Correction: Niclosamide and its analogs are potent inhibitors of Wnt/ß-catenin, mTOR and STAT3 signaling in ovarian cancer.

[This corrects the article DOI: 10.18632/oncotarget.13466.].

Niclosamide and its analogs are potent inhibitors of Wnt/ß-catenin, mTOR and STAT3 signaling in ovarian cancer.

Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer mortality worldwide. Platinum-based therapy is the standard first line treatment and while most patients initially respond, resistance to chemotherapy usually arises. Major signaling pathways frequently upregulated in chemoresistant cells and important in the maintenance of cancer stem cells (CSCs) include Wnt/ß-catenin, mTOR, and STAT3. The major objective of our study was to investigate the treatment of ovarian cancer with targeted agents that inhibit these three pathways. Here we demonstrate that niclosamide, a salicylamide derivative, and two synthetically manufactured niclosamide analogs (analog 11 and 32) caused significant inhibition of proliferation of two chemoresistant ovarian cancer cell lines (A2780cp20 and SKOV3Trip2), tumorspheres isolated from the ascites of EOC patients, and cells from a chemoresistant patient-derived xenograft (PDX). This work shows that all three agents significantly decreased the expression of proteins in the Wnt/ß-catenin, mTOR and STAT3 pathways and preferentially targeted cells that expressed the ovarian CSC surface protein CD133. It also illustrates the potential of drug repurposing for chemoresistant EOC and can serve as a basis for pathway-oriented in vivo studies.

S100A4 promotes pancreatic cancer progression through a dual signaling pathway mediated by Src and focal adhesion kinase.

S100A4 expression is associated with poor clinical outcomes of patients with pancreatic cancer. The effects of loss or gain of S100A4 were examined in pancreatic cancer cell lines. S100A4 downregulation remarkably reduces cell migration and invasion, inhibits proliferation, and induces apoptosis in pancreatic tumor cells. S100A4 downregulation results in significant cell growth inhibition and apoptosis in response to TGF-ß1, supporting a non-canonical role of S100A4 in pancreatic cancer. The role of S100A4 in tumor progression was studied by using an orthotopic human pancreatic cancer xenograft mouse model. Tumor mass is remarkably decreased in animals injected with S100A4-deficient pancreatic tumor cells. P27(Kip1) expression and cleaved caspase-3 are increased, while cyclin E expression is decreased, in S100A4-deficient pancreatic tumors in vivo. S100A4-deficient tumors have lower expression of vascular endothelial growth factor, suggesting reduced angiogenesis. Biochemical assays revealed that S100A4 activates Src and focal adhesion kinase (FAK) signaling events, and inhibition of both kinases is required to maximally block the tumorigenic potential of pancreatic cancer cells. These findings support that S100A4 plays an important role in pancreatic cancer progression in vivo and S100A4 promotes tumorigenic phenotypes of pancreatic cancer cells through the Src-FAK mediated dual signaling pathway.

Inhibition of Wnt/ß-catenin pathway by niclosamide: a therapeutic target for ovarian cancer.

Objective. The Wnt/ß-catenin pathway is known to regulate cellular proliferation and plays a role in chemoresistance. Niclosamide, an FDA approved salicyclamide derivative used for the treatment of tapeworm infections, targets the Wnt/ß-catenin pathway. Therefore, the objective of this study was to investigate niclosamide as a potential therapeutic agent for ovarian cancer. Methods. Tumor cells isolated from 34 patients' ascites with primary ovarian cancer were treated with niclosamide (0.1 to 5 µM) ± carboplatin (5 to 150 µM). Cell viability was assessed using the ATP-lite assay. LRP6, Axin 2, Cyclin D1, survivin and cytosolic free ß-catenin levels were determined using Western blot analysis. Tumorspheres were treated, and Wnt transcriptional activity was measured by the TOPflash reporter assay. ALDH and CD133 were analyzed by Flow cytometry and IHC. ALDH1A1 and LRP6 were analyzed by IHC in solid tumor and in ascites before and after treatment with niclosamide. Results. Combination treatment produced increased cytotoxicity compared to single agent treatment in 32/34 patient samples. Western blot analysis showed a decrease in Wnt/ß-catenin pathway proteins and the expression of target genes. A significant reduction of Wnt/ß-catenin signaling was confirmed by TOPflash assay. There was increased staining of ALDH1A1 and LRP6 in ascites compared to solid tumor which decreased after treatment. Conclusion. This study demonstrates that niclosamide is a potent Wnt/ß-catenin inhibitor. Targeting the Wnt/ß-catenin pathway led to decreased cellular proliferation and increased cell death. These findings warrant further research of this drug and other niclosamide analogs as a treatment option for ovarian cancer.

Effect of niclosamide on basal-like breast cancers.

