Docetaxel resistance-derived LINC01085 contributes to the immunotherapy of hormone-independent prostate cancer by activating the STING/MAVS signaling pathway.

Acquired docetaxel (doc) resistance, one of the major reasons for unfavorable prognosis in patients with aggressive hormone-independent prostate cancer (HIPC), is a major obstacle for patient treatment. Dysregulation of long non-coding RNAs promotes or suppresses chemoresistance in multiple cancers; however, the specific molecular mechanisms underlying HIPC remain unknown. In this study, we found that the LINC01085, as a tumor-suppressor, which showed significant clinically relevant in HIPC patients with doc-resistance. Mechanistically, in docetaxel-sensitive cells, LINC01085 could specifically bind to both TANK-binding kinase 1 (TBK1) and glycogen synthase kinase 3ß (GSK3ß), and higher LINC01085 RNA levels could inhibit TBK1 dimerization. Whereas, in doc-resistant cells, lower LINC01085 RNA level lost the strong binding with both, meanwhile, the interaction between TBK1 and GSK3ß enhanced which accelerated TBK1 phosphorylation at the Ser-172 site, resulting in decreased expression levels of PD-L1 and NF-κB as well as the secretion of type I/III interferons. Thus, Overexpression of LINC01085 combined with immune checkpoint blockade is an effective strategy for the treatment of HIPC patients.

LINC01510 suppresses cell proliferation and invasion by inhibiting Wnt/ß-catenin signaling in renal cell carcinoma.

The role of long non-coding RNA in Renal cell carcinoma (RCC) tumorigenesis and progression remains largely unknown. Here, we found that LINC01510 functions as a tumor suppressor in RCC tumorigenesis. We screened TCGA database and then found that LINC01510 is significantly down-regulated in malignant RCC tissues, and the lower expression of LINC01510 predicts poor prognosis. Moreover, the down-regulated LINC01510 was further confirmed in our fresh tissues and cell lines. Biological functions assays shown that Ectopic expression of LINC01510 not only inhibits RCC cell proliferation both in vitro and in vivo, but also impairs cell invasion ability. Moreover, we found overexpression of LINC01510 inhibits the expression of CCND1 and CCNE1, as well as MMPs (MMP2, MMP7 and MMP9), and thus affecting RCC cell cycle and invasion. Meanwhile, Western blot assays revealed that the expression of ß-catenin is regulated by LINC01510; overexpression of ß-catenin could partly rescue the cell viability and invasion ability caused by ectopic expression of LINC01510. Taken together, we found that LINC01510 regulates cell proliferation and invasion by modulating Wnt/ß-catenin signaling in RCC.

A characteristic biosignature for discrimination of gastric cancer from healthy population by high throughput GC-MS analysis.

Early diagnosis of gastric cancer is crucial to improve patient' outcome. A good biomarker will function in early diagnosis for gastric cancer. In order to find practical and cost-effective biomarkers, we used gas chromatography combined mass spectrometer (GC-MS) to profile urinary metabolites on 293 urine samples. Ninety-four samples are taken as training set, others for validating study. Orthogonal partial least squares discriminant analysis (OPLS-DA), significance analysis of microarray (SAM) and Mann-Whitney U test are used for data analysis. The diagnostic value of urinary metabolites was evaluated by ROC curve. As results, Seventeen metabolites are significantly different between patients and healthy controls in training set. Among them, 14 metabolites show diagnostic value better than classic blood biomarkers by quantitative assay on validation set. Ten of them are amino acids and four are organic metabolites. Importantly, proline, p-cresol and 4-hydroxybenzoic acid disclose outcome-prediction value by means of survival analysis. Therefore, the examination of urinary metabolites is a promising noninvasive strategy for gastric cancer screening.

