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 glycogen 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 2019 American Association for Cancer Research. attracted myeloid dendritic cells (cDC) of subtypes 1 and 2, which

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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 glycogen 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 in leukocytes. Following staining, samples were run on the the shHIF1A cells compared with EV cells, although not MACSQuant (Miltenyi Biotec) flow cytometer. statistically significant (P ¼ 0.07;Fig.2D).ASeahorseextra- cellular flux assay was used to measure glycolysis, showing that Extracellular flux assay for glycolysis under low oxygen conditions (2% O2) the glycolytic capability The XF Glycolysis Stress test was used according to the man- of shHIF1A cells was dramatically decreased compared with EV ufacturer protocol on the Seahorse Bioscience XF96e instrument, cells (Fig. 2E) with an increase in mitochondrial respiration to measure the rate of lactate formation and oxygen utilization. (Fig. 2F). These data indicate that an accumulation of glycogen is associated with slightly decreased glucose uptake, and a marked decrease in glucose utilization by glycolysis in the Results shHIF1A tumors, suggesting a nonmetabolic role of glycogen HIF1A-deficient cancer cells form fast growing tumors after a in these tumors. The source of energy metabolism in these cells delay period remains to be determined. EV and shHIF1A cells were injected in the flanks of Nod-scid mice. Tumors formed by EV cells grew rapidly to 1,000 mm3 scRNA-seq identifies hypoxic cell populations in tumors within 30 days, while the shHIF1A tumors persisted for up to In order to elucidate the mechanism underlying the accumu- 50 days before growing with kinetics that matched the EV tumors lation of glycogen in shHIF1A tumors, and to gain insights into (Fig. 1A). Only cells from EV tumors showed HRE promoter the pathways being utilized by the tumor to bypass the lack of activation (Fig. 1B). Cells obtained from EV and shHIF1A tumors HIF1A, scRNA-seq was utilized (Fig. 3A). Following sequencing were propagated in vitro, and used to establish second-generation and downstream analysis, 6 well-defined clusters were obtained tumors. The second-generation shHIF1A tumors grew after only a from the bioinformatics pipeline (Fig. 3B). Three clusters were short delay with growth kinetics that matched the EV tumors, found to express hypoxic genes, based on a hypoxia signature that indicating that HIF1A-independent growth was maintained includes non-HIF1A genes (5), with three different levels of ex vivo (Fig. 1C). IHC characterization of these tumors (Fig. 1D expression: "low," "medium," and "high." For analysis purposes,

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Loss of HIF1 Leads to Glycogen Accumulation and Angiogenesis

A Growth of shHIF1A xenografts B HRE-LUC C Reimplantation of shHIF1A xenografts Air 1% O2 )

3 **** 15,000 ) 2,000 Control shHIF1A 3 2,000 mm (

n 1,500 1,500 e 10,000 **** 1,000 1,000 5,000

umor burd 500 500 t n Luciferase activity Luciferase 0 0 0 Mea

A Mean tumor burden (mm 0 0 EV 1 EV 1A 10 2 30 F r HIF Days hHI s sh Days Tumo or m Tu

D Human HIF1A TUNEL αSMA Collagen I/III CD31 EV

200 μm 200 μm 100 μm 100 μm 100 μm shHIF1A

200 μm 200 μm 100 μm 100 μm 100 μm

HIF1A TUNEL αSMA Collagen I/III CD31 E ** ns 15 40 40 100 2.0

* 80 ** 30 30 1.5 10 ns 60 20 20 1.0 40 5 % Positive % Positive % Positive % Positive % Positive 10 10 0.5 20 ** 0 0 0 0 0.0 EV shHIF1A EV shHIF1A EV shHIF1A EV shHIF1A EV shHIF1A

