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CANCER RESEARCH | METABOLISM AND CHEMICAL BIOLOGY

KRAS Controls Pancreatic Cancer Cell Lipid Metabolismand Invasive Potential through the Lipase HSLCody N. Rozeveld1, Katherine M. Johnson2, Lizhi Zhang3, and Gina L. Razidlo2,4

ABSTRACT◥

Oncogene-induced metabolic reprogramming is a hallmark ofpancreatic cancer (PDAC), yet the metabolic drivers of metas-tasis are unclear. In PDAC, obesity and excess fatty acidsaccelerate tumor growth and increase metastasis. Here, we reportthat excess lipids, stored in organelles called lipid droplets (LD),are a key resource to fuel the energy-intensive process ofmetastasis. The oncogene KRAS controlled the storage andutilization of LD through regulation of hormone-sensitive lipase(HSL), which was downregulated in human PDAC. Disruption ofthe KRAS–HSL axis reduced lipid storage, reprogrammed tumorcell metabolism, and inhibited invasive migration in vitro and

metastasis in vivo. Finally, microscopy-based metabolic analysisrevealed that migratory cells selectively utilize oxidative metab-olism during the process of migration to metabolize stored lipidsand fuel invasive migration. Taken together, these results reveal amechanism that can be targeted to attenuate PDAC metastasis.

Significance: KRAS-dependent regulation of HSL biases cellstowards lipid storage for subsequent utilization during invasion ofpancreatic cancer cells, representing a potential target for thera-peutic intervention.

See related commentary by Man et al., p. 4886

IntroductionMetastases are responsible for 90% of cancer-related deaths and

represent a critical challenge for cancer therapy (1, 2). Becausemetastasis is a dynamic process (3), the mechanisms cancer cellsutilize during disseminationmust be adaptable for successful invasion,migration, and establishment of a secondary tumor. Metastatic inva-sion is a highly energy-intensive process, and the metabolic processesused by invading tumor cells are poorly defined. Although proliferativeprimary tumors rely heavily on glycolysis (4), metastatic tumor cellshave drastically different metabolic requirements (5) including reli-ance on mitochondrial integrity and increased oxidative phosphory-lation, likely to generate ATP for migration (6–8). The metabolicchanges that occur during tumor cell invasion may represent a noveltherapeutic target to reduce metastasis and improve outcomes forpatients with metastatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) ranks among the dead-liest cancers, due in part to a high incidence of metastasis at diagnosis.One of the few known risk factors for pancreatic cancer is obesity,which correlates with worse prognosis (9, 10). Corroborating theseepidemiologic studies, high-fat diets accelerate tumorigenesis andincrease metastasis in mouse models of PDAC (11, 12). In addition,increased intratumoral fatty acids (FA) may result from elevated denovo synthesis (9, 13–15). However, the mechanisms exploited by

PDAC cells to store and utilize fat to promote cancer progression andmetastasis are not well defined.

Excess FAs are stored as triglycerides within organelles called lipiddroplets (LD). These stored neutral lipids can be catabolized bylipolysis, which uses the sequential activity of lipases to liberate freeFAs (16). Best studied in adipocytes, adipose triglyceride lipase(ATGL), hormone-sensitive lipase (HSL), andmonoacylglycerol lipase(MGL) sequentially catabolize triacylglycerol to glycerol and freeFAs (17). Lipolysis, thus, mediates the release of bioavailable FAs frominert storage within LDs. Therefore, dysregulation of lipolysis, or anyof the crucial enzymes mediating this process, may be a novel mech-anism by which cancer cells fuel tumor progression and metastasis.

The GTPase KRAS is activated in over 90% of PDAC, and is a driverof tumorigenesis and metabolic reprogramming (18–20). OncogenicKRAS mutations instill a greater dependency on glycolysis and aminoacid metabolism, promoting biomass synthesis to support tumorgrowth (21–24). How KRAS-mediated metabolic regulation is alteredduring metastasis, and the mechanisms involved in this transientprocess, are not well understood.

Here, we describe a dynamic mechanism in which oncogenic KRASregulates HSL to control metabolism in metastatic pancreatic cancercells by regulating lipid storage and utilization. Oncogenic KRASsuppresses HSL expression as part of a metabolic shift away from FAoxidation in pancreatic cancer cells, leading to LD accumulation andpriming tumor cells for invasion. Live cell imaging revealed that thesestored LDs are then catabolized during invasion by the action oflipases, including residual HSL, resulting in increased FA oxidationand oxidative metabolism that drives tumor cell invasion. Thesefindings reveal a novel mechanism by which KRAS regulates tumorcell metabolism and controls metastatic invasion in pancreatic cancer.

Materials and MethodsCell culture

Panc-1, MiaPaCa-2, CFPAC, and mKPC cells were cultured inDMEM containing 10% FBS and Pen/Strep. BxPC3 cells were culturedin RPMI1640 containing 10% FBS and Pen/Strep. iKras cells werecultured in RPMI1640 containing 10% FBS, Pen/Strep, and 1 mg/mLdoxycycline hyclate (Sigma, D9891). HPDE cells were cultured in

1Mayo Graduate School of Biomedical Sciences, Mayo Clinic, Rochester,Minnesota. 2Division of Gastroenterology & Hepatology, Mayo Clinic, Rochester,Minnesota. 3Department of Anatomic Pathology, Mayo Clinic, Rochester,Minnesota. 4Department of Biochemistry & Molecular Biology, Mayo Clinic,Rochester, Minnesota.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Gina L. Razidlo, Mayo Clinic, 200 1st Street S.W.,Rochester, MN 55905. Phone: 507-284-0308; E-mail: [email protected]

Cancer Res 2020;80:4932–45

doi: 10.1158/0008-5472.CAN-20-1255

�2020 American Association for Cancer Research.

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keratinocyte-SFM containing epidermal growth factor, bovine pitui-tary extract, and Pen/Strep. DMEM, and RPMI1640 were fromCorning, and FBS was from Sigma. Cell lines were Mycoplasmatested using PCR (Southern Biotech), DAPI staining, or by IDEXXBioAnalytics. Atglistatin (15284), and CAY10499 (10007875) werefrom Cayman Chemical. U0126 (662005) was from Calbiochem,H89 (B1427) and inhibitors to DGAT1 (PZ0207) and DGAT2(PZ0233) were from Sigma-Aldrich. Cell lines were from ATCCor were provided by the laboratory of Dr. David Tuveson (mKPCT4–2D; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY),Dr. Marina Pasca di Magliano (iKRAS 4292; University of Michi-gan, Ann Arbor, MI), or Dr. Daniel Billadeau (HPDE; CFPAC,Mayo Clinic, Rochester, MN).

