Glycosyltransferase ST6Gal-I promotes the epithelial to mesenchymal transition in pancreatic cancer cells.

ST6Gal-I, an enzyme upregulated in numerous malignancies, adds α2-6-linked sialic acids to select membrane receptors, thereby modulating receptor signaling and cell phenotype. In this study, we investigated ST6Gal-I's role in epithelial to mesenchymal transition (EMT) using the Suit2 pancreatic cancer cell line, which has low endogenous ST6Gal-I and limited metastatic potential, along with two metastatic Suit2-derived subclones, S2-013 and S2-LM7AA, which have upregulated ST6Gal-I. RNA-Seq results suggested that the metastatic subclones had greater activation of EMT-related gene networks than parental Suit2 cells, and forced overexpression of ST6Gal-I in the Suit2 line was sufficient to activate EMT pathways. Accordingly, we evaluated expression of EMT markers and cell invasiveness (a key phenotypic feature of EMT) in Suit2 cells with or without ST6Gal-I overexpression, as well as S2-013 and S2-LM7AA cells with or without ST6Gal-I knockdown. Cells with high ST6Gal-I expression displayed enrichment in mesenchymal markers (N-cadherin, slug, snail, fibronectin) and cell invasiveness, relative to ST6Gal-I-low cells. Contrarily, epithelial markers (E-cadherin, occludin) were suppressed in ST6Gal-I-high cells. To gain mechanistic insight into ST6Gal-I's role in EMT, we examined the activity of epidermal growth factor receptor (EGFR), a known EMT driver. ST6Gal-I-high cells had greater α2-6 sialylation and activation of EGFR than ST6Gal-I-low cells. The EGFR inhibitor, erlotinib, neutralized ST6Gal-I-dependent differences in EGFR activation, mesenchymal marker expression, and invasiveness in Suit2 and S2-LM7AA, but not S2-013, lines. Collectively, these results advance our understanding of ST6Gal-I's tumor-promoting function by highlighting a role for ST6Gal-I in EMT, which may be mediated, at least in part, by α2-6-sialylated EGFR.

ST6Gal-I sialyltransferase promotes chemoresistance in pancreatic ductal adenocarcinoma by abrogating gemcitabine-mediated DNA damage.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a poor prognosis. Gemcitabine, as a single agent or in combination therapy, remains the frontline chemotherapy despite its limited efficacy due to de novo or acquired chemoresistance. There is an acute need to decipher mechanisms underlying chemoresistance and identify new targets to improve patient outcomes. Here, we report a novel role for the ST6Gal-I sialyltransferase in gemcitabine resistance. Utilizing MiaPaCa-2 and BxPC-3 PDAC cells, we found that knockdown (KD) of ST6Gal-I expression, as well as removal of surface α2-6 sialic acids by neuraminidase, enhances gemcitabine-mediated cell death assessed via clonogenic assays and cleaved caspase 3 expression. Additionally, KD of ST6Gal-I potentiates gemcitabine-induced DNA damage as measured by comet assays and quantification of γH2AX foci. ST6Gal-I KD also alters mRNA expression of key gemcitabine metabolic genes, RRM1, RRM2, hENT1, and DCK, leading to an increased gemcitabine sensitivity ratio, an indicator of gemcitabine toxicity. Gemcitabine-resistant MiaPaCa-2 cells display higher ST6Gal-I levels than treatment-naïve cells along with a reduced gemcitabine sensitivity ratio, suggesting that chronic chemotherapy selects for clonal variants with more abundant ST6Gal-I. Finally, we examined Suit2 PDAC cells and Suit2 derivatives with enhanced metastatic potential. Intriguingly, three metastatic and chemoresistant subclones, S2-CP9, S2-LM7AA, and S2-013, exhibit up-regulated ST6Gal-I relative to parental Suit2 cells. ST6Gal-I KD in S2-013 cells increases gemcitabine-mediated DNA damage, indicating that suppressing ST6Gal-I activity sensitizes inherently resistant cells to gemcitabine. Together, these findings place ST6Gal-I as a critical player in imparting gemcitabine resistance and as a potential target to restore PDAC chemoresponse.

The expression of MHC class II molecules on murine breast tumors delays T-cell exhaustion, expands the T-cell repertoire, and slows tumor growth.

