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Introduction: 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.Methods: 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.Results: 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. B7-H6 shedding by OAC tumour cells was induced by FLOT.Conclusion: 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. Finally, our data indicate that FLOT chemotherapy and obesity may promote B7-H6 shedding and immune evasion with detrimental consequences in OAC patients.

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.

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.

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.

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

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.

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-naive 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.

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.

Seven rhenium(I) complexes of the general formula fac-[Re(CO)(3)(NN)(OH(2))](+) where NN = 2,2′-bipyridine (8), 4,4′-dimethyl-2,2′-bipyridine (9), 4,4′-dimethoxy-2,2′-bipyridine (10), dimethyl 2,2′-bipyridine-4,4′-dicarboxylate (11), 1,10-phenanthroline (12), 2,9-dimethyl-1,10-phenanthroline (13), or 4,7-diphenyl-1,10-phenanthroline (14), were synthesized and characterized by (1)H NMR spectroscopy, IR spectroscopy, mass spectrometry, and X-ray crystallography. With the exception of 11, all complexes exhibited 50% growth inhibitory concentration (IC(50)) values that were less than 20 μM in HeLa cells, indicating that these compounds represent a new potential class of anticancer agents. Complexes 9, 10, and 13 were as effective in cisplatin-resistant cells as wild-type cells, signifying that they circumvent cisplatin resistance. The mechanism of action of the most potent complex, 13, was explored further by leveraging its intrinsic luminescence properties to determine its intracellular localization. These studies indicated that 13 induces cytoplasmic vacuolization that is lysosomal in nature. Additional in vitro assays indicated that 13 induces cell death without causing an increase in intracellular reactive oxygen species or depolarization of the mitochondrial membrane potential. Further studies revealed that the mode of cell death does not fall into one of the canonical categories such as apoptosis, necrosis, paraptosis, and autophagy, suggesting that a novel mode of action may be operative for this class of rhenium compounds. The in vivo biodistribution and metabolism of complex 13 and its (99m)Tc analogue 13* were also evaluated in naïve mice. Complexes 13 and 13* exhibited comparable biodistribution profiles with both hepatic and renal excretion. High-performance liquid chromatography inductively coupled plasma mass-spectrometry (HPLC-ICP-MS) analysis of mouse blood plasma and urine post administration showed considerable metabolic stability of 13, rendering this potent complex suitable for in vivo applications. These studies have shown the biological properties of this class of compounds and demonstrated their potential as promising theranostic anticancer agents that can circumvent cisplatin resistance.

Non-small cell lung cancer (NSCLC) accounts for a large proportion of cancer deaths and is characterized by low treatment response rates and poor overall prognosis. In the absence of specific treatable mutations, cisplatin-based chemotherapy plays an important role in the treatment of this disease. Unfortunately, the development of resistance has become a major therapeutic challenge in the use of this cytotoxic drug. Elucidating the mechanisms underlying this resistance phenotype, may result in the development of novel agents that enhance sensitivity to cisplatin in lung cancer patients. In this study, targeting the cancer stem cell activity of aldehyde dehydrogenase 1 (ALDH1) was investigated as a strategy to overcome chemoresistance in NSCLC. Tumors from NSCLC patients showed an increase in their profile of pluripotent stemness genes. Cisplatin exposure induced the emergence or expansion of an ALDH1-positive subpopulation in cisplatin sensitive and resistant NSCLC cell lines, respectively, further enhancing cisplatin resistance. Using the Aldefluor assay and FACS analysis, ALDH1 subpopulations were isolated and evaluated in terms of stem cell characteristics. Only ALDH1-positive cells exhibited asymmetric division, cisplatin resistance and increased expression of stem cell factors in vitro. Xenograft studies in NOD/SCID mice demonstrated efficient tumorigenesis from low cell numbers of ALDH1-positive and ALDH1-negative subpopulations. Targeting ALDH1 with Diethylaminobenzaldehyde (DEAB) and Disulfiram, significantly re-sensitized resistant lung cancer cells to the cytotoxic effects of cisplatin. Our data demonstrate the existence of a lung CSC population and suggest a role for targeting ALDH1 as a potential therapeutic strategy in re-sensitizing NSCLC cells to the cytotoxic effects of 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/β) 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.

