Inhibition of cytoplasmic GSK-3β increases cisplatin resistance through activation of Wnt/β-catenin signaling in A549/DDP cells

Cisplatin-based chemotherapy is recommended as the first-line therapy for advanced non-small cell lung cancer (NSCLC). However, acquired cisplatin resistance is ubiquitous in patients with NSCLC, but the molecular mechanism of such resistance remains ambiguous. The present study sought to examine the role of the Wnt/β-catenin signaling pathway in cisplatin resistance by assessing the phosphorylation and subcellular distribution of GSK-3β in a human lung adenocarcinoma cell line, A549, and its cisplatin-resistant subline, A549/DDP. Total GSK-3β, phosphorylated GSK-3β^ser9 and phosphorylated GSK-3β^tyr216 in cytoplasmic and nuclear fractions of A549/DDP and A549 cells were examined by western blot analysis. The regulation of cisplatin resistance, apoptosis, β-catenin and survivin protein expression by inhibition of cytoplasmic GSK-3β were determined by MTT assay, flow cytometry analysis, immunofluorescence technique and western blot analysis. In the present study, cytoplasmic levels of p-GSK-3β^ser9 were significantly increased in A549/DDP cells as compared with A549 cells (P < 0.01), and these levels were further increased by cisplatin treatment in A549/DDP cells (P < 0.01). In contrast, cytoplasmic levels of p-GSK-3β^ser9 were reduced in A549 cells after treatment with cisplatin (P < 0.01). However, cytoplasmic levels of p-GSK-3β^tyr216 were significantly decreased in A549/DDP cells as compared with A549 cells (P < 0.01), and these levels were further decreased by cisplatin treatment in A549/DDP cells (P < 0.01). Conversely, cytoplasmic levels of p-GSK-3β^tyr216 were raised in A549 cells after treatment with cisplatin (P < 0.01). Analysis of downstream effectors of the Wnt/β-catenin signaling pathway revealed upregulation of β-catenin and survivin expression in A549/DDP cells treated with cisplatin as compared to untreated cells. In A549 cells, cisplatin treatment decreased the expression of β-catenin and survivin. Furthermore, phosphorylation of GSK-3β at serine 9 by LiCl and transient interference of GSK-3β by siRNA increased β-catenin and survivin protein expression in A549/DDP cells. Low exogenous and endogenous cytoplasmic GSK-3β expression enhanced the IC50 and inhibited apoptosis. In conclusion, activation of the Wnt/β-catenin signaling pathway and upregulated survivin expression due to cytoplasmic GSK-3β inhibition might lead to cisplatin resistance in NSCLC.     

Glycogen synthase kinase-3: a new therapeutic target in renal cell carcinoma

Background:

Renal cell carcinoma (RCC) is highly resistant to chemotherapy because of a high apoptotic threshold. Recent evidences suggest that GSK-3β positively regulates human pancreatic cancer and leukaemia cell survival in part through regulation of nuclear factor (NF-κB)-mediated expression of anti-apoptotic molecules. Our objectives were to determine the expression pattern of GSK-3β and to assess the anti-cancer effect of GSK-3β inhibition in RCC.

Methods:

Immunohistochemistry and nuclear/cytosolic fractionation were performed to determine the expression pattern of GSK-3β in human RCCs. We used small molecule inhibitor, RNA interference, western blotting, quantitative RT–PCR, BrDU incorporation and MTS assays to study the effect of GSK-3β inactivation on renal cancer cell proliferation and survival.

Results:

We detected aberrant nuclear accumulation of GSK-3β in RCC cell lines and in 68 out of 74 (91.89%) human RCCs. We found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of renal cancer cells. We observed that inhibition of GSK-3 results in decreased expression of NF-κB target genes Bcl-2 and XIAP and a subsequent increase in renal cancer cell apoptosis. Moreover, we show that GSK-3 inhibitor and Docetaxel synergistically suppress proliferation and survival of renal cancer cells.

