Endocrine-Related Cancer (2008) 15 569–581

Amyloid precursor-like 1 is differentially upregulated in neuroendocrine tumours of the gastrointestinal tract

Yvonne Arvidsson1, Ellinor Andersson1, Anders Bergstro¨m1, Mattias K Andersson1,Gu¨lay Altiparmak1, Ann-Christin Illerskog1,Ha˚kan Ahlman2, Darima Lamazhapova1,3 and Ola Nilsson1

1Lundberg Laboratory for Cancer Research, Department of Pathology and 2Lundberg Laboratory for Cancer Research, Department of Surgery, Sahlgrenska University Hospital, Gula stra˚ket 8, SE-413 45 Go¨teborg, Sweden 3Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK (Correspondence should be addressed to Y Arvidsson; Email: [email protected])

Abstract We have examined the global expression profile of small intestinal carcinoids by microarray analysis. High expression of a number of was found including amyloid precursor-like protein 1 (APLP1). Quantitative real-time PCR and western blot analysis demonstrated higher expression of APLP1 in carcinoid metastases relative to primary tumours indicating a role of APLP1 in tumour dissemination. Tissue microarray analysis of gastroentero-pancreatic tumours demonstrated a high frequency of APLP1 expression and a low frequency of APLP2 expression in neuroendocrine (NE) tumours when compared with non-NE tumours at the same sites. Meta-analysis of data from a large number of tumours outside the gastrointestinal tract confirmed a correlationbetween APLP1 expression and NE phenotype where high expression of APLP1 was accompanied by downregulation of APLP2 in NE tumours. Cellular localization of APLP1, APLP2 and amyloid precursor protein (APP) in carcinoid cells (GOT1) by confocal microscopy demonstrated partial co-localization with synaptophysin. This suggests that the APP family of is transported to the cell membrane by synaptic microvesicles and that they may influence tumour cell adhesion and invasiveness. We conclude that APLP1 is differentially upregulated in gastrointestinal NE tumours and that APLP1 may be important for the dissemination of small intestinal carcinoids. Identification of APLP1 in NE tumours offers a novel target for treatment and may also serve as a tumour-specific marker. Endocrine-Related Cancer (2008) 15 569–581

Introduction involved in NE tumour formation and progression is Little is known about the causes and genetic changes needed. In an attempt to characterize the gene expression involved in the formation of gastrointestinal neuroendo- in gastrointestinal NE tumours, we performed global crine (NE) tumours. The majority of these tumours are gene expression analysis of metastatic lesions from sporadic with no family history and no known causative malignant ileal carcinoids. One of the differentially agents. Differentiated NE tumours generally grow slowly upregulated genes was amyloid precursor-like protein 1 but frequently metastasize to regional lymph nodes and (APLP1) which we choose to investigate further liver. Metastatic tumours may give rise to symptoms due regarding its role in NE tumour formation and to hormone overproduction (carcinoid syndrome). The progression. classification of NE gastrointestinal tumours is based on The highly conserved family of amyloid precursor the identification of characteristic growth patterns, proteins (APP) contains the three homologous proteins, expression of NE markers and specific hormone APP, APLP1 and APLP2 (Wasco et al. 1992, 1993, products. However, the prognosis of these tumours is Sprecher et al. 1993, Paliga et al. 1997). APP and difficult to predict. A better understanding of the genes APLP2 are expressed in virtually all tissues and cell

