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A Hypoxia-Inducible HIF1–GAL3ST1-Sulfatide Axis Enhances Ccrcc Immune Evasion Via Increased Tumor Cell–Platelet Binding Claire M

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A Hypoxia-Inducible HIF1–GAL3ST1-Sulfatide Axis Enhances Ccrcc Immune Evasion Via Increased Tumor Cell–Platelet Binding Claire M Signal Transduction and Functional Imaging Molecular Cancer Research A Hypoxia-Inducible HIF1–GAL3ST1-Sulfatide Axis Enhances ccRCC Immune Evasion via Increased Tumor Cell–Platelet Binding Claire M. Robinson1,2, Betty P.K. Poon1,2, Yoshihito Kano1,2, Fred G. Pluthero3, Walter H.A. Kahr2,3,4, and Michael Ohh1,2 Abstract Clear cell renal cell carcinoma (ccRCC) is the most common gene whose expression is induced upon VHL loss leading to form of kidney cancer and the major cause of mortality for the accumulation of its enzymatic product sulfatide. Notably, individuals with von Hippel-Lindau (VHL) disease. ccRCC is platelets bind more efficiently to renal cancer cells with high characterized most frequently by inactivation of VHL tumor GAL3ST1-sulfatide expression than to GAL3ST1-sulfatide– suppressor protein that mediates degradation of the alpha negative counterparts, which protects ccRCC cells against subunit of the hypoxia-inducible factor (HIF) transcription natural killer cell–mediated cytotoxicity. These results suggest factor family. HIF has been implicated in disease progression that GAL3ST1 is a HIF-responsive gene that may contribute to and the aim of this study was to identify novel HIF target genes ccRCC development via promoting cancer cell evasion of that may contribute to ccRCC. We show that GAL3ST1, an immune surveillance. enzyme that catalyzes the sulfonation of the plasma mem- brane sulfolipid sulfatide, is among the top 50 upregulated Implications: Cancer development is in part dependent on genes in ccRCC tissue relative to matched normal tissue. evasion of immune response. We identify a HIF target gene Increased expression of GAL3ST1 in primary ccRCC correlates product GAL3ST1 that may play a role in this critical with decreased survival. We show that GAL3ST1 is a HIF target process. Introduction Loss or mutation of von Hippel-Lindau (VHL) tumor sup- pressor gene is the most frequently occurring genetic aberration Renal cancer accounts for 2%–3% of all diagnosed cancers (1). in sporadic ccRCC, evident in more than 60% of cases (3). Several distinct types of renal cancer exist including clear cell renal Other mutations in sporadic ccRCC have been reported, pre- cell carcinoma (ccRCC), oncocytoma, chromophobe, and papil- dominantly of the genes controlling the epigenome, however lary cell RCC (PRCC). Of these, ccRCC is the most common, these occur at a much lower frequency (3). A rare, hereditary, accounting for approximately 80% of all renal cancer cases (2). If autosomal-dominant cancer syndrome called VHL disease also it is diagnosed early enough the cancerous kidney is surgically provides evidence of VHL involvement in ccRCC. VHL disease removed. However, approximately one third of patients have has several subcategories that depend on mutational status and locally advanced or metastatic cancer at diagnosis, and up to relative risk of developing various tumor types including highly 30% of patients develop metastases during therapy. Advanced vascularized hemangioblastoma of the brain, spinal cord, or ccRCC, stages 3 and 4, have low survival rates with a 5-year eye, and catecholamine secreting pheochromocytoma arising survival rate of 53% and 8%, respectively. from the adrenal glands. The primary cause of mortality in patients with VHL is, however, due to the development of ccRCC, underscoring the importance of VHL mutation or loss 1Department of Laboratory Medicine & Pathobiology, University of Toronto, 2 in ccRCC (4). Toronto, Ontario, Canada. Department of Biochemistry, University of Toronto, VHL is the substrate-conferring component of E3 ubiquitin Toronto, Ontario, Canada. 3Division of Haematology/Oncology and Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick ligase. Multiple targets of VHL have been proposed but its Children, Toronto, Ontario, Canada. 4Department of Paediatrics, University of involvement in the degradation of the alpha subunit of Toronto, Toronto, Ontario, Canada. transcription factors called hypoxia-inducible factors (HIF), a a a Note: Supplementary data for this article are available at Molecular Cancer of which there are three (HIF1 ,-2 ,and-3 )inmammals,is Research Online (http://mcr.aacrjournals.org/). the best described VHL target (5, 6). Under normal oxygen Corrected online April 2, 2021. tension, HIFs are rapidly hydroxylated at two proline sites (HIF1a, P402 and P564; HIF2a, P405 and P531) within the Corresponding Author: Michael Ohh, University of Toronto, Medical Science oxygen-dependent degradation domain (ODD) by prolyl Building, Room 6306, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. 