Galectin-3 expression is prognostic in diffuse type gastric adenocarcinoma, confers aggressive phenotype, and can be targeted by YAP1/BET inhibitors.

BACKGROUND: Overexpression of Galectin-3 (Gal-3), a ß-galactoside binding protein, has been noted in many tumour types but its functional significance and clinical utility in gastric adenocarcinoma (GAC) are not well known. METHODS: We studied 184 GAC patients characterised by histologic grade, sub-phenotypes (diffuse vs intestinal), and ethnicity (Asians vs North Americans). Immunohistochemistry was performed to assess the expression of Gal-3 in human GACs and we correlated it to the clinical outcomes. Cell proliferation, invasion, co-immunoprecipitation and kinase activity assays were done in genetically stable Gal-3 overexpressing GC cell lines and the parental counterparts to delineate the mechanisms of action and activity of inhibitors. RESULTS: Most patients were men, Asian, and had a poorly differentiated GAC. Gal-3 was over-expressed in poorly differentiated (P=0.002) tumours and also in diffuse sub-phenotype (P=0.02). Gal-3 overexpression was associated with shorter overall survival (OS; P=0.026) in all patients. Although, Gal-3 over-expression was not prognostic in the Asian cohort (P=0.337), it was highly prognostic in the North American cohort (P=0.001). In a multivariate analysis, Gal-3 (P=0.001) and N-stage (P=<0.001) were independently prognostic for shorter OS. Mechanistically, Gal-3 induced c-MYC expression through increasing RalA activity and an enhanced YAP1/RalA/RalBP complex to confer an aggressive phenotype. YAP1/BET bromodomain inhibitors reduced Gal-3-mediated aggressive phenotypes in GAC cells. CONCLUSIONS: Gal-3 is an independent prognostic marker of shorter OS and a novel therapeutic target particularly in diffuse type GAC in North American patients.

Nuclear expression of Gli-1 is predictive of pathologic complete response to chemoradiation in trimodality treated oesophageal cancer patients.

BACKGROUND: Predictive biomarkers or signature(s) for oesophageal cancer (OC) patients undergoing preoperative therapy could help administration of effective therapy, avoidance of ineffective ones, and establishment new strategies. Since the hedgehog pathway is often upregulated in OC, we examined its transcriptional factor, Gli-1, which confers therapy resistance, we wanted to assess Gli-1 as a predictive biomarker for chemoradiation response and validate it. METHODS: Untreated OC tissues from patients who underwent chemoradiation and surgery were assessed for nuclear Gli-1 by immunohistochemistry and labelling indices (LIs) were correlated with pathologic complete response (pathCR) or

Galectin-3 Cooperates with CD47 to Suppress Phagocytosis and T-cell Immunity in Gastric Cancer Peritoneal Metastases.

The peritoneal cavity is a common site of gastric adenocarcinoma (GAC) metastasis. Peritoneal carcinomatosis (PC) is resistant to current therapies and confers poor prognosis, highlighting the need to identify new therapeutic targets. CD47 conveys a "don't eat me" signal to myeloid cells upon binding its receptor signal regulatory protein alpha (SIRPα), which helps tumor cells circumvent macrophage phagocytosis and evade innate immune responses. Previous studies demonstrated that the blockade of CD47 alone results in limited clinical benefits, suggesting that other target(s) might need to be inhibited simultaneously with CD47 to elicit a strong antitumor response. Here, we found that CD47 was highly expressed on malignant PC cells, and elevated CD47 was associated with poor prognosis. Galectin-3 (Gal3) expression correlated with CD47 expression, and coexpression of Gal3 and CD47 was significantly associated with diffuse type, poor differentiation, and tumor relapse. Depletion of Gal3 reduced expression of CD47 through inhibition of c-Myc binding to the CD47 promoter. Furthermore, injection of Gal3-deficient tumor cells into either wild-type and Lgals3-/- mice led to a reduction in M2 macrophages and increased T-cell responses compared with Gal3 wild-type tumor cells, indicating that tumor cell-derived Gal3 plays a more important role in GAC progression and phagocytosis than host-derived Gal3. Dual blockade of Gal3 and CD47 collaboratively suppressed tumor growth, increased phagocytosis, repolarized macrophages, and boosted T-cell immune responses. These data uncovered that Gal3 functions together with CD47 to suppress phagocytosis and orchestrate immunosuppression in GAC with PC, which supports exploring a novel combination therapy targeting Gal3 and CD47. SIGNIFICANCE: Dual inhibition of CD47 and Gal3 enhances tumor cell phagocytosis and reprograms macrophages to overcome the immunosuppressive microenvironment and suppress tumor growth in peritoneal metastasis of gastric adenocarcinoma.