Endocrine-Related Cancer (2008) 15 569–581 Downloaded from Bioscientifica.comDOI: 10.1677/ERC-07-0145 at 09/28/2021 04:21:59AM 1351–0088/08/015–569 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access Y Arvidsson et al.: APLP1 expression in neuroendocrine tumours types, while APLP1 is mainly expressed in neural gastrointestinal tract. The aim of this study was to tissue (Tanzi et al. 1988, Slunt et al. 1994, Lorent et al. describe the expression profile and cellular localization 1995). of APLP1, APLP2 and APP in these NE tumours and to The functions of the APP-family proteins in non- relate these findings to tumour site, proliferative neuronal tissues are poorly understood. APP, the best activity and metastasis. characterized member of the family, has been associ- ated with cell adhesion, cell motility, cell proliferation, neuroprotection, neurite outgrowth, synapse formation, Material and methods vesicular transport and gene regulation (Saitoh et al. Cell culture 1989, Schubert et al. 1989, Milward et al. 1992, Mattson et al. 1993, Perez et al. 1997, De Strooper & Annaert Two different NE cell lines were used in expression 2000, Sabo et al.2001). In order to perform such diverse microarray, quantitative real-time PCR (qRT-PCR) functions, APP is proteolytically processed to generate and western blot (WB). The GOT1 cell line was several peptide fragments, each with specific functions. established from a liver metastasis of a small APP is digested by a-, b- and g-secretase to generate intestinal carcinoid (Ko¨lby et al. 2001) and the the extracellular peptides, b-amyloid peptide and BON cell line was derived from a metastasis of a soluble N-terminal domain (sAPP), as well as the APP malignant pancreatic carcinoid (Evers et al. 1991). intracellular C-terminal domain fragment (AID). The GOT1 cell line was cultured in RPMI-1640 APLP1 and APLP2 are processed in a similar way to medium with 5 mg/ml transferrin and 5 mg/ml , APP, leading to the secretion of the large N-terminal and the BON cell line was grown in Dulbecco’s domain and release of the intracellular peptides ALID1 Modified Eagle’s Medium (DMEM)/F-12 medium, and ALID2 respectively (Slunt et al. 1994, Paliga et al. both supplemented with 10% bovine serum, 200 IU/ 1997, Scheinfeld et al. 2002, Eggert et al. 2004). ml penicillin and 200 mg/ml streptomycin, and However, the b-amyloid domain is not present in the incubated in a 5% CO2 humidified atmosphere APLP1 and APLP2 proteins. Similarities in structure, at 37 8C. proteolytic processing and tissue distribution of the APP family members suggest that the proteins share common functions. To elucidate the individual functions of the RNA extraction APP proteins, knockout mice have been generated (Zheng Fresh biopsies of small intestinal (ileal) carcinoids, et al.1995, von Koch et al. 1997, Heber et al.2000). gastrointestinal stromal tumours (GIST), colorectal Double knockouts of the combinations APLP2/APP cancers (adenocarcinoma), as well as normal tissues and APLP2/APLP1 were found to be lethal; the mice from intestinal mucosa and brain were collected at the died shortly after birth demonstrating that APP proteins time of operation and stored at K135 8C. Tissue used have a key function in cell growth and survival. Mice with for gene expression microarray analysis was stabilized single knockouts of APP, APLP1 and APLP2 and the with RNAlater (Qiagen). To confirm the diagnosis and double knockout APLP1/APP were found to be viable, estimate the percentage of tumour cells in the biopsies, suggesting that the functions of the proteins are partially formalin-fixed and paraffin-embedded tissues were redundant. However, knockdown of APLP1 expression in taken in direct proximity to the biopsies, stained with cell culture reduced cell viability and neurite outgrowth hematoxylin and eosin, and examined microscopically. (Sakai & Hohjoh 2006). Biopsies with a density of at least 90% tumour cells The fundamental role of APP proteins in survival were used. Total RNA was prepared by homogenizing and proliferation of normal cells suggests that these tumour biopsies in TRIzol Reagent (Invitrogen) using a proteins may also play a role in tumour development. Tissuelyser (Qiagen), followed by RNA purification Increased expression of amyloid protein family using RNeasy Mini spin columns (Qiagen). Cultured members can influence cancer development in several cells were lysed in TRIzol and further purified using different tumour types (Nakagawa et al. 1999, Meng the RNeasy Mini spin columns. RNA was quantified by et al. 2001, Hansel et al. 2003, Quast et al. 2003, reading the absorbance at 260 nm (NanoDrop ND- Baldus et al.2004, Ko et al. 2004, Mauri et al. 2005, Li 1000; NanoDrop Technologies, Wilmington, DE, et al. 2006). Neuroblastoma, a malignant tumour of USA) and the ratio of absorbance at 260/280 nm was neural crest origin, is the only tumour type that has O1.9 in all samples. The quality of extracted RNA was been reported to express high levels of APLP1 (Wasco visually examined by gel electrophoresis (Agilent et al. 1992). Here, we demonstrate for the first time that 2100 Bioanalyzer; Agilent Technologies, Palo Alto, APLP1 is highly expressed in NE tumours of CA, USA).

