Small Cell Lung Cancer Phenotype Research Articles

Abstract A017: Identifying proliferative and immune-modulatory drivers of small cell lung cancer (SCLC)

Abstract Small cell lung cancer (SCLC) is a highly aggressive form of lung cancer that causes an estimated 200,000 patient deaths globally every year. Most (>70%) patients present with extensive stage (ES)-SCLC, and the disease primarily presents with a neuroendocrine (NE), immune-desert phenotype, with low expression of antigen-presenting machinery genes. Despite limited immune infiltration in the tumor, SCLC has reproducibly responded to immune checkpoint blockade. IMpower133 was a pivotal Phase III trial that added atezolizumab (anti-PD-L1) to platinum/etoposide chemotherapy for treatment of first-line ES-SCLC, which led to the first approval of an immune checkpoint blockade strategy in SCLC. While the inclusion of atezolizumab to the treatment regimen has extended the survival of some patients, SCLC remains an unmet medical need with a mOS of 12.3 months. Furthermore, we lack a deep molecular understanding of factors mediating response and resistance to standard of care therapies in SCLC. To understand the genetic drivers of SCLC that could be linked to immunotherapy resistance, we analyzed the matched whole-exome sequencing and RNA-seq data from baseline tumor samples collected from the IMpower133 trial. Previously, we applied the non-negative matrix factorization (NMF) approach to the RNA-seq data, which clustered patients into four subsets of SCLC, with varying degrees of immune infiltration, neuroendocrine program activation, and clinical benefit from immunotherapy. Notably, we identified an inflamed-neuroendocrine (I-NE) subset of patients that exhibit longer overall survival with chemoimmunotherapy compared to chemotherapy alone, and an inflamed-non-neuroendocrine (I-nonNE) subset of patients that did not show a similar benefit from the addition of immunotherapy to chemotherapy. Here, we interrogated the matched WES data for recurrent alterations that may identify subsets of tumors with distinct immunological features. We identified several genes whose alterations significantly affected the enrichment of immune-related gene signatures. In particular, we identified MANF and CSK, genes previously shown to be potential tumor suppressors, as genes whose alteration associates with an “immune desert” transcriptional program. Specifically, genetic deletions or low RNA expression was associated with significantly decreased gene expression of gene signatures associated with lymphocytes, macrophages, and antigen presentation machinery. These data suggest a potential mechanism by which commonly inactivated tumor suppressors may shape the “immune desert” phenotype of SCLC. Further work is needed to determine if and how these genes may directly or indirectly modulate the tumor-immune interaction. Citation Format: Myung Chang Lee, Velimir Gayevskiy, Stefanie Morris, Minu K Srivastava, Namrata S Patil, Stephen V Liu, Martin Reck, Sanjeev Mariathasan, David S Shames, David P MacPherson, Robert J Johnston, Barzin Y Nabet. Identifying proliferative and immune-modulatory drivers of small cell lung cancer (SCLC) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A017.

Signatures of plasticity, metastasis, and immunosuppression in an atlas of human small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively). To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 155,098 transcriptomes from 21 human biospecimens, including 54,523 SCLC transcriptomes. We observe greater tumor diversity in SCLC than lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discover a PLCG2-high SCLC phenotype with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival. SCLC exhibits greater immune sequestration and less immune infiltration than lung adenocarcinoma, and SCLC-N shows less immune infiltrate and greater Tcell dysfunction than SCLC-A. We identify a profibrotic, immunosuppressive monocyte/macrophage population in SCLC tumors that is particularly associated with the recurrent, PLCG2-high subpopulation.

Lineage-restricted neoplasia driven by Myc defaults to small cell lung cancer when combined with loss of p53 and Rb in the airway epithelium.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.

