Genomic Landscape of NSCLC in the Republic of Ireland.

Introduction: The identification of genomic "targets" through next-generation sequencing (NGS) of patient's NSCLC tumors has resulted in a rapid expansion of targeted treatment options for selected patients. This retrospective study aims to identify the proportion of patients with advanced NSCLC in the Republic of Ireland whose tumors harbor actionable genomic alterations through broad NGS panel testing. Methods: Institutional review board approval was obtained before study initiation. Patients with NSCLC whose tumors underwent genomic testing through the largest available NGS panel at a nationally funded Cancer Molecular Diagnostics laboratory (St. James's Hospital) between June 2017 and June 2022 were identified. Patient demographics and tumor-related data were collected by retrospective review from all cancer centers in Ireland, referring to the Cancer Molecular Diagnostics laboratory. A total of 203 (9%) tumor samples were excluded due to insufficient neoplastic cell content. Genomic data were collected through retrospective search of Ion Reporter software. The spectrum and proportion of patients with oncogenic driver mutations were evaluated using descriptive statistics (SPSS version 29.0). Results: In total, 2052 patients were identified. Patients were referred from 23 different hospital sites and all four geographic regions (Leinster = 1091, 53%; Munster = 763, 37.2%; Connacht = 191, 9.3%; Ulster = 7, 0.3%). Median age was 69 (range: 26-94) years; 53% were male. The most common tumor histologic subtype was adenocarcinoma (77%, n = 1577). An actionable genomic alteration was identified in 1099 cases (53%), the most common of which was KRAS (n = 657, 32%). Less frequently, NSCLC tumors harbored the following: MET exon 14 skipping (n = 53, 2.6%), MET amplification (n = 26, 1.3%), EGFR (n = 181, 8.8%), HER2 (n = 35, 1.7%), and BRAF (n = 72, 3.5%) mutations. Fusions were detected in 76 patients (3.7%) including ALK (n = 44, 58%), RET (n = 11, 14.5%), ROS1 (n = 16, 21%), and FGFR3 (n = 5, 6.6%), whereas no NTRK fusion was identified. Co-alterations were detected in 114 patients (5.6%), the most common of which was KRAS/PIK3CA (n = 19, 17%), EGFR/PIK3CA (n = 10, 8.5%), and KRAS/IDH1 (n = 9, 8%). Other co-alterations of interest identified included KRAS G12A/ROS1 fusion (n = 1) and KRAS G12C/BRAF G469A (n = 2). Conclusions: This is the first retrospective study to comprehensively characterize the genomic landscape of NSCLC in Ireland, using the broadest available NGS panel. Actionable alterations were identified in 53.4% of the patients, and KRAS was the most common oncogenic driver alteration. Our study revealed a lower prevalence of patients whose tumor harbors ALK, ROS1, and RET fusions, compared with similar data sets.

Developing an In Vitro Isogenic Model of Chemotherapy-Resistant Lung Cancer.

Despite improvements in therapies available for small subsets of patients affected by non-small cell lung cancer (NSCLC), the chemotherapy drug cisplatin is still one of the most commonly used treatments for advanced NSCLC patients in the absence of oncogenic driver mutations or immune checkpoints. Unfortunately, as in the case of many solid tumors, acquired drug resistance is a common phenomenon in NSCLC and presents a significant clinical challenge for oncologists. In order to study and elucidate the cellular and molecular mechanisms implicated in the development of drug resistance in cancer, the use of isogenic models provides a valuable in vitro tool for investigating novel biomarkers and the identification of potential targetable pathways involved in drug-resistant cancers.

Investigating the susceptibility of treatment-resistant oesophageal tumours to natural killer cell-mediated responses.