Mitochondrial aldehyde dehydrogenase 2 protects gastric mucosa cells against DNA damage caused by oxidative stress.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a member of the aldehyde dehydrogenase superfamily and is involved with the metabolic processing of aldehydes. ALDH2 plays a cytoprotective role by removing aldehydes produced during normal metabolism. We examined the cytoprotective role of ALDH2 specifically in gastric mucosa cells. Overexpression of ALDH2 increased the viability of gastric mucosa cells treated with H2O2, while knockdown of ALDH2 had an opposite effect. Moreover, overexpression of ALDH2 protected gastric mucosa cells against oxidative stress-induced apoptosis as determined by flow cytometry, Hoechst 33342, and TUNEL assays. Consistently, ALDH2 knockdown had an opposite effect. Additionally, DNA damage was ameliorated in ALDH2-overexpressing gastric mucosa cells treated with H2O2. We further identified that this cytoprotective role of ALDH2 was mediated by metabolism of 4-hydroxynonenal (4-HNE). Consistently, 4-HNE mimicked the oxidative stress induced by H2O2 in gastric mucosa cells. Treatment with 4-HNE increased levels of DNA damage in ALDH2-knockdown GES-1 cells, while overexpression of ALDH2 decreased 4-HNE-induced DNA damage. These findings suggest that ALDH2 can protect gastric mucosa cells against DNA damage caused by oxidative stress by reducing levels of 4-HNE.

mTORC1 maintains the tumorigenicity of SSEA-4(+) high-grade osteosarcoma.

Inactivation of p53 and/or Rb pathways restrains osteoblasts from cell-cycle exit and terminal differentiation, which underpins osteosarcoma formation coupled with dedifferentiation. Recently, the level of p-S6K was shown to independently predict the prognosis for osteosarcomas, while the reason behind this is not understood. Here we show that in certain high-grade osteosarcomas, immature SSEA-4(+) tumor cells represent a subset of tumor-initiating cells (TICs) whose pool size is maintained by mTORC1 activity. mTORC1 supports not only SSEA-4(+) cell self-renewal through S6K but also the regeneration of SSEA-4(+) TICs by SSEA-4(-) osteosarcoma cell dedifferentiation. Mechanistically, active mTORC1 is required to prevent a likely upregulation of the cell-cycle inhibitor p27 independently of p53 or Rb activation, which otherwise effectively drives the terminal differentiation of SSEA-4(-) osteosarcoma cells at the expense of dedifferentiation. Thus, mTORC1 is shown to critically regulate the retention of tumorigenicity versus differentiation in discrete differentiation phases in SSEA-4(+) TICs and their progeny.

Claudin-1 enhances tumor proliferation and metastasis by regulating cell anoikis in gastric cancer.

Claudin-1 (CLDN1) is overexpressed in gastric cancer and correlated with tumor invasion, metastasis and poor outcome. Here, we both down and up regulated CLDN1 expression in gastric cancer cells to elucidate its role in gastric carcinogenesis and tumor progression. We found that deficiency of CLDN1 inhibited cells migration, invasion, and colony formation in vitro and tumorigenicity, metastasis in vivo. Also, CLDN1 promoted cell aggregation and increased anoikis resistance. Down or up regulation of CLDN1 was accompanied with changes of membrane ß-catenin expression as well as Akt and Src activities. When ß-catenin was up-regulated in CLDN1-KD cells, cell aggregation and anoikis resistance were restored, and Akt and Src signal pathways were re-activated. Taken together, these findings suggest that CLDN1 is oncogenic in gastric cancer and its malignant potential may be attributed in part to regulation of anoikis, by mediating membrane ß-catenin-regulated cell-cell adhesion and cell survival.

The metastasis suppressor SOX11 is an independent prognostic factor for improved survival in gastric cancer.

SOX11 is involved in gastrulation and in malignant diseases. The aim of this study was to investigate the role of SOX11 in gastric cancer and its expression pattern and clinical significance. SOX11 overexpression cell model was used to examine in vitro and in vivo the role of SOX11 in cell growth and metastasis. Cell cycle analysis and Annexin V/PI double staining were used to investigate the effect of SOX11 on cell cycle progression and apoptosis. The expression of SOX11 in human gastric cancer was examined by immunohistochemistry. The correlation of SOX11 expression with clinicopathological characteristics and survival of patients was analyzed by Pearson's χ(2) and Kaplan-Meier analyses, respectively. Cox's proportional hazard model was employed in multivariate analysis. SOX11 overexpression did not inhibit cell growth but strongly suppressed cell migration/invasion in vitro and in vivo. We found a significant correlation between high SOX11 protein levels and Lauren's classification (intestinal type), differentiation status (high and medium), and early TNM stage. SOX11 is an independent prognostic factor for improved survival in gastric cancer patients. SOX11 was a potential tumor-suppressor and an independent positive prognostic factor in gastric cancer patients with less advanced clinicopathological features.