PI3K/Akt/mTOR signaling orchestrates the phenotypic transition and chemo-resistance of small cell lung cancer

Small cell lung cancer (SCLC) is a phenotypically heterogeneous disease with an extremely poor prognosis, which is mainly attributed to the rapid development of resistance to chemotherapy. However, the relation between the growth phenotypes and chemo-resistance of SCLC remains largely unclear. Through comprehensive bioinformatic analyses, we found that the heterogeneity of SCLC phenotype was significantly associated with different sensitivity to chemotherapy. Adherent or semiadherent SCLC cells were enriched with activation of the PI3K/Akt/mTOR pathway and were highly chemoresistant. Mechanistically, activation of the PI3K/Akt/mTOR pathway promotes the phenotypic transition from suspension to adhesion growth pattern and confers SCLC cells with chemo-resistance. Such chemo-resistance could be largely overcome by combining chemotherapy with PI3K/Akt/mTOR pathway inhibitors. Our findings support that the PI3K/Akt/mTOR pathway plays an important role in SCLC phenotype transition and chemo-resistance, which holds important clinical implications for improving SCLC treatment.

Defining the role of RUNX1T1 in "combined" small cell lung cancer.

e20101 Background: The RUNX1T1 gene, also known as ETO, was first identified in the fusion transcript AML1/ETO, generated by the translocation between chromosome 8 and 21, which occurs in 12%-15% of AML. We recently discovered that RUNX1T1 was specifically amplified only in the small cell lung cancer (SCLC) but not the non-small cell lung cancer (NSCLC) component of combined SCLC tumors. Here, we investigated if RUNX1T1 expression plays a role in transforming NSCLC to SCLC in combined SCLC tumor formation. Methods: We examined tumors from 92 of our SCLC patients by targeted exome and CNV analyses. RUNX1T1 mRNA expression was determined in various cancers by analysis of the CCLE and TCGA databases. RUNX1T1 mRNA expression was detected by RNAscope analysis of another 22 patient SCLC tumors. Stable overexpression of RUNX1T1 in NSCLC and SCLC cell lines used commercial RUNX1T1 lentiviral constructs. The resulting stable cell lines were analyzed by RT-qPCR, western blotting, Affymetrix gene array, and E2F-luciferase assays. Results: We found that 2 of 2 patients with combined SCLC demonstrated RUNX1T1 amplification in the SCLC component. Notably, only 2 of the remaining 90 patients with pure SCLC demonstrated RUNX1T1 amplification. RUNX1T1 mRNA is highly expressed in SCLC compared to the majority of other cancers, including NSCLC, using either CCLE or TCGA databases. Western blots confirmed higher RUNX1T1 protein expression in SCLC vs NSCLC cell lines, and RNAscope analysis confirmed greater RUNX1T1 mRNA expression in SCLC vs NSCLC patient tumors. When we stably overexpressed RUNX1T1 in NSCLC cell lines, there was no increase in neuroendocrine protein expression, however we did find an increased sensitivity to etoposide. Bioinformatic analysis of microarray data demonstrated significantly altered E2F pathway activity, and we could show a significant increase in E2F-luciferase activity after RUNX1T1 overexpression. Interestingly, this was associated with decreased p21 expression, which is a putative suppressor of E2F activity. Conclusions: RUNX1T1 expression is specifically upregulated in SCLC compared to NSCLC. E2F pathway activity is elevated after RUNX1T1 overexpression, consistent with an increased SCLC phenotype.

Small cell lung cancer, an epithelial to mesenchymal transition (EMT)-like cancer: significance of inactive Notch signaling and expression of achaete-scute complex homologue 1.