A new intronic quantitative PCR method led to the discovery of transformation from human ascites to murine malignancy in a mouse model.

Purpose: To establish a fast and accurate detection method for interspecies contaminations in the patient-derived xenograft (PDX) models and cell lines, and to elucidate possible mechanisms if interspecies oncogenic transformation is detected. Methods: A fast and highly sensitive intronic qPCR method detecting Gapdh intronic genomic copies was developed to quantify if cells were human or murine or a mixture. By this method, we documented that murine stromal cells were abundant in the PDXs; we also authenticated our cell lines to be human or murine. Results: In one mouse model, GA0825-PDX transformed murine stromal cells into a malignant tumorigenic murine P0825 cell line. We traced the timeline of this transformation and discovered three subpopulations descended from the same GA0825-PDX model: epithelium-like human H0825, fibroblast-like murine M0825, and main passaged murine P0825 displayed differences in tumorigenic capability in vivo. P0825 was the most aggressive and H0825 was weakly tumorigenic. Immunofluorescence (IF) staining revealed that P0825 cells highly expressed several oncogenic and cancer stem cell markers. Whole exosome sequencing (WES) analysis revealed that TP53 mutation in the human ascites IP116-generated GA0825-PDX may have played a role in the human-to-murine oncogenic transformation. Conclusion: This intronic qPCR is able to quantify human/mouse genomic copies with high sensitivity and within a time frame of a few hours. We are the first to use intronic genomic qPCR for authentication and quantification of biosamples. Human ascites transformed murine stroma into malignancy in a PDX model.

Evolution of immune and stromal cell states and ecotypes during gastric adenocarcinoma progression.

Understanding tumor microenvironment (TME) reprogramming in gastric adenocarcinoma (GAC) progression may uncover novel therapeutic targets. Here, we performed single-cell profiling of precancerous lesions, localized and metastatic GACs, identifying alterations in TME cell states and compositions as GAC progresses. Abundant IgA+ plasma cells exist in the premalignant microenvironment, whereas immunosuppressive myeloid and stromal subsets dominate late-stage GACs. We identified six TME ecotypes (EC1-6). EC1 is exclusive to blood, while EC4, EC5, and EC2 are highly enriched in uninvolved tissues, premalignant lesions, and metastases, respectively. EC3 and EC6, two distinct ecotypes in primary GACs, associate with histopathological and genomic characteristics, and survival outcomes. Extensive stromal remodeling occurs in GAC progression. High SDC2 expression in cancer-associated fibroblasts (CAFs) is linked to aggressive phenotypes and poor survival, and SDC2 overexpression in CAFs contributes to tumor growth. Our study provides a high-resolution GAC TME atlas and underscores potential targets for further investigation.

GRK3 is a poor prognosticator and serves as a therapeutic target in advanced gastric adenocarcinoma.

