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Oncogenic KRAS mutations are major drivers of lung adenocarcinoma (LAC), yet the direct therapeutic targeting of KRAS has been problematic. Here, we reveal an obligate requirement by oncogenic KRAS for the ADAM17 protease in LAC. In genetically engineered and xenograft (human cell line and patient-derived) KrasG12D-driven LAC models, the specific blockade of ADAM17, including with a non-toxic prodomain inhibitor, suppressed tumor burden by reducing cellular proliferation. The pro-tumorigenic activity of ADAM17 was dependent upon its threonine phosphorylation by p38 MAPK, along with the preferential shedding of the ADAM17 substrate, IL-6R, to release soluble IL-6R that drives IL-6 trans-signaling via the ERK1/2 MAPK pathway. The requirement for ADAM17 in KrasG12D-driven LAC was independent of bone marrow-derived immune cells. Furthermore, in KRAS mutant human LAC, there was a significant positive correlation between augmented phospho-ADAM17 levels, observed primarily in epithelial rather than immune cells, and activation of ERK and p38 MAPK pathways. Collectively, these findings identify ADAM17 as a druggable target for oncogenic KRAS-driven LAC and provide the rationale to employ ADAM17-based therapeutic strategies for targeting KRAS mutant cancers.Copyright © 2019 The Authors. Published under the terms of the CC BY 4.0 license

Our understanding of genomic heterogeneity in lung cancer is largely based on the analysis of early-stage surgical specimens. Here we used endoscopic sampling of paired primary and intrathoracic metastatic tumors from 11 lung cancer patients to map genomic heterogeneity inoperable lung cancer with deep whole-genome sequencing. Intra-patient heterogeneity in driver or targetable mutations was predominantly in the form of copy number gain. Private mutation signatures, including patterns consistent with defects in homologous recombination, were highly variable both within and between patients. Irrespective of histotype, we observed a smaller than expected number of private mutations, suggesting that ancestral clones accumulated large mutation burdens immediately prior to metastasis. Single-region whole-genome sequencing of from 20 patients showed that tumors in ever-smokers with the strongest tobacco signatures were associated with germline variants in genes implicated in the repair of cigarette-induced DNA damage. Our results suggest that lung cancer precursors in ever-smokers accumulate large numbers of mutations prior to the formation of frank malignancy followed by rapid metastatic spread. In advanced lung cancer, germline variants in DNA repair genes may interact with the airway environment to influence the pattern of founder mutations, whereas similar interactions with the tumor microenvironment may play a role in the acquisition of mutations following metastasis.Copyright © 2018, The Author(s).

Cigarette smoking is undoubtedly a risk factor for lung cancer. Moreover, smokers with genetic mutations on chromosome 3p21.3, a region frequently deleted in cancer and notably in lung cancer, have a dramatically higher risk of aggressive lung cancer. The RNA binding motif 5 (RBM5) is one of the component genes in the 3p21.3 tumour suppressor region. Studies using human cancer specimens and cell lines suggest a role for RBM5 as a tumour suppressor. Here we demonstrate, for the first time, an in vivo role for RBM5 as a tumour suppressor in the mouse lung. We generated Rbm5 loss-of-function mice and exposed them to a tobacco carcinogen NNK. Upon exposure to NNK, Rbm5 loss-of-function mice developed lung cancer at similar rates to wild type mice. As tumourigenesis progressed, however, reduced Rbm5 expression lead to significantly more aggressive lung cancer i.e. increased adenocarcinoma nodule numbers and tumour size. Our data provide in vivo evidence that reduced RBM5 function, as occurs in a large number of patients, coupled with exposure to tobacco carcinogens is a risk factor for an aggressive lung cancer phenotype. These data suggest that RBM5 loss-of-function likely underpins at least part of the pro-tumourigenic consequences of 3p21.3 deletion in humans.

