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PEA15 Promotes Liver Metastasis of Colorectal Cancer by Upregulating the ERK/MAPK Signaling Pathway

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ONCOLOGY REPORTS 41: 43-56, 2019 PEA15 promotes liver metastasis of colorectal cancer by upregulating the ERK/MAPK signaling pathway BO TANG1, WENJIN LIANG2, YONG LIAO2, ZEMING LI2, YAN WANG1 and CHAO YAN1 1School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240; 2Department of Hepatobiliary Surgery, Guilin Medical University, Affiliated Hospital, Guilin, Guangxi 541004, P.R. China Received March 30, 2018; Accepted September 24, 2018 DOI: 10.3892/or.2018.6825 Abstract. Liver metastasis is one of the major causes of death reason leading to the death of patients with colorectal cancer in patients with colorectal cancer, and although treatment has is distant invasion and metastasis such as liver, lung and bone improved recently, the long-term survival rate of patients has metastasis, while liver metastasis of colorectal cancer is the not improved significantly. In the present study, we used immu- most common (3). Although the diagnosis and treatment nohistochemistry to determine that phosphoprotein enriched in strategies for liver metastasis of colorectal cancer have greatly astrocytes-15 kDa (PEA15) was highly expressed in colorectal improved in recent years, to date, the overall survival rate of ca ncer tissues a nd liver metastatic ca ncer tissues. It was a lso h igh ly patients has not significantly improved. Therefore, exploring expressed in metastatic colorectal cancer patients compared to the mechanism of liver metastasis of colorectal cancer can non‑metastatic patients. Through clinicopathological data of provide effective guidance for potential clinical interventions, patients with liver metastasis of colorectal cancer, we found that thus improving the long‑term prognosis of patients. high expression of PEA15 was positively correlated with TNM PEA15 is a 15-kDa-sized small molecule protein containing staging, liver metastasis and poor prognosis of colorectal cancer an NH2 terminal death effector domain and a COOH terminal patients. Using confocal immunofluorescence microscopy, irregular structure distributed in the cytoplasm. Its DNA sequence western blotting and cell proliferation, migration and invasion is located on chromosome 1q21‑22 and it is highly conserved in assays, we also determined that PEA15 could promote cancer cell mammalian gene sequences (4,5). PEA‑15 is widely expressed in proliferation in vitro and in vivo, epithelial mesenchymal transi- human tissues and participates in the interaction between various tion (EMT) and the characteristics of cancer stem cells in vitro, proteins. It can regulate cell apoptosis, proliferation and glucose thus promoting the abilities of invasion and migration. In addi- metabolism by acting on the key functions of cellular effec- tion, we revealed that PEA15 promoted the liver metastasis of tors in vivo (6‑8). At present, it has been revealed that PEA‑15, colorectal cancer cells in a xenograft tumor metastasis model. In through its death-effector domain (DED) and ERK1/2-targeted addition, concerning the mechanism, we used gene chip analysis binding, inhibited ERK1/2 phosphorylation and nucleus transfer, to determine that PEA15 upregulated the ERK/MAPK signaling thus playing an anti‑apoptotic role (9). Some studies have pathway in colorectal cancer cells. Therefore, we concluded demonstrated that PEA15 is highly expressed in tumors such as that PEA15 may be a potential biomarker for liver metastasis liver (10), lung (11), breast (12) and ovarian cancer (13). However, of colorectal cancer therapy. Collectively, PEA15 promoted the it has a dual role in the regulation of tumors (14). It has been development of liver metastasis of colorectal cancer through the revealed that phosphorylation of PEA15 phosphorylated ERK ERK/MAPK signaling pathway. and promoted the proliferation and invasion of hepatocellular carcinoma cells (10), whereas the unphosphorylated state of Introduction PEA15 inhibited the phosphorylation of ERK and EGFR, thus inhibiting proliferation, invasion and metastasis of breast (12) Colorectal cancer is the third most common cancer in the and ovarian cancer (13). However, whether PEA15 plays a role in world and its annual fatality rate ranks third (1,2). The main liver metastasis of colorectal cancer is still unknown. In the present study, we revealed that PEA15 was highly expressed in colorectal cancer tissues and metastatic liver tissues. High expression of PEA15 was positively correlated with TNM stage, liver metastasis and indicated a poor prog- Correspondence to: Dr Chao Yan or Dr Yan Wang, School of nosis. In vitro and in vivo experiments confirmed that PEA15 Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P.R. China promoted the