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Expression microarray and reverse transcribed using the TaqMan Reverse Expression microarray analysis was performed on Transcription Reagents (Applied Biosystems, Foster biopsies from the liver metastasis of intestinal (ileal) city, CA, USA). A master mix was prepared for the ! carcinoids (five patients) and triplicate samples of GOT1 qRT-PCRs by mixing water, 2 TaqMan Universal and BON cell lines. cDNA was synthesized from total PCR Master Mix (Applied Biosystems) and TaqMan RNA (5 mg per sample) and labelled with Cy-3 gene expression assays (Applied Biosystems) ! (Amersham Biosciences), using the Pronto! Plus version containing a 20 solution of primer and probe 1.2 direct labelling and hybridization kit (Promega). specific for APLP1 (Hs00193069_m1), APLP2 cDNA of the universal human reference RNA (URR; (Hs00155778_m1), APP (Hs00169098_ml) and the Stratagene, La Jolla, CA, USA) was labelled with Cy-5. glyceraldehyde-3-phosphate dehydrogenase gene The reference RNA containing pooled total RNA from ((GAPDH) (Hs99999905_ml)). The comparison of 10 different cell lines was used to obtain broad gene primary carcinoid tumours with metastases was expression coverage. Microarray glass slides were performed in a 7700 Real-Time PCR System printed at the Swegene DNA Microarray Resource (Applied Biosystems). The comparison of different Center, Department of Oncology, Lund University, gastrointestinal tumour types was performed in a 7500 Sweden (http://swegene.onk.lu.se). The arrays consisted Fast Real-Time PCR System (Applied Biosystems). of 27 000 unique probes of the Human Array-Ready The samples were subjected to PCR cycling conditions oligonucleotide libraries version 2.1 and version 2.1.1 recommended by Applied Biosystems. The cycle (Operon Biotechnologies, Cologne, Germany) printed in threshold (Ct) of target genes and housekeeping duplicate. The microarray slides were hybridized with gene GAPDH was determined manually within the 50 pmol Cy-3-labelled sample cDNA and 50 pmol Cy-5- exponential phase of amplification (7700 Real-Time labelled reference cDNA at 62 8C for 18 h, then washed PCR System) or automatically (7500 Fast Real-Time to remove excess labelled cDNA and finally scanned PCR System) by the software. The expression levels of using an Agilent G2505B microarray scanner (Agilent target genes were related to endogenous expression Technologies). Fluorescence intensities were converted of GAPDH which was chosen as housekeeping to numeric data using Agilent G2567AA Feature gene since it showed low variability in expression Extraction 7.5 software (Agilent Technologies). Statisti- levels (Ct value) between the different tumour samples. cal analyses were performed using Genespring software Statistical significance was calculated by the two-tailed (Agilent Technologies). For each hybridization, fluor- unpaired t-test using GraphPad Prism 4 (GraphPad ! escence ratios (Cy-3/Cy-5) were normalized with the Software, San Diego, CA, USA) and a P value of 0.05 LOWESS algorithm. Differentially expressed genes was considered significant. related to the URR reference RNA passed a Student’s t-test with P value of !0.02 and Benjamin and Hochberg Antibodies false discovery rate. Antibodies raised against the C-terminal part of APLP1 (aa643–653), APLP2 (aa752–763) and APP Quantitative real-time PCR (qRT-PCR) (aa751–770) (Cat no: 171615, 171616, 171610; To analyse expression levels of APLP1, APLP2 and Calbiochem, San Diego, CA, USA) were used for APP in small intestinal carcinoids during tumour immunohistochemistry (IHC), immunofluorescence dissemination qRT-PCR analysis was performed on (IF) and WB. The dilutions used for these C-terminal biopsies from normal small intestinal mucosa (nZ3), antibodies were as follows: APLP1 1:1000 (IHC), 1:500 primary small intestinal (ileal) carcinoids (nZ6) and (IF) and 1:5000 (WB); APLP2 1:800 (IHC), 1:500 (IF) liver metastases of small intestinal carcinoids (nZ5) and 1:1000 (WB); and APP 1:400 (IHC), 1:2000 (IF) (the same biopsies as for expression microarray). As and 1:5000 (WB). Antibodies to the N-terminal part of controls served normal brain tissue (nZ1), and the APLP1 (cat no: NE1009; Oncogene, Sandiego, CA, GOT1 (nZ3) and BON (nZ3) cell lines. The primary USA), APLP2 (Cat no: 171617; Calbiochem) and tumours and metastases did not originate from the APP (aa44-62) (Cat no: ab15272; Abcam, Cambridge, same patients. To quantify the expression levels of UK) were used for IHC. The dilutions used for these APLP1, APLP2 and APP in different gastrointestinal N-terminal antibodies were as follows: APLP1 1:100, tumour types, we analysed primary colorectal adeno- APLP2 1:2000 and APP 1:200. Antibodies to chromo- carcinoma (nZ10), GIST (nZ10) and small intestinal granin A (CHGA) at 1:500 (Cat no: MAB319; carcinoids (nZ12). Total RNA was treated with Chemicon, Temecula, CA, USA), synaptophysin at DNase1 (DNA-free kit; Ambion, Austin, TX, USA) 1:20 (Cat no: MO776; DakoCytomation, Glostrup,

