Feng Wan

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.

Short hairpin RNA targeting FOXQ1 inhibits invasion and metastasis via the reversal of epithelial-mesenchymal transition in bladder cancer

The epithelial-mesenchymal transition (EMT) promotes cancer invasion and metastasis, however, the integrative mechanisms that coordinate the process are incompletely understood. In this study, we defined a pivotal functional role for the Forkhead transcription factor FOXQ1 in regulating EMT in bladder cancer. We initially investigated the expression of FOXQ1, TGF-β1 and EMT biomarkers E-cadherin, Vimentin in 65 cases of bladder transitional cell carcinoma (BTCC) specimens by reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis and immunohistochemistry. Search results indicated that FOXQ1 expression was inversely correlated to E-cadherin, but positively to TGF-β1 and Vimentin in patients with BTCC (P<0.05). Furthermore, we aimed to construct short hairpin RNA (shRNA) expression plasmids against the FOXQ1 gene and transfect shRNAs into high metastatic potential human bladder cancer T24 cells with Lipofectamine 2000. RNAi-mediated suppression of FOXQ1 expression reversed the EMT process accompanied by upregulation of E-cadherin, as well as a loss expression of Vimentin in highly invasive T24 cells (P<0.05). The inhibition of FOXQ1 expression with shRNA vector also led T24 cells to acquire an epithelial cobblestone phenotype, significantly reduced motility and subsequent invasiveness of bladder cancer cells (P<0.05). In conclusion that FOXQ1 may be a novel EMT-inducing transcription factor through controlling the expression of E-cadherin and aggressiveness of cancer cells and targeting the transcription factor FOXQ1 could hence serve as a novel therapeutic strategy for cancer patients.

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.

SATB1 Overexpression Regulates the Development and Progression in Bladder Cancer through EMT

The global gene regulator Special AT-rich sequence-binding protein-1 (SATB1) has been reported to induce EMT-like changes and be associated with poor clinical outcome in several cancers. This study aims to evaluate whether SATB1 affects the biological behaviors of bladder transitional cell carcinoma (BTCC) and further elucidate if this effect works through an epithelial-mesenchymal transition (EMT) pathway. The expression of SATB1, E-cadherin (epithelial markers), vimentin (mesenchymal markers) in BTCC tissues and adjacent noncancerous tissues, as well as in two cell lines of bladder cancer were investigated. Whether the SATB1 expression is associated with clinicopathological factors or not was statistically analyzed. Cell invasion and migration, cell cycle, cell proliferation and apoptosis were evaluated in SATB1 knockdown and overexpressed cell lines. Our results showed that the expression of SATB1 was remarkably up-regulated both in BTCC tissues and in bladder cancer cell lines with high potential of metastasis. The results were also associated with EMT markers and poor prognosis of BTCC patients. Moreover, SATB1 induced EMT processes through downregulation of E-cadherin, upregulation of E-cadherin repressors (Snail, Slug and vimentin). SATB1 also promoted cell cycle progression, cell proliferation, cell invasion and cell migration, but did not alter cell survival. In conclusion, our results suggest that SATB1 plays a crucial role in the progression of bladder cancer by regulating genes controlling EMT processes. Further, it may be a novel therapeutic target for aggressive bladder cancers.

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.

Neural stem cells preferentially migrate to glioma stem cells and reduce their stemness phenotypes.

Glioma stem cells (GSCs), characterized by self-renewal, multi-potentiality and tumorigenicity, are responsible for the tumor propagation, recurrence and resistance to traditional treatments, representing a critical therapeutic target. Neural stem cells (NSCs) possess inherent tropism to brain tumor cells and inhibit their growth. However, there is a limited understanding of the mechanism underlying NSC tropism and the effect of NSC migration on GSC stemness phenotypes. In the present study, we showed that GSCs exhibited enhanced chemotaxis for NSC tropism compared with their differentiated cells. Chemokines secreted by GSCs contributed to the targeted migration of NSCs. Hypoxia enhanced NSC tropism via the upregulated chemokine expression of GSCs, such as VEGF, EGF and bFGF. In vitro migration of NSCs induced GSC differentiation and reduced stem-like phenotypes. Moreover, in vivo data provided direct evidence that transplanted NSCs could migrate to GSCs from either the homolateral or contralateral brain injection site, which prolonged the survival of grafted mice. Taken together, these findings show that NSCs preferentially migrate to GSCs and reduce their stemness phenotypes, raising the intriguing possibility that the targeted migration of NSCs can be applied as a novel therapeutic strategy to target these intractable brain tumors.

