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Absence of HIF1A Leads to Glycogen Accumulation and an Inflammatory Response That Enables Pancreatic Tumor Growth

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Absence of HIF1A Leads to Glycogen Accumulation and an Inflammatory Response That Enables Pancreatic Tumor Growth Published OnlineFirst October 4, 2019; DOI: 10.1158/0008-5472.CAN-18-2994 Cancer Translational Science Research Absence of HIF1A Leads to Glycogen Accumulation and an Inflammatory Response That Enables Pancreatic Tumor Growth Marco Maruggi1, Fabiana Izidro Layng1, Robert Lemos Jr1, Guillermina Garcia1, Brian P. James1, Monica Sevilla1, Ferran Soldevilla2, Bas J. Baaten2, Petrus R. de Jong1, Mei Yee Koh3, and Garth Powis1 Abstract Cancer cells respond to hypoxia by upregulating the tumors identified hypoxic cancer cells with inhibited gly- hypoxia-inducible factor 1a (HIF1A) transcription factor, cogen breakdown, which promoted glycogen accumulation which drives survival mechanisms that include metabolic and the secretion of inflammatory cytokines, including adaptation and induction of angiogenesis by VEGF. Pan- interleukins 1b (IL1B) and 8 (IL8). scRNA-seq of the mouse creatic tumors are poorly vascularized and severely hypoxic. tumor stroma showed enrichment of two subsets of myeloid To study the angiogenic role of HIF1A, and specifically dendritic cells (cDC), cDC1 and cDC2, that secreted proan- probe whether tumors are able to use alternative pathways giogenic cytokines. These results suggest that glycogen in its absence, we created a xenograft mouse tumor model of accumulation associated with a clear-cell phenotype in pancreatic cancer lacking HIF1A. After an initial delay of hypoxic cancer cells lacking HIF1A can initiate an alternate about 30 days, the HIF1A-deficient tumors grew as rapidly pathway of cytokine and DC-driven angiogenesis. Inhibiting as the wild-type tumors and had similar vascularization. glycogen accumulation may provide a treatment for cancers These changes were maintained in subsequent passages of with the clear-cell phenotype. tumor xenografts in vivo and in cell lines ex vivo.Therewere many cancer cells with a "clear-cell" phenotype in the Significance: These findings establish a novel mecha- HIF1A-deficient tumors; this was the result of accumulation nism by which tumors support angiogenesis in an HIF1a- of glycogen. Single-cell RNA sequencing (scRNA-seq) of the independent manner. Introduction cancer (2). In that model, deletion of HIF1A promoted the formation of pancreatic intraepithelial neoplasms (PanIN) pre- Adaptation to hypoxia in pancreatic adenocarcinoma is medi- cursor lesions, but the role of HIF1A in tumor maintenance or ated through the stabilization and activation of the hypoxia- growth was not explored. As HIF1A is essential to adaptation of inducible factor (HIF) family of transcription factors. HIF1a cells to low oxygen, we set out to investigate the adaptive mechan- (hereafter HIF1A) drives the transcriptional activation of multiple isms cells may use to circumvent loss of HIF1A. Furthermore, due pathways, including a metabolic switch to promote glycolysis and to the role of HIF1A in promoting angiogenesis, and the depen- the expression of various proangiogenic cytokines including Vegf- dence of VEGFA expression on HIF1A (3), we discovered alter- A (VEGFA), which act on surrounding endothelial cells to induce native proangiogenic pathways that could be important in cancers the formation of new blood vessels (1). that become resistant to therapies targeting VEGFA such as bev- A previous study explored the dependence of tumor growth on acizumab, and in other diseases where anti-VEGFA therapy is HIF1A by selective deletion in a genetic model of pancreatic used, but likewise encounter high rates of resistance (4). Here, we studied the role of HIF1A in tumor growth and 1Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, maintenance in pancreatic cancer using a xenograft model of California. 2Infectious and Inflammatory Disease Center, Sanford Burnham MiaPaCa-2 cells with stable knockdown of HIF1A. We explored Prebys Medical Discovery Institute, La Jolla, California. 