Small Cell Lung Carcinoma Cells Research Articles

Epigenetic regulation of the X-chromosomal macrosatellite repeat encoding for the cancer/testis gene CT47

Macrosatellite repeats (MSRs) present an extreme example of copy number variation, yet their epigenetic regulation in normal and malignant cells is largely understudied. The CT47 cancer/testis antigen located on human Xq24 is organized as an array of 4.8 kb large units. CT47 is expressed in the testis and in certain types of cancer, but not in non-malignant somatic tissue. We used CT47 as a model to study a possible correlation between copy number variation, epigenetic regulation and transcription originating from MSRs in normal and malignant cells. In lymphoblastoid cell lines and primary fibroblasts, CT47 expression was absent, consistent with the observed heterochromatic structure and DNA hypermethylation of the CT47 promoter. Heterochromatinization of CT47 occurs early during development as human embryonic stem cells show high levels of DNA methylation and repressive chromatin modifications in the absence of CT47 expression. In small-cell lung carcinoma cell lines with low levels of CT47 transcripts, we observed reduced levels of histone 3 lysine 9 trimethylation (H3K9me3) and trimethylated lysine 27 of histone H3 (H3K27me3) without concomitant increase in euchromatic histone modifications. DNA methylation levels in the promoter region of CT47 are also significantly reduced in these cells. This supports a model in which during oncogenic transformation, there is a relative loss of repressive chromatin markers resulting in leaky expression of CT47. We conclude that some MSRs, like CT47 and the autosomal MSRs TAF11-Like, PRR20, ZAV and D4Z4, the latter being involved in facioscapulohumeral muscular dystrophy, seem to be governed by common regulatory mechanisms with their abundant expression mostly being restricted to the germ line.

Hsp72 mediates TAp73α anti-apoptotic effects in small cell lung carcinoma cells

The transcription factor p73, a member of the p53 family of proteins, is involved in the regulation of cell cycle progression and apoptosis. Due to alternative promoters and carboxy-terminal splicing, the P73 gene gives rise to a range of different isoforms. Interestingly, a particular increase in expression of the TAp73α isoform has been reported in various tumours. In addition, TAp73α has been shown to inhibit Bax activation and mitochondrial dysfunctions and thereby to confer small cell lung carcinoma (SCLC) cells resistance to drug-induced apoptosis. However, the precise mechanism by which TAp73α exerts its pro-survival effect is yet unclear. Here we report that TAp73α, but not TAp73β, regulates the expression of inducible Hsp72/HSPA1A. Hsp72 proved to be required for the survival effects of TAp73α as antisense knockdown of Hsp72 resulted in an abolishment of the anti-apoptotic effect of TAp73α in SCLC cells upon Etoposide treatment. Importantly, depletion of Hsp72 allowed activation of Bax, loss of mitochondrial membrane potential and lysosomal membrane permeabilization in SCLC cells even in the presence of TAp73α. Finally, we revealed that TAp73β counteracts the anti-apoptotic effect of TAp73α by preventing Hsp72 induction. Our results thus provide additional evidence for the potential oncogenic role of TAp73α, and extend the understanding of the mechanism for its anti-apoptotic effect.

MicroRNA Expression and Clinical Outcome of Small Cell Lung Cancer

The role of microRNAs in small-cell lung carcinoma (SCLC) is largely unknown. miR-34a is known as a p53 regulated tumor suppressor microRNA in many cancer types. However, its therapeutic implication has never been studied in SCLC, a cancer type with frequent dysfunction of p53. We investigated the expression of a panel of 7 microRNAs (miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a) in 31 SCLC tumors, 14 SCLC cell lines, and 26 NSCLC cell lines. We observed significantly lower miR-21, miR-29b, and miR-34a expression in SCLC cell lines than in NSCLC cell lines. The expression of the 7 microRNAs was unrelated to SCLC patients' clinical characteristics and was neither prognostic in term of overall survival or progression-free survival nor predictive of treatment response. Overexpression or downregulation of miR-34a did not influence SCLC cell viability. The expression of these 7 microRNAs also did not predict in vitro sensitivity to cisplatin or etoposide in SCLC cell lines. Overexpression or downregulation of miR-34a did not influence sensitivity to cisplatin or etoposide in SCLC cell lines. In contrast to downregulation of the miR-34a target genes cMET and Axl by overexpression of miR-34a in NSCLC cell lines, the intrinsic expression of cMET and Axl was low in SCLC cell lines and was not influenced by overexpression of miR-34a. Our results suggest that the expression of the 7 selected microRNAs are not prognostic in SCLC patients, and miR-34a is unrelated to the malignant behavior of SCLC cells and is unlikely to be a therapeutic target.

