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ROR1 Is Highly Expressed in Circulating Tumor Cells and Promotes Invasion of Pancreatic Cancer

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ROR1 Is Highly Expressed in Circulating Tumor Cells and Promotes Invasion of Pancreatic Cancer MOLECULAR MEDICINE REPORTS 18: 5087-5094, 2018 ROR1 is highly expressed in circulating tumor cells and promotes invasion of pancreatic cancer GUI-LI XU*, JIAN SHEN*, YUN-HUA XU, WAN-SHENG WANG and CAI-FANG NI Department of Interventional Radiology, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, P.R. China Received February 12, 2018; Accepted August 17, 2018 DOI: 10.3892/mmr.2018.9500 Abstract. Pancreatic cancer (PaC) is an aggressive malig- Introduction nancy, which is associated with high levels of metastasis. Circulating tumor cells (CTCs), which may be considered a Pancreatic cancer (PaC) is one of the most aggressive and functional biomarker and promising treatment strategy for lethal types of tumor, and is the fourth leading cause of metastasis, are associated with the prognosis and progression cancer-associated mortality worldwide (1). Despite improve- of various metastatic cancers, including PaC. Receptor tyrosine ment in cancer treatment, the prognosis for patients with PaC kinase-like orphan receptor 1 (ROR1) expression contributes remains poor, with a 5-year survival rate of ~5% (2). It has to cell metastasis and poor clinical outcomes in malignant been predicted that, with its current rise in incidence, PaC tumors. The present study aimed to explore the function of will become the second leading cause of cancer-associated ROR1 in PaC CTCs. Reverse transcription-quantitative poly- mortality by 2030 (3). The majority of patients with PaC are merase chain reaction and western blot analysis were used to not suitable for surgical resection due to local advancement examine the expression of ROR1, E-cadherin and N-cadherin. and metastasis, and the current chemotherapeutic regimens for Cell proliferative and invasive ability was assessed by MTT advanced PaC are limited (4). The high proclivity for partial and Transwell assays, respectively. The results revealed that invasion and early progression to distant metastases leads to the the mRNA and protein expression levels of ROR1 were poor outcome in PaC (5), thus resulting in the high mortality augmented in PaC tissues. Furthermore, the mRNA expres- rate of this malignancy. Furthermore, the current biomarkers sion levels of ROR1 were higher in CTCs compared with in poorly predict prognosis of patients with metastatic PaC (6). peripheral blood cells, and ROR1 was more highly expressed Therefore, for the clinical treatment of this disease, the selec- in CTCs than in cells. Notably, CTCs exhibited a markedly tion and identification of valid and reliable biomarkers as greater proliferative and invasive capacity than PANC-1 and prognostic indicators for patients with PaC are important. SW-1990 cells, whereas knockdown of endogenous ROR1 by Circulating tumor cells (CTCs) are tumor cells that induce small interfering RNA led to suppression of the invasion of metastasis, which is responsible for the majority of cases of CTCs. In addition, it was revealed that the mechanism under- cancer-associated mortality (7). CTCs have been widely lying the effects of ROR1 on PaC CTC metastasis may involve recognized as tumor- or metastasis-derived cells, which the epithelial-mesenchymal transition process. In conclusion, move into the circulatory system from the primary tumor site ROR1 may be considered a potential biomarker and thera- during surgical resection and lead to disease recurrence in peutic target for the treatment of PaC. patients with cancer (8-11). CTCs have already been widely used as a biomarker for the assessment of cancer prognosis and response to therapy (12). Previous studies have reported that CTC counts have an association with the prognosis and development of numerous metastatic diseases, including breast, colon, prostate and lung cancers (13-17). Therefore, Correspondence to: Professor Cai-Fang Ni or Dr Wan-Sheng Wang, studying CTCs may improve understanding of the potential Department of Interventional Radiology, First Affiliated Hospital mechanisms underlying tumorigenesis and metastasis, offer of Soochow University, 188 Shizi Street, Suzhou, Jiangsu 215006, promising clinical trials for patients with metastatic cancer, P. R. Ch i na and supply a target for designing effective and individualized E-mail: [email protected] cancer therapies. E-mail: [email protected] Receptor tyrosine kinase-like orphan receptor 1 *Contributed equally (ROR1) is an embryonic protein, and a member of the ROR receptor tyrosine kinase family, which serves key roles in Key words: pancreatic cancer, circulating tumor cells, receptor cell differentiation and proliferation, angiogenesis, tumor tyrosine kinase-like orphan receptor 1, cell