cancer cells. Thus, an Ahr-based exploration was performed to identify a new Ahr agonist with inhibitory potentials on cancer cell motility.
METHODS: For prediction of potential interactions between Ahr and candidate molecules, bioinformatics analysis was carried out. The interaction of the selected ligand with Ahr and its effects on migration and invasion were examined in vitro using the MDA-MB-231 and T47D cell lines. The silencing RNAs were transfected into cells by electroporation. Expressions of microRNAs (miRNAs) and coding genes were quantified by real-time PCR, and the protein levels were detected by western blot.
RESULTS: The in silico and in vitro results identified Flavipin as a novel Ahr agonist. It induces formation of Ahr/Ahr nuclear translocator (Arnt) heterodimer to promote the expression of cytochrome P450 family 1 subfamily A member 1 (Cyp1a1). Migration and invasion of MDA-MB-231 and T47D cells were inhibited with Flavipin treatment in an Ahr-dependent fashion. Interestingly, Flavipin suppressed the pro-metastatic factor SRY-related HMG-box4 (Sox4) by inducing miR-212/132 cluster. Moreover, Flavipin inhibited growth and adhesion of both cell lines by suppressing gene expressions of B-cell lymphoma 2 (Bcl2) and integrinα4 (ITGA4).
CONCLUSION: Taken together, the results introduce Flavipin as a novel Ahr agonist, and provide first evidences on its inhibitory effects on cancer cell motility, suggesting Flavipin as a candidate to control cell invasiveness in breast cancer patients.