// Susan Heavey 1 , Sinead Cuffe 1 , Stephen Finn 1 , Vincent Young 2 , Ronan Ryan 2 , Siobhan Nicholson 3 , Niamh Leonard 3 , Niall McVeigh 1 , Martin Barr 1 , Kenneth O’Byrne 4 , Kathy Gately 1 1 Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland 2 Department of Cardiothoracic Surgery, St. James’s Hospital, Dublin, Ireland 3 Department of Histopathology, St James Hospital, Dublin, Ireland 4 Cancer and Ageing Research Program, Institute of Health and Biomedical Innovation at the Translational Research Institute (TRI), Queensland University of Technology, Brisbane, Australia Correspondence to: Susan Heavey, email: heaveys@tcd.ie Keywords: PI3K, MEK, NSCLC, lung, co-target Received: September 29, 2015 Accepted: September 12, 2016 Published: October 19, 2016 ABSTRACT Clinical PI3K inhibition has been somewhat disappointing, due to both inadequate patient stratification and compensatory cell signalling through bypass mechanisms. As such, investigation of PI3K-MEK co-targeted inhibition has been recommended. With high mortality rates and a clear need for new therapeutic intervention strategies, non-small cell lung cancer (NSCLC) is an important setting to investigate the effectiveness of this approach. Here, 174 NSCLC tumours were screened for 150 mutations by Fluidigm technology, with 15 patients being profiled for phosphoprotein expression. The effects of GDC-0941 (a pan PI3K inhibitor), GDC-0980 (a dual PI3K/mTOR inhibitor) and GDC-0973 (a MEK inhibitor) alone and in combination were assessed in 3 NSCLC cell lines. PIK3CA was mutated in 5.17% of NSCLC patients. GDC-0941 and GDC-0980 treatment induced anti-proliferative and pro-apoptotic responses across all NSCLC cell lines, while GDC-0973 treatment induced only anti-proliferative responses. GDC-0980 and GDC-0973 combined treatment led to significant increases in apoptosis and synergistic reductions in proliferation across the panel of cell lines. This study found that the PI3K/MEK co-targeted inhibition strategy is synergistic in all 3 molecular subtypes of NSCLC investigated. Consequently, we would advocate clinical trials for NSCLC patients combining GDC-0980 and GDC-0973, each of which are separately under clinical investigation currently.

Background Aberrant activation of β-catenin and Yes-associated protein (YAP) signaling pathways has been associated with hepatocellular carcinoma (HCC) progression. The LIM domain protein Ajuba regulates β-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 β-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 β-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 β-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. Electronic supplementary material The online version of this article (10.1186/s13046-018-0806-3) contains supplementary material, which is available to authorized users.

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.

Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths worldwide. While partial or complete tumor regression can be achieved in patients, particularly with cisplatin-based strategies, these initial responses are frequently short-lived and are followed by tumor relapse and chemoresistance. Identifying the root of cisplatin resistance in NSCLC and elucidating the mechanism(s) of tumor relapse, is of critical importance in order to determine the point of therapeutic failure, which in turn, will aid the discovery of novel therapeutics, new combination strategies and a strategy to enhance the efficacy of current chemotherapeutics. It has been hypothesized that cancer stem cells (CSCs) may be the initiating factor of resistance. We have previously identified and characterized an aldehyde dehydrogenase 1 CSC subpopulation in cisplatin resistant NSCLC. BBI608 is a small molecule STAT3 inhibitor known to suppress cancer relapse, progression and metastasis. Here, we show that BBI608 can inhibit stemness gene expression, deplete CSCs and overcome cisplatin resistance in NSCLC.