The expression of MHC class II molecules (MHCII) on tumor cells correlates with survival and responsiveness to immunotherapy. However, the mechanisms underlying these observations are poorly defined. Using a murine breast tumor line, we showed that MHCII-expressing tumors grew more slowly than controls and recruited more functional CD4+ and CD8+ T cells. In addition, MHCII-expressing tumors contained more TCR clonotypes expanded to a larger degree than control tumors. Functional CD8+ T cells in tumors depended on CD4+ T cells. However, both CD4+ and CD8+ T cells eventually became exhausted, even in MHCII-expressing tumors. Treatment with anti-CTLA4, but not anti-PD-1 or anti-TIM-3, promoted complete eradication of MHCII-expressing tumors. These results suggest tumor cell expression of MHCII facilitates the local activation of CD4+ T cells, indirectly helps the activation and expansion of CD8+ T cells, and, in combination with the appropriate checkpoint inhibitor, promotes tumor regression.

Histone deacetylase inhibition promotes intratumoral CD8+ T-cell responses, sensitizing murine breast tumors to anti-PD1.

Histone deacetylase (HDAC) inhibitors impair tumor cell proliferation and alter gene expression. However, the impact of these changes on anti-tumor immunity is poorly understood. Here, we showed that the class I HDAC inhibitor, entinostat (ENT), promoted the expression of immune-modulatory molecules, including MHCII, costimulatory ligands, and chemokines on murine breast tumor cells in vitro and in vivo. ENT also impaired tumor growth in vivo-an effect that was dependent on both CD8+ T cells and IFNγ. Moreover, ENT promoted intratumoral T-cell clonal expansion and enhanced their functional activity. Importantly, ENT sensitized normally unresponsive tumors to the effects of PD1 blockade, predominantly through increases in T-cell proliferation. Our findings suggest that class I HDAC inhibitors impair tumor growth by enhancing the proliferative and functional capacity of CD8+ T cells and by sensitizing tumor cells to T-cell recognition.

Calmodulin antagonist enhances DR5-mediated apoptotic signaling in TRA-8 resistant triple negative breast cancer cells.

Patients with triple negative breast cancer (TNBC) have no successful "targeted" treatment modality, which represents a priority for novel therapy strategies. Upregulated death receptor 5 (DR5) expression levels in breast cancer cells compared to normal cells enable TRA-8, a DR5 specific agonistic antibody, to specifically target malignant cells for apoptosis without inducing normal hepatocyte apoptosis. Drug resistance is a common obstacle in TRAIL-based therapy for TNBC. Calmodulin (CaM) is overexpressed in breast cancer. In this study, we characterized the novel function of CaM antagonist in enhancing TRA-8 induced cytotoxicity in TRA-8 resistant TNBC cells and its underlying molecular mechanisms. Results demonstrated that CaM antagonist(s) enhanced TRA-8 induced cytotoxicity in a concentration and time-dependent manner for TRA-8 resistant TNBC cells. CaM directly bound to DR5 in a Ca2+ dependent manner, and CaM siRNA promoted DR5 recruitment of FADD and caspase-8 for DISC formation and TRA-8 activated caspase cleavage for apoptosis in TRA-8 resistant TNBC cells. CaM antagonist, trifluoperazine, enhanced TRA-8 activated DR5 oligomerization, DR5-mediated DISC formation, and TRA-8 activated caspase cleavage for apoptosis, and decreased anti-apoptotic pERK, pAKT, XIAP, and cIAP-1 expression in TRA-8 resistant TNBC cells. These results suggest that CaM could be a key regulator to mediate DR5-mediated apoptotic signaling, and suggests a potential strategy for using CaM antagonists to overcome drug resistance of TRAIL-based therapy for TRA-8 resistant TNBC.

The antitumor effects of entinostat in ovarian cancer require adaptive immunity.

BACKGROUND: Ovarian cancer is poorly immunogenic; however, increased major histocompatibility complex class II (MHCII) expression correlates with improved immune response and prolonged survival in patients with ovarian cancer. The authors previously demonstrated that the histone deacetylase inhibitor entinostat increases MHCII expression on ovarian cancer cells. In the current study, they evaluated whether entinostat treatment and resultant MHCII expression would enhance beneficial immune responses and impair tumor growth in mice with ovarian cancer. METHODS: C57BL/6 mice bearing intraperitoneal ID8 tumors were randomized to receive entinostat 20 mg/kg daily versus control. Changes in messenger RNA (mRNA) expression of 46 genes important for antitumor immunity were evaluated using NanoString analysis, and multicolor flow cytometry was used to measure changes in protein expression and tumor-infiltrating immune cells. RESULTS: Entinostat treatment decreased the growth of both subcutaneously and omental ID8 tumors and prolonged survival in immunocompetent C57BL/6 mice. NanoString analysis revealed significant changes in mRNA expression in 21 of 46 genes, including increased expression of the MHCI pathway, the MHCII transactivator (CIITA), interferon γ, and granzyme B. C57BL/6 mice that received entinostat had increased MHCII expression on omental tumor cells and a higher frequency of tumor-infiltrating, CD8-positive T cells by flow cytometry. In immunocompromised mice, treatment with entinostat had no effect on tumor size and did not increase MHCII expression. CONCLUSIONS: In the current murine ovarian cancer model, entinostat treatment enhances beneficial immune responses. Moreover, these antitumor effects of entinostat are dependent on an intact immune system. Future studies combining entinostat with checkpoint inhibitors or other immunomodulatory agents may achieve more durable antitumor responses in patients with ovarian cancer.