The majority of oesophageal adenocarcinoma (OAC) patients do not respond to multimodal treatment regimens and face dismal survival rates. Natural killer (NK) cells are crucial anti-tumour immune cells, and this study investigated the susceptibility of treatment-resistant OAC cells to these potent tumour killers. Natural killer receptor (NKR) ligand expression by OE33CisP (cisplatin-sensitive) and OE33CisR (cisplatin-resistant) cells was investigated. The immunomodulatory effects of OE33CisP and OE33CisR cells on NK cell phenotype and function were assessed. Finally, the impact of chemotherapy regimens on NKR ligand shedding was examined. Our data revealed significantly less surface expression of activating ligands B7-H6, MICA/B, ULBP-3 and activating/inhibitory ligands PVRL-1 and PVRL-4 by OE33CisR cells, compared to OE33CisP cells. Co-culture with OE33CisR cells reduced the frequencies of NKp30+ and NKp46+ NK cells and increased frequencies of TIGIT+, FasL+ and TRAIL+ NK cells. Frequencies of IFN-γ-producing NK cells increased while frequencies of TIM-3+ NK cells decreased after culture with OE33CisP and OE33CisR cells. Frequencies of circulating NKp30+ NK cells were significantly lower in OAC patients with the poorest treatment response and in patients who received FLOT chemotherapy, while B7-H6 shedding by OAC tumour cells was induced by FLOT. Overall, OE33CisR cells express less activating NKR ligands than OE33CisP cells and have differential effects on NKR expression by NK cells. However, neither cell line significantly dampened NK cell cytokine production, death receptor expression or degranulation. In addition, our data indicate that FLOT chemotherapy may promote B7-H6 shedding and immune evasion with detrimental consequences in OAC patients.

Prognostic value and immune regulatory role of dynamin 1-like in lung adenocarcinoma.

Background: Lung adenocarcinoma (LUAD) is the most common histological subtype of non-small cell lung cancer (NSCLC), with poor treatment outcomes worldwide. Dynamin-related protein 1 (DRP1), which is encoded by the dynamin 1-like (DNM1L) gene, acts as a regulator of mitochondrial fission and plays crucial roles in tumor initiation and progression. However, the clinical value and immune regulation of DNM1L in LUAD have not been explored. Methods: We comprehensively analyzed the expression of DNM1L in the LUAD cohort of the Human Protein Atlas (HPA) and the University of The ALabama at Birmingham CANcer data analysis Portal (UALCAN) databases. Kaplan-Meier plotter, in addition to the PrognoScan database, was used to estimate the correlation between DNM1L expression and survival outcome of LUAD patients. The association between the immune tumor microenvironment (TME) and DNM1L expression in LUAD was evaluated based on the Tumor IMmune Estimation Resource (TIMER)2.0 database. Finally, the functions of DNM1L were validated in vitro experiments, including reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot, wound healing assays, and transwell assays. Results: DNM1L was overexpressed in LUAD compared to healthy control tissues and was regarded as an independent prognostic factor. Overexpression of DNM1L was significantly related to clinical variables and poor survival outcomes of LUAD patients. Moreover, DNM1L expression was positively associated with the expression of key genes involved in the regulation of immune cell subsets, including T helper (Th)2 cells, Th cells, B cells, CD8 T cells, dendritic cells, and mast cells. In contrast, DNM1L was negatively correlated with the infiltrating levels of myeloid dendritic cells and B cells. Furthermore, DNM1L may play a role in regulating immune cell infiltration and have prognostic value in LUAD patients. Finally, the in vitro experiments showed that increased DNM1L significantly promoted the proliferation and migration of LUAD cells. Conclusions: This study suggested that DNM1L may play an important role in regulating the proliferation and migration of LUAD cells as well as the infiltration of tumor-related immune cells, which suggests DNM1L was a potential therapeutic target in LUAD. Further studies are however warranted to define its exact mechanism of action and potential therapeutic significance in LUAD patients.

Liquid Biopsy: A Multi-Parametric Analysis of Mutation Status, Circulating Tumor Cells and Inflammatory Markers in EGFR-Mutated NSCLC.

The liquid biopsy has the potential to improve patient care in the diagnostic and therapeutic setting in non-small cell lung cancer (NSCLC). Consented patients with epidermal growth factor receptor (EGFR) positive disease (n = 21) were stratified into two cohorts: those currently receiving EGFR tyrosine kinase inhibitor (TKI) therapy (n = 9) and newly diagnosed EGFR TKI treatment-naïve patients (n = 12). Plasma genotyping of cell-free DNA was carried out using the FDA-approved cobas® EGFR mutation test v2 and compared to next generation sequencing (NGS) cfDNA panels. Circulating tumor cell (CTC) numbers were correlated with treatment response and EGFR exon 20 p.T790M. The prognostic significance of the neutrophil to lymphocyte ratio (NLR) and lactate dehydrogenase (LDH) was also investigated. Patients in cohort 1 with an EGFR exon 20 p.T790M mutation progressed more rapidly than those with an EGFR sensitizing mutation, while patients in cohort 2 had a significantly longer progression-free survival (p = 0.04). EGFR exon 20 p.T790M was detected by liquid biopsy prior to disease progression indicated by computed tomography (CT) imaging. The cobas® EGFR mutation test detected a significantly greater number of exon 20 p.T790M mutations (p = 0.05). High NLR and derived neutrophil to lymphocyte ratio (dNLR) were associated with shorter time to progression and worse survival outcomes (p < 0.05). High LDH levels were significantly associated with shorter time to disease progression (p = 0.03). These data support the use of liquid biopsy for monitoring EGFR mutations and inflammatory markers as prognostic indicators in NSCLC.

CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma.

Background: Circular ribonucleic acids (circRNAs) play a key role in the development of different types of cancer. Ferroptosis is a type of programmed cell death that contributes to cancer progression. However, the role of circRNAs in lung adenocarcinoma (LUAD) ferroptosis remains unclear. Methods: The gene expression levels of circRNA P4HB (circP4HB), microRNA-1184 (miR-1184) and Solute carrier family 7 member 11 (Slc7a11), also known as Xct were detected using quantitative real-time polymerase chain reaction (qRT-PCR). Ferroptosis of established LUAD cells was induced by erastin. Cell viability was examined via Cell Counting Kit 8 assays. Ferroptosis was evaluated by malondialdehyde (MDA), Prostaglandin-endoperoxide Synthase 2 (Ptgs2), lipid reactive oxygen species (lipid ROS), and JC-1 detection. The mechanism of circP4HB/miR-1184/SLC7A11 was investigated by luciferase reporter assays, RNA immunoprecipitation, RNA pull-down, and western blot assays. A functional for circP4HB in vivo was determined using xenograft nude mice models. Results: CircP4HB expression levels were increased in LUAD. It triggered glutathione (GSH) synthesis and, therefore protected LUAD cells from ferroptosis induced by erastin. CircP4HB may function as a competing endogenous RNA by modulating miR-1184 to regulate SLC7A11. CircP4HB inhibited ferroptosis by regulating miR-1184/ SLC7A11-mediated GSH synthesis. In vivo, overexpression of circP4HB promoted tumor growth and inhibited ferroptosis. Conclusions: The circRNA, circP4HB acts as a novel ferroptosis suppressor in LUAD. Furthermore, circP4HB protects LUAD from ferroptosis via modulation of the miR-1184/SLC7A11 axis. Our findings identified circP4HB as a novel biomarker in LUAD and warrants further investigation in the early diagnosis and treatment of LUAD.

Cannabidiol Inhibits Tumorigenesis in Cisplatin-Resistant Non-Small Cell Lung Cancer via TRPV2.

Chemotherapy forms the backbone of current treatments for many patients with advanced non-small-cell lung cancer (NSCLC). However, the survival rate is low in these patients due to the development of drug resistance, including cisplatin resistance. In this study, we developed a novel strategy to combat the growth of cisplatin-resistant (CR) NSCLC cells. We have shown that treatment with the plant-derived, non-psychotropic small molecular weight molecule, cannabidiol (CBD), significantly induced apoptosis of CR NSCLC cells. In addition, CBD treatment significantly reduced tumor progression and metastasis in a mouse xenograft model and suppressed cancer stem cell properties. Further mechanistic studies demonstrated the ability of CBD to inhibit the growth of CR cell lines by reducing NRF-2 and enhancing the generation of reactive oxygen species (ROS). Moreover, we show that CBD acts through Transient Receptor Potential Vanilloid-2 (TRPV2) to induce apoptosis, where TRPV2 is expressed on human lung adenocarcinoma tumors. High expression of TRPV2 correlates with better overall survival of lung cancer patients. Our findings identify CBD as a novel therapeutic agent targeting TRPV2 to inhibit the growth and metastasis of this aggressive cisplatin-resistant phenotype in NSCLC.

