LIMS1 Promotes Pancreatic Cancer Cell Survival Under Oxygen

Published OnlineFirst January 24, 2019; DOI: 10.1158/1078-0432.CCR-18-3533

Translational Cancer Mechanisms and Therapy Clinical Cancer Research LIMS1 Promotes Pancreatic Cancer Cell Survival under Oxygen–Glucose Deprivation Conditions by Enhancing HIF1A Protein Translation Chongbiao Huang1,2, Yang Li1, Zengxun Li1, Yang Xu1,NaLi1,YiGe1, Jie Dong1, Antao Chang2, Tiansuo Zhao1, Xiuchao Wang1, Hongwei Wang1, Shengyu Yang3, Keping Xie4, Jihui Hao1, and He Ren1

Abstract

Purpose: Oxygen and glucose deprivation is a common sion was pivotal for tumor cells to survive in the oxygen– feature of the solid tumor. Regulatory network underlying the glucose deprivation conditions. Mechanistically, LIMS1 adaptation of cancer cells to the harsh microenvironment enhanced GLUT1 expression and membrane translocation, remains unclear. We determined the mechanistic role of LIM which facilitated tumor cell adaptation to the glucose dep- and senescent cell antigen-like–containing domain protein 1 rivation stress. Furthermore, LIMS1 promoted HIF1A pro- (LIMS1) in cancer cell survival under oxygen–glucose depri- tein translation by activating AKT/mTOR signaling, while vation conditions. hypoxia-inducible factor 1 (HIF1) transactivated LIMS1 Experimental Design: The expression level of LIMS1 was transcription, thus forming a positive feedback loop in determined by IHC staining and analyzing the mRNA expres- PDAC cell adaptation to oxygen deprivation stress. Inhibi- sion proﬁles from The Cancer Genome Atlas of three human tion of LIMS1 with jetPEI nanocarrier–delivered anti-LIMS1 solid tumors. Roles of LIMS1 in cancer cell metabolism and siRNAs signiﬁcantly increased cell death and suppressed growth were determined by molecular and cell biology meth- tumor growth. ods. A jetPEI nanocarrier was used as the vehicle for anti-LIMS1 Conclusions: LIMS1 promotes pancreatic cancer cell sur- siRNAs in mouse models of cancer therapeutics. vival under oxygen–glucose deprivation conditions by acti- Results: LIMS1 expression was drastically elevated in vating AKT/mTOR signaling and enhancing HIF1A protein pancreatic ductal adenocarcinoma (PDAC). High LIMS1 translation. LIMS1 is crucial for tumor adaptation to oxygen– level was associated with advanced TNM stage and poor glucose deprivation conditions and is a promising therapeutic prognosis of patients with tumor. Increased LIMS1 expres- target for cancer treatment.

Introduction causes major metabolic discrepancies between the microenviron- ments of solid tumor tissues and normal tissues. In normal tissues, In solid tumors, the poorly formed tumor vasculature leads to aerobic oxidation generates approximately 90% of the cell's energy, diffusion-limited hypoxia, accumulation of waste products, and whereas in tumor tissues, over 50% of the cellular ATP is generated lack of nutrients, including glucose (1). The oxygen shortage by anaerobic glycolysis (Warburg effect; ref. 2). Because of increased glucose consumption and limited supplies, the glucose concentra- 1Department of Pancreatic Cancer, Tianjin Medical University Cancer Institute tions in the tumor microenvironment are frequently 3- to 10-fold and Hospital, National Clinical Research Center for Cancer, Key Laboratory of lower than that in nontumor tissues (3). Hypoxia-inducible factor 1 Cancer Prevention and Therapy, Tianjin, China. 2School of Medicine, Nankai (HIF1) is the master regulator of tumor cell adaptation to the University, Tianjin, China. 3Department of Cellular and Molecular Physiology, oxygen–glucose deprivation microenvironment. HIF1 levels in 4 Penn State College of Medicine, Hershey, Pennsylvania. Departments of Inter- tumor tissues are elevated in response to hypoxia; activation of disciplinary Oncology and Internal Medicine, The University of Arizona College of Medicine, Phoenix, Arizona. some oncogenes, such as Ras and PI3K/Akt; and inactivation of tumor suppressors, such as VHL or PTEN (4). Elevated HIF1 levels, Note: Supplementary data for this article are available at Clinical Cancer in turn, enhance glucose influx by activating genes involved in Research Online (http://clincancerres.aacrjournals.org/). glucose uptake (such as GLUT1; ref. 5) and glycolysis (such as PFK1; C. Huang, Y. Li, and Z. Li are co-first authors of this article. ref. 6) and inhibit oxidative phosphorylation by transactivating Corresponding Authors: Keping Xie, University of Arizona College of Medicine genes such as PDK1 (7). Thus, HIF1 plays crucial roles in tumor at Phoenix, 550 E Van Buren Street, Phoenix, Arizona 85004. Phone: 832-398- glucose metabolism, facilitating cancer cell survival, and tumor 3103; E-mail: [email protected]; Jihui Hao, Department of Pancreatic growth in the oxygen–glucose deprivation microenvironment. Cancer, Tianjin Medical University Cancer Institute and Hospital, Hexi LIMS1 (also known as PINCH1) was originally identified as a District, Huanhuxi Road, Tianjin 300060, China. Phone/Fax: 8622-2334-0123; E-mail: [email protected]; and He Ren, [email protected] marker for senescent erythrocytes and is widely expressed in mammalian cells (8). As an adaptor protein that consists of five Clin Cancer Res 2019;25:4091–103 LIM domains and tandem nuclear localization signals (9), LIMS1 doi: 10.1158/1078-0432.CCR-18-3533 binds to integrin-linked kinase (ILK) and parvin to form a ternary 2019 American Association for Cancer Research. protein complex (10), which is essential for the control of cell–

