Citations

In vitro studies have proposed a tumor suppressor role for sulfatase 1 (SULF1) in hepatocellular carcinoma (HCC); however, high expression in human HCC has been associated with poor prognosis. The reason underlying this paradoxical observation remains to be explored. Using a transgenic (Tg) mouse model overexpressing Sulf1 (Sulf1‐Tg), we assessed the effects of SULF1 on the diethylnitrosamine model of liver carcinogenesis. Sulf1‐Tg mice show a higher incidence of large and multifocal tumors with diethylnitrosamine injection compared to wild‐type mice. Lung metastases were found in 75% of Sulf1‐Tg mice but not in wild‐type mice. Immunohistochemistry, immunoblotting, and reporter assays all show a significant activation of the transforming growth factor‐β (TGF‐β)/SMAD transcriptional pathway by SULF1 both in vitro and in vivo . This effect of SULF1 on the TGF‐β/SMAD pathway is functional; overexpression of SULF1 promotes TGF‐β‐induced gene expression and epithelial–mesenchymal transition and enhances cell migration/invasiveness. Mechanistic analyses demonstrate that inactivating mutation of the catalytic site of SULF1 impairs the above actions of SULF1 and diminishes the release of TGF‐β from the cell surface. We also show that SULF1 expression decreases the interaction between TGF‐β1 and its heparan sulfate proteoglycan sequestration receptor, TGFβR3. Finally, using gene expression from human HCCs, we show that patients with high SULF1 expression have poorer recurrence‐free survival (hazard ratio 4.1, 95% confidence interval 1.9‐8.3; P = 0.002) compared to patients with low SULF1. We also found strong correlations of SULF1 expression with TGF‐β expression and with several TGF‐β‐related epithelial–mesenchymal transition genes in human HCC. Conclusion : Our study proposes a novel role of SULF1 in HCC tumor progression through augmentation of the TGF‐β pathway, thus defining SULF1 as a potential biomarker for tumor progression and a novel target for drug development for HCC. (H epatology 2015;61:1269–1283)

Anoctamin‐1 (Ano1) is a widely expressed protein responsible for endogenous Ca 2+ ‐activated Cl 2 currents. Ano1 is overexpressed in cancer. Differential expression of transcriptional variants is also found in other diseases. However, the mechanisms underlying regulation of Ano1 are unknown. This study identifies the Ano1 promoter and defines a mechanism for regulating its expression. Next‐generation RNA sequencing (RNA‐seq) analysis in human gastric muscle found a new exon upstream of the reported exon 1 and identified a promoter proximal to this new exon. Reporter assays in human embryonic kidney 293 cells showed a 6.7 ± 2.1fold increase in activity over empty vector. Treatment with a known regulator of Ano1 expression, IL‐4, increased promoter activity by 1.6 ± 0.02‐fold over untreated cells. The promoter region contained putative binding sites for multiple transcription factors including signal transducer and activator of transcription 6 (STAT6), a downstream effector of IL‐4. Chromatin immunoprecipitation (ChIP) experiments on T84 cells, which endogenously express Ano1, showed a 2.1 ± 0.12‐fold increase in binding of STAT6 to P0 after IL‐4 treatment. These results were confirmed by mutagenesis, expression, and RNA interference techniques. This work allows deeper understanding of the regulation of Ano1 in physiology and as a potential therapeutic target in a variety of diseases—Mazzone, A., Gibbons, S. J., Bernard, C. E., Nowsheen, S., Middha, S., Almada, L. L., Ordog, T., Kendrick, M. L., Reid Lombardo, KM., Shen, K. R., Galietta, L. J. V., Fernandez‐Zapico, M. E., and Farrugia, G., Identification and characterization of a novel promoter for the human ANO1 gene regulated by the transcription factor signal transducer and activator of transcription 6 (STAT6). FASEB J. 29, 152–163 (2015). www.fasebj.org