Abstract 2058: The adaptation of small cell lung carcinoma (SCLC) cells to hypoxia is partially hypoxia inducible factor (HIF)-independent

Abstract Small cell lung carcinoma (SCLC) is an extremely aggressive tumor that is widely metastasized in early stages of the disease and the 5-year survival rate for these patients is only approximately 9-15%. Here we study the adaptive response of SCLC cells to lower oxygen levels, since tumor hypoxia (∼1% oxygen) is related to aggressive tumor behaviour and poor outcome in several tumor forms. Areas of hypoxia are a common characteristic in solid tumors and in response to hypoxia tumor cells induce expression of several genes involved in a variety of processes. This adaptive response is primarily regulated by the transcription factors hypoxia inducible factor (HIF)-1α and HIF-2α, which become stable at low oxygen levels. Studies using among others lung adenocarcinoma, neuroblastoma and breast carcinoma cells have demonstrated that HIF-1α primarily mediates the acute hypoxic response, whereas HIF-2α dominates during the more chronic phase of hypoxia. We investigated the viability of SCLC cells cultured at hypoxia and characterized the expression levels of HIF transcription factors in SCLC cells and specimens using quantitative real-time PCR, western blot and immunohistochemistry. Expression levels of well known hypoxia-driven genes were studied and to further characterize the hypoxic adaptive response, a gene expression profile of hypoxic SCLC cells was performed using microarray analyses. In all experiments a panel of non-SCLC cells and neuroblastoma cells were included for comparison. Our results show that SCLC cells have a high adaptive capacity to hypoxia, even though a modest induction of well known hypoxia-driven genes is observed. Interestingly, the HIF-1α and HIF-2α are differentially regulated in SCLC cells as compared to non-SCLC. In SCLC cells, no HIF-2α protein is detectable whereas HIF-1α protein levels are sustained and increased up to 96 hours. In addition, knock-down of HIF-1α at 0.1% oxygen are not affecting the viability of the SCLC cells and our gene expression profile of hypoxic SCLC cells has identified genes important for SCLC cells survival at hypoxia. Our data suggest that SCLC cells have an adaptive response to hypoxia that is partially HIF-independent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2058. doi:10.1158/1538-7445.AM2011-2058

Abstract 2566: Beneficial impact of valproate on the response of small cell lung cancer to cisplatin – etoposide therapy

Abstract Prognosis of small cell lung carcinoma (SCLC) is particularly poor, less than 5% of patients with extensive stage being alive after two years. We hypothesized that SCLC chemotherapy could be improved by using histone deacetylase (HDAC) inhibitors based on their ability to interfere with lysine acetylation and to alter gene expression. The goal of this study was to evaluate the anticancer efficacy of a HDAC inhibitor (valproate: VPA) on SCLC cells in combination with the standard chemotherapeutic first-line regimen (cisplatin + etoposide). We show that VPA induces apoptosis of small cell lung cancer cell lines and improves efficacy of cisplatin combined with etoposide. Both mitochondrial and death receptor pathways are involved in VPA-induced apoptosis. As expected for an HDAC inhibitor, VPA hyperacetylates histone H3. The mechanism of VPA pro-apoptotic activity involves induction of p21, inhibition of Bcl-XL, cleavage of Bid and phosphorylation of Erk and H2AX. In the presence of VPA, Bax is translocated from the cytoplasm to the mitochondria and cleaved in an 18 kDa isoform. Cytochrome c is released from the mitochondria into the cytosol. Transcriptomic analyses by microarray show that VPA modulates transcription of genes (Na+/ K+ ATPase, Bcl-XL) involved in chemoresistance to cisplatin and etoposide. Finally, the efficacy of VPA combined with cisplatin and etoposide is supported by preclinical models of SCLC cells engrafted into SCID mice. Together, these data demonstrate that VPA augments anticancer activity of cisplatin and etoposide, two components of the standard first-line chemotherapy of small cell lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2566. doi:10.1158/1538-7445.AM2011-2566

