Development Of Small Cell Lung Cancer Research Articles

Abstract 4254: Genetically engineered human lung organoid models for lung cancer evolution study

Abstract Initial cellular events in a transitional period from normal to cancer cells, such as transcriptomic or epigenetic dynamics, are poorly understood. Observing these is more challenging than in cancer progression, especially for humans. Mapping this phenomenon in early cancer evolution is crucial, as we can identify targetable molecules to inhibit cancer development at the precancerous stage. Immortalized human cancer cell lines hardly capture these appropriately as they have lost usual cellular characteristics. Normal organoids harbor stem cells and their differentiated cells, reflecting the epithelial physiology of tissues, and are more suitable models for cancer evolution studies. To establish a human cancer development model in vitro, we genetically engineered normal human airway organoids derived from adult stem cells into small cell lung cancer (SCLC). We used CRISPR-Cas9 systems to induce non-homologous end-joining in both TP53 and RB1, the most frequently mutated genes of SCLC. First, we knocked out TP53 of the airway organoids, selected by Nutlin-3a, an MDM2 antagonist, for 3-4 weeks, demonstrating 88-89% editing efficiencies calculated from Sanger sequencing results. Most mutations were one base pair deletion or insertion, which induced frameshift mutation in TP53. They did not show a significant difference from wild-type organoids morphologically at bright-field microscopy. Subsequently, we targeted RB1 on TP53-/- airway organoids to induce SCLC. We used Palbociclib, a CDK4/6 inhibitor, to enrich the mutated cells. They had 67.1% efficiency, and 20.4% had a 7-base pair deletion mutation. In the initial period, they seemed to have similar shapes to TP53-/- organoids. Unexpectedly and interestingly, however, we noticed more rapidly proliferating and disorganized organoids disrupting their original round shapes appeared in 4-5 weeks. The cells were indiscriminately piled up on the airway organoids' apical and basolateral surfaces, indicating their malignant traits. Their chaotic proliferation represented the rapidly progressing clinical feature of SCLC with a median survival of 7-16 months with treatment. This is the first work constructing the SCLC development model from the normal human airway organoid. By sequentially single-cell sequencing this organoid model changing to tumorous condition, we can capture the multi-omics landscape during the initial evolution of SCLC. Citation Format: Sungji Moon, Rokhyun Kim, Dakyung Lee, Kyunghyuk Park, Woochan Lee, Seyoon Lee, Young Jun Park, Jongil Kim. Genetically engineered human lung organoid models for lung cancer evolution study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4254.

Abstract 7335: Characterizing germline variants in patients with small cell lung cancer

Abstract Introduction: Small cell lung cancer (SCLC) has traditionally been considered to arise from environmental and lifestyle factors. Recent evidence has shown that germline mutations may also impact the development of SCLC, however, remains understudied. We sought to identify novel germline mutations in SCLC including unexplored copy-number variations (CNVs) in our cohort of patients. Methods: We applied a custom hybrid-capture gene panel of 191 cancer predisposition and commonly mutated genes in SCLC to a pre-treatment cohort of 67 patients with SCLC. Variant calls were performed using VarScan2 (2.3.8), Pindel (0.2.5b8) and CNVs were assessed using DeCON 1.0.2. Calls were annotated using the American College of Medical Genetics (ACMG) criteria and classified using gnomeAD (release 2.11), ESP6500 (ESP6500SI-V2), 1000Genomes, ExAC (release 0.3), COSMIC (v70), dbSNP (build 147), and ClinVar (NCBI ClinVar 20190305). We annotated variants of uncertain significance (VUS) with several in silico tools including MutationTaster, LRT, Polyphen2 HDIV, Polyphen2 HVAR, SIFT, PROVEAN, and Deepmind AlphaMissense. Kaplan-Meier survival analysis and log-rank tests were done to examine differences in overall survival (OS) and progression-free survival (PFS) from the date of diagnosis. Continuous variables were tested using Kruskal-Wallis H test and categorical variables using Fisher’s exact test. Results: The median age of our cohort was 68 years old, 64% (43/67) were male, 89% (60/67) were current/former smokers, and 58% (39/67) were Extensive-Stage. 64.6% (42/67) of patients self-reported a family history of cancer and 16% of the patients also self-reported personal history of cancer. In terms of self-reported ancestral background, 76% of were Caucasian, 1.5% were Middle-Eastern, 3% were Latin American, 14% were Asian, and 4.5% were Black. We identified pathogenic/likely pathogenic alterations in 7/67 patients. Five (71%) were novel alterations (BCORL1, FANCC, ATR, and BBC3) and a novel CNV (SLFN11) with the remaining two (29%) previously described mutations (CHEK1 and BRIP1). We identified 191 VUS in 60/67 patients. 4.7-14.1% of VUS were in silico predicted to be pathogenic. Patients with the 7 pathogenic alterations had a numerically larger OS (HR = 0.50, 95%CI = 0.18-1.39, p = 0.18) and PFS (HR = 0.45, 95%CI = 0.16 - 1.24, p = 0.11) compared to the rest of the cohort. We identified 20 variants at low variant allele fractions (0.05 to 0.24), likely somatic mutations. Some genes identified are implicated with clonal hematopoiesis such as GNAS, STAG, TET2, and MGA. Conclusion: In summary, we have identified several novel germline alterations (mutations and CNVs) in patients with SCLC. In addition, we utilized in silico prediction models to categorize potential high-risk VUS. Our findings suggest that beyond tobacco exposure germline alterations may also modulate development of tumorigenesis in SCLC patients. Citation Format: Sami Ul Haq, Gregory Downs, Luna Jia Zhan, Danielle Sacdalan, Janice J. Li, Vivek Philip, Raymond H. Kim, Geoffrey Liu, Scott V. Bratman, Peter J. Sabatini, Benjamin H. Lok. Characterizing germline variants in patients with small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7335.