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provided written consent for the use of their specimens and Translational Relevance disease information for future investigations according to the Pancreatic ductal adenocarcinoma (PDAC) is a deadly dis- ethics committee and in accordance with recognized ethical ease. The mechanisms underlying these aggressive behaviors guidelines of Declaration of Helsinki. are poorly understood. In this study, we demonstrated that LIMS1 was highly expressed in tumor tissues, and LIMS1 IHC and immunofluorescence overexpression was correlated with advanced TNM staging IHC was used to detect LIMS1, HIF1, pAKT (S473), and pAKT and poor survival of patients with cancer. We further identified (T308) in tumor tissues. Briefly, paraffin-embedded sections of a novel mechanistic role of LIMS1 in promoting tumor cell tumor tissues were deparaffinized and then heated in a pressure survival in the oxygen–glucose-deprived microenvironment pot for 3 minutes to retrieve the antigens. Then, the sections by activating AKT/mTOR signaling and enhancing HIF1A were incubated with primary antibodies (Supplementary Table protein translation. Our findings suggested that LIMS1 signal- S6) overnight at 4C. Antibody binding was detected using a ing promoted PDAC cell survival under oxygen–glucose dep- peroxidase-conjugated secondary antibody at 37C for 30 min- rivation conditions. Targeting LIMS1 signaling may be an utes. A DAB substrate kit was used to perform the chromogenic effective therapeutic strategy for PDAC. Therefore, our study reaction. The intensity of the staining was evaluated using the may have a significant effect on clinical management of PDAC. following criteria: 0, negative; 1, low; 2, medium; and 3, high. The extent of staining was scored as 0, 0% stained; 1, 1%–25% stained; 2, 26%–50% stained; and 3, 51%–100% stained. Five random fields (20 magnification) were evaluated under a extracellular matrix adhesion-mediated cell behavior (11). In light microscope. The final scores were calculated by multiply- addition, LIMS1 also binds to Nck2 (12), Ras1 (13), and thymo- ing the scores of the intensity with those of the extent and sin b4 (14). In response to transmembrane integrin and growth dividing the samples into four grades: 0, negative (); 1–2, low factor receptors, the LIMS1/ILK/parvin complex regulates cell staining (þ); 3–5, medium staining (þþ); and 6–9, high survival via the PI3K/PKB/Akt1 and Ras/MAPK signaling path- staining (þþþ; ref. 17). ways (13). LIMS1 was also shown to directly inhibit phosphatase Immunofluorescence staining was performed on the PDAC cell 1a (PP1a), an Akt1-regulating protein, to activate Akt1 lines. Briefly, PDAC cells seeded on coverslips were cultured with phosphorylation (15). anti-LIMS1 antibody at 4C overnight. Then, the cells were incu- Although LIMS1 was reported to be overexpressed in some bated with fluorescent dye–labeled secondary antibodies at room types of tumors (16), the roles of LIMS1 in tumors are not fully temperature for 1 hour. The cells were again incubated with anti- understood. In this study, we demonstrated that LIMS1 was highly fade DAPI solution (1:1,000) and images were captured with a expressed in tumor tissues, and LIMS1 overexpression was cor- confocal fluorescence microscope. related with advanced TNM staging and poor survival of patients with cancer. We further identified a novel role of LIMS1 in promoting tumor cell survival in the oxygen–glucose-deprived Tumor model microenvironment. All animal studies were approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) and conducted by skilled experimenters under an Materials and Methods approved protocol in accordance with the principles and proce- Cell culture and human sample collection dures outlined in the NIH Guide for the Care and Use of Labo- The human tumor cell lines CFPAC-1, BxPC-3, PANC-1, ratory Animals. Four-week-old female BALB/C nude mice were KYSE40, EC109, A549, and PC9 were obtained from the Type maintained in a barrier facility on high-efficiency particulate air– Culture Collection Committee of the Chinese Academy of filtered racks. Tumor cells were harvested by trypsinization, Sciences (Shanghai, China), and the MIA-PaCa-2 cell line was washed in PBS, and resuspended at 1 107 cells/mL in Matrigel. obtained from the ATCC in 2013. The cell lines were authenti- A total of 1 106 cells were subcutaneously or orthotopically cated through the short tandem repeat analysis method and injected into each mouse to develop tumors as described previ- Mycoplasma contamination was excluded in these cell lines at the ously (17, 18). Tumor size was measured weekly. beginning of this study. These cells were cultured in DMEM, RPMI1640, or IMDM basic medium supplemented with 10% In vivo experiments FBS at 37C in a humidified atmosphere of 95% air and 5% CO . 2 jetPEI-si scramble (40 mg), jetPEI-si LIMS1 (40 mg), or 5% A total of 109 sequential pancreatic ductal adenocarcinoma glucose control (200 mL) was intravenously injected into the (PDAC) tissues, 124 sequential esophageal carcinoma tissues, and corresponding mouse group every week from the 14th day. Six 113 sequential lung adenocarcinoma tissues were collected from weeks later, the tumors were harvested. Each group had 6 mice. patients who had received radical surgery at the Tianjin Medical University Cancer Institute and Hospital (Tianjin, China). Retro- spective clinicopathologic data of these patients, including age, The Cancer Genome Atlas data analysis sex, tumor size, regional lymph node status, TNM stage, patho- The Cancer Genome Atlas (TCGA) data of 179 patients with logic type, differentiation, and PET/CT scanning data were also pancreatic carcinoma, 184 patients with esophageal carcinoma, obtained. and 522 patients with lung adenocarcinoma were downloaded The usage of these specimens and the patient information were from TCGA website (http://cancergenome.nih.gov/). The normal- approved by the Ethics Committee of the Tianjin Medical Uni- ized RNA levels of LIMS1 and HIF1A, the clinicopathologic versity Cancer Institute and Hospital (Tianjin, China). All patients parameters, and the follow-up data were extracted and analyzed.