BACKGROUND: G protein-coupled receptor (GPCR) is the most targeted protein family by the FDA-approved drugs. GPCR-kinase 3 (GRK3) is critical for GPCR signaling. Our genomic analysis showed that GRK3 expression correlated with poor prognosis of gastric adenocarcinoma (GAC) patients. However, GRK3's functions and clinical utility in GAC progression and metastases are unknown. METHODS: We studied GRK3 expression in normal, primary, and metastatic GAC tissues. We identified a novel GRK3 inhibitor, LD2, through a chemical-library screen. Through genetic and pharmacologic modulations of GRK3, a series of functional and molecular studies were performed in vitro and in vivo. Impact of GRK3 on YAP1 and its targets was determined. RESULTS: GRK3 was overexpressed in GAC tissues compared to normal and was even higher in peritoneal metastases. Overexpression (OE) of GRK3 was significantly associated with shorter survival. Upregulation of GRK3 in GAC cells increased cell invasion, colony formation, and proportion of ALDH1+ cells, while its downregulation reduced these attributes. Further, LD2 potently and specifically inhibited GRK3, but not GRK2, a very similar kinase to GRK3. LD2 highly suppressed GAC cells' malignant phenotypes in vitro. Mechanistically, GRK3 upregulated YAP1 in GAC tissues and its transcriptional downstream targets: SOX9, Birc5, Cyr61 and CTGF. Knockdown (KD) YAP1 rescued the phenotypes of GRK3 OE in GAC cells. GRK3 OE significantly increased tumor growth but LD2 inhibited tumor growth in the PDX model and dramatically suppressed peritoneal metastases induced by GRK3 OE. CONCLUSIONS: GRK3, a poor prognosticator for survival, conferred aggressive phenotype. Genetic silencing of GRK3 or its inhibitor LD2 blunted GRK3-conferred malignant attributes, suggesting GRK3 as a novel therapeutic target in advanced GAC.

Patient-derived cell lines and orthotopic mouse model of peritoneal carcinomatosis recapitulate molecular and phenotypic features of human gastric adenocarcinoma.

BACKGROUND: Gastric adenocarcinoma with peritoneal carcinomatosis (PC) is therapy resistant and leads to poor survival. To study PC in depth, there is an urgent need to develop representative PC-derived cell lines and metastatic models to study molecular mechanisms of PC and for preclinical screening of new therapies. METHODS: PC cell lines were developed from patient-derived PC cells. The tumorigenicity and metastatic potential were investigated by subcutaneously (PDXs) and orthotopically. Karyotyping, whole-exome sequencing, RNA-sequencing, and functional studies were performed to molecularly define the cell lines and compare genomic and phenotypic features of PDX and donor PC cells. RESULTS: We established three PC cell lines (GA0518, GA0804, and GA0825) and characterized them in vitro. The doubling times were 22, 39, and 37 h for GA0518, GA0804, and GA0825, respectively. Expression of cancer stem cell markers (CD44, ALDH1, CD133 and YAP1) and activation of oncogenes varied among the cell lines. All three PC cell lines formed PDXs. Interestingly, all three PC cell lines formed tumors in the patient derived orthotopic (PDO) model and GA0518 cell line consistently produced PC in mice. Moreover, PDXs recapitulated transcriptomic and phenotypic features of the donor PC cells. Finally, these cell lines were suitable for preclinical testing of chemotherapy and target agents in vitro and in vivo. CONCLUSION: We successfully established three patient-derived PC cell lines and an improved PDO model with high incidence of PC associated with malignant ascites. Thus, these cell lines and metastatic PDO model represent excellent resources for exploring metastatic mechanisms of PC in depth and for target drug screening and validation by interrogating GAC for translational studies.

PPARδ Interacts with the Hippo Coactivator YAP1 to Promote SOX9 Expression and Gastric Cancer Progression.

Despite established functions of PPARδ in lipid metabolism and tumorigenesis, the mechanisms underlying its role in gastric cancer are undefined. Here, we demonstrate that SOX9 was dramatically induced by stably expressing PPARδ and by its agonist GW501516 in human gastric cancer cell lines. PPARδ knockdown in patient-derived gastric cancer cells dramatically reduced SOX9 expression and transcriptional activity, with corresponding decreases in invasion and tumor sphere formation. Mechanistically, PPARδ induced SOX9 transcription through direct interaction with and activation of the Hippo coactivator YAP1. PPARδ-YAP1 interaction occurred via the C-terminal domain of YAP1, and both TEAD- and PPARE-binding sites were required for SOX9 induction. Notably, CRISPR/Cas9-mediated genetic ablation of YAP1 or SOX9 abolished PPARδ-mediated oncogenic functions. Finally, expression of PPARδ, YAP1, and SOX9 were significantly correlated with each other and with poor survival in a large cohort of human gastric cancer tissues. Thus, these findings elucidate a novel mechanism by which PPARδ promotes gastric tumorigenesis through interaction with YAP1 and highlights the PPARδ/YAP1/SOX9 axis as a novel therapeutic target in human gastric cancer. IMPLICATIONS: Our discovery of a new model supports a distinct paradigm for PPARδ and a crucial oncogenic function of PPARδ in gastric cancer through convergence on YAP1/TEAD signaling. Therefore, PPARδ/YAP1/SOX9 axis could be a novel therapeutic target that can be translated into clinics.