2þ a Phone: 416-946-7922; Fax: 416-978-5959; E-mail: [email protected] hydroxylases that utilize Fe ,2- -ketoglutarate, and O2 to catalyze the prolyl-hydroxylation reaction (5, 7). If O2 levels Mol Cancer Res 2019;17:2306–14 become limited or VHL is mutated or lost, HIFa is rapidly doi: 10.1158/1541-7786.MCR-19-0461 stabilized. HIFa forms a heterodimer with the constitutively Ó2019 American Association for Cancer Research. expressed HIFb and binds to the hypoxia-responsive 2306 Mol Cancer Res; 17(11) November 2019 Downloaded from mcr.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. The HIF-Responsive Gene GAL3ST1 Promotes Immune Cell Evasion element (HRE; 50-ACGTG-30) to upregulate transcription of Cell culture and reagents genes involved in adaptation to hypoxia, including those that Cell lines (786-O, RCC4, HEK293A, HEK293T, and NK-92) promote angiogenesis (e.g., VEGF), glycolysis (e.g., GLUT1), were purchased from American Type Culture Collection (ATCC). and erythropoiesis (e.g., EPO). With the exception of NK-92 cells, cells were cultured in DMEM The HRE sequence is a commonly occurring sequence (Wisent Bio Products) supplemented with 10% foetal calf serum within the genome and the current list of HIF target genes and penicillin-streptomycin. NK-92 cells were cultured, according of which there are approximately 70 is not exhaustive (8). The to the manufacturer's instructions, in MEM supplemented best described HIF target genes promote survival, oxygen with 100 U/mL IL-2. 786-MOCK, 786-VHL, 786-VHL_C162F, delivery, and a switch from oxidative phosphorylation to 786-VHL_ L188V, RCC4-MOCK and RCC4-VHL were cultured in glycolysis. Many other genes have putative HREs, while HRE DMEM supplemented with FBS, penicillin streptomycin and sequences are also found in other genomic locations where 250 mg/mL G418. itsfunctionandtheimpactofHIF,ifany,areunknown(9).In Unless otherwise specified, cells were cultured at 37Cina this study, we identified a gene called galactosylceramide humidified incubator with 5% CO2. A hypoxic incubator was sulfotransferase (GAL3ST1 or CST) as a significantly upregu- used to grow cells in 1% O2. lated gene in ccRCC patient tissue compared with matched normal tissue. We found that GAL3ST1 contains two putative Generation of stable cell lines HREs in its promoter making it a putative hypoxia-responsive Stable cell lines were generated using the pGIPZ-turboGFP gene. This enzyme promotes the synthesis of sulfatides lentiviral vector containing an shSCR (Open Biosystems) by catalyzing the sulfonation of galactosylceramide and lac- or an shGAL3ST1 (a gift from Dr. Meredith Irwin, The tosylceramide to galactosylceramide sulfate (SM3) and lacto- Hospital for Sick Children, Toronto, Ontario, Canada). Stable sylceramide sulfate (SM4). Sulfatides (SM3 and SM4) are polyclonal cell lines using both lentiviruses were generated sulfoglycolipids that are multifunctional membrane-bound in both 786-O and RCC4 cells. Cells were selected using molecules involved in multiple cellular processes, including puromycin and stable cells were continually grown in low ligand binding, that are relevant to immune responses and dose puromycin for the duration of their time in culture. hemostasis. They comprise a significant proportion of the myelin sheath of neuronal cells but are also reported to Western blotting be upregulated in various cancer cells including in ccRCC To identify differences in protein expression, western (10).GiventhepresenceofputativeHREsinthisgene's blotting was performed. Protein lysates were separated by regulatory region, we hypothesized that this upregulation size on a SDS-PAGE gel. Following transfer onto PVDF was a VHL-dependent, HIF-mediated event, which may membrane, membranes were blocked and then incubated account for the increased levels of sulfatides reported to be with primary antibodies selective for proteins of interest. present in renal cancer cells (10). The aim of this study was to Following primary antibody incubation, membranes were determine the mechanism of transcriptional upregulation of washed, incubated with HRP conjugated secondary antibo- GAL3ST1 in ccRCC and investigate the potential functional dies and then washed again before developing. The following consequences of GAL3ST1-sulfatide in the pathogenesis primary antibodies were used for detection of proteins using of ccRCC. western blot. Anti-GAL3ST1 and anti-GLUT1 (Abcam), rabbit anti-HA (C29F4; Cell Signaling Technology), anti-HIF2a and anti-HIF1a (H1alpha67) (Novus Biologicals), anti-HIF1a Materials and Methods antibody (54/HIF-1a; BD Transduction Laboratories), anti- Donor consent and platelet isolation vinculin (V284; Upstate), anti-b-actin (AC-74; Sigma-Aldrich). This study was performed according to the guidelines of the Secondary horse radish peroxidase conjugated antibodies Research Ethics Board
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