P53 and Rb Aberrations in Small Cell Lung Cancer (SCLC): From Molecular Mechanisms to Therapeutic Modulation.

The genes coding for the tumor suppressors p53 and retinoblastoma (Rb) are inactivated in the vast majority of small cell lung cancer (SCLC) tumors. Data support the notion that these two deleterious genetic events represent the initial steps in the development of SCLC, making them essential for a lung epithelial cell to progress toward the acquisition of a malignant phenotype. With the loss of TP53 and RB1, their broad tumor suppressive functions are eliminated and a normal cell is able to proliferate indefinitely, escape entering into cellular senescence, and evade death, no matter the damage it has experienced. Within this setting, lung epithelial cells accumulate further oncogenic mutations and are well on their way to becoming SCLC cells. Understanding the molecular mechanisms of these genetic lesions and their effects within lung epithelial cells is of paramount importance, in order to tackle this aggressive and deadly lung cancer. The present review summarizes the current knowledge on p53 and Rb aberrations, their biological significance, and their prospective therapeutic potential, highlighting completed and ongoing clinical trials with agents that target downstream pathways.

Abstract A022: Dissecting patterns of small cell lung cancer evolution using deep whole genome sequencing of circulating tumor DNA

Abstract Background: Small cell lung cancer (SCLC) is the most lethal form of lung cancer. A key driver of this near universal lethality is SCLC’s recalcitrance to therapy. The standard of care for SCLC is a chemotherapy doublet of etoposide and cis/carboplatin in combination with immunotherapy (EP+IO). While most tumors respond to these therapies, >80% of SCLCs progress within one year of treatment. Additionally, the aggressive clinical course of SCLC has historically precluded collection of patient-matched treatment naïve and recurrent tumor tissue traditionally required to study evolution. In lieu of tissue, several groups have used circulating tumor DNA (ctDNA) to study SCLC evolution. However, these studies used targeted sequencing panels that analyze <0.04% of the SCLC genome. Using new methods to comprehensively trace how SCLCs rapidly evolve to therapy resistance is a significant unmet need. Methods: Medical records were reviewed to identify treatment naïve SCLC patients treated at MD Anderson that had both pre- and post-EP+IO liquid biopsies collected under IRB approved protocols. ctDNA was isolated from plasma using a MiniMax High Efficiency Cell-Free DNA Isolation Kit (Apostle Bio). Germline DNA was extracted from buffy coat samples using a QIAamp DNA Blood Midi Kit (Qiagen). Deep whole genome sequencing (WGS) (>100X) of ctDNA and WGS of germline DNA (>60X) was performed using an Illumina NovaSeq X sequencer. Reads were aligned to the hg38 reference genome using BWA-mem. Somatic mutations were called using MuTect2, and clonal and subclonal copy number alterations were identified using Battenberg. DPClust was used to reconstruct cancer cell populations, leveraging mutation and copy number calls. Results: We identified several deleterious genomic alterations impacting TP53 and RB1 tumor suppressor genes, known genetic drivers of SCLC development. We also detected recurrent, whole chromosome arm loss of heterozygosity events impacting many chromosomes in treatment naïve samples, some of which are known to be present in >80% of SCLCs. Additionally, we found that both treatment naïve and recurrent SCLCs were composed of multiple subclones, each defined by dozens to thousands of unique somatic mutations. COSMIC signature analysis identified high activity of tobacco carcinogens, APOBEC, and replicative-based processes across ctDNA samples. We also identified emergence of novel subclones carrying unique copy number alterations following EP+IO recurrence. Importantly, the affected regions encode key effectors that regulate anti-tumor immune responses and cell survival following DNA damage, both of which are