KRAS/NF-Kb/YY1/Mir-489 Signaling Axis Controls Pancreatic Cancer

Published OnlineFirst October 28, 2016; DOI: 10.1158/0008-5472.CAN-16-1898

Cancer Molecular and Cellular Pathobiology Research

KRAS/NF-kB/YY1/miR-489 Signaling Axis Controls Pancreatic Cancer Metastasis Peng Yuan1,2, Xiao-Hong He1,2, Ye-Fei Rong3, Jing Cao4, Yong Li5, Yun-Ping Hu6, Yingbin Liu6, Dangsheng Li7, Wenhui Lou3, and Mo-Fang Liu1,2,4

Abstract

KRAS activation occurring in more than 90% of pancreatic inhibited migration and metastasis by targeting the extracellular ductal adenocarcinomas (PDAC) drives progression and metas- matrix factors ADAM9 and MMP7. miR-489 downregulation tasis, but the underlying mechanisms involved in these processes elevated levels of ADAM9 and MMP7, thereby enhancing are still poorly understood. Here, we show how KRAS acts through the migration and metastasis of PDAC cells. Together, our inﬂammatory NF-kB signaling to activate the transcription factor results establish a pivotal mechanism of PDAC metastasis and YY1, which represses expression of the tumor suppressor gene suggest miR-489 as a candidate therapeutic target for their attack. miR-489. In PDAC cells, repression of miR-489 by KRAS signaling Cancer Res; 77(1); 100–11. 2016 AACR.

KRAS in pancreatic tumors, the most common of which is Introduction G12D KRAS (4). Mounting evidence indicates that such onco- Pancreatic cancer (PDAC) is one of the most lethal malignant genic mutations play critical roles in both the initiation and the tumors, with a 5-year survival rate less than 8% from 2005 to 2011 progression of pancreatic cancer via persistent activation of in United States (1). To date, lack of effective screening tool to KRAS signaling pathways (5). Sustained KRAS signaling leads detect asymptomatic premalignant or early-stage cancer results in to the activation of inflammatory signaling pathways that play the diagnosis of majority patients at their advanced stages. More critical roles in regulating the initiation of pancreatic intrae- than 90% of the patients with PDAC show distal metastasis at pithelial neoplasia (PanIN) and the progression of PDAC advanced stage, which is the major cause of mortality (2). Thus, (6, 7). Of note, NF-kB signaling, a major pathway that connects effective therapeutic interventions to stop PDAC metastasis are inflammation with cancers, is activated by KRAS signaling and urgently needed. However, our ability to design effective thera- has been shown to promote pancreatic cancer progression in peutic interventions aiming to stop PDAC metastasis is limited animal models (8–10). However, the downstream targets of because of our poor understanding of the molecular mechanisms NF-kB signaling that are directly involved in pancreatic cancer underlying pancreatic cancer metastasis (3). progression and metastasis still need to be defined. One prominent feature of PDAC is the high frequency microRNAs (miRNA) are a class of small, noncoding RNAs that (>90%) of KRAS mutations that generate oncogenic forms of negatively regulate protein-coding genes at the posttranscription- 1Center for RNA Research, State Key Laboratory of Molecular Biology–University al level and are involved in virtually all types of carcinogenesis of Chinese Academy of Sciences, CAS Center for Excellence in Molecular Cell (11). In particular, a number of miRNAs have been found to be Science, Shanghai, China. 2Shanghai Key Laboratory of Molecular Andrology, dysregulated in PDAC and involved in PDAC carcinogenesis (12). Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Nevertheless, how these miRNAs are dysregulated in PDAC and Sciences, Chinese Academy of Sciences, Shanghai, China. 3Department of how they interact with key regulatory signaling molecules in Pancreatic Surgery, Zhong Shan Hospital, Shanghai, China. 4School of Life Science and Technology, Shanghai Tech University, Shanghai, China. 5Depart- pancreatic cancer initiation and progression are questions that ment of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, still remain largely unanswered. Most importantly, despite the Ohio. 6Department of General Surgery, Xinhua Hospital Affiliated to Shanghai well-established role of oncogenic KRAS in PDAC, how KRAS Jiao Tong University School of Medicine, Shanghai, China. 7Shanghai Information signaling engages miRNAs to drive pancreatic cancer metastasis Center for Life Sciences, Shanghai Institutes for Biological Sciences, Chinese remains unknown. Academy of Sciences, Shanghai, China. In our attempt to identify important miRNAs involved in Note: Supplementary data for this article are available at Cancer Research pancreatic carcinogenesis, we identified KRAS signaling– Online (http://cancerres.aacrjournals.org/). repressed miR-489 as part of an important mechanism under- Corrected online September 16, 2020. pinning oncogenic KRAS-induced PDAC migration and metasta- P. Yuan, X.-H. He, and Y.-F. Rong contributed equally to this article. sis. Oncogenic KRAS signaling activates NF-kB, leading to Corresponding Authors: Mo-Fang Liu, Institute of Biochemistry and Cell enhanced expression of YY1, the transcription factor that directly MIR489 Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of suppresses transcription. Our results showed that miR- Sciences, 320 Yueyang Road, Shanghai 200031, China. Phone: 86-21- 489 decreases the migration of PDAC cells in cell cultures and 54921146; Fax: 86-21-54921011; E-mail: mfl[email protected]; and Wenhui Lou, inhibits lung and liver metastatic colonization of PDAC cells in Department of Pancreatic Surgery, Zhong Shan Hospital, 180 Fenglin Road, mice but contributes little to cell proliferation and anchorage- Shanghai 200032, China. Phone: 86-18-616881868; Fax: 86-21-64043947; independent growth. Mechanistically, we identified 2 metallo- E-mail: [email protected] proteinase genes, ADAM9 and MMP7, as novel targets of miR-489 doi: 10.1158/0008-5472.CAN-16-1898 that mediate its antimetastatic effect in these cells. Collectively, 2016 American Association for Cancer Research. our findings not only provide new mechanistic insights into how

