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A plausible role for actin gamma smooth muscle 2 (ACTG2) in small intestinal neuroendocrine tumorigenesis
© Edfeldt et al. 2016
Received: 14 July 2015
Accepted: 14 April 2016
Published: 23 April 2016
Small intestinal neuroendocrine tumors (SI-NETs) originate from the enterochromaffin cells in the ileum and jejunum. The knowledge about genetic and epigenetic abnormalities is limited. Low mRNA expression levels of actin gamma smooth muscle 2 (ACTG2) have been demonstrated in metastases relative to primary SI-NETs. ACTG2 and microRNA-145 (miR-145) are aberrantly expressed in other cancers and ACTG2 can be induced by miR-145. The aim of this study was to investigate the role of ACTG2 in small intestinal neuroendocrine tumorigenesis.
Protein expression was analyzed in SI-NETs (n = 24) and in enterochromaffin cells by immunohistochemistry. The cell line CNDT2.5 was treated with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep), the selective EZH2 inhibitor EPZ-6438, or 5-aza-2’-deoxycytidine, a DNA hypomethylating agent. Cells were transfected with ACTG2 expression plasmid or miR-145. Western blotting analysis, quantitative RT-PCR, colony formation- and viability assays were performed. miR-145 expression levels were measured in tumors.
Eight primary tumors and two lymph node metastases displayed variable levels of positive staining. Fourteen SI-NETs and normal enterochromaffin cells stained negatively. Overexpression of ACTG2 significantly inhibited CNDT2.5 cell growth. Treatment with DZNep or transfection with miR-145 induced ACTG2 expression (>10-fold), but no effects were detected after treatment with EPZ-6438 or 5-aza-2’-deoxycytidine. DZNep also induced miR-145 expression. SI-NETs expressed relatively low levels of miR-145, with reduced expression in metastases compared to primary tumors.
ACTG2 is expressed in a fraction of SI-NETs, can inhibit cell growth in vitro, and is positively regulated by miR-145. Theoretical therapeutic strategies based on these results are discussed.
Small intestinal neuroendocrine tumors (SI-NETs) are small, slow growing neoplasms that originate from the enterochromaffin cells in the ileum and jejunum. These rare tumors have an incidence about 1 case per 100 000. Metastases have often already occurred at time of diagnosis and the 5-year survival rate is around 65 %. Due to excess of tumor-secreted hormones; e.g. serotonin and tachykinins, patients can suffer from the carcinoid syndrome, causing cutaneous flushing, diarrhea, carcinoid heart disease and bronchoconstriction [1, 2]. The WHO classification from 2010 divides small intestinal neuroendocrine neoplasms in three grades; G1-NETs (Ki67 < 3 %), G2-NETs (Ki67 3–20 %) and NEC (neuroendocrine carinomas, Ki67 > 20 %) . SI-NETs (G1 and G2) are most often resistant to chemotherapy and radiation, and medical treatment is limited. Symptom relief can be obtained by somatostatin-analogues and interferon treatment. There is a great need of new therapeutic options that could be beneficial to the patients.
The knowledge of common genetic or epigenetic abnormalities is limited in SI-NETs. Loss of chromosome 18 is most frequently seen, but no tumor-associated mutations have been found on chromosome 18 [4–6]. A putative role for TCEB3C (elongin A3), located at 18q21, as tumor suppressor gene in SI-NETs was recently suggested . The mutation rate is overall low , and recently, exome- and genome sequencing found CDKN1B to be mutated in ~9 % of SI-NETs , implicating importance for this gene in tumorigenesis.
We have previously observed expression of actin gamma smooth muscle 2 (ACTG2) mRNA in a collection of primary SI-NETs, compared to undetectable expression levels in lymph node metastases . Actin proteins are involved in multiple intracellular processes, including maintenance of the cytoskeleton and cell motility , and ACTG2 is normally found in enteric tissue. Aberrant expression has been described in several different cancer types and this can affect chemotherapy sensitivity [12–14]. Lower expression levels of ACTG2 were detected in normal colon tissue compared to colon carcinoma . High expression levels of ACTG2 have been associated with improved disease-specific survival , and also with a more aggressive phenotype [17, 18]. Furthermore, microRNA-145 (miR-145) can positively regulate expression of ACTG2 [19, 20], and overexpression of this microRNA inhibits cell proliferation, cell invasion, tumor growth and can induce apoptosis in other cancer cell types [19, 21].
The aim of this study was to investigate a possible role of ACTG2 in small intestinal neuroendocrine tumorigenesis.