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Reverse transcription PCR Glucose uptake assays in vitro and in vivo The total RNA of the cells was extracted with TRizol (Invitrogen) In vitro. A glucose uptake assay kit was used to detect the uptake according to the manufacturer's instructions. Then, the mRNA was of 2-DG in the indicated tumor cells in vitro following the reverse transcribed to single-stranded cDNAs using a Reverse manual instructions (Abcam). In brief, the indicated tumor Transcription PCR (RT-PCR) System (Takara Bio Inc.). The pri- cells were starved in serum-free culture medium overnight mers are listed in Supplementary Table S7. Then, real-time fluo- followed by 40 minutes of incubation in Krebs–Ringer– rescent quantitative PCR or semiquantitative PCR was used to phosphate–HEPES buffer. Subsequently, cells were incubated analyze the cDNA levels. The products of semiquantitative PCR with 10 mmol/L 2-DG for 20 minutes. Cells were lysed by freezing were detected by agarose gel electrophoresis, and bactin was used and thawing procedures. The lysates were neutralized and then as a loading control. diluted with assay buffer. The colorimetric product generation was detected at 412 nm by a microplate reader (Bio-Rad). Western blotting In vivo. The indicated tumor cells were orthotopically trans- Whole-cell extracts were prepared by lysing the cells with RIPA planted into the pancreases of nude mice to develop tumors lysis buffer supplemented with a proteinase inhibitor cocktail (n ¼ 6 for each group). Four weeks later, the mice were intrave- (Sigma). A membrane and cytosol protein extraction kit was used nously injected with 18FDG (200 mCi in 0.2 mL), anesthetized, to extract membrane protein (Pierce). A total of 20-mg protein and then subjected to PET-CT scanning for 18FDG uptake in vivo. lysate was separated by SDS-PAGE, and then, the target proteins The concentrations of 18FDG uptake in tumor tissues were were detected by Western blotting with the antibodies to LIMS1, normalized to %ID/g. HIF1A, pAKT1 (S473), pAKT1 (T308), panAKT1, p-mTOR (s2481), pan-mTOR, p-4EBP1, pan-4EBP1, VEGFA, GLUT1, þ þ GLUT3, CA9, ETS1, Na /K -ATPase, and bactin (Supplementary In vitro survival assays under oxygen–glucose deprivation stress Table S6). Tumor cells were cultured in the oxygen deprivation conditions (oxygen concentration, 1%) and/or glucose deprivation conditions (glucose concentration, 0.75 mmol/L) as described previ- Plasmid construction and stable cell line establishment ously (3). The incubation time varied from 24 to 72 hours. The cell The complete coding sequence of the human LIMS1 gene morphology was observed by an inverted phase contrast micro- (NM_004987.5) was cloned into pLV-EF1-MCS-IRES-Bsd vec- scope. The cell survival was measured by CCK8 assays and flow tors (Biosettia). Lentiviruses were produced in 293T cells for the cytometry analysis. stable transfection of the cell lines, per the manufacturer's instructions, and an empty vector was transfected into cells to be used as a control. A total of 1 105 tumor cells in 2-mL Polysome profiling analysis medium with 8 mg/mL polybrene were infected with 1-mL Polysome RNA preparations and analysis were carried out as lentivirus supernatant. After 48 hours, blasticidin (InvivoGen) described previously (20). Briefly, cells were seeded in 10-cm was added for selection. dishes, washed with cold PBS containing 100 mg/mL cyclohexi- For the cell lines with stable knockdown, shRNA sequences mide, and then lysed in a hypotonic lysis buffer [5 mmol/L were designed with Biosettia's shRNA designer (http://biosettia. Tris-HCl (pH 7.5), 2.5 mmol/L MgCl2, 1.5 mmol/L KCl, 100 com/support/shrna-designer). Three recommended sequences mg/mL cycloheximide, 2 mmol/L DTT, 0.5% Triton X-100, and for each of the LIMS1 and HIF1A genes were synthesized and 0.5% sodium deoxycholate]. A lysate sample was used to isolate cloned into the pLV-hU6-EF1a-puro or pLV-mU6-EF1a-puro the cytoplasmic RNA using TRizol (Invitrogen). Lysates were vectors (Biosettia). Then, the lentiviruses were produced in loaded onto 10%–50% (w/v) sucrose density gradients [20 293T cells. Scrambled sequences were transfected into the cells mmol/L HEPES-KOH (pH 7.6), 100 mmol/L KCl, and 5 to be used as controls. Of the three stable cell lines, the most mmol/L MgCl2] and centrifuged at 36,000 rpm for 2 hours at efficient one was used for the relevant assays. 4C. Gradients were fractionated, and the optical density at 254 nm was continuously detected by an UV detector and fraction collector (Teledyne ISCO). RNA from each fraction was isolated Chromatin immunoprecipitation and luciferase analysis using TRizol (Invitrogen). Fractions with mRNA associated with Chromatin immunoprecipitation (ChIP) assays were per- polysomes were pooled (polysomal mRNA) for polysomal formed using a CHIP Kit (Millipore), according to the manufac- mRNA analysis of specific genes. turer's instructions. Briefly, PANC-1 cells were transiently transfected with or without pcDNA-HIF1A and then immunoprecipitated with anti-HIF1A antibody. The immunoprecipitated pro- Statistical analysis ducts were detected by RT-PCR assays. Statistical analyses were performed with the IBM SPSS Sta- Luciferase analysis was performed as described previously tistics Program. Each experiment was performed in triplicate, with minor changes (19). PANC-1 cells transfected with and the values are presented as the mean SD, unless other- pcDNA-HIF1A or control vector (pcDNA-vector) were trans- wise stated. The variance between the groups was statistically fected with pGL3-LIMS1-promoter, pGL3-LIMS1-promoter compared. Student t testwasusedtocomparethemeanvalues. mutation (MUT), or pGL3-empty vectors (pGL3.1 EV). Forty- Kaplan–Meier curves were analyzed for relevant variables. The eight hours later, cells were subjected to dual luciferase analysis. log-rank test was used to analyze the differences in survival The results are expressed as a fold induction relative to the cells times among the patient subgroups. All probability values had transfected with the control vector (pcDNA3.1) after normal- a statistical power level of 90%, and a two-sided level of 5%. ization to Renilla activity. P < 0.05 was significant.

