new, well-differentiated SSTR-expressing pancreatic neuroendocrine tumor (pNET) cell line NT-3 was used as control; expression of SSTRs was measured by ...
Antiproliferative effects of lanreotide in neuroendocrine tumors S. Lelek1*, C. von Hessert-Vaudoncourt1*, V. Bröker1, F. Briest1,3 , F. Jost-Brinkmann1, D. Sedding1 , J. Benecke1, H. Freitag1, H. Lammert2, B. Siegmund1, M. Hummel2, J. Schrader4 and P. Grabowski1,5 1Dept.
of Gastroenterology, Infectious Diseases and Rheumatology, CBF, Charité-Universitätsmedizin Berlin, Germany // 2Institute of Pathology, CBF, Charité-Universitätsmedizin Berlin, Germany // 3Department of Chemistry and Biochemistry, Freie Universität (FU) Berlin, Germany // 4Dept. of Gastroenterology, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany // 3Department of Gastroenterology and Endocrinology, Zentralklinik Bad Berka GmbH, Bad Berka, Germany.
*These authors contributed equally to this work.
have a 5-year survival of 60%2. Surgical resection is the first-line treatment, whenever possible with curative intent3. For intermediate proliferating BP-NETs, treatment options are limited and unsatisfactory. When the tumor becomes unresectable or relapses after surgery, patients can be treated with somatostatin
Methods: NCI-H720 (TC cell line) and NCI-H727 (ATC cell line) were incubated with PI3Kα selective inhibitor BYL719 (10 µM or 5 µM) for 48 h in order to re-express SSTRs, which become lost after continuous passaging6. The expression of membranebound sstr1, sstr2a, sstr3 and sstr5 was examined by immunocytochemistry. The new, well-differentiated SSTR-expressing pancreatic neuroendocrine tumor (pNET) cell line NT-3 was used as control; expression of SSTRs was measured by qPCR. To test the effect of lanreotide (provided by Ipsen Pharma), proliferation assays (WST-1) were performed after following an optimized protocol which consisted of growing cells under a prolonged incubation: lanreotide re-treatment every 48 h for 5 days. Prior to treating cells with lanreotide, they were pre-treated with 10 µM BYL719. Proliferation in control cells NT-3 was measured by MTT-assay after 120 h of treatment with re-treatment every 48 h. Signaling pathway changes in 800 cancer-related genes were analyzed in the lanreotide-treated BP-NET cells and control pNET cells making use of the novel Nanostring® technology (Fig. 1).
SSTR2a
SSTR3
SSTR5
Figure 2: Immunostaining of sstr1, sstr2a, sstr3 and sstr5 in two different BP-NET cell lines. Cells were treated for 48 h with 10 µM BYL719. NCI-H720 is an atypical carcinoid and NCI-H727 is a typical carcinoid cell line.
B
B
2.0
SSTR5 / GAPDH
4 3 2 1
1.5 1.0 0.5 0.0
N
T- NT3 3 + B N C NC YL I-H I-H 72 72 7 7 + B YL
0
T- NT3 3 + B N C NC YL I-H I-H 72 72 7 7 + B YL
SSTR2 / GAPDH
A
SSTR5
SSTR2
Figure 4: WST-1 cell proliferation assay after treatment with BYL719 + lanreotide. Cells were incubated with lanreotide for a period of 5 days. (A) Lanreotide in NCI-H720 shows an increasing antiproliferative effect at concentrations 0,01-10 µM. (B) NCIH727 displays a significant drop in viability at 10 µM.
N
A
SSTR1 NCI-H720 10 µM BYL719
Purpose: To establish a preclinical model (cell line-based) in order to study the antiproliferative effects of the somatostatin analog lanreotide on BP-NETs.
analogs (SSAs) such as octreotide and lanreotide, which bind somatostatin receptors (SSTRs) and initiate a signaling pathway leading to antiproliferative effects, as observed in the PROMID and CLARINET trials in gastroenteropancreatic neuroendocrine tumor disease4,5. However, their value in BP-NETs has yet to be defined.
NCI-H727 10 µM BYL719
Background: Broncho-pulmonary neuroendocrine tumors (BP-NETs) comprise 30% of NETS and differ greatly in their malignancy, ranging from slow proliferating carcinoids (typical carcinoids (TC) and atypical carcinoids (ATC)) to very aggressive large cell neuroendocrine carcinomas1. ATCs are more aggressive than TCs and reportedly
Figure 3: NCI-H727 and NT-3 qPCR. Cells were treated for 48 h with 10 µM BYL719 and expression of SSTR2 and SSTR5 was analyzed by qPCR.
cell survival (rel. to untreated)
NT-3
Fortina and Surrey, 2008
0.5
0.0
un tr e
at e 10 d 0n M 1µ M 10 µM
Figure 1: Nanostring gene expression analysis technology. (A) Three-step simplified workflow. (B) Overview of digital mRNA profiling7. Total RNA is mixed with reporter and capture probes, excess probes are removed, purified ternary complexes are bound to the imaging surface and reporter probes (representing individual copies of mRNA ) are tabulated for each gene.