Basal-like breast cancers (BLBC) are poorly differentiated and display aggressive clinical behavior. These tumors become resistant to cytotoxic agents, and tumor relapse has been attributed to the presence of cancer stem cells (CSC). One of the pathways involved in CSC regulation is the Wnt/ß-catenin signaling pathway. LRP6, a Wnt ligand receptor, is one of the critical elements of this pathway and could potentially be an excellent therapeutic target. Niclosamide has been shown to inhibit the Wnt/ß-catenin signaling pathway by causing degradation of LRP6. TRA-8, a monoclonal antibody specific to TRAIL death receptor 5, is cytotoxic to BLBC cell lines and their CSC-enriched populations. The goal of this study was to examine whether niclosamide is cytotoxic to BLBCs, specifically the CSC population, and if in combination with TRA-8 could produce increased cytotoxicity. Aldehyde dehydrogenase (ALDH) is a known marker of CSCs. By testing BLBC cells for ALDH expression by flow cytometry, we were able to isolate a nonadherent population of cells that have high ALDH expression. Niclosamide showed cytotoxicity against these nonadherent ALDH-expressing cells in addition to adherent cells from four BLBC cell lines: 2LMP, SUM159, HCC1187, and HCC1143. Niclosamide treatment produced reduced levels of LRP6 and ß-catenin, which is a downstream Wnt/ß-catenin signaling protein. The combination of TRA-8 and niclosamide produced additive cytotoxicity and a reduction in Wnt/ß-catenin activity. Niclosamide in combination with TRA-8 suppressed growth of 2LMP orthotopic tumor xenografts. These results suggest that niclosamide or congeners of this agent may be useful for the treatment of BLBC.

Monoclonal antibody-based immunotherapy of ovarian cancer: targeting ovarian cancer cells with the B7-H3-specific mAb 376.96.

OBJECTIVE: The high rate of relapse in patients with advanced ovarian cancer likely reflects the chemoresistance of cancer initiating cells (CICs). We evaluated the anti-tumor activity of monoclonal antibody (mAb) 376.96, which recognizes a B7-H3 epitope expressed on ovarian carcinoma cells (OCCs), in combination with the tyrosine kinase inhibitor Sunitinib and chemotherapy on chemosensitive and chemoresistant cells and CICs. METHODS: Eight ovarian cancer cell lines including platinum- and taxane-resistant cell lines were analyzed by flow cytometry to establish expression of the mAb 376.96-defined-B7-H3-epitope on differentiated ovarian cancer cells and CICs. Samples from 10 ovarian cancer patients were analyzed via immunohistochemistry for mAb 376.96-defined-B7-H3-epitope expression. In vitro studies assessed mAb 376.96 alone and in combination with Sunitinib on the growth of chemosensitive and chemoresistant cell lines and on the content of CICs. RESULTS: The mAb-376.96-defined-B7-H3 epitope is expressed on both differentiated cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines and 10 patient derived ovarian cancer tumors. In vitro treatment of chemoresistant cell lines with mAb 376.96 resulted in decreased cell viability. mAb 376.96 enhanced the cytotoxicity of Sunitinib and reduced the content of CICs. CONCLUSION: The mAb-376.96-defined-B7-H3-epitope was found to be expressed on both differentiated ovarian cancer cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines. mAb 376.96 inhibited the in vitro growth of chemosensitive and chemoresistant OCCs and reduced the content of CICs when used with Sunitinib. Further studies examining B7-H3 as a potential target of mAb-based immunotherapy for this type of malignancy are warranted.

The Wnt/ß-catenin pathway in ovarian cancer: a review.

OBJECTIVE: Ovarian cancer is the deadliest gynecologic malignancy and the fifth leading cause of death from cancer in women in the U.S. Since overall survival remains poor, there is a need for new therapeutic paradigms. This paper will review the Wnt/ß-catenin pathway as it relates to epithelial ovarian cancer, specifically its role in chemoresistance and its potential role as a target for chemosensitization. METHODS: A PubMed search was performed for articles published pertaining to Wnt/ß-catenin pathway specific to ovarian cancer. Wnt/ß-catenin signaling pathways play an active role in cancer stem cells (CSCs) and carcinogenesis of all ovarian cancer subtypes. Studies also have shown that ovarian CSCs are involved in chemoresistance, metastasis, and tumor recurrence. RESULTS: Wnt/ß-catenin target genes regulate cell proliferation and apoptosis, thereby mediating cancer initiation and progression. The Wnt/ß-catenin pathway is one of the major signaling pathways thought to be involved in epithelial-to-mesenchymal transition (EMT). Alterations affecting Wnt pathway proteins on the cell membrane, in the cytoplasm, and in the nucleus have been shown to play important roles in the tumorigenesis of ovarian cancer. CONCLUSIONS: Wnt signaling is activated in epithelial ovarian cancer. Given the role of the Wnt/ß-catenin pathway in carcinogenesis, more pre-clinical studies are warranted to further investigate other Wnt inhibitors in ovarian cancer. The Wnt pathway should also be investigated as a potential target in the development of new drugs for ovarian cancer as a single agent and in combination with chemotherapy or other targeted agents.

The C-terminal region Mesd peptide mimics full-length Mesd and acts as an inhibitor of Wnt/ß-catenin signaling in cancer cells.