LRIG1 enhances cisplatin sensitivity of glioma cell lines.

LRIG family shares similar structures that include a signal peptide, an extracellular region consisting of a leucine-rich repeat domain and three immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. After activation of EGFR, the extracellular LRR domain and immunoglobulin-like domains of LRIG1 can bind to the extracellular parts of EGFR, resulting in recruitment of c-Cbl to the cytoplasmic domains, and induction of EGFR degradation. This study investigated the effects of overexpression of leucine-rich repeats and LRIG1 on cisplatin (CDDP) sensitivity in the glioma cell line U251 and explored the possible mechanisms mediating this effect. We found that CDDP could inhibit the growth of U251 cell line and induced activation of the EGFR. Overexpression of LRIG1 increased the inhibitory effect of CDDP on the U251 cell line via the inhibition of proliferation and induction of apoptosis. The mechanisms underlying the effect of the combined treatment of LRIG1 and CDDP could be that LRIG1 blocked CDDP-induced EGFR activation and regulated the apoptosis proteins. These findings suggest that upregulation of LRIG1 expression enhances the CDDP sensitivity in the glioma cell line U251.

Effect of over-expressed LRIG3 on cell cycle and survival of glioma cells

This study examined the effects of over-expression of leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) on the cell cycle and survival of human glioma cell line U87 and U251 and explored the possible mechanisms. The LRIG3 gene was transduced into U87 and U251 cells respectively by using lentivirus and the transduced cells were selected by puromycin. The changes in LRIG3 mRNA and protein levels were measured by RT-PCR and Western blotting. The apoptosis rate was detected by Annexin V-FITC/PI double labeling and the cell cycle was flow cytometrically analyzed. Compared with control cells, LRIG3 mRNA expression in U251 and U87 cells transduced with pLVX-DsRed-LRIG3-Monomer-N1 were increased by 77.6% and 129.7%, and LRIG3 protein expression was raised by 141.3% and 322.7%, respectively. Cell cycle analysis showed that LRIG3 over-expression increased the percentage of cells at G0/G1 phase (P<0.01). Over-expressed LRIG3 could significantly promote the apoptosis of U87 and U251 cells (P<0.05). These findings suggest that the over-expression of LRIG3 could arrest the cell cycle in G0/G1 phase, and promote apoptosis of U87 and U251 cells.SummaryThis study examined the effects of over-expression of leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3) on the cell cycle and survival of human glioma cell line U87 and U251 and explored the possible mechanisms. The LRIG3 gene was transduced into U87 and U251 cells respectively by using lentivirus and the transduced cells were selected by puromycin. The changes in LRIG3 mRNA and protein levels were measured by RT-PCR and Western blotting. The apoptosis rate was detected by Annexin V-FITC/PI double labeling and the cell cycle was flow cytometrically analyzed. Compared with control cells, LRIG3 mRNA expression in U251 and U87 cells transduced with pLVX-DsRed-LRIG3-Monomer-N1 were increased by 77.6% and 129.7%, and LRIG3 protein expression was raised by 141.3% and 322.7%, respectively. Cell cycle analysis showed that LRIG3 over-expression increased the percentage of cells at G0/G1 phase (P<0.01). Over-expressed LRIG3 could significantly promote the apoptosis of U87 and U251 cells (P<0.05). These findings suggest that the over-expression of LRIG3 could arrest the cell cycle in G0/G1 phase, and promote apoptosis of U87 and U251 cells.

