MiRNA-34a inhibits EGFR-signaling-dependent MMP7 activation in gastric cancer

The molecular mechanism underlying cancer invasiveness and metastasis of gastric carcinoma remains elusive. Here, we reported significant decrease in microRNA (miRNA)-34a and significant increase in phosphorylated epidermal growth factor receptor (EGFR) and matrix metalloproteinase-7 (MMP7) in the resected gastric carcinoma from the patients, compared with adjacent normal tissue. Moreover, strong correlation was detected among these three factors. To examine whether a causal link exists, we used two human gastric carcinoma lines, SNU-5 and HGC27, to study the molecular basis of miRNA-34a, EGFR signaling, and MMP7 activation. We found that EGF-induced EGFR phosphorylation in SNU-5 or HGC27 cells activated MMP7 and consequently cancer invasiveness. Both an inhibitor for EGFR and an inhibitor for Akt significantly inhibited the EGF-induced activation of MMP7, suggesting a phosphatidylinositol 3-kinase (PI3K) signaling cascade dependent pathway. Moreover, miRNA-34a levels were not affected by EGF-induced EGFR phosphorylation. However, overexpression of miRNA-34a antagonized EGF-induced MMP7 activation without affecting EGFR phosphorylation in SNU-5 or HGC27 cells. Taken together, our data suggest that miRNA-34 inhibits EGFR signaling via downstream PI3K signaling cascades to regulate MMP7 expression in gastric carcinoma. Thus, miRNA-34a, EGFR, and MMP7 appear to be promising therapeutic targets for preventing the metastasis of gastric carcinoma.     

MiRNA-181c inhibits EGFR-signaling-dependent MMP9 activation via suppressing Akt phosphorylation in glioblastoma

As the most aggressive malignant primary human brain tumor, glioblastoma is noted with extremely poor patient survival. Previous studies have demonstrated that expression of matrix metalloproteinase-9 (MMP9) in glioblastoma cells is critical for cancer metastasis. However, the molecular signaling pathways that control MMP9 activation remain undefined. Here, we reported a strong negative correlation of microRNA (miRNA)-181c levels with either MMP9 levels or activation of epidermal growth factor receptor (EGFR) signaling in glioblastoma patients. EGF-induced activation of EGFR in a human glioblastoma line, A-172 cells, increased MMP9 expression through activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway, without affecting expression of miRNA-181c. On the other hand, overexpression of miRNA-181c in A-172 cells inhibited MMP9 expression by inhibiting Akt phosphorylation, but not phosphorylation of EGFR receptor. Taken together, these findings suggest that EGFR signaling activates downstream PI3K/Akt to increase MMP9 expression in glioblastoma, while phosphorylation of Akt is a control point by miRNA-181c. Our work thus provides new insights into the molecular basis underlying the metastasis of glioblastoma.     

Imbalance between MMP-2, 9 and TIMP-1 promote the invasion and metastasis of renal cell carcinoma via SKP2 signaling pathways

S-phase kinase-associated protein-2 (Skp2) is overexpressed in human cancers and acted as an oncogenic protein associated with poor prognosis by enhancing tumor metastasis. The present study has demonstrated that Skp2 overexpresses stable transfectants from 786-0 human renal cancer cells. We found that these stable transfectants exhibited increased migratory and invasive abilities. In addition, expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 was upregulated and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) was downregulated. In contrast, RNA interference-mediated knockdown Skp2 expression suppressed the ability of ACHN cells to migratory and invasive. Skp2 depletion increased P27 and decreased cyclin E activity, and then induced cell cycle arrest in the G0/G1 phase. Skp2 depletion also downregulated MMP-2 and MMP-9, while upregulated the TIMP-1 activity and expression. The results suggest that Skp2 signaling pathways promoted the ability to metastasize, by stimulating cell proliferation and increasing the ratio of MMP-2 and MMP-9/TIMP-1. So, in conclusion, we provide the first evidence that the imbalance of MMP/TIMP, including upregulation of MMP-2 and MMP-9 and downregulation of TIMP-1, is one of the mechanisms by which Skp2 promotes cell invasion.     

