DataSheet1_Concomitant inhibition of PI3K/mTOR signaling pathways boosts antiproliferative effects of lanreotide in bronchopulmonary neuroendocrine tumor cells.PDF

Introduction: Somatostatin analogues (SSAs) are commonly used in the treatment of hormone hypersecretion in neuroendocrine tumors (NETs), however the extent to which they inhibit proliferation is much discussed.Objective: We studied the antiproliferative effects of novel SSA lanreotide in bronchopulmonary NETs (BP-NETs). We focused on assessing whether pretreating cells with inhibitors for phosphatidylinositol 3-kinase (PI3K) and mammalian target for rapamycin (mTOR) could enhance the antiproliferative effects of lanreotide.Methods: BP-NET cell lines NCI-H720 and NCI-H727 were treated with PI3K inhibitor BYL719 (alpelisib), mTOR inhibitor everolimus and SSA lanreotide to determine the effect on NET differentiation markers, cell survival, proliferation and alterations in cancer-associated pathways. NT-3 cells, previously reported to express somatostatin receptors (SSTRs) natively, were used as control for SSTR expression.Results: SSTR2 was upregulated in NCI-H720 and NT-3 cells upon treatment with BYL719. Additionally, combination treatment consisting of BYL719 and everolimus plus lanreotide tested in NCI-H720 and NCI-H727 led to diminished cell proliferation in a dose-dependent manner. Production of proteins activating cell death mechanisms was also induced. Notably, a multiplexed gene expression analysis performed on NCI-H720 revealed that BYL719 plus lanreotide had a stronger effect on the downregulation of mitogens than lanreotide alone.Discussion/Conclusion: We report a widespread analysis of changes in BP-NET cell lines at the genetic/protein expression level in response to combination of lanreotide with pretreatment consisting of BYL719 and everolimus. Interestingly, SSTR expression reinduction could be exploited in therapeutic and diagnostic applications. The overall results of this study support the evaluation of combination-based therapies using lanreotide in preclinical studies to further increase its antiproliferative effect and ultimately facilitate its use in high-grade tumors.</p

Neuroendocrine marker expression was increased by BYL719 treatment.

<p>A: BON-1, H727 and QGP-1 cells were treated with 10 μM BYL719 for 96 h and mRNA expression of Chromogranin A <i>(CHGA)</i>, <i>SSTR1</i>, <i>SSTR2</i> and <i>SSTR5</i> was analyzed by qPCR. B: BON-1, H727 and QGP-1 cells were treated with 1 μM BYL719 for 96 h and mRNA expression of <i>SSTR2</i> was analyzed by qPCR; Values are summarized from three independent experiments of three replicates per data point. * p<0.05; ** p≤0.01; *** p≤0.001.</p

Representative western blots of the investigated signaling pathways, apoptosis and cell cycle markers in BON-1, QGP-1 and H727 cells: The means, standard deviations and p-values of the western blot quantification and statistical analysis from at least three independent replicates are given in S1 Table.

<p>Representative western blots of the investigated signaling pathways, apoptosis and cell cycle markers in BON-1, QGP-1 and H727 cells: The means, standard deviations and p-values of the western blot quantification and statistical analysis from at least three independent replicates are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182852#pone.0182852.s031" target="_blank">S1 Table</a>.</p

CgA secretion of NET cells was not altered by BYL719 treatment.

<p>BON-1, H727 and QGP-1 cells were treated with vehicle, PDBu (positive control) or BYL719 (1 μM, 10 μM) for 24 h; the supernatant was collected for CgA measurements (two independent experiments with three replicates per data point).</p

Cell lines were treated with BYL719 (IC₅₀ and IC₈₅) for 72 h.

<p>Cells were stained with PI (DNA content) analysis solely included and mitosis-specific Phospho(Ser10)-Histone H3 immunostain followed by flow cytometry assessment (single cells). According to the stain, events were divided into cells of several cell cycle phases or were classified as sub-G1-events indicating cell death (FlowJo software), given in percent of all detected cells (mean ± SD) of at least four independent experiments. A: Cell cycle. B: Mitotic index. * p<0.05; ** p≤0.01; *** p≤0.001; **** p≤0.0001.</p

Assessment of apoptosis: Caspase 3/7 assay results after 24 h treatment with different doses of BYL719 are shown as mean percentage of caspase 3/7 activity referred to the untreated control (100%) ± SD: Strongest apoptosis was induced in BON-1 cells, followed by H727 cells.

<p>QGP-1 cells showed the least but still significant apoptosis. * p<0.05; ** p≤0.01; *** p≤0.001 (two independent experiments with three replicates per data point).</p

The combination therapy with BYL719 plus everolimus strongly induced SSTR2 expression in BON-1 and QGP-1 cells.

<p>Cells have been treated with 5 μM BYL719, 5 nM everolimus, or the combination of both for 48h (A) or with 1 μM BYL719, 1 nM everolimus or the combination of both for 96h (B). SSTR2 expression was analyzed by Western Blot (A) and qPCR (B). Representative western blot of two replicates; qPCR results are summarized from three independent experiments of three replicates. * p<0.05; ** p≤0.01; *** p≤0.001.</p