The PI3K-mTOR pathway is involved in regulating all hallmarks of cancer, and is often dysregulated in NSCLC, making it an attractive therapeutic target in this setting. Acquired resistance to PI3K-mTOR inhibition is a major hurdle to overcome in the success of PI3K-mTOR targeted agents. H460, A549, and H1975 resistant cells were generated by prolonged treatment in culture with Apitolisib (GDC-0980), a dual PI3K-mTOR inhibitor over a period of several months, from age-matched parent cells. Resistance was deemed to have developed when a log fold difference in IC50 had been achieved. Resistant cell lines also exhibited resistance to another widely investigated PI3K-mTOR dual inhibitor; Dactolisib (BEZ235). Cell lines were characterised at the level of mRNA (expression array profiling expression of >150 genes), miRNA (expression array profiling of 2100 miRNAs), protein (bottoms-up label-free mass spectrometry) and phosphoprotein (expression array profiling of 84 phospho/total proteins). Key alterations were validated by qPCR and Western blot. H1975 cells were initially most sensitive to Apitolisib (GDC-0980), but developed resistance more quickly than the other cell lines, perhaps due to increased selective pressure from the impressive initial effect. In-depth molecular profiling suggested epithelial-mesenchymal transition (EMT) may play a role in resistance to PI3K-mTOR dual inhibition in NSCLC.

Lung cancer is the second most common type of cancer in the world and is the most common cause of cancer-related death in both men and women. Research into causes, prevention and treatment of lung cancer is ongoing and much progress has been made recently in these areas, however survival rates have not significantly improved. Therefore, it is essential to develop biomarkers for early diagnosis of lung cancer, prediction of metastasis and evaluation of treatment efficiency, as well as using these molecules to provide some understanding about tumour biology and translate highly promising findings in basic science research to clinical application. In this investigation, two-dimensional difference gel electrophoresis and mass spectrometry were initially used to analyse conditioned media from a panel of lung cancer and normal bronchial epithelial cell lines. Significant proteins were identified with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), pyruvate kinase M2 isoform (PKM2), Hsc-70 interacting protein and lactate dehydrogenase A (LDHA) selected for analysis in serum from healthy individuals and lung cancer patients. hnRNPA2B1, PKM2 and LDHA were found to be statistically significant in all comparisons. Tissue analysis and knockdown of hnRNPA2B1 using siRNA subsequently demonstrated both the overexpression and potential role for this molecule in lung tumorigenesis. The data presented highlights a number of in vitro derived candidate biomarkers subsequently verified in patient samples and also provides some insight into their roles in the complex intracellular mechanisms associated with tumour progression.

Platinum chemotherapeutic agents such as cisplatin are currently used in the treatment of various malignancies such as lung cancer. However, their efficacy is significantly hindered by the development of resistance during treatment. While a number of factors have been reported that contribute to the onset of this resistance phenotype, alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. The mode of action of cisplatin has been linked to its ability to crosslink purine bases on the DNA, thereby interfering with DNA repair mechanisms and inducing DNA damage. Following DNA damage, cells respond by activating a DNA-damage response that either leads to repair of the lesion by the cell thereby promoting resistance to the drug, or cell death via activation of the apoptotic response. Therefore, DNA repair is a vital target to improving cancer therapy and reduce the resistance of tumour cells to DNA damaging agents currently used in the treatment of cancer patients. To date, despite the numerous findings that differential expression of components of the various DNA repair pathways correlate with response to cisplatin, translation of such findings in the clinical setting are still warranted. The identification of alterations in specific proteins and pathways that contribute to these unique DNA repair pathways in cisplatin resistant cancer cells may potentially lead to a renewed interest in the development of rational novel therapies for cisplatin resistant cancers, in particular, lung cancer.