Conclusions:

Our results show nuclear accumulation of GSK-3β as a new marker of human RCC, identify that GSK-3 positively regulates RCC cell survival and proliferation and suggest inhibition of GSK-3 as a new promising approach in the treatment of human renal cancer.     

miR-34a regulates cisplatin-induce gastric cancer cell death by modulating PI3K/AKT/survivin pathway

The purposes of this study were to determine the expression profiles of microRNA-34a (miR-34a) in human gastric cancer cell line (SGC-7901) and cisplatin-resistant cell lines (SGC-7901/DDP), and to establish the correlation between miR-34a expression profile and the sensitivity of human gastric cancer cell to cisplatin-based pattern, thereby providing new methods and strategies for treating gastric cancer. Gastric cancer cell line (SGC-7901) and cisplatin-resistant cell line (SGC-7901/DDP) were cultivated in vitro, respectively. Quantitative real-time PCR (qRT-PCR) and Western blot were utilized to determine the expression profiles of miR-34a and survivin in both gastric cancer cell lines. With miR-34a mimic and miR-34a inhibitor transfected into SGC-7901 and SGC-7901/DDP for 48 h, post-transfection changes of miR-34a expression was determined; the effects of miR-34a ectopic expression on the viability of cisplatin-induce gastric cancer cell were assayed by the MTT method. The effects of miR-34a ectopic expression on apoptosis of cisplatin-induce gastric cancer cell were determined by Annexin V/propidium iodide (PI) double staining method and flow cytometry. The effects of miR-34a ectopic expression on the AKT and p-AKT expression of cisplatin-induce gastric cancer cells were determined by Western blot and flow cytometry with the PI3K pathway inhibitor Wortmannin. As shown by qRT-PCR and Western blot analyses, the expression of miR-34a in cisplatin-resistant cell lines decreased significantly in comparison to that of SGC-7901 cell line (p?<?0.05), while significant up-regulation of survivin expression was also observed (p?<?0.05). Compared with the control group, the expression of miR-34a increased significantly in SGC-7901 cells transfected with miR-34a mimic for 48 h (p?<?0.01). After miR-34a inhibitor transfection, the expression of miR-34a decreased significantly (p?<?0.05). The viability of cisplatin-induce gastric cancer cells increased significantly (p?<?0.05) with significant decrease of apoptosis after miR-34a expression inhibition, as demonstrated by MTT and flow cytometry with miR-34a over-expression, the viability of cisplatin-induce gastric cancer cells decreased significantly (p?<?0.05), with significant apoptosis increase (p?<?0.05). As shown by Western blot and flow cytometry, in comparison to the control group, Wortmannin could inhibit miR-34a inhibitor and DDP induced up-regulation of p-AKT significantly (p?<?0.05) and stimulated apoptosis. In conclusion, miR-34a expression was down-regulated in cisplatin-resistant cell lines. miR-34a over-expression could improve the sensitivity of gastric cancer cells against cisplatin-based chemotherapies, with PI3K/AKT/survivin signaling pathway possibly involved in the mechanism.     

The expression and function of microRNA-203 in lung cancer

We aimed to determine the expression of microRNA-203 (miR-203) in human lung cancer cell lines and to evaluate the effects of miR-203 by targeting survivin, on the lung cancer cell line 95-D to provide potential new strategies for treating lung cancer. The expression of miR-203 was detected using quantitative real-time PCR (qRT-PCR) in the in vitro cultured lung cancer cells A549, HCC827, NCI-H1299, and 95-D as well as in normal human bronchial epithelial cells. Following a 72-h transfection with the miR-203 precursor in 95-D lung cancer cells, the change in miR-203 expression was detected using qRT-PCR and the resulting effect on survivin protein expression was ascertained by Western blot analysis. The influence of miR-203 on the viability of 95-D lung cancer cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The effect of miR-203 on 95-D cell proliferation was analyzed using flow cytometry. The consequences of miR-203 expression on 95-D cell apoptosis were analyzed by Annexin V/propidium iodide double staining coupled with flow cytometry. The role of miR-203 in the invasive potential of 95-D cells was studied using a transwell chamber assay. A luciferase reporter gene system was used to verify that survivin is a target gene for miR-203. By qRT-PCR, the expression of miR-203 was lower in lung cancer cells than in normal bronchial epithelial cells (p?<?0.01), and the expression of miR-203 in 95-D lung cancer cells was significantly higher after a 72-h transfection with the miR-203 precursor (p?<?0.01). After a 72-h transfection with the miR-203 precursor, survivin protein levels in 95-D cells were significantly decreased (p?<?0.01). Cell viability, as assessed with an MTT assay, decreased following an increase in miR-203 expression (p?<?0.05). The flow cytometry results indicated that after miR-203 expression increased, the cell proliferation index decreased (p?<?0.05) and the number of apoptotic cells increased (p?<?0.01). Increased miR-203 expression led to a significant decrease in the number of cells that migrated through a transwell chamber membrane (p?<?0.01). The luciferase reporter gene system demonstrated that the relative luciferase activity significantly decreased after transfection with the miR-203 precursor (p?<?0.05). The expression of miR-203 is downregulated in lung cancer cells. miR-203 negatively regulates survivin protein expression and inhibits the proliferation and invasion of lung cancer cells. Therapeutic strategies that enhance miR-203 expression or silence survivin could potentially benefit lung cancer patients.     