The expression of claudin 1 correlates with ß-catenin and is a prognostic factor of poor outcome in gastric cancer.

Claudin 1 is one of the tight junction proteins, which are critical in the maintenance of epithelial integrity. Aberrant regulation of CLDN1 and its correlation with ß-catenin have been discovered in malignant tumors. The present study aimed to investigate the expression profile and clinical relevance of CLDN1 and ß-catenin. The protein levels of CLDN1 and ß-catenin were examined using immunohistochemical staining. The characteristics of expression profile and prognostic value were analyzed using Pearson's χ² test and Kaplan-Meier analysis, respectively. ß-catenin overexpression and knockdown were used to investigate its role in regulating CLDN1 expression. We showed that CLDN1 was overexpressed in intestinal-type, presence of lymph node metastasis, higher TNM stage in gastric cancer patients and correlated with decreased overall survival. The characteristics of CLDN1 expression were associated with that of ß-catenin. CLDN1 and ß-catenin showed similar prognostic value in intestinal-type gastric cancers. ß-catenin knockdown and overexpression in cell models revealed a positive relation between CLDN1 and ß-catenin. Our study demonstrated that CLDN1 is a biomarker for intestinal-type gastric cancer with shorter survival. The expression of CLDN1 was strongly associated with ß-catenin in gastric cancer patients and a gastric cancer cell model.

CEACAM6 promotes tumor migration, invasion, and metastasis in gastric cancer.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) shows increased expression in a wide variety of human cancers, and its over-expression is associated with enhanced migration, invasion, and in vivo metastasis. Here, we reported that CEACAM6 was up-regulated in gastric cancer (GC) cell lines and tumor tissues. Over-expression of CEACAM6 in MKN-45 and SGC-7901 GC cells promoted migration and invasion in vitro and metastasis in athymic mice, whereas migration and invasion of MKN-28 and SNU-16 GC cells were suppressed by knockdown of CEACAM6. We also observed that steroid receptor coactivator (C-SRC) phosphorylation was increased when CEACAM6 was over-expressed in SGC-7901 cells. Taken together, these results suggested that CEACAM6 functions as an oncoprotein in GC and may be an important metastatic biomarker and therapeutic target.

High levels of secreted frizzled-related protein 1 correlate with poor prognosis and promote tumourigenesis in gastric cancer.

BACKGROUND: Secreted frizzled-related protein 1 (sFRP1), Wnt signalling regulator, can positively or negatively regulate tumourigenesis and progression. We sought to determine the clinical relevance and the role of sFRP1 in gastric cancer development and progression. METHODS: We investigated the sFRP1 protein expression levels and its clinicopathological correlations using 85 cases of human gastric samples with survival information (JWCI cohort). mRNA levels of sFRP1 and coexpressed genes were analysed using 131-sample cDNA microarray data (Ruijin cohort). The effects of sFRP1 alteration were investigated using cell proliferation, colony formation, migration, and invasion and xenograft models. RESULTS: We show that sFRP1 is overexpressed in some human cancers and is significantly associated with lymph node metastasis and decreased overall survival in gastric cancer patients. Using gastric cancer cell models, we demonstrate that sFRP1 overexpression is correlated with the activation of TGFß (transforming growth factor-beta) signalling pathway and thereby induces cell proliferation, epithelial-mesenchymal transition (EMT), and invasion. Conversely, sFRP1 knockdown shows the opposite effects. Furthermore, sFRP1 overexpression promotes tumourigenesis and metastasis in a xenograft model. CONCLUSION: Our studies demonstrate that sFRP1 is a biomarker for aggressive subgroups of human gastric cancer and a prognostic biomarker for patients with poor survival. Our data provide insight into a crosstalk between Wnt and TGFß pathways which underlies gastric cancer development and progression.

Inactivation of tumor suppressor gene HIC1 in gastric cancer is reversed via small activating RNAs.