Harnessing Natural Killer Cells in Non-Small Cell Lung Cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide. There are two main subtypes: small cell lung cancer (SCLC), and non-small cell lung cancer (NSCLC). NSCLC accounts for 85% of lung cancer diagnoses. Early lung cancer very often has no specific symptoms, and many patients present with late stage disease. Despite the various treatments currently available, many patients experience tumor relapse or develop therapeutic resistance, highlighting the need for more effective therapies. The development of immunotherapies has revolutionized the cancer treatment landscape by enhancing the body's own immune system to fight cancer. Natural killer (NK) cells are crucial anti-tumor immune cells, and their exclusion from the tumor microenvironment is associated with poorer survival. It is well established that NK cell frequencies and functions are impaired in NSCLC; thus, placing NK cell-based immunotherapies as a desirable therapeutic concept for this malignancy. Immunotherapies such as checkpoint inhibitors are transforming outcomes for NSCLC. This review explores the current treatment landscape for NSCLC, the role of NK cells and their dysfunction in the cancer setting, the advancement of NK cell therapies, and their future utility in NSCLC.

MicroRNA expression profiling and biomarker validation in treatment-naïve and drug resistant non-small cell lung cancer.

BACKGROUND: In the absence of targetable mutations or immune checkpoints, cisplatin-doublet chemotherapy remains the standard of care in non-small cell lung cancer (NSCLC). Drug resistance has however become a significant clinical challenge. Exploring a role for small non-coding microRNAs (miRNA) as biomarker candidates in cisplatin resistant (CisR) lung cancer is lacking and warrants further investigation. METHODS: miRNA expression profiling was assessed in a panel of cisplatin sensitive and resistant NSCLC cell lines and validated by qPCR. Modulation of altered miRNAs was studied using antagomiRs and pre-miRs while functional assays were used to assess cisplatin response. The translational relevance of these miRNAs as potential biomarkers was assessed in serum and matched normal and tumour lung tissues from chemo-naïve NSCLC patients, in addition to xenograft formalin-fixed paraffin-embedded (FFPE) tumours derived from cisplatin sensitive and resistant cell lines. RESULTS: Differential expression of a 5-miR signature (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286) demonstrated their ability to distinguish between normal and tumour lung tissue and between NSCLC histologies. In squamous cell carcinoma (SqCC), tissue miRNA expression was associated with poor survival. miR-4286 showed promise as a blood-based diagnostic biomarker that could distinguish between adenocarcinoma and SqCC histologies. In a xenograft model of cisplatin resistance, using 7-9 week old female NOD/SCID mice (NOD.CB17-Prkdcscid/NCrCrl), a 5-miRNA panel showed altered expression between sensitive and resistant tumours. CONCLUSIONS: This study identified a panel of miRNAs which may have diagnostic and prognostic potential as novel biomarkers in lung cancer and furthermore, may have a predictive role in monitoring the emergence of resistance to cisplatin.

Exploitation of the vitamin A/retinoic acid axis depletes ALDH1-positive cancer stem cells and re-sensitises resistant non-small cell lung cancer cells to cisplatin.

Despite advances in personalised medicine and the emerging role of immune checkpoints in directing treatment decisions in subsets of lung cancer patients, non-small cell lung cancer (NSCLC) remains the most common cause of cancer-related deaths worldwide. The development of drug resistance plays a key role in the relapse of lung cancer patients in the clinical setting, mainly due to the unlimited renewal capacity of residual cancer stem cells (CSCs) within the tumour cell population during chemotherapy. In this study, we investigated the function of the CSC marker, aldehyde dehydrogenase (ALDH1) in retinoic acid cell signalling using an in vitro model of cisplatin resistant NSCLC. The addition of key components in retinoic acid cell signalling, all-trans retinoic acid (ATRA) and retinol to cisplatin chemotherapy, significantly reduced ALDH1-positive cell subsets in cisplatin resistant NSCLC cells relative to their sensitive counterparts resulting in the re-sensitisation of chemo-resistant cells to the cytotoxic effects of cisplatin. Furthermore, combination of ATRA or retinol with cisplatin significantly inhibited cell proliferation, colony formation and increased cisplatin-induced apoptosis. This increase in apoptosis may, at least in part, be due to differential gene expression of the retinoic acid (RARα/ß) and retinoid X (RXRα) nuclear receptors in cisplatin-resistant lung cancer cells. These data support the concept of exploiting the retinoic acid signalling cascade as a novel strategy in targeting subsets of CSCs in cisplatin resistant lung tumours.

The DNA damage repair-related gene PKMYT1 is a potential biomarker in various malignancies.

