Tiansuo Zhao

Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breast cancer.

Polymorphisms in the hypoxia-inducible factor-1α gene confer susceptibility to pancreatic cancer

The transcription factor hypoxia-inducible factor-1 (HIF-1) has α and β subunits. Recent studies have shown that the HIF-1α gene may have C1772T and G1790A single nucleotide polymorphisms (SNPs). These SNPs may increase the stability and activity of HIF-1α. In the present study, we looked for these SNPs by genotyping circulating mononuclear cells from 263 patients with pancreatic ductal adenocarcinoma (PDAC), using 271 healthy volunteers as controls. As a result, both SNPs were more frequent in PDAC patients than in healthy volunteers (C1772T: 21 vs. 11%, p < 0.01; G1790A: 25 vs. 8%, p < 0.01). Further, both SNPs were associated with higher risks for PDAC (C1772T: OR=2.156, 95% CI: 1.324–3.511, p < 0.05; G1790A: OR=3.716, 95% CI: 2.213–6.238, p < 0.01). We also stained HIF-1α by immunohistochemistry in 68 PDAC tumors to examine their HIF-1α expression levels. To this end, we designed a semi-quantitative method that was based on the staining intensity and frequency of HIF-1α-positive cells. As a result, the G1790A SNP, but not C1772T SNP, was associated with an increased HIF-1α expression. We also related genotyping data to patient’s survival times, serum CA19-9, and tumor’s volumes, grades, stages and lymph-node metastasis. The C1772T SNP was not associated with any of these parameters. In contrast, the G1790A SNP was associated with increases in serum CA19-9 and in tumor volumes. In conclusion, the C1772T and G1790A SNPs in the HIF-1α gene increase the susceptibility to pancreatic cancer. In addition, the G1790A SNP is associated with increases in tumor-produced HIF-1α and in the progression of the cancer.

Hypoxia-inducible factor-1α regulates chemotactic migration of pancreatic ductal adenocarcinoma cells through directly transactivating the CX3CR1 gene.

CX3CR1 is an important chemokine receptor and regulates the chemotactic migration of pancreatic ductal adenocarcinoma (PDAC) cells. Up to now, its regulatory mechanism remains largely undefined. Here, we report that hypoxia upregulates the expression of CX3CR1 in pancreatic cancer cells. When hypoxia-inducible factor (HIF)-1α expression was knocked down in vitro and in vivo, the expression of CX3CR1 was significantly decreased. Chromatin immunoprecipitation assay demonstrated that HIF-1α bound to the hypoxia-response element (HRE; 5′-A/GCGTG-3′) of CX3CR1 promoter under normoxia, and this binding was significantly enhanced under hypoxia. Overexpression of HIF-1α significantly upregulated the expression of luciferase reporter gene under the control of the CX3CR1 promoter in pancreatic cancer cells. Importantly, we demonstrated that HIF-1α may regulate cancer cell migration through CX3CR1. The HIF-1α/CX3CR1 pathway might represent a valuable therapeutic target to prevent invasion and distant metastasis in PDAC.

Hypoxia inducible factor (HIF)-1α directly activates leptin receptor (Ob-R) in pancreatic cancer cells.

The aim of this study is to investigate the regulatory mechanism of leptin receptors (Ob-R) in pancreatic cancer. We found that the over-expression of hypoxia inducible factor (HIF-1)α and hypoxia up-regulated the expression of Ob-R in pancreatic cancer cells. When HIF-1α gene was silenced in vitro, the expression of Ob-R was significantly decreased. Xenograft mouse models showed that the inhibition of HIF-1α resulted in the concomitant decrease of Ob-R in vivo. In addition, HIF-1α expression was correlated with Ob-R in pancreatic cancer tissues by immunohistochemical staining. Clinical data showed that over-expression of HIF-1 was associated with pathological tumor node metastasis stage, lymph node metastasis and overall survival. HIF-1α directly bound to the hypoxia-responsive element (HRE) located in Ob-R gene promoter (-828/-832) and activated the transcription. Finally, we demonstrated that the silence of HIF-1α gene reversed the inhibitory effect of leptin/Ob-R in pancreatic cancer cells. Taken together, our results indicate that HIF-1α directly regulated Ob-R expression in pancreatic cancer, which might be a valuable therapeutic target for pancreatic cancer.