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LIMS1 facilitates cancer cell survival in oxygen–glucose Results deprivation conditions LIMS1 is overexpressed in human cancer tissues To explore the roles of LIMS1 in tumor progression, we estab- To investigate the expression pattern of LIMS1 in cancers, we lished stable cell lines in which LIMS1 was overexpressed or performed LIMS1 IHC staining of PDAC tumor tissues and the knocked down (Fig. 2A). We subcutaneously transplanted PDAC paired adjacent nontumor tissues (pancreatic tissues 2–3cm cells with LIMS1 overexpression or knockdown into nude mice to around the tumor border). As shown in Fig. 1A, LIMS1 was generate tumors. The harvested tumors were assessed by an in situ overexpressed in tumor tissues and rarely expressed in the cell death detection kit to determine the cell mortality rate. As adjacent nontumor tissues. When comparing LIMS1 staining shown in Fig. 2B, the cell mortality rate of MIA PaCa-2 tissues with in 83 pairs of PDAC tumor tissues and the adjacent nontumor LIMS1 knockdown was strongly elevated (8.33 1.81 vs. 23.01 tissues, we discovered that LIMS1 protein was detected in the 6.14; P < 0.0001, unpaired t test). In contrast, LIMS1 overexpres- majority of PDAC tissues but not in the adjacent nontumor sion significantly reduced the mortality rate of CFPAC-1 tumors tissues (Z ¼6.058, P < 0.0001, Wilcoxon signed-rank (7.28 1.44 vs. 3.16 0.95; P ¼ 0.0001, unpaired t test). test; Fig. 1B). Furthermore, LIMS1 knockdown resulted in reduced tumor Subsequently, LIMS1 expression in eight pairs of fresh PDAC growth, whereas LIMS1 overexpression significantly enhanced tumor tissues and the adjacent nontumor tissues was analyzed the tumor growth (Fig. 2C). using qPCR (Fig. 1C) and Western blotting. Our data indicated LIMS1 was previously shown to contribute to apoptosis resis- that LIMS1 was abnormally overexpressed in PDAC tumor tance in cancer cells (21, 22). However, surprisingly, under tissues (P ¼ 0.019, paired t test; Fig. 1D). LIMS1 overexpression normal cell culture conditions, LIMS1 expression led to negligible wasalsodetectedinfive PDAC cell lines (Supplementary Fig. effect on cell viability in cancer cells (Fig. 2D; Supplementary Fig. S1). S4). Oxygen–glucose deprivation is a common characteristic of To elucidate the clinical significance of LIMS1 in PDAC, we used solid tumors (2). Therefore, we examined the effects of LIMS1 on IHC to determine the expression of LIMS1 in 109 human PDAC PDAC cell survival under oxygen–glucose deprivation. We used a specimens (Fig. 1E). As shown in Supplementary Table S1, high continuous flow culture system to maintain cancer cells in the expression of LIMS1 was strongly correlated with advanced tumor proliferative phase in a reduced but steady glucose concentration 2 2 size (P ¼ 0.021, x tests), T stage (P ¼ 0.036, x tests), regional (0.75 mmol/L) in a hypoxic incubator (1% oxygen) to mimic 2 lymph node involvement (P ¼ 0.019, x tests), pTNM staging (P ¼ oxygen–glucose deprivation conditions (ref. 3; Fig. 2E). As shown 2 2 0.017, x tests), and blood vessel infiltration (P ¼ 0.046, x tests). in Fig. 2F–H, LIMS1 overexpression strongly enhanced the sur- The overall survival times of patients with PDAC with low LIMS1 vival of BxPC-3 and CFPAC-1 cells in oxygen–glucose deprivation expression were significantly longer than those with high LIMS1 environments (unpaired t tests). In contrast, LIMS1 knockdown level (P ¼ 0.022, log-rank test; Fig. 1F). significantly reduced the survival of MIA PaCa-2 and PANC-1 cells To further investigate the clinical significance of LIMS1 in PDAC (unpaired t tests). Similar results were observed in esophageal progression, we analyzed the mRNA sequencing profiles and cancer cells (Supplementary Fig. S5) and lung adenocarcinoma clinical data of 179 patients with PDAC from TCGA. As shown cells (Supplementary Fig. S6), indicating a critical role of LIMS1 in in Supplementary Table S2, a high mRNA level of LIMS1 was tumor cell adaptation to oxygen–glucose deprivation. significantly correlated with advanced tumor size (P ¼ 0.044, unpaired t tests), T staging (P ¼ 0.024, unpaired t tests), regional LIMS1 facilitates the response to hypoxic stress by upregulating lymph node involvement (P ¼ 0.028, unpaired t tests), differen- HIF1A translation tiation (P ¼ 0.012, unpaired t tests), and pTNM staging (P ¼ HIF1 is a master regulator of tumor cell adaptation to oxygen– 0.005, unpaired t tests). A cohort of 130 patients with PDAC with glucose deprivation. We found that LIMS1 knockdown substan- a minimum follow-up time of 60 days was further studied. The tially inhibited the HIF1A protein level, whereas LIMS1 over- overall survival times of patients with PDAC with low LIMS1 expression significantly elevated the HIF1A protein level (Fig. 3A). mRNA levels (475.5 reads per kilobase per million mapped Upregulation of HIF1A protein in tumor tissues could be achieved reads, RPKM) were significantly longer than those with high by promoting transcription, inhibiting proteasomal degradation, LIMS1 mRNA levels (>475.5 RPKM; P ¼ 0.001, log-rank or enhancing mRNA translation (23). The upregulation or knock- test; Fig. 1G). Importantly, a multivariate Cox regression analysis down of LIMS1 did not change the mRNA level of HIF1A (Fig. 3B; revealed that the LIMS1 expression level was an independent unpaired t tests), indicating that LIMS1 did not enhance HIF1A prognostic factor for the overall survival (HR, 1.682; 95% con- levels by facilitating the transcriptional activity. To determine the fidence interval, 1.097–2.578; P ¼ 0.0170) of patients with PDAC effects of LIMS1 on HIF1A proteasomal degradation, we detected (Supplementary Table S3) the HIF1A protein level in the indicated cells cultured in hypoxic The expression and clinical significance of LIMS1 were also conditions. As shown in Fig. 3C, under hypoxic conditions, examined in two other cancers. Our analysis of esophageal overexpression of LIMS1 still significantly enhanced the protein carcinoma and lung adenocarcinoma data revealed that LIMS1 level of HIF1A. The proteasome inhibitor MG132 was used to was also frequently overexpressed in esophageal carcinoma (Sup- prevent HIF1A degradation and LIMS1 still increased HIF1A plementary Fig. S2A–S2B) and lung adenocarcinoma tissues expression (Fig. 3D). Furthermore, the results of pulse chase (Supplementary Fig. S3A–S3B). High LIMS1 expression was experiments demonstrated that LIMS1 expression did not alter significantly correlated with poor overall survival of patients the half-life of HIF1A (Supplementary Fig. S7). These results (Supplementary Figs. S2C–S2D and S3C–S3D), advanced tumor indicated that the regulation of HIF1A by LIMS1 was not through size, T staging, regional lymph node involvement, and pTNM inhibiting proteasomal degradation. staging both in esophageal carcinoma (Supplementary Table S4) To determine whether LIMS1 regulates HIF1A through mRNA and lung adenocarcinoma (Supplementary Table S5). translation, we next examined the impact of LIMS1 expression on

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Figure 1. The expression and clinical significance of LIMS1 in PDAC tissues. Sections of PDAC tissues were used to analyze the expression levels of LIMS1 in tumor tissues (Ca) and the corresponding adjacent nontumor (NT) tissues. Representative images are shown for absent, weak, moderate, and strong expression of LIMS1 in IHC staining of PDAC tumor and NT tissues (A). The differential expression of LIMS1 in 83 pairs of tumor and NT tissues is shown in a heatmap and was statistically analyzed by Wilcoxon signed rank tests (B). Eight pairs of fresh PDAC tumor tissues and NT tissues were subjected to RT-PCR analysis (C) and Western blotting (D) to compare the expression levels of LIMS1. E, The distribution of IHC results in 109 PDAC tissues. F, Kaplan–Meier analysis of overall survival of the 109 patients with PDAC according to different LIMS1 levels. G, mRNA profiles and follow-up data of 130 patients with PDAC from TCGA analyzed for the correlation of the LIMS1 mRNA expression and survival (the minimum follow-up time is 60 days; the cut-off line for the LIMS1-low and LIMS1-high groups is 475.5 RPKM). Paired t tests were used in D; log-rank tests were used in E and G. Tests of significance are two-sided; , P < 0.05; scale bars, 100 mm.