Hedgehog (Hh) signaling regulates cell fate and self-renewal in development and cancer. Canonical Hh signaling is mediated by Hh ligand binding to the receptor Patched (Ptch), which in turn activates Gli-mediated transcription through Smoothened (Smo), the molecular target of the Hh pathway inhibitors used as cancer therapeutics. Small cell lung cancer (SCLC) is a common, aggressive malignancy with universally poor prognosis. Although preclinical studies have shown that Hh inhibitors block the self-renewal capacity of SCLC cells, the lack of activating pathway mutations have cast doubt over the significance of these observations. In particular, the existence of autocrine, ligand-dependent Hh signaling in SCLC has been disputed. In a conditional Tp53;Rb1 mutant mouse model of SCLC, we now demonstrate a requirement for the Hh ligand Sonic Hedgehog (Shh) for the progression of SCLC. Conversely, we show that conditional Shh overexpression activates canonical Hh signaling in SCLC cells, and markedly accelerates tumor progression. When compared to mouse SCLC tumors expressing an activating, ligand-independent Smo mutant, tumors overexpressing Shh exhibited marked chromosomal instability and Smoothened-independent upregulation of Cyclin B1, a putative non-canonical arm of the Hh pathway. In turn, we show that overexpression of Cyclin B1 induces chromosomal instability in mouse embryonic fibroblasts lacking both Tp53 and Rb1. These results provide strong support for an autocrine, ligand-dependent model of Hh signaling in SCLC pathogenesis, and reveal a novel role for non-canonical Hh signaling through the induction of chromosomal instability.Copyright © The Autor(s) 2017.

Lung cancer is the leading cause of cancer death worldwide, and is frequently associated with the devastating paraneoplastic syndrome of cachexia. The potent immunomodulatory cytokine interleukin (IL)-6 has been linked with the development of lung cancer as well as cachexia; however, the mechanisms by which IL-6 promotes muscle wasting in lung cancer cachexia are illdefined. In this study, we report that the gp130F/F knock-in mouse model displaying hyperactivation of the latent transcription factor STAT3 via the common IL-6 cytokine family signalling receptor, gp130, develops cachexia during Kras-driven lung carcinogenesis. Specifically, exacerbated weight loss, early mortality and reduced muscle and adipose tissue mass were features of the gp130F/F:KrasG12D model, but not parental KrasG12D mice in which STAT3 was not hyperactivated. Gene expression profiling of muscle tissue in cachectic gp130F/F:KrasG12D mice revealed the upregulation of IL-6 and STAT3-target genes compared with KrasG12D muscle tissue. These cachectic features of gp130F/F:KrasG12D mice were abrogated upon the genetic normalization of STAT3 activation or ablation of IL-6 in gp130F/F:KrasG12D:Stat3- /+ or gp130F/F:KrasG12D:Il6-/- mice, respectively. Furthermore, protein levels of the soluble IL-6 receptor (sIL-6R), which is the central facilitator of IL-6 trans-signalling, were elevated in cachectic muscle from gp130F/F:KrasG12D mice, and the specific blockade of IL-6 trans-signalling, but not classical signalling, with an anti-IL-6R antibody ameliorated cachexia-related characteristics in gp130F/F:KrasG12D mice. Collectively, these preclinical findings identify transsignalling via STAT3 as the signalling modality by which IL-6 promotes muscle wasting in lung cancer cachexia, and therefore support the clinical evaluation of the IL-6 trans-signalling/STAT3 axis as a therapeutic target in advanced lung cancer patients presenting with cachexia.Copyright © 2017 Macmillan Publishers Limited, part of Spri

Malignant Rhabdoid Tumor (MRT) is a rare pediatric cancer of the kidney and CNS that is resistant to current treatment protocols. MRT is genetically characterized by homozygous inactivation of SMARCB1, a critical subunit of the SWI/SNF chromatin-remodeling complex. Next-generation sequencing data suggests that inactivation of SMARCB1 is the primary driver mutation, implicating epigenetic deregulation in the pathogenesis of MRT. Recently, we showed that sustained treatment of MRT cell lines with low-dose Panobinostat (LBH589), inhibited tumor growth by driving multi-lineage differentiation in vitro and in vivo. Furthermore, re-expression of physiological levels of SMARCB1 in G401 MRT cells phenocopied the low-dose LBH589 treatment and led to growth inhibition, senescence and terminal differentiation in vitro and in vivo. Enhancer of Zeste homolog 2 (EZH2), a core subunit of the Polycomb Repressive Complex 2 (PRC2), confers transcriptional silencing via the addition of methyl groups to Lysine 27 of Histone 3 (H3K27me ), and is a transcriptional target of SMARCB1. EZH2 expression and H3K27me were drastically reduced following sustained low-dose LBH589 treatment and re-expression of SMARCB1 in G401 MRT cells. Sustained siRNA knockdown of EZH2 in G401 cells resulted in reduced cell growth and changes in mRNA expression similar to those observed following low-dose LBH589 treatment and SMARCB1 re-expression. Treatment of MRT cells with the EZH2-catalytic domain inhibitor, GSK-126, had no effect on EZH2 expression and only partially reduced H3K27me and cell growth at doses 1nM-10muM suggesting important non-catalytic EZH2 function. However, MRT cells treated in combination with low-dose LBH589 and GSK-126, lost EZH2 and H3K27me expression and exhibited significantly reduced cell growth in vitro compared to single agent controls, revealing a synergistic relationship. Similar effects were observed in an in vivo xenograft model, with low-dose LBH589 and GSK-126 treatment leadi

