Fei Ye

Upregulation of LRIG1 suppresses malignant glioma cell growth by attenuating EGFR activity

Activated epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target for a variety of solid tumors, particularly malignant gliomas. A recently discovered transmembrane glycoprotein, LRIG1, antagonizes the activity of epidermal growth factor receptor family receptor tyrosine kinases and acts as a negative feedback loop of EGFR and proposed tumor suppressors. The aim of this study was to investigate the impact of LRIG1 on the biological features of glioma cells and the possible mechanisms of enhanced apoptosis induced by upregulation of LRIG1. We observed that the expression of LRIG1 was decreased, while the expression of EGFR was increased in the majority of astrocytomas, and the ratio of EGFR/LRIG1 was increased by sixfold in tumors versus corresponding non-neoplastic tissue. Upregulation of LRIG1, followed by a decrease of EGFR on the cytomembrane of the cells, induced cell apoptosis and cell growth inhibition, and further reversed invasion in glioma cell lines and primary glioma cells. Our study now clearly indicates that LRIG1 indeed affects cell fate and biology behaviors of the cells in vitro by inhibiting phosphorylation of downstream MAPK and AKT signaling pathway, and the elevated release level of caspase-8 might contribute to the enhanced apoptosis in LRIG1 transfected glioma cells. Taken together, these findings provide us with an insight into LRIG1 function, and we conclude that LRIG1 evolved in gliomas as a rare feedback negative attenuator of EGFR and could offer a novel therapeutic target to treat patients with malignant gliomas.

Abrogation of constitutive Stat3 activity circumvents cisplatin resistant ovarian cancer

The aim of the present study was to investigate the role of Stat3 in cisplatin resistant ovarian cancer. It was first demonstrated that higher activated Stat3 was detected in cisplatin-resistant ovarian cancer cell lines. To provide evidence that supported the hypothesis that phosphorylated-Stat3 expression may promote cisplatin resistance, ectopic Stat3 was expressed by IL-6 stimulation that partially abrogates Stat3, as opposed to the knock-down of Stat3 by specific siRNA that restores cisplatin sensitivity against ovarian cancer cells. This hypothesis was further confirmed by clinical tumor specimens of ovarian cancer obtained from patients with cisplatin-resistance. Based on these premises, Stattic, an effective small molecular inhibitor of Stat3, was used to inhibit Stat3 activation. The data presented here show that Stattic restored the sensitivity to cisplatin in chemoresistant ovarian cancer by significant reductions in the expression of the anti-apoptosis protein Bcl-2, Bcl-XL, Survivin protein and phosphorylated-Akt levels. Consistent with these observations, this experiment demonstrated the first evidence of Stattic circumvented cisplatin resistance of orthotopic xenograft ovarian cancer in vivo. Altogether, these findings emphasize the importance of Stat3 in cisplatin resistance in ovarian cancer and provide a further impetus to clinically evaluate biological modifiers that may circumvent cisplatin resistance in patients with chemoresistant ovarian cancer.

Inhibition of LRIG3 gene expression via RNA interference modulates the proliferation, cell cycle, cell apoptosis, adhesion and invasion of glioblastoma cell (GL15)

LRIG3 (leucine-rich repeats and immunoglobulin-like domains, LRIG) gene is both under-and over-expressed in human cancers and its role on tumor growth is not fully clarified. Here, we used a human U6 promoter-driven DNA template approach to induce short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block LRIG3 gene expression in the human glioma cell line GL15. Specific knockdown of LRIG3 by shRNA resulted in significantly increase of the GL15 invasion and adhesion activity in vitro and markedly promoted cell growth. LRIG3 repression also induced increment of the proportion of G0/G1 cells and inhibited apoptosis in GL15 cells. Our results demonstrated that RNAi against LRIG3 could effectively down regulate LRIG3 gene expression. LRIG3 might be involved in the regulation of EGFR signaling, and serve as a tumor suppressor gene in the pathogenesis of glioma.

Therapeutic targeting of EGFR-activated metabolic pathways in glioblastoma.