Epidermal growth factor induces FoxO1 nuclear exclusion to activate MMP7-mediated metastasis of larynx carcinoma

The molecular mechanism underlying cancer invasiveness and metastasis of larynx carcinoma remains elusive. Here we reported a strong correlation between phosphorylated epidermal growth factor receptor (EGFR) and matrix metalloproteinase-7 (MMP7) levels in larynx carcinoma patients. To examine whether a causal link exists, we used a human larynx carcinoma line, Hep-2, to study the molecular basis of EGFR signaling and MMP7 activation. We found that EGF-induced EGFR phosphorylation in Hep-2 cells resulted in activation of MMP7 and, consequently, an increase in cancer invasiveness. An EGFR inhibitor efficiently blocked this EGF-induced activation of MMP7. Moreover, an inhibitor for PI3 kinase (PI3K)/Akt, but not an inhibitor for mitogen-activated protein kinase (MAPK) or an inhibitor for c-Jun N-terminal kinase (JNK), significantly inhibited the EGF-induced activation of MMP7, suggesting that PI3K/Akt signaling cascades may be responsible for EGF-activated MMP7. Further dissection of the pathway revealed that nuclear exclusion of Akt downstream target, FoxO1, was induced by EGF-induced Akt activation and could be inhibited by either the EGFR inhibitor or by the PI3K/Akt inhibitor. Expression of a constitutive nuclear form of FoxO1 significantly inhibited MMP7 activation induced by EGF. Taken together, these findings suggest that EGF/EGFR signaling activates downstream PI3K/Akt to induce FoxO1 nuclear exclusion, which activates MMP7 to promote larynx carcinoma metastasis. Thus, Akt and FoxO1 appear to be promising therapeutic targets for preventing the metastasis of larynx carcinoma.     

Addition of bevacizumab enhances antitumor activity of erlotinib against non-small cell lung cancer xenografts depending on VEGF expression

Purpose

Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), and bevacizumab, an anti-vascular endothelial growth factor (VEGF) agent, are promising therapies for advanced non-small cell lung cancer (NSCLC). Our study was aimed to determine whether there were conditions under which the addition of bevacizumab would enhance the antitumor activity of erlotinib against NSCLC tumors in vitro and in vivo.

Methods

MTS was for NSCLC cell (PC9, 11–18, H1975, H157, H460 and A549) growth assay in vitro. ELISA was for VEGF protein assay in cells and tumor tissues. Mouse xenograft models were established with H157, H460 and A549 with primary resistance to erlotinib and treated with erlotinib plus bevacizumab or each agent alone. Erlotinib concentrations in tumors were determined by high-performance liquid chromatography.

Results

Bevacizumab alone did not inhibit NSCLC cell growth in vitro. In primarily erlotinib-resistant NSCLC cells, the levels of VEGF protein were highest in H157 cell followed in order by H460 and A549 cells. In vivo, bevacizumab alone significantly inhibited tumor growth only in xenograft models with high (H157) and/or moderate (H460) levels of VEGF protein. A combination of erlotinib and bevacizumab partially reversed resistance to erlotinib in H157 xenografts (high VEGF level) with increasing intratumoral erlotinib concentrations, but not in H460 (moderate) or A549 (low) xenografts.

Conclusions

These results support that combined with anti-VEGF therapy could enhance antitumor activity of anti-EGFR therapy and/or partially reverse resistance to EGFR TKI, by increasing EGFR TKI concentration in specific tumors that express high levels of VEGF protein.     

Interaction of PTPRO and TLR4 signaling in hepatocellular carcinoma

Protein tyrosine phosphatase receptor type O (PTPRO) has been identified as a tumor suppressor in a number of cancers including hepatocellular carcinoma (HCC). Toll-like receptor 4 (TLR4) plays diverse roles in HCC tumorigenesis and progression. The association between PTPRO and TLR4 signaling in HCC remains largely unknown. We aimed to clarify the interaction between PTPRO and TLR4 in HCC. Surprisingly, we found reduced and positive-related expression of TLR4 and PTPRO in 84 human HCC specimens. Increased TLR4 expression and activity was found in PTPRO-overexpressed HCC cells stimulated with lipopolysaccharide (LPS). The feedback regulation of PTPRO and TLR4 was dependent on nuclear factor-κB (NF-κB) activation, as suggested by NF-κB inhibition and luciferase reporter assay. Our study suggests that the effect of PTPRO on TLR4 signaling is dependent on NF-κB pathway, suggesting an interesting PTPRO/TLR4/NF-κB signaling feedback loop in HCC carcinogenesis and progression.     

