Molecular Network Pathway of ERBB In Diffuse-Type Gastric Cancer: Mesenchymal Stem Cells and Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is related to malignancy and metastasis in cancer. The molecular networks including tyrosine kinases are altered in gastric cancer (GC). This study aims to reveal the role of ERBBs (erb-b2 receptor tyrosine kinases) in EMT, and generate the molecular network pathway of ERBBs in diffusetype GC and mesenchymal stem cells (MSCs). The expression of ERBB genes was analyzed in MSCs and diffuse-type GC which has mesenchymal characteristics compared to intestinal-type GC. The signaling and molecular network of ERBB was analyzed using several databases, including cBioPortal for Cancer Genomics, Kyoto Encyclopedia of Genes and Genomes (KEGG), BioGRID and VaProS. ERBB2 and ERBB3 gene expression were up-regulated in diffuse-type GC compared to MSCs. The ERBB3 molecular network includes epidermal growth factor receptor (EGFR), cadherin 1 (CDH1), catenin beta 1 (CTNNB1) and EPH receptor A5 (EPHA5). These results demonstrate the importance of the ERBB network in cancer signaling, and revealed a ERBB3 network pathway model in diffuse-type GC and MSCs, and EMT.


Introduction
An abundance of molecular signaling pathways are linked between stem cells and cancer. As cellular phenotype changes, expression levels of specific genes in these signaling pathways are altered. We have previously investigated CTNNB1 networks in epithelial-mesenchymal transition (EMT), and revealed that the CTNNB1 network includes epidermal growth factor receptor (EGFR) and erb-b2 receptor tyrosine kinase (ERBB2) [1]. The ERBB family includes EGFR (ERBB1 or HER1), ERBB2 (HER2), ERBB3 (HER3) and ERBB4 (HER4) [2]. Previous studies have demonstrated that ERBB family members are important for cancer, and so in this article, we analyzed the ERBB network [3][4][5]. ERBB2 and ERBB3 were found to be overexpressed in an appendiceal adenocarcinoma in which p53 was inactivated and the Ras/RAF/MEK, Akt/mTOR, Wnt, and NOTCH pathways were activated [6]. Somatic mutations in ERBB3 were identified in a significant number of early-onset sporadic diffuse-type gastric

Gene expression analysis of MSCs and GC
Gene expression in MSCs (6 early-and 6 late-stage cultures, n=12) and diffuse-type GC (n=5) was analyzed with GeneChip® Human Genome U133 Plus 2.0 microarray (Affymetrix, Santa Clara, California, USA), as previously described [1,9,10]. Briefly, total purified cellular RNA was biotinylated and hybridized to the microarray. The signal intensity for each gene was analyzed and compared between MSCs and diffuse-type GC. The microarray data for MSCs and diffuse-type GC are available to the public in the NCBI Gene Expression Omnibus (GEO) database and are accessible via GEO Series accession number GSE7888 (https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE7888) and GSE42252 (https://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE42252), respectively [1,9,10].

Diffuse-Type GC tissues
Diffuse-type GC tissues were originally provided by the National Cancer Center Hospital after obtaining written informed consent from each patient and approval from the National Cancer Center Institutional Review Board. All cancer specimens were reviewed and classified histopathologically according to the Japanese Classification of Gastric Cancer. Following surgical extraction, tissue specimens were immediately frozen with liquid nitrogen and stored at -80°C until microarray analysis [1,9]. Existing, publicly available data were analyzed in the article.

Analysis with Cbioportal for Cancer Genomics
cBioPortal for Cancer Genomics (http://www.cbioportal.org) [11,12] was used to analyze cancer genomics data related to ERBB3. The term "ERBB3" was searched in the cBioPortal for Cancer Genomics, and the cross-cancer alteration summary for ERBB3 was obtained. Studies of stomach adenocarcinoma (TCGA, Nature 2014) were further analyzed for pathway network analysis [13].

Statistical Analysis
The gene expression data was shown in signal intensity or ratio to the average of MSC expression. The ratio to the mean of signal intensity in MSC samples normalized with GAPDH signal intensity was calculated and averaged. The data were shown as the mean ± SE. Student's t-test was performed in Microsoft Excel (Microsoft, Redmond, WA, USA). p<0.001 (n=12 in MSCs, n=5 in diffuse-type GC) was considered as statistically significant.

Gene expression of EGFR, ERBB2 and ERBB3 in MSCs and diffuse-type GC
Expression levels of the EGFR, ERBB2 and ERBB3 genes were analyzed in diffuse-type GC and MSCs ( Figure 1A). ERBB2 gene expression was up-regulated approximately 3-fold in diffusetype GC compared to MSCs ( Figure 1B). ERBB3 gene expression was up-regulated by approximately 150-fold in diffuse-type GC compared to MSCs ( Figure 1C). Signal Intensity of the ERBB3 gene expression in each sample is shown in Figure 1D. ERBB4 signal intensity was low.