Introduction: The highly divergent histological heterogeneities, aggressive invasion and extremely poor response to treatment make glioblastoma (GBM) one of the most lethal and difficult cancers in humans. Among key elements driving its behavior is epidermal growth factor receptor (EGFR), however, neither traditional therapy including neurosurgery, radiation, temozolomide, nor targeted EGFR therapeutics in clinic has generated promising results to date. Strategies are now focusing on blocking the downstream EGFR-activated metabolic pathways and the key phosphorylated kinases. Areas covered: Here, we review two major EGFR-activated downstream metabolic pathways including the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways and their key phosphorylated kinase alterations in GBMs. This review also discusses potential pharmacological progress from bench work to clinical trials in order to evaluate specific inhibitors as well as therapeutics targeting PI3K and RAS signaling pathways. Expert opinion: Several factors impede clinical progress in targeting GBM, including the high rates of acquired resistance, heterogeneity within and across the tumors, complexity of signaling pathways and difficulty in traversing the blood–brain barrier (BBB). Substantial insight into genetic and molecular pathways and strategies to better tap the potential of these agents include rational combinatorial regimens and molecular phenotype-based patient enrichment, each of which will undoubtedly generate new therapeutic approaches to combat these devastating disabilities in the near future.

Therapeutic targeting of EGFR in malignant gliomas

Importance of the field: Despite the improved prognosis for many cancer patients, the survival of those with malignant gliomas (MGs) remains dismal. Even with aggressive intervention, including surgery, chemotherapy and radiotherapy, the overall 2-year survival rate is only 25% in the most optimistic series, and 5-year survival rates are consistently in the low single digits. Therefore, it is evident that novel therapeutic paradigms are necessary to overcome the inherent limitations of conventional treatments. EGFR gene overexpression can be found in 40 – 50% of patients with MGs, whereas its expression is very low in normal brain. Therapeutic targeting of EGFR has indicated clinical success in the treatment of MGs. Areas covered in this review: The purpose of this review is to discuss the current status of several EGFR-targeted therapies in MGs patients and address the efficacy of these drugs as monotherapy or in combination with other drugs and/or treatments. We also emphasize the lessons learned and the future perspectives in the development of EGFR-targeted therapies for MGs. What the reader will gain: A more comprehensive understanding of the molecular, structural and biological characteristics of EGFR and the mechanisms of action of EGFR-targeted antagonists will most likely contribute to the successful use of strategies of EGFR-targeted therapy in the clinic. Take home message: Therapeutic targeting of EGFR include anti-EGFR mAbs, small-molecule EGFR tyrosine kinase inhibitors, peptide vaccination therapy and other therapeutic strategies. Each EGFR antagonist has its own advantages and limitations in terms of BBB crossing, ease of delivery, combination therapies and potential toxicity. Therefore, a multiple approach combining different agents that target EGFR signaling at multiple levels seems to have potential as future therapeutics for MGs, once the technical and safety issues unique to each of the approaches are overcome.

Downregulation of LRIG1 expression by RNA interference promotes the aggressive properties of glioma cells via EGFR/Akt/c-Myc activation

The LRIG1 [leucine-rich repeats and immunoglobulin-like domains (LRIG)] gene is not universally downregulated in human cancers, and its role in tumorigenesis and the development of glioma has not been well addressed. In this study, we used short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block LRIG1 gene expression in the GL15 human glioma cell line. Specific downregulation of LRIG1 by shRNA resulted in significantly enhanced capabilities of proliferation, inhibition of apoptosis and invasion in the GL15 cells. LRIG1 repression induced marked activation of epidermal growth factor receptor (EGFR), protein kinase B (Akt) and c-Myc signaling molecules. Our results demonstrated that RNAi against LRIG1 may effectively downregulate LRIG1 gene expression. LRIG1 functions as a tumor suppressor in the pathogenesis of glioma via EGFR/Akt/c-Myc activation.