microRNAs in cancer stem cells: current status and future directions

The presence of stem-like cells in cancer, popularly known as cancer stem cells, have been known for a long time but it was the research of Bonnet and Dick in leukemia which got cancer researchers interested in them. Over the past few years, a lot of research has gone into the characterization of cancer stem cells (CSCs) from different tumors. CSCs have been elucidated in almost all solid tumors. The growth of this field has not been without controversies as many researchers considered CSCs to be a transient population of little consequence. The field has nevertheless progressed providing us not only a better understanding of cancer and its related facets like proliferation, EMT, and metastasis but also generating a hope for new generation therapeutics with CSCs as their targets. This search for drugs which target CSCs has also focused on miRNAs. miRNAs are small non-coding regulatory RNA molecules capable of fine-tuning the gene expression. The miRNA profile of CSCs is remarkably different from non-stem cancer cells and many miRNAs have also been shown to regulate self-renewal and differentiation properties of CSCs. The differential miRNA profile in CSCs make them probable biomarkers for the prognosis of cancer and their specificity in targeting the properties of CSCs make them potential targets for therapeutic intervention. This review critically analyzes the advancement of the miRNA research in CSC context and also explores the prospect of miRNA therapies against CSCs.     

Novel microRNAs expression of patients with chemotherapy drug-resistant and chemotherapy-sensitive epithelial ovarian cancer

The aim of this study is to examine the microRNA (miRNA) expression of epithelial ovarian cancer (EOC) in both drug-resistant and drug-sensitive tissues and to explore the pathogenic characteristics of drug-resistant miRNAs in EOC. The samples with 10 cases of drug-resistant and drug-sensitive EOC tissue were obtained from undergoing surgical resection of ovarian cancer (OC). Total miRNAs were extracted and isolated, respectively. Hybridization was carried out on miRNA microarray chip. Real-time polymerase chain reaction (RT-PCR) was performed to confirm the difference of miRNA expression. Bioinformatic software was used to predict the possible target genes of each miRNA which expressed differently. The results indicated that four miRNAs related drug-resistance been identified, and the expression of hsa-miR-152 and hsa-miR-381 in drug-resistant OC tissue was significantly higher compared with those in drug-sensitive tissue (P?P?miRNA microarrays of differential expression with hsa-miR-106b-3p, hsa-miR-152, hsa-miR-200a-3p, hsa-miR-381, and hsa-miR-429 were confirmed by real-time PCR. There were 62 significantly different miRNAs, including 42 significant upregulated miRNAs and 20 significant downregulated miRNAs in the drug-resistant tissue. Five databases, including Target Scan, miRanda, miRDB, PicTar5, and RNA22, were used for bioinformatics prediction. In conclusion, miRNA microarray analysis has become a fast and efficient molecular biological technology for the study of biological information. hsa-miR-152, hsa-miR-200a-3p, hsa-miR-381, and hsa-miR-429 may participate in the formation of drug resistance in EOC through the target genes predicted.     

Analysis of the differences of serum protein mass spectrometry in patients with triple negative breast cancer and non-triple negative breast cancer

The objective of the present study was to investigate differences of serum protein mass spectrometry in patients with triple negative breast cancer (TNBC) and non-TNBC and thus to search for candidate serum protein biomarkers for identification and diagnosis of TNBC. Thirty serum samples from patients with TNBC without any treatment and 30 serum samples from patients with non-TNBC without any treatment were detected by using two-dimensional gel electrophoresis and matrix-assisted laser dissociation tandem time-of-flight mass spectrometry (MALDI-TOF-MS). PDQest 7.0 software of Bio-Rad was adopted to screen differentially expressed proteins. Protein ID retrieval was conducted by using Mascot software to confirm the results of differential proteins. Two-dimensional gel electrophoresis profiles were obtained successfully. A total of 16 differential protein loci were discovered by analyzing patient sera of the two groups using two-dimensional gel electrophoresis analysis software. Ten differential proteins were identified by mass spectrometric analysis in the 16 differential proteins. Combined with database and literature search results, it is speculated that the specifically upregulated proteins and downregulated proteins including transthyretin, haptoglobin, and antitrypsin may be the potential markers for early diagnosis of TNBC. Comparing the TNBC patients with the non-TNBC patients, there are differences in serum protein compositions. The ten differential proteins discovered in the present study provide reference basis for further improving early diagnosis and identification and diagnosis index of TNBC. Especially, transthyretin, haptoglobin, and antitrypsin show dramatic significances for the early diagnosis of TNBC.     