Identification of high-grade serous ovarian cancer miRNA species associated with survival and drug response in patients receiving neoadjuvant chemotherapy: a retrospective longitudinal analysis using matched tumor biopsies

BackgroundNeoadjuvant chemotherapy (NACT) has been recognized as a reliable therapeutic strategy in patients with unresectable advanced epithelial ovarian cancer (EOC). The molecular events leading to platinum (Pt) response in NACT settings have hitherto not been explored. In the present work, longitudinal changes of miRNA expression profile were investigated to identify miRNA families with prognostic role in high-grade serous EOC patients who received the NACT regimen.Patients and methodsOne hundred sixty-four matched tumor biopsies taken at initial laparoscopic evaluation and at interval-debulking surgery (IDS) after four courses of Pt-based therapy were selected from 82 stage IIIC–IV high-grade serous-EOC patients that were judged unsuitable for complete primary debulking and subjected the NACT protocol. miRNA profiling by microarray, real-time PCR and immuno-histochemical staining for Smad2 phosphorylation (P-Smad2) were used for data analysis.ResultsAnalysis revealed that 369 miRNAs were differentially expressed in matched biopsies (referred to as DEMs). DEMs were not scattered across the genome, but clustered into families: miR-199, let-7, miR-30, miR-181 and miR-29. Multivariate analysis showed that miR-199a-3p, miR-199a-5p, miR-181a-5p and let-7g-5p associated with overall and progression-free survival (P < 0.05); miR-199a-3p, miR-199a-5p and miR-181a-5p associated with residual tumor volume and Pt-free interval (P < 0.05). Immuno-histochemical staining confirmed an enrichment of P-Smad2, a marker of transforming growth factor-&bgr; activation, in tumors from patients with shorter PFS and OS, and with high levels of expression of miR-181a-5p (P < 0.05). Kaplan–Meier curves plotting concomitant expression of P-Smad2 and miR-181a-5p show significant differences in PFS and OS compared with those depicting the expression of each biomarker alone (P < 0.001).ConclusionsThis study describes several miRNA families with a prognostic role in the NACT setting. It also confirms that concomitant analysis of P-Smad2 and miR-181a-5p in surgical samples may be capable of identifying those ovarian cancer patients with poor outcome and little chance of response to Pt-based NACT.     

Positive prognostic impact of miR-210 in non-small cell lung cancer

Objectives

miR-210 is an important regulator of the cellular response to hypoxia. Therefore, we aimed to explore the prognostic significance of miR-210 in non-small cell lung cancer (NSCLC) patients with stage I-IIIA disease.

Materials and methods

In addition to clinicopathological and demograpic information, tumor tissues were collected and tissue micro arrays (TMAs) were constructed from 335 patients with stage I-IIIA NSCLC. Expression of miR-210 in cancer cells and stromal cells of the tumor was assessed by in situ hybridization.

Results

In univariate analyses, high cancer cell (p = 0.039) and high stromal cell expression (p = 0.008) of miR-210 were both significantly associated with an improved disease-spesific survival (DSS). High co-expression of miR-210 in cancer and stromal cells was also a positive prognostic factor for DSS (p = 0.010). In multivariate analysis, miR-210 in stromal cells (p = 0.011), and miR-210 co-expressed in cancer and stromal cells was an independent prognosticator for DSS (p = 0.011).

Conclusions

We show that miR-210 in stromal cells, and co-expressed in cancer cells and stromal cells mediates an independent prognostic impact. It is a candidate marker for prognostic stratification in NSCLC.     

Expression and regulatory function of miRNA-34a in targeting survivin in gastric cancer cells