proliferation, cell migration and metastasis (18-23). Numerous studies have invasion, epithelial-mesenchymal transition indicated that ROR1 is expressed at high levels in various blood and solid malignancies; however, it is lowly expressed in normal adult tissues (24-27). In this regard, ROR1 protein 5088 XU et al: DISRUPTION OF ROR1 INHIBITS CTC PROLIFERATION AND METASTASIS may be an ideal drug target for cancer therapy; however, the buffer (Beijing Leagene Biotech Co., Ltd., Beijing, China) underlying functions of ROR1 in PaC have yet to be elucidated. for 10 min at room temperature, and were then centrifuged On the basis of these findings, the present study explored at 800 x g for 8 min at room temperature. Subsequently, the the association between ROR1 and PaC, and revealed that cells were washed twice by resuspending the pellets in 6 ml ROR1 was increased in PaC tissues compared with in noncan- 1X PBS and were centrifuged at 800 x g for 3 min at room cerous tissues. In addition, the mRNA expression levels of temperature. Finally, the remaining cells were cultured in ROR1 were increased in CTCs compared with in peripheral RPMI 1640 medium (HyClone; GE Healthcare Life Sciences, blood cells from patients with PaC. Furthermore, the prolifera- Logan, UT, USA) supplemented with 10% FBS and incubated tive and invasive capacity was increased in CTCs with high in a cell incubator containing 5% CO2. levels of ROR1 compared with in PANC-1 and SW-1990 cells. Furthermore, the present study revealed that downregulating Reverse transcription‑quantitative polymerase chain ROR1 expression suppressed the invasive ability of CTCs. In reaction (RT‑qPCR). Total RNA was extracted from PaC addition, the results demonstrated that the epithelial-mesen- tissues or cells and peripheral blood cells using a HP Total chymal transition (EMT) process may participate in the RNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA) and Blood metastasis of CTCs from primary PaC tissue. Taken together, RNA kit (Omega Bio-Tek, Inc.), respectively, according to these results suggested that ROR1 may be a novel genetic the manufacturer's protocols. The RNA levels were analyzed modifier for the progression of PaC. using a NanoDrop spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). RNA was Materials and methods reverse transcribed into cDNA with an M-MLV First Strand kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, PaC tissues and blood samples. A total of 25 (male; mean USA) according to the manufacturer's protocol. The mRNA age, 52.6; age range 35-70 years) human PaC tissue and expression levels of ROR1 were quantified using a Platinum paired adjacent noncancerous pancreatic tissue samples were SYBR Green qPCR SuperMix-UDG kit (Invitrogen; Thermo acquired after written informed consent was obtained from Fisher Scientific, Inc.) on an ABI Prism 7500 Real-Time the participating patients. The patients did not receive partial PCR system (Applied Biosystems; Thermo Fisher Scientific, or systemic treatment prior to tissue sampling, and agreed Inc., Waltham, MA, USA), according to the manufacturer's to receive these treatments at the First Affiliated Hospital of protocol. Sequences of the primers were as follows: ROR1 Soochow University (Suzhou, China) between June 2006 and (forward), 5'-AGA TCA CAG CTG CCT TCA CTA T-3'; ROR1 January 2017. Paired adjacent noncancerous pancreatic tissues (reverse), 5'-GAC ATT CTC CAG GAT TTC ACA T-3'; β-actin (≤5 cm from the tumor site) were obtained from patients during (forward), 5'-GGC GGC ACC ACC ATG TAC CCT-3'; β-actin surgery. All tissue specimens were immediately snap-frozen (reverse), 5'-AGG GGC CGG ACT CGT CAT ACT-3'. The in liquid nitrogen following surgery. In addition, peripheral thermocycling conditions were as follows: 95˚C for 10 min, blood specimens were obtained from patients with PaC followed by 40 cycles of 50˚C for 20 sec and 60˚C for 1 min. using vacutainer tubes containing the anticoagulant EDTA. Quantification cycle (Cq) values of ROR1 mRNA were All peripheral blood samples were stored at 4˚C and were equilibrated to the internal control β-actin mRNA. Relative processed within 3 days. The present study was approved by expression was calculated using the ΔΔCq method (28). All the Ethics Review Committee of the First Affiliated Hospital experiments were performed in triplicate. of Soochow University. Western blot analysis. Protein lysates from cells and tissues PaC cell culture. Human PaC cell lines PANC-1 and SW-1990, were obtained using radioimmunoprecipitation lysis buffer and the normal human pancreatic cell line HPDE6-C7 were (Cell Signaling Technology, Inc., Danvers, MA, USA), which used in the present study from the Cell Bank of the Chinese contained protease and phosphatase inhibitors (Sangon Academy of Sciences (Shanghai, China). All cells were seeded Biotech Co., Ltd., Shanghai,
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