Small cell lung cancer (SCLC) is one of the most malignant neoplasms in common human cancers. The tumor is composed of small immature-looking cells with a round or fusiform shape, which possesses weak adhesion features among them, suggesting that SCLC shows the morphological characteristics of epithelial to mesenchymal transition (EMT). SCLC is characterized by high metastatic and recurrent rates, sensitivity to the initial chemotherapy, and easy acquirement of chemoresistance afterwards. These characters may be related to the EMT phenotype of SCLC. Notch signaling is an important signaling pathway, and could have roles in regulating neuroendocrine differentiation, proliferation, cell adhesion, EMT, and chemoresistance. Notch1 is usually absent in SCLC in vivo, but could appear after chemotherapy. Notch1 can enhance cell adhesion by induction of E-cadherin in SCLC, which indicates mesenchymal to epithelial transition. On the other hand, achaete-scute complex homologue 1 (ASCL1), negatively regulated by Notch signaling, is a lineage-specific gene of SCLC, and functions to promote neuroendocrine differentiation as well as EMT. ASCL1-transfected adenocarcinoma cell lines induced neuroendocrine phenotypes and lost epithelial cell features. SCLC is characterized by neuroendocrine differentiation and EMT-like features, which could be produced by inactive Notch signaling and ASCL1 expression. In addition, chemical and radiation treatments can activate Notch signaling, which suppress neuroendocrine differentiation and induces chemoradioresistance, accompanied by secession from EMT. Thus, the status of Notch signaling and ASCL1 expression may determine the cell behaviors of SCLC partly through modifying EMT phenotypes.

EPHA3 regulates the multidrug resistance of small cell lung cancer via the PI3K/BMX/STAT3 signaling pathway.

Multidrug resistance (MDR) is a major obstacle to the treatment of small cell lung cancer (SCLC). EPHA3 has been revealed to be the most frequently mutated Eph receptor gene in lung cancer with abnormal expression. Growing evidence indicates that the signaling proteins of EPHA3 downstream, including PI3K, BMX and STAT3, play crucial roles in tumorigenesis and cancer progression. To explore the possible role of EPHA3 in MDR, we assessed the influence of EPHA3 on chemoresistance, cell cycle, apoptosis, and tumor growth, as well as the relationship between EPHA3 and the expression of PI3K, BMX, and STAT3 in SCLC. We observed that overexpression of EPHA3 in SCLC cells decreased chemoresistance by increasing apoptosis and inducing G0/G1 arrest, accompanied by reduced phosphorylation of PI3K/BMX/STAT3 signaling pathway. Knockdown of EPHA3 expression generated a resistant phenotype of SCLC, as a result of decreased apoptosis and induced G2/M phase arrest. And re-expression of EPHA3 in these cells reversed the resistant phenotype. Meanwhile, increased phosphorylation of PI3K/BMX/STAT3 signaling pathway was observed in these cells with EPHA3 deficiency. Notably, both PI3K inhibitor (LY294002) and BMX inhibitor (LFM-A13) impaired the chemoresistance enhanced by EPHA3 deficiency in SCLC cell lines. Furthermore, EPHA3 inhibited growth of SCLC cells in vivo and was correlated with longer overall survival of SCLC patients. Thus, we first provide the evidences that EPHA3 is involved in regulating the MDR of SCLC via PI3K/BMX/STAT3 signaling and may be a new therapeutic target in SCLC.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-016-5048-4) contains supplementary material, which is available to authorized users.

Genomic characterisation of small cell lung cancer patient-derived xenografts generated from endobronchial ultrasound-guided transbronchial needle aspiration specimens.

Patient-derived xenograft (PDX) models generated from surgical specimens are gaining popularity as preclinical models of cancer. However, establishment of PDX lines from small cell lung cancer (SCLC) patients is difficult due to very limited amount of available biopsy material. We asked whether SCLC cells obtained from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) could generate PDX lines that maintained the phenotypic and genetic characteristics of the primary tumor. Following successful EBUS-TBNA sampling for diagnostic purposes, we obtained an extra sample for cytologic analysis and implantation into the flanks of immunodeficient mice. Animals were monitored for engraftment for up to 6 months. Histopathologic and immunohistochemical analysis, and targeted next-generation re-sequencing, were then performed in both the primary sample and the derivative PDX line. A total of 12 patients were enrolled in the study. EBUS-TBNA aspirates yielded large numbers of viable tumor cells sufficient to inject between 18,750 and 1,487,000 cells per flank, and to yield microgram quantities of high-quality DNA. Of these, samples from 10 patients generated xenografts (engraftment rate 83%) with a mean latency of 104 days (range 63–188). All but one maintained a typical SCLC phenotype that closely matched the original sample. Identical mutations that are characteristic of SCLC were identified in both the primary sample and xenograft line. EBUS-TBNA has the potential to be a powerful tool in the development of new targeting strategies for SCLC patients by providing large numbers of viable tumor cells suitable for both xenografting and complex genomic analysis.