Transfections and establishment of stable cell linesLipofectamine 2000 (11668-027, Invitrogen) was used for plasmid

transfection. The murine HSL-GFP plasmid was a gift from J. Liu(Mayo Clinic). The Lipe gene encoding HSL was cloned into thepLenti6.3 lentiviral vector by theMayo Clinic C-SiG Gene Editing andEpigenomics Core. To generate lentivirus, the HSL plasmid, or apLenti6.3.FLAG control vector, was cotransfected into HEK 293Tcells with VSV-G-pseudotyped viral packaging plasmids (provided byDr. Y. Ikeda,MayoClinic). After 72 hours, supernatants were collectedand passed through 0.45 mm filters to collect virus. mKPC cells weretransduced with virus for 72 hours with 10 mg/mL polybrene andselected with 5 mg/mL blasticidin for 14 days to obtain cells stablyoverexpressing control vector or HSL. Murine siGENOME SMART-pools (Dharmacon) for Lipe (HSL) and Cpt1a were used for knock-downs, with Lipofectamine RNAiMAX (13778-150).

ImmunoblottingFor Western blotting, cells were lysed in NP-40 lysis buffer (20

mmol/L Tris-Cl, pH 7.4, 137 mmol/L NaCl, 10% glycerol, 1% NP-40(IGEPAL), and 2 mmol/L EDTA, pH 8.0) with cOmplete ProteaseInhibitor Cocktail (11873580001, Roche). Protein levels were quan-tified by BCA assay (23225, Pierce), and equal amounts were resolvedon SDS-PAGE gels and transferred to PVDF for probing. Primaryantibodies were: HSL (4107, Cell Signaling Technology), phospho-HSL (4139, Cell Signaling Technology), ATGL (2439, Cell SignalingTechnology), MGL (sc-398942, Santa Cruz Biotechnology), phospho-ERK (4377, Cell Signaling Technology), ERK (9102, Cell SignalingTechnology), vimentin (5741, Cell Signaling Technology), Claudin-1(13255, Cell Signaling Technology), ZO-1 (8193, Cell Signaling Tech-nology), Snail (3879, Cell Signaling Technology), E-cadherin (610182,BD Biosciences), N-cadherin (610921, BD Biosciences), CPT1 (12252,Cell Signaling Technology), PLIN-2 (3121, LSBio), PLIN-3 (10694,Proteintech), PLIN-5 (46215, Invitrogen), Actin (A2066, Sigma), andGAPDH (5174, Cell Signaling Technology). Secondary antibodieswere conjugated to horseradish peroxidase (Biosource International)and immunoreactive signals were detected using SuperSignal WestPico or Femto substrates (Thermo Fisher Scientific) and HyBlot CLfilm (Denville Scientific), and developed using an X-Omat film pro-cessor. Densitometry values were quantified using ImageJ.

IHCFor human samples, 30 histologic samples from 10 de-identified

patients were obtained from the Mayo Clinic SPORE in PancreaticCancer. Patients provided written informed consent and approval ofthe Institutional Review Board was granted prior to investigation.From each patient, adjacent/normal, primary tumor, and tissue from ametastatic site were analyzed. For mouse experiments, tissue was fixedin 4% PFA and cryoprotected in 30% sucrose before embedding in

Tissue-Tek O.C.T. compound (4583). Four micrometer of humantissue sections were used and 10 mm murine sections were used forall applications. Anti-Rabbit HRP-DAB Tissue Staining Kit (R&DSystems, CTS005) was used. Primary antibodies used were: HSL(SAB4501762, Sigma), aSMA (ab5694, Abcam), and Ki67 (12202,Cell Signaling Technology). Semiquantitative scoring of human tissueswas performed by a pathologist (Dr. Lizhi Zhang) in the Mayo ClinicDivision of Anatomical Pathology. To label LDs, sections werewashed in 60% isopropanol for 30 seconds, 60% Oil Red O solution(5 mg/mL in isopropanol) for 15 minutes, 60% isopropanol for 30seconds, then washed in deionized water prior to hematoxylincounterstain. Further semiquantitative analysis of tissue sectionswas completed using color deconvolution in ImageJ to obtain anoptical density of the signal of interest.

Migration, invasion, and proliferation analysisFor transwell invasion analysis, 3� 105 cells were seeded in six-well

transwell chambers with 8-mm pores (MCEP06H48, Millipore) andcoated with 1mg/mLMatrigel (356231, Corning). Cells were seeded inthe upper chamber containing 0.1% serum and invaded towards thelower chamber containing 10% serum. Percent invasion was deter-mined by counting nuclei on the bottom and top of the filter for eachfield and dividing the invaded nuclei (bottom) over the total. Nucleiwere visualized using Hoechst 33342 (H3570, Invitrogen). For woundhealing assays, cells were grown to confluency on gridded glasscoverslips before a plastic pipette tip was used to scratch the mono-layer. Brightfield images were taken at the T ¼ 0 and a timepointdetermined for each cell line. Beginning and ending images wereoverlaid to determine migration distance using Adobe Photoshop.For matrix degradation assays cells were plated on fluorescently-labeled gelatin-coated coverslips and the percentage of cells thatdegraded the fluorescent gelatin was analyzed as described previ-ously (25). Invadopodia were counted manually based on over-lapping puncta of cortactin, actin, and gelatin degradation. Forproliferation and viability assays, the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, G5421) or propi-dium iodide (P4864, Sigma) were used.

Fluorescence and live-cell microscopyCells werewashed in PBS andfixed in 3% formaldehyde. Phalloidin-

TRITC (P1951, Sigma) was used to visualize actin. Anti-cortactin 4F11(05–180, Upstate Biotechnology) and Cy5 secondary antibody (115–175–166, Jackson ImmunoResearch) were used to visualize cortactin.To label LDs, Oil Red O (O0625, Sigma) and C12-BODIPY (D3822,Thermo Fisher Scientific) were used and quantitated as describedpreviously (26). Briefly, fixed samples were washed in 60% isopropanolfor 30 seconds, 60% Oil Red O solution (5 mg/mL in isopropanol) for1.75 minutes, and 60% isopropanol for an additional 30 seconds.Images were acquired on a Zeiss LSM 780 confocal microscope with a40� oil objective lens and Zeiss Zen software. LD quantitation wasdone using the auto local threshold and analyze particles tool withinImageJ as described previously (27).

qPCR mRNA analysisRNA isolated using the RNeasy Plus Mini Kit (74134, Qiagen) was

reverse-transcribed using oligo-(dT) primers and the Super Script IIIFirst Strand Kit (18080-51, Invitrogen). Quantitation of gene expres-sion was performed using SYBR green fluorescence on a LightCycler480 (04707516001, Roche). Primers used were: Lipe (HSL gene)forward: 50-GATTTACGCACGATGACACAGT-30, reverse: 50-ACC-TGCAAAGACATTAGACAGC-30. 18S ribosomal RNA: forward:

Stored Lipids Fuel Metastasis in Pancreatic Cancer

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50-CGCTTCCTTACCTGGTTGAT-30, reverse: 50-GAGCGACCA-AAGGAACCATA-30. Expression was determined by normalizingLipe to 18S by the DDCt method.

