EGFR induces expression of IRF-1 via STAT1 and STAT3 activation leading to growth arrest of human cancer cells

Recently, we reported that epidermal growth factor receptor (EGFR) induce expression of a module of genes known to be inducible by interferons and particularly interferon-gamma. Here we show that the module is tightly regulated by EGFR in the 2 human cancer cell lines that overexpress EGFR, A431 and HN5. The module of genes included the tumor suppressor IRF-1, which was used as a prototypical member to further investigate the regulation and function of the module. Ligand-activated EGFR induce expression of IRF-1 via phosphorylation of STAT1 and STAT3. In contrast, cells expressing the constitutively active cancer specific receptor EGFRvIII are unable to mediate phosphorylation of these STATs and thereby incapable of inducing IRF-1. We also demonstrate that IRF-1 is expressed in an EGF dose-dependent manner, which correlates with inhibition of cell proliferation, and that the regulation of IRF-1 is partially dependent on intracellular Src family kinase activity. Treatment with the dual specific Abl/c-Src kinase inhibitor AZD0530 significantly reduces the growth inhibitory effect of high EGF concentrations, signifying that EGFR induced IRF-1 is responsible for the observed growth inhibition. In addition, we show that media from these EGF treated cancer cells upregulate the activation marker CD69 on both B-cells and T-cells in peripheral blood. Taken together, these results suggest that cells acquiring sustained high activity of oncogenes such as EGFR are able to activate genes, whose products mediate growth arrest and activate immune effector cells, and which potentially could be involved in alerting the immune system in vivo leading to elimination of the transformed cells.     

Carcinogenic metalloid arsenic induces expression of mdig oncogene through JNK and STAT3 activation

Environmental or occupational exposure to arsenic, a chemical element classified as metalloid, has been associated with cancer of the lung, skin, bladder, liver, etc. Mdig (mineral dust-induced gene) is a newly identified oncogene linked to occupational lung diseases and lung cancer. It is unclear whether mdig is also involved in arsenic-induced malignant transformation of the lung cells. By using human bronchial epithelial cells and human lung cancer cell lines, we showed that arsenic was able to induce expression of mdig. We further demonstrated that this mdig induction by arsenic was partially dependent on the JNK and STAT3 signaling pathways. Disruption of the JNK or STAT3 by either chemical inhibitors or siRNAs diminished arsenic-induced accumulation of mdig mRNA and protein. Furthermore, we also showed that microRNA-21 (miR-21) and Akt were down-stream effectors of the JNK and STAT3 signaling pathways in arsenic-induced mdig expression. Transfection of the cells with anti-miR-21 or pre-treatment of the cells with Akt inhibitor blunted mdig induction by arsenic. Clinically, the levels of mdig can be applied to predict the disease progression, the first progression (FP), in non-small cell lung cancer (NSCLC) patients. Taken together, our data suggest that mdig may play important roles on the pathogenesis of arsenic-induced lung cancer and that JNK and STAT3 signaling pathways are essential in mediating arsenic-induced mdig expression.     

Hypoxia-induced CXC chemokine ligand 14 expression drives protumorigenic effects through activation of insulin-like growth factor-1 receptor signaling in glioblastoma

Hypoxic tumor microenvironment (HTM) promotes a more aggressive and malignant state in glioblastoma. However, little is known about the role and mechanism of CXC chemokine ligand 14 (CXCL14) in HTM-mediated glioblastoma progression. In this study, we report that CXCL14 expression correlated with poor outcomes, tumor grade, and hypoxia-inducible factor (HIF) expression in patients with glioblastoma. CXCL14 was upregulated in tumor cells within the hypoxic areas of glioblastoma. Hypoxia induced HIF-dependent expression of CXCL14, which promoted glioblastoma tumorigenicity and invasiveness in vitro and in vivo. Moreover, CXCL14 gain-of-function in glioblastoma cells activated insulin-like growth factor-1 receptor (IGF-1R) signal transduction to regulate the growth, invasiveness, and neurosphere formation of glioblastoma. Finally, systemic delivery of CXCL14 siRNA nanoparticles (NPs) with polysorbate 80 coating significantly suppressed tumor growth in vivo and extended the survival time in patient-derived glioblastoma xenografts. Together, these findings suggest that HIF-dependent CXCL14 expression contributes to HTM-promoted glioblastoma tumorigenicity and invasiveness through activation of the IGF-1R signaling pathway. CXCL14 siRNA NPs as an oligonucleotide drug can inhibit glioblastoma progression and constitute a translational path for the clinical treatment of glioblastoma patients.     