Novel Biomimetic Microphysiological Systems for Tissue Regeneration and Disease Modeling.

Biomaterials engineered to closely mimic morphology, architecture, and nanofeatures of naturally occurring in vivo extracellular matrices (ECM) have gained much interest in regenerative medicine and in vitro biomimetic platforms. Similarly, microphysiological systems (MPS), such as lab-chip, have drummed up momentum for recapitulating precise biomechanical conditions to model the in vivo microtissue environment. However, porosity of in vivo scaffolds regulating barrier and interface functions is generally absent in lab-chip systems, or otherwise introduces considerable cost, complexity, and an unrealistic uniformity in pore geometry. We address this by integrating electrospun nanofibrous porous scaffolds in MPS to develop the lab-on-a-brane (LOB) MPS for more effectively modeling transport, air-liquid interface, and tumor progression and for personalized medicine applications.

212Pb-Labeled Antibody 225.28 Targeted to Chondroitin Sulfate Proteoglycan 4 for Triple-Negative Breast Cancer Therapy in Mouse Models.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis. There is a clinical need for effective, targeted therapy strategies that destroy both differentiated TNBC cells and TNBC cancer initiating cells (CICs), as the latter are implicated in the metastasis and recurrence of TNBC. Chondroitin sulfate proteoglycan 4 (CSPG4) is overexpressed on differentiated tumor cells and CICs obtained from TNBC patient specimens, suggesting that CSPG4 may be a clinically relevant target for the imaging and therapy of TNBC. The purpose of this study was to determine whether α-particle radioimmunotherapy (RIT) targeting TNBC cells using the CSPG4-specific monoclonal antibody (mAb) 225.28 as a carrier was effective at eliminating TNBC tumors in preclinical models. To this end, mAb 225.28 labeled with 212Pb (212Pb-225.28) as a source of α-particles for RIT was used for in vitro Scatchard assays and clonogenic survival assays with human TNBC cells (SUM159 and 2LMP) grown as adherent cells or non-adherent CIC-enriched mammospheres. Immune-deficient mice bearing orthotopic SUM159 or 2LMP xenografts were injected i.v. with the targeted (225.28) or irrelevant isotype-matched control (F3-C25) mAbs, labeled with 99mTc, 125I, or 212Pb for in vivo imaging, biodistribution, or tumor growth inhibition studies. 212Pb-225.28 bound to adherent SUM159 and 2LMP cells and to CICs from SUM159 and 2LMP mammospheres with a mean affinity of 0.5 nM. Nearly ten times more binding sites per cell were present on SUM159 cells and CICs compared with 2LMP cells. 212Pb-225.28 was six to seven times more effective than 212Pb-F3-C25 at inhibiting SUM159 cell and CIC clonogenic survival (p < 0.05). Radiolabeled mAb 225.28 showed significantly higher uptake than radiolabeled mAb F3-C25 in SUM159 and 2LMP xenografts (p < 0.05), and the uptake of 212Pb-225.28 in TNBC xenografts was correlated with target epitope expression. 212Pb-225.28 caused dose-dependent growth inhibition of SUM159 xenografts; 0.30 MBq 212Pb-225.28 was significantly more effective than 0.33 MBq 212Pb-F3-C25 at inhibiting tumor growth (p < 0.01). These results suggest that CSPG4-specific 212Pb-225.28 is a useful reagent for RIT of CSPG4-expressing tumors, including metastatic TNBC.

Preferential Inhibition of Wnt/ß-Catenin Signaling by Novel Benzimidazole Compounds in Triple-Negative Breast Cancer.

