HIF-1α Regulates Cancer Stem Cell Phenotype And Radioresistance Through HIF-dependent-TAZ And-GLS2 Pathway In Cancer Cells

[Background]Cancers are characterised by dysregulated growth due to increased consumption of O2 by increased cell divisin leading to intratumoral hypoxia. Intratumoral hypoxia induces the expression of hypoxia-inducible factor-1α(HIF-1α), which is associated with the risk of tumor metastasis, relapse, and mortality in all kinds of clinical studies on thousands of cancer patients. HIF-1 is the key transcriptional activator response to hypoxia consisting of HIF-1α and HIF-1β subunits that regulate the expression of over 1000 target genes. Those genes induced by HIFs are correlated with multiple critical aspects of tumorigenesis, such as metabolic reprogramming, angiogenesis, invasion, metastasis, epithelial-mesenchymal transition, stem cell maintenance, and resistance to chemotherapy and radiation.A very small group of cancer cells in primary tumor which have self-renewal and tumor-initiation capacities, are necessary to form recurrent tumors, and are called tumor initiating cells or cancer stem cells(CSCs). Many cancers are now considered to contain cancer stem cells, whose numbers and phenotype are elevated and induced by hypoxia through the activiation of HIFs. However, the underlying mechanisms have not been well determined. It has been shown recently that one of the effectors of Hippo pathway named TAZ(transcriptional co-activator with PDZ binding motif), is required to promote self-renewal and tumor-initiation capacities in breast cancer stem cells(BCSCs). TAZ is highly expressed in more than 80% of high-grade breast cancers, but the amplification of its gene WWTR1 which encodes TAZ has been reported less than 10% in breast cancers. Meanwhile, high frequency of overlap between HIF-1α target genes and TAZ targets suggests that HIF-1α may play a critical role in the induction of TAZ transcriptional activity in the majority of cases.Adaptive metabolism switch in cancer development and progression is a most critical characteristic of different types of tumor. The activation of many oncogenes involved in metabolic networks leading to tumors undergo a metabolic alter and helping them response to tough environment such as radio- or chemo-therapy which also active HIF-1 expression. The upregulation of HIF-1 regulates GLUT-1 and PDK1 and promotes a switch from oxidative to glycolytic metabolism while limits the transformation into acetyl coenzyme A(acetyl-CoA). The decreased delivery of acetyl-CoA to the TCA cycle results in a switch from glucose to glutamine as the primery substrate for fatty acid synthesis. Our gene microarray analysis results from radioresistent cervical cancers show that the GLS2 enzyme is overexpressed in radioresistent groups compare to control group which remind us that the adaptive upregulation of glutamine metabolism might exist in those cancer cells. However, the potiential mechanism is unknown.[Objective]1. To investigate the role of HIF-1α in regulating BCSCs phenotype, and further explore the multiple crosstalk between HIF-1 and TAZ and molecular mechanism of HIF-1 regulating TAZ nuclear localization by affecting Hippo signaling activity.2. To investigate the regulatory effect of HIF-1α in glutamine metabolism of cervical cancer cells, and further uncover the critical role of glutaminase in causing the radioresistance in cervical cancer.[Methods]1. The correlations between TAZ gene WWTR1 and HIF metagene signature were analyzed by utilizing the Cancer Genome Atlas(TCGA) database. For hypoxic treatment, cells were cultured in hypoxia chamber with 1% O2, 5% N2 and 94% CO2. The expression levels of TAZ in breast cancer cells(BCCs) and tissues were measured by immunohistochemistry, real-time qPCR and immunobloting. The transcriptional mechanisms of HIF-1 on TAZ and SIAH1 gene promoter activity were detected by luciferase reporter assay and ChIP assay. CoIP assay was utilized to measuring the interaction between LATS2 and SIAH1. Immunofluorescent staining was used to determine the TAZ nuclear localization. Aldefluor assay, flow cytometry, mammosphere assay and limiting tumor injection assay were performed to determine the regulatory effect of HIF-1 and TAZ on BCSCs phenotype. To efficiently knockdown TAZ, SIAH1 or LATS2 expression, pLKO.1-puro lentiviral vectors encoding shRNA were established and transfected into 293 T cells with plasmid pCMV-dR8.91. Kaplan–Meier analysis and Wilcoxon rank sum test were used to analyze disease-specific survival for breast cancer patient stratified by TAZ and HIF signatures mRNA expression in their primary tumor survival data which were downloaded from the NCBI Gene Expression Omnibus.2. To investigate the metabolism of HIF-1 in regulating glutamine metabolism reprogramming and further radioresistance in cervical cancers, radioresistant Hela cell line(HelaR) was generated by continuous sublethal irradiation for 6 months, with a 2Gy radiation repeated 25 times to the total dose of 50 Gy. The protein level of GLS2 was measured by immunohistochemistry and immunobloting. GLS2-shRNA was designed against GLS2 and inserted into lentiviral pGCL-GFP vector. Clonogenic assay and flow cytometry were performed to measure the changes of radiosensitivity as well as apoptosis and cell cycle in different treatment groups. Immunofluorescent staining of dihydroethidium(DHE) was used to measure cellular ROS levels. Glutathione(GSH) and oxidative glutathione(GSSG) were determined by colorimetric microplate assay kits. NADH and NAD+ levels were measured by using the EnzyChromTM NAD+/NADH Assay Kit. The NADP+/NADPH ratio was determind by NADP+/NADPH quantitation colorimetric kit. Mouse tumor models were established to explore the proliferation ability in each treatment groups. Finally, cervical cancer tissues from patients were stained of GLS2 by IHC and used for statistical analysis of clinicopathological features.