Nuclear export signal mutation of epidermal growth factor receptor enhances malignant phenotypes of cancer cells.

Nuclear epidermal growth factor receptor (EGFR) has been shown to be correlated with drug resistance and a poor prognosis in patients with cancer. Previously, we have identified a tripartite nuclear localization signal (NLS) within EGFR. To comprehensively determine the functions and underlying mechanism of nuclear EGFR and its clinical implications, we aimed to explore the nuclear export signal (NES) sequence of EGFR that is responsible for interacting with the exportins. We combined in silico prediction with site-directed mutagenesis approaches and identified a putative NES motif of EGFR, which is located in amino acid residues 736-749. Mutation at leucine 747 (L747) in the EGFR NES led to increased nuclear accumulation of the protein via a less efficient release of the exportin CRM1. Interestingly, L747 with serine (L747S) and with proline (L747P) mutations were found in both tyrosine kinase inhibitor (TKI)-treated and -naïve patients with lung cancer who had acquired or de novo TKI resistance and a poor outcome. Reconstituted expression of the single NES mutant EGFRL747P or EGFRL747S, but not the dual mutant along with the internalization-defective or NLS mutation, in lung cancer cells promoted malignant phenotypes, including cell migration, invasiveness, TKI resistance, and tumor initiation, supporting an oncogenic role of nuclear EGFR. Intriguingly, cells with germline expression of the NES L747 mutant developed into B cell lymphoma. Mechanistically, nuclear EGFR signaling is required for sustaining nuclear activated STAT3, but not for Erk. These findings suggest that EGFR functions are compartmentalized and that nuclear EGFR signaling plays a crucial role in tumor malignant phenotypes, leading to tumorigenesis in human cancer.

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.

Epithelial SOX9 drives progression and metastases of gastric adenocarcinoma by promoting immunosuppressive tumour microenvironment.

OBJECTIVE: Many cancers engage embryonic genes for rapid growth and evading the immune system. SOX9 has been upregulated in many tumours, yet the role of SOX9 in mediating immunosuppressive tumour microenvironment is unclear. Here, we aim to dissect the role of SOX9-mediated cancer stemness attributes and immunosuppressive microenvironment in advanced gastric adenocarcinoma (GAC) for novel therapeutic discoveries. METHODS: Bulk RNAseq/scRNA-seq, patient-derived cells/models and extensive functional studies were used to identify the expression and functions of SOX9 and its target genes in vitro and in vivo. Immune responses were studied in PBMCs or CD45+ immune cells cocultured with tumour cells with SOX9high or knockout and the KP-Luc2 syngeneic models were used for efficacy of combinations. RESULTS: SOX9 is one of the most upregulated SOX genes in GAC and highly expressed in primary and metastatic tissues and associated with poor prognosis. Depletion of SOX9 in patient-derived GAC cells significantly decreased cancer stemness attributes, tumour formation and metastases and consistently increased CD8+ T cell responses when cocultured with PBMCs/CD45+ cells from GAC patients. RNA sequencing identified the leukaemia inhibitory factor (LIF) as the top secreted molecule regulated by SOX9 in tumour cells and was enriched in malignant ascites and mediated SOX9-induced M2 macrophage repolarisation and inhibited T cell function. CONCLUSION: Epithelial SOX9 is critical in suppressing CD8+ T cell responses and modified macrophage function in GAC through the paracrine LIF factor. Cotargeting LIF/LIFR and CSF1R has great potential in targeting SOX9-mediated cancer stemness, T cell immunosuppression and metastases suggesting the novel combination therapy against advanced GAC.

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.

Loss of ARID1A activates mTOR signaling and SOX9 in gastric adenocarcinoma-rationale for targeting ARID1A deficiency.