HIC1 is a tumor suppressor gene that is down-expressed in different malignancies, in part, because of promoter hypermethylation. However, the biological function of HIC1 in gastric cancer remains unclear. It is known that small double-stranded RNAs can induce gene expression by targeting promoter sequences. In the present study, we examined the expression levels of HIC1 in gastric cancer tissue. Several pieces of small double-stranded RNAs were used for the activation of HIC1. Tissue microarray analysis of gastric cancer indicated that down-regulation of HIC1 in gastric cancer tissue was dramatic compared with the adjacent gastric mucosa. Gastric cancer cell lines also showed down-regulated HIC1 expression compared with a human immortalized gastric mucosa cell line. One out of four dsRNAs produced activation of HIC1 as assessed by real-time PCR and Western blotting. Use of a cell counting kit 8 and clonogenicity assays indicated that dsRNA-mediated re-expression of HIC1 inhibited cell proliferation and clonogenicity in gastric cancer. Reactivation of HIC1 suppressed cell migration and induced cell cycle arrest in the G0/G1 phase, as well as induced apoptosis. These results suggest that HIC1 is a potential target of gene therapy against gastric cancer, and that dsRNAs could function as a therapeutic option for up-regulating tumor suppressor genes in gastric cancer and other malignancies.

CHD1L promotes tumor progression and predicts survival in colorectal carcinoma.

BACKGROUND: To evaluate the expression of chromodomain helicase/adenosine triphosphatase DNA binding protein 1-like gene (CHD1L) in colorectal carcinoma (CRC) and its clinical significance. Its oncogenic ability was also investigated. MATERIALS AND METHODS: CHD1L amplification and overexpression were detected by fluorescence in situ hybridization, real-time reverse transcriptase-polymerase chain reaction, and immunohistochemistry in 86 patients with CRC. The correlation between the clinical characteristics and prognosis was also determined. To evaluate the tumorigenic ability of CHD1L, it was cloned into expression vector pcDNA3.1(+) and transfected into CRC cell line SW1116. Next, the changes in the biologic behavior of the CRC cells, including cell proliferation, adhesion, migration, and invasion, were examined. Apoptosis and the cell cycle of the CRC cells were detected using flow cytometry. RESULTS: We have demonstrated that CHD1L is frequently amplified and overexpressed in CRC. Overexpression of CHD1L correlated with a large tumor size, deep tumor invasion, and a high histologic grade. It also conferred worse disease-free survival. CHD1L-transfected cells possessed a strong oncogenic ability, increasing the tumorigenicity in nude mice, which could be effectively suppressed by small interfering RNA against CHD1L. Functional studies showed that overexpression of CHD1L could promote G1/S-phase cells and inhibit apoptosis. CONCLUSIONS: Our results suggest that CHD1L is the target oncogene within the 1q21 amplicon and plays a pivotal role in CRC pathogenesis.

Oncogenic miR-544 is an important molecular target in gastric cancer.

MicroRNAs (miRNAs) and promoter hypermethylation are vital epigenetic mechanisms for transcriptional inactivation of tumor suppressor. IRX1 is a newly identified tumor suppressor gene and hypermethylation involves the decreased expression in gastric cancer. However, the microRNA regulatory mechanism on IRX1 expression is still unclear. In this study, we report an IRX1-targeting miRNA-544, which directly targets 3'-UTR of IRX1 gene by luciferase reporter assay. miR-544 suppresses the protein expression of IRX1 gene by Western blot and immunocytochemistry. Ectopic expression of miR-544 promotes cell proliferation and cell cycle progression significantly in vitro on gastric cancer cells. The study suggests that miR-544 is an oncogenic microRNA in gastric cancer. Over expression of miR-544 contributes to the inactivation and low-expression of IRX1 in gastric cancer. These findings are helpful for clarifying the molecular mechanisms involved in gastric carcinogenesis and indicate that miR-544 is a key regulator in switching cell cycle on or off. miR-544 may be a potential molecular target in miRNA-based strategy on gastric cancer.

RhoGDI2 confers resistance to 5-fluorouracil in human gastric cancer cells.