3Department of Phar- the tumor growth kinetics and observed a long delay in the macology, University of Utah, Salt Lake City, Utah. formation of tumors, followed by a rapid growth that was main- Note: Supplementary data for this article are available at Cancer Research tained through several tumor passages. Characterization of the Online (http://cancerres.aacrjournals.org/). tumors using multispecies single-cell RNA-seq (scRNA-seq), his- M. Maruggi and F.I. Layng share first authorship of this article. tology, and immunostaining indicated a dramatic accumulation of glycogen in tumors lacking HIF1A. This was found to be a result Corresponding Author: Garth Powis, Sanford Burnham Prebys Medical Discov- of the dependence on HIF1A for glycogen breakdown, leading to ery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: 858- 795-5195; E-mail: [email protected] an abundance of intracellular glycogen. We associated this gly- cogen accumulation with a proinflammatory signature in pan- Cancer Res 2019;79:5839–48 creatic cancer cells, characterized by several immunoattractant doi: 10.1158/0008-5472.CAN-18-2994 cytokines, including IL1b and IL8. These tumor-derived cytokines Ó2019 American Association for Cancer Research. attracted myeloid dendritic cells (cDC) of subtypes 1 and 2, which www.aacrjournals.org 5839 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst October 4, 2019; DOI: 10.1158/0008-5472.CAN-18-2994 Maruggi et al. in the context of the tumor microenvironment, acquired a proan- and E) showed no specific staining for HIF1A, thus excluding giogenic phenotype. This work indicates a novel nonmetabolic restoration of HIF1A as a mechanism of increased growth. There role for glycogen accumulation in driving a proinflammatory was no difference in apoptosis between EV and shHIF1A tumors, program, and sustaining solid tumor growth in the absence of as measured by TUNEL staining and no difference in gross HIF1A via cDC recruitment and proangiogenic cytokine release. necrosis. Extracellular matrix by collagen I/III staining was increased in the shHIF1A tumors, as was staining for a-smooth muscle actin, a marker for cancer-associated fibroblasts, tumor Materials and Methods hypoxia measured by pimonidazole staining was increased (Sup- Generation of shHIF1A and EV lines plementary Fig. S3), while staining for endothelial cell marker shRNA targeting HIF1A or glycogen synthase-1 GYS1 (Supple- CD31 showed that shHIF1A tumors were equally vascularized as mentary Fig. S1) were cloned into pSUPER backbone. MiaPaCa-2 the EV tumors. Overall, these data indicate that despite an initial cells (ATCC) with stable expression of hypoxia-response element delay in growth, HIF1A-deficient tumors adapt and grow rapidly (HRE)/luciferase, under neomycin selection, were transfected having normal angiogenesis despite increased hypoxia, and no with either pSUPER-HIF1A or pSUPER empty vector (EV). Cells increase in apoptosis. were selected using 2 mg/mL of puromycin for several passages, then single-cell sorted to establish clonal cultures. HIF1A knock- Tumors lacking HIF1A have a clear-cell phenotype down was verified by Western blot and decreased expression of characterized by intracellular glycogen accumulation downstream HIF1A genes (Supplementary Fig. S2). Cell lines Gross histochemical analysis of the tumor samples stained with were routinely tested to be Mycoplasma free, and the identity of Masson's trichrome showed a marked difference between the EV each line was authenticated at 2-month intervals while in culture and shHIF1A tumors, with shHIF1A tumors showing an abun- by the Genomics Shared Resource at SBP. Single-cell separation dance of clear cells throughout the whole tumor (Fig. 2A). Oil Red for scRNAeq is described in Supplementary Methods S1. siRNA O staining of the shHIF1A tumors was negative, suggesting that when used was Dharmacon SMARTpool used at 20–100 nmol/L the clear cells did not contain abundant lipids (Supplementary and validated by protein knockdown. Fig. S3), while Periodic acid–Schiff (PAS) staining was positive, indicating that the cells contained accumulated polysaccharides Mouse xenografts (Fig. 2B). To differentiate between glycogen, glycoproteins, and scid Five- to six-week-old Nod-scid (NOD.CB17-Prkdc /J) and mucins, PAS staining was performed in conjunction with diastase NOD-scid gamma (NOD-scid IL2Rgnull) mice were obtained from (PAS-D), an enzyme that specifically digests glycogen. Diastase the Jackson Laboratories. Tumor cells (107) were injected into the caused only marginal lightning of PAS staining in EV tumors, but flanks in 0.9% sterile saline. Tumor and body weight was mea- completely cleared the staining in shHIF1A tumors, indicating sured twice weekly. All studies beyond primary tumors (Fig. 1A) that the clear cells in shHIF1A tumors contain mainly glycogen. To were on tumors reimplanted once and cell lines derived there- validate these findings, glycogen content within the tumors was from. All animal studies were SBP ACUC approved. measured enzymatically, confirming a dramatic increase in gly- cogen content in the shHIF1A tumors (Fig. 2C). To explore a Flow cytometry potential metabolic role for increased glycogen content, the þ Following tumor dissociation, CD45 cells were magnetically glucose flux into the cells and its utilization by glycolysis was positively selected using the Miltenyi Biotec LS column. Samples measured using cell lines established from EV and shHIF1A were stained using a panel of antibodies against tumor-infiltrating tumors. Glucose uptake was found to be slightly decreased
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