BACKGROUND: Gastric adenocarcinoma (GAC) is a lethal disease with limited therapeutic options. Genetic alterations in chromatin remodelling gene AT-rich interactive domain 1A (ARID1A) and mTOR pathway activation occur frequently in GAC. Targeting the mechanistic target of rapamycin (mTOR) pathway in unselected patients has failed to show survival benefit. A deeper understanding of GAC might identify a subset that can benefit from mTOR inhibition. METHODS: Genomic alterations in ARID1A were analysed in GAC. Mouse gastric epithelial cells from CK19-Cre-Arid1Afl/fl and wild-type mice were used to determine the activation of oncogenic genes due to loss of Arid1A. Functional studies were performed to determine the significance of loss of ARID1A and the sensitivity of ARID1A-deficient cancer cells to mTOR inhibition in GAC. RESULTS: More than 30% of GAC cases had alterations (mutations or deletions) of ARID1A and ARID1A expression was negatively associated with phosphorylation of S6 and SOX9 in GAC tissues and patient-derived xenografts (PDXs). Activation of mTOR signalling (increased pS6) and SOX9 nuclear expression were strongly increased in Arid1A-/- mouse gastric tissues which could be curtailed by RAD001, an mTOR inhibitor. Knockdown of ARID1A in GAC cell lines increased pS6 and nuclear SOX9 and increased sensitivity to an mTOR inhibitor which was further amplified by its combination with fluorouracil both in vitro and in vivo in PDXs. CONCLUSIONS: The loss of ARID1A activates pS6 and SOX9 in GAC, which can be effectively targeted by an mTOR inhibitor. Therefore, our studies suggest a new therapeutic strategy of clinically targeting the mTOR pathway in patients with GAC with ARID1A deficiency.

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.

Targeting cancer stem cells with a pan-BCL-2 inhibitor in preclinical and clinical settings in patients with gastroesophageal carcinoma.

OBJECTIVE: Gastro-oesophageal cancers (GEC) are resistant to therapy and lead to poor prognosis. The cancer stem cells (CSCs) and antiapoptotic pathways often confer therapy resistance. We sought to elucidate the antitumour action of a BCL-2 inhibitor, AT101 in GEC in vitro, in vivo and in a clinical trial. METHODS: Extensive preclinical studies in vitro and in vivo were carried out to establish the mechanism action of AT101 on targeting CSCs and antiapoptotic proteins. A pilot clinical trial in patients with GEC was completed with AT-101 added to standard chemoradiation. RESULTS: Overexpression of BCL-2 and MCL-1 was noted in gastric cancer tissues (GC). AT-101 induced apoptosis, reduced proliferation and tumour sphere formation in MCL-1/BCL-2 high GC cells. Interestingly, AT101 dramatically downregulated genes (YAP-1/Sox9) that control CSCs in GEC cell lines regardless of BCL-2/MCL-1 expression. Addition of docetaxel to AT-101 amplified its antiproliferation and induced apoptosis effects. In vivo studies confirmed the combination of AT101 and docetaxel demonstrated stronger antitumour activity accompanied with significant decrease of CSCs biomarkers (YAP1/SOX9). In a pilot clinical trial, 13 patients with oesophageal cancer (EC) received AT101 orally concurrently with chemoradiation. We observed dramatic clinical complete responses and encouraging overall survival in these patients. Clinical specimen analyses revealed that AT-101 dramatically reduced the expression of CSCs genes in treated EC specimens indicating antitumour activity of AT101 relies more on its anti-CSCs activity. CONCLUSIONS: Our preclinical and clinical data suggest that AT-101 overcomes resistance by targeting CSCs pathways suggesting a novel mechanism of action of AT101 in patients with GEC.

YAP1 mediates gastric adenocarcinoma peritoneal metastases that are attenuated by YAP1 inhibition.