Tumor material and cell line
The patients included in the study (n = 28) were all diagnosed with SI-NET in the ileum and operated on at Uppsala University Hospital. This study was approved by the regional ethical review board in Uppsala (11-375/1.1.2011, Local ethical vetting board in Uppsala (Regionala etikprövningsnämnden i Uppsala)). Written informed consent for participation and publication of individual clinical details was obtained from all patients. All patients were above 18 years of age at time of inclusion. Fifteen tumors were classified as G1 NETs and 13 as G2 NETs. Patient characteristics are summarized in Additional file 1: Table S1. The tumors were snap frozen in liquid nitrogen and kept at −70 °C.
A SI-NET cell line, CNDT2.5, developed from a liver metastasis from a patient diagnosed with primary ileal SI-NET , was used in the experiments. These cells expressed neuroendocrine markers and somatostatin receptor 2 and responded to synthetic somatostatin analogue (octreotide) treatment [22, 23], although skepticism regarding the neuroendocrine authenticity of this cell line has also been raised . The growth medium for CNDT2.5 was DMEM-F12 complemented with 10 % fetal bovine serum (Sigma Aldrich), 1 % vitamins, 1 % L-glutamine, 1 % sodium pyruvate, 1 % nonessential amino acids and 1 % PEST (penicillin-streptomycin), and the cells were cultured at 37 °C in 5 % CO2.
Immunohistochemistry procedure is described in detail in previous research [25, 26]. Paraffin embedded tumor tissue (n = 24) sections (5 μm) were passed through descending alcohol concentrations and distilled water. Background staining was blocked with 3 % hydrogen peroxide and heated in citrate buffer. The tissues were treated with normal serum from goat (S-1000, Vector) and two different rabbit polyclonal anti-ACTG2 antibodies (NB100-91649 Novus Biologicals, diluted 1/200, and TA313418 Origene, diluted 1/80) and anti-chromogranin A antibodies (Ab-1, LK2H10, NeoMarkers, diluted 1/1000) were used and incubated. A biotinylated secondary antibody from goat anti-rabbit (BA-100 Vector, diluted 1/200) was added to the tissues and then treated with ABC complex. Visualization was done with DAB color reagent. Absence of primary antibody was used as a negative control. Consecutive sections from each tumor were incubated with anti-ACTG2 and anti-chromogranin A antibody. Also, consecutive sections of normal intestinal mucosa were treated with anti-ACTG2 (NB100-91649 Novus Biologicals, diluted 1/200) and anti-chromogranin A antibodies (Ab-1, LK2H10, NeoMarkers, diluted 1/1000).
Double immunofluorescence staining was done on sections of intestinal mucosa. Paraffin-embedded sections were deparaffinized, hydrated and subjected to pre-treatment (microwave heating for 10 min at 800 W, followed by 20 min at 450 W in citrate buffer, pH 6.0). The sections were blocked with normal goat serum (S-1000, Vector) for 30 min before incubation with primary antibody anti-chromogranin A (Ab-1, LK2H10, NeoMarkers, diluted 1/1000) for 90 min, followed by secondary antibody Alexa Fluor 488 goat anti-mouse for 30 min. Then, incubation with the next primary antibody anti-ACTG2 (NB100-91649 Novus Biologicals), for 90 min, was followed by the secondary antibody Alexa Fluor 594 goat anti-rabbit, for 30 min (Life Technologies). The sections were mounted with Vectashield with DAPI (Vector Laboratories Inc.) and evaluated under light microscope.
Western blotting analysis
Proteins were extracted from tumors or CNDT2.5 cells using Cytobuster™ protein extraction reagent (Novagen) supplemented with Complete mini protease inhibitor cocktail tablets (Roche Diagnostics). Analysis of ACTG2 in tumor tissue was done using a primary antibody; anti-actin gamma2 (NB100-91649). Anti-Actin antibody (sc 1616, Santa Cruz) or coomassie blue was used as loading controls, and for verification of transfection results a mouse monoclonal anti-DDK antibody (TA50011, Origene) was used. After incubation with the appropriate secondary antibody, bands were visualized using the enhanced chemiluminescence system (GE Healthcare).