Lung cancer is the leading cause of cancer-related deaths. Most patients develop resistance to platinum within several months of treatment. We investigated whether triggering lysosomal membrane permeabilization (LMP) or suppressing autophagy can restore cisplatin susceptibility in lung cancer with acquired chemoresistance. Cisplatin IC50 in A549Pt (parental) and A549cisR (cisplatin resistant) cells was 13 μM and 47 μM, respectively. Following cisplatin exposure, A549cisR cells failed to elicit an apoptotic response. This was manifested by diminished Annexin-V staining, caspase 3 and 9, BAX and BAK activation in resistant but not in parental cells. Chloroquine preferentially promoted LMP in A549cisR cells, revealed by leakage of FITC-dextran into the cytosol as detected by immunofluorescence microscopy. This was confirmed by increased cytosolic cathepsin D signal on Immunoblot. Cell viability of cisplatin-treated A549cisR cells was decreased when co-treated with chloroquine, corresponding to a combination index below 0.8, suggesting synergism between the two drugs. Notably, chloroquine activated the mitochondrial cell death pathway as indicated by increase in caspase 9 activity. Interestingly, inhibition of lysosomal proteases using E64 conferred cytoprotection against cisplatin and chloroquine co-treatment, suggesting that chloroquine-induced cell death occurred in a cathepsin-mediated mechanism. Likewise, blockage of caspases partially rescued A549cisR cells against the cytotoxicity of cisplatin and chloroquine combination. Cisplatin promoted a dose-dependent autophagic flux induction preferentially in A549cisR cells, as evidenced by a surge in LC3-II/α-tubulin following pre-treatment with E64 and increase in p62 degradation. Compared to untreated cells, cisplatin induced an increase in cyto-ID-loaded autophagosomes in A549cisR cells that was further amplified by chloroquine, pointing toward autophagic flux activation by cisplatin. Interestingly, this effect was less pronounced in A549Pt cells. Blocking autophagy by ATG5 depletion using siRNA markedly enhances susceptibility to cisplatin in A549cisR cells. Taken together, our results underscore the utility of targeting lysosomal function in overcoming acquired cisplatin refractoriness in lung cancer.

Chemoresistance is a major therapeutic challenge to overcome in NSCLC, in order to improve the current survival rates of

Research studies aimed at advancing cancer prevention, diagnosis, and treatment depend on a number of key resources, including a ready supply of high-quality annotated biospecimens from diverse ethnic populations that can be used to test new drugs, assess the validity of prognostic biomarkers, and develop tailor-made therapies. In November 2011, KHCCBIO was established at the King Hussein Cancer Center (KHCC) with the support of Seventh Framework Programme (FP7) funding from the European Union (khccbio.khcc.jo). KHCCBIO was developed for the purpose of achieving an ISO accredited cancer biobank through the collection, processing, and preservation of high-quality, clinically annotated biospecimens from consenting cancer patients, making it the first cancer biobank of its kind in Jordan. The establishment of a state-of-the-art, standardized biospecimen repository of matched normal and lung tumor tissue, in addition to blood components such as serum, plasma, and white blood cells, was achieved through the support and experience of its European partners, Trinity College Dublin, Biostór Ireland, and accelopment AG. To date, KHCCBIO along with its partners, have worked closely in establishing an ISO Quality Management System (QMS) under which the biobank will operate. A Quality Policy Manual, Validation, and Training plan have been developed in addition to the development of standard operating procedures (SOPs) for consenting policies on ethical issues, data privacy, confidentiality, and biobanking bylaws. SOPs have also been drafted according to best international practices and implemented for the donation, procurement, processing, testing, preservation, storage, and distribution of tissues and blood samples from lung cancer patients, which will form the basis for the procurement of other cancer types. KHCCBIO will be the first ISO accredited cancer biobank from a diverse ethnic Middle Eastern and North African population. It will provide a unique and valuable resource of high-quality human biospecimens and anonymized clinicopathological data to the cancer research communities world-wide.

Cisplatin remains the cornerstone of NSCLC management. Despite initial sensitivity tumours develop resistance, undermining the cytotoxic effect of cisplatin. Numerous mechanisms have been investigated in terms of cisplatin resistance and its development, however, the clinical issue of resistance remains. An avenue of interest is the CSC hypothesis, in which the survival and expansion of highly resistant CSCs during chemotherapy are thought to be a contributing factor of resistance. Specific inhibition of key CSC markers in combination with chemotherapy may subvert the inherent resistance of the CSCs thereby restoring therapeutic effeciency. One such CSC marker is aldehyde dehydrogenase 1 (ALDH1). We hypothesise that inhibition of the ALDH1-positive CSCs within cispaltin resistant NSCLC will resensitise the cells to cisplatin.

Despite advances in personalised medicine, cisplatin remains the mainstay in NSCLC management, however the develop-ment of pan-resistance to platinum agents has become a major clinical challenge. Novel drug design, preclinical and clinical trials working toward the approval of new drugs is a lengthy and costly process and in the interim research has turned its focus to overcoming resistance and repurposing drugs. Cancer stem cells (CSCs) have been hypothesised to be the initiating cells of resistance and recurrence. ALDH1-positive cells have been identified as a CSC subpopulation within cisplatin resistant (CisR) NSCLC sublines. ALDH1 converts retinal to retinoic acid during the metabolism of retinol (vitamin A), retinoic acid induces cell differentiation. All-trans retinoic acid (ATRA) is a well-established chemotherapeutic agent in the treatment of acute promyelocytic leukaemia; it induces the terminal differentiation of immature cells. We hypothesise that retinol and ATRA will deplete the ALDH1-positive CSC population thereby increasing or restoring cisplatin sensitivity.

