title: Analysis of Gene Expression in 3D Spheroids Highlights a Survival Role for ASS1 in Mesothelioma.
abstract: To investigate the underlying causes of chemoresistance in malignant pleural mesothelioma, we have studied mesothelioma cell lines as 3D spheroids, which acquire increased chemoresistance compared to 2D monolayers. We asked whether the gene expression of 3D spheroids would reveal mechanisms of resistance. To address this, we measured gene expression of three mesothelioma cell lines, M28, REN and VAMT, grown as 2D monolayers and 3D spheroids. A total of 209 genes were differentially expressed in common by the three cell lines in 3D (138 upregulated and 71 downregulated), although a clear resistance pathway was not apparent. We then compared the list of 3D genes with two publicly available datasets of gene expression of 56 pleural mesotheliomas compared to normal tissues. Interestingly, only three genes were increased in both 3D spheroids and human tumors: argininosuccinate synthase 1 (ASS1), annexin A4 (ANXA4) and major vault protein (MVP); of these, ASS1 was the only consistently upregulated of the three genes by qRT-PCR. To measure ASS1 protein expression, we stained 2 sets of tissue microarrays (TMA): one with 88 pleural mesothelioma samples and the other with additional 88 pleural mesotheliomas paired with matched normal tissues. Of the 176 tumors represented on the two TMAs, ASS1 was expressed in 87 (50%; staining greater than 1 up to 3+). For the paired samples, ASS1 expression in mesothelioma was significantly greater than in the normal tissues. Reduction of ASS1 expression by siRNA significantly sensitized mesothelioma spheroids to the pro-apoptotic effects of bortezomib and of cisplatin plus pemetrexed. Although mesothelioma is considered by many to be an ASS1-deficient tumor, our results show that ASS1 is elevated at the mRNA and protein levels in mesothelioma 3D spheroids and in human pleural mesotheliomas. We also have uncovered a survival role for ASS1, which may be amenable to targeting to undermine mesothelioma multicellular resistance.
authors.affiliation: Department of Medicine, Pulmonary, San Francisco General Hospital, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Dario
authors.initials: D
authors.lastname: Barbone
authors.affiliation: Department of Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Loes
authors.initials: L
authors.lastname: Van Dam
authors.affiliation: Department of Medicine, Pulmonary, San Francisco General Hospital, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Carlo
authors.initials: C
authors.lastname: Follo
authors.affiliation: Sidra Medical and Research Center, Doha, Qatar.
authors.forename: Puthen V
authors.initials: PV
authors.lastname: Jithesh
authors.affiliation: Center for Cancer Research and Cell Biology, Queen's University of Belfast, Belfast, United Kingdom.
authors.forename: Shu-Dong
authors.initials: SD
authors.lastname: Zhang
authors.affiliation: Division of Thoracic Surgery, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: William G
authors.initials: WG
authors.lastname: Richards
authors.affiliation: Division of Thoracic Surgery, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Raphael
authors.initials: R
authors.lastname: Bueno
authors.affiliation: Department of Cancer Studies, Cancer Research UK Leicester Centre, University of Leicester, Leicester, United Kingdom.
authors.forename: Dean A
authors.initials: DA
authors.lastname: Fennell
authors.affiliation: Department of Medicine, Pulmonary, San Francisco General Hospital, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: V Courtney
authors.initials: VC
authors.lastname: Broaddus
mesh_terms: D017314:Annexin A4 / Q000378:metabolism; D001124:Argininosuccinate Synthase / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008654:Mesothelioma / Q000235:genetics* / Q000473:pathology; D018874:Spheroids, Cellular; D020394:Vault Ribonucleoprotein Particles / Q000378:metabolism; D000099187:Major Vault Protein
chemical_list: D017314:Annexin A4; D001124:Argininosuccinate Synthase; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein