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S.G. Gray
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MINI 34 - RNA and miRNA (ID 162)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:C. Mascaux, L.(. Wang
- Coordinates: 9/09/2015, 18:30 - 20:00, 205+207
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MINI34.07 - A Novel microRNA Signature Associated with Cisplatin Resistance in NSCLC (ID 2709)
18:30 - 20:00 | Author(s): S.G. Gray
- Abstract
- Presentation
Background:
MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that range in size from 19 to 25 nucleotides. Alteration in miRNA expression can cause them to act as either tumour suppressor or oncogenes. They have also been shown to regulate a number of processes involved in tumour biology such as metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemo- and radio-resistance in many solid tumours, including lung cancer.
Methods:
An isogenic model of cisplatin resistance was established by chronically exposing a panel of NSCLC cell lines (MOR, H460, A549, SKMES-1, H1299) to cisplatin for 12 months, generating cisplatin resistant (CisR) sublines from their corresponding age-matched parental (PT) cells. MicroRNA expression profiling was carried out using 7th generation miRCURY LNA™ microRNA arrays consisting of 1,919 miRNAs (Exiqon). MicroRNAs that were significantly increased in CisR sublines were inhibited using antagomirs (Exiqon), while those that were significantly decreased were over-expressed using pre-miRs (Ambion). Functional studies examining clonogenic survival ability, proliferation (BrdU) and apoptosis (Annexin V/PI) were subsequently carried out in the presence or absence of cisplatin. To examine the translational relevance of these microRNAs, their expression was further examined in a cohort of pre-treatment matched normal and tumour lung tissues from NSCLC patients of different histological subtypes. Validation of this miRNA signature is currently being investigated in serum samples from this same cohort of patients and normal controls.
Results:
MicroRNA profiling analysis identified ten miRNAs which were significantly altered between parental and corresponding cisplatin resistant lung cancer cell lines. Validation of these miRNAs by real-time PCR (qPCR) identified a specific 5-miR signature that was significantly altered in CisR cells relative to their parental counterparts. Modification of these microRNAs altered the response of resistant cells to the cytotoxic effects of cisplatin and decreased the clonogenic survival of CisR cells when treated with increasing doses of cisplatin (0.1µM-10μM). Significant differential expression was found between normal and tumour tissues across each histological subtype, highlighting the potential use of these microRNAs as markers of response to cisplatin therapy in NSCLC patients. Three miRNAs (miR-A, B, C) belonging to the same family were significantly altered in tumour lung tissue of adenocarcinoma and squamous cell histology compared to matched normal lung tissue. MicroRNA-D expression was significantly altered in squamous cell carcinomas while miR-E was differentially expressed in adenocarcinomas only. Data relating to the expression of this novel signature in the circulation of our NSCLC patient cohort and normal controls will be presented at WCLC 2015.
Conclusion:
We have identified and validated a novel miRNA signature associated with cisplatin resistance in a panel of cisplatin resistant cell lines and in patient lung tumours. Genetic manipulation of these specific miRNAs in vitro altered the cisplatin resistant cell response to the cytotoxic effects of cisplatin chemotherapy. The data obtained from this study may provide a basis for the potential development of a companion diagnostic for lung cancer patients who are most likely to benefit, or not, from cisplatin chemotherapy.
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ORAL 42 - Drug Resistance (ID 160)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:R.C. Doebele, J.V. DeGregori
- Coordinates: 9/09/2015, 18:30 - 20:00, Mile High Ballroom 4a-4f
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ORAL42.06 - Cancer Stem Cells: Targeting Aldehyde Dehydrogenase 1 (ALDH1) as a Novel Strategy in Cisplatin Resistant Non-Small Cell Lung Cancer? (ID 2724)
18:30 - 20:00 | Author(s): S.G. Gray
- Abstract
- Presentation
Background:
Cisplatin is the backbone of chemotherapeutic treatment of lung cancer. Unfortunately the development of resistance has become a major challenge in the use of this cytotoxic drug. Understanding the mechanisms underlying this resistance phenotype may potentially result in the development of novel agents that may enhance the sensitivity of cisplatin chemotherapy in the clinical setting. The root of this resistance is hypothesized to be due to the presence of a rare cancer stem cell (CSC) population within the tumour that can reform a heterogenic tumour, resulting in recurrence and resistance following cisplatin chemotherapy.
