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L. Mac Donagh
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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)
19:05 - 19:10 | Author(s): L. Mac Donagh
- 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)
19:24 - 19:35 | Author(s): L. Mac Donagh
- 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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