In vivo experimentsAnimal experiments were performed under Institutional Animal

Care and Use Committee approval and in accordance with theapproved protocol. For pancreatic orthotopic injections, 6-week-oldfemale C57BL/6J mice were purchased from The Jackson Laboratory.Mice were anesthetized by intraperitoneal injection of ketamine andxylazine, and 1 � 104 mKPC cells stably expressing control vector orHSL suspended in 100 mL PBS were injected into the tail of thepancreas. Mice were weighed every other day. At 6 weeks, or upona weight loss of >10%, mice were sacrificed and both primary tumorsand macrometastases were isolated and analyzed. Genetic mousemodels, Ptf1a-Cre (023329) and KRas LSL-G12D (008179, both fromThe Jackson Laboratory) were crossed to achieve a pancreas-specificKRasG12D/þmouse that was compared with littermate Ptf1a-Cre mice.Both male and female mice were analyzed at 12 weeks, with no sex-specific differences in the results.

Metabolic analysesFor Seahorsemetabolic analysis, a Seahorse XF24Analyzerwas used

with Seahorse XF Cell Mito Stress Test Kit (103015-100, Agilent) andSeahorse XF24 FluxPak (100867-100, Agilent) reagents to measureoxygen consumption rate (OCR). Quantitation was normalized by cellcount, determined by Hoechst 33342 positive cells imaged on a Celigo(Nexcelom Bioscience). Glycolytic activity was determined using aGlycolysis Cell-Based Assay Kit (600450, CaymanChemical) andATPlevels were determined using an ATP Detection Assay Kit (700410,Cayman Chemical).

Optical redox ratio analysisTo obtain optical redox ratios, endogenous autofluorescence of

NADH and flavin adenine dinucleotide (FAD) were imaged on a ZeissLSM 510 confocal microscope with a 40� oil objective lens withexcitations of 364 and 458 nm and emissions of 460� 20 and>505 nm,respectively. Pixel-wise redox ratio maps were created in ImageJ usingfluorescence intensities as FAD/(FAD þ NADH). The mean redoxratio was acquired by averaging the redox ratio values within the cellcytoplasm, excluding the background and nucleus, when possible.

Statistical analysisMicroscopy images were processed using ImageJ andAdobe Photo-

shop. Any adjustments were made uniformly across the whole image.Data were analyzed using GraphPad Prism. Graphed data representmean � SEM, and statistical significance was determined using aStudent t test, aMann–WhitneyU test, or aWilcoxon signed rank test.For analysis of the The Cancer Genome Atlas (TCGA)-PancreaticCancer (PAAD) dataset, the UCSC Xenabrowser visualization tool(xenabrowser.net) was used to compare somatic mutation of KRASand the gene expression of LIPE. The log2 expression values weregraphed in GraphPad Prism as violin plots.

ResultsCatabolism of stored lipids promotes PDAC invasion andmigration

We first measured the basal LD content of pancreatic cancer cells,including Panc1, MiaPaCa2, BxPC3, and cells isolated from a mousemodel of pancreatic cancer (mKPC; ref. 28). Under basal conditions,

PDAC cells are capable of storing FAs in intracellular LDs (Fig. 1A;Supplementary Fig. S1A). Exogenous FAs in the form of oleic acid(OA) stimulated lipid accumulation in the PDAC cells and dramat-ically increased the LD content of the cells (Fig. 1A and B; Supple-mentary Fig. S1A). Thus, PDAC cells are capable of storing intracel-lular lipids in LDs, which is exacerbated when exposed to elevatedlevels of FAs.

As high fat diets correlate with increased metastasis and tumorprogression (11, 29), we tested how excess stored lipids impactedcellular invasion. Treatment of mKPC, BxPC3, and Panc04.03 cellswith exogenous FAs stimulated cellmigration in awoundhealing assay(Fig. 1C; Supplementary Fig. S1B) and invasive ability in a transwellinvasion assay (Fig. 1D; Supplementary Fig. S1C). A similar increasewas observed following addition of linoleic acid, indicating thatmultiple FA species augment PDAC cell migration (SupplementaryFig. S1D). Metastasis requires tumor cells to degrade extracellularmatrix for invasion into surrounding tissues. Following lipid-loadingwith exogenous FAs, we observed increased degradation of extracel-lular matrix as well as the formation of matrix-degrading invadopodia(Supplementary Figs. S1E–S1H). To test if lipid loading increased themetastatic capacity of PDAC cells by inducing an epithelial-to-mesenchymal transition (EMT), we analyzed the expression of a panelof EMT markers following OA treatment (Supplementary Fig. S1I).Although the expression of EMT markers was largely unchanged bytreatment with OA, there was a decrease in the tight junction markerZO-1. Thus, while not likely driven by an EMT program, lipidspromote multiple processes that support tumor cell invasion. Therewas not a significant change in mKPC cell proliferation or cell deathfollowing treatment with exogenous FAs (Fig. 1E; SupplementaryFigs. S1J and S1K), indicating that stored lipids aremore important forinvasive migration than proliferation of tumor cells. Together, theseresults indicate that PDAC cells can utilize exogenous FAs and storedlipids to enhance their metastatic capacity.

Importantly, increased invasion only occurred after preloading oflipids, resulting in lipid storage, and not while exogenous FAs wereavailable to the cells only during invasion (Supplementary Fig. S2A).We hypothesized that it was lipids stored in LDs that were required fortumor cell invasion. We therefore tested the requirement for diglyc-eride acyltransferases 1 and 2 (DGAT1/2) in tumor cell invasion,which are required for the packaging and storage of lipids in LDs.Inhibition of DGAT1/2 reduced the capacity to store lipids in LDs, andconsequently minimized the proinvasive effects of lipid loading onPDAC cells (Supplementary Figs. S2B and S2C). Thus, proper pack-aging of triglycerides into LDs is a crucial step in FA-augmentedPDAC cell invasion.

As lipases liberate free FAs from LDs, we tested their role in lipid-regulated metastatic processes in tumor cells. Strikingly, treatment ofmKPC cells with Atglistatin to inhibit ATGL, or CAY 10499 to inhibitHSL (with some inhibition of MGL), reduced transwell invasion(Fig. 1F), indicating that lipase activity is required for tumor cellinvasion. Treatment with lipase inhibitors had no significant impacton proliferation (Supplementary Fig. S2D), suggesting that storedlipids are largely contributing to invasive potential, rather than pro-liferative capacity. Together, these data suggest that lipase dependentliberation of stored lipids from LDs is required during tumor cellinvasion.

We hypothesized that LDs were undergoing lipolysis during theprocess of migration. Using live cell microscopy, we observed LDs inthe highlymotile leading-edge cells were lost as cellsmigrated (Fig. 2A;Supplementary Movie S1), consistent with LD catabolism (30). Incontrast, cells that were less motile and farther than two cell widths

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Cancer Res; 80(22) November 15, 2020 CANCER RESEARCH4934




from the migratory edge appeared to retain their stored LDs (Sup-plementary Fig. 2E; Supplementary Movie S2). Loss of LDs alsooccurred during invasion. Tumor cells preloaded with lipids wereseeded in a transwell assay, and the filters were analyzed by confocalmicroscopy to image the LD content on the top (noninvasive) andbottom (postinvasion). Invasive cells had fewer LDs remaining com-pared with noninvasive cells (Fig. 2B and C), consistent with LD lossand lipolysis during invasion.

