Long Yu

Secreted LOXL2 is a novel therapeutic target that promotes gastric cancer metastasis via the Src/FAK pathway

The purpose of this study was to investigate invasion- and metastasis-related genes in gastric cancer. To this end, we used the transwell system to select a highly invasive subcell line from minimally invasive parent cells and compared gene expression in paired cell lines with high- and low-invasive potentials. Lysyl oxidase-like 2 (LOXL2) was overexpressed in the highly invasive subcell line. Immunohistochemical analysis revealed that LOXL2 expression was markedly increased in carcinoma relative to normal epithelia, and this overexpression in primary tumor was significantly associated with depth of tumor invasion, lymph node metastasis and poorer overall survival. Moreover, LOXL2 expression was further increased in lymph node metastases compared with primary cancer tissues. RNA interference-mediated knockdown and ectopic expression of LOXL2 showed that LOXL2 promoted tumor cell invasion in vitro and increased gastric carcinoma metastasis in vivo. Subsequent mechanistic studies showed that LOXL2 could activate both the Snail/E-cadherin and Src kinase/Focal adhesion kinase (Src/FAK) pathways. However, secreted LOXL2 induced gastric tumor cell invasion and metastasis exclusively via the Src/FAK pathway. Expression correlation analysis in gastric carcinoma tissues also revealed that LOXL2 promoted invasion via the Src/FAK pathway but not the Snail/E-cadherin pathway. We then evaluated secreted LOXL2 as a target for gastric carcinoma treatment and found that an antibody against LOXL2 significantly inhibited tumor growth and metastasis. Overall, our data revealed that LOXL2 overexpression, a frequent event in gastric carcinoma progression, contributes to tumor cell invasion and metastasis, and LOXL2 may be a therapeutic target for preventing and treating metastases.

Profiling Tumor-Associated Autoantibodies for the Detection of Colon Cancer

Purpose: The purpose of the present study was to screen the autoantibody signature of colon cancers to develop serum markers for colon cancer detection. Experimental Design: A phage cDNA expression library of colon cancer was built. The library was sequentially screened by a pool of 10 colon cancer sera, goat antihuman IgG, and a pool of two healthy sera to identify phage-expressed antigens recognized by tumor-associated antibodies. The clones picked out by these screening were subjected to a training set with 24 colon cancer sera and 24 healthy sera. The antigen combination, which got the most satisfactory classification, was tested by an independent set of 24 colon cancer sera with equal number of sera from normal donors. The carcinoembryonic antigen (CEA) level of these sera was detected for the additional classification analysis with or without the antigen combination. Results: A cDNA expression library consisting of 2 × 106 primary clones was prepared. After three turns of screening, 24 antigens recognized by tumor-associated antibodies were picked out for serum marker identification. The training set showed that a six-marker combination got the most satisfactory classification in a logistic regression model; leave-one-out validation achieved 91.7% sensitivity and 91.7% specificity. In a testing set with this marker panel, we correctly predicted 85% of the samples. Although according to CEA level alone, we correctly predicted 75% of the samples with 42% of cancer patients misclassified. When CEA was combined with the six markers, the sensitivity and specificity increased to 91.7% and 95.8%, respectively. The six antigen sequences in the phage display system are relatively short peptides. Only two of them showed homology to known protein sequences. Conclusions: Autoantibodies against phage-expressed antigens derived from colon cancer tissues could be used as serum markers for the detection of colon cancer.

Tumor Cell-Microenvironment Interaction Models Coupled with Clinical Validation Reveal CCL2 and SNCG as Two Predictors of Colorectal Cancer Hepatic Metastasis