Resistance to 5-fluorouracil (5-FU) in patients with gastric cancer is a serious therapeutic problem and major efforts are underway to understand the underlying mechanisms. We have previously identified RhoGDI2 as a contributor to 5-FU resistance in colon cancer cells using 2D electrophoresis and mass spectrometry and the current study aimed to further investigate this role. The expression of RhoGDI2 in seven gastric cancer cell lines was positively correlated with resistance to 5-FU. Lower 5-FU sensitivity of isolated tumor cells from patients with gastric cancer was also associated with higher RhoGDI2 expression. Ectopic expression of RhoGDI2 in gastric cancer cells increased the resistance to 5-FU and reverted low dose 5-FU-induced G2/M phase arrest without affecting the population of sub-G1 cells. Overall, these findings suggest that RhoGDI2 is associated with 5-FU resistance and is a potential therapeutic target for enhancing chemotherapy efficacy in gastric cancer.

ABO blood group system and gastric cancer: a case-control study and meta-analysis.

This study focuses on the association between the ABO blood group system and the risk of gastric cancer or Helicobacter pylori infection. The data for the ABO blood group was collected from 1045 cases of gastric cancer, whereby the patient underwent a gastrectomy in Ruijin Hospital, Shanghai. The information on the ABO blood group from 53,026 healthy blood donors was enrolled as control. We searched the Pubmed database on the relationship between ABO blood groups and gastric cancer risk for meta-analysis. In our case-control study, the risk of gastric cancer in blood group A was significantly higher than that in non-A groups (O, B and AB) (odd ratio, OR1.34; 95% confidential interval, CI 1.25-1.44). Compared with non-O groups (A, B and AB), individuals with blood group O demonstrated a reduced risk of gastric cancer (OR = 0.80; 95% CI 0.72-0.88). The proportion of H. pylori infection in blood group A individuals was significantly higher than that in non-A blood groups (OR = 1.42; 95% CI 1.05-1.93). We further combined our data with the published data of others, and crossreferenced the risk of gastric cancer with the blood type, finding consistent evidence that gastric cancer risk in the blood A group was higher than that in the non-A groups (OR = 1.11; 95% CI 1.07-1.15), and that blood type O individuals were consistently shown gastric cancer risk reduction (OR = 0.91; 95% CI 0.89-0.94). Our study concluded that there was a slightly increased risk of gastric cancer in blood group A individuals, and people with blood type A are more prone to be infected by H. pylori than other ABO blood type individuals, whereas, a slightly decreased risk of gastric cancer was identified in blood type O individuals.

A unique feature of iron loss via close adhesion of Helicobacter pylori to host erythrocytes.

Iron deficiency anemia is an extra-stomach disease experienced in H. pylori carriers. Individuals with type A blood are more prone to suffering from H. pylori infection than other individuals. To clarify the molecular mechanisms underlying H. pylori-associated anemia, we collected erythrocytes from A, B, O, and AB blood donors and analyzed morphology, the number of erythrocytes with H. pylori colonies attached to them, and iron contents in erythrocytes and H. pylori (NCTC11637 and SS1 strains) by means of optical microscopy, scanning electron microscopy, and synchrotron radiation soft X-ray imaging. The number of type A erythrocytes with H. pylori attached to them was significantly higher than that of other erythrocytes (P<0.05). Far more iron distribution was observed in H. pylori bacteria using dual energy analysis near the iron L2, 3 edges by soft X-ray imaging. Iron content was significantly reduced in host erythrocytes after 4 hours of exposure to H. pylori. H. pylori are able to adhere more strongly to type A erythrocytes, and this is related to iron shift from the host to the bacteria. This may explain the reasons for refractory iron deficiency anemia and elevated susceptibility to H. pylori infection in individuals with type A blood.

DJ-1 promotes invasion and metastasis of pancreatic cancer cells by activating SRC/ERK/uPA.

