Elevated miR-16-5p induces somatostatin receptor 2 expression in neuroendocrine tumor cells

Somatostatin analogs, which are used to treat neuroendocrine tumors, inhibit hormone secretion or promote tumor shrinkage; however, their efficacy varies between patients, possibly because of differential expression of somatostatin receptors (SSTRs) in tumors. In this study, we evaluated the regulatory mechanism underlying the expression of SSTR2, the main octreotide target. Thirty miRNAs were found to be dysregulated in neuroendocrine cells (INS-1 cells) incubated with octreotide compared to that in placebo-treated cells. Among the upregulated miRNAs, miR-16-5p was elevated after short-term octreotide treatment. We conducted in vitro experiments to determine whether the expression of miR-16-5p was associated with the regulation of SSTR2 expression and affected octreotide sensitivity in INS-1 cells. Overexpression of miR-16-5p by transfected mimics induced upregulation of SSTR2 expression. Additionally, the expression of miR-16-5p further enhanced octreotide-induced reduction in cell proliferation in both two- and three-dimensional culture of INS-1 cells. Thus, our results reveal the mechanism underlying SSTR2 expression regulation and may aid in developing therapeutic approaches for enhancing the response to octreotide, particularly in patients unresponsive to SSTR2-targeted somatostatin analog treatment.


Introduction
Somatostatin (somatotropin release-inhibiting hormone, SST), a small polypeptide hormone produced in the hypothalamus [1], regulates endocrine function mainly by suppressing the release of hormones and neurotransmitters and inhibits tumor growth by regulating tumor cell survival and angiogenesis [2]. SST binds with high affinity to somatostatin receptors (SSTR1-5), which are specific G protein-coupled receptors present on cell surfaces [3]. Neuroendocrine tumors (NETs) are a heterogeneous group of neoplasms with a low growth rate in most cases; however, they occasionally secrete hormones that cause specific clinical syndromes, resulting in significant disability and decreased quality of life [4]. SSTR  protein expression patterns in NETs have been studied extensively, and SSTRs have been used as potential diagnostic and therapeutic targets because of their widespread expression and function in NETs. SST analogs (SSAs) have been developed and widely investigated [3]; for instance, octreotide and lanreotide are suitable for clinical applications because of their considerably longer half-life compared with that of natural SST. Similar to natural SST, SSAs bind to SSTRs; however, SSAs show different binding affinities for various receptors. Octreotide and lanreotide bind to SSTR2 with high affinity, followed by SSTR5, and activate inhibitory signals [5,6]. Thus, a positive correlation between the anticancer efficacy of octreotide and SSTR2 expression exists in NETs [2]. However, few studies have investigated mechanisms for enhancing SSTR expression and thereby increasing the anticancer effect of SSAs. Although acute administration of SST induces the activation of various inhibitory signals, the initial response against SSA is diminished. Continuous exposure to SSA reduces the initial drug effects via processes such as degradation, internalization, and phosphorylation of SSTRs [7].
MicroRNAs (miRNAs) are a class of non-coding RNAs comprising 21-25 nucleotides. miR-NAs have been reported to be closely associated with the regulation of different signaling pathways and several biological functions, including cell proliferation, differentiation, survival, and metabolism. miRNAs function by binding to complementary target mRNAs, resulting in mRNA translational inhibition or degradation [8]. Thus, several miRNA-targeting therapeutics have been developed and are being tested via clinical trials. Recently, miRNAs differentially expressed between SSA responders or non-responders in growth hormone-secreting pituitary adenomas have been identified [9]. However, the effects of miRNA-mediated control of SSTR2 expression and the subsequent effect on SST drug sensitivity in NETs remain poorly understood.
In this study, we identified novel miRNAs involved in the early response to octreotide treatment and investigated SSTR2 expression during this early response. This study provides insight into the mechanism of octreotide, which may be useful for enhancing its anticancer effects, particularly in octreotide non-responder patients with NETs.

