Somatostatin receptor 2 signaling promotes growth and tumor survival in small-cell lung cancer
Jonathan M. Lehman
Division of Medical Oncology, Vanderbilt Ingram Cancer Center, Nashville, TN
Cancer Early Detection and Prevention Initiative, Vanderbilt Ingram Cancer Center, Nashville, TN
Veterans Affairs, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorMegan D. Hoeksema
Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt Ingram Cancer Center, Nashville, TN
Cancer Early Detection and Prevention Initiative, Vanderbilt Ingram Cancer Center, Nashville, TN
Search for more papers by this authorJeremy Staub
Division of Medical Oncology, Vanderbilt Ingram Cancer Center, Nashville, TN
Search for more papers by this authorJun Qian
Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt Ingram Cancer Center, Nashville, TN
Cancer Early Detection and Prevention Initiative, Vanderbilt Ingram Cancer Center, Nashville, TN
Search for more papers by this authorBradford Harris
Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt Ingram Cancer Center, Nashville, TN
Search for more papers by this authorJ. Clay Callison
University of Tennessee Graduate School of Medicine, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorJennifer Miao
Vanderbilt University School of Medicine, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorChanjuan Shi
Department of Pathology, Microbiology and Immunology, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorRosana Eisenberg
Department of Pathology, Microbiology and Immunology, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorHeidi Chen
Vanderbilt University Department of Biostatistics, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorSheau-Chiann Chen
Vanderbilt University Department of Biostatistics, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorCorresponding Author
Pierre P. Massion
Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt Ingram Cancer Center, Nashville, TN
Cancer Early Detection and Prevention Initiative, Vanderbilt Ingram Cancer Center, Nashville, TN
Veterans Affairs, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Correspondence to: Pierre P. Massion, Vanderbilt Ingram Cancer Center, PRB 640, 2220 Pierce Avenue, Nashville, TN 37232, USA, E-mail: [email protected], Tel.: +1-615-936-2256Search for more papers by this authorJonathan M. Lehman
Division of Medical Oncology, Vanderbilt Ingram Cancer Center, Nashville, TN
Cancer Early Detection and Prevention Initiative, Vanderbilt Ingram Cancer Center, Nashville, TN
Veterans Affairs, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorMegan D. Hoeksema
Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt Ingram Cancer Center, Nashville, TN
Cancer Early Detection and Prevention Initiative, Vanderbilt Ingram Cancer Center, Nashville, TN
Search for more papers by this authorJeremy Staub
Division of Medical Oncology, Vanderbilt Ingram Cancer Center, Nashville, TN
Search for more papers by this authorJun Qian
Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt Ingram Cancer Center, Nashville, TN
Cancer Early Detection and Prevention Initiative, Vanderbilt Ingram Cancer Center, Nashville, TN
Search for more papers by this authorBradford Harris
Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt Ingram Cancer Center, Nashville, TN
Search for more papers by this authorJ. Clay Callison
University of Tennessee Graduate School of Medicine, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorJennifer Miao
Vanderbilt University School of Medicine, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorChanjuan Shi
Department of Pathology, Microbiology and Immunology, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorRosana Eisenberg
Department of Pathology, Microbiology and Immunology, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorHeidi Chen
Vanderbilt University Department of Biostatistics, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorSheau-Chiann Chen
Vanderbilt University Department of Biostatistics, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Search for more papers by this authorCorresponding Author
Pierre P. Massion
Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt Ingram Cancer Center, Nashville, TN
Cancer Early Detection and Prevention Initiative, Vanderbilt Ingram Cancer Center, Nashville, TN
Veterans Affairs, Tennessee Valley Healthcare System, Nashville Campus, Nashville, TN
Correspondence to: Pierre P. Massion, Vanderbilt Ingram Cancer Center, PRB 640, 2220 Pierce Avenue, Nashville, TN 37232, USA, E-mail: [email protected], Tel.: +1-615-936-2256Search for more papers by this authorAbstract
Somatostatin receptor 2 (SSTR2) is overexpressed in a majority of neuroendocrine neoplasms, including small-cell lung carcinomas (SCLCs). SSTR2 was previously considered an inhibitory receptor on cell growth, but its agonists had poor clinical responses in multiple clinical trials. The role of this receptor as a potential therapeutic target in lung cancer merits further investigation. We evaluated the expression of SSTR2 in a cohort of 96 primary tumors from patients with SCLC and found 48% expressed SSTR2. Correlation analysis in both CCLE and an SCLC RNAseq cohort confirmed high-level expression and identified an association between NEUROD1 and SSTR2. There was a significant association with SSTR2 expression profile and poor clinical outcome. We tested whether SSTR2 expression might contribute to tumor progression through activation of downstream signaling pathways, using in vitro and in vivo systems and downregulated SSTR2 expression in lung cancer cells by shRNA. SSTR2 downregulation led to increased apoptosis and dramatically decreased tumor growth in vitro and in vivo in multiple cell lines with decreased AMPKα phosphorylation and increased oxidative metabolism. These results demonstrate a role for SSTR2 signaling in SCLC and suggest that SSTR2 is a poor prognostic biomarker in SCLC and potential future therapeutic signaling target.
Abstract
What's new?