Retention of the In Vitro Radiosensitizing Potential of Gemcitabine Under Anoxic Conditions, in p53 Wild-Type and p53-Deficient Non–Small-Cell Lung Carcinoma Cells

Whereas radiosensitization by gemcitabine is well studied under normal oxygen conditions, little is known about its radiosensitizing potential under reduced oxygen conditions. Therefore, the present study evaluated the impact of anoxia on gemcitabine-mediated radiosensitization. The clonogenic assay was performed in three isogenic A549 cell lines differing in p53 status (24 h, 0-15 nM gemcitabine, 0-8 Gy irradiation, normoxia vs. anoxia). Using radiosensitizing conditions, cells were collected for cell cycle analysis and apoptosis detection. Whereas wild-type p53 A549-LXSN cells were more sensitive to radiation than p53-deficient A549-E6 cells, both cell lines showed similar radiosensitization by gemcitabine under normoxia and anoxia. Independent of p53 functionality, gemcitabine was able to overcome anoxia-induced G(0/1) arrest and established an (early) S phase block in normoxic and anoxic cells. The percentage early and late apoptotic/necrotic cells increased with the gemcitabine/radiation combination, with a significant difference between A549-LXSN and A549-E6. This study is the first to show that gemcitabine retains its radiosensitizing potential under low oxygen conditions. Although radiosensitization was observed in both p53 wild-type and p53-deficient cells, p53 status might influence induction of apoptosis after gemcitabine/radiation treatment, whereas no effect on cell cycle progression was noticed.

Quinazoline Sulfonamides as Dual Binders of the Proteins B-Cell Lymphoma 2 and B-Cell Lymphoma Extra Long with Potent Proapoptotic Cell-Based Activity

ABT-737 and ABT-263 are potent inhibitors of the BH3 antiapoptotic proteins, Bcl-x(L) and Bcl-2. This class of putative anticancer agents invariantly contains an acylsulfonamide core. We have designed and synthesized a series of novel quinazoline-based inhibitors of Bcl-2 and Bcl-x(L) that contain a heterocyclic alternative to the acylsulfonamide. These compounds exhibit submicromolar, mechanism-based activity in human small-cell lung carcinoma cell lines in the presence of 10% human serum. This comprises the first successful demonstration of a quinazoline sulfonamide core serving as an effective benzoylsulfonamide bioisostere. Additionally, these novel quinazolines comprise only the second known class of Bcl-2 family protein inhibitors to induce mechanism-based cell death.

Establishment and characterization of primary lung cancer cell lines from Chinese population

To establish and characterize primary lung cancer cell lines from Chinese population. Lung cancer specimens or pleural effusions were collected from Chinese lung cancer patients and cultured in vitro with ACL4 medium (for non-small cell lung carcinomas (NSCLC)) or HITES medium (for small cell lung carcinomas (SCLC)) supplemented with 5% FBS. All cell lines were maintained in culture for more than 25 passages. Most of these cell lines were further analyzed for oncogenic mutations, karyotype, cell growth kinetics, and tumorigenicity in nude mice. Eight primary cell lines from Chinese lung cancer patients were established and characterized, including seven NSCLC cell lines and one SCLC cell line. Five NSCLC cell lines were found to harbor epidermal growth factor receptor (EGFR) kinase domain mutations. These well-characterized primary lung cancer cell lines from Chinese population provide a unique platform for future studies of the ethnic differences in lung cancer biology and drug response.

Combined small-cell carcinoma of the lung with quadripartite differentiation of epithelial, neuroendocrine, skeletal muscle, and myofibroblastic type