A role for LRIG1 in the regulation of malignant glioma aggressiveness

The molecular mechanisms that drive the development and aggressive progression of malignant astrocytic tumors remain obscure. Recently, in the search for endogenous negative regulators of EGF receptor, LRIG1 was cloned and characterized as a putative tumor suppressor gene often downregulated in various human tumors, including astrocytic tumors. Although several studies have implicated the function of LRIG1 in the inhibition of tumorigenesis, its precise role and potential underlying mechanisms remain obscure. Therefore, we generated a full-length expression vector to overexpress LRIG1 in the U251 malignant glioma cell line. Introduction of exogenous LRIG1 into glioma cells inhibited cell proliferation manifested by MTT and soft agar clone assay in vitro and subcutaneously tumor xenografts. On the other hand, LRIG1 overexpression inhibited glioma growth by significantly changing the expression pattern of cyclins, resulting in delayed cell cycle. Employing transwell invasion and wound scratch assay and gelatin zymography, LRIG1 inhibited U-251 MG cell invasion and migration by attenuating MMP2 and MMP9 production. Under ligand-stimulated conditions, p-ERK levels did not change, whereas p-AKT levels were inhibited in cells with LRIG1 upregulation, indicating that LRIG1 exerts more inhibiting effects on the PI3K/AKT pathway. Our findings suggest that LRIG1 restricted glioma aggressiveness by inhibiting cell proliferation, migration and invasion. Restoration of LRIG1 to glioma cells could offer a novel therapeutic strategy.

Correlation between PTEN expression and PI3K/Akt signal pathway in endometrial carcinoma.

SummaryIn order to investigate the role of the PTEN expression in carcinogenesis and development of endometrial carcinoma and clarify whether and how PTEN and PI3K/Akt pathway relate to endometrial carcinoma, the expression of PTEN and phospho-Akt was detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) methods and Western-blot from 24 cases of endometrial carcinoma, 10 cases of endometrial atypical hyperplasia, 10 cases of endometrial hyperplasia, and 10 cases of normal endometrium. SP immunohistochemical methods were used to measure levels of PTEN protein expression in following 5 study groups: 31 cases of endometrium in proliferative phase, 30 cases of endometrium in secretory phase, 71 cases of endometrial hyperplasia, 25 cases of atypical hyperplasia and 73 cases of endometrial carcinoma. Immunostaining score of PTEN was 3.39±0.15 in proliferative phase, 1.90±0.21 in secretory phase, 3.34±0.29 in endometrial hyperplasia, 0.62±0.11 in atypical hyperplasia, and 0.74±0.19 in endometrial carcinoma, respectively. PTEN mRNA relative value in normal endometrium, endometrial hyperplasia, endometrial atypical hyperplasia, and endometrial carcinoma was 2.45±0.51, 2.32±0.32, 0.46±0.11, and 0.35±0.13 respectively. The expression levels of PTEN mRNA and protein in patients with endometrial carcinoma and atypical hyperplasia were significantly lower than in those of proliferative phase and with endometrial hyperplasia. The level of PTEN expression in patients with endometrial carcinoma was significantly related to tissue type (P<0.005), differentiation (P<0.05) and clinical stage (P<0.05), but not to depth of myometrium invasion (P>0.05). Western blot analysis revealed that Phospho-Akt level in PTEN negative cases was significantly higher, and there was a negative correlation between PTEN and phospho-Akt (r=−0.8973, P<0.0001). It was suggested that loss of PTEN expression was an early event in endometrial tumorigenesis. The phosphorylation of Akt induced by the loss of PTEN took part in the tumorigenesis and development of endometrial carcinoma.In order to investigate the role of the PTEN expression in carcinogenesis and development of endometrial carcinoma and clarify whether and how PTEN and PI3K/Akt pathway relate to endometrial carcinoma, the expression of PTEN and phospho-Akt was detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) methods and Western-blot from 24 cases of endometrial carcinoma, 10 cases of endometrial atypical hyperplasia, 10 cases of endometrial hyperplasia, and 10 cases of normal endometrium. SP immunohistochemical methods were used to measure levels of PTEN protein expression in following 5 study groups: 31 cases of endometrium in proliferative phase, 30 cases of endometrium in secretory phase, 71 cases of endometrial hyperplasia, 25 cases of atypical hyperplasia and 73 cases of endometrial carcinoma. Immunostaining score of PTEN was 3.39±0.15 in proliferative phase, 1.90±0.21 in secretory phase, 3.34±0.29 in endometrial hyperplasia, 0.62±0.11 in atypical hyperplasia, and 0.74±0.19 in endometrial carcinoma, respectively. PTEN mRNA relative value in normal endometrium, endometrial hyperplasia, endometrial atypical hyperplasia, and endometrial carcinoma was 2.45±0.51, 2.32±0.32, 0.46±0.11, and 0.35±0.13 respectively. The expression levels of PTEN mRNA and protein in patients with endometrial carcinoma and atypical hyperplasia were significantly lower than in those of proliferative phase and with endometrial hyperplasia. The level of PTEN expression in patients with endometrial carcinoma was significantly related to tissue type (P<0.005), differentiation (P<0.05) and clinical stage (P<0.05), but not to depth of myometrium invasion (P>0.05). Western blot analysis revealed that Phospho-Akt level in PTEN negative cases was significantly higher, and there was a negative correlation between PTEN and phospho-Akt (r=−0.8973, P<0.0001). It was suggested that loss of PTEN expression was an early event in endometrial tumorigenesis. The phosphorylation of Akt induced by the loss of PTEN took part in the tumorigenesis and development of endometrial carcinoma.