intimately related to the mechanism of action of EP+IO. Conclusions: Our data shows that deep WGS of ctDNA recapitulates known genetics of SCLC and allows for sensitive characterization of therapy resistant clones. Using this method, we have captured snapshots of both intrinsic and acquired resistance mechanisms to frontline EP+IO. Deep WGS of ctDNA is a promising approach to comprehensively dissect routes of SCLC evolution and therapy resistance. Citation Format: Benjamin B. Morris, Zhihui Zhang, Simon Heeke, Kyle Concannon, Bingan Zhang, Waree Rinsurongkawong, Vadeerat Rinsurongkawong, J. Jack Lee, Jianjun Zhang, Don L. Gibbons, Ara A. Vaporciyan, Carl M. Gay, Hai T. Tran, John V. Heymach, Lauren A. Byers, Peter Van Loo. Dissecting patterns of small cell lung cancer evolution using deep whole genome sequencing of circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A022.

Analysis of two-gene signatures and related drugs in small-cell lung cancer by bioinformatics.

Small-cell lung cancer (SCLC) has a poor prognosis and can be diagnosed with systemic metastases. Nevertheless, the molecular mechanisms underlying the development of SCLC are unclear, requiring further investigation. The current research aims to identify relevant biomarkers and available drugs to treat SCLC. The bioinformatics analysis comprised three Gene Expression Omnibus datasets (including GSE2149507, GSE6044, and GSE30219). Using the limma R package, we discovered differentially expressed genes (DEGs) in the current work. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were made by adopting the DAVID website. The DEG protein-protein interaction network was built based on the Search Tool for the Retrieval of Interacting Genes/Proteins website and visualized using the CytoHubba plugin in Cytoscape, aiming to screen the top ten hub genes. Quantitative real-time polymerase chain reaction was adopted for verifying the level of the top ten hub genes. Finally, the potential drugs were screened and identified using the QuartataWeb database. Totally 195 upregulated and 167 downregulated DEGs were determined. The ten hub genes were NCAPG, BUB1B, TOP2A, CCNA2, NUSAP1, UBE2C, AURKB, RRM2, CDK1, and KIF11. Ten FDA-approved drugs were screened. Finally, two genes and related drugs screened could be the prospective drug targets for SCLC treatment.

Bioinformatics-based screening of key genes for transformation of tyrosine kinase inhibitor-resistant lung adenocarcinoma to small cell lung cancer.

Lung adenocarcinoma (LUAD) is a common type of lung cancer. Cancer in a small number of patients with EGFR mutations will transform from LUAD to small cell lung cancer (SCLC) during epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapiesr. The purpose of the present study was to identify the core genes related to the transformation of LUAD into SCLC and to explore the associated molecular mechanisms. GSE29016, GSE1037, GSE6044 and GSE40275 mRNA microarray datasets from Gene Expression Omnibus (GEO) were analyzed to obtain differentially expressed genes (DEGs) between LUAD and SCLC tissues, and the results were used for network analysis of protein-protein interactions (PPIs). After identifying the hub gene by STRING and Cytoscape platform, we explored the relationship between hub genes and the occurrence and development of SCLC. Finally, the obtained hub genes were validated in treated LUAD cells. A total of 41 DEGs were obtained, four hub genes (EZH2, NUSAP1, TTK and UBE2C) were identified, and related prognostic information was obtained. The coexpressed genes of the hub gene set were further screened, and the analysis identified many genes related to the cell cycle. Subsequently, LUAD cell models with TP53 and RB1 inactivation and overexpression of ASCL1 were constructed, and then the expression of hub genes was detected, the results showed that the four hub genes were all elevated in the established cell model. EZH2, NUSAP1, TTK and UBE2C may affect the transformation of LUAD to SCLC and represent new candidate molecular markers for the occurrence and development of SCLC.