The combined variant of small-cell lung carcinoma (SCLC) refers to the variable admixture of small cell and non-small cell carcinoma, whereas the association with sarcoma or sarcoma-like elements is exceedingly rare. A 76-year-old Caucasian man underwent right upper lobectomy with regional lymphadenectomy because of a symptomatic 7cm-sized tumor mass. Formalin fixed-paraffin embedded material was used to highlight several differentiation cell lineages by means of immunohistochemistry, electron microscopy, and mutational assay. The tumor was discovered as being IIB stage (pT2b pN1(1/51) pM0) and featured biphasic appearance with close intermingling of SCLC (40%) and collagen-rich spindle cell sarcoma (60%). Epithelial (cytokeratins, TTF-1), neural (neurofilaments, GFAP), endocrine (chromogranin, synaptophysin, CD56), and skeletal muscle (desmin, sarcomeric actin, myogenin) markers were variably co-expressed by SCLC elements, whereas mesenchymal (vimentin), smooth muscle (actin, myosin, H-caldesmon, calponin), fibroblastic (CD10), and, more focally, skeletal muscle (desmin, sarcomeric actin and myogenin) markers were highlighted in the spindle cell sarcoma elements. TP53 codon V274F mutation in exon 8 was shared by either cell component. After undergoing adjuvant chemotherapy, the patient is currently alive and well at the 40-month follow-up. To the best of our knowledge, this is the first report of combined SCLC with quadripartite differentiation of epithelial, neuroendocrine, skeletal muscle, and myofibroblastic type, somewhere at the level of the same individual tumor cells. This tumor had probably derived for clonal evolution of a p53-mutated common ancestor lesion.

Selective M3 Muscarinic Receptor Antagonist Inhibits Small-Cell Lung Carcinoma Growth in a Mouse Orthotopic Xenograft Model

In small cell lung carcinoma (SCLC), acetylcholine (ACh) is synthesized and secreted, and it acts as an autocrine growth factor through activation of its receptors, muscarinic receptor (mAChR) and nicotinic receptor (nAChR). Alteration of tumor growth by blockade of M(3) mAChR in a human SCLC cell line, NCI-H82, was investigated in the present study. We used a highly selective M(3) muscarinic antagonist, N-(2-[3-([3R]-1-(cyclohexylmethyl)-3-piperidinyl]methylamino)-3-oxopropyl]amino-2-oxoethyl)-3,3,3-triphenyl-propioamide (J-115311). Our results show that J-115311 inhibited the increased intracellular calcium elicited by carbachol, a muscarinic agonist, in SCLC cells. J-115311 also inhibited SCLC cell growth in vitro. In a mouse orthotopic xenograft model, J-115311 dose-dependently reduced tumor growth when NCI-H82 cells were inoculated into the upper left lobe of the lung. These findings indicate that blockade of M(3) mAChR can suppress tumor growth in SCLC, suggesting the potential therapeutic utility of M(3) muscarinic antagonists as anti-cancer agents.

Binding of TS1, an anti-keratin 8 antibody, in small-cell lung cancer after 177Lu-DOTA-Tyr3-octreotate treatment: a histological study in xenografted mice

BackgroundSmall-cell lung carcinoma (SCLC) is an aggressive malignancy characterised by an early relapse, a tendency towards drug resistance, and a high incidence of metastasis. SCLC cells are of neuroendocrine origin and express high levels of somatostatin receptors; therefore, future treatment might involve targeting tumours with radiolabelled somatostatin analogues. This therapy induces abundant necrotic patches that contain exposed keratins; thus, keratin 8, which is one of the most abundant cytoskeletal proteins may represent an interesting secondary target for SCLC. This study aimed to investigate the effects of177Lu-DOTA-Tyr3-octerotate and the binding of the monoclonal anti-keratin 8 antibody, TS1, in vitro in treated SCLC- and midgut-xenografted mouse models.MethodsNCI-H69- and GOT1-xenotransplanted mice were treated with three doses of 30 MBq177Lu-DOTA-Tyr3-octreotate administered 24 h apart. Mice xenotransplanted with NCI-H69 were sacrificed 1, 5, 12, 20 and 150 days post-injection or when the tumour had regrown to its original size. GOT1-xenotransplanted mice were sacrificed 3 days post-injection. Immunohistochemistry was performed to evaluate TS1 staining in tumours and in seven human biopsies of primary SCLC from pulmonary bronchi. Central cell density and nucleus size were determined in NCI-H69 sections.ResultsTwelve days after177Lu-DOTA-Tyr3-octerotate treatment, the SCLC xenograft response was extensive. Twenty days after treatment, one of three analysed tumours displayed complete remission. The other two tumours showed 1/4 the cell density of untreated controls and cell nuclei were about three times larger than those of untreated controls. At 150 days after treatment, one of four mice exhibited complete remission. Treated tumours displayed increased TS1 antibody accumulation and high TS1 binding in necrotic patches. All seven human SCLC biopsies displayed necrotic areas with TS1 staining.ConclusionsRadiation treatment with three injections of 30 MBq177Lu-DOTA-Tyr3-octreotate had pronounced effects on tumour cell density and cell nuclei, which indicated mitotic catastrophe. Despite these anti-tumour effects, two of three SCLC tumours recurred. Further studies should investigate the nature of tumour cell survival and develop more effective treatments. High TS1 accumulation in tumour sections in vitro after177Lu-DOTA-Tyr3-octerotate treatment indicated that TS1 might represent a promising secondary therapeutic strategy.