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Denmark), FE65 at 1:50 (Cat no: 05-758; Upstate, Lake biopsies, 0.6 mm in diameter, were taken from each Placid, NY, USA) and Rab5 at 1:10 (Cat no: 610281; donor block. Each biopsy was mounted in a recipient BD Biosciences, San Jose´, CA, USA) were used for IHC paraffin block (NALLCRSET-1) using a robotized tissue and confocal laser scanning microscopy. Antibody puncher/arrayer (ATA-27; Beecher Instruments, Silver against Ki67 at 1:100 (MIB1, Cat no: M7240; Spring, MD, USA). Normal tissue samples from color- DakoCytomation) was used for IHC. Antibody to ectal mucosa/submucosa, gastric mucosa/submucosa, GAPDH at 1:4000 (Cat no: 4300; Ambion) was used exocrine and endocrine pancreas, small intestinal as a loading control for WB. mucosa/submucosa and cerebral cortex were obtained and placed at the start, the centre and at the end of the Western blot recipient block. These tissues served as negative and positive controls for the immunocytochemical staining. WB analysis was performed on primary small intestinal Tissue microarray was constructed in collaboration with (ileal) carcinoids (nZ4), liver metastases (nZ4), normal the Swegene National Tissue Microarray Center, Malmo¨, tissues from intestinal mucosa (nZ1), liver (nZ1) and Sweden. The tissue array was validated by examining the GOT1 and BON cell lines. Brain tissue (nZ1) served sections stained with hematoxylin and eosin, and as a positive control. The primary tumours and immunostained with antibodies against the NE markers metastases did not originate from the same patients. chromogranin A and synaptophysin. Whole-cell lysates were prepared as described previously and analyzed by western blotting (Jakobsen et al.2002). The chemiluminescent signals were Immunohistochemistry detected by an image reader (LAS 3000; Fujifilm, Sections of paraffin-embedded tissue were subjected to Tokyo, Japan) and quantified using MultiGauge version antigen retrieval by boiling in Tris–EDTA (pH 9) for 3.0 software (Fujifilm). GAPDH was analyzed to 15 min, followed by blocking with 0.5% non-fat milk and estimate the amount of protein transferred to membranes. incubation with primary antibodies to APLP1, APLP2 and APP. Bound antibodies were visualized by the Tissue microarray peroxidase-based EnVisionC Dual Link System (Dako- Cytomation). Immunohistochemical staining of tumour Formalin-fixed and paraffin-embedded tumour tissues biopsies in tissue microarrays was judged as being either were retrieved from the archives at the Department of positive or negative. A biopsy was considered to be Clinical Pathology and Cytology, Sahlgrenska University positive when more than 25% of tumour cells were Hospital, Go¨teborg, Sweden. Tissue blocks from the labelled in the cytoplasm, the nucleus or the cell following primary tumours were used: gastric carcinoids membrane. Similarly, nuclear labelling was judged to (7 enterochromaffin-like (ECL) cell tumours (benign) in be positive when more than 25% of tumour cell nuclei chronic atrophic gastritis, 4 sporadic ECL cell tumours were labelled. Tissue microarrays were evaluated by two (malignant)), small intestinal (ileal) carcinoids (32 entero- pathologists (A Bergstro¨mandONilsson). chromaffin cell tumours (malignant)), appendiceal carci- noids (18 classical type, 15 benign and 3 malignant; 3 mixed endocrine and exocrine type, 1 benign and Confocal laser scanning microscopy 2 malignant), rectal carcinoids (23 L cell type, 19 GOT1 cells were grown on chamber slides for 3 days and benign and 4 malignant), endocrine pancreatic tumours fixed in 4% paraformaldehyde in PBS. The slides were (12 non-functioning, 1 benign and 11 malignant; incubated with primary antibodies for 1 h at room 9 sporadic insulinoma, 6 benign and 3 malignant; 2 temperature in 1% BSA, 0.2% Triton X-100 and 0.1% sporadic glucagonoma (malignant); 1 sporadic sodium azide in PBS. The cells were incubated for 1 h VIPoma (malignant); 1 multiple endocrine neoplasia with a mixture of 0.006% Hoechst 33258 for nuclear type 1 (MEN1)-associated insulinoma (malignant); 2 staining and a 1:2000 dilution of secondary antibodies MEN1-associated non-functioning tumour (malignant); 1 conjugated with Alexa Fluor 594 (goat anti-rabbit, cat no: Von Hippel-Lindau disease (VHL)-associated non- A11037; Invitrogen) or Alexa Fluor 488 (goat anti- functioning tumour (malignant)), GIST (2 very low risk, mouse, cat no: A11029; Invitrogen). The primary 12 low risk, 2 intermediate risk and 12 high risk), gastric antibody was omitted in negative controls. Coverslips adenocarcinomas (9 diffuse type and 16 intestinal type), were mounted using ProLong Gold anti-fade reagent colorectal adenocarcinomas (24 moderately differentiated (Molecular Probes, Eugene, OR, USA). The fluorescent and 1 poorly differentiated) and pancreatic (ductal) cells were analyzed by confocal microscopy using a adenocarcinomas (1 well differentiated, 9 moderately Zeiss LSM 510 META system with the LSM-5 software differentiated and 11 poorly differentiated). Two core supplied by the manufacturer. Emission spectra were

Downloaded from Bioscientifica.com at 09/28/2021 04:21:59AM via free access 572 www.endocrinology-journals.org Endocrine-Related Cancer (2008) 15 569–581 recorded with the META multichannel detector using the (SYT13), DOPA decarboxylase (DDC), synapsin1 following settings: Hoechst 33258, diode laser 405 nm (SYN1), secretogranin III (SCG3) and vesicle- excitation, 422–476 nm emission detection; Alexa Fluor associated 2 (VAMP2) (Table 1). 488, argon laser 488 nm excitation, 497–550 nm Unexpectedly, we found high expression of APLP1, emission detection; and Alexa Fluor 594, DPSS-laser which has not been investigated previously in small 561 nm excitation, 583–668 nm emission detection. intestinal carcinoids. We further explored the Images were recorded with an optical section of expression pattern and possible function of the APP 0.8 mm using an aPlan-FLUAR 100X/1.45 N.A. oil gene family in NE tumours. immersion objective. Meta-analysis revealed differential upregulation Ethics of the APLP1 gene in NE tumours of the lung These studies were approved by the Regional Ethical To explore the APLP1 expression in a wider range of Review Board in Go¨teborg, Sweden. tumours, we performed meta-analysis of published data sets using the ONCOMINE database (www.onco- mine.org). The differential expression of the APLP1 Results gene in normal and cancer tissues was analyzed in Global gene expression analysis of small available data sets (April 2006) containing 35 different intestinal carcinoids demonstrated high microarray studies covering 14 different tumour types expression of APLP1 including tumours of the blood, brain, head and neck, salivary gland, thyroid, lung, adrenal gland, pancreas, In order to identify highly expressed genes in liver liver, prostate, ovary, uterus, kidney and urinary metastases of small intestinal carcinoids, we estab- bladder (Fig. 1B). The sets contained no gastrointes- lished a gene expression profile by microarray analysis. tinal tumours. There were significant changes in For comparison, the expression profile of the two expression of APLP1 (P value !0.02) in 8 out of the carcinoid cell lines, GOT1 and BON, was also 35 available comparisons. Three out of the eight determined. One thousand four hundred three signi- studies showed significant upregulation of APLP1. ficantly differentially expressed genes were analyzed These were all NE tumours of the lung, representing by hierarchical clustering using Pearson correlation the only NE tumour types within the 35 data sets. (Fig. 1A). The clustering of these samples showed These lung tumours also showed significant down- two main branches, one branch with the BON cell regulation of APLP2 (data not shown). Tumours of the line alone and the other branch containing a brain, exocrine pancreas and multiple myeloma group with the five intestinal carcinoids and a sub- displayed downregulation of APLP1. branch with the GOT1 cell line. The expression profiles of the intestinal carcinoids were highly correlated, indicating that these tumours as a group have great Tissue microarray demonstrated differential phenotypic similarities. When comparing the gene upregulation of APLP1 expression in NE expression profiles of the intestinal carcinoids with the tumours of the gastrointestinal tract two carcinoid cell lines GOT1 and BON, cluster To explore the expression of APLP1, APLP2 and APP analysis demonstrated the closest relationship between proteins in NE and non-NE tumours of the gastrointes- intestinal carcinoids and GOT1. This finding suggests tinal tract, we performed IHC on tissue microarrays that GOT1 cell line has an expression profile more comprising 214 primary tumours. NE tumourswere more similar to tissue samples of intestinal carcinoid than frequently positive for APLP1 than non-NE tumours; BON cells have. GOT1 cells may therefore be more 84% of NE tumours (gastric, small intestinal, appendi- representative for small intestinal carcinoids. This is in ceal and rectal carcinoids, and endocrine pancreatic agreement with the fact that GOT1 cell line was tumours) were positive, in contrast to 26% of non-NE established from an ileal carcinoid, while BON cells tumours (GIST, gastric, colorectal and pancreatic were derived from a pancreatic carcinoid. Analysis of adenocarcinoma) (Table 2). The frequency of positive differentially expressed genes in intestinal cacinoids APLP1 staining in NE tumours was similar in benign and revealed genes related to MAPK, Wnt, Hedgehog and malignant cases. APLP2 was frequently expressed in all Notch signalling, as well as a number of genes related types of tumours investigated except for small intestinal to NE functions. NE-related genes were found to carcinoid and endocrine pancreatic tumour; therefore, be highly expressed in carcinoid tumours, e.g. those NE tumours showed a significant lower frequency of for CHGA, secretagogin (SCGN), synaptotagminXIII APLP2 expression (77%) than non-NE tumours (97%).