BACKGROUND: Protein kinase membrane associated tyrosine/threonine 1 (PKMYT1) regulates cell cycle and is a part of DNA damage repair (DDR)-related signaling. Recent studies have identified a role for PKMYT1 in tumor immunity and DDR. Thus, we initiated this study aiming to characterize the molecular and immunological portrait of PKMYT1 in cancer. METHODS: Transcriptomic data extrapolated from Genotype-Tissue Expression (GTEx), The Cancer Genome Atlas (TCGA), and Cancer Cell Line Encyclopedia (CCLE) datasets were used to determine the mRNA expression levels of PKMYT1. PKMYT1 mRNA expression status was correlated with patients' prognosis as well as immune neoantigens, and immune checkpoints in 34 different tumors. The Tumor Immune Estimation Resource (TIMER) dataset was used to analyze immune infiltrating scores. RESULTS: PKMYT1 mRNA is differentially expressed in common tumors and high expression levels of PKMYT1 mRNA is associated with poor prognosis except for malignant thymoma (THYM). In addition, PKMYT1 mRNA expression was correlated with tumor-infiltrating immune cells particularly in lung squamous cell carcinoma, esophageal carcinoma, THYM, and lung adenocarcinoma. An upregulation of immune checkpoints and neoantigens was observed in tumors with a high PKMYT1 mRNA expression. Data from gene set enrichment analysis (GSEA) revealed that PKMYT1 is involved in tumor immunogenicity, metabolism, and cell cycle progression. CONCLUSIONS: PKMYT1 is differentially expressed in various cancers and exerts an important effect on tumor immunity and progression. The PKMYT1 gene holds the potential as a new potential biomarker. Therefore, further studies are clearly needed to elaborate our findings.

Thyroid receptor-interacting protein 13 and EGFR form a feedforward loop promoting glioblastoma growth.

Epidermal growth factor receptor (EGFR) amplification and EGFRvIII mutation drive glioblastoma (GBM) pathogenesis, but their regulation remains elusive. Here we characterized the EGFR/EGFRvIII "interactome" in GBM and identified thyroid receptor-interacting protein 13 (TRIP13), an AAA + ATPase, as an EGFR/EGFRvIII-associated protein independent of its ATPase activity. Functionally, TRIP13 augmented EGFR pathway activation and contributed to EGFR/EGFRvIII-driven GBM growth in GBM spheroids and orthotopic GBM xenograft models. Mechanistically, TRIP13 enhanced EGFR protein abundance in part by preventing Cbl-mediated ubiquitination and proteasomal degradation. Reciprocally, TRIP13 was phosphorylated at tyrosine(Y) 56 by EGFRvIII and EGF-activated EGFR. Abrogating TRIP13 Y56 phosphorylation dramatically attenuated TRIP13 expression-enhanced EGFR signaling and GBM cell growth. Clinically, TRIP13 expression was upregulated in GBM specimens and associated with poor patient outcome. In GBM, TRIP13 localized to cell membrane and cytoplasma and exhibited oncogenic effects in vitro and in vivo, depending on EGFR signaling but not the TRIP13 ATPase activity. Collectively, our findings uncover that TRIP13 and EGFR form a feedforward loop to potentiate EGFR signaling in GBM growth and identify a previously unrecognized ATPase activity-independent mode of action of TRIP13 in GBM biology.

Prostate cancer-derived holoclones: a novel and effective model for evaluating cancer stemness.

Prostate cancer accounts for approximately 13.5% of all newly diagnosed male cancer cases. Significant clinical burdens remain in terms of ineffective prognostication, with overtreatment of insignificant disease. Additionally, the pathobiology underlying disease heterogeneity remains poorly understood. As the role of cancer stem cells in the perpetuation of aggressive carcinoma is being substantiated by experimental evidence, it is crucially important to understand the molecular mechanisms, which regulate key features of cancer stem cells. We investigated two methods for in vitro cultivation of putative prostate cancer stem cells based on 'high-salt agar' and 'monoclonal cultivation'. Data demonstrated 'monoclonal cultivation' as the superior method. We demonstrated that 'holoclones' expressed canonical stem markers, retained the exclusive ability to generate poorly differentiated tumours in NOD/SCID mice and possessed a unique mRNA-miRNA gene signature. miRNA:Target interactions analysis visualised potentially critical regulatory networks, which are dysregulated in prostate cancer holoclones. The characterisation of this tumorigenic population lays the groundwork for this model to be used in the identification of proteomic or small non-coding RNA therapeutic targets for the eradication of this critical cellular population. This is significant, as it provides a potential route to limit development of aggressive disease and thus improve survival rates.

