Small Cell Lung Cancer (SCLC) typically responds well to initial chemotherapy with Etoposide and a platinum-containing agent. However survival is poor due to invariable relapse with chemoresistant disease. We used a unique series of SCLC cell lines (GLC-14, 16, 19), derived from a single patient at various time-points during her treatment, to identify genes involved in Etoposide resistance. We then attempted to determine their functional role and validate their importance using patient specimens.
Methods
The relationship of the cell lines to each other was confirmed using genomic methods. Genes whose expression pattern could explain the relative response of the cell lines to treatment with Etoposide were identified using cDNA microarray. These candidate Etoposide response genes were cloned from the cell line in which they were expressed at the highest level and transiently over-expressed in the cell line in which they were naturally expressed at the lowest level to determine whether this altered Etoposide resistance. Using immunohistochemistry, expression of the most promising candidates was examined using a tissue microarray (TMA).
Results
The two most promising candidate genes were identified as DNA Polymerase β, a DNA repair enzyme and NKX2.2, a neuroendocrine transcription factor. Specific inhibition of DNA Polymerase β with Pamoic Acid reduced the numbers of cells surviving treatment with Etoposide (p=0.029) and increased the amount of DNA damage in the cells (p<0.001). Stable overexpression of NKX2.2 significantly increased cell survival in response to Etoposide in two different SCLC cell lines. In keeping with this, we found that absence of nuclear staining for NKX2.2 in the TMA was an independent predictor of improved outcome in chemotherapy treated SCLC patients (HR 0.52, 95% CI 0.33 to 0.82, p=0.005).
Conclusion
Using a biologically plausible model of in vivo acquired resistance to Etopoide we have identified two novel Etoposide resistance factors—DNA Polymerase β and NKX2.2. Our in vitro data, in conjunction with the TMA results, provide justification for further prospective work to confirm the roles of these molecules in chemotherapy resistance in SCLC.
Abstract Background The Whey Acidic Protein domain is an evolutionarily conserved motif found in a number of proteins, the best studied of which are antiproteinases involved in the innate immune defence of multiple epithelia. We recently characterised the WFDC2 gene which encodes a two WAP domain-containing protein, initially suggested as a marker for epididymis, and showed that it is highly expressed in the lung and salivary gland. The precise location of WFDC2 protein in these sites has not been described. Methods We used immunohistochemistry to localise WFDC2 in normal tissues of the respiratory tract, naso- and oropharynx, as well as in chronically inflamed lung from Cystic Fibrosis and a range of pulmonary carcinomas. We have complemented these studies with molecular analysis of WFDC2 gene expression in primary human lung cell cultures. Results WFDC2 is expressed in some epithelial cells of the upper airways as well as in mucous cells and ducts of submucosal glands. No staining was seen in peripheral lung. Intense staining is found in major salivary glands and in minor glands of the nose, sinuses, posterior tongue and tonsil. Studies with the related protein Secretory Leukocyte Protease Inhibitor (SLPI) show that although both proteins are expressed in similar tissues, the precise cellular localisation differs. Significant increases in expression and localisation of WFDC2 are seen in patients with Cystic Fibrosis. SLPI expression was greatly reduced in the same samples. In cultures of tracheobronchial epithelial cells, expression of WFDC2 and SLPI are differentially regulated during differentiation yet WFDC2 is not induced by pro-inflammatory mediators. The majority of adenocarcinomas stain with WFDC2 whilst a significant minority of squamous, small cell and large cell carcinomas exhibit focal staining. There is no clear association with tumour grade. Conclusion We believe that these studies support the hypothesis that WFDC2 may be a component of the innate immune defences of the lung, nasal and oral cavities and suggest that WFDC2 functions in concert with related WAP domain containing proteins in epithelial host defence. We also suggest that WFDC2 re-expression in lung carcinomas may prove to be associated with tumour type and should be studied in further detail.