High‐grade neuroendocrine carcinoma of the lung shows increased thymidylate synthase expression compared to other histotypes

Thymidylate synthase (TS) expression has been reported in various tumors, including non-small-cell lung carcinoma (NSCLC), but not in high-grade neuroendocrine (HGNE) carcinoma of the lung. We measured TS expression in surgically resected pulmonary tumors, comparing HGNE carcinomas of the lung (13 large-cell neuroendocrine carcinomas, 8 small-cell lung carcinomas) with squamous cell carcinoma and adenocarcinoma of the lung using laser-capture microdissection for tissue isolation, real-time polymerase chain reaction (PCR), and immunohistochemistry. We also measured TS mRNA expression in small-cell lung carcinoma (SCLC) and NSCLC cell lines using real-time PCR. At both mRNA and protein levels, TS expression was significantly higher in squamous cell carcinoma compared to adenocarcinoma. Moreover, TS expression was significantly higher in HGNE carcinomas of the lung compared to squamous cell carcinoma. A significant correlation was found between mRNA and protein expression. TS mRNA expression in SCLC cell lines was significantly higher than in NSCLC cell lines. TS expression was higher in HGNE carcinomas of the lung than in squamous cell carcinoma, which was higher than in adenocarcinoma. This information may be useful in predicting the effects of TS-inhibiting agents in patients with NSCLC and HGNE carcinomas of the lung.

Znaczenie badania cytologicznego w rozpoznawaniu drobnokomórkowego raka płuca

Small cell lung carcinoma (SCLC) is a very aggressive neoplasm. Accurate and quick diagnosis is crucial to initiate proper treatment. The aim of this study was to establish the value of initial cytological diagnosis and to present typical cytological features of SCLC. We reviewed 116 cases of SCLC confirmed by cytology in: bronchial brushings, pleural fluids, and fine needle aspiration biopsies (FNAB). In 77% of SCLC cases, the diagnosis was established only by cytology; in 23% of cases, both cytological and histological recognition was possible. Cytology of SCLC was initially uncertain in 12%, and histology was uncertain in 30% of the cases. The morphology of SCLC cells was not uniform, and often a mixture of non-small atypical cells and bronchial epithelial cells with signs of metaplasia was observed. There were four cases of combined cell type with large cell carcinoma and two with adenocarcinoma. The main diagnostic problem was to distinguish small cell lung carcinoma from lymphomas, and from cancer consisting of small cells with the cytological features of non-small cell carcinoma. Diagnosis of SCLC in cytological smears is accurate, and final diagnosis is based on light microscopy. In the differential diagnosis, other tumours of small cells have to be taken into account.

Preclinical evidence for a beneficial impact of valproate on the response of small cell lung cancer to first-line chemotherapy

Prognosis of small cell lung carcinoma (SCLC) is particularly poor, less than 5% of patients with extensive stage being alive after two years. We hypothesized that SCLC chemotherapy could be improved by using histone deacetylase (HDAC) inhibitors based on their ability to interfere with lysine acetylation and to alter gene expression. The goal of this study was to evaluate the anticancer efficacy of a HDAC inhibitor (valproate: VPA) on SCLC cells in combination with the standard chemotherapeutic first-line regimen (cisplatin + etoposide). We show that VPA induces apoptosis of small cell lung cancer cell lines and improves efficacy of cisplatin combined with etoposide. Both mitochondrial and death receptor pathways are involved in VPA-induced apoptosis. As expected for an HDAC inhibitor, VPA hyperacetylates histone H3. The mechanism of VPA pro-apoptotic activity involves induction of p21, inhibition of Bcl-xL, cleavage of Bid and phosphorylation of Erk and H2AX. In the presence of VPA, Bax is translocated from the cytoplasm to the mitochondria and cleaved in an 18 kDa isoform. Cytochrome c is released from the mitochondria into the cytosol. Transcriptomic analyses by microarray show that VPA modulates transcription of genes (Na +/K + ATPase, Bcl-xL) involved in chemoresistance to cisplatin and etoposide. Finally, the efficacy of VPA combined with cisplatin and etoposide is supported by preclinical models of SCLC cells engrafted into SCID mice. Together, these data demonstrate that VPA augments anticancer activity of cisplatin and etoposide, two components of the standard first-line chemotherapy of small cell lung cancer.