Pancreatectomy is associated with significant morbidity and unpredictable outcome, with few diagnostic tools to determine, which patients gain the most benefit from this treatment, especially before the operation. This study aimed to define a preoperative signature panel of serum markers to indicate response to pancreatectomy for pancreatic cancer. Over 1000 patients with pancreatic cancer treated at two independent high‐volume institutions were included in this study and were divided into three groups, including resected, locally advanced and metastatic. Eight serum tumor markers most commonly used in gastrointestinal cancers were analyzed for patient outcome. Preoperative CA19‐9 independently indicated surgical response in pancreatic cancer. Patients with CA19‐9 ≥1000 U/mL generally had a poor surgical benefit. However, a subset of these patients still achieved a survival advantage when CA19‐9 levels decreased postoperatively. CEA and CA125 in the presence of CA19‐9 ≥1000 U/mL could independently predict the non‐decrease of CA19‐9 postoperatively. The combination of the three markers was useful for predicting a worse surgical outcome with a median survival of 5.1 months vs. 23.0 months (p < 0.001) for the training cohort and 7.0 months vs. 18.2 months (p < 0.001) for the validation cohort and also suggested a higher prevalence of early distant metastasis after surgery. Resected patients with this proposed signature showed no survival advantage over patients in the locally advanced group who did not receive pancreatectomy. Therefore, a preoperative serum signature of CEA+/CA125+/CA19‐9 ≥1000 U/mL is associated with poor surgical outcome and can be used to select appropriate patients with pancreatic cancer for pancreatectomy.

Abstract PURPOSE: Hedgehog (HH) pathway has been implicated in maintenace and survival of breast cancer and cancer stem cells, EMT regulation, breast carcinogenesis and resistance to chemoradiation in solid tumors. Dysregulation of this pathway occurs in more than 50% of breast cancers. We have already demonstrated that immune-induced epithelial to mesenchymal transition (EMT) leads to the generation of breast cancer stem cells (BCSCs) from a parental epithelial breast cancer cell line in an in vivo murine model (Cancer Research 2009). The aim of our study is to demonstrate that resistance in the BCSCs is linked to HH hyperactivation and that the selective blockade of this pathway could eradicate this subpopulation of BCSCs in preclinical models. METHODS: One parental epithelial (E) and three mesenchymal BCSCs cell lines (M1, M2 and M3) were used for all the in vitro experiments. qRT-PCR was used to quantify the expression of the gene components of the pathway and gene targets. Measurement of Gli1, caspase 3 and Cyclin D1 protein expression was determined by Western blot. Cell viability assays were performed with Paclitaxel and the HH inhibitor GDC-0449. Apoptosis assays were performed when cell lines were treated with paclitaxel (10nM), GDC-0449 (25uM) or the combination of them. For the in vivo xenografted mouse model, 106 cells (E and M2) were inoculated sc respectively into the flank of 6–8 week-old syngeneic female FVB/N-TgN (MMTVneu) 202Mul/J mice. Treatment was started on daily GDC-0449 (ip), paclitaxel (ip on days 1, 4 and 8) or both drugs when tumors reached 0.5 cm2. Statistical analysis was performed using one-way ANOVA with the Bonferroni multiple comparison test. RESULTS: Activity of the HH measured by the expression of Gli1 was increased in BCSC cell lines as compared to E cell line (p &lt; 0.001). This was antagonized as expected by treatment with GDC-0449. Reporter and expression assay showed that GDC-0449 decreases Gli1 transcriptional activity and levels of a HH downstream target gene, cyclin D1. In all BCSC lines (but not in the E cell line), a synergistic effect was showed with the combination treatment as compared to control (p ≤ 0.01) and to paclitaxel or GDC-0449 alone (p ≤ 0.05 respectively) as well as a highly increased apoptotic activity (p ≤ 0.001 with control and p ≤ 0.05 with monotherapy). Tumors derived from M2 injection regressed when the combination schedule was administered (p ≤ 0.05) at 2 weeks after the start of the treatment. CONCLUSION: The inhibition of the HH pathway with GDC-0449 plus paclitaxel demonstrates a synergistic therapeutic effect in comparison to monotherapy, as shown by increased apoptotic activity, cell cycle arrest and significant reduction of tumor size in vivo. Together these results provide the rationale for future clinical trials including the blockade HH and standard chemotherapy in order to eradicate the whole population of tumor cells (also BCSCs) within the tumors and avoid disease relapse and metastasis in breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-07.