Purpose: Malignant rhabdoid tumor (MRT) and atypical teratoid rhabdoid tumors (ATRT) are rare aggressive undifferentiated tumors primarily affecting the kidney and CNS of infants and young children. MRT are almost exclusively characterized by homozygous deletion or inactivation of the chromatin remodeling gene SMARCB1. SMARCB1 protein loss leads to direct impairment of chromatin remodeling and we have previously reported a role for this protein in histone acetylation. This provided the rationale for investigating the therapeutic potential of histone deactylase inhibitors (HDACi) in MRT. Experimental Design: Whereas previously HDACis have been used at doses and schedules that induce cytotoxicity, in the current studies we have tested the hypothesis, both in vitro and in vivo, that sustained treatment of human MRT with lowdose HDACi can lead to sustained cell growth arrest and differentiation. Result(s): Sustained low-dose panobinostat (LBH589) treatment led to changes in cellular morphology associated with a marked increase in the induction of neural, renal, and osteoblast differentiation pathways. Genome-wide transcriptional profiling highlighted differential gene expression supporting multilineage differentiation. Using mouse xenograft models, sustained lowdose LBH589 treatment caused tumor growth arrest associated with tumor calcification detectable by X-ray imaging. Histological analysis of LBH589-treated tumors revealed significant regions of ossification, confirmed by Alizarin Red staining. Immunohistochemical analysis showed increased TUJ1 and PAX2 staining suggestive of neuronal and renal differentiation, respectively. Conclusion(s): Low-dose HDACi treatment can terminally differentiate MRT tumor cells and reduce their ability to self-renew. The use of low-dose HDACi as a novel therapeutic approach warrants further investigation.Copyright © 2016 American Association for Cancer Research.

Introduction: Although neoadjuvant chemotherapy (NAC) for locally advanced breast cancer can improve operability and local disease control, there is a lack of reliable biomarkers that predict response to chemotherapy or long-term survival. Since expression of aldehyde dehydrogenase-1 (ALDH1) is associated with the stem-like properties of self-renewal and innate chemoresistance in breast cancer, we asked whether expression in serial tumor samples treated with NAC could identify women more likely to benefit from this therapy. Method(s): Women with locally advanced breast cancer were randomly assigned to receive four cycles of anthracycline-based chemotherapy, followed by four cycles of taxane therapy (Arm A), or the same regimen in reverse order (Arm B). Tumor specimens were collected at baseline, after four cycles, and then at surgical resection. ALDH1 expression was determined by immunohistochemistry and correlated with tumor response using Fisher's exact test while Kaplan-Meier method was used to calculate survival. Result(s): A hundred and nineteen women were enrolled into the study. Fifty seven (48%) were randomized to Arm A and 62 (52%) to Arm B. Most of the women (90%) had ductal carcinoma and 10% had lobular carcinoma. Of these, 26 (22%) achieved a pathological complete response (pCR) after NAC. There was no correlation between baseline ALDH1 expression and tumor grade, stage, hormone receptor, human epidermal growth factor receptor 2 (HER2) status and Ki67 index. ALDH1 negativity at baseline was significantly associated with pCR (P = 0.004). The presence of ALDH1(+) cells in the residual tumor cells in non-responding women was strongly predictive of worse overall survival (P = 0.024). Moreover, serial analysis of specimens from non-responders showed a marked increase in tumor-specific ALDH1 expression (P = 0.028). Overall, there was no survival difference according to the chemotherapy sequence. However, poorly responding tumours from women receiving docetaxel