Chemokine expression in tumor-to-tumor metastasis

Chemokines play an important role in cancer metastasis by modulating the directional cell movement and migration of tumor cells. The most commonly overexpressed chemokine receptor in human cancer is CXCR4. Once activated by its ligand CXCL12 (stromal cell-derived factor-1 ligand/SDF1), CXCR4 stimulates several key migratory, proliferative and survival signaling cellular pathways. CXCR4 is expressed in small-cell lung carcinoma (SCLC) cells and other tumors. To further characterize the role of chemokines in tumor-to-tumor metastasis, we analyzed the tissue expression of CXCR4 and CXCL12 in the surgical specimen of a patient with this phenomenon. We performed immunohistochemical analysis for the expression of CXCR4 and CXCL12 in metastatic tumor tissue of a 69-year-old Caucasian male with extensive SCLC metastatic to a renal oncocytoma. The oncocytoma tissue harboring SCLC showed CXCL12 expression, but not CXCR4. A high expression of the two molecules was found in a normal renal parenchymal control. Our results suggest that CXCR4 and CXCL12 plays a role in this condition, but their expression may be affected by the microenvironment of the harboring malignancy. Further characterization of these phenomena is needed to shed light on the biological mechanisms of tumor metastasis.

Abstract 452: The hypoxic response is differentially regulated in small cell lung carcinoma (SCLC) cells as compared to non-SCLC cells

Abstract Here we study the adaptive capacity of small cell lung carcinoma (SCLC) cells to hypoxia and the importance of activation of hypoxia-inducible factor (HIF) transcription factors in these cells. SCLC is an extremely aggressive tumor form and the 5-year survival rate for these patients is only approximately 9-15%. Regions of hypoxia (∼1% oxygen) often appear in solid tumors and are associated with aggressive tumor behaviour and poor outcome in several tumor forms. Tumor cells adapt to a hypoxic environment by changing the transcription of genes involved in a variety of processes, like angiogenesis, cell survival, and metabolism. This adaptive response is primarily regulated by the transcription factors HIF-1α and HIF-2α, which become stable at low oxygen levels. Studies using among others neuroblastoma, breast carcinoma and lung adenocarcinoma cells have demonstrated that HIF-1α primarily mediates the acute hypoxic response, whereas HIF-2α dominates during the more chronic phase of hypoxia. We investigated cell survival in a panel of SCLC cell lines cultured at different oxygen pressures. In these cells we also studied expression levels of well known hypoxia-driven genes using quantitative real-time PCR. Quantitative real-time PCR, Western blot and immunohistochemistry were used to characterise the expression of HIF transcription factors in SCLC cells as well as in SCLC specimens. In all experiments a panel of non-SCLC cells and neuroblastoma cells were included for comparison. Since tumor hypoxia correlates with poor prognosis it is important to elucidate how tumor cells adapt in response to hypoxia. Our results show that SCLC cells survive well at hypoxia, although the proliferation is somewhat reduced with lower oxygen pressure. Even though the SCLC cells have a high adaptive capacity, only a moderate up-regulation was observed for the investigated hypoxia-regulated genes at 1% oxygen. In addition, the expression patterns of HIF-1α and HIF-2α differ in SCLC as compared to non-SCLC, indicating that a “typical” adaptive response to hypoxia does not occur in SCLC cells. Based on these results a gene expression profile of hypoxic SCLC cells using microarray analyses are performed to further characterise the hypoxic adaptive response. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 452.