Abstract B27: Phenotypic plasticity and heterogeneity in small cell lung cancer (SCLC): Novel molecular subtypes and potential for targeted therapy.

Abstract Background: SCLC (15% of lung cancers) exhibits: 1) rapid growth and early fatal metastasis; 2) neuroendocrine features; 3) high initial responsiveness to chemotherapy and radiation; 4) aggressive recurrence with 5% 5-year patient survival. Gene expression and mutation profiling efforts to identify oncogenic mutations, gene amplifications or signatures with clinical utility in SCLC have thus far been unfruitful. In addition, prognostic or diagnostic markers for SCLC are scarce. Hence, there is a dire need for investigating molecular subtypes and oncogenic drivers in SCLC. We hypothesize that deregulated networks, rather than single genes, drive SCLC phenotype. Results: We previously identified a SCLC-specific gene co-expression network (Blue module, by Weighted Gene Co-expression Network Analysis - WGCNA) from a lung cancer patient dataset, and derived a SCLC-specific hub network (SSHN) signature that: 1) separated SCLC from other lung cancer types and normal lung in both genomic and proteomic independent datasets; 2) identified 2 SCLC subtypes with high and low SSHN expression in both patient specimens and cultured cell lines. Spleen tyrosine kinase (SYK) was validated as a candidate oncogenic driver of one subtype, as SYK targeted small-interfering RNA significantly decreased viability via increased death in high SYK-expressing SCLC cell lines. Due to the lack of larger SCLC patient datasets, we have now applied the SSHN classifier to the 53 SCLC cell lines from the Cancer Cell Line Encyclopedia (CCLE) and validated the SSHN-high and low subtypes. From this larger dataset, it is evident that the SSHN-defined subtypes are not totally separate. Rather, they are connected by gradual intermediate shades. This gradation became clearer by applying WGCNA to SCLC cell lines from CCLE, which identified 2 gene co-expression modules – Blue and Turquoise, that overlap with modules from patient datasets described above. The Blue module is enriched in neuroendocrine signaling, the Turquoise in mesenchymal adhesion-related pathways. Eigengene expression of the 2 modules (MEblue, MEturquoise) is anti-correlated, and all 53 SCLC cell lines are distributed along this anti-correlation diagonal. Expression of the neuroendocrine marker CD56 is highest in cells at one end of this diagonal (MEblue-high cell lines), and decreases towards the other end (MEturquoise-high cell lines), whereas the mesenchymal marker CD44 has an opposite trend. Multi-dimensional flow cytometry data, visualized with viSNE, indicated that SCLC cell lines are heterogeneous with respect to several additional cell surface and cytoplasmic markers and that, in general, there is a gradient of expression of these markers that tends to correlate with the neuroendocrine (e.g., SYK) to mesenchymal (e.g., TGFbeta receptor II) phenotype gradient. Finally, at the neuroendocrine end of the phenotypic spectrum (MEblue-high) cells grow in suspension, whereas they become increasingly adherent towards the mesenchymal end (MEturquoise-high). Conclusion: Our data provide strong evidence for a heterogeneous phenotypic space in SCLC that may define distinct subtypes. This heterogeneity was previously unsuspected in human SCLC, although evidence for it was reported in genetic mouse models of SCLC {Calbo J, et.al, Cancer Cell, 2011}. Classification of human SCLC cell lines along a neuroendocrine to mesenchymal differentiation gradient should apply to human tumors as well, since the WGCNA network classifiers overlap. However, further studies in patients are warranted to prove the existence of distinct SCLC subtypes, as well as to probe their translational value for biomarkers and targeted treatment. Citation Format: Akshata Ramrao Udyavar, Megan Hoeksema, Kirsten Diggins, Jonathan Irish, Pierre P. Massion, Vito Quaranta. Phenotypic plasticity and heterogeneity in small cell lung cancer (SCLC): Novel molecular subtypes and potential for targeted therapy. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B27.