Methods:
An isogenic model of cisplatin resistance was established by chronically exposing a panel of NSCLC cell lines (H460, SKMES, H1299) to cisplatin for 12months, thereby creating cisplatin resistant (CisR) sublines and their corresponding age-matched parental (PT) cells. To identify a CSC population within the resistant sublines, PT and CisR cell lines representing the three classifications of NSCLC were stained for ALDH1 using the Aldefluor kit (Stemcell Technologies). ALDH1 positive (+ve) and negative (-ve) subpopulations were isolated and their functional characteristics assessed. Proliferation and survival of ALDH1+ve fractions in response to cisplatin was assessed using BrdU and clonogenic survival assays relative to ALDH1-ve cells. ALDH1 subpopulations were examined for asymmetric division and expression of the human embryonic stem cell markers Nanog, Oct-4, Sox-2, Klf-4 and c-Myc and CD133. To confirm that this ALDH1+ve population is associated with cisplatin treatment, PT and CisR cells were chronically exposed to high dose cisplatin for 2 weeks and stained for ALDH1 and re-assessed for stemness qualities. Apoptosis and clonogenic survival of PT and CisR cells was assessed in response to selective inhibition of ALDH1 using diethylaminobenzaldehyde (DEAB) in combination with cisplatin. Xenograft studies in NOD/SCID mice are currently under investigation to examine the tumourigenic potential of isolated subpopulations of ALDH1.
Results:
A significant ALDH1+ve population was detected in CisR sublines, but not in their PT counterparts. Characterisation of the ALDH1+ve subpopulation confirmed enhanced expression of stemness markers, increased resistance and clonogenic survival in response to cisplatin compared to their ALDH1-ve counterparts, and the ability to asymmetrically divide. Chronic cisplatin treatment of the PT cell lines for 2 weeks increased resistance to cisplatin, increased stemness marker expression and induced the emergence of an ALDH1+ve population. Chronic high dose cisplatin treatment significantly expanded the ALDH1+ve population in the CisR cell lines. Importantly, inhibition of ALDH1 activity, with DEAB, decreased the mean cell viability, clonogenic survival capacity and increased cisplatin-induced apoptosis of the CisR cells when used in combination with cisplatin, an effect not seen in the PT cells.
Conclusion:
In this study, we have demonstrated the existence of a putative CSC population within our model of isogenic cisplatin resistant cell lines and suggest a role for ALDH1 inhibition as a potential therapeutic strategy in re-sensitizing chemoresistant lung cancer cells to the cytotoxic effects of cisplatin. Further studies will focus on re-purposing of FDA-approved ALDH1 inhibitor, Disulfiram (Antabuse), used in the treatment of chronic alcoholism as a potential combination therapy to prime chemoresistant cells to cisplatin.
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-088 - Lung Cancer Cells Can Alter the Behaviour of Normal Bronchial Epithelial Cells Through Multiple Mechanisms (ID 1312)
09:30 - 17:00 | Author(s): S.G. Gray
- Abstract
Background:
Lung cancer is one of the most heterogeneous of all solid cancers. This may in part be due to hi-jacking and additional bystander affects that are exerted on the normal lung cell population by the cancer cells. A number of pathways may be stimulated through soluble factors or effector filled vesicles such as exosomes secreted by cancer cells. The aim of this project was to evaluate the effects of non-small cell lung cancer (NSCLC) cells on an immortalised normal bronchial epithelial cell line.
Methods:
A normal bronchial epithelial cell line (HBEC4) was exposed to adenocarcinoma, large cell and squamous NSCLC cell lines and a number of phenotypic and genotypic characterisations were undertaken. These included cellular proliferation (BrdU ELISA), gene (RT-PCR) and miRNA expression screening (Nanostring). The effect of cancer exosome fractions was also determined.
Results:
Exposure to various subtypes of NSCLC significantly increased the cellular proliferation rate of the immortalised cell line in a number of models. Expression of a number of miRNAs were altered in the normal cells pre- and post exposure to the cancer cells. Various stem cell factor markers (KLF4, Oct, c-myc) were also significantly changed at the mRNA level. In addition, exosome fractions altered the behaviour of the normal cell line, likewise stimulating cell proliferation.
Conclusion:
Lung cancer cells may influence normal cell behaviour in both a direct and indirect manner using multiple mechanisms. Normal bronchial epithelial cells with stem like features may be induced to proliferate and behave in a malignant manner. This, akin to Hodgkin’s lymphoma, may contribute significantly to the composition of the tumour. Furthermore this observation may contribute to the heterogeneity of lung cancer tumours and affect treatment response. Ongoing studies are evaluating these effects in novel 2D and 3D culture systems.