We predicted that FAs liberated from LDs during lipolysis wereacting as a fuel source for invasion. Etomoxir, an inhibitor of carnitinepalmitoyltransferase 1 (CPT1), perturbs beta-oxidation of FAs bypreventing FA mitochondrial import. Importantly, etomoxir reducedthe migration of mKPC cells, suggesting that lipid import and oxida-tion are required formigration (Fig. 2D). Similar results were observedfollowing a knockdown of CPT1 (Supplementary Figs. S2F and S2G).As treatment with either lipase inhibitors (Fig. 1F) or a CPT1 inhibitorreduced tumor cell invasion andmigration, the data suggest that tumorcells utilize lipolysis of LDs and subsequent FA uptake into mito-chondria to promote invasive migration.

The lipase HSL is suppressed by the oncogenic GTPase KRASThese data suggest that lipase activity is required for tumor cell

invasion. Because lipolysis has beenprimarily studied in adipocytes, wefirst determined the levels of lipase expression in a panel of pancreatic

cancer cell lines. Strikingly, we observed a correlation betweenHSL expression and the KRAS status of cultured pancreatic cancercells (Fig. 3A). Both non-neoplastic HPDE cells and BxPC3 PDACcells harbor wild-type KRAS (31, 32) and showed significantly higherlevels of HSL expression when compared with PANC1, CFPAC, andPanc04.03 cells, which have an activatingmutation ofKRAS (Fig. 3A).This suggested that HSL may be specifically downregulated in cellswith oncogenic KRAS. Interestingly, the correlation between KRASand expression level does not extend toATGLorMGL (Fig. 3A andB).

Activating mutations of KRAS play a crucial role in the metabolismof pancreatic cancer cells (18, 21, 24, 33). Therefore, we investigated thecorrelation between HSL and oncogenic KRAS using a doxycycline-inducible cell culture system (iKras; ref. 34). Culture of these mousepancreatic cancer cells in the presence of doxycycline drives expressionof KRASG12D. Following removal of doxycycline, the cells revert towild-type KRAS, as indicated by decreased phosphorylated ERK(Fig. 3C). Strikingly, reversion to wild-type Kras also resulted in asignificant increase in total and phosphorylated HSL (Fig. 3C), but notATGL, consistent with the concept that oncogenic KRAS specificallysuppresses HSL expression. To determine if expression of oncogenicKras was sufficient to downregulate HSL in vivo, we examinedpancreatic tissue from mice expressing KRASG12D specifically in thepancreas using Ptf1a-Cre (“KC”mice). Indeed, IHC forHSL revealed astriking downregulation of HSL in KC mice compared with mice

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Excess lipid promotes PDAC invasion and migration through lipolysis. A, Control and OA-loaded (200 mmol/L) mKPC cells were visualized by staining for LDs usingOil Red O. Scale bar, 10 mm. B, Quantitation of total LD area per cell from the conditions in A. More than 125 cells scored in at least five fields for three independentbiological replicates. C,Quantitation of distance migrated of control and OA-loaded mKPC cells in a wound healing assay over 12 hours. Ten measurements of threefields for three independent biological replicates represented. D, Quantitation of control and OA-loaded mKPC cells in a transwell invasion assay. Cells invaded for16 hours through a Matrigel-coated filter toward high-serum media. More than 300 cells scored in each of three independent biological replicates. E, Quantitationof control andOA-loadedmKPCcell proliferation over the indicatednumberof days. Eight independent biological replicates, normalized toDay 1 for each experiment.F, Quantitation of control or drug-treated mKPC cells in a transwell invasion assay. Cells attached for 2 hours prior to drug treatment and invaded for 14 hours. Tenmmol/L Atglistatin (ATGLi) and 10 mmol/L CAY 10499 (HSL/MGLi) were used. More than 300 cells scored in each of three independent biological replicates. Graphsindicate mean � SEM, analyzed by Student t test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






expressing Ptf1a-Cre alone (Fig. 3D; Supplementary Fig. S3A). Tofurther examine the mechanism of KRAS-mediated HSL downregula-tion, we determined if KrasG12D decreased HSL transcription. qRT-PCR revealed that HSL mRNA was decreased in cells with oncogenicKras (þDox) compared with cells with wild-type Kras (�Dox),suggesting that oncogenic KRAS suppresses HSL transcription(Fig. 3E).

KRAS signaling occurs through several downstream pathways (18).To determine which signaling pathway mediated the suppression ofHSL by KRAS, we utilized the pharmacologic MEK inhibitor U0126.Interestingly,MEK inhibition reversed the Kras-mediated suppressionof HSL and increased HSL expression after 24 hours (Fig. 3F),

indicating that KRAS-mediated HSL suppression occurs through theMEK/ERK signaling pathway. Together, these data reveal that expres-sion of the lipase HSL is suppressed by oncogenic KRAS, and mayrepresent a novel mechanism by which KRAS controls tumor cellmetabolism.

HSL regulates the metastatic impact of oncogenic KRASWe hypothesized that KRAS-mediated HSL regulation is a crucial

mechanism for modulating lipid storage and metastatic ability.Decreased levels ofHSLwould be predicted to lead to LDaccumulationdue to decreased lipolysis. Indeed, cells with oncogenic Kras hadsignificantly higher levels of stored LDs than cells with wild-type Kras

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LDs are catabolized during invasive migration. A, Still images taken from Supplementary Video S1 depicting Panc04.03 cells at indicated time points migrating in awound healing assay, with LDs visualized by BODIPY. Brightfield, LDs (white), and composite images are shown. Scale bar, 10 mm.B,Representative images of LDs inmKPC cells in an invasion assay and preloaded with OA (200 mmol/L). Noninvasive cells (left) did not invade, whereas Invasive cells (right) did invade andwere imaged on the bottom of the transwell. Cells were stained for nuclei (Hoechst, top) and LDs (Oil Red O, bottom). Scale bar, 10 mm. C, Quantitation of lipidarea per cell from B. More than 50 cells scored in at least five fields from six independent biological replicates. D, Migration quantitation of control or etomoxir-treated (10 mmol/L) mKPC cells. More than 10 measurements of three fields for three independent biological replicates shown. Graphs indicate mean � SEMdata, analyzed by Student t test. � , P < 0.01; �� , P < 0.01; �� , P < 0.001.

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(Fig. 4A andB), consistent with lower levels ofHSL and increased lipidstorage. Similarly, oncogenic Kras led to increased levels of the LDprotein PLIN-2, which is also consistent with lipid storage (Supple-mentary Fig. S3B). This was specific to Plin2, as we observed no changein Plin3 or Plin5 by oncogenic Kras (Supplementary Fig. S3B).