Genomic Deregulation of the E2F/Rb Pathway Leads to Activation of the Oncogene EZH2 in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is a highly aggressive lung neoplasm with extremely poor clinical outcomes and no approved targeted treatments. To elucidate the mechanisms responsible for driving the SCLC phenotype in hopes of revealing novel therapeutic targets, we studied copy number and methylation profiles of SCLC. We found disruption of the E2F/Rb pathway was a prominent feature deregulated in 96% of the SCLC samples investigated and was strongly associated with increased expression of EZH2, an oncogene and core member of the polycomb repressive complex 2 (PRC2). Through its catalytic role in the PRC2 complex, EZH2 normally functions to epigenetically silence genes during development, however, it aberrantly silences genes in human cancers. We provide evidence to support that EZH2 is functionally active in SCLC tumours, exerts pro-tumourigenic functions in vitro, and is associated with aberrant methylation profiles of PRC2 target genes indicative of a “stem-cell like” hypermethylator profile in SCLC tumours. Furthermore, lentiviral-mediated knockdown of EZH2 demonstrated a significant reduction in the growth of SCLC cell lines, suggesting EZH2 has a key role in driving SCLC biology. In conclusion, our data confirm the role of EZH2 as a critical oncogene in SCLC, and lend support to the prioritization of EZH2 as a potential therapeutic target in clinical disease.

Abstract A29: Genomic alteration of E2F/Rb activates EZH2 in small cell lung cancer

Abstract Introduction: Small Cell Lung Cancer (SCLC) is a highly aggressive lung tumor with a 5 year survival rate of only 5% for extensive stage disease, which has only modestly improved over the last few decades. Identification of new molecular diagnostic and therapeutic targets is thus imperative. The purpose of this study was to employ genomic profiling of SCLC tumors to identify novel genomic alterations responsible for driving the aggressive biology of SCLC. Methods: DNA was extracted from a panel of 14 formalin fixed paraffin embedded SCLC tumors and used to perform high resolution array comparative genomic hybridization (aCGH) on a tiling array to identify recurrent copy number alterations. RNA was harvested from a series of SCLC cell lines and used to perform qRT-PCR on candidate genes. Publicly available SCLC tumor microarray data was also analyzed to interrogate the expression of candidate genes. Results: Through application of integrated genome and transcriptome microarray profiling, and comparison of SCLC to less aggressive non-small cell lung cancers (NSCLC) we have identified novel patterns of genomic alteration mediated pathway disruption specific to SCLC. This includes activation of the cell cycle through deregulation of downstream pathway components, as opposed to upstream deregulation of receptors such as EGFR which is characteristic of NSCLC. Strikingly we observed direct genomic activation of E2F transcription factors, in addition to the classically described loss of the Rb tumor suppressor. Analysis of targets of the E2F/Rb pathway identified EZH2, a polycomb repressive complex 2 (PRC2) member involved in epigenetic silencing of genes involved in differentiation. EZH2 has been characterized as a target of genomic amplification in prostate and breast cancers, however, no genomic amplifications were detected in our SCLC samples, thus any overexpression is likely regulated by upstream elements of the E2F/Rb pathway. qRT-PCR confirmed EZH2 as being specifically hyper-activated with a mean 42 fold over-expression (range 10 to 74 fold) in SCLC compared to 13 fold in NSCLC. This pattern was verified from an analysis of an independent array study of SCLC. Moreover, shRNA-mediated knockdown demonstrated a significant reduction in cell viability in SCLC cell lines. Conclusion: We conclude that EZH2 activation through genomic alteration of E2F/Rb contributes to the malignant phenotype of SCLC and may represent a potential therapeutic target.