Wnt/ß-catenin signaling is upregulated in triple-negative breast cancer (TNBC) compared to other breast cancer subtypes and normal tissues. Current Wnt/ß-catenin inhibitors, such as niclosamide, target the pathway nonspecifically and exhibit poor pharmacokinetics/pharmacodynamics in vivo. Niclosamide targets other pathways, including mTOR, STAT3 and Notch. Novel benzimidazoles have been developed to inhibit Wnt/ß-catenin signaling with greater specificity. The compounds SRI33576 and SRI35889 were discovered to produce more cytotoxicity in TNBC cell lines than in noncancerous cells. The agents also downregulated Wnt/ß-catenin signaling mediators LRP6, cyclin D1, survivin and nuclear active ß-catenin. In addition, SRI33576 did not affect mTOR, STAT3 and Notch signaling in TNBC and noncancerous cells. SRI35889 inhibited mTOR signaling less in noncancerous than in cancerous cells, while not affecting STAT3 and Notch pathways. Compounds SRI32529, SRI35357 and SRI35361 were not selectively cytotoxic against TNBC cell lines compared to MCF10A cells. While SRI32529 inhibited Wnt/ß-catenin signaling, the compound also mitigated mTOR, STAT3 and Notch signaling. SRI33576 and SRI35889 were identified as cytotoxic and selective inhibitors of Wnt/ß-catenin signaling with therapeutic potential to treat TNBC in vivo.

Characterization of the Interactions between Calmodulin and Death Receptor 5 in Triple-negative and Estrogen Receptor-positive Breast Cancer Cells: AN INTEGRATED EXPERIMENTAL AND COMPUTATIONAL STUDY.

Activation of death receptor-5 (DR5) leads to the formation of death inducing signaling complex (DISC) for apoptotic signaling. Targeting DR5 to induce breast cancer apoptosis is a promising strategy to circumvent drug resistance and present a target for breast cancer treatment. Calmodulin (CaM) has been shown to regulate DR5-mediated apoptotic signaling, however, its mechanism remains unknown. In this study, we characterized CaM and DR5 interactions in breast cancer cells with integrated experimental and computational approaches. Results show that CaM directly binds to DR5 in a calcium dependent manner in breast cancer cells. The direct interaction of CaM with DR5 is localized at DR5 death domain. We have predicted and verified the CaM-binding site in DR5 being (354)WEPLMRKLGL(363) that is located at the α2 helix and the loop between α2 helix and α3 helix of DR5 DD. The residues of Trp-354, Arg-359, Glu-355, Leu-363, and Glu-367 in DR5 death domain that are important for DR5 recruitment of FADD and caspase-8 for DISC formation to signal apoptosis also play an important role for CaM-DR5 binding. The changed electrostatic potential distribution in the CaM-binding site in DR5 DD by the point mutations of W354A, E355K, R359A, L363N, or E367K in DR5 DD could directly contribute to the experimentally observed decreased CaM-DR5 binding by the point mutations of the key residues in DR5 DD. Results from this study provide a key step for the further investigation of the role of CaM-DR5 binding in DR5-mediated DISC formation for apoptosis in breast cancer cells.

Calmodulin Binding to Death Receptor 5-mediated Death-Inducing Signaling Complex in Breast Cancer Cells.

Activation of death receptor-5 (DR5) leads to the formation of death-inducing signaling complex (DISC) for apoptotic signaling. TRA-8, a DR5 specific agonistic antibody, has demonstrated significant cytotoxic activity in vitro and in vivo without inducing hepatotoxicity. Calmodulin (CaM) that is overexpressed in breast cancer plays a critical role in regulating DR5-mediated apoptosis. However, the mechanism of CaM in regulating DR5-mediated apoptotic signaling remains unknown. In this study, we characterized CaM binding to DR5-mediated DISC for apoptosis in TRA-8 sensitive breast cancer cell lines using co-immunoprecipitation, fluorescence microscopic imaging, caspase signaling analysis, and cell viability assay. Results show that upon DR5 activation, CaM was recruited into DR5-mediated DISC in a calcium dependent manner. CaM antagonist, trifluoperazine (TFP), inhibited CaM recruitment into the DISC and attenuated DISC formation. DR5 oligomerization is critical for DISC formation for apoptosis. TFP decreased TRA-8 activated DR5 oligomerization, which was consistent with TFP's effect on DR5-mediated DISC formation. TFP and Ca2+ chelator, EGTA, impeded TRA-8-activated caspase-dependent apoptotic signaling, and TFP decreased TRA-8-induced cell cytotoxicity. These results demonstrated CaM binding to DR5-mediated DISC in a calcium dependent manner and may identify CaM as a key regulator of DR5-mediated DISC formation for apoptosis in breast cancer. J. Cell. Biochem. 118: 2285-2294, 2017. © 2017 Wiley Periodicals, Inc.