To determine the functional consequence ofHSLdownregulation inPDAC cells, we disrupted the Kras-mediated suppression by over-expressingHSL in bothmKPC and iKras cells (Fig. 4C; SupplementaryFig. S3C). BecauseHSL catabolizes intracellular LDs, we first examinedthe LDcontent of the cells followingHSL overexpression. As predicted,overexpression of the lipase HSL dramatically depleted the storedlipids (Fig. 4D and E; Supplementary Figs. S3D and S3E). Thus,bypassing the downregulation of HSL in cells with oncogenic Krasreduces the capacity to store lipids in LDs.

Because stored lipids promote invasive potential (Fig. 1D),and HSL overexpression depletes stored lipids, we hypothesizedthat HSL may affect invasion. Indeed, HSL overexpression dramat-ically reduced the invasive potential of both the mKPC cells and

the iKras cells (Fig. 4F and G). Although KrasG12D significantlyincreased invasion in vitro, this effect was completely blocked by re-expression of HSL. Thus, even in the presence of oncogenic Kras,overexpression of HSL depleted stored lipids and reduced invasionto the level of cells expressing wild-type Kras. If the reduction ininvasive ability caused by HSL overexpression is due to depletion ofstored lipids available for the invasive cell to utilize, then providingadditional lipids to the cell should overcome this inhibitory effect.Indeed, when HSL overexpressing cells were preloaded with excessOA, the defect in invasive ability was rescued (Fig. 4G). Thesedata support the model that lipolysis of stored LDs promotes tumorcell invasion.

Although HSL is downregulated by oncogenic KRAS, its expressionis not completely lost (Fig. 3A and C). Therefore, we tested ifKRASG12D-expressing cells were still sensitive to lipase inhibition.Indeed, pharmacologic inhibition of the lipases ATGL or HSL/MGLblocked invasion in iKras cells, and reduced invasion to the level ofWTKras cells (Fig. 4H). Similar to pharmacologic inhibition, knockdown

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HSL is regulated by oncogenic KRAS.A,Western blot analysis of the indicated cells for HSL, ATGL, andGAPDH fromwhole-cell lysates.B,Western blot analysis of theindicated cells for MGL and actin. C,Western blot analysis of HSL, phospho-HSL, ATGL, phospho-ERK, ERK, and actin expression fromwhole-cell lysates. iKras cellswere cultured in doxycycline, or with doxycycline withdrawn for 24, 48, or 72 hours prior to lysis. Quantitative densitometry values for HSL expression from blotshown (both bands) are indicated above. D, IHC for HSL expression in pancreatic tissue sections isolated from Ptf1a-Cre (Cre) or littermate Ptf1a-Cre–drivenKRasG12D (KC) mice. n ¼ 3 mice per condition. Scale bar, 10 mm. E, Relative HSL mRNA levels by qRT-PCR from iKras cells cultured with doxycycline (G12D) orfollowing 72-hour doxycycline withdrawal (WT). Three technical replicates for six independent biological replicates are shown. Data analyzed by Wilcoxonsigned rank test. F,Western blot analysis of mKPC cells treated with vehicle control (Con) or the MEK inhibitor U0126 (10 mmol/L) for the indicated time. Blotsare representative of three independent experiments. Graphs indicate mean � SEM. � , P < 0.05.






of HSL also reduced tumor cell invasion (Fig. 4I; SupplementaryFig. S3F). Therefore, even though cells with oncogenic Kras promotelipid storage via decreased HSL expression, residual lipase activity isstill required to utilize stored lipids during invasion. In contrast toHSLoverexpression, the invasive defect in the HSL knockdown cells could

not be rescued by preloading with excess OA as there was not sufficientHSL to catabolize this excess lipid (Fig. 4I). It initially seemedparadoxical that both HSL overexpression and pharmacologic inhi-bition similarly inhibited tumor cell invasion (Figs. 1F and 4F–I).However, the net result of both treatments is the same in the reduced

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HSL regulates the invasive impact of oncogenic KRAS. A, Representative images of iKras cells cultured in doxycycline (KrasG12D) or following 72-hour doxycyclinewithdrawal (WT) and stained for LDs (Oil Red O). Scale bar, 10 mm. B, Quantitation of total LD area per cell described in A. More than 50 cells scored in each of fivefields for three independent biological replicates. C,Western blot analysis of HSL and actin in mKPC whole-cell lysates constitutively overexpressing HSL or vectorcontrol. D, Quantitation of total LD area per cell of mKPC cells overexpressing HSL or a vector control. More than 75 cells scored in each of five fields for threeindependent biological replicates. E, Representative immunofluorescence images from cells described in D stained for LDs (Oil Red O). Scale bar, 10 mm.F, Quantitation of iKras cells overexpressing GFP or HSL-GFP in the presence of doxycycline (G12D) or following 72-hour doxycycline withdrawal (WT) prior toseeding on aMatrigel-coated filter and allowed to invade for 16 hours. More than 40 cells scored in each of three independent biological replicates.G,Quantitation ofmKPC overexpressing HSL or vector control and loaded with 200 mmol/L BSA-conjugated OA or BSA control prior to invasion for 16 hours. More than 245 cellsscored in each of three independent biological replicates. H, Quantitation of iKras cells cultured in the presence of doxycycline (G12D) or following 72-hourdoxycycline withdrawal (WT) prior to seeding on a Matrigel-coated filter and allowed to invade. Cells attached for 2 hours prior to drug treatment and invadedfor 14 hours. Ten mmol/L Atglistatin (ATGLi) and 10 mmol/L CAY 10499 (HSL/MGLi) were used. More than 220 cells scored in each of three independentbiological replicates. I, mKPC cells transfected with siRNA targeting HSL and loaded with 200 mmol/L BSA-conjugated OA or BSA control prior to seeding ona Matrigel-coated filter and invading for 16 hours. More than 260 cells scored in each of three independent biological replicates. Graphs indicate mean � SEMand were analyzed by Student t test. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.

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HSL regulates metastasis in vivo. A, The average total number of metastases from C57BL/6 mice following pancreatic orthotopic injections with mKPC cellsconstitutively overexpressing HSL or a vector control. n ¼ 10 mice per group. B, Average weights of the primary tumors isolated from the experimentdescribed in A. C, Hematoxylin and eosin staining of primary tumor sections isolated from the experiment described in A. Scale bar, 20 mm. D, LD staining ofprimary tumor sections isolated from the experiment described in A using Oil Red O and hematoxylin as counterstain. Scale bar, 10 mm. E, Quantitation ofhistological sections stained for LDs and depicted in D. Data represent the relative change in the optical density (OD) of lipid content visualized by Oil Red Ostaining in three distinct fields from three independent biological replicates. F, IHC analysis of patient-matched human pancreatic tissue sections stained forHSL. Data represent adjacent normal, primary tumor, and metastatic tissues isolated from each patient. G, Representative IHC images of the tissuesdescribed in F. Scale bar, 20 mm. Graphs indicate mean � SEM and were analyzed by Student t test. H, Analysis of the TCGA-Pancreatic Cancer datasetcomparing LIPE (HSL) gene expression in tumors with wild-type or mutant KRAS. Mean log2 expression values are indicated by a dashed line and significancewas determined by a Mann–Whitney U test. � , P < 0.05; ��, P < 0.01; �� , P < 0.001; �� , P < 0.0001.