Gli transcription factors of the Hedgehog (Hh) pathway have been reported to be drivers of malignant mesothelioma (MMe) cell survival. The Gli inhibitor GANT61 induces apoptosis in various cancer cell models, and has been associated directly with Gli inhibition. However various chemotherapeutics can induce cell death through generation of reactive oxygen species (ROS) but whether ROS mediates GANT61-induced apoptosis is unknown. In this study human MMe cells were treated with GANT61 and the mechanisms regulating cell death investigated. Exposure of MMe cells to GANT61 led to G1 phase arrest and apoptosis, which involved ROS but not its purported targets, GLI1 or GLI2. GANT61 triggered ROS generation and quenching of ROS protected MMe cells from GANT61-induced apoptosis. Furthermore, we demonstrated that mitochondria are important in mediating GANT61 effects: (1) ROS production and apoptosis were blocked by mitochondrial inhibitor rotenone; (2) GANT61 promoted superoxide formation in mitochondria; and (3) mitochondrial DNA-deficient LO68 cells failed to induce superoxide, and were more resistant to apoptosis induced by GANT61 than wild-type cells. Our data demonstrate for the first time that GANT61 induces apoptosis by promoting mitochondrial superoxide generation independent of Gli inhibition, and highlights the therapeutic potential of mitochondrial ROS-mediated anticancer drugs in MMe.

Background: Although it has been shown that Gli transcription factors are the major intracellular target of the small molecule inhibitor GANT61, the underlying mechanism by which GANT61 inhibits cancer cell growth remains unclear. In this study, we aimed to elucidate the mechanisms that underlie the induction of mesothelioma cell apoptosis by GANT61. Method: Human mesothelioma cells were treated with GANT61. Levels of apoptosis and reactive oxygen species (ROS) were quantified by flow cytometry. Results: Our study showed that GANT61 induces growth inhibition and apoptosis in mesothelioma cells through the induction of oxidative stress. Exposure of mesothelioma cells to GANT61 resulted in the rapid production of ROS. Quenching of ROS by the antioxidants N-acetylcysteine and L-glutathione blocked the induction of apoptosis and depolarization of mitochondrial membrane potential by GANT61, indicating that ROS overproduction is the primary driver for the pro-apoptotic activity of GANT61. Although GANT61 could inhibit Gli transcriptional activity, the inhibition of Gli was not sufficient to initiate apoptosis in mesothelioma cells. Further study revealed that the ROS species generated was superoxide from the mitochondria, and that mitochondrial electron transport chain inhibition by rotenone significantly suppressed the pro-apoptotic effect of GANT61. Conclusion: Our study provides evidence that GANT61 is a potent anti-mesothelioma therapeutic agent, which induces apoptosis in mesothelioma cells through the generation of mitochondrial superoxide.

The landscape of advanced non-small lung cancer (NSCLC) therapies has rapidly been evolving beyond chemotherapy over the last few years. The discovery of oncogenic driver mutations has led to new ways in classifying NSCLC as well as offered novel therapeutic targets for anticancer therapy. Targets such as epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) gene rearrangements have successfully been targeted with appropriate tyrosine kinase inhibitors (TKIs). Other driver mutations such as ROS, MET, RET, BRAF have also been investigated with targeted agents with some success in the early phase clinical setting. Novel strategies in the field of immune-oncology have also led to the development of inhibitors of cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 receptor (PD-1), which are important pathways in allowing cancer cells to escape detection by the immune system. These inhibitors have been successfully tried in NSCLC and also now bring the exciting possibility of long term responses in advanced NSCLC. In this review recent data on novel targets and therapeutic strategies and their future prospects are discussed.Copyright © 2015 by the authors; licensee MDPI, Basel, Switzerland.

Background: Preclinical studies in small cell lung cancer cell lines and xenograft models have shown that Hyaluronic acid (HA) can be effectively used to deliver Irinotecan (IR) and selectively decrease CD44 expressing (stem cell-like) tumour cells and prolong duration of response. This "proof of principle" study aims to replicate these findings in the clinical setting and obtain data on safety and response rates. Method(s): Extensive Small Cell Lung Cancer (ESCLC) patients with measurable disease (suitable for biopsy), PS 0-2, medically fit and able to give informed consent were screened for this study. A safety cohort (n=5) were treated with HA-IR (150mg/m2) and Carboplatin(C) at 5 AUC, q3 weekly with subsequent patients stratified as 1st or 2nd line. All 2nd line patients received open label HA-IR+C while 1st line patients were randomized to receive either HA-IR+C or equivalent dosing regimen of IR+C. Sequential tumour biopsies were obtained at baseline and after 1 or 2 cycles. Tumour response was measured by CT/PET scan at baseline, after 1 cycle and every 2 cycles subsequently. A final biopsy at disease progression was planned. Blood samples for circulating tumour cells (CTCs) were obtained at baseline, and at every cycle. Result(s): Patients N=16, Age: median= 60; Range 39-78. Three 2nd line patients were not evaluated due to rapid early disease progression after Cycle 1. Overall toxicity profile of HA-IR+C was similar to IR+C with grade III/IV diarrhoea and neutropenia seen in 15% and 20% respectively. One patient (7%) had grade III anaemia, while no grade III/IV nausea or vomiting was observed. No biopsy related complications were observed. Of 13 patients evaluated for tumour response, the overall response rate was 60% with 1 (7%) complete and 7 (53%) partial responses. Three patients (24%) achieved stable disease while 2 patients (16%) progressed during the treatment. To date median progression-free survival is 5.9 months (7.6 months in first line and 3.1 m