Wood dust exposure and small cell lung cancer: a systematic review and meta-analysis.

Occupational exposure role on small cell lung cancer (SCLC) onset has been little studied. Wood dust has been recognized as a human carcinogen, and many occupations have high wood-dust exposure. The aim of this study was therefore to perform a systematic review and meta-analysis of the scientific literature to summarize and analyse the risks of wood dust-related occupations on development of SCLC, taking tobacco use into account. We conducted a literature search of PubMed, EMBASE, Web of Science and Cochrane using a predefined strategy and including case-control and cohort studies assessing occupational exposure to wood dust or wood dust-related occupations. To perform the meta-analysis, the odds ratio (OR) and 95% confidence interval (CI) of each of the studies were extracted. A random-effects model was fitted using the DerSimonian Laird method. Sensitivity and subgroup analyses were performed. Quality was assessed using the Office and Health Assessment and Translation (OHAT) for human and animal studies instrument. Eleven studies with a total of 2,368 SCLC cases and 357,179 controls were included. Overall, exposure to wood dust significantly increases risk of SCLC (RR = 1.41, 95% CI 1.11-1.80), with low heterogeneity between studies (I2 = 40%). The association was maintained in studies conducted on males (RR = 1.41, 95% CI 1.12-1.78) but not in those conducted on females/both sexes (RR = 1.37, 95% CI 0.35-3.44). Sensitivity analysis showed that none of the studies significantly modified the results. Our results support that exposure to wood-dust can increase the risk of SCLC. Although the level of evidence is low, there are strong arguments to recommend the implementation of effective control measures to reduce exposure in occupational settings, as a means of preventing SCLC. The results of this study support that exposure to wood-dust can increase the risk of developing small cell lung cancer. Determining the impact of occupational exposure on workers is essential to improve their individual protection and prevention. There is a strong case for recommending the implementation of control measures to reduce occupational exposure to wood dust, specifically for highly exposed occupations such as carpenters and sawmills, in order to prevent small cell lung cancer.

NEMO- and RelA-dependent NF-κB signaling promotes small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive type of lung cancer driven by combined loss of the tumor suppressors RB1 and TP53. SCLC is highly metastatic and despite good initial response to chemotherapy patients usually relapse, resulting in poor survival. Therefore, better understanding of the mechanisms driving SCLC pathogenesis is required to identify new therapeutic targets. Here we identified a critical role of the IKK/NF-κB signaling pathway in SCLC development. Using a relevant mouse model of SCLC, we found that ablation of NEMO/IKKγ, the regulatory subunit of the IKK complex that is essential for activation of canonical NF-κB signaling, strongly delayed the onset and growth of SCLC resulting in considerably prolonged survival. In addition, ablation of the main NF-κB family member p65/RelA also delayed the onset and growth of SCLC and prolonged survival, albeit to a lesser extent than NEMO. Interestingly, constitutive activation of IKK/NF-κB signaling within the tumor cells did not exacerbate the pathogenesis of SCLC, suggesting that endogenous NF-κB levels are sufficient to fully support tumor development. Moreover, TNFR1 deficiency did not affect the development of SCLC, showing that TNF signaling does not play an important role in this tumor type. Taken together, our results revealed that IKK/NF-κB signaling plays an important role in promoting SCLC, identifying the IKK/NF-κB pathway as a promising therapeutic target.