Secreted protein acidic and rich in cysteine inhibits the growth of human pancreatic cancer cells with G1 arrest induction

Aberrant secreted protein acidic and rich in cysteine (SPARC) expression has been reported to play an important role in the tumor development. However, the pattern and the role of SPARC in pancreatic cancer remain largely unknown. Therefore, we further deciphered the role of SPARC played in pancreatic cancer. We first evaluated the SPARC expression in human pancreatic cancer tissues and pancreatic cancer cells. Then we forced expression and silenced SPARC expression in pancreatic cancer cell lines MIA PaCa2 and PANC-1, respectively, using lentivirus vectors. We characterized the stable cells in vitro. In this study, we found that SPARC expression was weak in cancer cells in specimens which negatively correlated with the expression level of phosphorylated pRB and poorer outcome. Moreover, our results demonstrated that SPARC negatively regulated pancreatic cell growth in vitro. Furthermore, we disclosed that the activation of p53 and p27^Kip1 may involve in the effect of SPARC on pancreatic cancer cells. SPARC is downregulated in pancreatic cancer cells and retards the growth of pancreatic cancer cell. Taken together, these results indicate SPARC may be a potential target for pancreatic cancer therapy.     

MiR-7-5p is frequently downregulated in glioblastoma microvasculature and inhibits vascular endothelial cell proliferation by targeting RAF1

The aberrant expression of microRNAs (miRNAs) is always associated with tumor development and progression. Microvascular proliferation is one of the unique pathologic features of glioblastoma (GBM) . In this study, the microvasculature from GBM or normal brain tissue derived from neurosurgeries was purified and total RNA was isolated from purified microvasculature. The difference of miRNA expression profiles between glioblastoma microvasculature and normal brain capillaries was investigated. It was found that miR-7-5p in GBM microvessels was significantly reduced compared with that in normal brain capillaries. In the in vitro experiments, overexpression of miR-7-5p significantly inhibited human umbilical vein endothelial cell proliferation. Forced expression of miR-7-5p in human umbilical vein endothelial cells in vitro significantly reduced the protein level of RAF1 and repressed the activity of the luciferase, a reporter vector carrying the 3′-untranslated region of RAF1. These findings indicate that RAF1 is one of the miR-7-5p target genes. Furthermore, a significant inverse correlation between miR-7-5p expression and RAF1 protein level in GBM microvasculature was found. These data suggest that miR-7-5p functions as a tumor suppressor gene to regulate GBM microvascular endothelial cell proliferation potentially by targeting the RAF1 oncogene, implicating an important role for miR-7-5p in the pathogenesis of GBM. It may serve as a guide for the antitumor angiogenesis drug development.     

Decreasing relapse in colorectal cancer patients treated with cetuximab by using the activating KRAS detection chip

The KRAS oncogene was among the first genetic alterations in colorectal cancer (CRC) to be discovered. Moreover, KRAS somatic mutations might be used for predicting the efficiency of anti-epidermal growth factor receptor therapeutic drugs. Because the KRAS mutations are similar in the primary CRC and/or the CRC metastasis, KRAS mutation testing can be performed on both specimen types. The purpose of this study was to investigate the clinical advantage of using a KRAS pathway-associated molecule analysis chip to analyze CRC patients treated with cetuximab. Our laboratory developed a KRAS pathway-associated molecule analysis chip and a weighted enzymatic chip array (WEnCA) technique, activating KRAS detection chip, which can detect KRAS mutation status by screening circulating cancer cells in the bloodstream. We prospectively enrolled 210 stage II–III CRC patients who received adjuvant oxaliplatin plus infusional 5-fluorouracil/leucovorin (FOLFOX)-4 chemotherapy with or without cetuximab. We compared the chip results of preoperative blood specimens with disease control status in these patients. Among the 168 CRC patients with negative chip results, 119 were treated with FOLFOX-4 plus cetuximab chemotherapy, and their relapse rate was 35.3 % (42/119). In contrast, the relapse rate was 71.4 % among the patients with negative chip results who received FOLFOX-4 treatment alone (35/49). Negative chip results were significantly correlated with better treatment outcomes in the FOLFOX-4 plus cetuximab group (P?KRAS detection chip is a potential tool for predicting clinical outcomes in CRC patients following FOLFOX-4 treatment with or without cetuximab therapy.     

