Inactivation of the WASF3 gene in prostate cancer cells leads to suppression of tumorigenicity and metastases

Background:

The WASF3 protein is involved in cell movement and invasion, and to investigate its role in prostate cancer progression we studied the phenotypic effects of knockdown in primary tumors and cell lines.

Methods:

ShRNA was used to knockdown WASF3 function in prostate cell lines. Cell motility (scratch wound assay), anchorage independent growth and in vivo tumorigenicity and metastasis were then compared between knockdown and wild-type cells.

Results:

Increased levels of expression were seen in high-grade human prostate cancer and in the PC3 and DU145 cell lines. Inactivation of WASF3 using shRNAs reduced cell motility and invasion in these cells and reduced anchorage independent growth in vitro. The loss of motility was accompanied by an associated increase in stress fiber formation and focal adhesions. When injected subcutaneously into severe combined immunodeficiency (SCID) mice, tumor formation was significantly reduced for PC3 and DU145 cells with WASF3 knockdown and in vivo metastasis assays using tail vain injection showed a significant reduction for PC3 and DU145 cells. The loss of the invasion phenotype was accompanied by down-regulation of matrix metalloproteinase 9.

Conclusions:

Overall, these observations demonstrate a critical role for WASF3 in the progression of prostate cancer and identify a potential target to control tumorigenicity and metastasis.     

HIF1A可作为乳腺癌的预后标志物且与免疫细胞浸润相关

目的：借助多种癌症生物信息数据库研究乳腺癌组织中缺氧诱导因子1亚基α(HIF1A)的表达水平及其与乳腺癌患者预后及肿瘤免疫细胞浸润的关系。方法：利用Oncomine、人类蛋白质图谱、基因表达谱交互式分析（GEPIA）及TCGA数据库分析HIF1A基因在乳腺癌组织中的表达及其与患者预后、临床病理特征的关系,并在中国人乳腺癌组织标本（选用2011年1月至2015年12月中国武汉大学人民医院手术切除的93例乳腺癌组织和14例良性乳腺疾病组织）中进行验证。对HIF1A高低表达组间的差异基因进行基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析,用Cibersort R软件评估HIF1A高低表达样本中免疫细胞浸润丰度差异。结果：生物信息数据显示,HIF1A在乳腺癌组织中高表达,预示着患者DFS预后更好（P<0.05）。HIF1A的表达与雌激素受体（ERP）、孕激素受体（PR）和人表皮生长因子受体2(HER2)表达相关（均P<0.05）。GO生物功能及KEGG通路富集分析结果提示,HIF1A可能参与肿瘤免疫调节等生物活动。使用Cibersort分析结果显示,HIF1A与肿瘤...     

Opposing effects of HIF1α and HIF2α on chromaffin cell phenotypic features and tumor cell proliferation: Insights from MYC‐associated factor X

Pheochromocytomas and paragangliomas (PPGLs) are catecholamine‐producing chromaffin cell tumors with diverse phenotypic features reflecting mutations in numerous genes, including MYC‐associated factor X (MAX). To explore whether phenotypic differences among PPGLs reflect a MAX‐mediated mechanism and opposing influences of hypoxia‐inducible factor (HIF)s HIF2α and HIF1α, we combined observational investigations in PPGLs and gene‐manipulation studies in two pheochromocytoma cell lines. Among PPGLs from 140 patients, tumors due to MAX mutations were characterized by gene expression profiles and intermediate phenotypic features that distinguished these tumors from other PPGLs, all of which fell into two expression clusters: one cluster with low expression of HIF2α and mature phenotypic features and the other with high expression of HIF2α and immature phenotypic features due to mutations stabilizing HIFs. Max‐mutated tumors distributed to a distinct subcluster of the former group. In cell lines lacking Max, re‐expression of the gene resulted in maturation of phenotypic features and decreased cell cycle progression. In cell lines lacking Hif2α, overexpression of the gene led to immature phenotypic features, failure of dexamethasone to induce differentiation and increased proliferation. HIF1α had opposing actions to HIF2α in both cell lines, supporting evolving evidence of their differential actions on tumorigenic processes via a MYC/MAX‐related pathway. Requirement of a fully functional MYC/MAX complex to facilitate differentiation explains the intermediate phenotypic features in tumors due to MAX mutations. Overexpression of HIF2α in chromaffin cell tumors due to mutations affecting HIF stabilization explains their proliferative features and why the tumors fail to differentiate even when exposed locally to adrenal steroids.     