A multiplexed in vivo approach to identify driver genes in small cell lung cancer

Small cell lung cancer (SCLC) is a lethal form of lung cancer. Here, we develop a quantitative multiplexed approach on the basis of lentiviral barcoding with somatic CRISPR-Cas9-mediated genome editing to functionally investigate candidate regulators of tumor initiation and growth in genetically engineered mouse models of SCLC. We found that naphthalene pre-treatment enhances lentiviral vector-mediated SCLC initiation, enabling high multiplicity of tumor clones for analysis through high-throughput sequencing methods. Candidate drivers of SCLC identified from a meta-analysis across multiple human SCLC genomic datasets were tested using this approach, which defines both positive and detrimental impacts of inactivating 40 genes across candidate pathways on SCLC development. This analysis and subsequent validation in human SCLC cells establish TSC1 in the PI3K-AKT-mTOR pathway as a robust tumor suppressor in SCLC. This approach should illuminate drivers of SCLC, facilitate the development of precision therapies for defined SCLC genotypes, and identify therapeutic targets.

NDR1 activates CD47 transcription by increasing protein stability and nuclear location of ASCL1 to enhance cancer stem cell properties and evasion of phagocytosis in small cell lung cancer.

Small cell lung cancer (SCLC) is one of the most malignant types of lung cancer. Cancer stem cell (CSC) and tumor immune evasion are critical for the development of SCLC. We previously reported that NDR1 enhances breast CSC properties. NDR1 might also have a role in the regulation of immune responses. In the current study, we explore the function of NDR1 in the control of CSC properties and evasion of phagocytosis in SCLC. We find that NDR1 enhances the enrichment of the ALDEFLUORhigh and CD133high population, and promotes sphere formation in SCLC cells. Additionally, NDR1 upregulates CD47 expression to enhance evasion of phagocytosis in SCLC. Furthermore, the effects of NDR1 enhanced CD47 expression and evasion of phagocytosis are more prominent in CSC than in non-CSC. Importantly, NDR1 promotes ASCL1 expression to enhance NDR1-promoted CSC properties and evasion of phagocytosis in SCLC cells. Mechanically, NDR1 enhances protein stability and the nuclear location of ASCL1 to activate the transcription of CD47 in SCLC. Finally, CD47-blocking antibody can be used to target NDR1 enhanced CSC properties and evasion of phagocytosis by suppressing EGFR activation in SCLC. In summary, our data indicate that NDR1 could be a critical factor for modulating CSC properties and phagocytosis in SCLC.

WNT5A-RHOA Signaling Is a Driver of Tumorigenesis and Represents a Therapeutically Actionable Vulnerability in Small Cell Lung Cancer.

The p130-WNT5A-RHOA pathway drives SCLC progression and is a potential target for the development of therapeutic interventions and biomarkers to improve patient treatment.

Repression of linc01555 up-regulates angiomotin-p130 via the microRNA-122-5p/clic1 axis to impact vasculogenic mimicry-mediated chemotherapy resistance in small cell lung cancer

ABSTRACT Long non-coding ribonucleic acid 01555 (linc01555) is a brand-new long non-coding RNA (lncRNA) that acts a carcinogenic function in various cancers. However, its role in small cell lung cancer (SCLC) is uncertain. This research was to figure out the role of linc01555 in cisplatin (DDP) resistance of SCLC cells and its possible latent mechanism. After establishment of the resistant sub-strain H446/DDP or DMS-53/DDP, detection of linc01555, microRNA (miR)-122-5p and CLICl was done in the H446/DDP or DMS-53/DDP cell line. After intervention, cell biological functions were determined, as well as tube formation ability. The detection of angiomotin (Amot)-p130 and the validation of the regulatory mechanism were performed. Furthermore, tumor xenografts were applied in nude mice to evaluate the effect of linc01555 on DDP resistance in SCLC in vivo. Linc01555 was elevated in SCLC tissues and cells, and in H446/DDP cells or DMS-53/DDP vs. its parental cells; Restraining linc01555 or elevating miR-122-5p repressed the proliferation and metastasis of H446/DDP or DMS-53/DDP cells and vasculogenic mimicry (VM) formation. CLIC1 mediated miR-122-5p to influence the occurrence and development of SCLC. Linc01555 competitively combined with miR-122-5p, which targeted CLIC1. Refrained linc01555 elevated Amot-p130 via the miR-122-5p/CLIC1 axis. Reduced linc01555 refrained tumor growth and DDP resistance in vivo.In short, linc01555 may cause changes in DDP resistance via miR-122-5p/CLIC1 in SCLC. The finding may offer drug targets for SCLC resistance.