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oncogenic KRAS-induced inflammatory signaling promotes pore size inserts, 3422; Corning) in the Transwell assays according PDAC metastasis but also indicate that miR-489 is a robust to the manufacturer's instructions. The nonmigrating cells were inhibitor of metastasis and a potential therapeutic target for detached, and the migrating cells were stained with 40,6-diamidio- treating PDAC. 2-phenylindole (DAPI) and counted after 24 hours using a fluorescence microscope. All fields were selected in a blind Materials and Methods manner. Cell lines Three-dimensional cell culture assay The human PDAC cell lines BxPC-3 and PANC-1 cells were The 3D cell culture assay was performed as recently described obtained from ATCC (May, 2011) and cultured according to their (17). Transfected and viable cancer cells (1,000) in culture medi- guidelines. All the cell lines were mycoplasma-free and recently um containing 2% Matrigel were seeded on the solidified 100% authenticated by cellular morphology and the STR analysis at Ji- Matrigel-precoated chamber (177402; Nunc). The sphere mor- Ying Inc. (January 2014 and May 2016) according to the guide- phology was observed with a microscope after 19 days. lines from ATCC (13). The 2 cell lines stably expressing luciferase (PANC-1-luc and BxPC-3-luc) were generated from their parental cell lines as previously reported (14). Xenograft assays in mice NOD/SCID mice were purchased from SLAC Corporation and were housed under standard housing conditions at the animal Antibodies and reagents facilities in the Institute of Biochemistry and Cell Biology, Shang- The antibodies used in this study included: anti-KRAS (60309- hai Institutes for Biological Sciences, Chinese Academy of 1; Proteintech), anti-PCNA (#2586; Cell Signaling Technology), Sciences. The lung and liver metastasis assay was performed as anti-YY1 (ab38422; Abcam), anti-ETS-1 (sc-350; Santa Cruz Bio- described previously (14, 18). PANC-1-luc cells (4 104)or technology), anti-p65 (10745-1-AP, Proteintech), anti-ADAM9 BxPC-3-luc cells (1 105) in 100 mL Dulbecco PBS (D-PBS) were (ab186833, Abcam), anti-MMP7 (10374-2-AP, Proteintech), injected into 6- to 8-week NOD/SCID mice through the tail vein (n anti-b-actin (A3854; Sigma-Aldrich), anti-mouse secondary anti- ¼ 5–6), and the luciferase activity in lungs was analyzed 50 days body (A9044, Sigma-Aldrich), and anti-rabbit secondary anti- later. PANC-1-luc cells (1 105) orBxPC-3-luc cells (5 105)in body (A9169, Sigma-Aldrich). These antibodies were diluted 50 mL D-PBS were injected into the spleens of 6- to 8-week-old according to the manufacturers' instructions. The NF-kB signaling NOD/SCID mice (n ¼ 4–5), and the tumor numbers in livers were inhibitor BAY 11-7082 (S2913; Selleck) was first diluted in cancer analyzed 60 days later. For bioluminescence imaging, mice were cell culture medium and then used as in previous studies with given luciferin 5 minutes before imaging and were then anesthe- some modifications (i.e., incubation at 5 mmol/L for 24 hours; tized (3% isoflurane). Luminescence