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P1.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 224)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 2
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.08-013 - Long Non-Coding RNAs Associated with Lysine Demethylases Are Overexpressed and Epigenetically Regulated in Malignant Pleural Mesothelioma (ID 1757)
09:30 - 17:00 | Author(s): S.G. Gray
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is an aggressive rare cancer affecting the pleura and is predominatly associated with prior exposure to asbestos. Treatment options are limited, and most patients die within 24 months of diagnosis. The current standard of care for MPM patients is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed), yet most patients die within 24 months of diagnosis. Lysine Demethylases (KDMs) containing a JmjC domain regulate gene expression by “erasing or removing” methylation on histones in chromatin. Members of this family are frequently found to have aberrant expression in cancer and currently are actively pursued as candidate pharmaceutical therapeutic targets. We have shown that various members of the JmjC family of KDMs have significantly altered expression in MPM. Long non-coding RNAs (lncRNAs) belong to a group of RNAs that are usually more than 200 nucleotides long and play important roles in different regulatory processes, including regulation of gene expression. Several lncRNAs have also been shown to play a role as oncogenic molecules in different cancer cells (one example being HOTAIR). Altered expression of lncRNAS therefore make them candidate biomarkers with diagnostic and therapeutic potential in cancer. Several such lncRNAs have now been shown to locate to the same chromosomal region as various KDMs. These are KDM4A/KDM4A-AS1, KDM5B/KDM5B-AS1 (also known as PCAT6), KDM5C/ AY927613.1 and JARID2/JARID2-AS1. We therefore examined the expression of these lncRNAs in MPM.
Methods:
A panel of MPM cell lines were screened for expression of KDM4A-AS1, KDM5B-AS1, AY927613.1 and JARID2-AS1 by RT-PCR. lncRNA transcript levels were subsequently examined by RT-PCR in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies. The effects of KDM and HDAC inhibitors on their expression was also examined.
Results:
The expression of the various lncRNAs was detectable across our panel of cell lines. In primary tumours the expression of these lncRNAs were significantly elevated in malignant MPM compared to benign pleura (p<0.05), and significant differences were also observed when samples were analysed across different histological subtypes.
Conclusion:
The expression of these lncRNAs are significantly altered in MPM. We have cloned KDM4A-AS1 and PCAT6 into overexpression constructs and future studies will assess the effects of these lncRNAs overexpression on mesothelioma proliferation, cellular health and gene expression to determine their potential role in mesothelioma.
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P1.08-014 - The Small Molecule Inhibitor, LCRF004, Is Effective in Targeting the RON/MST1R Pathway in Malignant Pleural Mesothelioma (ID 1311)
09:30 - 17:00 | Author(s): S.G. Gray
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer. We have previously identified RON as frequently activated in MPM patient samples and cell lines. RON is a member of the MET proto-oncogene family and is bound by macrophage stimulating protein (MSP). High positivity for total RON by IHC was an independent predictor of favourable prognosis. Additionally, elevated expression levels of MSP correlated with better survival. The aim of this study was to further examine the MSP-RON signalling axis in MPM using a RON inhibitor, LCRF004.
Methods:
MPM cell lines and a normal mesothelial cell line were screened for the expression of RON and MSP at the protein (Western) and mRNA (RT-PCR) level. Downstream mediators affected by MSP stimulation and LCRF004 were identified using a proteome profiler array. The effect of LCRF004 and MSP were examined using proliferation (BrdU ELISA), viability (High Content Analysis), migration (xCELLigence), apoptosis and cell cycle (HCA) assays. A xenograft study was also completed.
Results:
Treatment with LCRF004 resulted in a significant decrease in proliferation, viability and migration in vitro and reduced tumour growth in vivo (p<0.05, compared with vehicle control). In addition, LCRF004 significantly increased apoptosis. In terms of cell cycle, drug treatment decreased cells in 2n, whilst increasing cells in the G0/G1 phase. Experiments are on going to further characterise the mechanism of action of LCRF004.
Conclusion:
The in vivo and in vitro data generated in this study, indicates that the MSP-RON signalling axis is a potential target in MPM. Targeting the RTK domain of the RON receptor with a small molecule inhibitor is an effective interventional strategy in MPM.