An improved strategy for CRISPR/Cas9 gene knockout and subsequent wildtype and mutant gene rescue.

A fluorescence marker mOrange was inserted to the popular pLentiCrispr-V2 to create pLentiCrispr-V2-mOrange (V2mO) that contained both a puromycin selection and a fluorescent marker, making viral production and target transduction visible. Lentiviruses packaged with this plasmid and appropriate guide RNAs (gRNAs) successfully knocked out the genes RhoA, Gli1, and Gal3 in human gastric cancer cell lines. Cas9-gRNA editing efficiency could be estimated directly from Sanger electropherograms of short polymerase chain reaction products around the gRNA regions in Cas9-gRNA transduced cells. Single cloning of transduced target cell pools must be performed to establish stable knockout clones. Rescue of wildtype (RhoA and Gal3) and mutant (RhoA.Y42C) genes into knockout cells was successful only when cDNAs, where gRNAs bind, were modified by three nucleotides while the amino acid sequences remained unchanged. Stringent on-target CRISPR/Cas9 editing was observed in Gal3 gene, but not in RhoA gene since RhoA.Y42C already presented a nucleotide change in gRNA5 binding site. In summary, our improved strategy added these advantages: adding visual marker to the popular lentiviral system, monitoring lentiviral production and transduction efficiencies, cell-sorting Cas9+ cells in target cells by fluorescence-activated cell sorting, direct estimation of gene editing efficiency of target cell pools by short PCR electropherograms around gRNA binding sites, and successful rescue of wildtype and mutant genes in knockout cells, overcoming Cas9 editing by modifying cDNAs.

LncRNA PVT1 Is a Poor Prognosticator and Can Be Targeted by PVT1 Antisense Oligos in Gastric Adenocarcinoma.

Gastric adenocarcinoma (GAC) is inherently resistant or becomes resistant to therapy, leading to a poor prognosis. Mounting evidence suggests that lncRNAs can be used as predictive markers and therapeutic targets in the right context. In this study, we determined the role of lncRNA-PVT1 in GAC along with the value of inhibition of PVT1 using antisense oligos (ASOs). RNA scope in situ hybridization was used to analyze PVT1 expression in tumor tissue microarrays (TMAs) of GAC and paired normal tissues from 792 patients. Functional experiments, including colony formation and invasion assays, were performed to evaluate the effects of PVT1 ASO inhibition of PVT1 in vitro; patient-derived xenograft models were used to evaluate the anti-tumor effects of PVT1 ASOs in vivo. LncRNA-PVT1 was upregulated in GACs compared to the matched adjacent normal tissues in the TMA. LncRNA PVT1 expression was positively correlated with larger tumor size, deeper wall invasion, lymph node metastases, and short survival duration. Inhibition of PVT1 using PVT1 ASOs significantly suppressed tumor cell growth and invasion in vitro and in vivo. PVT1 expression was highly associated with poor prognosis in GAC patients and targeting PVT1 using PVT1 ASOs was effective at curtailing tumor cell growth in vitro and in vivo. Thus, PVT1 is a poor prognosticator as well as therapeutic target. Targeting PVT1 using PVT1 ASOs provides a novel therapeutic strategy for GAC.

Targeting Hippo coactivator YAP1 through BET bromodomain inhibition in esophageal adenocarcinoma.