HIF-1α siRNA leads to apoptosis of pancreatic cancer cells under hypoxic conditions

OBJECTIVE To explore the role （HIF-1α） in the proliferation and cells under hypoxic conditions. of hypoxic inducible factor-1α apoptosis of pancreatic cancer METHODS A cassette encoding small interference RNA （siRNA） targeting HIF-1α mediated by recombinant adeno-associated virus （rAAV） was constructed, giving rAAV-siHIE rAAV-siHIF or rAAV- hrGFP was transfected into exponentially growing MiaPaCa2 cells under hypoxic conditions. Then, the expression of HIF-1α mRNA and protein, the proliferation and apoptosis of MiaPaCa2 cells were examined, using real-time PCR, Western Blot, MTT and TUNEL, respectively. RESULTS Under hypoxic conditions, rAAV-siHIF inhibited the expression of HIF-1α mRNA and protein in MiaPaCa2 cells. At the same time, rAAV-siHIF decreased MiaPaCa2 cell proliferation and induced apoptosis. However, rAAV-hrGFP had no effect on the expression of HIF-1α as well as the proliferation and apoptosis of MiaPaCa2 cells under hypoxic conditions. CONCLUSION Under hypoxic conditions, HIF-1α plays a key role in the proliferation of MiaPaCa2 cells, and inhibition of HIF- 1α expression can lead to MiaPaCa2 cell apoptosis.     

Overcoming evasive resistance from vascular endothelial growth factor a inhibition in sarcomas by genetic or pharmacologic targeting of hypoxia‐inducible factor 1α

Increased levels of hypoxia and hypoxia‐inducible factor 1α (HIF‐1α) in human sarcomas correlate with tumor progression and radiation resistance. Prolonged antiangiogenic therapy of tumors not only delays tumor growth but may also increase hypoxia and HIF‐1α activity. In our recent clinical trial, treatment with the vascular endothelial growth factor A (VEGF‐A) antibody, bevacizumab, followed by a combination of bevacizumab and radiation led to near complete necrosis in nearly half of sarcomas. Gene Set Enrichment Analysis of microarrays from pretreatment biopsies found that the Gene Ontology category “Response to hypoxia” was upregulated in poor responders and that the hierarchical clustering based on 140 hypoxia‐responsive genes reliably separated poor responders from good responders. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (Dox), was recently found to block HIF‐1α binding to DNA at low metronomic doses. In four sarcoma cell lines, HIF‐1α shRNA or Dox at low concentrations blocked HIF‐1α induction of VEGF‐A by 84–97% and carbonic anhydrase 9 by 83–93%. HT1080 sarcoma xenografts had increased hypoxia and/or HIF‐1α activity with increasing tumor size and with anti‐VEGF receptor antibody (DC101) treatment. Combining DC101 with HIF‐1α shRNA or metronomic Dox had a synergistic effect in suppressing growth of HT1080 xenografts, at least in part via induction of tumor endothelial cell apoptosis. In conclusion, sarcomas respond to increased hypoxia by expressing HIF‐1α target genes that may promote resistance to antiangiogenic and other therapies. HIF‐1α inhibition blocks this evasive resistance and augments destruction of the tumor vasculature.     

HIF-1与肿瘤相关性研究进展

转录因子－缺氧诱导因子－1（ HIF－1）在细胞适应从常氧（21％O2）到低氧（1％O2）的过程中扮演了重要角色。研究表明,HIF－1与人类多种恶性肿瘤的发生发展有着密切关系。本文就HIF－1在肿瘤的增殖、转移、凋亡和血管生成中的作用进行综述。     

Hypoxia/pseudohypoxia‐mediated activation of hypoxia‐inducible factor‐1α in cancer

Since the first identification of hypoxic cells in sections of carcinomas in the 1950s, hypoxia has been known as a central hallmark of cancer cells and their microenvironment. Indeed, hypoxia benefits cancer cells in their growth, survival, and metastasis. The historical discovery of hypoxia‐inducible factor‐1α (HIF1A) in the early 1990s had a great influence on the field as many phenomena in hypoxia could be explained by HIF1A. However, not all regions or types of tumors are necessarily hypoxic. Thus, it is difficult to explain whole cancer pathobiology by hypoxia, especially in the early stage of cancer. Upregulation of glucose metabolism in cancer cells has been well known. Oxygen‐independent glycolysis is activated in cancer cells even in the normoxia condition, which is known as the Warburg effect. Accumulating evidence and recent advances in cancer metabolism research suggest that hypoxia‐independent mechanisms for HIF signaling activation is a hallmark for cancer. There are various mechanisms that generate pseudohypoxic conditions, even in normoxia. Given the importance of HIF1A for cancer pathobiology, the pseudohypoxia concept could shed light on the longstanding mystery of the Warburg effect and accelerate better understanding of the diverse phenomena seen in a variety of cancers.     