Purpose: This study aimed to identify novel biological markers for the prediction of colorectal cancer liver metastasis. Experimental Design: We established two models that mimicked the interactions between colorectal tumor cells and the liver microenvironment. From these models we established subcell lines that had an enhanced ability to metastasize to the liver. Genes that related to hepatic metastasis were screened by microarray. The candidate markers were tested by immunohistochemistry, and their predictive accuracy was assessed by the cross-validation method and an independent test set. Results: Highly metastatic colon cancer cell sublines SW1116p21 and SW1116v3 were established from the tumor cell-microenvironment interaction models. Seven of the up-regulated genes in the sublines were selected as candidate markers for predicting metastatic potential. A total of 245 colorectal cancer samples were divided into a training set containing 117 cases and a test set containing 128 cases. In the training set, immunohistochemical analysis showed CCL2 and SNCG expression was higher in the hepatic metastasis group than in the nonmetastasis group, and was correlated with poor survival. Logistic regression analysis revealed that CCL2 and SNCG levels in primary tumors, serum carcinoembryonic antigen level, and lymph node metastasis status were the only significant (P < 0.05) parameters for detecting liver metastasis. In leave-one-out-cross-validation, the two markers, when combined with clinicopathologic features, resulted in 90.5% sensitivity and 90.7% specificity for hepatic metastasis detection. In an independent test set, the combination achieved 87.5% sensitivity and 82% specificity for predicting the future hepatic metastasis of colorectal cancer. Conclusion: Our results suggest that these models are able to mimic the interactions between colorectal cancer cells and the liver microenvironment, and may represent a promising strategy to identify metastasis-related genes. CCL2 and SNCG, combined with clinicopathologic features, may be used as accurate predictors of liver metastasis in colorectal cancer. (Clin Cancer Res 2009;15(17):5485–93)

TM4SF3 promotes esophageal carcinoma metastasis via upregulating ADAM12m expression

Esophageal cancer is characterized by rapid clinical progression and poor prognosis due to adjacent tissue invasion and distant organs metastasis at a very early stage. TM4SF3 (transmembrane 4 superfamily 3), a member of tetraspanin family, has been reported as a metastasis associated gene in many types of tumors. Herein, we described new properties of TM4SF3 in tumor metastasis, which suggested that this gene might be involved in esophageal carcinoma metastasis. Western blotting revealed that TM4SF3 was overexpressed in 57.1% (8/14) of esophageal carcinomas and esophageal carcinoma cell lines with high-invasive potential. Exogenous expression of TM4SF3 in two low-invasive esophageal carcinoma cell lines, KYSE150 and EC9706, significantly promoted cell migration and invasion. Upregulating TM4SF3 expression in EC9706 cells promoted xenograft tumor invading into surrounding tissues, enhanced lung metastasis, and shortened the lifespan of mice (median survival EC9706-TM4SF3 106.5xa0days versus EC9706-Vector 169.0xa0days, Pxa0<xa00.0001) in a spontaneous metastasis model. Further studies demonstrated that ADAM12m was upregulated by TM4SF3 overexpression in vitro and in vivo. Abrogating up-expression of ADAM12m by siRNA significantly suppressed TM4SF3-mediated invasion. Together, these data from our studies indicated that overexpression of TM4SF3 in esophageal cancer conferred advantage to the invasion and metastasis of this destructive disease. Upregulated expression of ADAM12m by TM4SF3 might play a key role in TM4SF3-mediated invasion and metastasis. TM4SF3 and ADAM12m might be potential targets of esophageal carcinoma for anti-metastasis therapy.

Antibody Library-based Tumor Endothelial Cells Surface Proteomic Functional Screen Reveals Migration-stimulating Factor as an Anti-angiogenic Target

Angiogenesis is critical for cancer development and metastasis. Here we have employed a functional antibody library-based proteomic screen to identify proteins that participate in and might be used as therapeutic targets for tumor-related angiogenesis. Mice were immunized with human esophageal cancer endothelial cells (HECEC). The antibody library was established with the mouse spleen cells the serum of which had most anti-angiogenic effect. Monoclonal antibodies were subjected to an immunoreactive and functional screen and monoclonal antibodies that reacted strongly with cell surface antigens of HECECs and influenced their behavior were selected. Antigens that recognized by the antibodies were obtained by immunoprecipitation and then identified by mass spectrometry analysis. Migration-stimulating factor (MSF), the antigen of 1D2 antibody was identified using this approach. Further studies demonstrated that the 1D2 antibody suppressed MSF-effected migration and adhesion of HECECs on fibronectin matrix. Biodistribution assay showed that MSF targeting antibody 1D2 could specifically home to the xenograft with humanized blood vessel. Targeting treatment with 1D2 antibody significantly suppressed tumor growth through inhibition of human tumor-related angiogenesis. These results indicate that the functional antibody library-based proteomic screen can successfully identify proteins that involved in tumor-related angiogenesis and MSF may be a target for the anti-angiogenic treatment of the esophageal cancer.