Figure 1. Characterization and in vivo growth of MIAPaCa-2 shHIF1A cells. A, Growth of shHIF1A tumors (red), as compared with EV (blue) in Nod-scid mice. B, HRE luciferase activation was measured in EV (blue) and shHIF1A (red) cells derived from tumors and cultured for four passages ex vivo, after incubation in air and

hypoxia (1% O2) for 48 hours. Error bars, SD of three technical replicates. C, Tumor growth of cells derived from first-passage shHIF1A tumors, cultured ex vivo for four passages, and reinjected in the flanks of Nod-scid mice. D, Tumors of the reimplanted xenografts in log phase growth were stained for HIF1A, TUNEL, a-smooth muscle actin (aSMA; fibroblasts), Sirius Red (Collagen I/III), and CD31. Panels show typical fields. E, Aperio quantification of the staining using a complete section from at least 6 tumors. , P < 0.05; , P < 0.01; , P < 0.001; ns, nonsignificant, P > 0.05. EV, blue; shHIF1A, red.

these three clusters were grouped into a single "hypoxic" group, HIF1A-deﬁcient tumors show suppressed glycogen breakdown while all others were grouped "normoxic." Comparison of the at the transcriptional level hypoxic and normoxic clusters using a GSEA signature for hypoxia To elucidate the mechanism of glycogen accumulation in the validated the classiﬁcation of the cells (Fig. 3C). Within these shHIF1A cells, clusters from the scRNA-seq were analyzed for clusters, it was possible to compare the expression of transcripts expression of key glycogen synthesis and breakdown enzymes. between cells from EV and shHIF1A tumors. Expression of gly- A heat map was generated of the relative expression values colytic enzymes, broadly reported to be HIF1A dependent, was within normoxic and hypoxic clusters in EV and shHIF1A enriched in the EV cells within the hypoxic clusters, and repressed tumors showing increased glycogen synthesis and breakdown in the shHIF1A cells (Fig. 3D). enzymes in hypoxia, in both the EV and shHIF1A tumors

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A Trichrome B PAS PAS-D EV EV

2 mm 100 μm 100 μm 10 μm 10 μm shHIF1A shHIF1A

2 mm 100 μm 100 μm 10 μm 10 μm

C Glycogen DEGlucose uptake rate Glycolytic function F Mitochondrial function

20 in 60 40,000 2-DG 30 EV FCCP EV m Glucose ) Oligomycin Oligomycin Rotenone & ** shHIF1A Antimycin A shHIF1A 30,000 15 40 20 ns ** 10

20,000 mpH/min ( RFU R glucose/cell/ 20 10 10,000 5 A ** C OCR (pmoL/min) E moL μ 0 0 0 0 EV shHIF1A EV shHIF1A 020406080 0 20 40 60 80 Time (minutes) Time (minutes)

Figure 2. Increased glycogen content of shHIF1A tumors and ability to sustain increased glucose uptake in hypoxia. A, Masson's trichrome staining of shHIF1A and EV tumors. Two representative EV and shHIF1A tumors are shown, with the boxed areas enlarged to show a higher magniﬁcation of the tumor cells. B, PAS for detection of polysaccharides in EV and shHIF1A tumors, with added preincubation of the tumors with diastase (PAS-D) that breaks down glycogen. C, Glycogen content of the tumors measured using an assay based on glycogen hydrolase. Five tumors from each group were used, and data are shown as relative ﬂuorescent units (RFU) per mg tumor (n ¼ 5). D, Glucose uptake assay was performed using 2-deoxyglucose (2-DG) in cells from xenografted tumors cultured

for 5 passages ex vivo and incubated in hypoxia (1% O2) for 48 hours (n ¼ 3). E and F, Cells from xenograft tumors were cultured ex vivo and used for Seahorse- based glycolysis measurements in air (F), and oxygen consumption rate in hypoxia (2% O2; both n ¼ 10; F). Data, means SE (C, D,andE). EV, blue; shHIF1A, red (C, D, E,andF). , P < 0.001; ns, nonsigniﬁcant, P > 0.05.

