Somatostatin receptor 2 signaling promotes growth and tumor survival in small-cell lung cancer

Abstract

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.

Keywords: cancer progression; somatostatin; survival.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest:

Jonathan M. Lehman’s institution receives funding from IPSEN for an upcoming pre-clinical study directed by him.

© 2018 UICC.

Figures

Figure 1.. SSTR2 expression in lung Carcinoma tumors and cell lines.

A) Normalized expression data on 53 SCLC cell lines from CCLE on selected Neuroendocrine and SST components with overexpression of SSTR2 in most cell lines. B) RNA seq data and FPKM (fragments per kilobase per million mapped reads) showing SST pathway expression in 81 primary SCLC, data released by C) Immunoblotting for SSTR2 and loading control beta actin in cell lines from CCLE with overexpression of SSTR2 in most cell lines and low level expression in SCC or adenocarcinoma lines. D) SSTR2A staining of primary Lung Carcinoma including small cell lung carcinoma (SCLC) tumors, adenocarcinoma, squamous cell carcinoma, and carcinoid with staining intensity used in IHC scoring.

Figure 2:. Improved Median Survival in Limited Stage SCLC patients with low SSTR2 expression.

Results from a 96 SCLC patient cohort assembled in a TMA and stained for SSTR2. Panel (A) demonstrates prolonged survival with low/no SSTR2 expression compared to high/observed SSTR2 expression in limited stage disease n=52, (HR: 0.23–0.87 (95% confidence) based on multivariant Cox Proportional hazards analysis (adjusted for age, smoking status, and race). Panel (B) represents the same analysis in patients with extensive stage disease n=44, and fails to demonstrate any survival advantage in this population.

Figure 3:. Loss of SSTR2 expression leads to decreased viability in multiple high grade neuroendocrine cell lines.

A) Loss of SSTR2 leads to reduced cell viability in multiple cell lines and constructs (error bars are 95% Cl *=p

Figure 4.. Loss of SSTR2 leads to increased cleaved caspase and apoptosis.

A) Immunoblotting of HI 048 in vitro cells with increased p-AKT, stable p-ERK, and increased caspase activity consistent with increased apoptosis with loss of SSTR2 (note SSTR2 knockdown for these clones is shown in Fig. 3). B) H727 immunoblots with stable p-AKT and mildly increased cleaved caspase. C) Propidium Iodide based flow cytometry with an increased pre Gl phase noted in HI048 SSTR2 KD cells (right) compared to shRNA controls (left) consistent with increased apoptosis.

Figure 5.. Loss of SSTR2 slows in vivo SCLC growth.

A) Log transform and nonlinear modeling with REML fit (bar connotes 95% confidence) for 9 HI 048 control shRNA xenografts and matched 10 SSTR2 shRNA KD xenografts. Note the initial growth delay in the shRNA KD xenografts until day 20 post implantation. B) Immunoblotting of the 19 tumors and initial SSTR2 expression in control and KD cell lines C) Tumor imaging of representative shRNA CTL (top) and shSSTR2 xenografts (bottom) with maintained knockdown (1 cm scalebar) with Hematoxylin and Eosin, Ki67, and Cleaved Caspase 3 staining for tumor #267 both shRNA control xenograft (top), and SSTR2 shRNA knockdown xenograft (bottom) show similar Ki67 and increased cleaved caspase 3 in the SSTR2 KD xenograft.

Figure 6.. Loss of SSTR2 expression leads to changes in oxygen metabolism in SCLC lines.

A) Mitochondrial Stress Testing of SSTR2 shRNA Ctrl and SSTR2 knockdown (KD) HI 048 cell lines show increased basal 02 consumption (B) in the SSTR2 KD lines as well as significantly increased maximal respiration with FCCP Treatment (C). ECAR/OCR ratios show increased overall mitochondrial activity at baseline (B) as well as with FCCP treatment (C).