We aimed to investigate the expression of microRNA-34a (miR-34a) in human gastric cancer cells and to evaluate the effects of miR-34a, acting via its gene survivin, on gastric cancer cell HGC-27 to provide potential new strategies for treating gastric cancer. In vitro cultures of the human gastric cancer cell lines MGC80-3, HGC-27, NCI-N87, and SGC-7901 and the normal human gastric epithelial cell line GES-1 were established. The expression of miR-34a in each gastric cancer cell line and GES-1 normal human gastric epithelial cell line was detected using quantitative real-time polymerase chain reaction (qRT-PCR). After the HGC-27 cells were transfected with a miR-34a mimic for 48 h, the changes in the expression levels of miR-34a were detected using qRT-PCR. The effect of miR-34a on HGC-27 cell viability was measured using a tetrazolium-based colorimetric [?(4,5)-dimethylthiahiazo-(?z-y1)-3,5-di-phenytetrazoliumromide (MTT)] assay. Flow cytometry was used to analyze the effects of miR-34a on HGC-27 cell proliferation. Annexin V/propidium iodide double staining and flow cytometry were used to analyze the effects of miR-34a on HGC-27 cell apoptosis. A Transwell invasion chamber was used to detect the effects of miR-34a on HGC-27 cell invasion. Finally, western blotting was used to analyze the effects of miR-34a on survivin protein expression. The qRT-PCR test determined that miR-34a expression in gastric cancer cells was significantly reduced compared to the normal gastric epithelial cell line GES-1 (p?<?0.01). Compared to the control group, cellular miR-34a expression levels were significantly increased in HGC-27 human gastric carcinoma cells after transfection with a miR-34a mimic for 48 h (p?<?0.01). The MTT assay demonstrated that after overexpressing miR-34a in HGC-27 cells, cellular viability was significantly reduced (p?<?0.05). Flow cytometry analysis determined that upon miR-34a overexpression, the proliferation index decreased significantly (p?<?0.05), and cellular apoptosis was significantly increased (p?<?0.01). The Transwell invasion chamber assay illustrated that after increasing the expression of miR-34a, the number of cells passing through the Transwell chamber was significantly reduced (p?<?0.01). Based on western blotting, compared with the control group, survivin protein expression levels were significantly decreased in the HGC-27 cells transfected with the miR-34a mimic for 48 h (p?<?0.01). In conclusion, the expression level of miR-34a was downregulated in human gastric cancer cell lines. miR-34a can negatively regulate survivin protein expression and inhibit gastric cancer cell proliferation and invasion. Therapeutically enhancing miR-34a expression or silencing the survivin gene may benefit patients with gastric cancer.     

Overexpression of DLC-1 induces cell apoptosis and proliferation inhibition in the renal cell carcinoma

The lack of effective anti-tumor therapy for renal cell carcinoma (RCC) has stimulated the search for novel target whose inhibition could block tumorigenesis. Recently, reduced DLC-1 has been shown to be associated with aggressive and highly metastatic renal cell carcinoma. In this study, the biological role of DLC-1 on cell growth, migration and cell cycle progression in RCC cells was investigated. Over-expression of DLC-1 was associated with a marked inhibition of cell growth (P < 0.01). The inhibitory effect was partly due to the induction of apoptosis and cell cycle arrest in G₀/G₁ accompanied by up-regulation of the intracellular signal proteins of p27 and down-regulation of cyclin D1 and cyclin E. Furthermore, DLC-1 induced FAK dephosphorylation of focal adhesion proteins inhibited cell migration (P < 0.05). Decreased DLC-1 expression strongly correlated with proliferative activity, as indicated by the elevated levels of Ki67. Restoration of DLC-1 expression in RCC cells led to Bcl-2 and caspase-3 mediated apoptosis as well as attenuated the ability of the cells to form RCC tumors in athymic nude mice (P < 0.05). Taken together, these results suggest that DLC-1 plays a crucial role in signal transduction pathway regulating the cell proliferation, migration, and carcinogenesis of human RCC.     

Epigenetic silencing of microRNA-199b-5p is associated with acquired chemoresistance via activation of JAG1-Notch1 signaling in ovarian cancer

Epithelial ovarian cancer is a highly lethal and aggressive gynecological malignancy. The high mortality rate is due in part to the fact that many advanced cancer patients become refractory to current chemotherapeutic agents, leading to tumor recurrence and death. However, the underlying mechanisms leading to chemoresistance remain obscure. Here, we report that the loss of miR-199b-5p due to progressive epigenetic silencing leads to the activation of the JAG1-mediated Notch1 signaling cascade, thereby leading to the development of acquired chemoresistance in ovarian cancer. Using miRCURY LNA™ microRNA array and Q-PCR analyses of two pairs of cisplatin-sensitive and –resistant ovarian cancer cell lines, we identified miR-199b-5p as significantly down-regulated in cisplatin-resistant ovarian cancer cells and confirmed that miR-199b-5p is clinically associated with advanced and poor survival ovarian cancers. Interestingly, the loss of miR-199b-5p could be restored by 5-Aza-dC-mediated demethylation, and methylated specific PCR (MS-PCR), bisulfite-sequencing and pyrosequencing revealed that the promoter region of miR-199b-5p was hypermethylated. Computational and mechanistic analyses identified JAG1 as a primary target of miR-199b-5p. Notably, the reduced expression of miR-199b-5p was found to be inversely correlated with the increased expression of JAG1 using an ovarian cancer tissue array. Enforced expression of miR-199b-5p sensitized ovarian cancer cells to cisplatin-induced cytotoxicity both in vitro and in vivo. Conversely, re-expression of miR-199b-5p and siRNA-mediated JAG1 knockdown or treatment with Notch specific inhibitor γ-secretase (GSI) attenuated JAG1-Notch1 signaling activity, thereby enhancing cisplatin-mediated cell cytotoxicity. Taken together, our study suggests that the epigenetic silencing of miR-199b-5p during tumor progression is significantly associated with acquired chemoresistance in ovarian cancer through the activation of JAG1-Notch1 signaling.     