(Fig. 3E). When analyzing the hypoxic groups only, and com- HIF1A-deficient cancer cells express a proinflammatory paring the shHIF1A to EV cells, the synthetic glycogen enzyme signature that is dependent on glycogen GYS1 was found to remain elevated in the shHIF1A groups. In order to explore the mechanism by which increased glycogen However, glycogen branching by GBE1, debranching by accumulation could contribute to HIF1A-independent tumor AGL, and breakdown as regulated by the PYGL cofactors growth, we analyzed the signaling pathways upregulated in PHKA1, PHKA2, and PHKB, were decreased in the shHIF1A shHIF1A cells. To this end, bulk RNA-seq was performed on two tumors (6). Figure 3F depicts the glycogen synthesis and independently derived resistant shHIF1A tumors, and two EV breakdownintheshHIF1Atumors, in hypoxia. This transcrip- tumors. Pathway analysis of the top transcripts indicated a sig- tional pattern suggests a mechanism by which, in the absence nificant upregulation of various inflammation-associated tran- of HIF1A, glucose is converted into glycogen, resulting in the scripts, including IL1A, IL1B, CXCL8 (IL8), and GCSF, as well as accumulation of glycogen in shHIF1A cells. We next investi- various MMP family members (Fig. 4A), consisting an inflam- gated the effect of GYS1 knockdown on tumor formation in matory signature present solely in the shHIF1A tumors. Indeed, Nod-scid mice finding that by itself it had only a small effect the most upregulated member of this signature, IL1B, was also delaying tumor growth, less than by HIF1A inhibition, while shown by IHC to be only expressed at the protein level in the both GSY1 and HIF1A inhibition completely prevented shHIF1A tumors (Fig. 4B). Importantly, IL1B remained upregu- tumor formation (Fig. 3G and Supplementary Fig. S4). Taken lated during ex vivo propagation of the cells, and continued to be together, the results suggest that an increase in glycogen in expressed in subsequent xenograft experiments (Supplementary HIF1A-deficient cells, which gives rise to a clear-cell phenotype, Fig. S5). To understand the upstream driver of this transcriptional is a key factor in the ability of the tumors to overcome pattern in cancer cells, pathway analysis was used, which pre- dependence on HIF1A for growth. dicted that IL1B and NF-kB were activated (Fig. 4C).

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Loss of HIF1 Leads to Glycogen Accumulation and Angiogenesis

Figure 3. Analysis of glycogen transcripts and pathway using scSeq. A, Workflow for single-cell separation from EV and shHIF1A tumors, which were combined for scSeq. B, SIMLR-based dimensionality reduction of the human transcriptome. Red, the three hypoxic clusters, labeled as "high," "medium," and "low." Blue, all other clusters. C, GSEA analysis was performed comparing the three hypoxic clusters against the normoxic clusters (P < 0.001). The green line indicates the enrichment score for each gene in the data set. Identification of hypoxic clusters was based on a common hypoxia signature (21). D, GSEA analysis for glycolysis by cells from EV and shHIF1A tumors within the hypoxia clusters (P < 0.001) using the Molecular Signatures Database (MSigDB) hallmark gene set for glycolysis. E, Key transcripts involved in glycogen synthesis and breakdown were probed against the scSeqRNA data set, focusing on the comparison of normoxic versus hypoxic clusters in EV or shHIF1A tumors. Shades of red are indicative of relative transcript expression, and no color indicates that the transcript was not detected. F, Pathway from extracellular glucose to intracellular glucose, and glycogen synthesis/branching and breakdown/debranching. Red arrows, paths based on transcript expression used in both EV and shHIF1A tumors. Gray arrows, paths solely expressed in the EV and absent from the shHIF1A tumors (, decreased transcript). G, The effect of GYS1 inhibition on tumor formation following injection of 107 MiaPaCa-2 cells with EV, stable shHIF1A, shGYS1, or shHIF1A together with shGYS1 in Nod-scid mice. There were 6 female mice per group; vertical bars on symbols are SE. Block arrows show the mean day SE tumor growth was first detected; , P 0.01 for shGYS1 and shHIF1A, compared with EV. Right panel shows tumor glycogen is significantly elevated by shHIF1A only.