Quercetin induces protective autophagy and apoptosis through ER stress via the p-STAT3/Bcl-2 axis in ovarian cancer

Quercetin (3,3′,4′,5,7-pentahydroxyflavone, Qu) is a promising cancer chemo-preventive agent for various cancers because it inhibits disease progression and promotes apoptotic cell death. In our previous study, we demonstrated that Qu could evoke ER stress to enhance drug cytotoxicity in ovarian cancer (OC). However, Qu-induced ER stress in OC is still poorly understood. Here, we demonstrated that Qu evoked ER stress to involve in mitochondria apoptosis pathway via the p-STAT3/Bcl-2 axis in OC cell lines and in primary OC cells. Unexpectedly, inhibition of ER stress did not reverse Qu-induced cell death. Further functional studies revealed that Qu-induced ER stress could activate protective autophagy concomitantly by activating the p-STAT3/Bcl-2 axis in this process. Moreover, the autophagy scavenger 3-MA was shown to enhance Qu’s anticancer effects in an ovarian cancer mice xenograft model. These findings revealed a novel role of ER stress as a “double edge sword” participating in Qu-induced apoptosis of OC and might provide a new angle to consider in clinical studies of biological modifiers that may circumvent drug resistance in patients by targeting protective autophagy pathways.

Identification of U251 glioma stem cells and their heterogeneous stem‑like phenotypes

Glioblastoma, the most common and lethal type of intracranial tumor, is characterized by extensive heterogeneity at the cellular and molecular levels. The discovery of glioma stem cells (GSCs) lends support to a new paradigm in tumor biology. In the present study, we aimed to clarify the validity of using U251 glioma cells as a source of GSC culture and critically evaluate the heterogeneous stem-like phenotypes of these cells when grown under various culture conditions. The findings suggested that U251 cells (U251-Adh, U251-SC-Sph and U251-SC-Adh) showed distinctive growth patterns and self-renewal capacity. The U251 glioma cell line is endowed with certain GSC phenotypes that may be moderately enriched in vitro when transferred into stem cell culture conditions, although this is not sustainable and reproducible in vivo. Notably, glioma cells are plastic in response to their environment. The reversible adaptive plasticity contributes to the GSC heterogeneity, which may lead to the heterogeneity of glioblastoma and the differing responses to current therapies. Therefore, an improved understanding of GSC heterogeneity is urgently required for designing more effective therapies against this highly malignant brain tumor.