GHRP-6 induces CREB phosphorylation and growth hormone secretion via a protein kinase Cσ-dependent pathway in GH3 cells

This study examined the effect of GHRP-6, a known GHSs receptor agonist, on the phosphorylation of cAMP-responsive element-binding protein (CREB) and the underly mechanism. GH3 cells were cultured and subjected to different treatments as follows: GHRP-6, GHRP-6 plus GHRH, phorbol ester (PMA), an activator of PKC, alone or in combination with GHRP-6, Gö6983, a general inhibitor of PKCs, in the presence or absence of GHRP-6, rottlerin, an inhibitor of PKCs, alone or plus GHRP-6. The cells were transiently transfected with PKCσ-specific siRNA and then treated with GHRP-6. GH level was measured by enzyme-linked immunosorbent assay (ELISA). The expression of phosphor-CREB, PKCσ, PKCθ and phosphor-PKCσ was determined by Western blotting. The results showed that GHRP-6 stimulated GH secretion in both time- and dose-dependent manners and enhanced the effect of GHRH on GH secretion. GHRP-6 was also found to induce CREB phosphorylation. Moreover, GH secretion was enhanced by the PKC activator PMA and reduced by the PKC inhibitors (Gö6983, rottlerin) and knockdown of PKCσ. PKCσ could be activated by GHRP-6. It is concluded that PKC, especially PKCσ, mediates CREB phosphorylation and GHRP-6-induced GH secretion.SummaryThis study examined the effect of GHRP-6, a known GHSs receptor agonist, on the phosphorylation of cAMP-responsive element-binding protein (CREB) and the underly mechanism. GH3 cells were cultured and subjected to different treatments as follows: GHRP-6, GHRP-6 plus GHRH, phorbol ester (PMA), an activator of PKC, alone or in combination with GHRP-6, Gö6983, a general inhibitor of PKCs, in the presence or absence of GHRP-6, rottlerin, an inhibitor of PKCs, alone or plus GHRP-6. The cells were transiently transfected with PKCσ-specific siRNA and then treated with GHRP-6. GH level was measured by enzyme-linked immunosorbent assay (ELISA). The expression of phosphor-CREB, PKCσ, PKCθ and phosphor-PKCσ was determined by Western blotting. The results showed that GHRP-6 stimulated GH secretion in both time- and dose-dependent manners and enhanced the effect of GHRH on GH secretion. GHRP-6 was also found to induce CREB phosphorylation. Moreover, GH secretion was enhanced by the PKC activator PMA and reduced by the PKC inhibitors (Gö6983, rottlerin) and knockdown of PKCσ. PKCσ could be activated by GHRP-6. It is concluded that PKC, especially PKCσ, mediates CREB phosphorylation and GHRP-6-induced GH secretion.

Radiation-induced G2/M arrest rarely occurred in glioblastoma stem-like cells

Abstract Purpose: The purpose of this study is to systematically study the cell-cycle alterations of glioblastoma stem-like cells (GSLCs) after irradiation, possibly enriching the mechanisms of radioresistance of GSLCs. Materials and methods: GSLCs were enriched and identified, and then the radioresistance of GSLCs was validated by analyzing cell survival, cell proliferation, and radiation-induced apoptosis. The discrepancy of the cell-cycle distribution and expression of cell-cycle-related proteins between GSLCs and glioblastoma differentiated cells (GDCs) after irradiation was completely analyzed. Results: The survival fractions and the cell viabilities of GSLCs were significantly higher than those of GDCs after irradiation. Radiation-induced apoptosis was less prominent in GSLCs than in GDCs. After irradiation with high-dose X-rays, the percentages of GDCs in G2/M phase was evidently increased. However, radiation-induced G2/M arrest occurred less frequently in GSLCs, but S-phase arrest occurred in GSLCs after irradiation with 8 Gy. Further mechanistic studies showed that the expressions levels of Cdc25c, Cdc2, and CyclinB1 in GSLCs were not apparently changed after irradiation, while those of p-ATM and p-Chk1 were sharply increased after irradiation in GSLCs. The basal level of Cdc25c expression in GSLCs was much higher than that in GDCs. Conclusions: We explored the cell-cycle alterations and cell-cycle-related proteins expression levels in GSLCs after irradiation, providing a novel mechanism of radioresistance of GSLCs.