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Figure 2. LIMS1 facilitates PDAC survival in the oxygen–glucose deprivation microenvironment. A, The indicated PDAC cells were transfected with lentiviruses to upregulate or knockdown LIMS1 expression. Western blotting was performed to verify the stable cell lines. The indicated tumor cells were subcutaneously transplanted into nude mice to develop tumors (n ¼ 6–7 for each group). The harvested tumors were sliced and then detected by an in situ cell death detection kit to determine the cell mortality rate (B). Tumor size was measured weekly, and the tumor growth curve was generated based on the mean tumor volume (C). D, The indicated cells cultured in whole medium were stained with 7-AAD/Annexin-V and then subjected to ﬂow cytometry analysis to determine the cell viability. The indicated cells were cultured in the oxygen–glucose deprivation conditions (glucose concentration, 0.75 mmol/L and oxygen concentration, 1%). The incubation time varied from 24 hours to 72 hours. Schematic of the culture conditions (E); morphology of the cells was observed by an inverted phase contrast microscope (F); CCK8 assays (G); and ﬂow cytometry analysis (H) were used to detect the cell viability. Unpaired t tests were used in B, C, D, G,andH; data are shown as the mean value SD; , P < 0.05; scale bars, 100 mm.

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overall mRNA translation by studying polysome formation. The LIMS1 led to an increase in the polysome/monosome ratio number of ribosomes engaged in polysomes is directly propor- compared with the control treatment (Fig. 3E and F). The poly- tional to the translation initiation speed (21). Overexpression of somal mRNA levels of HIF1A were signiﬁcantly elevated in

Figure 3. LIMS1 facilitates the response to hypoxic stress by upregulating HIF1A translation. The indicated PDAC cells with LIMS1 upregulation or downregulation were subjected to Western blotting (A) and RT-PCR (B) to detect the expression level of HIF1A. C, The indicated PDAC cells were incubated in hypoxic conditions (1%

O2) for 48 hours and then subjected to Western blotting to detect the level of HIF1A. D, The indicated PDAC cells were incubated with or without 10 nmol/L MG132 for 24 hours and then subjected to Western blotting to detect the level of HIF1A. Polysome proﬁles from the indicated cells. Absorbance at 254 nm is shown as a function of sedimentation (E). The AUC for polysomes and the 80S peak were calculated, and the ratio is shown (F). The fractions of polysomes were mixed together, and the RNA of the mixture was isolated and subjected to real-time PCR assays to determine the polysomal mRNA level of HIF1A (G). H, The indicated PDAC cells were incubated with or without 10 nmol/L MK2206/0.1 nmol/L rapamycin and then subjected to Western blotting to detect the expression level of p-AKT1, p-mTOR (S2481), p-4EBP1, and HIF1A. The incubation times are as follows: MK2206, 24 hours; rapamycin, 30 minutes for detection of p-4EBP1, and 24 hours for detection of HIF1A. I, The indicated cells were subjected to Western blotting to detect the expression levels of three markers of HIF1 signaling: VEGFA, CA9, and ETS1. J–K, The indicated tumor cells were incubated in hypoxic conditions (oxygen concentration, 0.5%) for 48 hours and then subjected to ﬂow cytometry analysis to detect cell viability. Unpaired t tests were used in B; data are shown as the mean value SD; , P < 0.05.

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LIMS1-overexpresing cells (Fig. 3G), suggesting that the transla- PDAC cells and tissues. Consequently, we explored whether tional activity of HIF1A was enhanced by LIMS1. LIMS1 promotes HIF1A translation by activating AKT signaling. LIMS1 was previously shown to activate the PI3K/PKB/Akt1 In Fig. 3H, the data showed that LIMS1 overexpression enhanced signaling pathway (13), which plays essential roles in HIF1A the phosphorylation of AKT1, mTOR, and 4EBP1. The AKT1 translation (24). In Supplementary Fig. S8, we conﬁrmed that inhibitor MK2206 completely abrogated the LIMS1-induced LIMS1 was crucial in maintaining AKT signaling activation in phosphorylation of AKT1, mTOR, and 4EBP1, as well as

Figure 4. LIMS1 promotes glucose uptake by enhancing GLUT1 expression and membrane translocation. A, A glucose uptake assay kit was used to detect the uptake of 2-DG (structurally similar to glucose) in the indicated tumor cells in vitro. B, The indicated tumor cells were orthotopically transplanted into the nude mouse pancreases to develop tumors (n ¼ 6 for each group). Four weeks later, the mice were intravenously injected with 18FDG, anesthetized, and then subjected to PET-CT scanning for 18FDG uptake in vivo. The representative images of PET-CT scans of each group were shown (left top). Then the abdomen of mice was opened to harvest the tumors. Representative images of tumors in situ were shown (left bottom). The concentrations of 18FDG in tumor tissues were normalized to %ID/g (right bottom). C, The indicated tumor cells were subjected to Western blotting to detect the expression levels of GLUT1, GLUT3, mGLUT1, and mGLUT3; Naþ/Kþ-ATPase was used as a loading control for membrane protein; bactin was used as a loading control for whole protein. D, Immunoﬂuorescence images showing the expression and localization of GLUT1 in the indicated tumor cells. The indicated tumor cells were subjected to real-time PCR (E) and Western blotting (F) to detect GLUT1 levels. G, the polysomal mRNA level of GLUT1 in the indicated tumor cells was determined as in Fig. 3G. H, the indicated tumor cells were incubated with/without 10 nmol/L MK2206 for 24 hours and then subjected to Western blotting to detect the levels of GLUT1 and mGLUT1. The indicated tumor cells were transiently transfected with anti-GLUT1 silencing RNA (GLUT1 siRNA) or scramble control RNA (SC) and then subjected to glucose uptake assays (I) or incubated in glucose-deprived conditions (glucose concentration, 0.75 mol/L) for 48 hours and then subjected to ﬂow cytometry analysis to detect cell viability (J). Unpaired t tests were used in A, B, E, G, I, and J; data are shown as the mean value SD; , P < 0.05; scale bars, 100 mm.