Abstract 188: Interferon stimulated gene expression as a potential resistance mechanism in DNA damage from epigenetic inhibitors

Abstract Since epigenetic inhibitors, such as inhibitors of histone deacetylases (HDAC) and DNA methyltransferases (DNMT) can induce inhibition of cancer cell proliferation we tested the hypothesis that concurrent inhibition of DNA methylation by 5-AZA-dC and histone acetylation by LBH589 or MGCD0103 could synergistically reduce the viability of human small cell lung carcinoma (SCLC) cell lines. SCLC cells were treated with 5-AZA-dC in combination with HDAC inhibitors. Growth inhibition of cells was assessed after 72 h with Alamar blue assay. Synergistic effect was determined by Chou and Talalay analysis. Inhibition of DNMT1 expression by 5-AZA-dC was verified by Western blot. TUNEL and Caspase 3 cleavage assays were used to detect apoptosis in SCLC. DNA damage was assessed by Comet assay and analyzed using CometScoreTM software. Illumina microarray analysis was performed to compare basal gene expression in sensitive H526 and resistant H196 cells. Real-time RT-PCR was used to confirm gene array data. Although sub-IC50 concentrations of 5-AZA-dC and the HDAC inhibitors (LBH589 or MGCD0103) synergistically reduced proliferation of 4/6 SCLC cell lines, loss of viability of SCLC cells did not correlate with the inhibition of either DNMT1 or HDACs suggesting non-epigenetic mechanisms for the synergy. Since combinations of 5-AZA-dC and HDAC inhibitors had marginal effects on cell cycle profiles or apoptosis index, comet assay was undertaken to assess DNA damage. MGCD0103 and 5AZA-dC co-treatment augmented DNA damage in SCLC cells resulting in increased tail length or moment in comet assays by 24 hrs in sensitive cell lines. Comparison of ratio of basal gene expression profiles of resistant H196 to sensitive H526 cells identified expressed (≥2x) mRNA differences of 2197 genes in H196 cells. Of these, >x10x expression of 273 genes was detected in resistant H196 cells compared to sensitive H526. More than 17% of genes from highly expressed genes (>60 fold) belonged to interferon stimulated gene (ISG) pathways. IFI27 was the most highly expressed gene (379 fold difference) in H196 cells compared to H526 cells; other constitutively expressed ISGs included IFIT1, IFI44, IFI35, MX1 and GBP2. In confirmation of this unexpected microarray result, qRT-PCR identified marked upregulation of 15 selected genes in H196 cells. To further confirm correlation between upregulation ISGs with DNA damage resistance, basal expression of ISGs in sensitive (H526, H82, H146 and DMS114) and resistant (H196, SW1271) cells were compared. Box and Whiskers analysis identified markedly higher median mRNA levels for most of ISGs in the resistant cells. Constitutive expression of IFN-stimulated genes may predict SCLC cells resistance to epigenetic modulators or other DNA damage agents; these unexpected findings may contribute to enhance mechanistic understanding of cell growth inhibition by both IFNs and epigenetic inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 188.

Abstract 2592: Host dependency factors for binding and internalization of oncolytic Seneca Valley Virus