A Novel Role for Tissue Factor Pathway Inhibitor-2 in the Therapy of Human Esophageal Carcinoma

Esophageal cancer is characterized by rapid clinical progression and poor prognosis, due to early-stage invasion of adjacent tissues and metastasis. Tissue factor pathway inhibitor-2 (TFPI-2) has been implicated as a metastasis-associated gene in many types of tumors. Here we describe the potential involvement of TFPI-2 in the development of esophageal carcinoma. Western blotting revealed that TFPI-2 was downregulated in 75% of esophageal carcinomas and in most esophageal carcinoma cell lines. Immunohistochemistry revealed that TFPI-2 was significantly downregulated in tumor tissues and in lymph node metastases. Experimental overexpression of TFPI-2 in KYSE450, KYSE510, YES2, and EC9706 cells significantly inhibited their invasive ability. Overexpression of TFPI-2 in EC9706 cells inhibited xenograft tumor growth and invasion into surrounding tissues, as well as reduced lung metastasis. Further studies demonstrated that recombinant TFPI-2 protein significantly inhibited the activity of matrix metalloproteinases and tumor-related angiogenesis. Parenteral treatment with recombinant TFPI-2 protein significantly suppressed xenograft growth and metastasis. Together, these data indicate that TFPI-2 inhibits tumor invasion and angiogenesis both in vitro and in vivo, and suggest a potentially important therapeutic role for recombinant TFPI-2 in the treatment of malignant esophageal carcinomas.

Derlin-1 Is Overexpressed on the Tumor Cell Surface and Enables Antibody-Mediated Tumor Targeting Therapy

Purpose: Tumor targeting therapy is one of the most promising strategies for anticancer treatment. Derlin-1 has been reported to participate in misfolded protein dislocation and integrates into the endoplasmic reticulum (ER) membrane to survey for such protein aggregates. We elucidate herein that Derlin-1 can leak to the plasmalemma from the ER in tumor cells and may have clinical application as a novel cancer target in the hope of developing a new tumor targeting therapy. Experimental Design: The cell surface expression of Derlin-1 was shown by immunofluorescence analysis of nonpermeabilized cells and Western blotting of fractional proteins of tumor cells. Derlin-1 expression in cancerous tissues was also shown by immunohistochemistry. Biodistribution analysis and γ-scintigraphic imaging were done using 125I-labeled Derlin-1 targeting antibody in isogenic mice models. Finally, tumor-bearing mice were treated by the anti-Derlin-1 polyclonal antibody and monoclonal antibodies. Results: Derlin-1 was expressed on various tumor cell surfaces and adopted a homodimer conformation. Robust cytoplasmic and membrane expression of Derlin-1 was detected in various types of human cancers tissues but was not correlated with any clinicopathologic features of pancreatic cancer. Derlin-1 directed antibodies specifically targeted to colon tumors and significantly suppress tumor growth in isogenic mice. Conclusions: These preclinical data show that Derlin-1 protein is a functional molecular target expressed on the tumor cell surface and is a candidate therapeutic target that may be translated into clinical applications.

Serum haptoglobin as a novel molecular biomarker predicting colorectal cancer hepatic metastasis.

Early detection of liver metastasis is important for improving colorectal cancer (CRC) patient survival. Our previous studies showed haptoglobin was highly expressed in primary CRC tissues, especially in heterochronous metastatic cases. Here, we assessed the potential of serum haptoglobin (sHP) as a biomarker for early detection of CRC liver metastasis by evaluating the sHP in 475 CRC patients and 152 healthy volunteers. In the training set (250 cases), sHP level in CRC‐M1 (1773.18u2009±u2009690.25 ng/mL) were significantly increased as compared to in CRC‐M0 (1544.37u2009±u20091497.65 ng/mL) or healthy (917.76u2009±u2009571.59 ng/mL). And the high sHP level was correlated with poor survival. Logistic regression analysis revealed that sHP, serum carcinoembryonic antigen (sCEA) and serum carbohydrate antigen 19.9 (sCA19.9) level were the significant parameters for detecting liver metastasis. In leave‐one‐out‐cross‐validation, these three markers resulted in 89.1% sensitivity and 85.8% specificity for hepatic metastasis detection. In an independent test set (225 cases), receiver operating characteristic curve analysis of sHP in CRC liver metastasis showed an area under the curve of 0.735, with a sensitivity of 87.2% and a specificity of 59.9%. Combination of sHP, sCEA and sCA19.9 improved diagnostic accuracy to 0.880, with a sensitivity of 88.5% and a specificity of 87.8%. Silencing of HP by specific shRNA significantly inhibited the LOVO and SW620 cell invasion, and suppressed xenograft tumor invasive growth. In summary, these results demonstrate that sHP is associated with poor prognosis of CRC patients and that HP promotes colorectal cancer cell invasion. sHP combining with sCA19.9 and sCEA may be used as accurate predictors of CRC liver metastasis.