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ability to use stored LDs: pharmacologic inhibition or knockdown ofHSL blocks mobilization of stored lipids from LDs, whereas HSLoverexpression depletes LD content, resulting in a lack of stored lipidsfor subsequent utilization during invasion, but can be rescued byintroduction of additional lipids. This important finding furtheremphasizes that the lipolysis-based utilization of the stored lipids isnecessary for tumor cell invasion.

Previous investigations have established thatHSL is phosphorylatedand activated by protein kinase A (PKA) to promote lipolysis inadipocytes and hepatocytes (35). In support of this concept, inhibitionof PKA signaling with H89 led to an accumulation of LDs consistentwith defective lipolysis, and decreased PDAC cell invasion (Supple-mentary Figs. S3G and S3H). Although PKA is known to affect cellmigration by multiple mechanisms, these findings are consistent withthe concept that HSL expression and activity are important for tumorcell invasion.

Interestingly, although the Kras activation state profoundly impactsthe proliferation of the iKras cells (Supplementary Fig. S3I), HSLoverexpression did not significantly change the proliferative capacityof either cell model (Supplementary Figs. S3J and S3K). These obser-vations indicate that lipid storage and utilization by the lipase HSL iscritical for regulating the invasive ability of PDAC cells.

These data demonstrate the importance of HSL expression andactivity for metastatic potential in vitro. We next utilized HSL over-expression to disrupt Kras-mediated downregulation of HSL in vivo.mKPC cells constitutively overexpressing HSL (SupplementaryFig. S4A) were orthotopically injected into the pancreas of syngeneicC57BL/6 mice, and tumor progression and metastasis were comparedwith mice injected with vector control cells. Similar to our in vitroobservations (Fig. 4F and G), overexpression of HSL blunted tumorprogression and metastasis in vivo (Fig. 5A and B). HSL overexpres-sion dramatically reducedmetastasis, as the total number ofmetastaseswas reduced when compared with mice injected with control cells(Fig. 5A; Supplementary Fig. S4B). Interestingly, although HSL over-expression did not significantly alter cellular proliferation in vitro(Supplementary Fig. S3K), the primary tumor weight was reduced inmice injected with HSL overexpressing cells compared with that ofcontrol cells (Fig. 5B), suggesting additional systemic factors related totumor lipid metabolism can contribute to tumor growth. These resultsindicated that KRAS-mediated HSL regulation is crucial to maintainmetastatic ability in vivo.

Hematoxylin and eosin staining revealed that both control andHSL-overexpressing tumors showed a similarly heterogeneous duc-tal and desmoplastic phenotype (Fig. 5C). Staining for intracellularLDs revealed reduced LD content in primary tumor tissue with HSLoverexpression (Fig. 5D and E), similar to our in vitro observationsand consistent with depletion of stored lipids (Fig. 4D and E).Staining for HSL confirmed increased expression in the primary

and metastatic tissues of the mice injected with HSL overexpressingcells (Supplementary Fig. 4C). Ki67 staining indicated similarlevels of proliferation in the primary and metastatic tumors ofboth experimental groups (Supplementary Fig. S4D). Finally, stain-ing for a-smooth muscle actin revealed similar levels of activatedfibroblasts in both experimental groups (Supplementary Fig. S4E).Thus, the primary difference resulting from HSL overexpression inthe tumor cells is the depletion of stored lipids, and a reduction intumor progression and metastasis.

To better understand the regulation of HSL in vivo, we assessed HSLprotein levels inmatchedprimary tumor,normal adjacent, andmetastaticpancreatic tumor tissue from patients with human pancreatic cancer.Quantitation and imaging of these tissues revealed downregulated HSLexpression inboth theprimaryandmetastatic tissuewhencomparedwithbenign (tumor-adjacent) tissue (Fig. 5F andG). In addition, analysis ofthe TCGA-Pancreatic Cancer dataset revealed a significant inversecorrelation between expression of the HSL gene (LIPE) and KRASmutation (Fig. 5H). This is consistent with our previous observa-tions that HSL expression is downregulated in tumor cells withoncogenic KRAS (Fig. 3A, C, and E). Together, these resultsindicate that KRAS-dependent HSL expression is a crucial regulatorof lipid storage, tumor progression, and metastasis in vivo.

HSL expression shifts PDAC cells to oxidative metabolismOncogenic KRAS controls metabolic reprogramming in cancer

cells (19), including preferential utilization of glycolysis rather thanoxidative respiration (21). Because of the profound impact of HSLin vitro and in vivo, we hypothesized that KRAS-mediated HSLregulation may play an uncharacterized role in PDAC metabolism.Because KRAS can shift cells towards glycolysis, we tested if thedownregulation of HSL was a component of this metabolic shift bymeasuring the effects of HSL overexpression on both glycolysis andoxidative respiration. First, HSL overexpression in mKPC cellsdecreased glycolytic activity compared with control cells, as measuredby lactate production (Fig. 6A). These suggest that HSL downregula-tion may contribute to the increased reliance on glycolysis. We nexttested if HSL expression also impacts oxidative metabolism usingSeahorse metabolic analysis. Interestingly, HSL overexpressionresulted in increased basal and maximal respiration in both mKPCand doxycycline inducible iKras cell models (Fig. 6B; SupplementaryFig. S5A), suggesting that high HSL induces a metabolic shift towardsoxidative respiration.

To further analyze this phenomenon, we utilized the autofluores-cence of flavin adenine dinucleotide (FAD) andNADH to gain insightsinto the metabolic pathways being utilized by the cells (36). From theendogenous fluorescence of these two cofactors an optical redox ratiocan be calculated as FAD/(NADHþ FAD), with a higher optical redoxratio consistent with increased oxidative phosphorylation (37). As a

Figure 6.HSL shifts PDAC cells to oxidative metabolism. A, Analysis of glycolytic activity determined by lactate production of mKPC cells overexpressing HSL or a vectorcontrol. Data represent six independent biological replicates. B, Quantitation of the normalized OCR of mKPC cells overexpressing HSL or a vector control viaSeahorse metabolic analysis. Data represent three independent biological replicates. C, Representative composite images of the optical redox ratio of iKrascells overexpressing HSL or vector control. Cells were cultured in the presence of doxycycline (G12D) or following a 72-hour withdrawal of doxycycline (WT)prior to imaging. Scale bar, 10 mm. D, Optical redox ratio quantitation of the conditions depicted in B. More than 40 cells scored in each of five fields for threeindependent biological replicates. E, Optical redox ratio quantitation of mKPC cells overexpressing HSL or vector control. More than 40 cells scored in each offive fields for three independent biological replicates. F, Optical redox ratio quantitation of mKPC cells in a wound healing assay. Cells were treated withDMSO (control), 10 mmol/L etomoxir, or CAY 10499 (HSL/MGLi) for 4 hours prior to imaging. Cells on the edge of the wound were classified as “Motile Edge,”whereas cells at least four frames away from the edge were classified as “Non-Motile.” More than 35 cells scored in each of five fields for three independentbiological replicates shown. G, mKPC cells treated with 200 mmol/L OA, 10 mmol/L etomoxir, or grown under control conditions prior to lysis and use in anATP detection assay. H, Representation of lipid storage and catabolism during metastatic invasion. Graphs indicate mean � SEM and were analyzed byStudent t test. � , P < 0.05; ��, P < 0.01; �� , P < 0.001; �� , P < 0.0001.






