Up-regulation of glyceraldehyde-3-phosphate dehydrogenase gene expression by HIF-1 activity depending on Sp1 in hypoxic breast cancer cells

Hypoxia up-regulates the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a cell type-specific manner. It is unknown whether this occurs in breast cancer. Here, we report that hypoxia up-regulates the GAPDH gene expression through breast cancer-specific molecular mechanisms in MCF-7 cells. Mutation analysis identified a novel hypoxia response element (HRE), in addition to the HRE found previously in prostate cancer LNCaP cells. Knockdown and overexpression of hypoxia-inducible factor (HIF)-1α indicated that HIF-1 contributed to the up-regulation of GAPDH gene expression by hypoxia. Although chromatin immunoprecipitation (ChIP) and plasmid immunoprecipitation analyses revealed the presence of HIF-1α on the novel HRE in both hypoxic cell lines, a mutation in either the novel HRE or its 3′-flanking GC-box resulted in a reduction of hypoxia-increased GAPDH promoter activity only in MCF-7 cells. ChIP analysis showed that Sp1 bound to the GC-box in MCF-7 cells, but not in LNCaP cells, in normoxia and hypoxia. Knockdown of Sp1 reduced hypoxia-increased promoter activity and expression level of GAPDH in MCF-7 cells. These results indicate that in MCF-7 cells, the activation of HIF-1 on the novel HRE contributes to the breast cancer-specific hypoxic induction of GAPDH gene expression and absolutely depends on the presence of Sp1 on the GC-box.     

Dual regulation of macrophage migration inhibitory factor (MIF) expression in hypoxia by CREB and HIF-1

Macrophage migration inhibitory factor (MIF) is a well-described pro-inflammatory mediator that has also been implicated in the process of oncogenic transformation and tumor progression. However, despite the compelling evidence that MIF is overexpressed in, and contributes to, the pathology of inflammatory and malignant diseases the mechanisms that contribute to exaggerated expression of MIF have been poorly described. Here we show that hypoxia, and specifically HIF-1alpha, is a potent and rapid inducer of MIF expression. In addition, we demonstrate that hypoxia-induced MIF expression is dependent upon a HRE in the 5'UTR of the MIF gene but is further modulated by CREB expression. We propose a model where hypoxia-induced MIF expression is driven by HIF-1 but amplified by hypoxia-induced degradation of CREB. Given the importance of MIF in inflammatory and malignant diseases these data reveal a HIF-1-mediated pathway as a potential therapeutic target for suppression of MIF expression in hypoxic tissues.     

Rhein inhibits angiogenesis and the viability of hormone-dependent and -independent cancer cells under normoxic or hypoxic conditions in vitro

Hypoxia is a hallmark of solid tumors, including breast cancer, and the extent of tumor hypoxia is associated with treatment resistance and poor prognosis. Considering the limited treatment of hypoxic tumor cells and hence a poor prognosis of breast cancer, the investigation of natural products as potential chemopreventive anti-angiogenic agents is of paramount interest. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid), the primary anthraquinone in the roots of Cassia alata L., is a naturally occurring quinone which exhibits a variety of biologic activities including anti-cancer activity. However, the effect of rhein on endothelial or cancer cells under hypoxic conditions has never been delineated. Therefore, the aim of this study was to investigate whether rhein inhibits angiogenesis and the viability of hormone-dependent (MCF-7) or -independent (MDA-MB-435s) breast cancer cells in vitro under normoxic or hypoxic conditions. Rhein inhibited vascular endothelial growth factor (VEGF₁₆₅)-stimulated human umbilical vein endothelial cell (HUVEC) tube formation, proliferation and migration under normoxic and hypoxic conditions. In addition, rhein inhibited in vitro angiogenesis by suppressing the activation of phosphatidylinositol 3-kinase (PI3K), phosphorylated-AKT (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) but showed no inhibitory effects on total AKT or ERK. Rhein dose-dependently inhibited the viability of MCF-7 and MDA-MB-435s breast cancer cells under normoxic or hypoxic conditions, and inhibited cell cycle in both cell lines. Furthermore, Western blotting demonstrated that rhein inhibited heat shock protein 90alpha (Hsp90α) activity to induce degradation of Hsp90 client proteins including nuclear factor-kappa B (NF-κB), COX-2, and HER-2. Rhein also inhibited the expression of hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF₁₆₅), epidermal growth factor (EGF), and the phosphorylation of inhibitor of NF-κB (I-κB) under normoxic or hypoxic conditions. Taken together, these data indicate that rhein is a promising anti-angiogenic compound for breast cancer cell viability and growth. Therefore, further studies including in vivo and pre-clinical need to be performed.     