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Figure 4. scRNA-seq indicates a proinflammatory signature in shHIF1A tumors. A, Bulk RNA-seq and IPA pathway analysis of the human transcriptome of two shHIF1A tumors in log phase growth shows the presence of a number of inflammatory mediators. The shade of red indicates the differential expression of the gene when compared with the baseline, measured as gene expression normalized to two EV tumors. B, IHC staining of EV and shHIF1A tumors for human IL1B, counterstained with hematoxylin. C, Upstream analysis performed on the two shHIF1A tumors for key members of proinflammatory pathways, IL1B and NFkB. The positive z-score reflects a predicted activation based on gene transcript patterns found in the transcriptomes. D, Effect of siRNA knockdown of genes regulating glycogen synthesis and branching enzymes (GYS1 and GBE1) and breakdown and debranching (PYGL and AGL) on IL1B expression in cells cultured from EV and shHIF1A tumors. Individual siRNAs were transfected at 20 nmol/L and scrambled siRNA as control at 40 nmol/L for 24 hours, and then exposed to air or hypoxia for 48 hours. Data, means SE (C and D). n ¼ 3. , P < 0.05.

Given a previous report of the mechanistic link between gly- included several top hits from the human transcriptome: IL1A, cogen accumulation and a proinflammatory stress (7), the con- IL1B, CXCL8, and IL6 (Fig. 5A). This suggests that the transcrip- nection between the transcriptional regulation of the glycogen tional changes in the mouse stroma are in part caused by the enzymes and the inflammatory signature was further explored. human cytokines secreted by the tumor. Because a number of the siRNA-mediated knockdown of members of the glycogen syn- proinflammatory cytokines that were expressed by the tumor are thesis and breakdown pathway was performed, with IL1B used as thought to play a role in immune cell recruitment, the mouse a readout for the proinflammatory signature in cancer cells. stroma transcripts were analyzed for evidence of immune path- Inhibition of the glycogen synthesis and branching enzymes way activation with the caveat that NOD-scid mice are deficient in GYS1 and GBE1 resulted in a dramatic decrease in IL1B expres- a number of immune cells, including B and T cells. A number of sion, both in the EV and shHIF1A cells, while inhibition of the pathways related to myeloid cells were found to be upregulated in glycogen breakdown and debranching enzymes PYGL1 and AGL the shHIF1A tumors, including chemotaxis and activation, and had no effect (Fig. 4D). However, IL1B antibody given to the mice specifically myeloid DC were predicted to have migrated into had no effect on tumor growth, suggesting multiple cytokines shHIF1A tumors (Fig. 5B). To validate this finding, flow cytometry may be involved. These data indicate that the shHIF1A tumors was used to quantify the percentage of a panel of tumor- acquire a proinflammatory transcriptional signature, most likely infiltrating lymphocytes (TIL) within EV and shHIF1A tumors as a result of the accumulation of intracellular glycogen. (Fig. 5C and Supplementary Table S1). Consistent with previous studies on the role of HIF1A in pancreatic cancer (2), the shHIF1A þ Increased transcriptional myeloid signature correlates with DC tumors displayed an increased percentage of CD45 cells, and infiltration consistent with the predictions from the bulk RNA-seq and þ þ þ To explore the interplay between tumor and stroma, upstream pathway analysis, CD11c cDC, identified as CD11b CD11c þ regulator analysis was performed on the bulk RNA-seq of the Class II Ly-6C/G cells were identified. Next, we used Nod-scid þ mouse tumor stroma transcriptome. From this pathway analysis, gamma mice whose CD11c DCs are deficient in cytokine pro- a number of predicted positive regulators of mouse transcription duction compared with Nod-scid mice (8), and found that