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Table 1 Significantly upregulated neuroendocrine and neuronal genes in small intestinal carcinoid tumours in comparison to Universal human reference RNA (URR)

Fold GeneBank Ranka change Gene accession no.

4 41.9 SCGN NM 006998 8 34.5 SYT13 NM 020826 9 30.7 DDC NM 000790 10 27.9 CHGA NM 001275 51 9.6 APLP1 NM 005166 63 8.9 NCALD NM 032041 68 8.6 CDK5R2 NM 003936 69 8.6 SCG3 NM 013243 135 6.6 CRMP1 NM 001014809 152 6.3 CPLX2 NM 006650 182 5.7 BEX1 NM 018476 241 4.9 NBEAL1 NM 198945 245 4.9 CPE NM 001873 251 4.8 NFASC NM 015090 272 4.4 SCG2 NM 003469 328 4.0 MTAC2D1 NM 152332 329 4.0 SYT4 NM 020783 368 3.6 INA NM 032727 387 3.4 MAPK8IP1 NM 005456 394 3.4 SYN1 NM 133499 457 2.9 NBEA NM 015678 461 2.9 NRXN3 NM 004796 494 2.7 VAMP2 NM 014232 495 2.7 VGF NM 003378 580 2.4 OPTN NM 001008211

aPosition in gene list based on expression level.

APP was also frequently expressed in gastrointestinal tumours, but with no significant difference between NE tumours (85%) and non-NE tumours (92%). APLP1, APLP2 and APP labelling of tumours were confined to tumour cells, while tumour stroma was consistently negative. The tumour cells showed cytoplasmic labelling using antibodies recognizing the C-terminal domain (Fig. 2A). Positive labelling of tumour cell nuclei was detected in some NE and non-NE tumours using the antibody to the C-terminal domain of APLP2. Nuclear labelling was most prominent in pancreatic adenocar- cinomas (80%), but was also seen in gastric adenocarci- nomas (43%) and gastric carcinoids (14%). N-terminal

APLP1 in NE tumours of the lung. The differential expression of APLP1 in normal tissue compared with cancer tissue is Figure 1 Global gene expression analysis. (A) Hierarchical depicted using box plots. Normalized expression units are given clustering of carcinoid tumours reveals homogenous on the x-axis (log transformed, array median set to zero and expression patterns. Gene expression data from five small array standard deviation set to one). Eight analyses with intestinal carcinoid tumours and the carcinoid cell lines GOT1 significant differences (i.e. with P value !0.02) were found. For and BON using 1403 significantly expressed genes. Each every class, there is a box with three vertical lines corres- column in the diagram represents a tumour sample and each ponding to the median, lower quartile and upper quartile of the row a gene. Gene expression values are represented as expression values. The ends of the whiskers correspond to the colours; red represents relatively higher expression and blue tenth and ninetieth percentiles. Dots correspond to minimum relatively lower expression than the reference URR. (B) Data and maximum values. The number of samples in each class search of the database ONCOMINE reveals upregulation of compared (normal versus cancer) is given in parentheses.