imaging was performed and ref. 15). The sequences of chemically synthesized DNA and RNA analyzed using the Xenogen IVIS Imaging System (Xenogen, a oligonucleotides are listed in Supplementary Table S1. subsidiary of Caliper Life Sciences). Cell proliferation and soft agar colony formation assays Statistical analyses The assays were performed as described previously (16). In All results are presented as the mean SD. The Student t test brief, cancer cells were subjected to transfection for 24 hours. For was performed to judge the significance of differences between cell proliferation assay, 3,000 viable cells were seeded into each treated groups and their paired controls. P values indicate the level well of 96-well plates. Cell growth was determined by MTT assays of statistical significance (, P < 0.05; , P < 0.01; , P < 0.001). and verified by counting the cells excluding trypan blue. For soft The fold changes of the relative miRNA or mRNA levels were agar colony formation assay, 5,000 viable cancer cells were triply performed using Mev-HCL software algorithm analyses (19). seeded in 1.5 mL of tissue culture medium with 1% glutamine and antibiotics, and 0.4% soft agar was layered onto 0.8% solidified agar in tissue culture medium in 6-well plates. After incubation for Study approval 15 days, the colony foci were stained with 0.005% crystal violet PDAC specimens and paired normal adjacent tissues were and counted using a dissecting microscope. Experiments were collected during surgery from Zhong Shan Hospital, which is fi carried out in triplicate. af liated with Fudan University (Shanghai, China) with written informed consent from patients. Samples were immediately snap- frozen and stored at 80C. The specimen collection was Wound-healing assay approved by the Medical Ethical Committee of the hospital. All Wound-healing assays were performed as previously described animal experiments were performed under protocols approved by (14). Cancer cells were transfected for 24 hours and starved in the Institute of Biochemistry and Cell Biology, Shanghai Institutes culture medium containing 0.2% FBS for 12 hours. The mono- for Biological Sciences, Chinese Academy of Sciences and in layer of confluent cells was scratched using a 10-mL Pipetman tip. accordance with the Guide for the Care and Use of Laboratory Cells were then photographed at different time points with an Animals (NIH publication nos. 80–23, revised 1996). Olympus IX81 microscope. The relative wound areas were then measured using ImageJ software (NIH, Bethesda, MD). Results Transwell assay Downregulation of miR-489 is crucial for oncogenic KRAS to Cancer cells were transfected for 24 hours, following by star- promote cell migration in human PDAC cells vation for 12 hours. PANC-1 cells (2 104) or BxPC-3 cells (5 To gain new insight into the role of miRNAs in oncogenic 104) were used in each 20% (in culture medium without FBS) KRAS-initiated pancreatic tumorigenesis, we compared the Matrigel-precoated (356231BD; BD Biosciences) insert (8.0-mm expression of 53 miRNAs that are dysregulated during PDAC