Background: Lung cancer has the highest cancer incidence and mortality worldwide. Small cell lung cancer (SCLC) accounts for 15% of all cases. Platinum-based chemotherapy induces responses in up to 70%. However, treatment-resistant recurrence is near universal, and 5-year survival remains poor at 1-2%. Therefore, there is urgent need for pre-clinical models that accurately recapitulate the parent tumour and allow testing for predictive biomarkers of response and resistance to drugs, and also screening of novel anticancer agents. Furthermore, as the vast majority of SCLC are inoperable, it is crucial that the mode of tumour tissue acquisition be minimally invasive and repeatable in cases of recurrence. Here we describe a novel pre-clinical model using samples obtained by the minimally invasive technique of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to develop primary xenografts of SCLC. Method(s): Cell suspensions from samples of SCLC obtained by EBUS-TBNA were implanted directly into the flanks of NSG (Non- Obese Diabetic, Severe Combined Immune Deficient, IL2Rgamma knockout) mice to generate primary xenografts. The mice were monitored for tumour growth, and if engraftment was successful, pre-graft and post-graft tumours were compared in terms of morphology, immunohistochemistry and molecular characteristics. Result(s): Thus far, 14 SCLC specimens have been implanted, with 7 cases completing 6 months of tumour monitoring. Of these, 6 have undergone successful engraftment (86%). Samples typically contained over 1 million tumour cells with minimal stromal contamination. Mean engraftment lag time was 96 days. In all cases of engraftment, histological and molecular fidelity to the original tumour was demonstrated. Conclusion(s): This is the first report of the generation of a primary xenograft model of lung cancer using a new method of tissue acquisition by EBUS-TBNA. Furthermore, it is the largest reported group of primary xenografts o

Background: Originating from Primordial Germ Cells/gonocytes and developing via a precursor lesion called Carcinoma In Situ (CIS), Germ Cell Cancers (GCC) are the most common cancer in young men, subdivided in seminoma (SE) and non-seminoma (NS). During physiological germ cell formation/maturation, epigenetic processes guard homeostasis by regulating the accessibility of the DNA to facilitate transcription. Epigenetic deregulation through genetic and environmental parameters (i.e. genvironment) could disrupt embryonic germ cell development, resulting in delayed or blocked maturation. This potentially facilitates the formation of CIS and progression to invasive GCC. Therefore, determining the epigenetic and functional genomic landscape in GCC cell lines could provide insight into the pathophysiology and etiology of GCC and provide guidance for targeted functional experiments. Results: This study aims at identifying epigenetic footprints in SE and EC cell lines in genome-wide profiles by studying the interaction between gene expression, DNA CpG methylation and histone modifications, and their function in the pathophysiology and etiology of GCC. Two well characterized GCC-derived cell lines were compared, one representative for SE (TCam-2) and the other for EC (NCCIT). Data were acquired using the Illumina HumanHT-12-v4 (gene expression) and HumanMethylation450 BeadChip (methylation) microarrays as well as ChIP-sequencing (activating histone modifications (H3K4me3, H3K27ac)). Results indicate known germ cell markers not only to be differentiating between SE and NS at the expression level, but also in the epigenetic landscape. Conclusion: The overall similarity between TCam-2/NCCIT support an erased embryonic germ cell arrested in early gonadal development as common cell of origin although the exact developmental stage from which the tumor cells are derived might differ. Indeed, subtle difference in the (integrated) epigenetic and expression profiles indicate TCam-2 to