Cell culture
The INS-1 rat insulinoma cell line and GH3 rat pituitary GH-and PRL-producing cell line were purchased from Addexbio (San Diego, CA, USA) and the Korean Cell Line Bank. Both cell lines expressed SSTR2 (S1 Fig). INS-1 cells and GH3 cells were cultured in RPMI-1640 (Gibco, Grand Island, NY, USA) and Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 0.05 mmol/L 2-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin and maintained at 5% CO 2 and 37˚C. HeLa cells were purchased from the Korean Cell Line Bank and grown in Dulbecco's Modified Eagle Medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin. Octreotide was purchased from TOCRIS Bioscience (Bristol, UK) and dissolved in deionized water to prepare a 1 mM stock solution. For 3-dimensioanl culture, Costar1 Ultra-Low attachment multi-well plates were purchased from Sigma-Aldrich Korea (St. Louis, MO, USA) and used as described previously [10].

RNA extraction and miRNA synthesis
Total RNA was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA concentrations were measured using a NanoDrop spectrophotometer (DeNovix, Wilmington, DE, USA). miRNAs were synthesized from 500 ng of total RNA using an miScript II RT Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

Library preparation, sequencing, and data analysis
For control and test RNAs, a library was constructed using the NEBNext Multiplex Small RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions. Briefly, 1 μg of total RNA from each sample was used to ligate the adaptors, after which cDNA was synthesized using reverse-transcriptase with adaptor-specific primers. PCR was performed for library amplification, and then the libraries were purified using a QIAquick PCR Purification Kit (Qiagen) and AMPure XP beads (Beckman Coulter, Brea, CA, USA). The yield and size distribution of the small RNA libraries were assessed by high-sensitivity DNA analysis on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). High-throughput sequences were produced by single-end 75 sequencing using the NextSeq 500 system (Illumina, San Diego, CA, USA). Sequence reads were mapped using the Bowtie 2 software tool to obtain the BAM file (alignment file). A mature miRNA sequence was used as a reference for mapping. Read counts mapped onto the mature miRNA sequence were extracted from the alignment file using bedtools (v2.25.0) and Bioconductor which uses R (version 3.2.2) statistical programming language (R development Core Team, 2011). The read counts were then used to determine the expression levels of miRNAs. The quantile normalization method was used to compare samples. miRWalk 2.0 was used for miRNA target analysis. Functional gene classification was performed using DAVID (http://david.abcc.ncifcrf.gov/).

Cell viability assay and flow cytometry analysis
Cell viability was assessed using the CCK8 assay. INS1 cells were seeded at 1 × 10 5 cells/ml in a 96-well plate, incubated for 24 h, and treated under various conditions. To measure cell viability, 10 μL/well of CCK8 reagent (Dojindo Laboratories, Kumamoto, Japan) was added to each well and the plates were incubated in a humidified incubator at 37˚C for 30 min. Absorbance at 440 and 640 nm was measured using a Spectra Max190 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cell viability for 3D spheroids was assessed in a CellTiter-Glo1 3D Cell Viability Assay (Promega, Madison, WI, USA). To quantify apoptosis, double staining was performed following the protocol as described in Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen™, Franklin Lakes, NJ, USA). INS1 cells were collected after incubation with mimic miR-15-5p and octretodie and analyzed as described previously [11].

Immunocytochemistry
Immunocytochemistry was performed as previously described [10]. Cells fixed in 4% paraformaldehyde were blocked with 3% bovine serum albumin for 30 min at room temperature (25-27˚C) and incubated with SSTR2 primary antibody (clone UMBI; 1:500; Abcam, Cambridge, UK) at 4˚C overnight. The cells were washed, incubated with the appropriate fluorescenceconjugated secondary antibody (1:200; Invitrogen) for 1 h at room temperature, and counterstained with Hoechst (Invitrogen). The coverslips were then mounted, and the cells were observed under a light microscope. Images were recorded using an Olympus BX53 microscope with Olympus Cell Sens software (Tokyo, Japan).

Statistical analysis
A paired two-tailed Student's t-test was used to detect significant differences between two sets of data. The differences were considered as statistically significant when p values were <0.05.