Hippo/YAP1 signaling is a major regulator of organ size, cancer stemness, and aggressive phenotype. Thus, targeting YAP1 may provide a novel therapeutic strategy for tumors with high YAP1 expression in esophageal cancer (EC). Chromatin immunoprecipitation (ChiP) and quantitative ChiP-PCR were used to determine the regulation of the chromatin remodeling protein bromodomain-containing protein 4 (BRD4) on YAP1. The role of the bromodomain and extraterminal motif (BET) inhibitor JQ1, an established BRD4 inhibitor, on inhibition of YAP1 in EC cells was dissected using western blot, immunofluorescence, qPCR, and transient transfection. The antitumor activities of BET inhibitor were further examined by variety of functional assays, cell proliferation (MTS), tumorsphere, and ALDH1+ labeling in vitro and in vivo. Here, we show that BRD4 regulates YAP1 expression and transcription. ChiP assays revealed that BRD4 directly occupies YAP1 promoter and that JQ1 robustly blocks BRD4 binding to the YAP1 promoter. Consequently, JQ1 strongly suppresses constitutive or induced YAP1 expression and transcription in EC cells and YAP1/Tead downstream transcriptional activity. Intriguingly, radiation-resistant cells that acquire strong cancer stem cell traits and an aggressive phenotype can be effectively suppressed by JQ1 as assessed by cell proliferation, tumorsphere formation, and reduction in the ALDH1+ cells. Moreover, effects of JQ1 are synergistically amplified by the addition of docetaxel in vitro and in vivo. Our results demonstrate that BRD4 is a critical regulator of Hippo/YAP1 signaling and that BRD4 inhibitor JQ1 represents a new class of inhibitor of Hippo/YAP1 signaling, primarily targeting YAP1 high and therapy-resistant cancer cells enriched with cancer stem cell properties.

YAP1-Mediated CDK6 Activation Confers Radiation Resistance in Esophageal Cancer - Rationale for the Combination of YAP1 and CDK4/6 Inhibitors in Esophageal Cancer.

PURPOSE: Esophageal cancer is a lethal disease that is often resistant to therapy. Alterations of YAP1 and CDK6 are frequent in esophageal cancer. Deregulation of both molecules may be responsible for therapy resistance. EXPERIMENTAL DESIGN: Expressions of YAP1 and CDK6 were examined in esophageal cancer cells and tissues using immunoblotting and immunohistochemistry. YAP1 expression was induced in esophageal cancer cells to examine YAP1-mediated CDK6 activation and its association with radiation resistance. Pharmacologic and genetic inhibitions of YAP1 and CDK6 were performed to dissect the mechanisms and assess the antitumor effects in vitro and in vivo. RESULTS: YAP1 expression was positively associated with CDK6 expression in resistant esophageal cancer tissues and cell lines. YAP1 overexpression upregulated CDK6 expression and transcription, and promoted radiation resistance, whereas treatment with the YAP1 inhibitor, CA3, strongly suppressed YAP1 and CDK6 overexpression, reduced Rb phosphorylation, as well as sensitized radiation-resistant/YAP1high esophageal cancer cells to irradiation. CDK4/6 inhibitor, LEE011, and knock down of CDK6 dramatically inhibited expression of YAP1 and sensitized resistant esophageal cancer cells to irradiation indicating a positive feed-forward regulation of YAP1 by CDK6. In addition, suppression of both the YAP1 and CDK6 pathways by the combination of CA3 and LEE011 significantly reduced esophageal cancer cell growth and cancer stem cell population (ALDH1 + and CD133 + ), sensitized cells to irradiation, and showed a strong antitumor effect in vivo against radiation-resistant esophageal cancer cells. CONCLUSIONS: Our results document that a positive crosstalk between the YAP1 and CDK6 pathways plays an important role in conferring radiation resistance to esophageal cancer cells. Targeting both YAP1 and CDK6 pathways could be a novel therapeutic strategy to overcome resistance in esophageal cancer.

A Novel YAP1 Inhibitor Targets CSC-Enriched Radiation-Resistant Cells and Exerts Strong Antitumor Activity in Esophageal Adenocarcinoma.