Genetic Variations in the HIF1A Gene Modulate Response to Adjuvant Chemotherapy after Surgery in Patients with Colorectal Cancer

Background: Hypoxia-inducible factor 1α (HIF-1α) plays an important role in regulating cell survivaland angiogenesis, which are critical for tumor growth and metastasis. Genetic variations of HIF1A have beenshown to influence the susceptibility to many kinds of human tumors. Increased expression of HIF-1α has alsobeen demonstrated to be involved in tumor progression. However, the prognostic value of single nucleotidepolymorphisms (SNPs) inthe HIF1A gene remains to be determined in most cancer types, including colorectalcancer (CRC). In this study, we sought to investigate the predictive role of HIF1A SNPs in prognosis of CRCpatients and efficacy of chemotherapy. Materials and Methods: We genotyped two functional SNPs in HIF1Agene using the Sequenom iPLEX genotyping system and then assessed their associations with clinicopathologicalparameters and clinical outcomes of 697 CRC patients receiving radical surgery using Cox logistic regressionmodel and Kaplan Meier curves. Results: Generally, no significant association was found between these 2 SNPsand clinical outcomes of CRC. In stratified analysis of subgroup without adjuvant chemotherapy, patientscarrying CT/TT genotypes of rs2057482 exhibited a borderline significant association with better overallsurvival when compared with those carrying CC genotype [Hazard ratio (HR), 0.47; 95% confidence interval(95% CI): 0.29-0.76; P < 0.01]. Moreover, significant protective effects on CRC outcomes conferred by adjuvantchemotherapy were exclusively observed in patients carrying CC genotype of rs2057482 and in those carryingAC/CC genotype of rs2301113. Conclusions: Genetic variations in HIF1A gene may modulate the efficacy ofadjuvant chemotherapy after surgery in CRC patients.     

ALDH1A1增强乳腺癌细胞血管生成因子表达并促进共培养HUVEC细胞小管形成和侵袭能力

目的:探讨干细胞标志物醛脱氢酶1A1（ALDH1A1）对乳腺癌细胞血管生成因子表达的影响,以及对与乳腺癌细胞共培养的HUVEC细胞小管形成和侵袭能力的影响。方法:采用免疫组化检测了乳腺癌组织和乳腺增生组织中ALDH1A1的表达。使用ALDH1A1 shRNA或过表达ALDH1A1的pcDNA3.1质粒转染乳腺癌细胞（MCF-7和MDA-MB-231）,通过qRT-PCR和Western blot检测敲低或过表达ALDH1A1对乳腺癌细胞中血管内皮生长因子（VEGF）、缺氧诱导因子-1α（HIF-1α）和白细胞介素-12（IL-12）表达的影响。通过用1 μmol/L 的外源性RA和RAR阻断剂（AGN 193109）处理乳腺癌细胞48 h来考察视黄酸信号通路是否参与ALDH1A1对VEGF和HIF-1α的调控过程。将乳腺癌细胞（MCF-7和MDA-MB-231）和HUVEC细胞共培养来模拟肿瘤形成的微环境,并检测HUVEC的小管形成能力和细胞侵袭能力。结果:乳腺癌组织的ALDH1A1染色平均光密度显著高于乳腺增生组织,并且淋巴结转移的乳腺癌组织显著高于未淋巴结转移的乳腺癌组织（P＜0.05）。敲低ALDH1A1可显著降低MCF-7和MDA-MB-231细胞中VEGF和HIF-1α蛋白表达,并上调IL-12蛋白表达。然而,上调ALDH1A1表达则可逆转上述变化。外源性RA处理可显著上调MCF-7和MDA-MB-231细胞中VEGF和HIF-1α的表达,然而,RAR阻断剂处理可抑制MCF-7和MDA-MB-231细胞中VEGF和HIF-1α的上调。敲低乳腺癌细胞中ALDH1A1的表达可导致共培养的HUVEC细胞的小管形成能力和侵袭能力显著降低。而上调乳腺癌细胞中ALDH1A1的表达则可显著促进共培养的HUVEC细胞的小管形成能力和侵袭能力。结论:在乳腺癌细胞中,ALDH1A1通过激活HIF-1α和视黄酸信号通路来上调血管生成因子的表达并提高共培养的内皮细胞的血管生成能力,从而增加肿瘤的侵袭性。     

Sulforaphane inhibited expression of hypoxia-inducible factor-1alpha in human tongue squamous cancer cells and prostate cancer cells