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Figure 5. Upstream analysis of mouse transcriptome matches the proinflammatory signature previously found in the human transcriptome. A, The bulk RNA- sequenced mouse transcriptome was analyzed using IPA and a list of upstream regulators obtained based on genes predicted to be activated by the transcript patterns found in the data set. This list included proinflammatory transcripts found in the human transcriptome shown as a heat map, with shades of blue and red depicting negative and positive z-scores of activation, respectively. B, IPA pathway analysis of z-scores showing activation of key myeloid cell pathways. P values of overlap of significant genes with the pathways are indicated. Four tumors were used for the EV and four tumors for the shHIF1A. C, The percentage of CD45-positive cells in single-cell suspensions derived from EV and shHIF1A xenografts was measured by flow cytometry (n ¼ 4; , P < 0.05; , P < 0.001). Analysis of various cells types showed CD11Cþ conventional (or myeloid) dendritic cells (cDC) were the most significantly altered between EV and shHIF1A groups (, P < 0.05). Bars, SD. EV, blue; shHIF1A, red. D, Tumor growth following injection of 107 MiaPaCa- 2 EV or shHIF1A cells into the flanks of Nod-scid gamma mice. n ¼ 6 female mice per group; vertical bars on symbols are SD. Block arrows show the mean day SD tumor growth was first detected. , P 0.01 for shHIF1A compared with EV. E, SIMLR clustering of mouse transcriptome showing circled two populations of cDC, subtypes 1 and 2. F, Analysis of expression within the two cDC subtypes, comparing cells found within EV and shHIF1A tumors, identifies a panel of angiogenesis- associated transcripts. G, Conditioned media from CD11Cþ and CD11C fractions of shHIF1A tumor were compared with spleen CD11Cþ for angiogenic ability. Left, typical patterns of HUVEC angiogenic tube formation; right, quantification of HUVEC tube length.

implanted siHIF1A MiaPaCa-2 cells took signiﬁcantly longer to Single-cell analysis of stroma unveils an angiogenic signature begin to form tumors, 54 days (Fig. 5D), than we had seen for in cDC1 and cDC2 DC subtypes Nod-scid mice, 28 days, P ¼ < 0.01 (Figs. 1A and 3G). Taken Because of a previously reported role for cDC in promoting together, these results suggest that human cytokines secreted by angiogenesis (9), and the absence of HIF1A/VEGF-driven the HIF1A inhibited tumor cells lead to the recruitment of mouse angiogenesis in the shHIF1A tumors, the increase in cDC could immune cells into the tumor, the most signiﬁcant being DCs that be responsible for allowing the shHIF1A tumor to become contribute to tumor formation in the absence of HIF1A. vascularized. To understand if cDC were responsible for an

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Figure 6. TCGA analysis indicates a differential effect of glycogen synthesis and breakdown genes on patient survival. A, The pan-cancer TCGA data set containing 9,184 patients was analyzed for survival against the expression of key transcripts identiﬁed in the study as important in driving the accumulation of glycogen in the absence of HIF1A expression. Left, Kaplan–Meier plots for GYS1 and GBE1 involved in synthesis and branching, respectively, indicate that high expression of either enzyme correlates with poor patient outcome, as measured by overall survival. Right, Kaplan–Meier plots for patient expression of the glycogen phosphorylase cofactors PHKA1/PHKA2/PHKB and the branching enzyme AGL indicate that high expression of these enzymes correlates with improved patient survival. All plots P < 0.001 difference in overall survival between high and low expression groups. B, A glycogen accumulation signature was developed that includes high expression of GYS1 and GBE1, and low expression of PHKA1/PHKA2/PHKB and AGL. This signature correlates with lower overall survival in pan-cancer TCGA, with a median survival difference of 1,818 days. C and D, Kaplan–Meier plots developed from TCGA data using the signature, showing decreased survival for ovarian cancer (P ¼ 0.021; C) and no signiﬁcant effect on survival for pancreatic cancer (D). E, Regression analysis of the glycogen signature and 63 HIF1A target genes from GSEA for HIF1A activation in pan-cancer TCGA patients showing a negative correlation between the two signatures.