[Results]1. Multiplex crosstalk between HIF-1α and TAZ regulates the breast cancer stem cell phenotype1.1 Heat map revealed that TAZ mRNA levels were correlated with the 10-gene HIF signature. Analysis of expression of the individual genes revealed that TAZ expression was significantly correlated with 8 out of 10 genes. Real-time PCR and immunobloting results showed TAZ mRNA and protein levels increased under hypoxic conditions in all 5 different breast cancer cell lines especially in metastatic lines. And this induction was blocked in HIF-1α but not HIF-2α knockdown cell lines. Moreover, analysis of the human WWTR1 gene from UCSC gene database revealed the sequence 5’-GCGTGGCACACA-3’ on the antisense strand within intron 2. ChIP assays were performed to demonstrate hypoxia-inducible binding of HIF-1α and HIF-1β at this site. To test whether this putative HRE was functional, luciferase activity was found increased significantly in hypoxic cells but was lost in the HRE mutant group.1.2 Knockdown of TAZ did not affect HIF-1α or HIF-2α protein levels. ChIP assay showed that knockdown of HIF-1α or HIF-1β decreased TAZ transcriptional activity. The induction of CTGF, PAI-1 and Survivin mRNA expression under hypoxia were also in a HIF-1α and TAZ dependent manner in BCCs. Additionally, immunofluorescence was preformed to evaluate the subcellular localization of TAZ under hypoxia and then confirmed with immunobloting. Data showed that TAZ was localized primarily in cytoplasm under nomoxia, but moved to nuclei with increased TAZ expression and with decreased levels in the cytoplasm under hypoxia.1.3 To investigate the role of hypoxia on Hippo pathway, immunoblot was performed to detect LATS1/2. Data showed only LATS2 decreased under hypoxia with HIF-dependent manner. Analysis of E3 ubiquitin-protein ligases associated with hypoxia revealed SIAH1 was regulated by HIF-1, and it degradates LATS2 protein under hypoxia in an ubiquitin-proteasome-dependent degradation. Moreover, ChIP assay was used to explore the HRE-binding site of HIF-1 on SIAH1 gene.1.4 Aldefluor assay, flow cytometry and mammosphere assay were performed to explore the amount of BCSCs and its self-renewal ability at hypoxia. Results showed the percentage of ALDH+ cells were increased in hypoxia but reduced in HIF-1α and TAZ knockdown cells. Similar results were found in mammosphere assay. Knockdown of SIAH1 or LATS2 also affected the BCSCs phenotype and TAZ subcellular localization. Next, the limiting tumor injection assay was performed to detect the tumor-initiating potential of breast cancer cells in vivo. Data revealed only in the HIF-1α knockdown cells, the tumor-seeding ability was completely loss. Finally, Kaplan–Meier analysis suggested that breast cancer patient with overexpression of both HIF and TAZ signatures were associated with significantly decreased patient survival.2. The role of HIF-1 on glutamine metabolism reprogramming induced radioresistance in cervical cancer.2.1 The radioresistant Hela cell line(HelaR) was established and confirmed its radioresistent property by performing clonogenic, apoptosis and cell cycle assay. Microarray showed the glutaminase 2 was highly upregulated in HelaR compared to Hela cells. Then we knockdown GLS2 and performed clonogenic, apoptosis and flow cytometry assay to invesgated the changes of radiosensitivity in each groups. However, GLS2 knockdown cells displayed weaker colony formation capacity compared to other control groups. In addition, more apoptotic cells were detected in GLS2 knockdown group than radioresistant or control group at 48 h after 6 Gy irradiation.2.2 Reduced GLS2 expressions led to a vast increase in ROS levels in both unstressed and ionizing radiation-treated cells by flow cytometry. Moreover, knockdown of GLS2 resulted in increased oxidation of DHE to 2-OH-E+ when compared to HelaR and control cells. Then, we measured the GSH levels and GSH/GSSG ratio in different groups. Results showed HelaR cells displayed higher levels of GSH and GSH/GSSG ratio, whereas knocking down of GLS2 expression decreased GSH levels and the GSH/GSSG ratio in cells. Additionally, silencing GLS2 singnificantly decreased NADH and NADPH levels in cells.2.3 Mouse xenografts were established to determine whether the role of GLS2 on radioresistant cervical cancer to radiation in vivo. The size of the xenografts in GLS2 knockdown group decreased more dramatically than those in radioresistant group and other groups. Moreover, IHC staining of clinical patient samples revealed positive expressions of GLS2 in tumor tissues of radioresistant patient were much higher than those of radiosensitive patients. The levels of GLS2 staining were highly correlated with radiosensitivity of cervical cancer. Finally, correlation analysis showed GLS2 mRNA expression was significantly correlated with HIF signature. Real-time PCR and immunobloting results showed GLS2 was regulated by HIF-1α.[Conclusion]1. TAZ is a HIF-target gene; HIF-1 affects TAZ trancriptional activity; HIF-1α regulates SIAH1-dependent LATS2 degradation and increases TAZ nuclear localization; HIF-1-TAZ axis plays an important role in affecting breast cancer stem cell phenotype in vitro and in vivo.2. The activation of HIF-1α under irradiation treatment upregulates the glutamine metabolism and GLS2 expression in cervical cancer cells which leading to more GSH but less ROS production and increases the radioresistance of those cells; while block GLS2 increases the radiosensitivity in cervical cancer response to radio therapy.