Abstract Background Platinum-based chemotherapy is commonly used for the treatment of non-small cell lung cancer (NSCLC), yet clinical outcomes and survival rates remain very poor and continues to be a cancer of unmet need. Recent evidence points to cellular senescence, a response to oncogenic- and therapy-induced genotoxic stress, and its associated proinflammatroy secretory phenotype (SASP) as an emerging hallmark of cancer. Hence, targeting senescence and its associated tumor-promoting activities is emerging as a novel and promising therapeutic strategy but largely unexplored in lung cancer. Experimental Procedures Our study includes a variety of methodologies, including: - Functional in vitro analyses: proliferation, colony formation, tumor spheres, migration assays. High throughput unbiased analyses: RNAseq, proteomics and microenvironment microarrays (MEMA). - In vivo models of lung cancer: xenografts, orthotopic and genetically engineered mouse models. Longitudinal tumor burden by IVIS and microCT and mouse survival. - Clinical samples: Histological and in silico analyses. Results Here we show that cisplatin-derived SASP enhances the malignant phenotype of lung cancer cells. Using xenograft, orthotopic and KrasG12V-driven murine NSCLC models, we demonstrate that cisplatin-induced senescent cells strongly promote tumor progression. Mechanistically, we find that a TGF-β-enriched SASP drives pro-proliferative effects through TGFβR1 and Akt/mTOR pathway activation. We validate the translational relevance of chemotherapy-induced SASP using clinical NSCLC samples from a trial with patients who received neoadjuvant platinum-based chemotherapy. Importantly, TGFβR1 inhibition with galunisertib or senolytic treatment significantly reduces tumor promotion driven by cisplatin-induced senescence. Finally, we demonstrate, using distinct murine NSCLC models, that addition of TGFBR1 inhibitors to platinum-based chemotherapy reduces tumor burden and improves survival, providing pre-clinical proof-of-concept for future trial designs on combination therapies. Conclusions and Impact We regard this work as a major conceptual dissection of tumor-promoting activities of therapy-induced senescence, and a preclinical advance in the management of lung cancer with potential wide therapeutic applications in precision medicine. Of note, we expect our findings to have implications for multiple cancer types, including ovarian, breast, mesothelioma, oesophageal, head and neck, bladder and brain cancers, where platinum-based therapies remain important standard-of-care treatments. Citation Format: Estela González-Gualda, David Macias, Samir Morsli, José Ezequiel Martín, Hui-Ling Ou, Mary Denholm, Ioana Olan, Reuben Hoffmann, Mark Dane, Dimitris Veroutis, Guillermo Medrano, Francisca Mulero, Carla P. Martins, Mariano Barbacid, Vassilis Gorgoulis, James E. Korkola, Doris M. Rassl, Gary J. Doherty, Robert C. Rintoul, Masashi Narita, Daniel Muñoz-Espín. A tumor-promoting senescent secretome triggered by platinum chemotherapy exploits a targetable TGFβR1/Akt-mTOR axis in lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5504.
<div>Abstract<p>The nuclear deubiquitylase BRCA1-associated protein 1 (BAP1) is frequently inactivated in malignant pleural mesothelioma (MPM) and germline <i>BAP1</i> mutation predisposes to cancers including MPM. To explore the influence on cell physiology and drug sensitivity, we sequentially edited a predisposition mutation (<i>w-</i>) and a promoter trap (<i>KO</i>) into human mesothelial cells. <i>BAP1<sup>w-/KO</sup></i> MeT5A cells express less BAP1 protein and phenocopy key aspects of BAP1 loss in MPM. Stable isotope labeling with amino acids in cell culture–mass spectrometry revealed evidence of metabolic adaptation, with concomitant alteration of cellular metabolites. In MeT5A, BAP1 deficiency reduces glycolytic enzyme levels but increases enzymes involved in the tricarboxylic acid cycle and anaplerotic pathways. Notably both argininosuccinate synthase 1 (ASS1), essential for cellular synthesis of arginine, and its substrate aspartate, are elevated in <i>BAP1<sup>w-/KO</sup></i> MeT5A cells. Likewise, ASS1 expression is higher in BAP1-altered MPM cell lines, and inversely correlates with BAP1 in The Cancer Genome Atlas MESO dataset. Elevated ASS1 is also evident by IHC staining in epithelioid MPM lacking nuclear BAP1 expression, with improved survival among patients with BAP1-negative/ASS1-expressing tumors. Alterations in arginine metabolism may sensitize cells to metabolic drugs and we find that BAP1-negative/ASS1-expressing MPM cell lines are more sensitive to ASS1 inhibition, although not to inhibition of purine synthesis by mizoribine. Importantly, <i>BAP1<sup>w-/KO</sup></i> MeT5A become desensitized to arginine deprivation by pegylated arginine deiminase (ADI-PEG20), phenocopying BAP1-negative/ASS1-expressing MPM cell lines.</p>Implications:<p>Our data reveal an interrelationship between BAP1 and arginine metabolism, providing a potential means of identifying patients with epithelioid MPM likely to benefit from ADI-PEG20.</p></div>
Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by limited treatment options and high mortality. A better understanding of the molecular drivers of IPF progression is needed.
The Cancer Genome Atlas (TCGA) cancer genomics dataset includes over 10,000 tumor-normal exome pairs across 33 different cancer types, in total >400 TB of raw data files requiring analysis. Here we describe the Multi-Center Mutation Calling in Multiple Cancers project, our effort to generate a comprehensive encyclopedia of somatic mutation calls for the TCGA data to enable robust cross-tumor-type analyses. Our approach accounts for variance and batch effects introduced by the rapid advancement of DNA extraction, hybridization-capture, sequencing, and analysis methods over time. We present best practices for applying an ensemble of seven mutation-calling algorithms with scoring and artifact filtering. The dataset created by this analysis includes 3.5 million somatic variants and forms the basis for PanCan Atlas papers. The results have been made available to the research community along with the methods used to generate them. This project is the result of collaboration from a number of institutes and demonstrates how team science drives extremely large genomics projects.
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