angiogenic stimulus, the mouse component of the xenograft cancer cells could be related to patient survival. The PANCAN scRNA was annotated using a panel of transcript data for database of 9,184 solid cancers was compared with the various cell types found within the stroma (Supplementary glycogen pathway members identified in this study as Table S2), and two clusters were identified as cDC. Further contributing to glycogen accumulation. As previously reported stratification based on transcription allowed for the identifica- for hematologic malignancies (11), elevated expression of tion of DC subtypes cDC1 and cDC2 (Fig. 5E and Supplemen- GYS1 and branching enzyme gene GBE1 expression was tary Table S3; ref. 10). Angiogenic transcript expression was correlated with poor patient survival (Fig. 6A). In contrast, analyzed within these two subtypes and compared between the expression of the glycogen phosphorylase cofactor genes EV and shHIF1A tumors and indicated that both cDC1 and PHKA1, PHKA2, and PHKB, as well as the debranching cDC2 subtypes expressed more angiogenic transcripts in the enzyme gene AGL, was associated with improved survival. A shHIF1A tumors versus the EV (Fig. 5F). A functional HUVEC signature for glycogen accumulation was therefore developed assay was then performed to quantify the angiogenic potential based on the increased glycogen accumulation observed in our þ of the CD11c cells found within the shHIF1A tumors. The model system, with upregulated GYS1/GBE and downregu- þ CD11c fraction of the tumor was significantly more proan- lated PHKA1/PHKA2/PHKB/AGL (Fig. 6B). By combining þ giogenic than either tumor cells alone, or the CD11c fraction synthesis and breakdown, a signature that favors glycogen þ from the spleen (Fig. 5G). These data indicate that host CD11c accumulation in pan-TCGA data were correlated with shorter cells found in tumors can promote angiogenesis in the absence patient survival, consistent with our findings implicating of cancer cell–driven angiogenesis. glycogen accumulation in tumor growth. However, for individual tumor types, only ovarian cancer showed a significant Analysis of TCGA data for glycogen accumulation and patient correlation and pancreatic cancer was nonsignificant (Fig. 6C survival and D). Finally, TCGA was used to show a negative correlation A search of The Cancer Genome Atlas (TCGA) was between the glycogen accumulation signature and an HIF1A performed in order to explore if glycogen accumulation in activation signature (Fig. 6E).