Insulin-Like Growth Factor 2 mRNA-Binding Protein 3 and Its Related Molecules as Potential Biomarkers in Small-Cell Lung Cancer.

Background Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) plays a key role in tumorigenesis and tumor progression. Lung cancer is the leading cause of cancer-related death in men and women all over the world. However, the relationship between IGF2BP3 and small-cell lung cancer (SCLC) has not been reported yet. Methods SCLC and normal samples (GSE19945 and GSE149507) were obtained in the Gene Expression Omnibus (GEO) dataset. Differential genes were screened by R software, and functional analysis and signal pathway enrichment analysis were carried out. In addition, we used the survival analysis database to analyze the relationship between prognosis and gene expression. Besides, immunohistochemistry (IHC) and quantitative real-time PCR (qPCR) were used for further research. Results Five differentially expressed miRNAs and 9 differentially expressed mRNAs were selected by using R software. Survival analysis database results show that C7, CLIC5, PRDX1, IGF2BP3, and LDB2 were related the overall survival of patients with SCLC. Furthermore, multivariate analysis included that IGF2BP3 was independent risk factors for SCLC patients. Besides, gene function and signal pathway enrichment analysis showed that differentially expressed miRNAs were involved in the process of tumorigenesis and development. Furthermore, IHC and qPCR outcomes showed that the expression level of hsa-miR-182, hsa-miR-183, and IGF2BP3 was differentially expressed in normal lung tissues (NLTs) and SCLC tissues (SCLCTs). Conclusions Our results concluded that hsa-miR-182, hsa-miR-183, and IGF2BP3 may take part in the development of SCLC.

Bioinformatics Analysis of Key Genes and miRNAs Associated with Small-cell Lung Cancer

Small-cell lung cancer (SCLC) is a type of lung cancer, accounting for approximately 15% of lung cancers. SCLC is significantly associated with early recurrence, metastasis, and poor prognosis, with a 2-year survival rate after therapy, which is <5%. The molecular mechanisms underlying the development of SCLC are not clear. In this study, we identified SCLC-specific biomarkers by evaluating the differential expression of mRNA and microRNA (miRNA) profiles in SCLC tissue compared with normal lung tissue. A non-coding RNA sequence dataset (GSE19945) and transcriptome sequencing dataset (GSE6044) were downloaded from the GEO database. We identified 445 DEGs (differentially expressed genes) and 128 DE-miRNAs (differentially expressed miRNAs) using the GEO2R tool of the GEO and R limma software package. Furthermore, using the KEGG database, we identified 15 enrichment pathways, mostly associated with DNA replication, cell cycle, and oocyte meiosis mismatch repair, and the GO function was considerably enriched for 26 items. To investigate the molecular processes of key signaling pathways and cellular activity in SCLC, we used Cytoscape software to construct protein-protein interaction (PPI) networks. Using miRNAWalk, we identified 598 target genes of the 1380 miRNAs and constructed miRNA target networks. In addition, we identified eighteen overlapping genes that are regulated by 28 different miRNAs. The identified hub genes are important because they may be used as biomarkers for prognosis, diagnosis, and therapeutic target for SCLC.

TLR4 regulatory region variants reduce the susceptibility of small-cell lung cancer in Chinese population.

Toll-like receptors (TLRs) participate in the induction and regulation of immune responses and are closely related to the occurrence and development of small-cell lung cancer (SCLC). This study aimed to investigate the impact of polymorphisms in the regulatory regions of TLRs on the susceptibility of SCLC. The case-control study included 304 SCLC patients and 304 healthy controls. TLRs gene polymorphisms were genotyped by PCR-restrictive fragment length polymorphism analysis and TaqMan assay. Unconditional logistic regression was used to estimate odds ratio (OR) and its 95% confidence interval (95% CI). Our results showed that TLR4 rs1927914 GG genotype and TLR4 rs7869402 TT genotype reduced the risk of SCLC with OR (95% CI) of 0.54 (0.32-0.90) and 0.47 (0.28-0.80), respectively. Stratified analysis suggested that TLR4 rs1927914 GG genotypes significantly reduced the risk of SCLC among male (OR = 0.35; 95% CI, 0.18-0.69; P < 0.01), the younger patients (OR = 0.49; 95% CI, 0.26-0.94; P = 0.03) and non-drinkers (OR = 0.47; 95% CI, 0.24-0.89; P = 0.02). TLR4 rs7869402 CT or TT genotype significantly reduced the susceptibility to SCLC among male patients (OR = 0.37; 95% CI, 0.19-0.71, P < 0.01), the younger patients (OR = 0.41; 95% CI, 0.22-0.79; P < 0.01), smokers (OR = 0.25; 95% CI, 0.10-0.60; P < 0.01) and drinkers (OR = 0.31; 95% CI, 0.11-0.88; P = 0.03). TLR3 rs5743303, TLR4 rs11536891, TLR5 rs1640816 and TLR7 rs3853839 had no significant correlation with the risk of SCLC. These findings emphasized the important role of TLR4 in the development of SCLC.