proliferation, invasion, migration and EMT E‑mail: [email protected] of colorectal cancer cells. To determine its mechanism, we E‑mail: [email protected] used gene chip analysis and found that PEA15 regulated the ERK/MAPK signaling pathway to promote the invasion and Key words: PEA15, clinicopathological correlation, liver metastasis migration of colorectal cancer cells. Concurrently, xenograft of colorectal cancer, ERK/MAPK pathway tumor experiments also confirmed that PEA15 promoted liver metastasis of colorectal cancer cells in vivo. 44 TANG et al: PEA15 PROMOTES LIVER METASTASIS OF COLORECTAL CANCER Materials and methods P‑40, and 5 mM protease inhibitor cocktail; pH 7.4] was added to extract total proteins. Then the protein concentration was Experimental reagents and antibodies. Lipofectamine assessed by BCA (bicinchoninic acid) kit (Beyotime Institute 2000 liposomes and TRIzol reagent were purchased from of Biotechnology) and the appropriate amount of loading buffer Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, was added. Protein sample (30 µg) was added to 10% sodium USA). Primary antibodies for PEA15 (cat. no. ab133217), dodecyl sulfate-polyacrylamide gel electrophoresis system PLD1 (cat. no. ab50695), ERK (cat. no. ab184699), p38 (SDS-PAGE) for electrophoresis separation and then trans- (cat. no. ab170099), MAP2K6 (cat. no. ab154901), JNK ferred to polyvinylidene difluoride (PVDF) membranes (cat. no. ab47337), PRKX (cat. no. ab109389) and FLNB (Bio‑Rad Laboratories, Inc., Hercules, CA, USA). Then, 5% (cat. no. ab97457) proteins were purchased from Abcam non‑fat milk was used to incubate the PVDF membranes, (Cambridge, MA, USA). Primary antibodies for E‑cadherin and peroxidase-labeled secondary antibodies (HRP-labeled (cat. no. #14472), N‑cadherin (cat. no. 13116), vimentin goat anti‑rabbit IgG; cat. no. A0208; Beyotime Institute of (cat. no. 5741) and β‑actin (cat. no. 3700) were purchased Biotechnolgy) at a 1:10,000 were used for incubation for 1 h from Cell Signaling Technology (Danvers, MA, USA). Other at room temperature. Subsequently, chemiluminescence was reagents were purchased from Sigma-Aldrich (Merck KGaA, used to display the imprinting, and the results were analyzed Darmstadt, Germany). using the Tannon 5200 chemiluminescent imaging system (Tanon Science and Technology, Shanghai, China). Patient and samples. The present study included 40 cases of colorectal cancer and corresponding clinicopathological data, Quantitative reverse transcription polymerase chain which contained 19 cases of liver metastasis tissue specimens. reaction (qRT‑PCR). The collected cells and tissues were These specimens were all paraffin specimens and obtained added to TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) from the Department of General Surgery of the Affiliated and total RNA was extracted according to the RNAiso Plus Hospital of Guilin Medical University from August 2000 to kit instructions (Takara Bio, Inc., Otsu, Japan). The RNA August 2016 and all the specimens were clinically diagnosed by concentration was assessed using the NanoDrop spectro- the Department of Pathology. All clinical cases and specimen photometer (Thermo Fisher Scientific, Inc.). The FastQuant tissues were reviewed and approved by the Ethics Committee cDNA First‑Strand Synthesis kit (Tiangen Biotech, Co., of Guilin Medical University, and informed consents were Ltd., Beijing, China) was used for reverse transcription. A signed by the patients according to the Declaration of Helsinki. qRT‑PCR kit FastStart Universal SYBR Green Master Mix Clinicopathological data included sex, age, tumor diameter, (Rox) (Roche Diagnostics GmbH, Mannheim, Germany) tumor differentiation, TNM staging and liver metastasis. was used to produce the reaction system and amplification Follow‑up of patients was performed until March 2018. according to the manufacturer's instructions. The reaction cycling conditions were performed as follows: 1 cycle at Immunohistochemistry. The surgically resected paraffin 95˚C for 15 min, followed by 40 cycles at 95˚C for 10 sec specimens were collected from the Department of Pathology and at 60˚C for 60 sec, and then relative mRNA expression of the Affiliated Hospital of Guilin Medical University and was analyzed using the comparative quantification cycle (Cq) were prepared into 4‑mm thick slices. According to the method followed by normalization to β-actin expression EnVision two‑step procedure (14), dewaxing and hydration (Applied Biosystems; Thermo Fisher Scientific, Inc.) (15). were carried out, after EDTA buffer high pressure antigen The primers used for amplification were as follows: PEA15 retrieval. Subsequently a primary antibody against PEA15 forward, 5'-GTT CTG TAG TCA ACC ACC AT-3' and reverse, (Abcam) with a dilution of 1:100 was added, and the speci- 5 ' ‑ A C C AAC AAC
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