Abstract Background Seneca Valley Virus (SVV-001), an experimental oncolytic picornavirus, is the prototype member of the genus Senecavirus of the family Picornaviridae. SVV-001 is remarkable for its exquisite tumor selectivity for human cancers with neuroendocrine features including small cell lung carcinoma (SCLC) and numerous pediatric cancers while being non-cytotoxic in normal human cells in vitro and in vivo. The mechanism of tropism and neuroendocrine tumor specificity of SVV-001 has not been elucidated previously. Identification of host factors involved in infection would have immediate benefit for ongoing clinical trials by providing potential biomarker candidates to predict efficacy. Here we present data implicating α4 integrin as a key component of SVV-001 infection and demonstrating that the virus is dependent on caveloae for internalization and trafficking. Results Microarray analysis comparing permissive with non-permissive cell lines and membrane sub-proteome analysis of permissive H446 SCLC cells predict α4 integrin is a potential receptor candidate for SVV-001. To test SVV-001 dependency on α4 integrin, stable shRNA knockdown cell lines were generated from the permissive parental SCLC cell line H446. Viral infection was studied by single virus particle tracking, GFP reporter virus infection, and FACS analysis. α4 integrin knockdown cells showed reduced viral internalization and intracellular motility and were resistant to infection. Heterologous expression of human α4 integrin in chinese hamster ovary (CHO) cells significantly increased binding of fluorescently-labeled virus to the cell surface, but did not result in productive infection. Tracking of fluorescently-labeled SVV-001 in permissive cells expressing either clathrin-GFP, caveolin-1-GFP, or flotillin-1-GFP revealed a striking and long-lasting colocalization of internalized viral particles with caveolin-rich vesicles. Dependency on caveolin-1 for internalization was supported by results from pharmacological inhibition experiments. Pre-treatment of cells with nystatin, a cholesterol sequestration drug, resulted in a reduction in virus-induced cytopathic effect when compared to untreated cells. Stable shRNA caveolin-1 knockdown cells showed significantly reduced internalization, reduced infection by recombinant GFP reporter virus and significant impairment in SVV-001 genomic RNA release. Conclusions Defining the mechanism of tropism for SVV-001 is a key factor in successful clinical application. α4 integrin and caveolin-1 represent the first host cell factors identified in the mechanism of tropism of SVV-001 leading to a basic understanding of how the virus binds and enters permissive cells. These findings may have direct relevance for current clinical trials of SVV-001 in adult and pediatric settings and for development of second-generation derivatives of this agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2592.

Activation of Corticotropin-Releasing Factor Receptor 2 Inhibits the Growth of Human Small Cell Lung Carcinoma Cells

ABSTRACTPreviously, we found the activation of corticotropin-releasing factor receptor 2 (CRFR2) could inhibit tumor growth via an anti-angiogenic pathway, implying CRFR2 may be a tumor therapeutic target. Here, CRFR2 expression in human neuroendocrine small cell lung carcinoma (SCLC) tissues and cell lines NCI-H446 and NCI-H1688 were detected. Meanwhile, UCNs could directly inhibit the proliferation and promote the apoptosis of SCLC cells via CRFR2. It was also shown that the activation of CRFR2 could inhibit p38 and Akt phosphorylation to suppress the secretion of VEGF in SCLC cells. These observations implied CRFR2 might be a therapeutic target in human SCLC.

MET in small cell lung carcinoma (SCLC): Effects of a MET inhibitor in SCLC cell lines and prognostic role of MET status in patients

e14617 Purpose: HGF/MET pathway is aberrantly activated by receptor overexpression and mutations in SCLC preclinical models, enhancing their oncogenicity. The significance of MET expression in SCLC remains unclear. Our aim was to analyze the effects of MET inhibition in chemosensitive/refractory SCLC models and to study the expression pattern and prognostic impact of total and phosphorylated (p) MET in SCLC patients. Methods: Total and p-MET expression (Western Blot), gene copy number (FISH), and exon 14 activating mutations (sequencing) were evaluated in H69 and H69AR SCLC cell lines. PHA-665752 (PHA), alone or combined with doxorubicin, was used to study the effects of pathway inhibition on viability, colony formation and invasion assays in basal/stimulated conditions (HGF). Fifty-eight SCLC cases were evaluated for MET and p-MET expression by immunohistochemistry. Survival analyses were performed. Results: H69 and H69AR (both R988C mutated) expressed MET at basal conditions, but not p-MET. HGF induced MET phosphorylation, increased proliferation (20%) and protected cells from doxorubicin cytotoxicity. PHA 0.5μM blocked MET phosphorylation, decreased colony formation by 50% in H69, counteracted the cytoprotective effect of HGF and inhibited invasion in H69AR. MET expression was found in 98% normal bronchial epithelia, and 78% tumor samples (overexpression 38%). Activated MET was focally detected in normal and metaplastic mucosa and expressed in 22% tumors. MET expression was associated with improved overall and disease free survival (p: 0.06 and 0.051, respectively). All p-MET positive cases within MET expressing tumors, showed relapsed disease (83% in negative p-MET samples, p=0.065), suggesting MET activation may revert the good prognosis linked to total MET expression. Conclusions: MET activation, results in a more aggressive phenotype in SCLC cells. PHA at MET inhibiting concentrations reverses this phenotype. In SCLC specimens, MET expression was more prevalent than p-MET and associated with raised prognosis. All these data suggest that studies with MET inhibitors should focus on p-MET positive SCLC. [Table: see text]