Phyllodes tumor of the breast: role of Axl and ST6GalNAcII in the development of mammary phyllodes tumors

Phyllodes tumor exhibits an aggressive growth. The expression of many biological markers has been explored to discriminate between different grades of phyllodes tumor and to predict their behavior. The purpose of this study was to evaluate the implications of Axl and ST6GalNAcII in phyllodes tumors. Real-time PCR, Western blot, and immunohistochemical were used to analyze differential expression of ST6GalNAcII and Axl in phyllodes tumor (PT) cell lines and tissue specimens. RNAi assay, ECM invasion assay, and tumorigenicity assay were used to analyze the altered expression of ST6GalNAcII gene effects on the expression of Axl and invasive ability of phyllodes tumor cells in vitro and in vivo. Compared to benign tumors, borderline and malignant ones showed a remarkable increase in mRNA levels of Axl and ST6GalNAcII gene, and it was higher in malignant tumor cells than in borderline tumor cells. When ST6GalNAcII was silenced, compared to the control, the expression level of Axl was significantly reduced in malignant tumor cell transfectants and knockdown of ST6GalNAcII gene significantly inhibited invasive activity in malignant tumor cells. The high expression of ST6GalNAcII and Axl was significantly correlated with tumor grade and distance metastasis by immunohistochemical analysis. Axl and ST6GalNAcII expression increases with increasing tumor grade in mammary phyllodes tumors. ST6GalNAc II might be participated in the glycosylation of Axl, and this Axl glycosylation may mediate the tumorigenicity, invasion, and distant metastasis of PT cells.     

Use of risk-reducing surgeries in a prospective cohort of 1,499 BRCA1 and BRCA2 mutation carriers

Inherited mutations in BRCA1 or BRCA2 (BRCA1/2) confer very high risk of breast and ovarian cancers. Genetic testing and counseling can reduce risk and death from these cancers if appropriate preventive strategies are applied, including risk-reducing salpingo-oophorectomy (RRSO) or risk-reducing mastectomy (RRM). However, some women who might benefit from these interventions do not take full advantage of them. We evaluated RRSO and RRM use in a prospective cohort of 1,499 women with inherited BRCA1/2 mutations from 20 centers who enrolled in the study without prior cancer or RRSO or RRM and were followed forward for the occurrence of these events. We estimated the age-specific usage of RRSO/RRM in this cohort using Kaplan–Meier analyses. Use of RRSO was 45 % for BRCA1 and 34 % for BRCA2 by age 40, and 86 % for BRCA1 and 71 % for BRCA2 by age 50. RRM usage was estimated to be 46 % by age 70 in both BRCA1 and BRCA2 carriers. BRCA1 mutation carriers underwent RRSO more frequently than BRCA2 mutation carriers overall, but the uptake of RRSO in BRCA2 was similar after mutation testing and in women born since 1960. RRM uptake was similar for both BRCA1 and BRCA2. Childbearing influenced the use of RRSO and RRM in both BRCA1 and BRCA2. Uptake of RRSO is high, but some women are still diagnosed with ovarian cancer before undergoing RRSO. This suggests that research is needed to understand the optimal timing of RRSO to maximize risk reduction and limit potential adverse consequences of RRSO.     

CXCL10 mRNA expression predicts response to neoadjuvant chemoradiotherapy in rectal cancer patients

Chemoradiotherapy has been commonly used as neoadjuvant therapy for rectal cancer to allow for less aggressive surgical approaches and to improve quality of life. In cancer, it has been reported that CXCL10 has an anti-tumor function. However, the association between CXCL10 and chemoradiosensitivity has not been fully investigated. We performed this study to investigate the relationship between CXCL10 expression and chemoradiosensitivity in rectal cancer patients. Ninety-five patients with rectal cancer who received neoadjuvant chemoradiotherapy (NCRT) were included. Clinical parameters were compared with the outcome of NCRT and CXCL10 messenger RNA (mRNA) expression between the pathological complete response (pCR) group and non-pathological complete response (npCR) group. CXCL10 mRNA and protein expressions between groups were analyzed using the Student’s t test and chi-square test. The mean mRNA level of CXCL10 in the pCR group was significantly higher than that in the npCR group (p?=?0.010). In the pCR group, 73.7 % of the patients had high CXCL10 mRNA expression, and 61.4 % of the patients in the npCR group had low CXCL10 mRNA expression. Subjects with high CXCL10 mRNA expression demonstrated a higher sensitivity to NCRT (p?=?0.011). The receiver operating characteristic curve showed that the diagnostic performance of CXCL10 mRNA expression had an area under the curve of 0.720 (95 % confidence interval, 0.573–0.867). There were no differences between the pCR and npCR groups in CXCL10 protein expression (p?>?0.05). High CXCL10 mRNA expression is associated with a better tumor response to NCRT in rectal cancer patients and may predict the outcome of NCRT in this malignancy.