miRNA profiling in metastatic renal cell carcinoma reveals a tumour-suppressor effect for miR-215

Background:

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney. Metastatic RCC is difficult to treat. The 5-year survival rate for metastatic RCC is ⩽10%. Recently, microRNAs (miRNAs) have been shown to have a role in cancer metastasis and potential as prognostic biomarkers in cancer.

Method:

We performed a miRNA microarray to identify a miRNA signature characteristic of metastatic compared with primary RCCs. We validated our results by quantitative real-time PCR. We performed experimental and bioinformatic analyses to explore the involvement of miR-215 in RCC progression and metastasis.

Results:

We identified 65 miRNAs that were significantly altered in metastatic compared with primary RCCs. We validated our results by examining the expression of miR-10b, miR-126, miR-196a, miR-204 and miR-215, in two independent cohorts of patients. We showed that overexpression of miR-215 decreased cellular migration and invasion in an RCC cell line model. In addition, through gene expression profiling, we identified direct and indirect targets of miR-215 that can contribute to tumour metastasis.

Conclusion:

Our analysis showed that miRNAs are altered in metastatic RCCs and can contribute to kidney cancer metastasis through different biological processes. Dysregulated miRNAs represent potential prognostic biomarkers and may have therapeutic applications in kidney cancer.     

MicroRNA-451 induces epithelial–mesenchymal transition in docetaxel-resistant lung adenocarcinoma cells by targeting proto-oncogene c-Myc

Epithelial–mesenchymal transition (EMT) has been reported to play a significant role in tumour metastasis as well as chemoresistance. However, the molecular mechanisms involved in chemotherapy-induced EMT are still unclear. MicroRNA (miRNA) expression and functions have been reported to contribute to phenotypic features of tumour cells. To investigate the roles of miRNAs in chemotherapy-induced EMT, we established two docetaxel-resistant lung adenocarcinoma (LAD) cell models (SPC-A1/DTX and H1299/DTX), which display EMT-like properties and gain increased invasion or migration activity. MiR-451 was found to be significantly downregulated in docetaxel-resistant LAD cells, and re-expression of miR-451 could reverse EMT to mesenchymal–epithelial transition (MET) and inhibit invasion and metastasis of docetaxel-resistant LAD cells both in vitro and in vivo. The proto-oncogene c-Myc was identified as a direct and functional target of miR-451, and further researches confirmed that overexpression of c-Myc which induced extracellular-signal-regulated kinase (ERK)-dependent glycogen synthase kinase-3 beta (GSK-3β) inactivation and subsequent snail activation is essential for acquisition of EMT phenotype induced by loss of miR-451. Furthermore, c-Myc was significantly upregulated in docetaxel-non-responding LAD tissues in comparison with docetaxel-responding tissues, and its expression was inversely correlated with miR-451 expression. This study first reported the involvement of miR-451/c-Myc/ERK/GSK-3β signalling axis in the acquisition of EMT phenotype in docetaxel-resistant LAD cells, suggesting that re-expression of miR-451 or targeting c-Myc will be a potential strategy for the treatment of chemoresistant LAD patients.     