Single nucleotide polymorphism in the microRNA-199a binding site of HIF1A gene is associated with pancreatic ductal adenocarcinoma risk and worse clinical outcomes

Hypoxia-inducible factor-1 alpha (HIF-1α) is over-expressed in many cancers including pancreatic ductal adenocarcinoma (PDAC) and correlated with poor prognosis. We aim to determine the effect of germline genetic variants on the regulation of the homeostasis of the miRNA-gene regulatory loop in HIF1A gene and PDAC risk. HIF1A rs2057482 single nucleotide polymorphism (SNP) was genotyped in 410 PDAC cases and 490 healthy controls. The CC genotype SNP HIF1A is significantly correlated with PDAC risk (OR = 1.719, 95% CI: 1.293–2.286) and shorter overall survival (OS, P<0.0001) compared with the CT/TT alleles group. The C/T variants of rs2057482, a SNP located near the miR-199a binding site in HIF1A, could lead to differential regulation of HIF1A by miR-199a. Specifically, the C allele of rs2057482 weakened miR-199a–induced repression of HIF-1α expression on both mRNA and protein levels. In the PDAC tissue, individuals with the rs2057482-CC genotype expressed significantly higher levels of HIF-1α protein than those with the rs2057482-CT/TT genotype (P<0.0001). Both the CC genotype of SNP HIF1A and increased HIF-1α expression are significantly associated with shorter OS of patients with PDAC. After adjusted by TNM staging, differentiation grade, and the levels of CA19-9, both SNP HIF1A and HIF-1α expression retained highly significance on OS (P<0.0001). Taken together, our study demonstrates that host genetic variants could disturb the regulation of the miR-199a/HIF1A regulatory loop and alter PDAC risk and poor prognosis. In conclusion, the rs2057482-CC genotype increases the susceptibility to PDAC and associated with cancer progression.

Tumour-derived Interleukin 35 promotes pancreatic ductal adenocarcinoma cell extravasation and metastasis by inducing ICAM1 expression

Interleukin 35 (IL-35) is a novel member of the IL-12 family, consisting of an EBV-induced gene 3 (EBI3) subunit and a P35 subunit. IL-35 is an immune-suppressive cytokine mainly produced by regulatory T cells. However, the role of IL-35 in cancer metastasis and progression is not well understood. Here we demonstrate that IL-35 is overexpressed in human pancreatic ductal adenocarcinoma (PDAC) tissues, and that IL-35 overexpression is associated with poor prognosis in PDAC patients. IL-35 has critical roles in PDAC cell extravasation and metastasis by facilitating the adhesion to endothelial cells and transendothelial extravasation. Mechanistically, IL-35 promotes ICAM1 overexpression through a GP130-STAT1 signalling pathway, which facilitates endothelial adhesion and transendothelial migration via an ICAM1–fibrinogen–ICAM1 bridge. In an orthotopic xenograft model, IL-35 promotes spontaneous pancreatic cancer metastasis in an ICAM1-dependent manner. Together, our results indicate additional functions of IL-35 in promoting PDAC metastasis through mediating ICAM1 expression.

Stem cell factor is a novel independent prognostic biomarker for hepatocellular carcinoma after curative resection

Stem cell factor (SCF), a ligand of c-kit, is a hematopoietic growth factor. Uncontrolled activity of SCF/c-kit signaling pathway contributes to the formation of a variety of human malignancies. In this study, we determined whether SCF expression could risk-stratify patients with hepatocellular carcinoma (HCC) after curative resection. HCC tissues from 160 patients were collected during curative resection and stained with SCF and CD34, a marker for microvessel density (MVD), using immunohistochemistry. Two statistical analyses were performed: an independent continuous and a multivariate categorical analysis, with test/validation set-defined cut points, and Kaplan-Meier estimated outcome measures of overall survival (OS) and relapse-free survival (RFS). We found that higher levels of SCF confer worse OS (continuous P = 0.014; and categorical P = 0.009), and RFS (continuous P = 0.002; categorical P = 0.003) of patients with HCC. SCF varies independently from MVD-CD34, tumor node metastasis, histologic grade, age and gender, and retains prognostic significance when analysed as a categorical variable in a multivariate analysis . We confirmed that MVD-CD34 is also an independent prognostic marker for patients with HCC. The levels of SCF and CD34 showed a positive and significant correlation (P < 0.0001) and double low expression confers superior OS (median = 48 months) and RFS (median = 24 months), whereas double high expression confers shortest RFS (median = 10.5 months) compared with single measurements. The prognostic values of SCF and CD34 were independently determined in this study and we propose that both of them are independent prognostic markers for HCC.