Surveying the serologic proteome in a tissue-specific kras(G12D) knockin mouse model of pancreatic cancer.

We have applied a serologic proteomic workflow involving three complementary MS approaches to a tissue-specific Kras(G12D) -knockin mouse model of pancreatic cancer that consistently forms precancerous lesions by 4 months of age. The three proteomics applications were highly complementary and allowed us to survey the entire range of low to high molecular weight serologic proteins. Combined, we identified 121 (49↓, 72↑) unique and statistically relevant serologic biomarkers with 88% previously reported to be associated with cancer and 38% specifically correlated with pancreatic cancer. Four markers, lysozyme C2, cytokeratin 19, Serpina1A and Pcf11, were further verified by Western blotting. When applying systems analysis, the top-associated gene ontology functions were tied to wound healing, RXR signaling, growth, differentiation and innate immune activation through the JAK/STAT pathway. Upon further investigation of the apparent immune response using a multiplex cytokine screen, we found that IFN-γ, VEGF and GM-CSF were significantly increased in serum from the Kras(G12D) animals compared to littermate controls. By combining three complementary MS applications, we were able to survey the native intact peptidome and the global proteome in parallel, unveiling pathways that may be biologically relevant to promotion of pancreatic cancer progression and serologic markers of noninvasive early-stage neoplasia.

Targeting the Wnt/ß-catenin pathway in primary ovarian cancer with the porcupine inhibitor WNT974.

Preclinical studies in ovarian cancer have demonstrated upregulation of the Wnt/ß-catenin pathway promoting tumor proliferation and chemoresistance. Our objective was to evaluate the effect of the Wnt/ß-catenin pathway inhibitor, WNT974, in primary ovarian cancer ascites cells. Ascites cells from patients with papillary serous ovarian cancer were isolated and treated with 1 µM WNT974±100 µM carboplatin. Viability was evaluated with the ATPlite assay. The IC50 was calculated using a dose-response analysis. Immunohistochemistry (IHC) was performed on ascites cells and tumor. Expression of R-spondin 2 (RSPO2), RSPO3, PORCN, WLS, AXIN2, and three previously characterized RSPO fusion transcripts were assessed using Taqman assays. Sixty ascites samples were analyzed for response to WNT974. The ascites samples that showed a decrease in ATP concentration after treatment demonstrated no difference from the untreated cells in percent viability with trypan blue staining. Flow cytometry demonstrated fewer cells in the G2 phase and more in the G1 and S phases after treatment with WNT974. Combination therapy with WNT974 and carboplatin resulted in a higher percentage of samples that showed ≥30% reduction in ATP concentration than either single drug treatment. IHC analysis of Wnt pathway proteins suggests cell cycle arrest rather than cytotoxicity after WNT974 treatment. QPCR indicated that RSPO fusions are not prevalent in ovarian cancer tissues or ascites. However, higher PORCN expression correlated to sensitivity to WNT974 (P=0.0073). In conclusion, WNT974 produces cytostatic effects in patient ascites cells with primary ovarian cancer through inhibition of the Wnt/ß-catenin pathway. The combination of WNT974 and carboplatin induces cytotoxicity plus cell cycle arrest in a higher percentage of ascites samples than with single drug treatment. RSPO fusions do not contribute to WNT974 sensitivity; however, higher PORCN expression indicates increased WNT974 sensitivity.

Ovarian cancer and the immune system - The role of targeted therapies.

The majority of patients with epithelial ovarian cancer are diagnosed with advanced disease. While many of these patients will respond initially to chemotherapy, the majority will relapse and die of their disease. Targeted therapies that block or activate specific intracellular signaling pathways have been disappointing. In the past 15years, the role of the immune system in ovarian cancer has been investigated. Patients with a more robust immune response, as documented by the presence of lymphocytes infiltrating within their tumor, have increased survival and better response to chemotherapy. In addition, a strong immunosuppressive environment often accompanies ovarian cancer. Recent research has identified potential therapies that leverage the immune system to identify and destroy tumor cells that previously evaded immunosurveillance mechanisms. In this review, we discuss the role of the immune system in ovarian cancer and focus on specific pathways and molecules that show a potential for targeted therapy. We also review the ongoing clinical trials using targeted immunotherapy in ovarian cancer. The role of targeted immunotherapy in patients with ovarian cancer represents a field of growing research and clinical importance.