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Table 2 Tissue microarray analysis of amyloid precursor-like protein 1 (APLP1), APLP2 and amyloid precursor protein (APP) expression in gastrointestinal tumours

APLP1 APLP2 APP

Positive Positive Positive tumoursa Total number tumoursa Total number tumoursa Total number of tumours of tumours of tumours Tumour type (%) (n)(n) (%) (n)(n) (%) (n)(n)

Carcinoid, gastric 82 9 11 91 10 11 64 7 11 Carcinoid, small intestinal 75 24 32 81 25 31 90 28 31 Carcinoid, appendiceal 83 15 18 90 17 19 86 18 21 Carcinoid, rectal 83 19 23 100 16 16 94 15 16 Endocrine pancreatic tumour 96 27 28 24 6 25 93 25 27 All NE tumours 84b 94 112 77b 74 102 85 93 106 GIST 7 2 28 96 26 27 100 28 28 Adenocarcinoma, gastric 24 6 25 100 23 23 88 21 24 Adenocarcinoma, colorectal 56 14 25 100 25 25 100 25 25 Adenocarcinoma, pancreatic 16 3 19 90 18 20 76 16 21 All non-NE tumours 26b 25 97 97b 92 95 92 90 98

GIST, gastrointestinal stromal tumour. Data for tissue microarray were obtained by incubation with C-terminal directed antibodies against APLP1, APLP2 and APP. aTumours were judged to be positive when R25% of tumour cells were stained. bSignificant difference in immuno-positive staining between NE and non-NE tumours determined by Binomial two sample test. domain-specific antibodies gave similar results regarding and appendiceal (81%) carcinoids in contrast to gastric the expression of APLP1, APLP2 and APP as antibodies (0%) and rectal (0%) carcinoids. directed to the C-terminal domain (data not shown). To quantify the differential expression of APLP1, However, N-terminal domain-specific antibodies APLP2 and APP in gastrointestinal tumours, we per- clearly labelled tumour cell membrane in ileal (94%) formed qRT-PCR analysis of small intestinal carcinoids,

Figure 2 Expression of APLP1, APLP2 and APP in gastrointestinal tumours. (A) APLP1, APLP2 and APP are expressed in small intestinal carcinoids. Immunohistochemical analysis of a primary small intestinal carcinoid demonstrating strong cytoplasmic expression of APLP1, APLP2 and APP in tumour cells with no expression in stroma cells. (B) APLP1 is highly expressed in small intestinal carcinoids. The expression levels of RNA encoded by the APLP1, APLP2 and APP genes in primary tumours of colorectal adenocarcinoma (nZ10), GIST (nZ10) and small intestinal carcinoid (nZ12) were determined by qRT-PCR. The values given are relative to the housekeeping gene GAPDH. Asterisks indicate statistically significant changes (unpaired t-test): *P!0.05 and **P!0.01.

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GIST and colorectal adenocarcinoma (Fig. 2B). APLP1 range 0.0–22.2%; nZ5). However, there was no showed the greatest difference in expression between the significant correlation between proliferation index three tumour types, with the highest expression in small and APLP1 expression level in tumour biopsies. intestinal carcinoids and lowest expression in colorectal adenocarcinoma and GIST. APLP2 also showed differences in expression levels between the three tumour APP family proteins are localized to synaptic-like types, with the highest expression in GIST followed by microvesicles and early endosomes, and carcinoids and colorectal adenocarcinoma. The level of co-localized with the adaptor protein FE65 APP expression was found to be high in all three tumour in carcinoid tumour cells types with least expression in colorectal cancer. In order to determine whether APLP1, APLP2 and APP may function as adhesion molecules, secretory APLP1 is upregulated in disseminated small proteins or regulators of gene transcription, we studied intestinal carcinoids the cellular localization of these proteins in carcinoid In order to explore APLP1, APLP2 and APP tumour cells (GOT1) by confocal laser scanning expression in small intestinal carcinoids during tumour microscopy. C-terminal directed antibodies against dissemination, we performed qRT-PCR and WB APLP1, APLP2 and APP were used in order to analysis on biopsies from normal intestinal mucosa, visualize the full-length proteins as well as the primary carcinoid tumoursandlivermetastases. processed CTFs. Using antibodies directed against qRT-PCR analysis revealed elevated expression of the C-terminal part of APLP1, APLP2 and APP, a APLP1 and APP in both primary and metastatic predominant cytoplasmic distribution was seen, but carcinoid tumours relative to the expression in normal with minor fractions located at or in the nucleus. The intestinal mucosa (Fig. 3A). On the other hand, APLP2 cytoplasmic location of APLP1, APLP2 and APP was gene expression was reduced in primary and metastatic further studied in relation to markers for large dense tumours compared with intestinal mucosa. Comparison core vesicles (CHGA), synaptic-like microvesicles of primary carcinoids with metastatic tumours revealed (synaptophysin) and early endosomes (Rab5) (Fig. 4). a significant upregulation of APLP1 expression in All three APP proteins partly co-localized with metastatic lesions, while APLP2 and APP expression synaptophysin and Rab-5, indicating localization to was downregulated, clearly demonstrating a differ- synaptic-like microvesicles and early endosomes. ential upregulation of APLP1 gene expression during There was only weak co-localization of APLP1, carcinoid tumour dissemination. GOT1 cells showed APLP2 and APP with CHGA. The adaptor molecule an expression pattern that was similar to that observed FE65, which interacts with the CTF of APP to regulate in metastatic carcinoid tumours, i.e. high expression of transcription (Borg et al. 1996), was found to be APLP1 and APP but low expression of APLP2. BON highly expressed in GOT1 cells and co-localized with cells showed an expression pattern that was different all three amyloid proteins in both the cytoplasm and the from those of GOT1 cells and intestinal tumour, nucleus (Fig. 4D). i.e. they showed low expression of APLP1.WB confirmed high protein levels of APLP1, APLP2 and APP in small intestinal carcinoids and upregulation of APLP1 in metastatic lesions (Fig. 3B). WB also Discussion verified the difference in APLP1 protein levels In this paper, the expression pattern of the APP protein between GOT1 and BON cells and demonstrated family in tumours of the gastrointestinal tract is reported intracellular C-terminal fragments (CTFs) in the two for the first time. We found that APP proteins are highly cell lines. Faint bands of CTF were also detected by the expressed in gastrointestinal tumours, with differential anti-APLP2 and anti-APP antibodies, suggesting upregulation of APLP1 in NE tumours. APLP1 was proteolytic processing of APP family proteins in found to be highly expressed in intestinal carcinoids, with carcinoid tumours and involvement of APP proteins enhanced expression in metastatic lesions compared in gene regulation. Proliferation index (percentage of with primary tumours, which suggests that APLP1 may Ki67 positive tumour cell nuclei) was also analysed in be upregulated during tumour dissemination. Upregula- biopsies of primary tumours and metastases used for tion of APLP1 in intestinal carcinoids was accompanied qRT-PCR. In primary tumours, the Ki67 labelling by downregulation of APLP2, indicating differential index was 0.4% (mean, range 0.0–1.4%; nZ6) while gene regulation of the APP family in NE tumours. metastatic lesions had a labelling index of 5.4% (mean, This observation was further supported by a search in