Regulation of the NRSF/REST gene by methylation and CREB affects the cellular phenotype of small-cell lung cancer

The neuron-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST) is a negative regulator of gene expression restricting the expression of neuronal genes to the nervous system. NRSF/REST is highly expressed in non-neuronal tissues like the lung. In previous work, we identified small-cell lung cancer (SCLC) cell lines with no detectable NRSF/REST expression that, as a consequence, expressed neuronal markers like L1-cell adhesion molecule (L1-CAM) and neural cell adhesion molecule (NCAM). The loss of NRSF/REST expression was linked to malignant progression; however, its mechanistic role remained elusive. Here, we show that NRSF/REST itself, rather than one of its regulated genes, acts like a classic tumour suppressor, being in part regulated by methylation. In SCLCs, NRSF/REST is positively regulated by CREB, with an NRSF/REST promoter fragment showing cell type specificity. Downstream, NRSF/REST directly regulates AKT2, in which NRSF/REST loss leads to an epidermal growth factor-mediated de-regulation of AKT-Serine473 phosphorylation, important for cellular proliferation and survival. Assaying anchorage-independent growth, we observed that with reduced NRSF/REST expression, proliferation was significantly enhanced, whereas NRSF/REST rescue decreased the potential of cells to grow anchorage independently. Our observations support the fact that NRSF/REST may act as an important modulator of malignant progression in SCLC.

Repetitive Nicotine Exposure Leads to a More Malignant and Metastasis-Prone Phenotype of SCLC: A Molecular Insight into the Importance of Quitting Smoking during Treatment

Cigarette smoking is strongly correlated with the onset of lung cancer. Nicotine, a major component in cigarette smoke, has been found to promote tumor growth and angiogenesis, as well as protect cancer cells from apoptosis. Among all lung cancer cases, small cell lung cancer (SCLC) is found almost exclusively in smokers; metastasis and chemoresistance are the main reasons for the high mortality rates associated with SCLC. Retrospective studies have shown that patients with tobacco-related cancers who continue to smoke after their diagnosis display lower response rates and a shorter median survival compared with those who stop smoking. In the current work, we examined the effects of acute and repetitive exposure to nicotine, in the concentrations found in the lungs of active smokers, on the malignant properties of N417 SCLC cells in vitro. We observed that repetitive nicotine exposure induced a neuronal-like appearance in N417 cells along with increased adhesion to the extracellular matrix and chemoresistance. These changes were accompanied by enhanced migration through collagen matrices and adhesion to and transmigration across lymphatic endothelial cell monolayers. SCLC differentiation reverted after cessation of nicotine exposure. Here, we provide evidence for the leading role of the CXCR4/CXCL12 axis in these phenomena. Finally, we show how nicotine-differentiated N417 cells produced bigger and more vascularized tumors in mice, with lower apoptotic rates, than their nondifferentiated counterparts. In short, these findings identify the mechanisms through which nicotine increases SCLC malignancy and provide further evidence that CXCR4 is a potential anticancer target for nicotine-associated SCLC.

Expression of tetraspanins in human lung cancer cells: frequent downregulation of CD9 and its contribution to cell motility in small cell lung cancer.

Small cell lung cancer (SCLC) invades locally and metastasizes distantly extremely early when compared with nonsmall cell lung cancer (NSCLC). The underlying molecular mechanisms, however, have not been elucidated. Accumulating evidence suggests that downregulation of several members of tetraspanins is associated with progression of solid tumors, thus indicating poor prognosis. Here we screened 30 lung cancer cell lines for expression of tetraspanins, CD9, CD63, CD81, CD82, CD151, and NAG-2. Flow cytometry revealed that, among these proteins, CD9 is broadly expressed in NSCLC lines, but is absent or highly reduced in most SCLC lines (P<0.0001). Using the Boyden chamber and videomicroscopic cell motility assays, we showed that stable transfection of CD9 into an SCLC line, OS3-R5, reduced cell motility on fibronectin. Furthermore, by transient transfection of green fluorescent protein (GFP)-tagged CD9 into three other SCLC lines, we observed that SCLC cells expressing GFP-CD9 were uniformly less motile than untransfected cells. CD9 or GFP-CD9 was associated with beta1 integrins and distributed at the tumor cell periphery and cell-cell contacts, suggesting that CD9 modifies beta1 integrin function to reduce motility. These findings suggest that low expression of CD9 may contribute to the highly invasive and metastatic phenotype of SCLC.