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G12D progression in the Kras transgenic animal model and in strongly enhanced miR-489 expression in these cells, whereas G12D KRAS -transfected human pancreatic ductal epithelial cells knockdown of ETS1 had little effect (Fig. 2C). We further found (20, 21) between 2 human PDAC cell lines, namely, wild-type that overexpression of YY1, but not ETS1, potently decreased miR- G12D KRAS-containing BxPC-3 and KRAS -containing PANC-1 cells 489 expression in PANC-1 cells (Fig. 2D). These results strongly (Supplementary Fig. S1A, left). Our qRT-PCR assays showed that suggest that YY1 is a repressor for MIR489. To further substantiate 17 miRNAs were differentially expressed between the 2 PDAC cell this conclusion, we constructed 2 luciferase reporters under con- lines (Supplementary Fig. S1B, left column), including 3 miRNAs trol of either the wild-type human MIR489 promoter or a mutant (miR-489, miR-889, and miR-574) enriched in BxPC-3 cells and 4 version with 2 putative YY1-binding sites deleted (termed the WT miRNAs (miR-125b, miR-491, miR-21, and miR-23b) enriched in PMIR489 reporter and the mut PMIR489 reporter, respectively; Fig. PANC-1 cells (by >3.0-fold). To examine the effect of oncogenic 2A, bottom). As expected, the activity of the WT PMIR489 reporter KRAS signaling on miRNA expression in these PDAC cells, we was potently enhanced by knockdown of YY1 but reduced by YY1 knocked down KRAS in PANC-1 cells and ectopically expressed overexpression in PANC-1 cells (Fig. 2E). The mut PMIR489 reporter G12D KRAS in BxPC-3 cells, respectively (Supplementary Fig. S1C). displayed about 4-fold activity in PANC-1 cells compared with G12D miR-21 was the most markedly upregulated by KRAS over- WT, and it was not affected by either YY1 knockdown or overexpression in BxPC-3 cells (Supplementary Fig. S1B, middle expression (Fig. 2E). Collectively, these results support the notion column) and downregulated by KRAS knockdown in PANC-1 that YY1 is a direct transcriptional repressor for MIR489. cells (right column), consistent with previous studies reporting an We next examined whether KRAS signaling represses MIR489 important role for miR-21 in oncogenic KRAS–induced cell pro- via YY1 in PDAC cells. The level of YY1 protein was significantly liferation (22, 23). Interestingly, miR-489, among all the tested lower in wild-type KRAS-containing BxPC-3 cells than in G12D G12D miRNAs, showed the greatest reduction in KRAS -transfected KRAS -containing PANC-1 cells (Fig. 2F, left, lane 1 vs. lane BxPC-3 cells (Supplementary Fig. S1B, middle column) and the 4). Interestingly, YY1 mRNA and protein were markedly increased G12D greatest increase in KRAS siRNA–transfected PANC-1 cells (right by ectopic expression of KRAS in BxPC-3 cells (Fig. 2F, left, column). lane 6) and significantly reduced by KRAS knockdown in PANC-1 The strong negative correlation between KRAS signaling and cells (Fig. 2F, left, lane 3), indicating that oncogenic KRAS acti- miR-489 prompted us to examine whether downregulation of vates YY1 expression in PDAC cells. Then, we searched and found miR-489 is involved in KRAS signaling–mediated pancreatic that NF-kB family member was predicted to be transcription G12D tumorigenesis. As expected, ectopic expression of KRAS in factor in YY1 promoter, which is consistent with the study in BxPC-3 cells substantially increased cell proliferation (Fig. 1A), myoblasts (28). As NF-kB is a well-documented downstream anchorage-independent growth (Fig. 1B), and cell migration effector of KRAS signaling (8, 10), we examined whether the in vitro (Fig. 1C and D). Intriguingly, restoration of miR-489 YY1/miR-489 axis is regulated by KRAS/NF-kB signaling in expression in these cells by transfection of the miR-489 expression human PDAC cells. Both KRAS knockdown and treatment with vector pSIF-miR-489 barely affected cell proliferation and soft an NF-kB inhibitor, BAY 11-7082, significantly elevated miR-489 agar colony formation (Fig. 1A and B; Supplementary Fig. S1D) expression, with a concomitant reduction of YY1 expression in G12D but rescued the KRAS expression–induced cell migration in a PANC-1 cells, whereas the elevation of miR-489 expression by