Identification of differentially expressed miRNAs in INS-1 cells during the early response to octreotide treatment
To identify the miRNAs involved in the early response of SSTR2 to octreotide treatment, INS-1 cells were treated with 1 μM octreotide for various incubation times. As shown in Fig 1A, SSTR2 expression was downregulated after 5 min of treatment with 1 μM octreotide. Immunocytochemistry revealed that membranous SSTR2 expression was reduced, and punctate perinuclear staining of SSTR2 was observed in the cytoplasm, suggesting that a 5-min incubation time is sufficient for identifying miRNAs associated with the early drug response of SSTR2 ( Fig 1B). Thus, we analyzed the change in miRNA after 5 min octreotide treatment. miRNA array analysis showed that 21 miRNAs were downregulated and 9 were upregulated in INS-1 cells after 5-min treatment with octreotide compared with 0-min treatment (absolute fold-change >1.5 ; Fig 2A and 2B). Furthermore, KEGG pathway enrichment analysis revealed that the targets of these miRNAs were associated with cancer, mitogen-activated protein kinase (MAPK) signaling pathway, and endocrine and other factor-regulated calcium reabsorption (S1 Table). The expression of miR-16-5p, which showed the largest number of target genes among the 30 differentially expressed miRNAs, was verified by qPCR. Similar to the miRNA array results, miR-16-5p was upregulated after 5 min of octreotide treatment in INS-1 cells (Fig 2C). To confirm these results, we employed other neuroendocrine cells (GH3 cells). As shown in Fig 3D, miR-16-5p was upregulated in GH3 cells after 5 min incubation with octreotide. These results confirmed the effect of octreotide on the expression of miR-16-5p. Next, we investigated the function of miR-16-5p to SSTR2 expression after octreotide treatment.

miR-16-5p upregulation increases sensitivity to octreotide
Because SSTR2 expression was found to be regulated by miR-16-5p, we assessed the effect of miR-16-5p on SSTR2 expression after octreotide treatment. INS-1 cells transfected with a miR-16-5p mimic or control miRNA were treated with octreotide for a short incubation time. Compared with control miRNA-transfected INS-1 cells, INS-1 cells exhibiting upregulation of miR-16-5p showed higher SSTR2 levels before and after octreotide treatment (Fig 4A and 4B). We next evaluated whether the increased SSTR2 levels induced by the miR-16-5p mimic affected the anti-cancer properties of octreotide. INS-1 cells transfected with the miR-16-5p mimic showed reduced cell proliferation after octreotide treatment (Fig 5A). To confirm the synergistic anti-cancer effect of combined treatment, annexin V-PI staining was performed. Annexin V-PI staining demonstrated that combined treatment with the mimic miR-16-5p and octreotide increased early and late apoptosis (S4 Fig). Spheroids from 3-dimensional (3D) culture systems have been used as models to evaluate drug sensitivity [13,14]. Thus, we used spheroids to examine the role of miR-16-5p in octreotide sensitivity. The proliferation of miR-16-5p mimic-transfected INS-1 cells was reduced compared to that of spheroids from control miRNA-transfected INS-1 cells after 48 h octreotide treatment, demonstrating an increased sensitivity to octreotide (Fig 5B). In parallel, immunostaining for SSTR2 was performed. Similar to 2D culture, SSTR2 expression was upregulated and maintained in spheroids from miR-16-5p mimic-transfected INS-1 cells (S5 Fig). These findings suggest a role for miR-16-5p expression in mediating the antiproliferative action of octreotide.

Discussion
The absence or low expression of SSTRs in NETs has been identified as the main cause of NET resistance to the current generation of SSAs. Therefore, to enhance NET cell sensitivity to octreotide, we used miRNA profiling and identified the miRNA associated with the initial response to octreotide treatment. Notably, our results showed that modulation of miR-16-5p levels affects SSTR2 expression and cell sensitivity to octreotide, suggesting that it can be used in combination with SSAs to improve their therapeutic effect via regulation of SSTR2 expression.
Few studies have determined the expression patterns of SSA-related miRNAs. Recently, Døssing et al. identified distinct dysregulation of miRNAs, including miR-7 and miR-148a, in the human neuroendocrine tumor cell line NCI-H727 after treatment with SSA [15]. Another