Ganglioside expression in lung cancer cell lines

Gangliosides are complex glycolipid constituents of cell membranes. They are involved in many biological functions including cell–cell recognition, cell–matrix attachment, cell growth and cell differentiation. Analysis of tumour associated gangliosides may aid in the characterisation of tumour cells and their degree of malignant transformation. We have characterised a total of eight lung cancer cell lines (four small cell and four non-small cell lung cancer) with respect to ganglioside and αv integrin receptor expression. Ganglioside GD3 was detected using the monoclonal antibody R24. Ganglioside GM1 was detected using the β-subunit of cholera toxin. Ganglioside 9-O-acetyl GD3 and the αv integrin receptor were measured using commercially available monoclonal antibodies. Our results indicate that small cell lung cancer cell lines express significant levels of GD3 and 9-O-acetyl GD3. Ganglioside GM1 and αv integrin receptor were not specific to any histological subtype. The expression of ganglioside GM1 and GD3 was independent of cell–cycle phase. We conclude that GD3 and 9-O-acetyl GD3 expression may be additional markers of the Small Cell Lung Cancer phenotype, but their significance is unknown. Therefore a characteristic ganglioside pattern cannot be defined according to histological subtype. αv integrin receptor expression is not unique to cells expressing GD3.

Human small cell lung cancer expresses the octamer DNA-binding and nervous system-specific transcription factor N-Oct 3 (brain-2) : Schreiber E, Himmelmann A, Malipiero U, Tobler A, Stahel R. Fontana A. Section of Clinical Immunology, Department of Internal Medicine, University Hospital, Haldeliweg 4, CH-8044 Zurich. Cancer Res 1992;52:6121–6124

Small cell lung cancer (SCLC) cells express several characteristics of neuronal cells, including synthesis of neuropeptides and expression of the respective receptors. Establishment and maintenance of the neuronal phenotype of SCLC may depend on expression of gene transcription factors inherent to the central nervous system. The present study shows the nervous system-specific transcription factor N-Oct 3 (brain-2) to be expressed in all 13 SCLC cell lines investigated. Furthermore, N-Oct 3 (brain-2) was also found in SCLC-derived skin metastasis. In contrast, in extracts and RNA of non-SCLC cell lines and non-SCLC tumor tissues, such as lung squamous, large cell, and adenocarcinoma, expression of N-Oct 3 (brain-2) was not detectable. These data support the concept that SCLC cells derive from the neuroectodermal cell lineage since expression of N-Oct 3 (brain-2) protein is highly abundant at the neural tube stage and in the adult restricted to the neuroectodermal cell lineage.

Chapter 24 Serological and biochemical analysis of four antigens associated with small cell lung cancer

Abstract Four IgG1 monoclonal antibodies (Moab) reactive with cell surface antigens of small cell lung cancer (SCLC) were produced and serologically analysed. NE-25 and NE-150 Moabs showed an almost identical specificity, reacting with SCLC and other tumours of neural and/or (neuro)endocrine origin. NE-25 Moab precipitated an antigen of 25 kilodaltons (kD). In addition, a band of 150 kD was also recognized on certain cell lines. In contrast, NE-150 Moab precipitated only a band of 150kD. OE-130 Moab was produced to distinguish SCLC from non-SCLC. This Moab precipitated a main band of 130kD and three other molecules. PE-35 Moab detected a 35kD pan-epithelial antigen. Sixteen SCLC and 38 non-SCLC were studied with these 4 Moabs. Typical phenotype of SCLC was NE-25(NE-150) + /PE-35 + /OE-130 − , whereas that of non-SCLC was NE-25(NE-150) − /PE-35 + /OE-130 + . These Moabs were found to be useful for studying the heterogeneity and cell lineage of SCLC.