KIX domain determines a selective tumor-promoting role for EP300 and its vulnerability in small cell lung cancer.

EP300, a transcription coactivator important in proliferation and differentiation, is frequently mutated in diverse cancer types, including small cell lung cancer (SCLC). While these mutations are thought to result in loss of EP300 function, the impact on tumorigenesis remains largely unknown. Here, we demonstrate that EP300 mutants lacking acetyltransferase domain accelerate tumor development in mouse models of SCLC. However, unexpectedly, complete Ep300 knockout suppresses SCLC development and proliferation. Dissection of EP300 domains identified kinase inducible domain-interacting (KIX) domain, specifically its interaction with transcription factors including MYB, as the determinant of protumorigenic activity. Ala627 in EP300 KIX results in a higher protein-binding affinity than Asp647 at the equivalent position in CREBBP KIX, underlying the selectivity of KIX-binding partners for EP300. Blockade of KIX-mediated interactions inhibits SCLC development in mice and cell growth. This study unravels domain-specific roles for EP300 in SCLC and unique vulnerability of the EP300 KIX domain for therapeutic intervention.

Pathways in small cell lung cancer and its therapeutic perspectives.

Small cell lung cancer (SCLC) is known for its rapid growth with high metastatic spread. Its treatment remains a major challenge for oncologists due to the high mutation rate and other clinical disadvantages. The survival rate of these patients is very poor but there is no significant progress over the last few decades in the treatment protocols. Hence there is an urgency to design new clinical trials with novel drug combinations that can specifically and effectively target key pathways for proper treatment of SCLC. There have been several indications that developmental signalling pathways are involved in tumor growth, progression, metastasis and invasion of SCLC. Thus, it is anticipated that deciphering the signalling cascades of these embryonic signalling pathways may reveal novel therapeutic breakthroughs in SCLC. In this context, we have tried to assemble all the relevant information to give an updated overview of the various signalling pathways involved in the development of SCLC.

Sox2 Is an Oncogenic Driver of Small-Cell Lung Cancer and Promotes the Classic Neuroendocrine Subtype.

Although many cancer prognoses have improved in the past 50 years due to advancements in treatments, there has been little improvement in therapies for small-cell lung cancer (SCLC). One promising avenue to improve treatment for SCLC is to understand its underlying genetic alterations that drive its formation, growth, and cellular heterogeneity. RB1 loss is one key driver of SCLC, and RB1 loss has been associated with an increase in pluripotency factors such as SOX2. SOX2 is highly expressed and amplified in SCLC and has been associated with SCLC growth. Using a genetically engineered mouse model, we have shown that Sox2 is required for efficient SCLC formation. Furthermore, genome-scale binding assays have indicated that SOX2 can regulate key SCLC pathways such as NEUROD1 and MYC. These data suggest that SOX2 can be associated with the switch of SCLC from an ASCL1 subtype to a NEUROD1 subtype. Understanding this genetic switch is key to understanding such processes as SCLC progression, cellular heterogeneity, and treatment resistance. IMPLICATIONS: Understanding the molecular mechanisms of SCLC initiation and development are key to opening new potential therapeutic options for this devastating disease.

Identification of Key Biomarkers and Pathways in Small-Cell Lung Cancer Using Biological Analysis.