Mounting evidence suggests that the Hippo coactivator Yes-associated protein 1 (YAP1) is a major mediator of cancer stem cell (CSC) properties, tumor progression, and therapy resistance as well as often a terminal node of many oncogenic pathways. Thus, targeting YAP1 may be a novel therapeutic strategy for many types of tumors with high YAP1 expression, including esophageal adenocarcinoma. However, effective YAP1 inhibitors are currently lacking. Here, we identify a small molecule (CA3) that not only has remarkable inhibitory activity on YAP1/Tead transcriptional activity but also demonstrates strong inhibitory effects on esophageal adenocarcinoma cell growth especially on YAP1 high-expressing esophageal adenocarcinoma cells both in vitro and in vivo Remarkably, radiation-resistant cells acquire strong cancer stem cell (CSC) properties and aggressive phenotype, while CA3 can effectively suppress these phenotypes by inhibiting proliferation, inducing apoptosis, reducing tumor sphere formation, and reducing the fraction of ALDH1+ cells. Furthermore, CA3, combined with 5-FU, synergistically inhibits esophageal adenocarcinoma cell growth especially in YAP1 high esophageal adenocarcinoma cells. Taken together, these findings demonstrated that CA3 represents a new inhibitor of YAP1 and primarily targets YAP1 high and therapy-resistant esophageal adenocarcinoma cells endowed with CSC properties. Mol Cancer Ther; 17(2); 443-54. ©2017 AACR.

The Hippo Coactivator YAP1 Mediates EGFR Overexpression and Confers Chemoresistance in Esophageal Cancer.

PURPOSE: Esophageal cancer is an aggressive malignancy and often resistant to therapy. Overexpression of EGFR has been associated with poor prognosis of patients with esophageal cancer. However, clinical trials using EGFR inhibitors have not provided benefit for patients with esophageal cancer. Failure of EGFR inhibition may be due to crosstalk with other oncogenic pathways. EXPERIMENTAL DESIGN: In this study, expression of YAP1 and EGFR were examined in EAC-resistant tumor tissues versus sensitive tissues by IHC. Western blot analysis, immunofluorescence, real-time PCR, promoter analysis, site-directed mutagenesis, and in vitro and in vivo functional assays were performed to elucidate the YAP1-mediated EGFR expression and transcription and the relationship with chemoresistance in esophageal cancer. RESULTS: We demonstrate that Hippo pathway coactivator YAP1 can induce EGFR expression and transcription in multiple cell systems. Both YAP1 and EGFR are overexpressed in resistant esophageal cancer tissues compared with sensitive esophageal cancer tissues. Furthermore, we found that YAP1 increases EGFR expression at the level of transcription requiring an intact TEAD-binding site in the EGFR promoter. Most importantly, exogenous induction of YAP1 induces resistance to 5-fluorouracil and docetaxcel, whereas knockdown of YAP1 sensitizes esophageal cancer cells to these cytotoxics. Verteporfin, a YAP1 inhibitor, effectively inhibits both YAP1 and EGFR expression and sensitizes cells to cytotoxics. CONCLUSIONS: Our data provide evidence that YAP1 upregulation of EGFR plays an important role in conferring therapy resistance in esophageal cancer cells. Targeting YAP1-EGFR axis may be more efficacious than targeting EGFR alone in esophageal cancer.

Metformin sensitizes chemotherapy by targeting cancer stem cells and the mTOR pathway in esophageal cancer.

Our clinical study indicates esophageal adenocarcinoma patients on metformin had a better treatment response than those without metformin. However, the effects of metformin and the mechanisms of its action in esophageal cancer (EC) are unclear. EC cell lines were used to assess the effects of metformin alone or in combination with 5-fluorouracil on survival and apoptosis. RPPA proteomic array and immunoblots were used to identify signaling affected by metformin. Standard descriptive statistical methods were used. Reduction in cell survival and induction of apoptosis by metformin were observed in several EC cell lines. The use of metformin in combination with 5-FU significantly sensitized EC cells to the cytotoxic effect of 5-FU. RPPA array demonstrated that metformin decreased various oncogenes including PI3K/mTORsignaling and survival/cancer stem cell-related genes in cells treated with metformin compared with its control. Immunoblots and transcriptional analyses further confirm that metformin downregulated these CSC-related genes and the components of the mTOR pathway in a dose­dependent manner. Sorted ALDH-1+ cell tumor sphere forming capacity was preferentially reduced by metformin. Finally, metformin reduced tumor growth in vivo and when combined with FU, there was synergistic reduction in tumor growth. Metformin inhibits EC cell growth and sensitizes EC cells to 5-FU cytotoxic effects by targeting CSCs and the components of mTOR. The present study supports our previous clinical observations that the use of metformin is beneficial to EC patients. Metformin can complement other therapeutic combinations to effectively treat EC patients.