Quantitative real-time RT-PCR
For extraction and purification of RNA, RNeasy Plus Mini Kit (Qiagen) was used according to manufacturer’s instructions, and for microRNA, miRNeasy Mini Kit (Qiagen) was used. Quantity was measured using NanoDrop. Reverse transcription of DNA-free RNA with random hexamer primers was performed using the “First strand cDNA Synthesis kit” according to manufacturer’s instructions (Fermentas) or MicroRNA RT kit (Life Technologies) using 10 ng RNA. Successful DNase I treatment of all RNA preparations was established by PCR analysis of the MYC promoter. qRT-PCR reactions were performed on the Step I qRT-PCR system (Applied Biosystems) using TaqMan Gene Expression Master Mix and assays for ACTG2 (Hs00242273_m1), GAPDH (Hs02758991_g1), hsa-miR-145 (002278) and RNU48 (001006) (Applied Biosystems). All samples were amplified in triplicates, and non-template controls were included. Each sample’s mean threshold value was corrected for the corresponding mean value for GAPDH mRNA or RNU48 miRNA, used as endogenous controls.
CNDT2.5 cells were seeded onto 6 well plates and treated with different concentrations of 5-aza-dC (5-aza-2’-deoxycytidine, Sigma Chemical Co., St. Louis, MO, USA, A3656) (0.025, 0.5, 1.0, 1.25, 1.5 μM) and DZNep (3-deazaneplanocin A, 2.5, 5.0, 10.0, 12.5, 15 μM) and cell viability was accessed using WST-1 (Roche Diagnostics GmbH). Not toxic concentrations were chosen; 1 μM for 5-aza-dC and 10 μM DZNep. Freshly prepared 5-aza-dC was used in the experiments. DZNep was kindly provided by Dr. Victor Marques .
2 × 105 CNDT2.5 cells were seeded onto 6 well plates. After 24 h 10 μM DZNep or 1 μM 5-aza-dC was added in triplicates or 1, 2.5, or 5 μM EPZ-6438 (Selleckchem, Houston, TX, USA), a specific EZH2 inhibitor , was added to the wells and fresh medium and compounds were added every 24 h. The cells were harvested after 72 h, 96 h for EPZ-6438 treated cells, for RNA preparations. The DZNep treatment was repeated three times and 5-aza-dC and EPZ-6438 twice.
CNDT2.5 cells (1 × 105) were distributed onto 6 well plates. After 24 h hsa-miR-145 or negative control miR (mirVana™miRNA mimics, Ambion) was transfected in triplicates using 20 mM miRNA and 8 μl INTERFERin siRNA transfection reagent (Polyplus Transfection). The cells were harvested and RNA prepared after 72 h. Transfections were repeated three times and successful transfection was determined by qRT-PCR using miR-145 assay. Apoptosis was measured in transfected cells using the Cell Death Detection ELISA kit (Roche Molecular Biochemicals), and as a positive control cells were incubated with 0.1 μg/ml Camptothecin (Sigma-Aldrich), a specific inhibitor of DNA topoisomerase I that induces apoptosis.
Frozen tumor sections from 24 tumors; 8 primary tumors, 9 lymph node metastases and 7 liver metastasis, were when needed macro-dissected to obtain at least 80 % tumor cells (in most cases more than 90 %) and RNA was extracted using TriZol reagent (Invitrogen), according to manufacturer’s instructions. cDNA synthesis followed by qRT-PCR was performed as described above.
Proliferation and viability assays
A colony formation assay was performed and repeated three times; CNDT2.5 cells (1 × 105) were seeded onto 6 well plates and transfected with 4 μg ACTG2-plasmid expression vector using 8 μl Lipofectamine 2000 reagent (Life Technologies) according to manufacturer’s instructions. The ACTG2 expression vector consisted of an expression-validated cDNA in pCMV6-Entry (TrueORF Gold, catalog no. RC203151. Origene Technologies, Inc., Rockville, MD, USA) and empty pcDNA3.1 was used as control. Six hours after transfection fresh medium was added complemented with 1 % PEST and 0.2 mg/ml Geneticin (G418, Sigma Aldrich). After 24 h 2000 cells were distributed onto 6 well plates and fresh medium with 0.2 mg/ml Geneticin was added every 72 h. After 8 days in selection the cells were fixed with 10 % acetic acid/10 % methanol, stained with 0.4 % crystal violet, and visible colonies were photographed and counted. Successful transfection was verified by western blotting after 24 h.
To analyze effect of ACTG2 on viability, CNDT2.5 cells were transiently transfected and 1000 cells were seeded in a 96 well plate in triplicates. After 72 h cell viability was measured using the cell proliferation reagent WST-1 (Roche Diagnostics GmbH) according to manufacturer’s instructions.