Rhabdoid Tumour (RT) is a rare, malignant tumour of infancy arising mainly in the kidney or CNS. RTs are highly resistant to conventional treatments and outcomes remain poor despite aggressive multimodal therapy. The sole recurrent genetic abnormality in RT is homozygous deletion or inactivation of the chromatin-remodelling gene, SMARCB1 thus providing an ideal model for exploring epigenetic therapies such as the use of histone deacetylase inhibitors (HDACi). This study investigates the effects of HDACi on cell growth and differentiation in RT cell lines and mouse xenografts and analyses resultant changes in gene expression. METHOD(S): In vitro based cell proliferation, cell cycle and colony forming assays were undertaken and the altered gene expression profiles upon HDACi treatment were analysed using Illumina expression beadchip arrays and quantitative real-time PCR. The effects of HDACi on RT cell differentiation, both in vitro and in a mouse xenograft tumour model, were determined and post-treatment, cells or tissue were stained with various differentiation markers. RESULT(S): HDACi treatment inhibited RT cell growth and self-renewal in vitro and halted tumour growth in vivo. After 21 days of continuous, low dose treatment with HDACi LBH589, gene expression signatures and qualitative differentiation marker staining of cells or tissue showed evidence of osteoblast differentiation and bone formation, as well as neuronal differentiation. CONCLUSION(S): Our data suggest that low dose HDACi treatment has the potential to inhibit RT cell growth and drive differentiation. This makes differentiation therapy an exciting avenue to explore and avoids the challenges of achieving a cytotoxic response in patients. The ability of HDACi to differentiate tumour cells and reduce their ability to self-renew warrants further investigation, as it provides an appealing means of tackling the issue of tumour recurrence.

Renal primary cilia are microscopic sensory organelles found on the apical surface of epithelial cells of the nephron and collecting duct. They are based upon a microtubular cytoskeleton, bounded by a specialized membrane, and contain an array of proteins that facilitate their assembly, maintenance and function. Cilium-based signalling is important for the control of epithelial differentiation and has been implicated in the pathogenesis of various cystic kidney diseases and in renal repair. As such, visualizing renal primary cilia and understanding their composition has become an essential component of many studies of inherited kidney disease and mechanisms of epithelial regeneration. Primary cilia were initially identified in the kidney using electron microscopy and this remains a useful technique for the high resolution examination of these organelles. New reagents and techniques now also allow the structure and composition of primary cilia to be analysed in detail using fluorescence microscopy. Primary cilia can be imaged in situ in sections of kidney, and many renal-derived cell lines produce primary cilia in culture providing a simplified and accessible system in which to investigate these organelles. Here we outline microscopy-based techniques commonly used for studying renal primary cilia. There is a growing recognition of the importance of cilia in kidney biology and of the impact of mutations of proteins associated with the primary cilia as the unifying feature of cystic kidney diseases. This review focuses on current methodologies for visualizing renal primary cilia. © 2012 The Authors. Nephrology © 2012 Asian Pacific Society of Nephrology.

The cancer stem cell (CSC) model is based on a myriad of experimental and clinical observations suggesting that the malignant phenotype is sustained by a subset of cells characterized by the capacity for self-renewal, differentiation and innate resistance to chemotherapy and radiation. CSC may be responsible for disease recurrence after definitive therapy and may therefore be functionally synonymous with minimal residual disease. Similar to other solid tumours, several putative surface markers for lung CSC have been identified, including CD133 and CD44. In addition, expression and/or activity of the cytoplasmic enzyme aldehyde dehydrogenase ALDH and capacity of cells to exclude membrane permeable dyes (known as the 'side population') correlate with stem-like function in vitro and in vivo. Embryonic stem cell pathways such as Hedgehog, Notch and WNT may also be active in lung cancers stem cells and therefore may be therapeutically targetable for maintenance therapy in patients achieving a complete response to surgery, radiotherapy or chemotherapy. This paper will review the evidence regarding the existence and function of lung CSC in the context of the experimental and clinical evidence and discuss some ongoing controversies regarding this model. © 2013 Asian Pacific Society of Respirology.

Aim: To examine the role of deregulated gp130 signalling in the development of lung tumours and cancer-related cachexia in two models of lung cancer. Method(s): We used the gp130F/F (FF) mouse which carries a knock-in mutation in gp130, the critical co-receptor for the IL-6 cytokine family. These mice display elevated IL-6 levels and hyper-activated Stat3 in the absence of gp130-mediated PI3K/Akt and Mapk signalling. Two separate models were undertaken using FF and gp130+/+ (WT) mice commencing at 6 weeks of age. Model 1: Mice were exposed to a cigarette carcinogen (NKK) and observed over 16 weeks prior to the cellular and molecular evaluation of lung tumourigenesis. Model 2: FF and WT mice harbouring a conditionally activated oncogenic Kras allele were observed over 6 weeks after Kras activation. In addition to assessing development of lung tumours, mouse weight and muscle and fat mass were examined. Result(s): In NNK-treated animals there was a significant reduction in the number of tumours in FF mice compared to WT mice. This appeared to be independent of Stat3 as the number of tumours was unchanged from FF levels in mice with genetically normalized Stat3 levels. PI3K/Akt and Mapk pathway specific PCR arrays showed deregulation of a number of key oncogenes and tumour suppressor genes in the FF mouse suggesting a role for gp130- mediated PI3K/Akt and Mapk signalling. In the Kras model the key finding was marked weight loss in the FF mouse, with loss of both fat and lean tissue mass. This weight loss was attenuated by genetically normalizing Stat3. Conclusion(s): Our data suggest that deregulated IL-6/gp130 signalling contributes to both the development of lung cancer and its complications.