Aberrant expression of fibroblast growth factor receptor-1 in prostate epithelial cells allows induction of promatrilysin expression by fibroblast growth factors

Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins, and there is evidence that they play a role in tumor cell growth, invasion and metastasis. Matrilysin (MMP-7) is over-expressed in prostate cancer cells and increases prostate cancer cell invasion. Prostate stromal fibroblasts secrete a factor(s), including fibroblast growth factor-1 (FGF-1), which induces promatrilysin expression in the prostate carcinoma cell line LNCaP but not in normal prostate epithelial cells (PrECs). Since FGF-1 is present in the prostate, an altered sensitivity to FGF-1 might explain the up-regulation of matrilysin expression in prostate cancer cells compared to normal prostate epithelium. FGF receptor-1 (FGFR-1) is not normally expressed by normal prostate epithelial cells; however, aberrant expression of this receptor has been reported in prostate cancer cells, including the LNCaP cell line. We hypothesized that aberrant expression of FGFR-1 in PrECs would render them sensitive to induction of promatrilysin expression by recombinant FGF-1. To test this hypothesis, we transiently transfected PrECs with an FGFR-1 expression vector, which resulted in over-expression of FGFR-1 protein in approximately 40% of cells. FGF-1 increased promatrilysin expression in FGFR-1-transfected PrECs 4-fold over mock-transfected cells, and this induction was inhibited by a specific FGFR-1 inhibitor, SU5402, and by co-expression of a dominant negative FGFR-1 protein. Our results demonstrate that aberrant FGFR-1 expression, an epigenetic phenomenon that has been associated with prostate cancer progression, allows induction of promatrilysin expression by FGF-1 in PrECs.     

Characterization of STAT3 activation and expression in canine and human osteosarcoma

Background

MUTYH-associated polyposis (MAP) is a recessively inherited disorder which predisposes biallelic carriers for a high risk of polyposis and colorectal carcinoma (CRC). Since about one third of the biallelic MAP patients in population based CRC series has no adenomas, this study aimed to identify specific clinicopathological characteristics of MAP CRCs and compare these with reported data on sporadic and Lynch CRCs.

Methods

From 44 MAP patients who developed ≥ 1 CRCs, 42 of 58 tumours were analyzed histologically and 35 immunohistochemically for p53 and beta-catenin. Cell densities of CD3, CD8, CD57, and granzyme B positive lymphocytes were determined. KRAS2, the mutation cluster region (MCR) of APC, p53, and SMAD4 were analyzed for somatic mutations.

Results

MAP CRCs frequently localized to the proximal colon (69%, 40/58), were mucinous in 21% (9/42), and had a conspicuous Crohn's like infiltrate reaction in 33% (13/40); all of these parameters occurred at a higher rate than reported for sporadic CRCs. Tumour infiltrating lymphocytes (TILs) were also highly prevalent in MAP CRCs. Somatic APC MCR mutations occurred in 14% (5/36) while 64% (23/36) had KRAS2 mutations (22/23 c.34G>T). G>T tranversions were found in p53 and SMAD4, although the relative frequency compared to other mutations was low.

Conclusion

MAP CRCs show some similarities to micro-satellite unstable cancers, with a preferential proximal location, a high rate of mucinous histotype and increased presence of TILs. These features should direct the practicing pathologist towards a MAP aetiology of CRC as an alternative for a mismatch repair deficient cause. High frequent G>T transversions in APC and KRAS2 (mutated in early tumour development) but not in P53 and SMAD4 (implicated in tumour progression) might indicate a predominant MUTYH effect in early carcinogenesis.     

Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression

Although HER2-targeting antibody trastuzumab confers a substantial benefit for patients with HER2-overexpressing breast and gastric cancer, overcoming trastuzumab resistance remains a large unmet need. In this study, we revealed a STAT3-centered positive feedback loop that mediates the resistance of trastuzumab. Mechanistically, chronic exposure of trastuzumab causes the upregulation of fibronection (FN), EGF and IL-6 in parental trastuzumab-sensitive breast and gastric cells and convergently leads to STAT3 hyperactivation. Activated STAT3 enhances the expression of FN, EGF and IL-6, thus constituting a positive feedback loop which amplifies and maintains the STAT3 signal; furthermore, hyperactivated STAT3 signal promotes the expression of MUC1 and MUC4, consequently mediating trastuzumab resistance via maintenance of persistent HER2 activation and masking of trastuzumab binding to HER2 respectively. Genetic or pharmacological inhibition of STAT3 disrupted STAT3-dependent positive feedback loop and recovered the trastuzumab sensitivity partially due to increased apoptosis induction. Combined trastuzumab with STAT3 inhibition synergistically suppressed the growth of the trastuzumab-resistant tumor xenografts in vivo. Taken together, our results suggest that feedback activation of STAT3 constitutes a key node mediating trastuzumab resistance. Combinatorial targeting on both HER2 and STAT3 may enhance the efficacy of trastuzumab or other HER2-targeting agents in HER2-positive breast and gastric cancer.     