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Discussion A significant finding from our studies is that of apparently normal angiogenesis in the absence of VEGFA expression in the HIF1A has long been thought to play an essential role in cancer shHIF1A xenograft. Although a number of agents targeting the cells' tolerance and adaptation to hypoxic stress through activities VEGFA pathway are approved for use in cancer and in ocular such as promotion of glycolysis, and the formation of angiogenic disorders, tumor inhibition and ocular improvement is short- factors (e.g., VEGFA; refs. 12, 13). We found that inhibition of lived due to acquired resistance in the form of either VEGFA target HIF1A in a transplanted human pancreatic cancer cell mouse and receptor overexpression, or a shift to other proangiogenic xenograft model, after initially showing growth, ultimately factors (26). In this work, we identified several proangiogenic returned to the same growth rate of the parental cell line. This cytokines secreted by DCs including PROK2/Bv8, whose secretion effect was maintained through subsequent implantation and in þ by CD11c cells was previously reported to be involved in cell lines. The HIF1A inhibited tumor cells acquired a "clear-cell" VEGFA-independent angiogenesis, and speculated to drive resis- phenotype due to accumulation of glycogen associated with loss tance to bevacizumab (27). Thus, the tumor-inflammatory cyto- of HIF1A-dependent glycogenolytic enzymes, while glycolysis kine/stroma angiogenic cytokine mechanism we identified may was inhibited. At the same time, the formation of immunoat- help explain how VEGFA-independent angiogenesis is possible. tractant inflammatory cytokines led to accumulation in the tumor Finally, an analysis of the TCGA database indicates that our microenvironment of conventional DCs that through release of findings may have clinical relevance. A pan-cancer analysis of proangiogenic factors sustained the tumor vascularization nec- more than 9,000 patients showed a correlation between patients essary for tumor growth. with low HIF1A activation and a signature for glycogen Glycogen accumulation is observed in various cancers, but is accumulation. most well established in renal clear-cell carcinoma. Here, loss of Thus, key enzymes involved in the glycogen pathway may the Von Hippel–Lindau tumor suppressor E3 ubiquitin ligase provide targets for drug discovery to modulate the elevated prevents both HIF1A and HIF2A degradation allowing elevated glycogen phenotype, which patient data suggest may be correlated levels and activity in both air and hypoxia. However, the inter- with decreased survival, and as a way to overcome resistance to action between the two HIFs in driving renal clear-cell carcinoma anti-VEGF therapy. growth, whether as tumor suppressors or activators remains unclear (14). In ovarian clear-cell carcinoma, HIF1A is thought Disclosure of Potential Conflicts of Interest to be responsible for the accumulation of glycogen by increasing M.Y. Koh has ownership interest (including patents) in Kuda Therapeutics, the expression of the glycogen synthesis enzyme GYS1 (15). Inc. No potential conflicts of interest were disclosed by the other authors. Although clear-cell carcinomas of the pancreas have been observed, this phenotype is not typically associated with Authors' Contributions increased intracellular levels of glycogen (16). However, glycogen Conception and design: M. Maruggi, B.P. James, F. Soldevilla, P.R. de Jong, is seen in serous microcystadenoma, a benign or indolent form M.Y. Koh, G. Powis representing 5% to 10% of pancreatic neoplasms (17, 18). As Development of methodology: M. Maruggi, B.J. Baaten, M.Y. Koh, G. Powis pancreatic cancer can express both HIF1A and HIF2A (19), it was Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Maruggi, F.I. Layng, R. Lemos Jr, B.P. James, for simplicity that we chose to study a cell line that expresses only F. Soldevilla, B.J. Baaten, G. Powis HIF1A. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Currently, there is no definitive mechanistic connection computational analysis): M. Maruggi, F.I. Layng, B.P. James, B.J. Baaten, P.R. de between glycogen accumulation and the transcription of proin- Jong, M.Y. Koh, G. Powis flammatory genes observed in our pancreatic cancer model. Writing, review, and/or revision of the manuscript: M. Maruggi, F.I. Layng, Hypoxia is known to activate the unfolded protein response (20), P.R. de Jong, G. Powis Administrative, technical, or material support (i.e., reporting or organizing which can increase tumor growth through the formation of fl data, constructing databases): R. Lemos Jr, G. Powis proin ammatory cytokines (21). It is thus possible that glycogen Study supervision: P.R. de Jong, M.Y. Koh accumulation could further enhance this pathway (22). The Other (histology): G. Garcia inflammatory signature expressed by the shHIF1A tumor cells was correlated with an increase in myeloid DC accumulation, Acknowledgments which our findings suggest results in a proangiogenic phenotype. This study was supported by NIH grant 5F31CA203286 (M. Maruggi), Although NOD-scid mice used in our study of tumor growth lack CA216424 (G. Powis), and CCSG grant P30CA030199. The help of the SBP a number of immune cells, including B and T cells, they do have Cancer Center Animal, Genomics, Histology, and Flow Cytometry Services is gratefully acknowledged. DCs (23). This infiltration of DCs in tumors has been reported (24), and their role in promoting angiogenesis is estab- The costs of publication of this article were defrayed in part by the lished (25). Notably, in Nod-scid gamma mice that have dys- payment of page charges. This article must therefore be hereby marked functional DCs with decreased cytokine release (8), the appear- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate ance of shHI1A tumors was considerably delayed compared with this fact. Nod-scid mice, consistent the importance of DCs to tumor formation. In immune-competent animals, it is possible that Received September 21, 2018; revised May 15, 2019; accepted September 25, other immune cells may play a similar role. 2019; published first October 4, 2019.

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5848 Cancer Res; 79(22) November 15, 2019 Cancer Research

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

Marco Maruggi, Fabiana Izidro Layng, Robert Lemos, Jr, et al.

Cancer Res 2019;79:5839-5848. Published OnlineFirst October 4, 2019.

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