Cancer-FOXP3 directly activated CCL5 to recruit FOXP3 + Treg cells in pancreatic ductal adenocarcinoma

Forkheadbox protein 3 (FOXP3), initially identified as a key transcription factor for regulatory T cells (Treg cells), was also expressed in many tumors including pancreatic ductal adenocarcinoma (PDAC). However, its role in PDAC progression remains elusive. In this study, we utilized 120 PDAC tissues after radical resection to detect cancer-FOXP3 and Treg cells by immunohistochemistry and evaluated clinical and pathological features of these patients. Cancer-FOXP3 was positively correlated with Treg cells accumulation in tumor tissues derived from PDAC patients. In addition, high cancer-FOXP3 expression was associated with increased tumor volumes and poor prognosis in PDAC especially combined with high levels of Treg cells. Overexpression of cancer-FOXP3 promoted the tumor growth in immunocompetent syngeneic mice but not in immunocompromised or Treg cell-depleted mice. Furthermore, CCL5 was directly trans-activated by cancer-FOXP3 and promoted the recruitment of Treg cells from peripheral blood to the tumor site in vitro and in vivo. This finding has been further reinforced by the evidence that Treg cells recruitment by cancer-FOXP3 was impaired by neutralization of CCL5, thereby inhibiting the growth of PDAC. In conclusion, cancer-FOXP3 serves as a prognostic biomarker and a crucial determinant of immunosuppressive microenvironment via recruiting Treg cells by directly trans-activating CCL5. Therefore, cancer-FOXP3 could be used to select patients with better response to CCL5/CCR5 blockade immunotherapy.

SCF, regulated by HIF-1α, promotes pancreatic ductal adenocarcinoma cell progression.

Stem cell factor (SCF) and hypoxia-inducible factor-1α (HIF-1α) both have important functions in pancreatic ductal adenocarcinoma (PDAC). This study aims to analyze the expression and clinicopathological significance of SCF and HIF-1α in PDAC specimens and explore the molecular mechanism at PDAC cells in vitro and in vivo. We showed that the expression of SCF was significantly correlated with HIF-1α expression via Western blot, PCR, chromatin immunoprecipitation (ChIP) assay, and luciferase assay analysis. The SCF level was also correlated with lymph node metastasis and the pathological tumor node metastasis (pTNM) stage in PDAC samples. The SCF higher-expression group had significantly lower survival rates than the SCF lower-expression group (p<0.05). Hypoxia up-regulated the expression of SCF through the hypoxia-inducible factor (HIF)-1α in PDAC cells at the protein and RNA levels. When HIF-1α was knocked down by RNA interference, the SCF level decreased significantly. Additionally, ChIP and luciferase results demonstrated that HIF-1α can directly bind to the hypoxia response element (HRE) region of the SCF promoter and activate the SCF transcription under hypoxia. The results of colony formation, cell scratch, and transwell migration assay showed that SCF promoted the proliferation and invasion of PANC-1 cells under hypoxia. Furthermore, the down-regulated ability of cell proliferation and invasion following HIF-1α knockdown was rescued by adding exogenous SCF under hypoxia in vitro. Finally, when the HIF-1α expression was inhibited by digoxin, the tumor volume and the SCF level decreased, thereby proving the relationship between HIF-1α and SCF in vivo. In conclusion, SCF is an important factor for the growth of PDAC. In our experiments, we proved that SCF, a downstream gene of HIF-1α, can promote the development of PDAC under hypoxia. Thus, SCF might be a potential therapeutic target for PDAC.

Wnt2 protein plays a role in the progression of pancreatic cancer promoted by pancreatic stellate cells

This study aimed to investigate the expression of Wnt2 protein in pancreatic cancer tissues and pancreatic stellate cells (PSCs), and determine its effect on the biological functions of pancreatic cancer cells. Immunohistochemistry was used to study the expression pattern of Wnt2 in pancreatic cancer tissues. The relationship between Wnt2 protein expression level and patient prognosis was analyzed. PSCs were isolated and cultured. The expression of Wnt2 in activated PSCs was investigated using Western blot and immunofluorescence. We also analyzed the effect of Wnt2 recombinant protein and stellate cell culture supernatant on the Wnt/β-catenin signaling pathway, as well as the effect of Wnt2 recombinant protein on the biological functions of pancreatic cancer cells. The expression of Wnt2 in interstitial cells of pancreatic cancer was correlated with the prognosis of pancreatic cancer. Wnt2 protein was expressed in activated PSCs. Both stellate cell culture supernatant and Wnt2 recombinant protein could activate the classic Wnt/β-catenin signaling pathway. Wnt2 protein enhanced the migration, invasion, and metastasis of pancreatic cancer cells. These results suggested that Wnt2 protein secreted by PSCs promoted the progression of pancreatic cancer by activating the classic Wnt/β-catenin signaling pathway.