Small-cell lung cancer (SCLC) is a highly aggressive and metastatic neuroendocrine carcinoma with no therapeutic improvement in decades. SSTR2 is canonically viewed as an inhibitory receptor on cell growth, but trials have shown poor clinical responses to agonists. This work shows that SCLC cell lines and primary tumors express high levels of SSTR2, and high SSTR2 expression is correlated with worse patient survival in SCLC. Furthermore, SSTR2 signaling rather serves as an important protumor survival signal in a subset of SCLC cell lines/tumor tissues, with loss of SSTR2 expression leading to profound effects on apoptosis with significant clinical implications.
Supporting Information
| Filename | Description |
|---|---|
| ijc31771-sup-0001-FigureS1.tifTIFF image, 384.4 KB | Supporting Information, Figure 1 Validation of novel SSTR2 antibody. (a, top) Serial dilution of SSTR2 protein and blotting with novel anti-SSTR2 antibody (a, bottom) Incubation with blocking peptide and SSTR2 antibody leads to no 42 kD SSTR2 band from cell lysates with high baseline expression of SSTR2. (b) SSTR2 expression in multiple SCLC primary tumors compared to normal lung tissue. |
| ijc31771-sup-0002-FigureS2.tifTIFF image, 6.7 MB | Supporting Information, Figure 2 Somatostatin receptor 2 is expressed in most small-cell lung carcinoma cell lines. CCLE expression and mutation data on SSTR family genes including somatostatin (SST) in small-cell lung carcinoma. mRNA expression from the CCLE SCLC cell lines was determined using the Affymetrix CEL file converted using robust multi-array average followed by quantile normalization (not normalized across the SCLC lines) (red, overexpression; blue, reduced expression). Note the increased SSTR2 and SST expression in many of the 53 cell lines assessed. |
| ijc31771-sup-0003-FigureS3.tifTIFF image, 5.1 MB | Supporting Information, Figure 3 Reactome pathway analysis of overexpression associated with SSTR2 expression in CCLE SCLC lines and primary SCLC tumors. Spearman correlation analysis was used to generate a gene list of genes with a statistically significant correlation factor of >0.5. An overexpression analysis was performed using Reactome software. This graphical representation represents in yellow enriched pathways found in the analysis of primary SCLC tumors (top) and 53 CCLE SCLC cell lines (bottom). Both sets show enrichment of neuroendocrine pathways and enriched developmental pathways including Ephrin/FYN related signaling in primary SCLC tumors. |
| ijc31771-sup-0004-FigureS4.tifPDF document, 4.7 MB | Supporting Information, Figure 4 High SSTR2 expression is associated with reduced long-term survival in a 98 patient SCLC cohort. Results from a 98 SCLC patient cohort assembled in a TMA (tumor micro-array) and stained for SSTR2. Panel demonstrates prolonged survival with low/no SSTR2 expression compared to high/observed SSTR2 expression. |
| ijc31771-sup-0005-FigureS5.tifTIFF image, 693.8 KB | Supporting Information, Figure 5 Bcl family reduced expression and increased cleaved caspase in multiple H1048 cell lines. (Left) Bcl family expression is reduced coinciding with reduced SSTR2 expression in H1048 SCLC cell lines compared to shRNA controls. (Right) Cleaved caspases 3, 7 and 9 are increased with SSTR2 knockdown in H1048 cells. |
| ijc31771-sup-0006-FigureS6.tifTIFF image, 7.6 MB | Supporting Information, Figure 6 Reduction in SSTR2 leads to downstream changes in pAMPKa. (a) Average luminance for each antibody doublet in the phospho-protein array. (b) Phospho-protein array data showing increased phosphorylation of multiple proteins. |
| ijc31771-sup-0007-TableS1.tifTIFF image, 4.3 MB | Supporting Information, Table 1 Patient characteristics for 98 patient SCLC cohort. No significant differences were observed between SSTR2 high- or low-expressing tumors and sex, age, smoking hx or tumor stage. |
| ijc31771-sup-0008-TableS2.xlsxExcel 2007 spreadsheet , 398.7 KB | Supporting Information, Table 2 Spearman correlations of primary SCLC tumor RNAseq and expression chip analysis of CCLE data with SSTR2 expression. Each tab corresponds to SCLC cell line (CL) data from CCLE and correlation to SSTR2 expression compared among all genes or an NE panel. Additional tabs correspond to spearman correlation data for primary SCLC tumor (PT) and all genes analyzed or the NE panel. |
| ijc31771-sup-0009-TableS3.xlsxExcel 2007 spreadsheet , 145.7 KB | Supporting Information, Table 3 Reactome pathway analysis result table. Spearman correlation analysis was used to generate a gene list of genes with a statistically significant correlation factor of >0.5. An overexpression analysis was performed using Reactome software. This is the Reactome output including the pathways, identified genes from the gene list in the pathway and the overrepresentation analysis. |
| ijc31771-sup-0010-TableS4.pdfPDF document, 56.5 KB | Supporting Information, Table 4 Cox proportional hazard table. Cox proportional hazard techniques were used in a multi-variable analysis including age and smoking status. Hazard ratios, 95% confidence intervals and corresponding p-values are reported for the limited stage and extensive stage analyses. Of note, a significant link was found between observed stage and effect of SSTR2 expression on the hazards model for the complete cohort. There was no significant difference in SSTR2 status based on tumor stage (Supporting Information, Table 1). |
| ijc31771-sup-0011-supinfo1.TIFTIFF image, 32 MB | Supporting Information 1 |
| ijc31771-sup-0012-supinfo2.tifTIFF image, 35.1 MB | Supporting Information 2 |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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