Functional Screen for Secreted Proteins by Monoclonal Antibody Library and Identification of Mac-2 Binding Protein (Mac-2BP) as a Potential Therapeutic Target and Biomarker for Lung Cancer

Identification of secreted proteins of lung cancer could provide new candidates of serum biomarkers for cancer diagnosis or targets for therapeutic intervention. In this study, we developed a novel strategy that combined functional monoclonal antibody library screening technique and mass spectrometry to identify functional secreted proteins. BALB/c mice were immunized with cancer cells isolated from fresh human lung cancer tissues. The monoclonal antibody library containing 1160 mAbs was established with the mouse spleen cells, whose serum had most anti-proliferative effect on lung cancer cells. Monoclonal antibodies were subjected to an immunoreactive and functional screen and monoclonal antibodies that reacted strongly with secreted proteins in condition medium and lung cancer tissues with high inhibotion of cell proliferation were selected. Antigens that recognized by antibodies were obtained by immunoprecipitation and then identified by mass spectrometry. Mac-2-binding protein (Mac-2BP), the antigen of 13H3 antibody, was identified using this approach. Functional studies demonstrated that the 13H3 antibody suppressed lung cancer cell lines ANIP-973 and A549 proliferation in vitro and inhibit ANIP973 xenograft tumors growth in vivo by inducing cell-cycle arrest at G1 phase, with up-regulation of p27 and down-regulation of cyclin D1. Moreover, the serum level of Mac-2BP was significantly higher in lung cancer patients than healthy controls. At a cutoff value of 6 μg/ml, Mac-2BP might be a diagnostic biomarker of lung cancer, especially for SCLC. Mac-2BP concentrations of 6 μg/ml or higher was associated with poor overall survival in univariate analysis, and was an independent predictor in the multivariate COX analysis. Together, these results firstly demonstrated that Mac-2BP can be used as a therapeutic target and potential biomarker for lung cancer. Our strategy is feasible, which may facilitate the identification of novel secreted biomarkers of lung cancer.

miR‑196a‑5p modulates gastric cancer stem cell characteristics by targeting Smad4

Cancer stem cells (CSCs) are undifferentiated cancer cells with a high tumorigenic activity, the ability to undergo self-renewal, and a multilineage differentiation potential. Clinical evidence suggests that CSCs in a tumor mass are the cellular determinants to promote cancer invasion and metastasis. MicroRNAs (miRNAs) have emerged as important modulators of cancer stem cell characteristics. Unveiling the candidate miRNAs that regulate CSCs may provide novel therapeutic targets against cancer. We analyzed the miRNA expression profiles regulating the cancer stem-like cell characteristics in gastric cancer. Gastric cancer stem cells (GCSCs) were sorted using the stem cell marker CD44 by fluorescence-activated cell sorting. Functional studies revealed that CD44(+) cells formed more sphere colonies and showed higher invasiveness than CD44(−) cells. miRNA microarray analysis revealed that miR-196a-5p was significantly upregulated in CD44(+) cells than CD44(−) cells. Suppression of miR-196a-5p led to decreased colony formation and invasion of GCSCs. miR-196a-5p decreased the expression of Smad4 by targeting 3′-UTR of the mRNA. The expression of Smad4 in gastric cancer tissues was correlated with differentiation state of tumors, TNM stage and depth of invasion. The stimulation of epithelial-mesenchymal transition (EMT) by miR-196a-5p in cancer stem-like cells was abolished by overexpression of Smad4. Collectively, these data demonstrate that miR-196a-5p has a key role in EMT and invasion by targeting Smad4 in GCSCs. miR-196a-5p may serve as a potential target for gastric cancer therapy.