While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/ß-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/ß-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer.

ST6Gal-I protein expression is upregulated in human epithelial tumors and correlates with stem cell markers in normal tissues and colon cancer cell lines.

The ST6Gal-I sialyltransferase adds an α2-6-linked sialic acid to the N-glycans of certain receptors. ST6Gal-I mRNA has been reported to be upregulated in human cancer, but a prior lack of antibodies has limited immunochemical analysis of the ST6Gal-I protein. Here, we show upregulated ST6Gal-I protein in several epithelial cancers, including many colon carcinomas. In normal colon, ST6Gal-I localized selectively to the base of crypts, where stem/progenitor cells are found, and the tissue staining patterns were similar to the established stem cell marker ALDH1. Similarly, ST6Gal-I expression was restricted to basal epidermal layers in skin, another stem/progenitor cell compartment. ST6Gal-I was highly expressed in induced pluripotent stem (iPS) cells, with no detectable expression in the fibroblasts from which iPS cells were derived. On the basis of these observations, we investigated further an association of ST6Gal-I with cancer stem cells (CSC). Selection of irinotecan resistance in colon carcinoma cells led to a greater proportion of CSCs compared with parental cells, as measured by the CSC markers CD133 and ALDH1 activity (Aldefluor). These chemoresistant cells exhibited a corresponding upregulation of ST6Gal-I expression. Conversely, short hairpin RNA (shRNA)-mediated attenuation of ST6Gal-I in colon carcinoma cells with elevated endogenous expression decreased the number of CD133/ALDH1-positive cells present in the cell population. Collectively, our results suggest that ST6Gal-I promotes tumorigenesis and may serve as a regulator of the stem cell phenotype in both normal and cancer cell populations.

Basal-like breast cancer stem cells are sensitive to anti-DR5 mediated cytotoxicity.

Breast cancer stem cells (BrCSC) are resistant to common therapeutic modalities including chemotherapy, radiation, and hormonal agents. They are thought to contribute to treatment resistance, relapse, and metastases. This study examines the effect of a monoclonal anti-DR5 antibody (TRA-8) and chemotherapy (adriamycin, taxol) on BrCSC populations from basal-like breast cancer cell lines. Doubly enriched BrCSC (CD44(+), CD24(-), ALDH(+)) cells were exposed to TRA-8 and control reagents and examined for cytotoxicity, caspase activation, tumorsphere formation and tumorigenicity. Doubly enriched BrCSC populations expressed cell surface DR5 and were sensitive to TRA-8 mediated cytotoxicity with induction of caspase 8 and 3 activation. TRA-8 at sub-nanomolar concentrations inhibited 2LMP and SUM159 BrCSC tumorsphere formation and was more than 50-fold more inhibitory than TRAIL or anti-DR4 at equimolar concentrations. Chemotherapy treatment of 2LMP and SUM159 cell lines resulted in a relative increase of BrCSC, whereas TRA-8 produced a decrease in the percentage of BrCSC. TRA-8 exposure to 2LMP and SUM159 BrCSC preparations produced significant inhibition of tumorigenicity. DR5 maybe a therapeutic target on the surface of basal-like BrCSC which is amenable to agonistic monoclonal anti-DR5 therapy.

The impact of novel retinoids in combination with platinum chemotherapy on ovarian cancer stem cells.

OBJECTIVE: Retinoids are important modulators of cell growth, differentiation, and proliferation. 9cUAB30, 9cUAB124, and 9cUAB130 are three novel retinoid compounds that show cytotoxic effects in other malignancies. We evaluated these novel retinoids in combination with chemotherapy against ovarian cancer stem cells (CSCs) in vitro and in an ex vivo model. METHODS: A2780 cells were plated in 96-well plates and treated with retinoid, carboplatin, or combination therapy. Cell viability was evaluated using ATPLite assay. The A2780 cell line was also analyzed for CSCs by evaluating ALDH activity using flow cytometry. A2780 cells treated ex vivo with retinoids and chemotherapy were injected into the flank of athymic nude mice in order to evaluate subsequent tumor initiating capacity. RESULTS: A2780 cells were sensitive to treatment with retinoids and carboplatin. The best treatment resulted from the combination of retinoid 9cUAB130 and carboplatin. Untreated A2780 cells demonstrated ALDH activity in 3.3% of the cell population. Carboplatin treatment enriched ALDH activity to 27.3%, while 9cUAB130±carboplatin maintained the ALDH positive levels similar to untreated controls (2.3% and 6.7%, respectively). Similar results were found in tumorsphere-forming conditions. Flank injections of ex vivo treated A2780 cells resulted in 4/4 mice developing tumors at 40 days in the untreated group, while 0/4 tumors developed in the 9cUAB130 and carboplatin treated group. CONCLUSION: Combination treatment with carboplatin and retinoids reduced cell-viability, reduced CSC marker expression, and inhibited tumorigenicity, making it a more effective treatment when compared with carboplatin alone.