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Figure 3 APLP1 is upregulated in metastases of small intestinal carcinoids. Expression levels of RNA encoding (A) APLP1, (B) APLP2 and (C) APP relative to GAPDH in intestinal mucosa (nZ3, circles), primary tumour of carcinoid (nZ6, squares), metastases of carcinoid (nZ5, up triangles), BON (nZ3, down triangles), GOT1 (nZ3, crosses) and brain (nZ1, diamonds) as determined by qRT-PCR analysis. APLP1 RNA was significantly upregulated in metastases of carcinoid when compared with intestinal mucosa (***P!0.001) and primary tumour (*P!0.02). The average is indicated by a horizontal line. Asterisks indicate statistically significant changes (unpaired t-test). Normal intestinal mucosa, liver, primary tumour of carcinoid (nZ4), metastasis of carcinoid (nZ4), BON and GOT1 were immunoblotted with antibodies against (D) APLP1, (E) APLP2, (F) APP and (G) GAPDH. Brain tissue was used as positive control. Two C-terminal APLP1 fragments (CTFs) in BON and GOT1 are clearly visible. The relative densities of APLP1, APLP2, APP and GAPDH were quantified by MultiGauge version 3.0 (Fujifilm, Tokyo, Japan). the ONCOMINE database, which demonstrated upregu- that APP proteins may have a role in both cell adhesion lation of APLP1 and downregulation of APLP2 in NE and gene regulation. tumours of the lung. We also studied the cellular APP has previously been shown to be involved in co-localization of the APP protein family in a model several fundamental functions related to cancer. The system with carcinoid cells (GOT1) and found that they secreted N-terminal fragment of APP (sAPP) has been co-localized to synaptic-like microvesicles, early endo- shown to act as an autocrine growth factor. It promotes somes and the adaptor protein FE65, which suggests cell proliferation in fibroblasts, thyroid epithelial cells

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and pancreatic cancer cells (Saitoh et al. 1989, Pietrzik et al. 1998, Hansel et al. 2003). The activities of the two proteases b- and g-secretase that are involved in the cleavage of the APP family of proteins can influence tumour growth and angiogenesis (Paris et al. 2005). It was recently shown that homo- and heterocomplexes of the APP family of proteins promote cell adhesion via transcellular interactions. This finding supports the view that the APP proteins may have cell adhesion properties, such as trans- dimerization similar to cadherins and nectins (Soba et al. 2005). Deregulation of adhesion molecules, and also switching from one set of adhesion molecules to another, e.g. the E-cadherin to N-cadherin switch, have been shown to be important events in tumour dissemination (Hazan et al. 2004). The switch between APLP1 and APLP2 during carcinoid tumour dissemi- nation may be of equal relevance. The intracellular C-terminal domain of members of the APP protein family participates in the regulation of transcription by a multi-protein complex consisting of AID, ALID1 or ALID2, the histone acetyltransfer- ase Tip60 and also other proteins containing phospho- tyrosine-binding domains, the adaptor proteins FE65, X11, Dab1 and the non-receptor tyrosine kinase ABL (Borg et al. 1996, Homayouni et al. 1999, Cao & Sudhof 2001, Zambrano et al. 2001, Scheinfeld et al. 2002). To date, only a few genes have been shown to be regulated by AID, namely tetraspanin-27/KAI-1, which is linked to the regulation of NFkb-related proteins, and the two major acetylcholinesterases, ace-1 and ace- 2, of the nematode Caenorhabditis elegans (Baek et al. 2002, Bimonte et al. 2004). It is not presently known whether ALID1 and ALID2 regulate the same genes as AID. We found that in carcinoid tumours all three members of the APP family are processed by g-secretase to produce the CTFs crucial for signal transduction and gene regulation. We also detected high expression of FE65 in carcinoid tumour cells, co-localized with APP, APLP1 and APLP2 in the cytoplasm and nuclei, although the nuclear staining was faint for APP and APLP1. Together, these findings Figure 4 APLP1, APLP2 and APP co-localize with synaptic vesicle protein synaptophysin, early endosome protein Rab5 indicate that APP-dependent gene regulation may be of and the adaptor protein FE65. GOT1 cells were subjected to importance in carcinoid tumours. double-label immunofluorescence staining to determine any One of the characteristics of NE tumours is that they co-localization of APLP1, APLP2 and APP with (A) Chromo- graninA (CHGA), (B) Synaptophysin (SYP), (C) Rab5 and have regulatory secretory pathways by which hor- (D) FE65. APLP1, APLP2 and APP were immunostained with mones are released upon stimulation. Two types antibodies to the C-terminal part followed by Alexa Fluor of secretory organelles have been characterized in 594-conjugated secondary antibodies. Antibodies to CHGA, SYP, Rab5 and FE65 were visualized with Alexa Fluor NE tumours, large dense core vesicles containing 488-conjugated secondary antibodies. The nuclei were stained chromogranin A, and synaptic-like microvesicles blue with Hoechst 33258. Scale bars represent 5 mm. containing, e.g. synaptophysin, synapsin and synapto- The histograms illustrate co-localization by comparing the intensities of red (Ch1) and green (Ch2) fluorescent signals brevin. In this study, we examined the cellular in each pixel. Yellow pixels symbolize co-localization. localization of APP proteins by C-terminal-directed