OBJECTIVE: Peritoneal carcinomatosis (PC; malignant ascites or implants) occurs in approximately 45% of advanced gastric adenocarcinoma (GAC) patients and associated with a poor survival. The molecular events leading to PC are unknown. The yes-associated protein 1 (YAP1) oncogene has emerged in many tumour types, but its clinical significance in PC is unclear. Here, we investigated the role of YAP1 in PC and its potential as a therapeutic target. METHODS: Patient-derived PC cells, patient-derived xenograft (PDX) and patient-derived orthotopic (PDO) models were used to study the function of YAP1 in vitro and in vivo. Immunofluorescence and immunohistochemical staining, RNA sequencing (RNA-Seq) and single-cell RNA-Seq (sc-RNA-Seq) were used to elucidate the expression of YAP1 and PC cell heterogeneity. LentiCRISPR/Cas9 knockout of YAP1 and a YAP1 inhibitor were used to dissect its role in PC metastases. RESULTS: YAP1 was highly upregulated in PC tumour cells, conferred cancer stem cell (CSC) properties and appeared to be a metastatic driver. Dual staining of YAP1/EpCAM and sc-RNA-Seq revealed that PC tumour cells were highly heterogeneous, YAP1high PC cells had CSC-like properties and easily formed PDX/PDO tumours but also formed PC in mice, while genetic knockout YAP1 significantly slowed tumour growth and eliminated PC in PDO model. Additionally, pharmacologic inhibition of YAP1 specifically reduced CSC-like properties and suppressed tumour growth in YAP1high PC cells especially in combination with cytotoxics in vivo PDX model. CONCLUSIONS: YAP1 is essential for PC that is attenuated by YAP1 inhibition. Our data provide a strong rationale to target YAP1 in clinic for GAC patients with PC.

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.

The Long Noncoding RNA CCAT2 Induces Chromosomal Instability Through BOP1-AURKB Signaling.

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic. METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, γ-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients. RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients. CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.

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.

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.

Author Correction: CDK2-mediated site-specific phosphorylation of EZH2 drives and maintains triple-negative breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multiplex profiling of peritoneal metastases from gastric adenocarcinoma identified novel targets and molecular subtypes that predict treatment response.

OBJECTIVE: Peritoneal carcinomatosis (PC) occurs frequently in patients with gastric adenocarcinoma (GAC) and confers a poor prognosis. Multiplex profiling of primary GACs has been insightful but the underpinnings of PC's development/progression remain largely unknown. We characterised exome/transcriptome/immune landscapes of PC cells from patients with GAC aiming to identify novel therapeutic targets. DESIGN: We performed whole-exome sequencing (WES) and whole transcriptome sequencing (RNA-seq) on 44 PC specimens (43 patients with PC) including an integrative analysis of WES, RNA-seq, immune profile, clinical and pathological phenotypes to dissect the molecular pathogenesis, identifying actionable targets and/or biomarkers and comparison with TCGA primary GACs. RESULTS: We identified distinct alterations in PC versus primary GACs, such as more frequent CDH1 and TAF1 mutations, 6q loss and chr19 gain. Alterations associated with aggressive PC phenotypes emerged with increased mutations in TP53, CDH1, TAF1 and KMT2C, higher level of 'clock-like' mutational signature, increase in whole-genome doublings, chromosomal instability (particularly, copy number losses), reprogrammed microenvironment, enriched cell cycle pathways, MYC activation and impaired immune response. Integrated analysis identified two main molecular subtypes: 'mesenchymal-like' and 'epithelial-like' with discriminating response to chemotherapy (31% vs 71%). Patients with the less responsive 'mesenchymal-like' subtype had high expression of immune checkpoint T-Cell Immunoglobulin And Mucin Domain-Containing Protein 3 (TIM-3), its ligand galectin-9, V-domain Ig suppressor of T cell activation (VISTA) and transforming growth factor-ß as potential therapeutic immune targets. CONCLUSIONS: We have uncovered the unique mutational landscape, copy number alteration and gene expression profile of PC cells and defined PC molecular subtypes, which correlated with PC therapy resistance/response. Novel targets and immune checkpoint proteins have been identified with a potential to be translated into clinics.

CDK2-mediated site-specific phosphorylation of EZH2 drives and maintains triple-negative breast cancer.