Introduction: Despite advances in NSCLC therapeutics, including small molecule and antibody based targeted therapies, the majority of patients with advanced disease will ultimately relapse. Lung cancer is characterized by genomic instability leading to tissue invasion, metastasis and resistance to systemic treatments. hSSB1 has a key role in the detection and repair of DNA damage (double- strand breaks, replication fork arrest and oxidative stress). Recently we demonstrated that hSSB1 is phosphorylated by DNA-PK in response to replication stress to promote cellular survival. Here we establish that hSSB1 is a prognostic factor for NSCLC a potential novel target for therapy. Methods: We analyzed the prognostic significance of hSSB1 mRNA expression from public on line databases and from protein expression in an NSCLC tissue macro-array (TMA) through hSSB1 immunohistochemistry. hSSB1 mRNA levels were analyzed in matched normal, tumour adenocarcinoma and squamous cell tumour samples. We explored the impact of hSSB1 expression on NSCLC cell line proliferation by depleting hSSB1 with specific small interfering (si)RNA. We also measured the impact of hSSB1 inhibition on NSCLC cells proliferation with the use of a DNA-PK inhibitor and a novel agent, DKLS02. Results: hSSB1 expression was associated with poor prognosis for lung cancer, high levels of mRNA and protein expression correlating with a worse overall survival. We also observed that hSSB1 depletion leads to increased lung cancer cell death. The same result was observed when inhibiting hSSB1 by treating lung cancer cells with a DNA-PK inhibitor. Targeting hSSB1 with DKLS02 induces cell death in vitro and inhibits tumour growth in vivo in platinum sensitive and resistant tumours. Conclusion: Our results indicate that hSSB1 is a prognostic factor in NSCLC. Moreover, targeting hSSB1 may prove to be an effective therapeutic strategy for the treatment of chemosenstive and resistant NSCLC in the future. Disclosure: All authors have declared no conflicts of interest.

Introduction: Lung cancer is the leading cause of all cancer death worldwide. It is characterized by genomic instability leading to tissue invasion, metastasis and resistance to chemotherapy, notably cisplatin. Being the protein guardian of the genome, hSSB1 has a key role in the detection and repair of DNA damage (double-strand breaks, replication fork arrest and oxidative stress). Recently we have shown that hSSB1 is phosphorylated by DNA-PK at serine residue 134 in response to replication stress to promote cellular survival. We wanted to check is hSSB1 could be involved in the mechanism of non-small cell lung cancer (NSCLC) proliferation, establishing hSSB1 not only as a novel prognostic factor for NSCLC but also as a potential target for a new therapy strategy. Methods: We analyzed the prognostic significance of hSSB1 mRNA expression from public on line database and from protein expression in an NSCLC tissue macro-array (TMA) through hSSB1 immunohistochemistry staining. hSSB1 mRNA levels were analyzed in matched normal, tumour adenocarcinoma and squamous cell tumour samples. We explored the impact of hSSB1 expression on NSCLC cell lines proliferation by depleting hSSB1 with specific small interfering (si)RNA. We also measured the impact of hSSB1 inhibition on NSCLC cells proliferation with the use of a DNA-PK inhibitor. Results: hSSB1 expression was associated with poor prognosis for lung cancer, high levels of mRNA and protein expression correlating with a worse overall survival. We also observed that hSSB1 depletion leads to increased lung cancer cell death. The same result was observed when inhibiting hSSB1 by treating lung cancer cells with a DNA-PK inhibitor. Conclusion: Our results set hSSB1 as a prognostic factor in non-small cell lung cancer. Moreover, targeting hSSB1 proved to be effective to stop lung cancer cells proliferation, showing the potential as a new treatment for NSCLC. Disclosure: All authors have declared no conflicts of interest.