Expression and regulatory function of miRNA-182 in triple-negative breast cancer cells through its targeting of profilin 1

We aimed to evaluate the expression of microRNA-182 (miR-182) in triple-negative breast cancer (TNBC) tissues and the TNBC cell line MDA-MB-231 and to investigate the effects of mirR-182 on the cellular behavior of MDA-MB-231 and the expression of the target gene profilin 1 (PFN1), thus providing new methods and new strategies for the treatment of TNBC. Quantitative real-time PCR (qRT-PCR) was utilized to evaluate the expression of miR-182 in TNBC tissues, relatively normal tissues adjacent to TNBC and the TNBC cell line MDA-MB-231. Forty-eight hours after the MDA-MB-231 cells were transfected with the miR-182 inhibitor, qRT-PCR was utilized to detect the changes in miR-182 expression levels, and an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was utilized to determine the effects of miR-182 on cell viability. Flow cytometry was adopted to determine whether miR-182 affects the proliferation rates and apoptosis levels of the MDA-MB-231 cells. The transwell migration assay method was used to investigate the effects of miR-182 on the migration of the MDA-MB-231 cells. A luciferase reporter gene system was applied to validate that PFN1 was the target gene of miR-182. Western blot was used to measure the effects of miR-182 on the PFN1 protein expression levels in the cells. qRT-PCR results showed that compared with the relatively normal tissues adjacent to TNBC, miR-182 expression was significantly increased in the TNBC tissues and the MDA-MB-231 cells (p?<?0.01). Compared with the control group, MDA-MB-231 cells transfected with the miR-182 inhibitor and incubated for 48 h showed significantly decreased miR-182 expression (p?<?0.01). The results of an MTT assay showed that inhibition of miR-182 in MDA-MB-231 cells led to significantly reduced cell viability (p?<?0.05). Flow cytometry analysis indicated that inhibition of miR-182 expression resulted in significantly decreased cell proliferation (p?<?0.05) and significantly increased levels of apoptosis (p?<?0.05). The results of a transwell migration assay showed that after inhibited of miR-182 expression, the number of cells passing through the transwell membranes was significantly decreased (p?<?0.05). The results from a luciferase reporter gene system showed that compared with the control group, the relative luciferase activity of the group transfected with the miR-182 inhibitor was significantly increased (p?<?0.05). Western blot analysis showed that compared with the control group, PFN1 protein expression levels were significantly increased in the MDA-MB-231 cells transfected with the miR-182 inhibitor and incubated for 48 h (p?<?0.05). In conclusion, miR-182 is upregulated in TNBC tissues and cells. It promotes the proliferation and invasion of MDA-MB-231 cells and could negatively regulate PFN1 protein expression. Treatment strategies utilizing inhibition of miR-182 expression or overexpression of the PFN1 gene might benefit patients with TNBC.     

miR-199a-3p inhibits hepatocyte growth factor/c-Met signaling in renal cancer carcinoma

MicroRNAs (miRNAs) are a class of small non-coding RNAs that bind protein-coding mRNAs and negatively regulate protein expression by translation repression or mRNA cleavage. Accumulating evidence suggests that miRNAs are involved in cancer development and progression, acting as either tumor suppressors or oncogenes. It has been shown that miR-199a-3p was significantly down-regulated in several types of cancers. However, its role and relevance in renal cell carcinoma (RCC) are still largely unknown. Here, we show that miR-199a-3p is significantly down-regulated in human RCC primary tumors and cell lines compared to their non-tumor counterparts. Moreover, the down-regulation of miR-199a-3p is correlated with the histological grade and TNM (tumor–lymph node–metastasis) stage of RCC. Reintroducing miR-199a-3p in RCC cell lines 769-P and Caki-1 inhibited cell proliferation and caused G1 phase arrest. We found that c-Met was up-regulated in RCC cell lines and its expression could be repressed by miR-199a-3p. Moreover, c-Met was up-regulated in RCC primary tumors and reversely correlated with miR-199a-3p expression in the same paired RCC tissues. Reintroducing miR-199a-3p inhibited c-Met expression and led to attenuated activation of c-Met downstream signaling pathways including STAT3, mTOR and ERK1/2. We found that the concentrations of serum hepatocyte growth factor (HGF), the ligand of c-Met receptor, were significantly elevated in RCC patients compared to healthy persons. In addition, HGF treatment could promote proliferation of RCC cells, and the increased cell proliferation was abrogated by miR-199a-3p. Our findings indicated that miR-199a-3p target HGF/c-Met signaling pathway which is crucial for RCC development and suggest that miR-199a-3p may serve as a potential target miRNA for RCC therapy.     

Tumor-suppressive microRNA-449a induces growth arrest and senescence by targeting E2F3 in human lung cancer cells

MicroRNA-449a (miR-449a) was significantly downregulated in 156 lung cancer tissues (p < 0.001). We found that the low expression of miR-449a was highly correlated with cancer recurrence and survival of lung cancer patients. The transient introduction of miR-449a caused cell cycle arrest and cell senescence in A549 and 95D cells. Further studies revealed that E2F3 was a direct target of miR-449a in lung cancer cells. miR-449a also suppressed tumor formation in vivo in nude mice. These results suggest that miR-449a plays an important role in lung cancer tumorigenesis and that miR-449a might predict cancer recurrence and survival of lung cancer patients.     