Background Expression of aldehyde dehydrogenase 1A1 (ALDH1A1) and CD133 has been functionally associated with a stem cell phenotype in normal and malignant cells. The prevalence of such cells in solid tumours should therefore correlate with recurrence and/ or metastasis following definitive surgical resection. The aim of this study was to evaluate the prognostic significance of ALDH1A1 and CD133 in surgically resected, early stage non-small cell lung cancer (NSCLC). Methods A retrospective analysis of ALDH1A1 and CD133 expression in 205 patients with pathologic stage I NSCLC was performed using immunohistochemistry. The association between the expression of both markers and survival was determined. Results We identified 62 relapses and 58 cancer-related deaths in 144 stage 1A and 61 stage 1B patients, analysed at a median of 5-years follow-up. Overexpression of ALDH1A1 and CD133, detected in 68.7% and 50.7% of primary tumours, respectively, was an independent prognostic indicator for overall survival by multivariable Cox proportional hazard model (p=0.017 and 0.039, respectively). Overexpression of ALDH1A1, but not of CD133, predicted poor recurrence-free survival (p=0.025). When categorised into three groups according to expression of ALDH1A1/CD133, patients with overexpression of both ALDH1A1 and CD133 belonged to the group with the shortest recurrence-free and overall survival (p=0.015 and 0.017, respectively). Conclusions Expression of ALDH1A1 and CD133, and coexpression of ALDH1A1 and CD133, is strongly associated with poor survival in early-stage NSCLC following surgical resection. These data are consistent with the hypothesis that expression of stem cell markers correlates with recurrence as an indirect measure of selfrenewal capacity.

Oncogenic driver mutations frequently occur in lung cancer and play role in carcinogenesis. These mutations are usually associated with distinct clinical and histological features and are attractive targets for anticancer therapy. Recently, several molecularly distinct phenotypes of NSCLC based on specific and mutually exclusive genetic derangements have been described. Few targets like epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) gene rearrangements have successfully been targeted with EGFR tyrosine kinase inhibitors (TKIs) and crizotinib, respectively. Many more inhibitors of specific driver mutations involving genes like ROS, c-MET, FGFR, mTOR, IGFR and RET are currently under development. However, efforts to target some mutated genes like K-RAS have been unsuccessful. Moreover, the emerging challenge of acquired resistance to initially effective therapy is becoming another major concern. In this review recent data on novel molecular targets and their future prospects are discussed. © 2013 Elsevier Ltd. All rights reserved.

Background The Hedgehog (HH) signaling pathway is critical for embryonic development and adult homeostasis. Recent studies have identified regulatory roles for this pathway in certain cancers with mutations in the HH pathway genes. The extent to which mutations of the HH pathway genes are involved in the pathogenesis of malignant mesothelioma (MMe) is unknown. Methodology/Principal Findings Real-time PCR analysis of HH pathway genes PTCH1, GLI1 and GLI2 were performed on 7 human MMe cell lines. Exon sequencing of 13 HH pathway genes was also performed in cell lines and human MMe tumors. In silico programs were used to predict the likelihood that an amino-acid substitution would have a functional effect. GLI1, GLI2 and PTCH1 were highly expressed in MMe cells, indicative of active HH signaling. PTCH1, SMO and SUFU mutations were found in 2 of 11 MMe cell lines examined. A non-synonymous missense SUFU mutation (p.T411M) was identified in LO68 cells. In silico characterization of the SUFU mutant suggested that the p.T411M mutation might alter protein function. However, we were unable to demonstrate any functional effect of this mutation on Gli activity. Deletion of exons of the PTCH1 gene was found in JU77 cells, resulting in loss of one of two extracellular loops implicated in HH ligand binding and the intracellular C-terminal domain. A 3-bp insertion (69_70insCTG) in SMO, predicting an additional leucine residue in the signal peptide segment of SMO protein was also identified in LO68 cells and a MMe tumour. Conclusions/Significance We identified the first novel mutations in PTCH1, SUFU and SMO associated with MMe. Although HH pathway mutations are relatively rare in MMe, these data suggest a possible role for dysfunctional HH pathway in the pathogenesis of a subgroup of MMe and help rationalize the exploration of HH pathway inhibitors for MMe therapy.