Downloaded from Bioscientifica.com at 09/28/2021 04:21:59AM via free access 578 www.endocrinology-journals.org Endocrine-Related Cancer (2008) 15 569–581 antibodies and found mainly cytoplasmic distribution, which would interfere with the proteolytic processing of localized to synaptic-like microvesicles and early APP family proteins in tumour cells and block the endosomes. Only small amounts of APP proteins formation of soluble amyloid peptides and ALID were associated with large dense core vesicles. (van Es et al.2005). This would inhibit APLP1 However, N-terminal-directed antibodies clearly signalling in tumour cells, which may in turn inhibit demonstrated membrane labelling of tumour cells in tumour dissemination. However, this type of therapy ileal and appendiceal carcinoids. The results suggest should be targeted to tumour cells to avoid side effects in that members of the APP protein family are transported normal tissue. Another therapeutic option is to utilize to the cell membrane by microvesicles and may act as APLP1, being a membrane-bound protein, for targeting adhesion proteins, influencing tumour migration and with radionuclide or antibody therapy. Cleavage of invasiveness. APLP1 generates N-terminal fragments that may be In this study, we found elevated expression of APLP1 detectable in serum and serve as diagnostic markers. in NE tumours of the gastrointestinal tract relative to its We conclude that NE tumours of the gastrointestinal expression in non-NE tumours. A search of the tract express all three members of the APP family. ONCOMINE database also showed upregulation of APLP1 is highly expressed in NE tumours when APLP1 and downregulation of APLP2 in NE tumours compared with non-NE tumours with marked upregula- of the lung. One may speculate that APLP1 in NE tion in metastatic small intestinal carcinoids suggesting tumours controls the degree of NE differentiation by a role in tumour dissemination. Identification of APLP1 interfering with Notch signalling. Notch has been shown in NE tumours offers a novel target for treatment and it to control differentiation in endocrine cells and in the may also serve as a tumour-specific marker. endocrine pancreatic cell line BON (Nakakura et al. 2005, Bjerknes & Cheng 2006). In the developing endoderm, Notch signalling inhibits NE differentiation Acknowledgements by repressing the expression of basic helix-loop-helix We thank Prof. Go¨ran Stenman and Prof. Pierre A˚ man transcription factors. Similarities in proteolytic acti- for critically reading the manuscript. We also wish to vation by g-secretase and signal transduction of the three thank Siw Tuneberg and Pa¨r Bjo¨rklund for their expert members of the APP family and Notch raise the technical assistance. This work was supported by the possibility that signals from these two protein families Swedish Research Council, Sahlgrenska Academy (the may interact. In vitro and genetic studies in Drosophila government ALF agreement), the Swedish Cancer have confirmed that there is cross-talk between the APP Society, the Inga-Britt and Arne Lundberg Research family and Notch through interactions between AID and Foundation, the Assar Gabrielsson Research Foun- the inhibitors of the Notch receptor Numb and Numb-like dation, and Sahlgrenska University Hospital Research proteins (Roncarati et al.2002, Merdes et al.2004). Foundations. Microarrays and protocols were obtained A second way of interaction between APP and Notch from the Swegene DNA Microarray Resource Center is by heterodimerization resulting in activation of in Lund, supported by the Knut and Alice Wallenberg reciprocal target genes (Fischer et al.2005). Foundation through the Swegene consortium. The Recently, a tumour suppressor effect of Notch-1 has authors declare that there is no conflict of interest that been demonstrated in a number of NE-tumours (Kunni- would prejudice the impartiality of this scientific work. malaiyaan & Chen 2007) suggesting that APLP1– Notch1 interaction in carcinoids may interfere with tumour growth. Further studies of APLP1 processing and References Notch signalling in carcinoid tumours will help to Baek SH, Ohgi KA, Rose DW, Koo EH, Glass CK & elucidate the functional significance of APLP1 for NE Rosenfeld MG 2002 Exchange of N-CoR corepressor and growth and differentiation in carcinoid tumours. Tip60 coactivator complexes links gene expression by Current strategies for treatment of carcinoid tumours NF-kappaB and beta-amyloid precursor protein. 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