MiR-125b,miR-100 and miR-99a co-regulate vincristine resistance in childhood acute lymphoblastic leukemia

MicroRNA-125b (miR-125b), miR-99a and miR-100 are overexpressed in vincristine-resistant acute lymphoblastic leukemia (ALL). Cellular viability of ETV6-RUNX1-positive Reh cells significantly increased in presence of 9 ng/mL vincristine upon co-expression of miR-125b/miR-99a (91 ± 4%), miR-125b/miR-100 (93 ± 5%) or miR-125b/miR-99a/miR-100 (82 ± 17%) compared with miR-125b-transduced cells (38 ± 13%, P < 0.05). Co-expression of these miRNAs resulted in downregulation of DNTT, NUCKS1, MALAT1, SNRPE, PNO1, SET, KIF5B, PRPS2, RPS11, RPL38 and RPL23A (fold-change 1.3–1.9, p < 0.05). Similarly, 7 out of these genes are lower expressed in vincristine-resistant ALL cells of children (p < 0.05). The concerted function of miR-125b in combination with miR-99a and/or miR-100 illustrates the complexity of vincristine-resistant pediatric ALL.     

Type I interferons as radiosensitisers for pancreatic cancer

Background

Radiotherapy is an established treatment for malignant localised disease. Pancreatic cancer however seems relatively insensitive to this form of therapy.

Methods

Pancreatic cancer cell lines MiaPaca-2 and Panc-1 were pre-treated with 3000 IU/ml IFNα or 100 IU/ml IFNβ followed by 0, 2, 4, or 6 Gray (Gy) irradiation. Colony forming assay was used to assess the effects on cellgrowth. To measure the surviving fraction at the clinically relevant dose of 2 Gy (SF2), cells were pre-treated with 1000-10.000 IU/ml IFNα or 50-500 IU/ml IFNβ followed by 2 Gy irradiation.

Results

The plating efficiency was 49% for MiaPaca-2 and 22% for Panc-1. MiaPaca-2 was more radiosensitive than Panc-1 (surviving fraction of 0.28 versus 0.50 at 4 Gray). The SF2 of MiaPaca-2 was 0.77 while the SF2 of Panc-1 was 0.70. The SF2 significantly decreased after pretreatment with IFNα 1000 IU/ml (p < 0.001) and IFNβ 100 IU/ml (p < 0.001) in MiaPaca-2 and with IFNα 5000 IU/ml (p < 0.001) and IFNβ 100 IU/ml (p < 0.01) in Panc-1. The sensitising enhancement ratio (SER) for IFNα 3000 IU/ml was 2.15 in MiaPaca-2 and 1.90 in Panc-1. For IFNβ 100 IU/ml the SER was 1.72 for in MiaPaca-2 and 1.51 in Panc-1.

Conclusions

Type I interferons have radiosensitising effects in pancreatic cancer cell lines. This radiosensitising property might lead to an improved response to treatment in pancreatic cancer. Interferon β is the most promising drug due to its effect in clinically obtainable doses.     

Markers of tumour angiogenesis and tumour cells in bone marrow in gastric cancer patients

Aims

Vascular endothelial growth factors VEGF-A, VEGF-C and VEGF-D are considered to be potentially angiogenetic and lymphangiogenetic. “Minimal residual disease” is responsible for cancer progression and recurrence. In this study, we investigated the relation between expressions of VEGF-A, VEGF-C and VEGF-D in gastric cancer tissue and the presence of tumour cells in bone marrow.

Methods

A total of 50 resected primary gastric adenocarcinomas, 44 non-cancerous gastric mucosa and 36 lymph node metastases were analyzed by immunohistochemistry for VEGF-A, VEGF-C and VEGF-D. The specimens used were drawn from a previous study cohort, where the presence of ITC in bone marrow was confirmed with immunohistochemical assay with cytokeratin (CK)-18.

Results

The levels of expression of VEGF-A, VEGF-C and VEGF-D were highest in tumour (p < 0.001), and the level in lymph node metastases was significantly higher (p < 0.01) than in mucosa. The expression of VEGF-A was correlated significantly with venous tumour invasion (p < 0.05) and the presence of tumour cells in bone marrow (p < 0.05). Tumours expressing high levels of VEGF-D showed significantly advanced stages of tumour infiltration (p < 0.05) and lymph node metastasis (p < 0.01).

