Abstract
Cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), contribute to tumorigenesis, resistance to chemoradiotherapy and recurrence in human cancers, suggesting targeting CSCs may represent a potential therapeutic strategy. In the current study, we found family with sequence similarity 83, member A (FAM83A) is significantly overexpressed and associated with poorer overall survival and disease-free survival in pancreatic cancer. Overexpression of FAM83A markedly promoted, whereas inhibition of FAM83A decreased, CSC-like traits and chemoresistance both in vitro and in an in vivo mouse model of pancreatic cancer. Furthermore, overexpression of FAM83A activated the well-characterized CSC-associated pathways transforming growth factor-β (TGF-β) signaling and Wnt/β-catenin signaling. Importantly, the FAM83A locus was amplified in a number of human cancers and silencing FAM83A in associated cancer cell lines inhibited activation of the WNT/β-catenin and TGF-β signaling pathways and reduced tumorigenicity. Taken together, these results indicate that FAM83A has a vital oncogenic role to promote pancreatic cancer progression and may represent a potential clinical target.
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Introduction
Pancreatic cancer is the seventh leading cause of cancer-related mortality.1, 2 Despite advances in modern medical technology, pancreatic cancer has benefited from marginal improvements in survival outcomes; the 5-year overall survival rate of patients with pancreatic cancer is only 6% and the median survival time is <9 months.3, 4 Failure of conventional chemotherapy, including both intrinsic and acquired chemoresistant behavior, is a major factor that significantly decreases the clinical efficacy of chemotherapy for pancreatic cancer.5, 6 The response rates to common chemotherapeutic drugs, such as gemcitabine, erlotinib and 5-fluorouracil (5-FU), in pancreatic cancer have been reported to be lower than 25%.5, 7, 8 Therefore, better understanding the molecular mechanisms that underlie drug resistance in pancreatic cancer could lead to the development novel therapeutic strategies for this highly lethal malignancy.
The intrinsic resistance of cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), to conventional therapy is currently regarded as a potential therapeutic target.9 For instance, it has recently been reported that the high rates and patterns of therapeutic failure observed in ovarian cancer are closely associated with stable accumulation of drug-resistant CSCs.10 Li et al.11 found that the percentage of the CD44+CD24–/low CSC sub-population, which exhibits intrinsic resistance to chemotherapy, was significantly increased in patients with breast cancer treated with chemotherapeutic drugs such as docetaxel, doxorubicin or cyclophosphamide. Similarly, CD133+ pancreatic CSCs have been demonstrated to be exclusively tumorigenic and highly resistant to chemotherapy and radiation therapy, and the CD133+ CXCR4+ sub-population of pancreatic CSCs is critical for tumor metastasis,12, 13, 14 suggesting that CSCs have important roles in pancreatic cancer progression. Therefore, targeting pancreatic CSCs could potentially increase chemosensitivity and thus improve the response to treatment.