Network Pathway Analysis of ERBB3
cBioPortal for Cancer Genomics network pathway analysis revealed the ERBB3-associated network and ERBB3 cross-cancer alterations ( Figure 3A and B). The ERBB3 molecular network was analyzed in stomach adenocarcinoma (TCGA, Nature 2014, tumor samples with sequencing and CNA data 287 samples / 1 gene) [13]. The network contained 51 nodes and included the 1 query gene ERBB3 as well as the 50 most frequently altered neighboring genes (out of a total of 420) in cBioPortal ( Table 1). The network shown in Figure 4 includes the 1 originally queried core gene and the 50 neighboring genes. Analysis of molecular interactions in VaProS demonstrated that ERBB3 interacts with neuregulin 1 (NRG1) and SH2B adaptor protein 3 (SH2B3).

Model Network of ERBB3
The model ERBB3 interaction network is shown in Figure 4. Nodes are colored based on gene expression alteration in diffuse-type GC compared to MSCs. Pink nodes indicate up-regulated (UR) genes and blue nodes indicate down-regulated (DR) genes with FC>3, p value <0.01. UR genes with 2<FC<3, p value <0.01 are highlighted with orange.

KEGG pathway and Gene Ontology of ERBB3
According to KEGG pathway analysis, ERBB3 is involved with EGFR tyrosine kinase inhibitor resistance, the ErbB signaling pathway, the calcium signaling pathway, endocytosis, proteoglycans in cancer, and microRNAs in cancer. The GO of ERBB3 analyzed in AmiGO 2 includes the ERBB3 signaling pathway, the ERBB4-ERBB3 complex, the ERBB2-ERBB3 signaling pathway, and regulation of the ERBB3 signaling pathway (http://amigo.geneontology.org/ amigo/search/ontology?q=ERBB3). GO biological process of ERBB3 included transmembrane receptor protein tyrosine kinase signaling pathway and regulation of cell proliferation.

DAVID analysis of ERBB3 network molecules
Molecules of ERBB3 network were analyzed in Functional Annotation Tool of DAVID Bioinformatics Resources 6.8, NIAID/ NIH (https://david.ncifcrf.gov/home.jsp). The Gene Ontology and Online Mendelian Inheritance in Man (OMIM) disease for the molecules of ERBB3 network were analyzed. Molecules in ERBB3 network are involved in cell adhesion or mitogen-activated protein kinase (MAPK) cascade.

Enrichment Analysis of ERBB3
The ERBB3 enrichment analysis using cBioPortal for Cancer Genomics revealed that the mRNA expression levels of DLG associated protein 3 (DLGAP3) and RNA polymerase II subunit J2 (POLR2J2) were altered in ERBB3-altered cases and ERBB3unaltered cases in stomach adenocarcinoma (TCGA, Nature 2014) [13].

Discussion
In this study, we investigated the ERBB signaling role in EMT and cancer. Gene expression of ERRB3, as well as ERBB2, was upregulated in diffuse-type GC compared to MSCs. ERBB3 network pathway model generated in this study included the EMT-related molecules such as CTNNB1 and CDH1. ERBB3 plays an important role in the PI3K/AKT pathway and is thus a target for the treatment of solid tumors [18]. Cell survival can be inhibited by a mouse monoclonal antibody against ERBB3 [18]. Expression of ERBB3 is correlated with intestinal stem cell markers such as LGR5, EPHB2, CD44 in colorectal cancer, although ERBB3 expression is observed in the differentiated cell population at the top of the crypts [19]. Furthermore, ERBB3 is targeted by miR-205-5p, a tumor suppressor in breast cancer [20]. Knockdown of ERBB3 suppressed migration and invasion of bladder cancer cells [21]. miR-148a-3p targets both ERBB3 and AKT2 and may repress ERBB3-and AKT-mediated proliferation and EMT [21].  [26]. It is also demonstrated that ERBB3 is involved in resistance to EGFR or ERBB2 targeted therapy for cancer [27]. Furthermore, the study of nanoparticles targeting ERBB2 and microRNA21 suggests the importance of ERBB signaling in cancer [28]. These insights indicate the significance of regulation of ERBB2 and ERBB3 signaling in cancer.
The down-regulation of ERBB3 following EMT leads to a reduction in serum-independent proliferation and an enhancement of PI3K-dependent proliferation [29]. Molecules related to intercellular adhesion and metastatic processes (such as PTEN) and endocytosis (such as ERBB3) are involved in liver metastasis [30]. In ERBB2-positive cancers, expression of EGFR, ERBB3 and CDKN1B are demonstrated to predict and distinguish the PI3K and MAPK signaling axes [31]. The ERBBs are inhibited by N-myc downstream regulated gene 1 (NDRG1) [32]. Since ERBBs are involved in oncogenic pathways, such as the PI3K, Akt and STAT3 pathways, further investigation of targeted molecules in the ERBB network is needed. Considering that the cancer cells migrating and metastasizing exhibit EMT-like phenotype, the investigation of ERBB3 signaling in bone-marrow derived MSCs showing mesenchymal phenotype and diffuse-type GC that exhibits more EMT-like phenotype compared to intestinal-type GC may lead to the novel discoveries for the overcome of the therapeutic resistance. Although the current finding showing ERBB3 gene up-regulation in diffuse-type GC compared to MSCs needs to be further examined in terms of the discussion whether the ERBB3 plays crucial role in EMT or not, it is suggested that the ERBB3 signaling is activated in gastric cancer. To further investigate the role of ERBB3 signaling in EMT and CSCs, the analysis in diffusetype GC and intestinal-type GC would be needed in future.