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Cell-surface heparan sulfate proteoglycans (HSPGs) function as receptors or co-receptors for ligand binding and mediate the transmission of critical extracellular signals into cells. The complex and dynamic modifications of heparan sulfates on the core proteins are highly regulated to achieve precise signaling transduction. Extracellular endosulfatase Sulf1 catalyzes the removal of 6-O sulfation from HSPGs and thus regulates signaling mediated by 6-O sulfation on HSPGs. The expression of Sulf1 is altered in many cancers. Further studies are needed to clarify Sulf1 role in tumorigenesis, and new tools that can expand our knowledge in this field are required. We have developed and validated novel SULF1 monoclonal antibodies (mAbs). The isotype and subclass for each of these antibodies were determined. These antibodies provide invaluable reagents to assess SULF1- tissue and blood levels by immunohistochemistry and ELISA assays, respectively. This study reports novel mAbs and immunoassays developed for sensitive and specific human Sulf1 protein detection. Using these SULF1 mAbs, we developed an ELISA assay to investigate whether blood-derived SULF1 may be a useful biomarker for detecting cancer early. Furthermore, we have demonstrated the utility of these antibodies for Sulf1 protein detection, localization, and quantification in biospecimens using various immunoassays. This study describes novel Sulf1 mAbs suitable for various immunoassays, including Western blot analysis, ELISA, and immunohistochemistry, which can help understand Sulf1 pathophysiological role. New tools to assess and clarify SULF1 role in tumorigenesis are needed. Our novel Sulf1 mAbs and immunoassays assay may have utility for such application. • New tools to assess and clarify the SULF1 role in tumorigenesis. • Novel SULF1 mouse monoclonal antibodies suitable for a variety of immunoassays. • SULF1 is present at the invasive front of tumors from NSCLC patients. • Novel SULF1 ELISA to measure its blood levels in healthy and disease. • SULF1 can be detected and quantified in plasma by a novel ELISA assay. [ABSTRACT FROM AUTHOR]

Aims: Lung cancer is one of the most deadly cancers; median survival from diagnosis is less than one year in those with advanced disease. Novel lung cancer biomarkers are desperately needed. In this study, we evaluated SULF2 expression by immunohistochemistry and its association with overall survival in a cohort of patients with non-small cell lung cancer (NSCLC). We also looked for the presence of SULF2 protein in plasma to evaluate its potential as an early detection biomarker for NSCLC. Methods: We identified patients who underwent surgical resection for pulmonary adenocarcinoma or squamous cell carcinoma at our institution. A section from each paraffin-embedded specimen was stained with a SULF2 antibody. A pathologist determined the percentage and intensity of tumor cell staining. Survival analysis was performed using a multivariate Cox proportional hazards model. Using a novel SULF2 ELISA assay, we analyzed plasma levels of SULF2 in a small cohort of healthy donors and patients with early stage NSCLC. Results: SULF2 staining was present in 82% of the lung cancer samples. Squamous cell carcinomas had a higher mean percentage of staining than adenocarcinomas (100% vs. 60%; p<0.0005). After adjusting for age, sex, race, histologic type, stage, and neoadjuvant therapy, there was a non-significant (31%; p = 0.65) increase in the risk of death for patients with adenocarcinoma with SULF2 staining in tumor cells. In contrast, there was a significant decrease in the risk of death (89%; p = 0.02) for patients with squamous cell carcinoma with SULF2 staining in tumor cells. SULF2 protein was present in plasma of patients with early stage NSCLC, and soluble SULF2 levels increased with age. Finally, plasma SULF2 levels were significantly elevated in early stage NSCLC patients, compared to healthy controls. Conclusions: Tumor expression of SULF2 may affect prognosis in NSCLC, while blood SULF2 levels may have a significant role in the diagnosis of this fatal disease. [ABSTRACT FROM AUTHOR]

Background Lung cancer is the leading cause of morbidity and death worldwide. Although the available lung cancer animal models have been informative and further propel our understanding of human lung cancer, they still do not fully recapitulate the complexities of human lung cancer. The pathogenesis of lung cancer remains highly elusive because of its aggressive biologic nature and considerable heterogeneity, compared to other cancers. The association of Cul4 A amplification with aggressive tumor growth and poor prognosis has been suggested. Our previous study suggested that Cul4 A is oncogenic in vitro, but its oncogenic role in vivo has not been studied. Methods Viral delivery approaches have been used extensively to model cancer in mouse models. In our experiments, we used Cre-recombinase induced overexpression of the Cul4A gene in transgenic mice to study the role of Cul4A on lung tumor initiation and progression and have developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A. Results Here we show that the use of a recombinant adenovirus expressing Cre-recombinase (' Adeno Cre') to induce Cul4A overexpression in the lungs of mice allows controls of the timing and multiplicity of tumor initiation. Following our mouse models, we are able to study the potential role of Cul4A in the development and progression in pulmonary adenocarcinoma as well. Conclusion Our findings indicate that Cul4A is oncogenic in vivo, and this mouse model is a tool in understanding the mechanisms of Cul4A in human cancers and for testing experimental therapies targeting Cul4A. [ABSTRACT FROM AUTHOR]