PLOS ONE
Elevated miR-16-5p induces SSTRs in neuroendocrine tumor cells study revealed that circulating miR-200a is involved in metastasis during SSA-treated small intestine NET progression [16]. However, little is known about the dysregulation of miRNAs during the early response to SSA. Thus, our study focused on miRNA expression patterns following treatment of cells with SSA under a shorter incubation period than that used in the two previous studies cited above. Only a few dysregulated miRNAs overlapped between our study and previous studies. Although the incubation time with octreotide was short, upregulation of miRNAs associated with the anticancer effect was found by KEGG analysis in our study. Moreover, our results showed that the predicted target genes of the 30 differentially expressed

PLOS ONE
Elevated miR-16-5p induces SSTRs in neuroendocrine tumor cells miRNAs were enriched in nine pathways, of which "microRNAs in cancer" and "MAPK signaling pathway" were the most prominent. Inhibition of cell proliferation by SSA through SSTRs involves several signal transduction pathways including control of the ERK/MAPK pathway [17]. Induction of ERK/MAPK pathway activation by the miR-16-5p-induced upregulation of SSTR2 was also observed in this study, suggesting that regulation of SSTR2 by miR-16-5p affects downstream signaling. Thus, we identified a miRNA involved in both early regulation of SSTR2 expression and the anticancer effect of octreotide.

PLOS ONE
Elevated miR-16-5p induces SSTRs in neuroendocrine tumor cells Internalization of SSTR2 and intracellular trafficking are involved in SSTR desensitization. Recently, Gatto et al. suggested that low expression of ARRB1/β-arrestin 1 in pituitary adenoma is associated with reduced SSTR2 desensitization, leading to an improved response to SSA [18]. In our study, experiments focused on SSTR2 internalization and intracellular trafficking have not been performed. However, according to immunostaining, miR-16-5p overexpression may not only induce an increase in basal SSTR2 expression but also the internalization of SSTR2 under exposure to octreotide compared with that in the control. Indeed, miR-16-5p regulation in INS-1 cells also influenced the levels of Arrb1 mRNA, suggesting that miR-16-5p is involved in SSTR2 internalization and desensitization (S3 Fig). SSTR2 regulation by chemical compounds has not been widely examined. Previous studies showed that histone deacetylase inhibitors and DNA methyltransferase inhibitors upregulate Sstr2 in NET cells [19]. Our study revealed that upregulation of SSTR2 occurs partly via miR-16-5p in NET cells. Although we did not investigate the direct chemical compound targets of miR-16-5p, enrichment analysis suggested that some genes targeted by miR-16-5p are associated with the proteasome-ubiquitin system, including USP25. Thus, chemical compounds targeting the proteasome are potential candidates for modulating the expression of miR-16-5p. Therefore, further studies are needed to verify whether miR-16-5p directly binds to proteasome-related genes and investigate the effect of proteasome-related chemicals on miR-16-5p.
Several preclinical studies have shown that proteasome inhibitors impair proliferation and induce apoptosis in other neuroendocrine tumors such as pituitary tumors [20][21][22]. Thus, our results may be easy to apply in clinical settings to inhibit the reduction in SSTR2 expression by SSA. Moreover, SSTRs have been exploited as targets for NET diagnosis [23,24]. Positron emission tomography imaging targeting SSTRs, such as imaging using 68 Ga-labeled SSAs (DOTATOC, DOTATATE, and DOTANOC), was recently reported as a useful imaging modality for diagnosing NET and selecting patients for SSA treatment [25]. Thus, upregulation of SSTR2 by miR-16-5p may be effective in patients with low SSTR2 levels by enhancing positron emission tomography imaging-based diagnosis.
Collectively, our study highlights a novel role for miR-16-5p in regulating the expression of SSTR2 in NET cells. We also demonstrated that upregulation of mir16-5p enhances the sensitivity of octreotide in INS-1 cells, suggesting the potential of using octreotide with miR-16-5p as a novel combination therapy for NET treatment. Moreover, as desensitization and downregulation of SSTR2 after SSA treatment is an important clinical issue, the ability to modulate SSTR expression via miR-16-5p in patients with NET is clinically relevant, as it would improve the therapeutic response or detection sensitivity.