A major hallmark of pancreatic ductal adenocarcinoma (PDAC) is extensive local tumor invasion and early systemic dissemination. DJ-1 has been shown to prevent cell death via the Akt pathway, thereby playing an important role in cancer progression and Parkinson's disease development. Here, we investigated the role of DJ-1 in tumor invasion and metastasis of pancreatic cancer and showed that DJ-1 is upregulated in 68.5% of pancreatic cancer specimens, correlated with tumor stage and predictive of short overall survival. Knockdown of DJ-1 expression in two PDAC cell lines reduced cell migration and invasion potential in vitro and inhibited metastasis in vivo. Knockdown of DJ-1 led to cytoskeleton disruption and diminished urokinase plasminogen activator (uPA) activity and expression, without affecting plasminogen activator inhibitor-1 and uPA receptor (uPAR) expression. All these effects were reversed by restoration of DJ-1 expression. In determining the pathway through which DJ-1 regulated cell migration and invasion, DJ-1 was found not to regulate Akt phosphorylation. Rather, it promoted extracellular signal-regulated kinase (ERK) and SRC phosphorylation. Inhibition of the ERK pathway in PDAC mimicked the effects of DJ-1 on cell migration, invasion, actin cytoskeleton and uPA/uPAR system and abolished the effects on promoting PDAC cell invasion and migration. These data represent the first identification of an important function of DJ-1, which is to regulate the invasion and metastasis properties of PDAC through the ERK/uPA cascade.

[Establishment of experimental angiogenic models with applications of quantitative digital image analysis].

OBJECTIVE: To establish experimental models for tumor neovascularization and to apply quantitative digital imaging analysis in the study. METHODS: An endothelial tube formation model was established by human umbilical vein endothelial cells (HUVECs). A vasculogenic mimicry model was established by SGC-7901 gastric cancer cell line. Fertilized eggs were used to establish a chorioallantoic membrane angiogenesis model. Using gene transfection experiment, IRX1 tumor suppressor gene was chosen as a therapeutic target. Image Pro Plus (IPP) analysis software was used for digital vascular images analysis with parameters including points, lines, angles and integral absorbance (IA) for the tubular formation or vasculogenic mimicry. RESULTS: Digital image analysis by IPP showed that HUVEC tubular formation was significantly inhibited in IRX1 transfectant, compared with controls. The tubular numbers in three groups were 12.80 +/- 3.83, 29.00 +/- 5.34 and 28.20 +/- 4.32 (P<0.01). The connection points of tubules in three groups were 13.20 +/- 2.59, 25.00 +/- 2.24 and 24.60 +/- 3.21 (P<0.01). The tubular lengths of three groups were (821.5 +/- 12.5), (930.9 +/- 13.5) and (948.4 +/- 18.1) microm (P=0.022). The IA values of PAS stain in three groups were 3606 +/- 363, 14 200 +/- 1251 and 15 043 +/- 1220 (P<0.01). In chick chorioallantoic membrane model, the angular numbers of tubules in three groups were 6.41 +/- 2.60, 10.27 +/- 2.65 and 9.18 +/- 1.99 (P<0.01). CONCLUSIONS: The endothelial tube formation model, vasculogenic mimicry model and chorioallantoic membrane angiogenesis model are useful for gene therapy and drug screening with targeting neoplastic vascularization. Professional image analysis software may greatly facilitate the quantitative analysis of tumor neovascularization.

[Promoter cloning and activity analysis for iroquois homeobox gene 1 in gastric mucosa cell line].

OBJECTIVE: To clone core promoter regions of iroquois homeobox gene 1 (IRX1) gene and evaluate the regulatory mechanism of IRX1 transcription. METHODS: Upstream sequence from transcriptional start site was predicted using bioinformatics methods. Serial deleted fragments from IRX1 promoter sequences were amplified by PCR and luciferase reporter plasmids were constructed. The luciferase intensity was analyzed after transferring reporters into GES-1 gastric mucosa cell line. RESULTS: The promoter of IRX1 was predicted to be within 700 bp from the 5'-flanking region of IRX1 gene. Eight serial deleted luciferase reporter plasmids were constructed. The transcriptional activity of different fragments was expressed as following: p-416>p-584>p-715>p-350>p-687>p-320>p-188>p-92. Except p-320 and p-188, the transcriptional activity of other 6 fragments was higher than that of PGL3-basic plasmid. The transcriptional activity was the highest in p-416 and decreased sharply from p-320 to p-188. CONCLUSIONS: The fragment p-416 shows the strongest promoter activity. The sequence from -320 bp to -188 bp is identified as core promoter region, which is focused as key sequence for further regulatory analysis, since some binding sites for important transcriptional factors such as Sp1 and TFII D are predicted.