Background: Over 40% of lung cancers present as inoperable, locally advanced disease. Current diagnosis is based on endobronchial biopsy or cytology, which results in advanced stage lung cancer being markedly under-represented in conventional tissue banks of surgically resected stage 1 tumours. Accurate preclinical models of advanced stage disease are needed for testing of new therapeutic agents, as well as for the development of biomarkers. Here we describe a novel preclinical model using samples obtained by the minimally invasive technique of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in patients with locally advanced lung cancer. Method(s): Cell suspensions from samples obtained by EBUS-TBNA were implanted directly into the flanks of NSG (Non-Obese Diabetic, Severe Combined Immune Deficient, IL2Rgamma knockout) mice to generate primary xenografts. Once engrafted, serial passage was performed in nude athymic mice. Result(s): Freshly obtained samples from EBUS-TBNA typically contained over 100,000 viable tumour cells, with less then 10% stromal contamination. Engraftment and serial passage were achieved in 5 of 15 (33%) cases. The mean duration from implantation to engraftment was 99 days. In all 5 cases, the histological subtype was identical in patient and xenograft tumours despite serial passage. Key conclusions: This is the first report of the generation of a mouse xenograft model using fresh human EBUS-TBNA samples. The primary xenograft lines derived from these specimens may provide the much-needed basis for more accurate preclinical modeling of locally advanced lung cancer and a means to investigating targeted therapeutic regimens.

Drug carrier particles composed of poly(ethylene glycol)-co-poly(sebacic acid) (PEG-PSA) have been shown capable of efficient aerosolization into model lungs and the ability to rapidly penetrate human mucus. Here, we develop PEG-PSA particles (Etop/PEG-PSA) that encapsulate up to 40% etoposide by weight in a one step process, release it continuously for 6 days in vitro, and maintain its cytotoxic activity against a human lung tumor cell line in vitro. We further show that Etop/PEG-PSA injected intratumorally effectively suppress human lung tumor growth in a xenograft mouse model, with 100% survival after 31 days. In contrast, 0% survival was observed by day 24 in animals that received free etoposide (either intratumoral or intraperitoneal administration) or placebo particles intratumorally. These findings support PEG-PSA as a drug delivery platform for improved local therapy of cancer. © 2009 Elsevier Ltd. All rights reserved.

Hypermethylated in cancer-1 (HIC1) is a tumor suppressor frequently targeted for promoter hypermethylation in medulloblastoma, an embryonal tumor of the cerebellum. Recently, we showed that HIC1 is a direct transcriptional repressor of ATOH1, a proneural transcription factor required for normal cerebellar development, as well as for medulloblastoma cell viability. Because demethylating agents can induce reexpression of silenced tumor suppressors, restoring HIC1 function may present an attractive therapeutic avenue in medulloblastoma by exploiting an apparent addiction to ATOH1. ©2008 American Association for Cancer Research.

Background: Angiogenesis is the formation of neovasculature from a pre-existing vascular network. Progression of solid tumors including lung cancer is angiogenesis-dependent. We previously introduced a bioinformatics-based methodology to identify endogenous anti-angiogenic peptide sequences, and validated these predictions in vitro in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays. Method(s): One family of peptides with high activity is derived from the alpha-fibrils of type IV collagen. Based on the results from the in vitro screening, we have evaluated the ability of a 20 amino acid peptide derived from the alpha5 fibril of type IV collagen, pentastatin-1, to suppress vessel growth in an angioreactor-based directed in vivo angiogenesis assay (DIVAA). In addition, pentastatin-1 suppressed tumor growth with intraperitoneal peptide administration in a small cell lung cancer (SCLC) xenograft model in nude mice using the NCI-H82 human cancer cell line. Result(s): Pentastatin-1 decreased the invasion of vessels into angioreactors in vivo in a dose dependent manner. The peptide also decreased the rate of tumor growth and microvascular density in vivo in a small cell lung cancer xenograft model. Conclusion(s): The peptide treatment significantly decreased the invasion of microvessels in angioreactors and the rate of tumor growth in the xenograft model, indicating potential treatment for angiogenesis-dependent disease, and for translational development as a therapeutic agent for lung cancer. © 2010 Koskimaki et al; licensee BioMed Central Ltd.