proof of principle for this approach, we determined the impact ofhypoxia on PDACcells, which selectively blocks oxidativemetabolism,resulting in enhanced utilization of glycolysis (38). This block inmitochondrial oxidative capacity would result in an increase ofmitochondrial NADH due to lack of consumption by the electrontransport chain, decreasing the optical redox ratio. Indeed, weobserved a reduced optical redox ratio in hypoxic cells when comparedwith control cells (Supplementary Figs. S5B and S5C). Therefore, theoptical redox ratio is a suitable method for the examination of PDACcell metabolism.

Optical redox ratio analysis reveals that HSL overexpression inmKPC and iKras cells leads to an elevated redox ratio compared withcontrol cells (Fig. 6C–E), consistent with the Seahorse-based analysisand suggesting increased utilization of oxidative metabolism withinPDAC cells. This is consistent with the concept that elevated HSLactivity results in increased FA oxidation following lipolysis fromstored LDs. Subsequently, increased oxidative phosphorylation by theelectron transport chain results in production of FAD and consump-tion of NADH, leading to the observed elevation in the optical redoxratio. Thus, HSL overexpression shifts cells away from glycolysis andtowards oxidative metabolism.

Although metabolic analysis in populations of tumor cells high-lights global metabolic changes, our data suggest that metabolism ofstored lipids is crucially important during the acute process of tumorcell invasion. Themechanisms by which cancer cells transiently utilizemetabolic resources during invasion and metastasis remain nearlycompletely undefined. To address this, we used the optical redox ratioto determine the metabolic state of tumor cells during migration usinglive cell imaging. Interestingly, leading-edge migratory cells had asignificantly higher optical redox ratio than non-motile cellsmore thanfive cell widths within the cell mass (Fig. 6F; Supplementary Fig. S5D).Consistent with increased LD turnover in the leading-edge cells(Fig. 2A), these data indicate that tumor cells can catabolize andutilize stored lipids during the process of migration to transiently shifttheir metabolic program towards oxidative phosphorylation to fuelmetastasis.We hypothesized that lipid utilization is critical for the shifttowards oxidative metabolism in leading-edge migratory cells. To thatend, we treated cells with pharmacological inhibitors to mitochondriallipid uptake (etomoxir) and HSL/MGL lipolytic activity, and imagedtheir redox ratio during migration. Both inhibition of mitochondriallipid uptake and HSL/MGL ablated the utilization of oxidative metab-olism in leading-edge cells (Fig. 6F; Supplementary Fig. S5D), indi-cating that lipolysis and lipid utilization is a critical component of themetabolic shift in highly dynamic, migratory cells. Thus, althoughKRAS-driven cancers may downregulate FA metabolism to rely onglycolysis for steady-state proliferation, a transient switch to oxidativemetabolism occurs to support the use of stored lipids to enableinvasion and metastasis.

A metabolic advantage of increased FA oxidation and oxidativephosphorylation could be increased generation of ATP, which is inhigh demand during tumor cell migration (8, 39–41). We thereforemeasured ATP levels following HSL overexpression and in the pres-ence of excess lipids. HSL overexpression, which depletes stored lipidsand impairs invasive migration, also reduced ATP generation. Impor-tantly, ATP production in the HSL overexpressing cells was signifi-cantly increased when cells were treated with excess lipid (Fig. 6G).These findings are consistent with Fig. 4G, which showed thatproviding excess lipids replenished lipid stores and restored tumorcell invasion. Inhibition of mitochondrial lipid uptake with etomoxirdecreased ATP generation (Fig. 6G). Interestingly, ATP levels were

not significantly altered by lipid loading in control conditions. Takentogether, these results indicate that both lipids and HSL are requiredfor augmented ATP generation.

DiscussionAltered cellular metabolism is a feature of cancer cells that

enables unrestrained growth and motility. To date, the primary focusof metabolic rewiring has been on glycolysis (42), although recentstudies have also implicated the importance of amino acids andlipids (14, 43, 44). Although metabolic reprogramming in cancer hasbeen extensively studied, the majority of these investigations analyzedproliferating cells rather than metastatic cells (45, 46). The focus ofthis study was to elucidate the resources metastatic cells utilize toenable invasive migration, and the mechanisms controlling the con-sumption of those resources. In PDAC, we observe that stored lipidscan promote the prometastatic processes of migration, invasion, andmatrix degradation. LDs are catabolized during invasive migrationthrough the action of lipases, which allows mitochondrial import ofFAs and upregulation of oxidative metabolism to promote metastaticmigration (Fig. 6H). Although oxidative metabolism is upregulated inactively migrating cells, it is important to note that overexpressionof HSL did not increase tumor cell migration, even though it led toincreased steady-state oxidative metabolism. Thus, elevated oxida-tive phosphorylation is not sufficient to drive invasive migration.Rather, we propose that tumor cell migration requires both thepresence of stored LDs and a dynamic metabolic shift, allowingselective activation of oxidative phosphorylation coupled with LDbreakdown in actively migrating cells.

Activating mutations ofKRAS drive andmaintain greater than 90%of pancreatic cancers (18, 34, 47). KRAS has been thought to promote ashift to aerobic glycolysis and anabolic glucose metabolism (48).However, our understanding of KRAS-driven metabolic reprogram-ming has evolved to include alterations in scavenging pathways, aminoacidmetabolism, and lipidmetabolism (20, 33, 49). Here, we identifieda novel mechanism by which KRAS modulates lipid metabolismthrough downregulation of HSL in both cell culture and in mice,consistent with decreased expression of HSL in human pancreaticcancers. We also report that oncogenic KRAS leads to increasedprotein levels of the LD protein PLIN-2, suggesting it may drive alarger program promoting lipid storage. Oncogenic KRAS led todecreased transcription of HSL through the MAPK/ERK signalingpathway, though themechanismof this downregulation is unknown. Ithas been reported that ERK regulates lipolysis through phosphoryla-tion of HSL (50); however, determining how this regulatory pathwaycontrols HSL gene expression through transcriptional or epigeneticregulatory mechanisms requires further investigation. Importantly,this KRAS–HSL regulatory axis acts to promote lipid storage in PDACcells for subsequent utilization during invasive migration. Re-expression of HSL depletes stored lipids, shifts cells to upregulatedoxidative metabolism and decreased glycolysis, and reverses theinvasive effects of oncogenic KRAS. It is likely that this shift isparticularly important during the process of cell migration, whenpancreatic tumor cells utilize LD turnover and increased oxidativemetabolism. Understanding the metabolic requirements of metastaticcells remains extremely challenging due to the transient nature ofinvasive migration. Therefore, the optical redox ratio emerged as apowerful tool to analyze the relative metabolic pathways utilized bymigratory and static cells. HSL, then, is a novel component of KRAS-mediated metabolic rewiring in pancreatic cancer.