Correction: Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature.

[This corrects the article DOI: 10.1371/journal.pone.0054193.].

Correction to: Vascular endothelial growth factor is an autocrine growth factor, signaling through neuropilin-1 in non-small cell lung cancer.

Since the publication of this work [1] and in response to a recent query that was brought to our attention in relation to the Western Blot in Figure 1(C) for NP2, protein lysates prepared around the same time as those presented in the manuscript in question, were run by SDS-PAGE under similar experimental conditions and probed using the same primary antibodies to NP1 and NP2 that were used originally.

Targeting NF-κB-mediated inflammatory pathways in cisplatin-resistant NSCLC.

OBJECTIVES: The majority of patients with non-small cell lung cancer (NSCLC) present with advanced stage disease, at which time chemotherapy is usually the most common treatment option. While somewhat effective, patients treated with platinum-based regimens will eventually develop resistance, with others presenting with intrinsic resistance. Multiple pathways have been implicated in chemo-resistance, however the critical underlying mechanisms have yet to be elucidated. The aim of this project was to determine the role of inflammatory mediators in cisplatin-resistance in NSCLC. MATERIALS AND METHODS: Inflammatory mediator, NF-κB, and its associated pathways were investigated in an isogenic model of cisplatin-resistant NSCLC using age-matched parental (PT) and corresponding cisplatin-resistant (CisR) sublines. Pathways were assessed using mass spectrometry, western blot analysis and qRT-PCR. The cisplatin sensitizing potential of an NF-κB small molecule inhibitor, DHMEQ, was also assessed by means of viability assays and western blot analysis. RESULTS: Proteomic analysis identified dysregulated NF-κB responsive targets in CisR cells when compared to PT cells, with increased NF-κB expression identified in four out of the five NSCLC sub-types examined (CisR versus PT). DHMEQ treatment resulted in reduced NF-κB expression in the presence of cisplatin, and re-sensitized CisR cells to the cytotoxic effects of the drug. CONCLUSION: This study identified NF-ĸB as a potential therapeutic target in cisplatin-resistant NSCLC. Furthermore, inhibition of NF-ĸB using DHMEQ re-sensitized chemo-resistant cells to cisplatin treatment.

When RON MET TAM in Mesothelioma: All Druggable for One, and One Drug for All?

Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer with a poor survival rate. Treatment options are limited at best and drug resistance is common. Thus, there is an urgent need to identify novel therapeutic targets in this disease in order to improve patient outcomes and survival times. MST1R (RON) is a trans-membrane receptor tyrosine kinase (RTK), which is part of the c-MET proto-oncogene family. The only ligand recognized to bind MST1R (RON) is Macrophage Stimulating 1 (MST1), also known as Macrophage Stimulating Protein (MSP) or Hepatocyte Growth Factor-Like Protein (HGFL). In this study, we demonstrate that the MST1-MST1R (RON) signaling axis is active in MPM. Targeting this pathway with a small molecule inhibitor, LCRF-0004, resulted in decreased proliferation with a concomitant increase in apoptosis. Cell cycle progression was also affected. Recombinant MST1 treatment was unable to overcome the effect of LCRF-0004 in terms of either proliferation or apoptosis. Subsequently, the effect of an additional small molecular inhibitor, BMS-777607 (which targets MST1R (RON), MET, Tyro3, and Axl) also resulted in a decreased proliferative capacity of MPM cells. In a cohort of MPM patient samples, high positivity for total MST1R by IHC was an independent predictor of favorable prognosis. Additionally, elevated expression levels of MST1 also correlated with better survival. This study also determined the efficacy of LCRF-0004 and BMS-777607 in xenograft MPM models. Both LCRF-0004 and BMS-777607 demonstrated significant anti-tumor efficacy in vitro, however BMS-777607 was far superior to LCRF-0004. The in vivo and in vitro data generated by this study indicates that a multi-TKI, targeting the MST1R/MET/TAM signaling pathways, may provide a more effective therapeutic strategy for the treatment of MPM as opposed to targeting MST1R alone.