Src family kinases promote AML cell survival through activation of signal transducers and activators of transcription (STAT)

We investigated the role of Src family kinases (SFKs) in the regulation of STAT activation in myeloid leukemia cells. Two of 6 AML cell lines displayed constitutive STAT5 activation, whereas four cell lines had constitutive SFK activity. Treatment with the SFK inhibitors suppressed STAT5 activation and decreased viability. Akt phosphorylation and Mcl-1 expression decreased after SFK inhibition accompanied by apoptosis induction. In primary AML specimens, SFK inhibitors suppressed proliferation in 5 of 14 specimens. These data indicate that Src-STAT5 and Src-Akt pathways are integral survival signal pathways in AML cells. Src inhibition may represent a novel treatment strategy for investigation in AML.     

Stromal cell-derived factor 1alpha stimulates human glioblastoma cell growth through the activation of both extracellular signal-regulated kinases 1/2 and Akt

In this paper, we describe the role of chemokine receptor CXCR4 activation by its natural ligand, the chemokine stromal cell-derived factor (SDF-1) (CXCL12), in glioblastoma cell growth in vitro. We show that both CXC chemokine receptor 4 (CXCR4) and SDF-1 mRNA are expressed in several human glioblastoma multiforme tumor tissues and in two human glioblastoma cell lines, U87-MG and DBTRG-05MG. These cells are able to secrete SDF-1 under basal conditions, and the rate of secretion is highly increased after lipopolysaccharide or 1% fetal bovine serum treatment. Exogenous SDF-1alpha induces proliferation in a dose-dependent manner in both cell lines. Moreover, we observed that SDF-1alpha-dependent proliferation is correlated with phosphorylation and activation of both extracellular signal-regulated kinases 1/2 and Akt and that these kinases are independently involved in glioblastoma cell proliferation. The role of CXCR4 stimulation in glioblastoma cell growth is further demonstrated by the ability of human monoclonal CXCR4 antibody (clone 12G5) to inhibit the SDF-1alpha-induced proliferation as well as the proliferation induced by SDF-1-releasing treatments (lipopolysaccharide and 1% fetal bovine serum). These data support a role for SDF-1alpha in the regulation of glioblastoma growth in vitro, likely through an autocrine/paracrine mechanism.     

乙肝病毒x蛋白激活缺氧诱导因子-1通路促进血管内皮生长因子表达的研究

目的:探讨乙肝病毒x蛋白(HBx)是否可以通过激活缺氧诱导因子-1α(HIF-1α)通路促进血管内皮生长因子(VEGF)的表达。方法：将pIRES-EGFP-HBx和pTK-Hyg质粒共同转染人肝癌细胞系SMMC-7721,筛选出荧光阳性和阴性的抗性克隆,检测两种克隆中VEGF和加HIF-1α在mRNA和蛋白水平的表达。结果：荧光阳性细胞有HBx的稳定整合和表达,并有HIF-1α和VEGF的上调表达;荧光阴性细胞无HBx的表达,HIF-1α和VEGF的表达亦为阴性。结论：HBx可以通过激活HIF-1通路促进VEGF的表达。     

Fibroblast growth factor-2 enhances NK sensitivity of hepatocellular carcinoma cells

The roles of fibroblast growth factor-2 (FGF-2) in the hepatocellular carcinoma (HCC) development are still controversial. In this study, we investigated the expression of FGF-2 in chronic hepatitis (CH) type C patients with or without HCC and the immunoregulation of FGF-2 in NK sensitivity of HCC cells. The FGF-2 expressions were detected in the liver tissues of patients, but not in normal liver. The serum FGF-2 levels of the patients with CH, liver cirrhosis (LC) or HCC were significantly higher than those of healthy volunteers. The serum FGF-2 levels of patients decreased with the progression of chronic liver disease. HCC occurrence of LC patients with high levels of serum FGF-2 was significantly lower than that with low levels of serum FGF-2. Proinflammatory cytokines, such as IL-1β and IL-6, induced FGF-2 expressions in HCC cells and normal hepatocytes. FGF-2 stimulation resulted in increasing the expression of the membrane-bound major histocompatibility complex class I-related chain A (MICA), an NK activating molecule, and decreasing that of human leukocyte antigen (HLA) class I, an NK inhibitory molecule, on HCC cells. This did not occur with normal hepatocytes. Adding anti-FGF receptor-2 neutralizing antibody resulted in inhibiting the change of MICA and HLA class I expressions on FGF-2 stimulated HCC cells. FGF-2 stimulation on HCC cells resulted in increasing NK sensitivity against HCC cells. These findings indicate that FGF-2 produced by HCC cells or normal hepatocytes of chronic liver disease may play critical roles in eliminating HCC cells by innate immunity.