Family with sequence similarity 83, member A (FAM83A), also known as BJ-TSA-9, is located on chromosome 8q24 and was originally identified as a potential tumor-specific gene by a bioinformatics approach.15 Furthermore, FAM83A is overexpressed in multiple human tumors, including lung, breast, testis and bladder cancer,16, 17, Primers and oligonucleotides Cloning primer human FAM83A-ORF, forward: 5′-AGCCGGTCAAGGCACCTGGG-3′ and reverse 5′-TCAGAAGTGAGGGGAGGCCTGCAGGAAGGGCCTCCAGGTT-3′; real-time PCR primer: FAM83A, forward: 5′-CCCATCTCAGTCACTGGCATT-3′ and reverse: 5′-CCGCCAACATCTCCTTGTTC-3′; ABCG2 forward: 5'-TGGTGTTTCCTTGTGACACTG-3′ and reverse: 5′-TGAGCCTTTGGTTAAGACCG-3′; BMI1 forward: 5′-TCGTTGTTCGATGCATTTCT-3′ and reverse: 5′-CTTTCATTGTCTTTTCCGCC-3′; SOX2 forward: 5′-GCTTAGCCTCGTCGATGAAC-3′ and reverse: 5′-AACCCCAAGATGCACAACTC-3′; OCT4 forward: 5′-GGTTCTCGATACTGGTTCGC-3′ and reverse: 5′-GTGGAGGAAGCTGACAACAA-3; NANOG forward: 5′-ATGGAGGAGGGAAGAGGAGA-3′ and reverse: 5′-GATTTGTGGGCCTGAAGAAA-3′; GAPDH forward: 5′-AATGAAGGGGTCATTGATGG-3′ and reverse: 5′-AAGGTGAAGGTCGGAGTCAA-3′. FAM83A primer used for genomic copy number detection: forward: 5′-CGCCACTGTGTACTTCCAGA-3′ and reverse: 5′-TCCACATCCGTGAACACATC-3′, FAM83A RNAi#1: 5′-GCACAACAACATCAGAGACCT-3′; FAM83A RNAi#2: 5′-GACTGGAGATTTGTCCTGTCT-3′. The human FAM83A gene was PCR-amplified from cDNA and cloned into pMSCV retroviral vector (Clontech, Mountain View, CA, USA). ShRNAs targeting FAM83A were cloned into the pSuper-retroviral vector. Transfection of plasmids was performed using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. Stable cell lines expressing FAM83A and FAM83A shRNA(s) were generated via retroviral infection as previously described,45 and were selected with 0.5 μg/ml puromycin for 10 days. Immunohistochemistry analysis was performed on the 103 paraffin-embedded pancreatic adenocarcinoma tissues, using anti-FAM83A antibody (Sigma). In brief, paraffin-embedded specimens were cut into 4-μm sections and baked at 65 °C for 30 min. The sections were deparaffinized with xylenes and rehydrated. Sections were submerged into EDTA antigenic retrieval buffer and microwaved for antigenic retrieval. The sections were treated with 3% hydrogen peroxide in methanol to quench the endogenous peroxidase activity, followed by incubation with 1% bovine serum albumin to block the nonspecific binding. Rabbit anti- FAM83A (1:500; Sigma) was incubated with the sections overnight at 4 °C. For negative controls, the IgG antibody or normal goat serum was co-incubation at 4 °C overnight preceding the immunohistochemical staining procedure. After washing, the tissue sections were treated with biotinylated anti-rabbit secondary antibody (Zymed, San Francisco, CA, USA), followed by further incubation with streptavidin-horseradish peroxidase complex (Zymed). The tissue sections were immersed in 3-amino-9-ethyl carbazole and counterstained with 10% Mayer's hematoxylin, dehydrated, and mounted in Crystal Mount. The degree of immunostaining were reviewed and scored separately by two independent pathologists blindly. The scores were determined by combining the proportion of positively stained tumor or normal pancreatic epithelial cells and the intensity of staining. Cell proportions were scored as follows: 0, no positive cells; 1, <10% positive cells; 2, 10–35% positive cells; 3, 35–75% positive cells; 4, >75% positive cells. Staining intensity was graded according to the following standard: 1, no staining; 2, weak staining (light yellow); 3, moderate staining (yellow brown); 4, strong staining (brown). The staining index (SI) was calculated as the product of the staining intensity score and the proportion of positive cells. Using this method of assessment, we evaluated protein expression in benign pancreatic epithelia and malignant lesions by determining the SI, with possible scores of 0, 2, 3, 4, 6, 8, 9, 12 and 16. Then the median value, which SI=8, was chosen as the cut off value. Therefore, samples with a SI⩾8 were determined as high expression and samples with a SI<8 were determined as low expression. Five hundred cells were seeded in six-well ultralow cluster plates (Corning, NY, USA) for 10 days. Spheres were cultured in Dulbecco’s modified Eagle’s medium/F12 serum-free medium (Invitrogen, Grand Island, NY, USA) supplemented with 2% B27 (Invitrogen, Grand Island, NY, USA), 20 ng/ml of EGF, and 20 ng/ml of bFGF (PeproTech, Offenbach, Germany), 0.4% bovine serum albumin (Sigma) and 5 μg/ml insulin. Gemcitabine (Gemzar, Lilly SA, Alcobendas, Spain) and 5-FU (Sigma; 03738) were dissolved in phosphate-buffered saline with concentration of 50 μM. β-Catenin/TCF inhibitor (FH535)(S7484), TGF-β inhibitor (S2704) were purchased from Selleck (Houston, TX, USA). The male/female BALB/c nude mice (6–7 weeks of age, 18–20 g) were randomly divided into 15 groups (n=6 per group). The indicated cells were inoculated with Matrigel subcutaneously into the inguinal folds of nude mice. Tumor volume was determined using external caliper and calculated using the equation (L × W2)/2. The mice were killed 31 days after inoculation, tumors were excised and subjected to pathologic examination. In the experiment testing, the chemoresistance effect of FAM83A, the BALB/c nude mice were implanted subcutaneously with the indicated cells (1 × 106) in order to rapidly induce exponentially growing tumors. When tumors reached a volume of approximate 100 mm3, animals were randomly assigned to five groups (n=6 per group), followed by intraperitoneal injection of Gemcitabine (80 mg/kg) twice a week. On day 43, animals were killed, and tumors were excised, weighed and subjected to pathological examination. All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and conformed to our institutional ethical guidelines for animal experiments. Cells were dissociated with trypsin and re-suspended at 1 × 106 cells/ml in Dulbecco’s modified Eagle’s medium containing 2% fetal bovine serum and then pre-incubated at 37 °C for 30 min with or without 100 μM verapamil (Sigma-Aldrich, Deisenhofen, Germany) to inhibit ABC transporters. The cells were subsequently incubated for 90 min at 37 °C with 5 μg/ml Hoechst 33342 (Sigma-Aldrich). Finally, the cells were incubated on ice for 10 min and washed with ice-cold phosphate-buffered saline before flow cytometry analysis. The data were analyzed by Summit5.2 (Beckman Coulter, Indianapolis, IN, USA). Ten thousand cells were seeded in triplicate in 48-well plates and allowed to settle for 24 h. One hundred nanograms of luciferase reporter plasmids or the control-luciferase plasmid, plus 5 ng of pRL-TK renilla plasmid (Promega, Madison, WI, USA), were transfected into pancreatic adenocarcinoma cells using the Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendation. Luciferase and renilla signals were measured 48 h after transfection using the Dual Luciferase Reporter Assay Kit (Promega) according to a protocol provided by the manufacturer. Three independent experiments were performed, and the data are presented as mean±s.d. Statistical tests for data analysis included Fisher’s exact test, log-rank test, chi-square test and Student’s two-tailed t-test. Multivariate statistical analysis was performed using a Cox regression model. Statistical analyses were performed using the SPSS 11.0 statistical software package for Windows SPSS Inc. (Chicago, IL, USA). Data represent mean±s.d. P<0.05 was considered statistically significant.Plasmids, retroviral infection and transfection
Immunohistochemistry
Sphere formation assay
Chemical reagents
Xenografted tumor
Flow cytometric analysis
Luciferase assay
Statistical analysis
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Acknowledgements
This work was supported by the Science and Technology Program of Guangdong Province (no 2014A020212155 and 2014A030313008), the PhD Start-up Fund of Natural Science Foundation of Guangdong Province (no 2015A030310052), the Natural Science Foundation of China (81325013 and 91529301), the Medical Scientific Research Foundation of Guangdong Province (no B2013127), the Natural Science Foundation of Guangdong Province (no 2015A030313044).
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Chen, S., Huang, J., Liu, Z. et al. FAM83A is amplified and promotes cancer stem cell-like traits and chemoresistance in pancreatic cancer. Oncogenesis 6, e300 (2017). https://doi.org/10.1038/oncsis.2017.3
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DOI: https://doi.org/10.1038/oncsis.2017.3
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