Oncolytic Newcastle disease virus induces autophagy-dependent immunogenic cell death in lung cancer cells.

In addition to direct oncolysis, oncolytic viruses trigger immunogenic cell death (ICD) and primes antitumor immunity. We have previously shown that oncolytic Newcastle disease virus (NDV), strain FMW (NDV/FMW), induces apoptosis and/or autophagy in cancer cells. In this study, we investigated whether oncolytic NDV can induce ICD in lung cancer cells and whether apoptosis or autophagy plays a role in NDV-triggered ICD. To this end, we examined cell surface expression of calreticulin (CRT) on NDV-infected lung cancer cells and measured ICD determinants, high mobility group box 1 (HMGB1), heat shock protein 70/90 (HSP70/90) and ATP in supernatants following viral infection. Flow cytometric analysis using anti-CRT antibody and PI staining of NDV-infected lung cancer cells showed an increase in the number of viable (propidium iodide-negative) cells, suggesting the induction of CRT exposure upon NDV infection. In addition, confocal and immunoblot analysis using anti-CRT antibody showed that an enhanced accumulation of CRT on the cell surface of NDV-infected cells, indicating the translocation of CRT to the cell membrane upon NDV infection. We further demonstrated that NDV infection induced the release of secreted HMGB1 and HSP70/90 by examining the concentrated supernatants of NDV-infected cells. Furthermore, pre-treatment with either the pan-caspase inhibitor z-VAD-FMK or the necrosis inhibitor Necrostain-1, had no impact on NDV-induced release of ICD determinants in lung cancer cells. Rather, depletion of autophagy-related genes in lung cancer cells significantly inhibited the induction of ICD determinants by NDV. Of translational importance, in a lung cancer xenograft model, treatment of mice with supernatants from NDV-infected cells significantly inhibited tumour growth. Together, these results indicate that oncolytic NDV is a potent ICD-inducer and that autophagy contributes to NDV-mediated induction of ICD in lung cancer cells.

Ajuba inhibits hepatocellular carcinoma cell growth via targeting of ß-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation.

BACKGROUND: Aberrant activation of ß-catenin and Yes-associated protein (YAP) signaling pathways has been associated with hepatocellular carcinoma (HCC) progression. The LIM domain protein Ajuba regulates ß-catenin and YAP signaling and is implicated in tumorigenesis. However, roles and mechanism of Ajuba expression in HCC cells remain unclear. The E3 ligase Hakai has been shown to interact with other Ajuba family members and whether Hakai interacts and regulates Ajuba is unknown. METHODS: HCC cell lines stably depleted of Ajuba or Hakai were established using lentiviruses expressing shRNAs against Ajuba or Hakai. The effects of Ajuba on HCC cells were determined by a number of cell-based analyses including anchorage-independent growth, three dimension cultures and trans-well invasion assay. In vivo tumor growth was determined in a xenograft model and Ajuba expression in tumor sections was examined by immunohistochemistry. Co-immunoprecipitation, confocal microscopy and immunoblot assay were used to examine the expression and interaction between Ajuba and Hakai. RESULTS: Depletion of Ajuba in HCC cells significantly enhanced anchorage-independent growth, invasion, the formation of spheroids and tumor growth in a xenograft model, suggesting a tumor suppressor function for Ajuba in HCC. Mechanistically, Ajuba depletion triggered E-cadherin loss and ß-catenin translocation with increased Cyclin D1 levels. In addition, depletion of Ajuba upregulated the levels of YAP and its target gene CYR61. Furthermore, siRNA-mediated knockdown of either ß-catenin or YAP attenuated the pro-tumor effects by Ajuba depletion on HCC cells. Notably, Ajuba stability in HCC cells was regulated by Hakai, an E3 ligase for E-cadherin. Hakai interacted with Ajuba via its HYB domain and induced Ajuba neddylation, which was antagonized by the neddylation inhibitor, MLN4924, but not MG132. We further show that overexpression of Hakai in HCC cells markedly increased anchorage-independent growth, spheroid-formation ability and tumor growth in xenografts whereas Hakai depletion resulted in these opposite effects, indicating an oncogenic role for Hakai in HCC. Hakai also induced ß-catenin translocation with increased levels of Cyclin D1. CONCLUSIONS: Our data suggest a role for Ajuba and Hakai in HCC, and uncover the mechanism underlying the regulation of Ajuba stability.