All data are presented as arithmetical mean ± standard deviation. Unpaired t test was used for statistical analysis and p < 0.05 was considered significant.
ACTG2 protein is variably expressed in 42 % of analyzed SI-NETs
ACTG2 protein is not detected in enterochromaffin cells of the normal small intestine
ACTG2 expression is induced by DZNep in vitro
Involvement of miR-145
Growth inhibition by ACTG2 in vitro
ACTG2 is often aberrantly expressed in multiple cancers [15, 17, 18], and low levels have been associated with worse disease-specific survival . Previously, low mRNA expression levels of ACTG2 were demonstrated in metastases relatively to primary SI-NETs . To investigate a possible role and function of ACTG2 in SI-NET tumorigenesis this finding was first confirmed by immunohistochemistry, demonstrating absence of protein expression in the majority of investigated SI-NETs. Interestingly, eight primary tumors and two lymph node metastases displayed positive staining for ACTG2 in tumor cells, albeit at variable level and appearance. We could not detect ACTG2 expression in the enterochromaffin cells of the normal intestinal mucosa, suggesting that expression of ACTG2 can be induced at some point during tumor progression representing a dedifferentiated phenotype, rather than being normally expressed in this cell type. Induction of ACTG2 at some point during primary tumor growth may have beneficial effects as ACTG2 showed growth inhibitory effects, at least in vitro. Expression of ACTG2 was detected in stromal cells and whether ACTG2 can display growth effects here remains to be investigated.
This study demonstrated that expression of ACTG2 can be induced by DZNep treatment or miR-145 transfection of the human SI-NET cell line CNDT2.5. Treatment with DZNep also induced expression of miR-145, indicating a possibility that induction of ACTG2 by DZNep may be due to the effects on miR-145 expression. DZNep is a potential drug in cancer treatment . DZNep can inhibit the histone methyltransferase EZH2, which is the catalytic subunit of polycomb repressive complex 2 and is responsible for methylation of lysine 27 on histone 3, a repressive mark . A role of EZH2 was however excluded here since EPZ-6438, a newly developed specific drug inhibiting EZH2 enzymatic activity , was not able to induce ACTG2 expression. MiR-145 is often deregulated in cancer cells [31, 32] and is known to induce ACTG2 expression in breast cancer . Here, it is demonstrated that this occurs also in SI-NET cells; overexpression of miR-145 increased expression of ACTG2 in vitro. There was a decrease of miR-145 expression in metastasis compared to primary tumors, as observed for ACTG2 . Low levels of ACTG2 are correlated to chemotherapy resistance [12, 14] and inducing this gene in SI-NETs would, not only have a growth inhibitory effect, but also potentially make the tumors more sensitive to treatment. SI-NETs are difficult to cure due to their resistance to chemotherapy and radiation, and new treatment strategies are warranted. MicroRNAs are involved in gene regulation and cancer development, and thus, have a potential role as therapeutic targets. miR-145 has been suggested to be a candidate for RNA medicine in colon tumors with a reduced expression . miR-145 have multiple gene targets, and seems to be able to act as both a tumor suppressor and an oncogene depending on tumor type. Ruebel et al.  detected a difference in expression levels of miR-145 between primary SI-NETs and metastases, and here we confirmed a decrease in expression by tumor progression. These results suggest that miR-145 may be a tumor suppressor and may be important for the ability to metastasize. Inducing or introducing miR-145 may be a potential new therapeutic strategy in SI-NETs.
Involvement of ACTG2 in small intestinal neuroendocrine tumorigenensis has not been investigated previously. Here, we demonstrate that ACTG2 protein expression can be detected in a fraction of SI-NETs and absent in others, and that it is regulated by miR-145. Overexpression of ACTG2 inhibited cell growth and reduced cell viability in vitro. Further investigation is needed to determine if introducing miR-145 in SI-NETs could have therapeutic advantages.
Ethics and consent to participate statement
This study was approved by the regional ethical review board in Uppsala (11-375/1.1.2011, Local ethical vetting board in Uppsala (Regionala etikprövningsnämnden i Uppsala)). Written informed consent for participation and publication of individual clinical details was obtained from all patients.
Consent to publish statements
Written informed consent for participation and publication of individual clinical details was obtained from all patients.
Availability of data and materials statement
The data is presented in the main manuscript and in an Additional file 1: Table S1.
The authors are grateful to B Bondeson and E Persson for skillful technical assistance. The authors thank Dr. Lee Ellis for making the CNDT2.5 cell line available to them.
This study was supported Medical Research Council.
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