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Interestingly, genetic knockout of HSL was recently reported toincrease pancreatic cancer incidence inmice (51). AsKC;HSL�/�miceexhibited enhanced inflammation in the adipose tissue and pancreas,the prior study focused on the importance of HSL in the context ofadipose tissue, whereas we investigated the tumor-cell specific effectsofHSL. Interestingly, the authors reported enhanced survival in PDACpatients with higher expression of HSL based on transcript data frompancreatic tumor tissue. Although this approach does not distinguishtumor cells from stromal adipocytes or islets, they are consistent withour findings that overexpression of HSL in pancreatic tumor cellsdecreased tumor burden and metastasis. Thus, both studies implicateHSL as an important regulator of pancreatic cancer progression,perhaps through multiple mechanisms.

Pancreatic cancer cells could access lipids from multiple sources.Obesity elevates the levels of available FAs, is commonly associatedwith pancreatic cancer, and worsens prognosis of patients and elevatesmetastatic burden (9, 13, 29). Further, mouse models of PDAC hadearlier onset and more invasive pancreatic cancer when given anobesity-inducing diet (11, 12, 52, 53). Cachexia is an additionalcomorbidity often present in advanced pancreatic cancer patients.This wasting condition promotes adipose breakdown, leading toelevated levels of FAs available for utilizationby the tumor cells (15, 54).The tumor microenvironment may also provide lipids for use bypancreatic cancer cells. Other cell types, such as adipocytes, cancer-associated fibroblasts, and activated stellate cells, may be in closeproximity to tumor cells and facilitate tumor progression throughelevated lipid availability (55, 56). In these contexts, lipid uptake intoPDAC cells would be required for excess lipids to impact metastasis,although the specific lipid transporters and mechanisms of uptake arestill under investigation. Previous investigations in breast cancer havedemonstrated that uptake of exogenous lipids promoted aggressivecellular phenotypes similar to our observations in pancreatic cancercells (57). Therefore, import of FAs into PDAC cells represents acritical aspect upstream of our observations. Finally, PDAC cells haveelevated levels of lipid synthesis (58), and can accumulate storedlipids in response to reactive oxygen species or hypoxia (59). Eachof these factors may impact the availability of lipids to PDAC cells.Regardless of the source or uptake of the excess lipids, the datapresented here support the hypothesis that lipid storage and utilizationis a key mechanism that facilitates invasive migration of pancreaticcancer cells.

Our data indicate that coordinated breakdown of LDs is impor-tant for tumor cell migration and invasion. In other tissues,stimulation of lipolysis occurs following activation of PKA (35).Indeed, we observed changes in stored LDs and invasive migrationfollowing inhibition of PKA signaling, implicating the importanceof PKA as an upstream signaling mechanism for the stimulation oflipid utilization and invasion. Further, the changes we observed inPLIN-2 levels following KRAS activation may also impact lipidutilization. While the specific stimulus to activate lipase-mediatedbreakdown during migration is unknown, our data support thatstored lipids may be used for energy production to promote invasivemigration, as inhibition of lipid transport into the mitochondria

hinders migration. This is consistent with prior studies demon-strating increased oxidative phosphorylation and ATP demand inmigratory cells (6–8) and that augmented ATP generation is criticalto several aspects of invasive migration (8, 40, 41), although thesource of this ATP is still not well defined. Interestingly, our dataindicate that excess HSL levels and lipid are both required foraugmented ATP production. This does not exclude the possibilitythat lipids may also be used for membrane dynamics and repair, oras signaling molecules (60). In addition, KRAS activation, lipidmetabolism, and mitochondrial respiration can all impact the levelsof reactive oxygen species (61–63), which may contribute to tumorprogression metastasis (64). Although we observed lipolysis andHSL regulation to be critical to metastatic potential, other lipasesand mechanisms of lipid utilization may also prove critical tometastasis. ATGL is thought to be the rate-limiting lipolyticenzyme (17), and MGL has been previously implicated in tumoraggressiveness (65). Indeed, pharmacologic inhibition of all lipolyticenzymes perturbed tumor cell invasion, however the regulation ofHSL by the oncogene KRAS may indicate its specific importance inthe context of pancreatic cancer metastasis.

Taken together, the integrity ofKRAS-mediatedHSL regulation andthe subsequent modulation of lipid storage, utilization, and metabo-lism reveal novel targets for suppression of metastasis and increasedpatient survival.

Disclosure of Potential Conflicts of InterestG.L. Razidlo reports grants from NCI, Hirshberg Foundation for Pancreatic

Cancer Research, and NIH during the conduct of the study. No potential conflictsof interest were disclosed by the other authors.

Authors’ ContributionsC.N. Rozeveld: Conceptualization, formal analysis, investigation, methodology,

and writing-original draft. K.M. Johnson: Formal analysis, investigation, writing-review and editing. L. Zhang: Pathology review. G.L. Razidlo: Conceptualization,supervision, funding acquisition, writing-review and editing.

AcknowledgmentsThis research was supported by the Mayo Clinic SPORE in Pancreatic Cancer

(P50 CA102701), the Mayo Clinic Center for Cell Signaling in Gastroenterology(P30 DK084567), the Mayo Clinic Cancer Center (P30 CA015083), the NCI (R01CA104125), the Hirshberg Foundation for Pancreatic Cancer Research, the MayoClinic Center for Biomedical Discovery, and the Mayo Clinic Graduate School ofBiomedical Sciences (to C.N. Rozeveld). We acknowledge Dr. Marina Pasca diMagliano for the inducible KRAS cell line (iKRAS), Dr. David Tuveson for themouse KPC cell line (mKPC), and Dr. Kun Ling, Dr. Gregory Gores, Dr. EugeniaTrushina, Dr. Jason Doles, and Camden Daby for reagents, equipment, andtechnical support. We thank Mark McNiven, all members of the McNiven lab, andO. Kuiper for technical support, helpful insight, and fortitude.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 19, 2020; revised July 16, 2020; accepted August 14, 2020;published first August 19, 2020.

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2020;80:4932-4945. Published OnlineFirst August 19, 2020.Cancer Res Cody N. Rozeveld, Katherine M. Johnson, Lizhi Zhang, et al. Invasive Potential through the Lipase HSLKRAS Controls Pancreatic Cancer Cell Lipid Metabolism and

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