Conclusions

VEGF-A is a significant marker for the presence of tumour cells in the bone marrow of gastric cancer patients. Our results confirm VEGF-D as a predictor for the lymphatic spread of tumour cells. Therefore, the route of metastatic spread of gastric cancer could be determined, at least in part, by the profile of VEGF family members expressed in the primary tumour of gastric cancer patients.     

Ribosomal s6 protein kinase 4: a prognostic factor for renal cell carcinoma

Background:

The expression and function of ribosomal s6 protein kinase 4 (RSK4) in renal cell carcinoma (RCC) are unknown.

Methods:

Immunohistochemistry was used to detect the expression of RSK4 in RCC, and the relationship between RSK4 expression and clinicopathological features as well as prognosis of RCC patients was statistically analysed. Ectopic RSK4 expression in RCC cell lines was performed to determine its effect on cell cycle regulation, tumour invasiveness, and metastatic capability.

Results:

RSK4 was overexpressed in RCCs (P=0.003), compared with normal tissues, and the expression varied in different RCC subtypes (P=0.021), especially in two subtypes of papillary RCCs (P=0.001). RSK4 expression was positively correlated with high pT stage (P<0.001), high Fuhrman grade (P<0.001), lymph node involvement (P<0.001), and presence of distant metastasis (P=0.039), and could predict poor outcome in RCC patients. Molecular studies showed that overexpression of RSK4 could promote cell cycle progression and enhance the invasive and metastatic capability of RCC cell lines and vice versa.

Conclusion:

The expression pattern and molecular mechanisms of RSK4 in RCCs indicate that it could be a potential independent prognostic factor and serve as a new potential therapeutic target for RCC patients.     

Identification of a microRNA expression signature for chemoradiosensitivity of colorectal cancer cells,involving miRNAs-320a, -224, -132 and let7g

Background and purpose

Preoperative chemoradiotherapy (CRT) represents the standard treatment for locally advanced rectal cancer. Tumor response and progression vary considerably. MicroRNAs represent master regulators of gene expression, and may therefore contribute to this diversity.

Material and methods

Genome-wide microRNA (miRNA) profiling was performed for 12 colorectal cancer (CRC) cell lines and an individual in vitro signature of chemoradiosensitivity was established. Functional relevance of selected miRNAs was established by transfecting miRNA-mimics into SW480 and SW837 cells. The prognostic value of selected miRNAs was assessed in 128 pretherapeutic patient biopsies.

Results

Thirty-six miRNAs were identified to significantly correlate with sensitivity to CRT (Q < 0.05) including miR-320a and other miRNAs involved in the MAPK-, TGF- and Wnt-pathway. Transfection of selected miRNAs (let-7g, miR-132, miR-224, miR-320a) each induced a shift of sensitivity. High expression of let-7g was associated with a good prognosis in rectal cancer patients (P = 0.03).

Conclusions

This is the first report of a miRNA expression signature for in vitro chemoradiosensitivity of CRC cell lines. Many of the identified miRNAs have not been linked to the response to CRT and may represent potential molecular targets to sensitize resistant cancers. If further validated, let7g expression may serve as predictive biomarker.     

Nanomicellar paclitaxel increases cytotoxicity of multidrug resistant breast cancer cells

Multidrug resistance (MDR) of breast cancer cells still represents an unmet medical need in chemotherapy. To this end, the purpose of this study was to determine efficacy of paclitaxel loaded in sterically stabilized, biocompatible and biodegradable sterically stabilized mixed phospholipid nanomicelles (SSMM; size, ∼15 nm) and actively targeted vasoactive intestinal peptide-grafted SSMM (SSMM-VIP) in circumventing P-gp-mediated paclitaxel resistance in BC19/3 cells, a human breast cancer cell line that expresses >10-fold higher P-gp than its parental sensitive cell line, MCF-7. We found that in drug sensitive MCF-7 cells, paclitaxel loaded in SSMM (P-SSMM) and SSMM-VIP (P-SSMM-VIP) significantly inhibited cell growth in dose-dependent fashion (p < 0.05). Both formulations were ∼7-fold more potent than paclitaxel dissolved in DMSO (P-DMSO). Efficacy of P-SSMM and P-SSMM-VIP was similar (p > 0.5). By contrast, in drug resistant BC19/3 cells, P-SSMM-VIP was significantly more effective than either P-SSMM or P-DMSO (∼2- and 5-fold, respectively; p < 0.05). Collectively, these data indicate that actively targeted paclitaxel-loaded SSMM-VIP overcomes multiple drug resistance of BC19/3 cells. We suggest this formulation should be further developed to treat MDR breast cancer.