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LIMS1 is Crucial for Solid Cancer Cell Survival

LIMS1-induced HIF1A expression. Moreover, the mTOR inhibitor HIF1 transactivates LIMS1 transcription rapamycin also completely inhibited the LIMS1-induced phos- We next explored the mechanism underlying LIMS1 overex- phorylation of mTOR and 4EBP1, as well as LIMS1-induced pression in cancer cells. We found that in hypoxic conditions, HIF1A expression. In vivo, MK2206 and rapamycin showed sim- LIMS1 expression was significantly elevated (Fig. 5A and B). To ilar growth-inhibiting effects, as well as LIMS1 knockdown (Sup- determine whether HIF1 transcriptionally regulates LIMS1 expres- plementary Fig. S9). These results suggested that LIMS1 enhanced sion in cancer cells, we established stable cell lines in which HIF1A HIF1A translation by activating AKT1/mTOR/4EBP1 signaling. was overexpressed or knocked down (Fig. 5D). HIF1A knockdown When HIF1A was knocked down, the LIMS1-induced HIF1A significantly inhibited the mRNA and protein levels of LIMS1. signaling and survival were completely inhibited (Fig. 3I–K), Moreover, when HIF1A was overexpressed, LIMS1 expression was suggesting that LIMS1 played crucial roles in the response to substantially increased (Fig. 5C and D; unpaired t tests). hypoxic stress by activating HIF1A translation in tumor cells. We analyzed the promoter region of the human LIMS1 gene and identified two hypoxia response elements (HRE; Fig. 5E, top). LIMS1 promotes glucose uptake by enhancing GLUT1 Then, we analyzed a ChIP sequencing profile deposited in the expression and membrane translocation GEO database (GEO-GSE59937; Fig. 5E, bottom) and found that Because LIMS1 facilitates the survival of tumor cells in oxygen– both HIF1A and HIF1b can bind to the promoter region of LIMS1. glucose deprivation conditions, we examined the effects of LIMS1 ChIP assays were performed in PANC-1 cells with or without on PDAC glucose uptake using 2-deoxyglucose (2-DG) in vitro.As HIF1A overexpression. In DNA fractions pulled down by an anti- shown in Fig. 4A, LIMS1 overexpression significantly increased HIF1A antibody, only the HRE of the LIMS1 promoter located at the uptake of 2-DG in PDAC (unpaired t test). Conversely, LIMS1 1267 to 1262 was detected when HIF1A was upregulated knockdown substantially reduced the uptake of 2-DG in PDAC (Fig. 5F), suggesting that HIF1A can bind to the promoter of cells. This was well supported by the evidence that tumor cells LIMS1. with high LIMS1 expression had an elevated extracellular acidi- To determine whether HIF1A binding activates LIMS1 tran- fication rate in vitro (Supplementary Fig. S10). The role of LIMS1 in scription, we constructed a full-length LIMS1 luciferase promoter glucose uptake was also assessed in esophageal carcinoma cells vector (containing HRE1) and cotransfected this reporter with or (Supplementary Fig. S5B) and lung adenocarcinoma cells (Sup- without the HIF1A overexpression vector (pcDNA-HIF1A) into plementary Fig. S6B). To determine whether LIMS1 regulates PANC-1 cells. Luciferase analysis showed that HIF1A overexpres- glucose uptake in vivo, we measured the uptake of 18fluorine- sion substantially increased the transcriptional activity of the fluorodeoxyglucose (18FDG) by orthotopically implanted PDAC LIMS1 promoter. To determine whether HRE1 is necessary for in nude mice by PET/CT scanning. Our data indicated that LIMS1 HIF1A to transactivate the LIMS1 promoter, we mutated this significantly enhanced the uptake of 18FDG (Fig. 4B; unpaired t HIF1A binding site from ACGTG to ACATG. As shown tests) in orthotopic PDAC xenograft models. To determine wheth- in Fig. 5G, the mutation of HRE1 almost abrogated the HIF1A- er LIMS1 correlates with glucose uptake in human cancer patients, induced transactivation of the LIMS1 promoter. we used PET/CT scanning to compare the standardized 18FDG Taken together, our data indicated that LIMS1 and HIF1 mutu- uptake between LIMS1-high and LIMS1-low patients. As shown in ally upregulated the expression of each other, thus forming a Supplementary Fig. S11, PDAC, patients with esophageal carci- positive feedback signaling loop. noma and lung adenocarcinoma with high-LIMS1 expression levels also had substantially higher standardized 18FDG uptake The LIMS1-HIF1–positive feedback loop is pivotal to oxygen– values. GLUT1 and GLUT3 are the two major glucose transporters glucose deprivation stress resistance that are overexpressed in cancer (25, 26). Our data showed that Given that LIMS1 and HIF1A mutually regulate the expression LIMS1 enhanced the expression and membrane transport of of each other, forming a positive feedback loop, we explored their GLUT1 but not GLUT3 in tumor cells (Figs. 4C and D). Next, relationship in vivo. First, the data from TCGA were analyzed. As we investigated the mechanism underlying LIMS1 activation of shown in Supplementary Fig. S12A, the correlation between the GLUT1 expression to facilitate glucose uptake. We found that mRNA levels of LIMS1 and HIF1A reached 0.702 (P < 0.0001, LIMS1 overexpression increased the mRNA and protein levels of Spearman correlation analysis). The LIMS1 mRNA level in HIF1A- GLUT1, and LIMS1-mediated upregulation of GLUT1 was inhib- high patients was significantly higher than that in HIF1A-low ited by HIF1A knockdown, suggesting that LIMS1 increases patients (Supplementary Fig. S12B; 564.80 228.10 vs. 290.06 GLUT1 transcription by activating HIF1 signaling (Fig. 4E and 158.28; P < 0.0001, unpaired t test), suggesting that HIF1 F). Intriguingly, LIMS1 also increased the GLUT1 mRNA levels in increased LIMS1 expression in PDAC. Next, we examined the polysomes (Fig. 4G), suggesting that GLUT1 translation was correlation between the protein levels of LIMS1 and HIF1A by promoted by LIMS1. Both LIMS1-mediated GLUT1 transcription IHC staining in a cohort of 114 PDAC specimens. As shown in and translation were directly or indirectly mediated by AKT Supplementary Fig. S12C, LIMS1 expression colocalized with signaling, which was further confirmed by blocking assays. As HIF1A in consecutive sections of the PDAC tissues. LIMS1 over- shown in Fig. 4H, LIMS1-mediated GLUT1 upregulation was expression leads to elevated HIF1A expression both in normoxia completely abrogated by the AKT inhibitor MK2206 (Fig. 4H). area and hypoxia area of tumor tissues (Supplementary Fig. S13). In addition, when AKT signaling was inhibited by MK2206, the The LIMS1 expression level in PDAC tissues was significantly LIMS1-induced GLUT1 overexpression (Fig. 4H), enhanced 2-DG associated with the HIF1A expression level (r ¼ 0.505; P < uptake (Fig. 4I), and elevated tumor survival (Fig. 4J) were 0.0001, Spearman correlation analysis), that is, tumor tissues completely abrogated. with a high LIMS1 level usually had high HIF1A expression These results indicated that LIMS1 played a crucial role in (Supplementary Fig. S12D and S12E). Then, in transplanted response to glucose deprivation stress by enhancing GLUT1 tumor tissues from mice, HIF1A overexpression significantly expression and membrane translocation. increased LIMS1 level and vice versa (Supplementary Fig. S12F).