SUMMARY Non-small-cell lung cancer (NSCLC) is the most common cause of cancer-related deaths among men and women worldwide. Despite the development of molecular targeted therapies, platinum-based combination chemotherapy remains the most effective systemic chemotherapy for NSCLC patients. Unfortunately, the outcomes of platinum-based therapies, in particular those containing cisplatin, have reached a plateau due to the development of both intrinsic and acquired resistance. While significant variations in response to platinum-based chemotherapeutic regimens exist, defining molecular features that may determine resistance or response to chemotherapy is critical. This review will focus on some of the emerging biomarkers that are predictive of response to such treatments that may offer potential in the future management of NSCLC patients.

1 ISSN 1758-1966 10.2217/LMT.12.51 © 2013 Future Medicine Ltd Lung Cancer Manage. (2013) 2(1), 1–4 Lung cancer, in particular non-smallcell lung cancer (NSCLC), is the most common cause of cancer-related death in the world [1]. Rates of lung cancer will continue to rise owing to high levels of cigarette consumption in Asia and the developing world, and aging populations. Despite increased surgical resection rates, optimization of chemoradiotherapy and the introduction of targeted therapies, the treatment outcome for NSCLC remains poor [1]. The current standard of care for the majority of patients suitable for therapy involves treatment with a platinum-based chemotherapy regimen [1]. However, only a proportion of patients benefit and the remainder develop resistance to platinum-based therapy. Ongoing research aims to understand both the molecular mechanisms underpinning this resistance and identify agents or therapies that could sensitize or even resensitize patients to cisplatin-based therapeutic regimens. Targeting epigenetic mechanisms may represent one such approach. At its simplest, epigenetics can be described as stable and heritable changes in gene expression that are not due to changes in the primary DNA sequence, and is most commonly used to describe chromatinbased regulation of gene expression [1,2]. The main mechanisms used involve the laying down of modifications to DNA and histones, that are then utilized by various chromatin-modifying enzymes which can be categorized as ‘readers’, ‘writers’ and ‘erasers’ [2]. More recently, miRNAs, a form of noncoding RNA, have also been shown to affect epigenetic regulation by directly targeting the epigenetic machinery, leading them to be described as ‘epi-miRNAs’ [1]. Aberrant epigenetic regulation is now well established as a frequent event in NSCLC, with all known forms of epigenetic regulation playing a role [1]. This ‘epigenetic landscape’ can potentially be targeted and may have important implications for the management and treatment of NSCLC [1,2]. Critically, an emerging body of evidence now links some of these epigenetic events with cisplatin resistance.

The cancer stem-cell (CSC) hypothesis suggests that there is a small subset of cancer cells that are responsible for tumor initiation and growth, possessing properties such as indefinite self-renewal, slow replication, intrinsic resistance to chemotherapy and radiotherapy, and an ability to give rise to differentiated progeny. Through the use of xenotransplantation assays, putative CSCs have been identified in many cancers, often identified by markers usually expressed in normal stem cells. This is also the case in lung cancer, and the accumulated data on side population cells, CD133, CD166, CD44 and ALDH1 are beginning to clarify the true phenotype of the lung cancer stem cell. Furthermore, it is now clear that many of the pathways of normal stem cells, which guide cellular proliferation, differentiation, and apoptosis are also prominent in CSCs; the Hedgehog (Hh), Notch, and Wnt signaling pathways being notable examples. The CSC hypothesis suggests that there is a small reservoir of cells within the tumor, which are resistant to many standard therapies, and can give rise to new tumors in the form of metastases or relapses after apparent tumor regression. Therapeutic interventions that target CSC pathways are still in their infancy and clinical data of their efficacy remain limited. However Smoothened inhibitors, gamma-secretase inhibitors, anti-DLL4 antagonists, Wnt antagonists, and CBP/β-catenin inhibitors have all shown promising anticancer effects in early studies. The evidence to support the emerging picture of a lung cancer CSC phenotype and the development of novel therapeutic strategies to target CSCs are described in this review.