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P2.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 225)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.08-006 - The JmjC Family of Lysine Demethylases Are Overexpressed and Potential Therapeutic Targets in Malignant Pleural Mesothelioma (ID 1753)
09:30 - 17:00 | Author(s): S.G. Gray
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is an aggressive rare cancer affecting the pleura and is predominatly associated with prior exposure to asbestos. Treatment options are limited, and most patients die within 24 months of diagnosis. The current standard of care for MPM patients is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed), yet most patients die within 24 months of diagnosis. There is therefore an urgent unmet need to identify new therapeutic options for the treatment of MPM. Asbestos fibres contain of transition metals and their ability to both adsorb and accumulate these metals was one of the first mechanisms suggested for explaining the toxic and particularly carcinogenic effects of asbestos. One of the transition metals in asbestos fibres is iron, and therefore asbestos fibres may cause an alteration of iron homeostasis in the tissue. In addition, asbestos fibres have also been shown to have high affinity for histones, and therefore may result in high accumulation of iron around chromatin. Lysine Demethylases (KDMs) containing a JmjC domain require both Fe2+ and 2-oxoglutarate as co-factors to regulate gene expression by “erasing or removing” methylation on histones in chromatin. Members of this family are frequently found to have aberrant expression in cancer and currently are actively pursued as candidate pharmaceutical therapeutic targets. Given that asbestos increases iron levels, this may result in aberrant KDM activity, and these KDMs could therefore be novel candidate targets in mesothelioma. We therefore examined the expression of several JmjC containing KDMs in MPM and assessed their potential for therapeutic intervention in mesothelioma using existing small molecule inhibitors.
Methods:
A panel of MPM cell lines were screened for expression of KDM4A-D, KDM5A/B and KDM6A/B by RT-PCR. mRNA levels were subsequently examined by RT-PCR in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies. IHC was performed for KDM4A on a cohort of FFPE specimens. The effects of treatments with small molecule inhibitors targeting these proteins on both cellular health and gene expression were assessed.
Results:
The expression of the various KDMs was detectable across our panel of cell lines. In primary tumours the expression of these KDMs were significantly elevated in malignant MPM compared to benign pleura (p<0.05), and significant differences were also observed when samples were analysed across different histological subtypes. Treatment of mesothelioma cell lines with various small molecule inhibitors caused significant effects on cellular health and on the expression of a panel of genes.
Conclusion:
The expression of KDMs are significantly altered in MPM. Small molecule inhibitors directed against these KDMs show potential therapeutic efficacy with significant anti-proliferative effects. We continue to assess the effects of these compounds on gene expression and cellular health to confirm their potential utility as novel therapies for the treatment of MPM.
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P3.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 226)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.08-008 - Tip60 (KAT5) May Be a Candidate for Therapeutic Targeting in Malignant Pleural Mesothelioma (ID 1760)
09:30 - 17:00 | Author(s): S.G. Gray
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is a rare aggressive cancer of the pleura. The most well established risk factor for this disease is exposure to asbestos. The current standard of care for patients suffering from MPM is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed). Most patients however, die within 24 months of diagnosis. New therapies are therefore urgently required for this disease. KAT5 (also known as Tip60) is the catalytic subunit of the NuA4 histone acetyltransferase complex which is involved in transcriptional activation of select genes. This complex may be required for the activation of transcriptional programs associated with oncogene and proto-oncogene mediated growth induction, tumor suppressor mediated growth arrest and replicative senescence, apoptosis, and DNA repair. KAT5 has also been linked with the development of cisplatin resistance. KAT5 may therefore be a significant element in MPM with respect to responses to cisplatin based therapy and could represent a novel candidate target for intervention.
Methods:
KAT5 has 4 variant mRNAs. Primers were designed to distinguish between all variants, and a panel of MPM cell lines was screened for KAT5 expression by RT-PCR. Levels of KAT5 were subsequently examined in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies by RT-PCR. The effects of a small molecule inhibitor of KAT5 (MG-149) on cellular proliferation were examined.
Results:
Semi-quantitative densitometric analysis showed that the expression of KAT5 is dramatically increased in MPM for all mRNA variants. When separated according to histological subtype, significant differences were also observed between the various histologies. A small molecule inhibitor of KAT5 exists and treatment of cells with this small molecule inhibitor (MG-149) resulted in a significant inhibition of cellular proliferation (p < 0.001) in the NCI-H226 cell line. We continue to assess this compound by other methodologies to confirm its potential utility in the treatment of MPM.
Conclusion:
KAT5, a lysine acetyltransferase associated with cisplatin resistance in cancer is significantly altered in MPM. A small molecule inhibitor of this protein shows significant anti-proliferative effects in MPM cell lines. Targeting this protein may have important future implications for the management of MPM.