Ovarian and cervical cancer patient derived xenografts: The past, present, and future.

Preclinical research in gynecologic malignancies has largely relied upon cloned cancer-derived cell lines and tumor xenografts derived from these cell lines. Unfortunately, the use of cell lines for translational research has disadvantages because genetic and phenotypic alterations from serial passaging have resulted in expression profiles that are different from the original patient tumors. The patient-derived xenograft (PDX) model derived from human tumor not previously cultured has shown better representation of the heterogeneity of gynecologic malignancies and the human tumor microenvironment with preservation of cytogenetics, cellular complexity, and vascular and stromal tumor architecture. Studies have shown promise with these models to analyze tumor development and adaptation, test drug efficacy, and predict clinical outcomes. Their ultimate value may be seen with preclinical drug screening including novel targeted therapies, biomarker identification, and the development of individualized treatment plans. This article reviews PDX model development, current studies testing chemotherapeutics and targeted therapies, and limitations of the PDX model in gynecologic malignancies.

Niclosamide Analogs for Treatment of Ovarian Cancer.

OBJECTIVE: Niclosamide has shown activity against ovarian cancer in vitro; however, it has low bioavailability in vivo. Therefore, we investigated the cytotoxicity of niclosamide analogs in combination with carboplatin against ovarian cancer patient ascites cells and tissue slices. MATERIALS/METHODS: Tumorspheres were isolated from ascites collected from patients undergoing ovarian cancer surgery and plated at 10,000 cells per 50 µL into low attachment plates. Tumor slices were also processed at the time of surgery. These were treated concurrently with niclosamide or analogs (0.1-5 µM) and carboplatin (5-150 µM). At 48 hours, cell viability was assessed with ATPlite assay. Western blotting was used to determine expression of Wnt/ß-catenin proteins in ascites cells. RESULTS: Cytotoxicity of niclosamide and its analogs in combination with carboplatin was demonstrated in 24 patient ascites samples. Increased cytotoxicity was seen with 2 analogs in 23 patient ascites samples when compared with niclosamide. Similar cytotoxicity was produced in an ex vivo tumor slice model. Western blot analysis showed decreased expression of Wnt/ß-catenin proteins with niclosamide and analog treatment in a dose-dependent fashion. CONCLUSIONS: The niclosamide-like analogs produced cytotoxicity both alone and in combination with carboplatin against tumorspheres from patient ascites and slices from solid tumor samples. Tumor slices showed similar cytotoxicity to matched ascites samples. Western blots showed down-regulation of Wnt pathway-associated proteins in patient samples treated with niclosamide analogs. These results suggest that more soluble niclosamide analogs may be useful for the treatment of ovarian cancer in combination with chemotherapy.

Recurrent read-through fusion transcripts in breast cancer.

Read-through fusion transcripts that result from the splicing of two adjacent genes in the same coding orientation are a recently discovered type of chimeric RNA. We sought to determine if read-through fusion transcripts exist in breast cancer. We performed paired-end RNA-seq of 168 breast samples, including 28 breast cancer cell lines, 42 triple negative breast cancer primary tumors, 42 estrogen receptor positive (ER+) breast cancer primary tumors, and 56 non-malignant breast tissue samples. We analyzed the sequencing data to identify breast cancer associated read-through fusion transcripts. We discovered two recurrent read-through fusion transcripts that were identified in breast cancer cell lines, confirmed across breast cancer primary tumors, and were not detected in normal tissues (SCNN1A-TNFRSF1A and CTSD-IFITM10). Both fusion transcripts use canonical splice sites to join the last splice donor of the 5' gene to the first splice acceptor of the 3' gene, creating an in-frame fusion transcript. Western blots indicated that the fusion transcripts are translated into fusion proteins in breast cancer cells. Custom small interfering RNAs targeting the CTSD-IFITM10 fusion junction reduced expression of the fusion transcript and reduced breast cancer cell proliferation. Read-through fusion transcripts between adjacent genes with different biochemical functions represent a new type of recurrent molecular defect in breast cancer that warrant further investigation as potential biomarkers and therapeutic targets. Both breast cancer associated fusion transcripts identified in this study involve membrane proteins (SCNN1A-TNFRSF1A and CTSD-IFITM10), which raises the possibility that they could be breast cancer-specific cell surface markers.

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.

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.