Hepatocellular carcinoma (HCC) is refractory to chemotherapies, necessitating novel effective agents. The lysosome inhibitor Bafilomycin A1 (BafA1) at high concentrations displays cytotoxicity in a variety of cancers. Here we show that BafA1 at nanomolar concentrations suppresses HCC cell growth in both 2 dimensional (2D) and 3D cultures. BafA1 induced cell cycle arrest in the G1 phase and triggered Cyclin D1 turnover in HCC cells in a dual-specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B) dependent manner. Notably, BafA1 induced caspase-independent cell death in HCC cells by impairing autophagy flux as demonstrated by elevated LC3 conversion and p62/SQSTM1 levels. Moreover, genetic ablation of LC3 significantly attenuated BafA1-induced cytotoxicity of HCC cells. We further demonstrate that pharmacological down-regulation or genetic depletion of p38 MAPK decreased BafA1-induced cell death via abolishment of BafA1-induced upregulation of Puma. Notably, knockdown of Puma impaired BafA1-induced HCC cell death, and overexpression of Puma enhanced BafA1-mediated HCC cell death, suggesting a role for Puma in BafA1-mediated cytotoxicity. Interestingly, pharmacological inhibition of JNK with SP600125 enhanced BafA1-mediated cytotoxicity both in vitro and in xenografts derived from HCC cells. Taken together, our data suggest that BafA1 may offer potential as an effective therapy for HCC.

Background In the absence of specific treatable mutations, platinum-based doublet chemotherapy remains the gold standard treatment for NSCLC patients. However, its clinical efficacy is hindered in many patients due to both intrinsic and acquired resistance to these agents, in particular, cisplatin. Alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. Methods DNA Repair Pathway RT2 Profiler Arrays were used to elucidate key DNA repair genes in H460 cisplatin resistant (CisR) and corresponding parental (PT) NSCLC cell lines previously generated in our laboratory. DNA repair genes significantly altered in CisR cells were validated using RT-PCR and western blot analysis, respectively. Loss of function studies were carried out using siRNA technology. The effect of gene knockdown on apoptosis was assessed by Annexin V/propidium iodide (PI) staining using the Cytell® Imaging System. DNA damage and repair in response to cisplatin following gene knockdown was investigated using the γH2AX foci formation assay. The translational relevance of these genes was examined in a cohort of chemo-naive matched normal and tumour lung tissues (n = 20). Results We identified a number of important DNA repair genes differentially regulated between H460 PT and CisR NSCLC cells. These included XRCC6BP1, TOP3A, XPA, PMS1 and hSSB1. XRCC6BP1 mRNA was significantly increased (19.4-fold) in H460 CisR cells relative to their PT counterparts. Gene silencing of XRCC6BP1 re-sensitized CisR lung cancer cells to the pro-apoptotic effects of cisplatin and significantly reduced the DNA repair capacity of these cells. Relative to matched normal lung tissues, XRCC6BP1 mRNA was significantly increased in adenocarcinoma tissues (AD), while hSSB1 mRNA was significantly increased in both AD and squamous cell carcinoma (SCC) tissues. Conclusions We have identified XRCC6BP1, in addition to hSSB1, as key DNA repair genes implicated in cisplatin resistant NSCLC. Our data highlights the potential of targeting XRCC6BP1, at least in part, in re-sensitizing chemoresistant lung cancer cells to the cytotoxic effects of cisplatin chemotherapy.

Background In addition to their involvement in mechanisms underlying cancer initiation and progression, emerging evidence has highlighted a key role for microRNAs in resistance to a number of anti-cancer therapies. Methods MicroRNA profiling and validation was carried out on a panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines using 7th generation miRCURY LNA™ arrays (Exiqon). Transient transfections were used to either inhibit or over-express these microRNAs using antagomirs or pre-miRs, respectively. The effects of modulating these miRNAs on cell proliferation, apoptosis and clonogenic survival were examined in response to cisplatin. The translational relevance of these miRNAs in lung cancer was confirmed in a cohort of chemo-naive matched normal and tumour lung tissues and sera from NSCLC patients. MicroRNAs were also assessed in mouse xenograft FFPE tissues from H460 CisR-derived ALDH1+ and ALDH1– cancer stem cell subpopulations. Results A 5-miRNA signature consisting of members of the miR-30 family, miR-34a and miR-4286 was identified and validated. MicroRNAs were significantly up-regulated in all CisR cell lines relative to their parental counterparts, in contrast to miR-4286 which was significantly down-regulated. Inhibition of the miR-30 family and miR-34a and forced over-expression of miR-4286 significantly reduced the clonogenic survival ability of CisR cells in response to cisplatin. The miR-30 family was significantly decreased in AD and SCC tissues. MiR-34a expression was significantly lower in SCC tissues while miR-4286 levels were significantly increased in AD tissues only. A significant-fold increase in miR-4286 was found in sera from SCC lung cancer patients. Similar to that found at the in vitro level, the miR-30 family and miR-34a were up-regulated in mouse xenograft FFPE tissues derived from CisR and PT cell lines in vivo. Conclusions In this study, a 5-microRNA signature was identified using an in vitro model of cisplatin resistance. Differential expression of these microRNAs was found between matched normal and tumour lung tissues from NSCLC patients. These may offer potential as diagnostic markers in NSCLC and in predicting response to cisplatin chemotherapy.