Previous studies show that a number of natural compounds from our diet have anticancer effects. Sulforaphane is the most characterized isothiocyanates (ITCs), which are identified in cruciferous vegetables. Sulforaphane is viewed as a conceptually promising agent in cancer prevention. Because of its ability to induce cancer cell apoptosis, it inhibits progression of benign tumors to malignant tumors and interrupts metastasis. However, the effect of sulforaphane on tongue cancer cell proliferation has not yet been reported, and the mechanisms that sulforaphane inhibits cancer development are still unclear. Hypoxia-inducible factor 1 (HIF-1) expression is associated with tumorigenesis and angiogenesis. It regulates the expression of many genes including vascular endothelial growth factor (VEGF), inducible nitric oxide synthase, and lactate dehydrogenase A. In our study, we investigated the effects of sulforaphane on expression of hypoxia-inducible factor-1alpha (HIF-1alpha), which was overexpressed in many human malignant tumors, human tongue squamous cell carcinoma and prostate cancer DU145 cells. Sulforaphane inhibited hypoxia induced expression of HIF-1alpha via inhibiting synthesis of HIF-1alpha. Sulforaphane was also found to inhibit hypoxia induced HIF-1alpha expression through activating JNK and ERK signaling pathways, but not AKT pathway. Inhibition of HIF-1alpha by sulforaphane resulted in decreasing expression of VEGF. Taken together, these results suggest that sulforaphane is an effective chemopreventive compound against tongue cancers and prostate cell angiogenesis in vitro, and that the HIF-1alpha target provides a new sight into the mechanisms of sulforaphane's inhibition against tumor cell proliferation.     

FAT1 modulates EMT and stemness genes expression in hypoxic glioblastoma

Glioblastoma (GBM) is characterized by the presence of hypoxia, stemness and local invasiveness. We have earlier demonstrated that FAT1 promotes invasiveness, inflammation and upregulates HIF‐1α expression and its signaling in hypoxic GBM. Here, we have identified the role of FAT1 in regulating EMT (epithelial‐mesenchymal transition) and stemness characteristics in GBM. The expression of FAT1, EMT (Snail/LOX/Vimentin/N‐cad), stemness (SOX2/OCT4/Nestin/REST) and hypoxia markers (HIF‐1α/VEGF/PGK1/CA9) was upregulated in ≥39% of GBM tumors (n = 31) with significant positive correlation (p ≤ 0.05) of the expression of FAT1 with LOX/Vimentin/SOX2/HIF‐1α/PGK1/VEGF/CA9. Furthermore, positive correlation (p ≤ 0.01) of FAT1 with Vimentin/N‐cad/SOX2/REST/HIF‐1α has been observed in TCGA GBM‐dataset (n = 430). Analysis of cells (U87MG/A172) exposed to severe hypoxia (0.2%O₂) revealed elevated mRNA expression of FAT1, EMT (Snail/LOX/Vimentin/N‐cad), stemness (SOX2/OCT4/Nestin/REST) and hypoxia markers (HIF‐1α/PGK1/VEGF/CA9) as compared to their normoxic (20%O₂) counterparts. FAT1 knockdown in U87MG/A172 maintained in severe hypoxia and in normoxic primary glioma cultures led to significant reduction of EMT/stemness markers as compared to controls. HIF‐1α knockdown in U87MG cells markedly reduced the expression of all the EMT/stemness markers studied except for Nestin and SOX2 which were more under the influence of FAT1. This indicates FAT1 has a novel regulatory effect on EMT/stemness markers both via or independent of HIF‐1α. The functional relevance of our study was corroborated by significant reduction in the number of soft‐agar colonies formed in hypoxic‐siFAT1 treated U87MG cells. Hence, our study for the first time reveals FAT1 as a novel regulator of EMT/stemness in hypoxic GBM and suggests FAT1 as a potential therapeutic candidate.     

原代培养大鼠心肌细胞低氧损伤时琥珀酸G蛋白偶联受体通路的变化

目的：探讨琥珀酸G蛋白偶联受体（GPR91）通路与低氧环境心肌细胞损伤之间的关系。方法：体外培养乳鼠原代心肌细胞，建立低氧模型，再利用siRNA干扰技术降低细胞内GPR91的表达，随机分为阴性对照组（CTL）、CTL+siGPR91组、琥珀酸盐组、琥珀酸盐+siGPR91组、低氧CTL组、低氧CTL+siGPR91组、低氧琥珀酸盐组和低氧琥珀酸盐+siGPR91组。10%氧含量下培养5 d后，CCK-8法检测心肌细胞的存活率、原代心肌细胞低氧模型的细胞ATP含量，Western blot法检测心肌损伤相关蛋白钙调素依赖蛋白激酶Ⅱ（CaMK Ⅱ）、B型尿钠肽（BNP）、GPR91以及PI3K/Akt蛋白通路Akt及P-Akt的表达变化。结果：与CTL组比较，低氧环境下可见心肌细胞存活率降低，ATP含量减少，BNP及CaMK Ⅱ表达上调（均为P < 0.05）；siRNA干扰组心肌细胞GPR91的表达水平下调（P < 0.05），心肌细胞存活率上升、ATP含量增多、BNP及CaMK Ⅱ蛋白表达下调、PI3K及下游信号分子Akt磷酸化水平降低（均为P < 0.05）。结论：GPR91可能通过调控PI3K/Akt通路的磷酸化介入心肌缺血损伤的病理过程。