过表达DNALI1抑制低氧环境中鼻咽癌细胞株CNE-2z侵袭与迁移

目的探讨模拟肿瘤内低氧微环境下，动力蛋白轴突轻链中间体1 （DNALI1）对鼻咽癌细胞株CNE-2z增殖、迁移、侵袭和凋亡的影响。 方法R语言软件分析GEO数据库中鼻咽癌组织的3个数据集，分析其差异基因（DEGs）。低氧是肿瘤微环境基本特征，体外细胞研究均在低氧工作站（1﹪O₂）中进行，以模拟体内低氧微环境。将DNALI1过表达质粒和空载体质粒包装成慢病毒后感染CNE-2z细胞，构建DNALI1基因稳定过表达的CNE-2z细胞株。将CNE-2z细胞分别进行正常培养（空白对照，BC），感染空载体慢病毒（阴性对照，NC）和感染过表达DNALI1慢病毒（过表达DNALI1，DNALI1-OE）。实时荧光定量聚合酶链式反应（RT-qPCR）和Western blot检测DNALI1 mRNA和蛋白表达水平；集落形成实验检测细胞增殖能力；Transwell实验检测细胞迁移和侵袭能力；流式细胞术检测细胞凋亡水平。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。 结果生物信息学分析发现，与健康人鼻咽组织相比，鼻咽癌组织中DNALI1基因低表达。在低氧环境中，BC组与NC组细胞增殖、凋亡、迁移和侵袭比较，差异均无统计学意义；与BC组和NC组比较，DNALI1-OE组细胞增殖能力和凋亡水平比较，差异均无统计学意义（P均> 0.05）。与BC组和NC组比较，DNALI1-OE组细胞迁移能力[（198.67±3.22），（199.00±3.00）比（75.00±7.55）个]和侵袭能力[（240.67±16.01），（259.67±3.06）比（83.33±9.50）个]降低，差异具有统计学意义（P均< 0.001）。 结论低氧环境中，DNALI1过表达可抑制CNE-2z细胞侵袭与迁移能力，但对细胞增殖、凋亡无明显影响。     

miR-138-5p靶向缺氧诱导因子1α对胰腺癌PANC-1细胞生长和转移的调节作用

目的探索miR-138-5p对胰腺癌细胞PANC-1生长、转移的影响及其相关机制。方法应用荧光实时定量PCR (real-time quantitative PCR, RT-PCR)检测miR-138-5p及其缺氧诱导因子1α(hypoxia inducible factor 1 alpha, HIF-1α)在PANC-1细胞中的表达。应用荧光素酶报告检测验证miR-138-5p与HIF-1α之间的生物学关系。通过体外试验研究miR-138-5p、HIF-1α在PANC-1细胞中的生物学功能,Western blot检测蛋白表达情况;CCK-8检测PANC-1细胞增殖能力;Transwell试验检测PANC-1细胞侵袭能力;划痕试验检测PANC-1细胞迁移能力。结果 miR-138-5p表达明显下调HIF-1α表达水平(P<0.01),生物信息学预测和荧光素酶报告试验证明miR-138-5p通过直接结合HIF-1α 3′-未翻译区域(3′-UTR)抑制HIF-1α。在PANC-1细胞中,miR-138-5p过表达可抑制HIF-1α表达及细胞增殖、侵袭、迁移,且差异有统计学意义(P<0.01)。结论 miR-138-5p结合HIF-1α 3′-UTR的沉默HIF-1α;miR-138-5p通过打靶HIF-1α而抑制胰腺癌细胞PANC-1增殖和转移。HIF-1α可能是胰腺癌的治疗靶点。     

Long noncoding RNA FEZF1-AS1 predicts poor prognosis and modulates pancreatic cancer cell proliferation and invasion through miR-142/HIF-1α and miR-133a/EGFR upon hypoxia/normoxia

Nowadays, pancreatic cancer (PC) remains the most lethal tumor, partially due to the invasive and treatment-resistant phenotype induced by the extent of hypoxic stress within the tumor tissue. According to previous studies, miR-142/HIF-1α and miR-133a/EGFR could modulate PC cell proliferation under hypoxic and normoxic conditions, respectively. In the present study, FEZF1-AS1, a recently described oncogenic long noncoding RNA, was predicted to target both miR-142 and miR-133a; thus, we hypothesized that FEZF1-AS1 might affect PC cell proliferation through these two axes under hypoxic or normoxic conditions. In PC cell lines, FEZF1-AS1 acted as an oncogene via promoting PC cell proliferation and invasion through miR-142/HIF-1α axis under hypoxic condition; however, FEZF1-AS1 failed to affect the protein levels of HIF-1α and VEGF under the normoxic condition, suggesting the existence of another signaling pathway under normoxic condition. As predicted by an online tool, FEZF1-AS1 could target miR-133a to inhibit its expression; under the normoxic condition, FEZF1-AS1 exerted its effect on PC cell lines through miR-133a/EGFR axis. Taken together, FEZF1-AS1 might be a promising target in controlling the aberrant proliferation and invasion of PC cell lines.     