Background Squamous cell carcinomas (SCC) account for approximately 30% of non-small cell lung cancer. Investigation of the mechanism of invasion and metastasis of lung SCC will be of great help for the development of meaningful targeted therapeutics. This study is intended to understand whether the activation of Hedgehog (Hh) pathway is involved in lung SCC, and whether activated Hh signaling regulates metastasis through epithelial-mesenchymal transition (EMT) in lung SCC. Methods Two cohorts of patients with lung SCC were studied. Protein expression was examined by immunohistochemistry, Western blot, or immunofluorescence. Protein expression levels in tissue specimens were scored and correlations were analyzed. Vismodegib and a Gli inhibitor were used to inhibit Shh/Gli activity, and recombinant Shh proteins were used to stimulate the Hh pathway in lung SCC cell lines. Cell migration assay was performed in vitro. Results Shh/Gli pathway components were aberrantly expressed in lung SCC tissue samples. Gli1 expression was reversely associated with the expression of EMT markers E-Cadherin and β-Catenin in lung SCC specimens. Inhibition of the Shh/Gli pathway suppressed migration and up-regulated E-Cadherin expression in lung SCC cells. Stimulation of the pathway increased migration and down-regulated E-Cadherin expression in lung SCC cells. Conclusions Our results suggested that the Shh/Gli pathway may be critical for lung SCC recurrence, metastasis and resistance to chemotherapy. Inhibition of the Shh/Gli pathway activity/function is a potential therapeutic strategy for the treatment of lung SCC patients. [ABSTRACT FROM AUTHOR]

Malignant pleural mesothelioma (MPM) is a highly aggressive tumor with poor prognosis. Current treatment is rarely curative, thus novel meaningful therapies are urgently needed. Inhibition of Hedgehog (Hh) signaling at the cell membrane level in several cancers has shown anti-cancer activity in recent clinical studies. Evidence of Hh-independent Gli activation suggests Gli as a more potent therapeutic target. The current study is aimed to evaluate the potential of Gli as a therapeutic target to treat MPM. The expression profiles of Gli factors and other Hh signaling components were characterized in 46 MPM patient tissue samples by RT-PCR and immunohistochemistry. Cultured cell lines were employed to investigate the requirement of Gli activation in tumor cell growth by inhibiting Gli through siRNA or a novel small molecule Gli inhibitor (Gli-I). A xenograft model was used to evaluate Gli-I in vivo. In addition, a side by side comparison between Gli and Smoothened (Smo) inhibition was conducted in vitro using siRNA and small molecule inhibitors. Our study reported aberrant Gli1 and Gli2 activation in a large majority of tissues. Inhibition of Gli by siRNAs or Gli-I suppressed cell growth dramatically both in vitro and in vivo. Inhibition of Gli exhibited better cytotoxicity than that of Smo by siRNA and small molecule inhibitors vismodegib and cyclopamine. Combination of Gli-I and pemetrexed, as well as Gli-I and vismodegib demonstrated synergistic effects in suppression of MPM proliferation in vitro. In summary, Gli activation plays a critical role in MPM. Inhibition of Gli function holds strong potential to become a novel, clinically effective approach to treat MPM. [ABSTRACT FROM AUTHOR]

The histopathological diagnosis of melanoma can be challenging. No currently used molecular markers accurately distinguish between nevus and melanoma. Recent transcriptome analyses have shown the differential expression of several genes in melanoma progression. Here, we describe a multi-marker diagnostic assay using 5 markers (ARPC2, FN1, RGS1, SPP1, and WNT2) overexpressed in melanomas. Immunohistochemical marker expression was analyzed in 693 melanocytic neoplasms comprising a training set (tissue microarray of 534 melanomas and nevi), and 4 independent validation sets: tissue sections of melanoma arising in a nevus; dysplastic nevi; Spitz nevi; and misdiagnosed melanocytic neoplasms. Both intensity and pattern of expression were scored for each marker. Based on the differential expression of these 5 markers between nevi and melanomas in the training set, a diagnostic algorithm was obtained. Using this algorithm, the lesions in the validation sets were diagnosed as nevus or melanoma, and the results were compared with the known histological diagnoses. Both the intensity and pattern of expression of each marker were significantly different in melanomas compared to nevi. The diagnostic algorithm exploiting these differences achieved a specificity of 95% and a sensitivity of 91% in the training set. In the validation sets, the multi-marker assay correctly diagnosed a high percentage of melanomas arising in a nevus, Spitz nevi, dysplastic nevi, and misdiagnosed lesions. The multimarker assay described here can aid in the diagnosis of melanoma. [ABSTRACT FROM AUTHOR]