1.0
lanreotide
Results:
Figure 6: Heat map of gene regulation after treatment with BYL719 vs. BYL719 + lanreotide in NCI-H720 cells. Differentially upregulated genes appear in red and differentially downregulated genes appear in blue.
Figure 5: NT-3 proliferation assay. NT-3 cells were treated for 120 h with indicated concentrations of lanreotide. Cell numbers were measured by MTT-assay.
B
A
SSTR immunocytochemistry: BYL719 exerted an effect on NCI-H720 and NCIH727 cells in terms of SSTR expression in most cases (n = 3 different experiments) (Fig. 2). Expression of sstr1 and sstr5 were the strongest, while sstr3 expression was virtually absent in both cell lines. NCI-H727 cells responded better to BYL719. The comparison between the 10 and 5 µM dosage did not produce any significant differences in SSTR expression (not shown). 48 h incubation was as effective as 96 h incubation (not shown). SSTR qPCR: Treatment of both NCI-H727 and NT-3 cells with BYL719 at 10 µM resulted in an upregulation of SSTR2 after 48 h of treatment. In contrast, SSTR5 expression decreased upon treatment with BYL719 in both cell lines (Fig. 3). Proliferation assays: lanreotide was shown to inhibit growth more effectively in NCI-H720 cells, where dosages 0,01-10 µM produced a 30% reduction in viability (Fig. 4). NCI-727 cells responded fairly similar to the different concentrations of lanreotide, where only 10 µM lanreotide managed to reduce cell viability significantly. In NT-3 cells, lanreotide reduced growth after 120 h of treatment only at a dose of 10 µM by 23% (Fig. 5). No significant effects were observed at lower dosages. Nanostring Analysis: combination treatment of BYL719 and lanreotide resulted in a marked downregulation of genes strongly associated with cell proliferation and cancer stem cell potential (Wnt, Notch, HH, TGFB, among others.) (Fig. 6). Moreover, combination treatment more than doubled the number of genes being significantly altered at the transcriptional level in comparison to single treatment with BYL719 only (Fig. 7 and Table 1).
Figure 7: Volcano Plot showing most important gene expression changes after treatment with BYL719 (A) and BYL719 + lanreotide (B) in NCI-H720 cells.
B References: A
Table 1. (A) All significantly altered transcripts with 1,5 fold after BYL719 treatment. (B) All significantly altered transcripts 1,5 fold after BYL719 + lanreotide treatment.
1. Travis et al. (2015). WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart. 2. Rekhtman, N. (2010). Arch Pathol Lab Med. 134 (11): 1628-38. 3. Pusceddu et al. (2016). Crit Rev Oncol Hematol. 100: 167-76. 4. Caplin et al. (2015). Ann Oncol. 26: 160420. 5. Rinke et l. (2009). J Clin Oncol. 27: 465663. 6. Nölting et al. (submitted 2/2017). Neuroendocrinology. 7. Fortina and Surrey (2008). Nature Biotech. 26 (3): 293-4. 8. Jost-Brinkmann et al. (2016). Poster at DACH Tagung München 2016. 9. Kharmate et al. (2013). Cancer Cell Int. 13 (1): 93 10. Buscail et al. (1995). Proc Natl Acad Sci. 92: 1580-84.
Conclusions: The effects of combination treatment of BYL719 and lanreotide largely contributed to the enhancement of effects observed in single lanreotide treatments (previous study8), as demonstrated here in BP-NETs cells. Single drug treatment for 9 days resulted in a cell proliferation reduction that was here obtained in only 5 days of combination treatment. Re-expression of SSTRs via BYL719 pre-treatment was also obtained. Receptors sstr2 and sstr5 show a moderate to strong signal in the immunostainings. These
two receptors are the most predominantly expressed in NETs, including those of the lung,9,10 which makes our cell line-based model suitable for studying SSA therapy in BP-NETs. Pre-treatment with BYL719 has been optimised in terms of concentration and incubation periods: both 5 vs. 10 µM dosage and 5 vs. 10 day incubation produced the same results in SSTR reexpression.
proliferation genes and cancer stem cell markers, but that it also has a much wider effect on RNA transcription than lanreotide single drug treatment. Combination treatment altered transcripts for a broader array of signalling key players: genes acting in cell mitosis and apoptosis (CDKN2D, MAP2K1, AKT3) and cell-cell attachment, tumor initiation and metastasis (Notch, LEFTY1, WNT8) pathways.
The Nanostring analysis revealed that not only combination treatment downregulates important cell
We are currently working on optimised protocols to enhance downregulation of cancer genes further.
Funding: This project is funded by a research grant from Ipsen Pharma.