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Figure 5.

HIF1 transactivates LIMS1 transcription. The indicated PDAC tumor cells were incubated in normoxic conditions (N) or hypoxic conditions (H; 1% O2) for 16 hours and subjected to Western blotting (A) and RT-PCR assays (B). The indicated PDAC tumor cells with HIF1A upregulated or knocked down were subjected to RT- PCR assays (C) and Western blotting (D). E, A HIF1 CHIP sequencing data profile (GEO-GSE59937) was downloaded and analyzed in UCSC Genome Browser (http://genome.ucsc.edu/; top). Red rectangle, HIF1A-binding site; black rectangle, HIF1b-binding site; and blue rectangle, the first exon of LIMS1. Schematic of the human LIMS1 promoter, two HREs were identified (UCSC gene ID: uc002teg.4.; bottom). F, ChIP analysis of PANC-1 cells transfected with or without HIF1A- overexpressing plasmids (pcDNA-HIF1A). Chromatin was immunoprecipitated with anti-HIF1A antibody and then subjected to PCR analysis. G, Luciferase analysis of Panc-1 cells. Panc-1 cells transfected with pcDNA-HIF1A or control vector (pcDNA-vector) were transfected with pGL3-LIMS1-promoter, pGL3-LIMS1- promoter mutation (MUT), or pGL3-empty vectors (pGL3.1 EV). Forty-eight hours later, cells were subjected to dual luciferase analysis. The results are expressed as a fold induction relative to the cells transfected with the control vector (pcDNA3.1) after normalization to Renilla activity. Unpaired t tests were used in B, C, and G; data are shown as the mean value SD; , P < 0.05.

We then explored the roles of the HIF1-LIMS1 loop under test), whereas LIMS1 overexpression relieved the inhibitory effect glucose–oxygen deprivation conditions, a characteristic stressor of HIF1A knockdown on cell survival and tumor growth (Sup- in solid tumors. The ectopic expression of HIF1A signiﬁcantly plementary Fig. S12H and S12J; unpaired t test). Vice versa, HIF1A promoted PDAC cell survival, and HIF1A knockdown strongly had important roles in LIMS1-induced tumor growth in mice inhibited survival under oxygen–glucose deprivation stress. The models (Supplementary Fig. S14). prosurvival effects of HIF1A were abrogated by LIMS1 knock- Taken together, these results suggested that the HIF1-LIMS1– down. LIMS1 overexpression in HIF1A-knockdown cells rescued positive feedback loop was pivotal for cancer cells to survive in the the survival under oxygen–glucose deprivation (Supplementary oxygen–glucose deprivation microenvironment. Fig. S12G; unpaired t test). These data indicated that LIMS1 is crucial for HIF1A-mediated resistance to oxygen–glucose depri- LIMS1-targeted therapy strikingly inhibits tumor growth in vivo vation. We further examined the HIF1-LIMS1 loop in oxygen– To investigate the antitumor effects of LIMS1-targeted therapy glucose deprivation resistance in vivo. The results of in vivo experi- in vivo, we used a jetPEI nanocarrier (27, 28) as the vehicle for anti- ments were consistent with those of in vitro assays. LIMS1 knock- LIMS1 siRNAs to inhibit LIMS1 expression in mouse models down abrogated the HIF1-induced increase in cell survival and (Fig. 6A). Our data showed that the jetPEI-delivered anti-LIMS1 tumor growth (Supplementary Fig. S12H and S12I; unpaired t siRNA reagent efﬁciently inhibited LIMS1 expression (Fig. 6B).

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LIMS1 is Crucial for Solid Cancer Cell Survival

Figure 6. jetPEI nanocarrier–delivered anti-LIMS1 siRNAs signiﬁcantly inhibit tumor growth in PDAC mouse models. A, jetPEI nanocarrier was used as the vehicle of anti- LIMS1 siRNAs to inhibit LIMS1 expression in vivo. Schematic of the nanoparticle (left); the time line of the drug administration (right). B–E, CFPAC-1 cells were orthotopically transplanted into the nude mice to develop tumors (n ¼ 6 for each group). jetPEI-si scramble or jetPEI anti-LIMS1 siRNAs nanoparticles were intravenously injected into mice. The 5% glucose group was used as a control group. When the mice were sacriﬁced, the tumors were sliced and then detected by an in situ cell death detection kit to determine the cell mortality rate (B and C). The tumor volumes were analyzed (D and E). F, Schematic of the roles of LIMS1 in mediating tumor cell survival in oxygen–glucose deprivation conditions. The hypoxia-glucose deprivation microenvironment is a general feature of solid tumors. In hypoxic conditions, HIF1 directly activates the transcription of LIMS1. However, by activating AKT-mTOR signaling, LIMS1 increases the translation of HIF1A in response to hypoxic stress. LIMS1 and HIF1 form a positive feedback loop, which is essential for the abnormally elevated levels of LIMS1 and HIF1 in tumors. More importantly, by activating AKT signaling and HIF1A translation, LIMS1 enhances GLUT1 production and membrane translocation, promotes glucose uptake, and facilitates tumor cell survival in the glucose deprivation environment. Unpaired t tests were used in B and C; data are shown as the mean value SD; , P < 0.05; scale bars, 100 mm.