Hippo coactivator YAP1 upregulates SOX9 and endows esophageal cancer cells with stem-like properties.

Cancer stem cells (CSC) are purported to initiate and maintain tumor growth. Deregulation of normal stem cell signaling may lead to the generation of CSCs; however, the molecular determinants of this process remain poorly understood. Here we show that the transcriptional coactivator YAP1 is a major determinant of CSC properties in nontransformed cells and in esophageal cancer cells by direct upregulation of SOX9. YAP1 regulates the transcription of SOX9 through a conserved TEAD binding site in the SOX9 promoter. Expression of exogenous YAP1 in vitro or inhibition of its upstream negative regulators in vivo results in elevated SOX9 expression accompanied by the acquisition of CSC properties. Conversely, shRNA-mediated knockdown of YAP1 or SOX9 in transformed cells attenuates CSC phenotypes in vitro and tumorigenicity in vivo. The small-molecule inhibitor of YAP1, verteporfin, significantly blocks CSC properties in cells with high YAP1 and a high proportion of ALDH1(+). Our findings identify YAP1-driven SOX9 expression as a critical event in the acquisition of CSC properties, suggesting that YAP1 inhibition may offer an effective means of therapeutically targeting the CSC population.

EGFR-targeted hybrid plasmonic magnetic nanoparticles synergistically induce autophagy and apoptosis in non-small cell lung cancer cells.

BACKGROUND: The epidermal growth factor receptor (EGFR) is overexpressed in 80% of non-small cell lung cancer (NSCLC) and is associated with poor survival. In recent years, EGFR-targeted inhibitors have been tested in the clinic for NSCLC. Despite the emergence of novel therapeutics and their application in cancer therapy, the overall survival rate of lung cancer patients remains 15%. To develop more effective therapies for lung cancer we have combined the anti-EGFR antibody (Clone 225) as a molecular therapeutic with hybrid plasmonic magnetic nanoparticles (NP) and tested on non-small cell lung cancer (NSCLC) cells. METHODOLOGY/PRINCIPAL FINDINGS: Cell viability was determined by trypan-blue assay. Cellular protein expression was determined by Western blotting. C225-NPs were detected by electron microscopy and confocal microscopy, and EGFR expression using immunocytochemistry. C225-NP exhibited a strong and selective antitumor effect on EGFR-expressing NSCLC cells by inhibiting EGFR-mediated signal transduction and induced autophagy and apoptosis in tumor cells. Optical images showed specificity of interactions between C225-NP and EGFR-expressing NSCLC cells. No binding of C225-NP was observed for EGFR-null NSCLC cells. C225-NP exhibited higher efficiency in induction of cell killing in comparison with the same amount of free C225 antibody in tumor cells with different levels of EGFR expression. Furthermore, in contrast to C225-NP, free C225 antibody did not induce autophagy in cells. However, the therapeutic efficacy of C225-NP gradually approached the level of free antibodies as the amount of C225 antibody conjugated per nanoparticle was decreased. Finally, attaching C225 to NP was important for producing the enhanced tumor cell killing as addition of mixture of free C225 and NP did not demonstrate the same degree of cell killing activity. CONCLUSIONS/SIGNIFICANCE: We demonstrated for the first time the molecular mechanism of C225-NP induced cytotoxic effects in lung cancer cells that are not characteristic for free molecular therapeutics thus increasing efficacy of therapy against NSCLC.