Background The root of therapeutic resistance is hypothesized to be due to the presence of a rare CSC population within the tumour population. When challenged with chemotherapy, this tumour cell subset survives the insult and has the potential to recapitulate a heterogenic tumour. Aldehyde dehydrogenase 1 (ALDH1) is involved in the catalytic conversion of vitamin A (retinol) to retinoic acid and has been identified as a CSC marker in a number of solid malignancies. Methods FACS analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines of different histologies was used to examine ALDH1 activity. The H460 CisR subline was FACS-sorted (MoFlo®) into ALDH1-positive and ALDH1-negative fractions and subcutaneously injected into NOD/SCID mice to assess tumour initiation and growth. The effects of inhibiting ALDH1 on cell proliferation (BrdU), apoptosis (Annexin-V/propidium iodide) and colony formation (crystal violet) in PT and CisR cell lines was assessed in vitro using two ALDH1 inhibitors, DEAB (N,N-diethylaminobenzaldehyde) and disulfiram (Antabuse), alone and in combination with cisplatin. Lung cancer cells were treated with retinol and All-trans retinoic acid (ATRA) to exploit the role of ALDH1 in the vitamin A/retinoic acid axis in NSCLC. Results ALDH1 activity was significantly increased in all CisR cell lines relative to their parental counterparts in which little or no ALDH1 expression was detected. Xenograft studies using ALDH1+ and ALDH1– fractions from H460 CisR cells showed no significant difference in tumour growth in mice. ALDH1 inhibition, in combination with cisplatin, significantly decreased cell viability, proliferation and clonogenic survival of CisR sublines only and increased apoptotic cell death compared to cells treated with cisplatin alone. Similarly, treatment of cells with retinol or ATRA, in combination with cisplatin, showed similar re-sensitizing effects. Conclusions In this study, we show that inhibition of the CSC marker, ALDH1, re-sensitizes cisplatin resistant NSCLC cells to the cytotoxic effects of cisplatin. Furthermore, our data suggest a role for ALDH1 as a key enzyme in the vitamin A/retinoic acid axis.

Lung cancer is the leading cause of cancer mortality worldwide. Despite advances in anti-cancer therapies such as chemotherapy, radiotherapy and targeted therapies, five-year survival rates remain poor (

In the absence of specific treatable mutations, platinum-based chemotherapy remains the gold standard of treatment for lung cancer patients. However, 5-year survival rates remain poor due to the development of resistance and eventual relapse. Resistance to conventional cytotoxic therapies presents a significant clinical challenge in the treatment of this disease. The cancer stem cell (CSC) hypothesis suggests that tumors are arranged in a hierarchical structure, with the presence of a small subset of stem-like cells that are responsible for tumor initiation and growth. This CSC population has a number of key properties such as the ability to asymmetrically divide, differentiate and self-renew, in addition to having increased intrinsic resistance to therapy. While cytotoxic chemotherapy kills the bulk of tumor cells, CSCs are spared and have the ability to recapitulate the heterogenic tumor mass. The identification of lung CSCs and their role in tumor biology and treatment resistance may lead to innovative targeted therapies that may ultimately improve clinical outcomes in lung cancer patients. This review will focus on lung CSC markers, their role in resistance and their relevance as targets for future therapies.

Alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating resistance to chemotherapeutic agents.