Triple-negative breast cancer (TNBC), which lacks estrogen receptor α (ERα), progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expression, is closely related to basal-like breast cancer. Previously, we and others report that cyclin E/cyclin-dependent kinase 2 (CDK2) phosphorylates enhancer of zeste homolog 2 (EZH2) at T416 (pT416-EZH2). Here, we show that transgenic expression of phospho-mimicking EZH2 mutant EZH2T416D in mammary glands leads to tumors with TNBC phenotype. Coexpression of EZH2T416D in mammary epithelia of HER2/Neu transgenic mice reprograms HER2-driven luminal tumors into basal-like tumors. Pharmacological inhibition of CDK2 or EZH2 allows re-expression of ERα and converts TNBC to luminal ERα-positive, rendering TNBC cells targetable by tamoxifen. Furthermore, the combination of either CDK2 or EZH2 inhibitor with tamoxifen effectively suppresses tumor growth and markedly improves the survival of the mice bearing TNBC tumors, suggesting that the mechanism-based combination therapy may be an alternative approach to treat TNBC.

LncRNA PVT1 up-regulation is a poor prognosticator and serves as a therapeutic target in esophageal adenocarcinoma.

BACKGROUND: PVT1 has emerged as an oncogene in many tumor types. However, its role in Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) is unknown. The aim of this study was to assess the role of PVT1 in BE/EAC progression and uncover its therapeutic value against EAC. METHODS: PVT1 expression was assessed by qPCR in normal, BE, and EAC tissues and statistical analysis was performed to determine the association of PVT1 expression and EAC (stage, metastases, and survival). PVT1 antisense oligonucleotides (ASOs) were tested for their antitumor activity in vitro and in vivo. RESULTS: PVT1 expression was up-regulated in EACs compared with paired BEs, and normal esophageal tissues. High expression of PVT1 was associated with poor differentiation, lymph node metastases, and shorter survival. Effective knockdown of PVT1 in EAC cells using PVT1 ASOs resulted in decreased cell proliferation, invasion, colony formation, tumor sphere formation, and reduced proportion of ALDH1A1+ cells. Mechanistically, we discovered mutual regulation of PVT1 and YAP1 in EAC cells. Inhibition of PVT1 by PVT1 ASOs suppressed YAP1 expression through increased phosphor-LATS1and phosphor-YAP1 while knockout of YAP1 in EAC cells significantly suppressed PVT1 levels indicating a positive regulation of PVT1 by YAP1. Most importantly, we found that targeting both PVT1 and YAP1 using their specific ASOs led to better antitumor activity in vitro and in vivo. CONCLUSIONS: Our results provide strong evidence that PVT1 confers an aggressive phenotype to EAC and is a poor prognosticator. Combined targeting of PVT1 and YAP1 provided the highest therapeutic index and represents a novel therapeutic strategy.

Author Correction: FGL2 promotes tumor progression in the CNS by suppressing CD103+ dendritic cell differentiation.

The original version of this Article contained errors in the author affiliations. Qingnan Zhao, Xueqing Xia, Longfei Huo and Shulin Li were incorrectly associated with Beijing Institute for Brain Disorders, 100069, Beijing, China.This has now been corrected in both the PDF and HTML versions of the Article.

FGL2 promotes tumor progression in the CNS by suppressing CD103+ dendritic cell differentiation.

Few studies implicate immunoregulatory gene expression in tumor cells in arbitrating brain tumor progression. Here we show that fibrinogen-like protein 2 (FGL2) is highly expressed in glioma stem cells and primary glioblastoma (GBM) cells. FGL2 knockout in tumor cells did not affect tumor-cell proliferation in vitro or tumor progression in immunodeficient mice but completely impaired GBM progression in immune-competent mice. This impairment was reversed in mice with a defect in dendritic cells (DCs) or CD103+ DC differentiation in the brain and in tumor-draining lymph nodes. The presence of FGL2 in tumor cells inhibited granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced CD103+ DC differentiation by suppressing NF-κB, STAT1/5, and p38 activation. These findings are relevant to GBM patients because a low level of FGL2 expression with concurrent high GM-CSF expression is associated with higher CD8B expression and longer survival. These data provide a rationale for therapeutic inhibition of FGL2 in brain tumors.

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.