Background Small-cell lung cancer (SCLC) is a major cause of carcinoma-related deaths worldwide. The aim of this study was to identify the key biomarkers and pathways in SCLC using biological analysis. Methods Key genes involved in the development of SCLC were identified by downloading three datasets from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were screened using the GEO2R online analyzer; for the functional annotation and pathway enrichment analysis of genes, Funrich software was used. Construction of protein-to-protein interaction (PPI) networks was accomplished using the Search Tool for the Retrieval of Interacting Genes (STRING), and network visualization and module identification were performed using Cytoscape. Results A total of 268 DEGs were ultimately obtained. The enriched functions and pathways of the upregulated DEGs included cell cycle, mitotic, and DNA replication, and the downregulated DEGs were enriched in epithelial-to-mesenchymal transition, serotonin degradation, and noradrenaline. Analysis of significant modules demonstrated that the upregulated genes are primarily concentrated in functions related to cell cycle and DNA replication. Kaplan-Meier analysis of hub genes revealed that they may promote the carcinogenesis and progression of SCLC. The result of ONCOMINE demonstrated that these 10 hub genes were significantly overexpressed in SCLC compared with normal samples. Conclusion Identification of the molecular functions and signaling pathways of participating DEGs can deepen the current understanding of the molecular mechanisms of SCLC. The knowledge gained from this work may contribute to the development of treatment options and improve the prognosis of SCLC in the future.

P66.03 The Functional Role of TGF-β Signaling in SCLC Heterogeneity and Metastasis

Small cell lung cancer (SCLC) is the most aggressive lung cancer and highly heterogeneous phenotypes have been observed in human and mouse SCLC cells. Tumor heterogeneity has been proven to contribute to SCLC malignant progression and/or metastasis in RP-based models. And the unique therapeutic vulnerabilities across different SCLC subtypes have been linked with their specific gene expression profile, indicating the need of personal therapeutic strategy in clinic. The TGF-β signaling consists of secreted TGF-β family members, receptors and SMAD proteins, and is implicated in multiple cell biological processes, such as cell proliferation and survival, epithelial to mesenchymal transition (EMT), cell fate control, immune response, and tumorigenesis. Dysregulation of the TGF-β signaling pathway has been reported in different cancer types and dual role of this pathway in cancer development and progression has been documented. Activation of this pathway results in cell cycle arrest and apoptosis at the early-stage of cancer development, indicating a tumor suppressive function. On the other hand, TGF-β could promote EMT as well as tumor malignant progression at the late-stage disease. Up to date, the contribution of the TGF-β pathway in SCLC development and progression remains elusive. Using the Rb1L/L/Trp53 L/L (RP) mouse model, we find that mouse SCLC cells with adherent growth pattern are composed of mesenchymal-like (Mes) and epithelial-like (Epi) subpopulations. Take advantage of in vitro functional assay and in vivo allograft assay, we find that the Mes cells harbor higher malignant transformation and metastatic capabilities than Epi cells. Through the analysis of microarray data, we find that the TGF-β signaling is enriched in Mes cells. And the inhibition of TGF-β signaling in Mes cells significantly abrogates its metastatic ability in allograft assay. Moreover, genetic deletion of the key components of the TGF-β signaling pathway, dramatically attenuated SCLC metastasis in the RP mouse model. In this study, we find that mouse SCLC cells derived from RP autochthonous mouse model with adherent growth pattern are composed of Mes and Epi subpopulations. The Mes cells have increased ability to form colonies in soft agar and harbored stronger metastatic capability in vivo when compared to the Epi cells. Gene Set Enrichment Analysis (GSEA) reveals that the TGF-β signaling is enriched in the Mes cells. Inhibition of the TGF-β signaling through either ectopic expression of dominant negative Tgfbr2 (Tgfbr2-DN) or treatment with Tgfbr1 inhibitor SD-208 consistently greatly abrogates the tumor metastasis in allograft assays. Moreover, genetic deletion of Tgfbr2 or Smad4, key components of the TGF-β signaling pathway, dramatically attenuates SCLC metastasis in the RP autochthonous mouse model of SCLC. Our results suggest the critical role of TGF-β signaling in SCLC heterogeneity and metastasis. Importantly, inhibition of TGF-β signaling significantly abrogates SCLC metastasis, providing a potential therapeutic avenue for SCLC management in clinic.