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The inhibition of LIMS1 blocked AKT/mTOR signal pathway and lational apparatus and did not affect HIF1A translation through resulted in decreased HIF1A and GLUT1 expression in tumor the Cap-independent translation (Supplementary Fig. S16). tissues (Supplementary Fig. S15), leading to a remarkable increase Our data showed that LIMS1 overexpression significantly of cell mortality (Fig. 6C; unpaired t test) and significant tumor increased the protein level of HIF1A in tumor cells. When inhibitory effect (Fig. 6D and E; unpaired t test). These results LIMS1 was knocked down, HIF1A expression was substantially suggested that LIMS1 could be a promising therapeutic target for reduced, suggesting that LIMS1 plays crucial roles in HIF1A cancer therapy. expression. Instead of promoting the transcription and stabi- lization of HIF1a, LIMS1 facilitates the translation of HIF1A, which is mediated by the AKT/mTOR pathway. Thus, LIMS1 Discussion andHIF1formapositivefeedback loop, which is a crucial link In solid tumors, the poorly formed tumor vasculature and the between HIF1 and AKT signaling. elevated glucose consumption result in a characteristic oxygen– As a transcription factor, HIF1 can initiate the transcription glucose deprivation microenvironment. To survive, cancer cells of GLUT1 and facilitate glucose uptake in the oxygen–glucose have evolved complicated regulatory network to adapt to the deprivation microenvironment (2). In this study, we found that harsh conditions. LIMS1 promoted GLUT1 transcription via HIF1A and transla- In this study, we identified LIMS1 as a critical regulator of tion by activating AKT signaling. LIMS1/AKT signaling was tumor cell resistance to oxygen–glucose deprivation. LIMS1 can important in maintaining the HIF1A translational expression form a ternary protein complex with ILK and a-parvin in focal in tumor cells. LIMS1 overexpression significantly rescued the complexes and focal adhesions (10). LIMS1 is required for the inhibitory effect of HIF1A knockdown on GLUT1 expression. maintenance of ILK protein expression level and is indispens- These results suggest that LIMS1/AKT signaling strongly con- able for some integrin-dependent functions (11, 29). Although tributes to GLUT1 expression and glucose uptake in tumor LIMS1 has been implicated in cell spreading (11) and surviv- cells. Hence, LIMS1 is probably a key molecule in maintaining al (21), the roles of LIMS1 in cancer progression are poorly glucose homeostasis of cancer cells in the oxygen–glucose understood. Here, we demonstrated that LIMS1 expression was deprivation microenvironment. significantly elevated in multiple cancer tissues, including pan- The important roles of LIMS1 in glucose uptake of tumor creatic adenocarcinoma, lung adenocarcinoma, and esophageal cells were verified in vivo. When we inhibited LIMS1 expression, carcinoma. High levels of LIMS1 correlated with advanced the tumor cell viability and growth speed decreased significant- tumor size, tumor staging, nodal involvement, and poor sur- ly, suggesting that LIMS1 could be a promising therapeutic vival of patients with tumor. Although knockdown of LIMS1 target for cancer treatment. However, as LIMS1 is not ubiqui- had negligible effects on cancer cell apoptosis under normal tously overexpressed in all tumor tissues, anti-LIMS1 therapy culture conditions, LIMS1 was critical for cancer cell survival might only be efficacious in patients with tumor with LIMS1 under oxygen–glucose deprivation. overexpression. As the SUVmax in PET-CT scanning of patients Mechanistically, LIMS1 enhances glucose uptake by increasing with tumor was positively correlated with the expression level the expression levels of GLUT1 (30), possibly through ILK- of LIMS1 in tumor tissues, PET-CT scanning is a potential mediated phosphorylation and activation of PKB/AKT (31). noninvasive approach to identify the potential beneficiaries of LIMS1 was shown to be crucial for the stability and localization anti-LIMS1 therapy. Further investigations are needed to verify of ILK (10) and is important for ILK-induced phosphorylation of this hypothesis. AKT (15, 29). Our results showed that when AKT signaling was Several limitations to this study are noted. As no anti-LIMS1 inhibited, the LIMS1-induced glucose uptake was completely inhibitors are currently available, in the translational experi- abrogated, indicating that LIMS1-induced glucose uptake is medi- ments, we used the anti-LIMS1 siRNA to inhibit LIMS1 expres- ated by AKT/GLUT1 signaling. sion in vivo. Although the anti-LIMS1 siRNA significantly inhib- In the oxygen–glucose deprivation microenvironment, HIF1 ited LIMS1 expression and tumor growth, siRNAs were not yet signaling is another master regulator that plays crucial roles in an ideal agent for clinical usage. Specific inhibitors for LIMS1 tumor angiogenesis, tumor metastasis, and survival. are demanded to verify these results before launch a clinical There are complicated correlations between HIF1 signaling study. Second, as a single agent LIMS1 might be not strong and the AKT pathway. PKB/AKT was shown to be activated by enough to suppress tumor in clinical application, considering hypoxia in various tumor types (32). Several studies demon- pancreatic cancer is such a tenacious disease. The combination strated that hypoxia-induced resistance to apoptosis was largely of anti-LIMS1 agents and chemotherapy drugs might be more mediated by the AKT pathway (33, 34). Nevertheless, how AKT effective for curing pancreatic cancer. Further investigations are signaling is activated by hypoxia is currently unclear. In this needed to confirm this. study, we reported a new link between these two pathways: the HIF1–LIMS1–AKT signaling-HIF1 axis transcriptionally initiat- Disclosure of Potential Conflicts of Interest ed LIMS1 expression to activate AKT signaling. In addition, the No potential conflicts of interest were disclosed. hypoxia-induced AKT pathway activation is cell specific (35). This might be due to the differential expression of LIMS1 in Authors' Contributions different tumor cells. AKT signaling is considered the major Conception and design: C. Huang, Y. Li, K. Xie, J. Hao, H. Ren regulator of HIF1a synthesis in cancer cells (27). AKT initiates Development of methodology: C. Huang, Y. Li, X. Wang, H. Wang the mTOR pathway, which regulates HIF1A protein biosynthe- Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Huang, Y. Li, Z. Li, N. Li, Y. Ge, A. Chang, sis by regulating cap-dependent translation and ribosome bio- T. Zhao, X. Wang, H. Wang genesis(36).Thiswasfurtherverified by the evidence that Analysis and interpretation of data (e.g., statistical analysis, biostatistics, LIMS1 did not have a direct interaction with the basal trans- computational analysis): C. Huang, Y. Li, K. Xie, J. Hao, H. Ren, J. Dong

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Writing, review, and/or revision of the manuscript: C. Huang, Y. Li, S. Yang, dation of Tianjin (grant Nos. 16JCQNJC09900, 13YCYBYC37400, and K. Xie, J. Hao, H. Ren 11JCZDJC18400), and by the NIH grants CA175741 (to S. Yang); and Administrative, technical, or material support (i.e., reporting or organizing CA173322, CA195651, CA198090, and CA220236 (to K. Xie). data, constructing databases): Y. Li, Y. Xu, J. Dong, X. Wang Study supervision: Y. Li, H. Ren The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in Acknowledgments accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported in part by the National Natural Science Foundation of China (grant Nos. 81871978, 81602017, 81772633, 81720108028, 81525021, 81502067, 81302082, 81272685, 31301151, 81172355, Received October 27, 2018; revised January 4, 2019; accepted January 22, 31471340, 31470957, 81472264, and 81401957), the Natural Science Foun- 2019; published ﬁrst January 24, 2019.

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www.aacrjournals.org Clin Cancer Res; 25(13) July 1, 2019 4103

LIMS1 Promotes Pancreatic Cancer Cell Survival under Oxygen− Glucose Deprivation Conditions by Enhancing HIF1A Protein Translation

Chongbiao Huang, Yang Li, Zengxun Li, et al.

Clin Cancer Res 2019;25:4091-4103. Published OnlineFirst January 24, 2019.

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