The interplay between epigenetic silencing,oncogenic KRas and HIF-1 regulatory pathways in control of BNIP3 expression in human colorectal cancer cells

Bcl-2/adenovirus E1B-19 kDa-interacting protein 3 (BNIP3) is an important mediator of cell survival and a member of the Bcl-2 family of proteins that regulate programmed cell death and autophagy. We have previously established a link between the expression of oncogenic HRas and up-regulation of BNIP3 and the control of autophagy in cancer cells. However, in view of varied expression of BNIP3 in different tumor types and emerging uncertainties as to the role of epigenetic silencing, oncogenic regulation and the role of BNIP3 in cancer are still poorly understood.     

Hepatitis B virus x gene-downregulated growth-arrest specific 5 inhibits the cell viability and invasion of hepatocellular carcinoma cell lines by activating Y-box-binding protein 1/p21 signaling

The long noncoding RNA growth-arrest specific 5 (GAS5) is a suppressor of many cancers. However, the role and mechanism of action of GAS5 in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remain unclear. Here, the expression of hepatitis B virus x gene (HBx) mRNA and GAS5 was assessed by qRT–PCR, and western blot analysis was performed to determine the protein expression levels. In addition, the cell viability and invasion of cells were confirmed using  MTT assay and Transwell assay, respectively. The DNA methylation level of GAS5 was measured by methylation-specific PCR. Moreover, RIP assay and RNA pull down assay were carried out to examine the combination of Y-box-binding protein 1 (YBX1) and GAS5. First, our data proved that HBx is increased, while GAS5 is decreased in HCC cell lines. Subsequently, we found that HBx facilitates HCC cell viability and invasion by inhibiting GAS5 expression. Then, we further clarified that HBx induces the DNA methylation of GAS5 by promoting methyltransferase expression, thereby suppressing GAS5 expression. Furthermore, GAS5 binds YBX1 and promotes YBX1 and p21 expression. Finally, the functional analysis revealed that the upregulation of GAS5 could attenuate cell viability and invasion by boosting p21 expression via binding YBX1. Overall, our results demonstrated that HBx promotes HCC progression by inducing GAS5 methylation to reduce its expression. The upregulation of GAS5 suppressed HBV-related HCC by activating YBX1/p21 signaling. Our data provide novel evidence supporting the potential of GAS5 as a treatment target in HBV-related HCC.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00645-z.     

The role of the ATPase inhibitor factor 1 (IF1) in cancer cells adaptation to hypoxia and anoxia

The physiological role of the mitochondrial ATP synthase complex is to generate ATP through oxidative phosphorylation. Indeed, the enzyme can reverse its activity and hydrolyze ATP under ischemic conditions, as shown in isolated mitochondria and in mammalian heart and liver. However, what occurs when cancer cells experience hypoxia or anoxia has not been well explored. In the present study, we investigated the bioenergetics of cancer cells under hypoxic/anoxic conditions with particular emphasis on ATP synthase, and the conditions driving it to work in reverse. In this context, we further examined the role exerted by its endogenous inhibitor factor, IF₁, that it is overexpressed in cancer cells. Metabolic and bioenergetic analysis of cancer cells exposed to severe hypoxia (down to 0.1% O₂) unexpectedly showed that Δψ_m is preserved independently of the presence of IF₁ and that ATP synthase still phosphorylates ADP though at a much lower rate than in normoxia. However, when we induced an anoxia-mimicking condition by collapsing Δμ_Η⁺ with the FCCP uncoupler, the IF₁-silenced clones only reversed the ATP synthase activity hydrolyzing ATP in order to reconstitute the electrochemical proton gradient. Notably, in cancer cells IF₁ overexpression fully prevents ATP synthase hydrolytic activity activation under uncoupling conditions. Therefore, our results suggest that IF₁ overexpression promotes cancer cells survival under temporary anoxic conditions by preserving cellular ATP despite mitochondria dysfunction.