dosage-dependent manner (Fig. 1C and D). Conversely, knock- KRAS knockdown or BAY 11-7082 treatment was completely down of KRAS in PANC-1 cells markedly decreased cell prolifer- suppressed by ectopic expression of Flag-YY1 in these cells (Fig. ation (Fig. 1E), anchorage-independent growth (Fig. 1F), and cell 2G). These results suggest that the YY1/miR-489 axis is controlled migration in vitro (Fig. 1G and H), whereas concurrent suppres- by KRAS/NF-kB signaling in PDAC cells. To further substantiate sion of miR-489 by anti-miR-489 showed no significant effect on this conclusion, we examined the effect of KRAS knockdown or cell proliferation (Fig. 1E and F; Supplementary Fig. S1E) but BAY 11-7082 treatment on the activity of the PMIR489reporter in significantly reversed the impact of KRAS knockdown on cell PANC-1 cells. In a similar manner, both KRAS knockdown and migration in these KRAS-mutant cells (Fig. 1G and H). These treatment with NF-kB inhibitor significantly enhanced the activity results together suggest that downregulation of miR-489 contri- of the WT PMIR489reporter in PANC-1 cells, and their effects were butes little to KRAS signaling–driven cell proliferation but is an completely abrogated by ectopic expression of Flag-YY1 (Fig. 2H). important mechanism underlying KRAS signaling–driven cell In sharp contrast, KRAS knockdown or BAY 11-7082 treatment migration of PDAC cells. barely affected the activity of the mut PMIR489reporter (Fig. 2H). Taken together, these results indicate that the MIR489 gene is miR-489 is downregulated by the KRAS/NF-kB/YY1 axis in repressed by the novel KRAS/NF-kB/YY1 axis in human PDAC PDAC cells cells. We next asked how KRAS signaling downregulates miR-489 in PDAC cells. To this end, we first searched for potential transcrip- miR-489 inhibits the migration and metastasis of PDAC cells tion factor–binding sites in MIR489 promoter using the PROMO We next examined the role of miR-489 in PDAC tumorigenesis. and TransFac programs (24, 25). Interestingly, ETS1 and YY1, 2 Modulation of miR-489 expression in PDAC cells, including transcription factors that have been shown to control miRNA transfection of pSIF-miR-489 in PANC-1 cells (which have lower expression in cancer cells (26, 27), were predicted as candidate endogenous miR-489 expression) or anti-miR-489 in BxPC-3 cells transcription factors for MIR489 (Fig. 2A, top). To explore whether (which show a high level of endogenous miR-489 expression; ETS1 or YY1 directly regulates MIR489,wefirst performed a Supplementary Fig. S1A, right), barely altered cell proliferation chromatin immunoprecipitation (ChIP) assay in PANC-1 cells. and anchorage-independent growth (Supplementary Fig. S2). We found that the PMIR489(promoter of MIR489) fragment was Western blot analyses confirmed that the proliferation marker effectively enriched by anti-YY1, but not by anti-ETS1, compared proliferating cell nuclear antigen (PCNA) was little altered in pSIF- with the IgG control (Fig. 2B). Moreover, RNAi knockdown of YY1 miR-489–transfected PANC-1 or anti-miR-489–transfected BxPC-

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Figure 1. Downregulation of miR-489 is crucial for oncogenic KRAS to promote cell migration in human PDAC cells. A–D, Restoration of miR-489 expression overrode the effect of ectopic KRASG12D on wound-healing (C) and Transwell cell migration (D) but only slightly altered oncogenic KRAS signaling–promoting cell proliferation (A) and anchorage-independent growth (B) in BxPC-3 cells. The cells were transfected with pCMV-myc-G12D-KRAS (G12D-KRAS) or cotransfected with G12D-KRAS and the indicated amounts of pSIF-miR-489, with pCMV-myc and pSIF-H1 vectors serving as negative controls. A, Cell proliferation assays. Top, MTT assays; bottom, Western blot analyses of KRAS and PCNA proteins. B, Soft agar colony formation assays. Top, the number of soft agar foci per field; bottom, representative images (scale bar, 100 mm). C, Wound-healing assays. Top, quantitative results of wound closure 16 hours after wounding; bottom, representative images at 0 and 16 hours after wounding (scale bar, 100 mm). D, Transwell migration assays. Top, number of migratory cells per field 24 hours after the cells were plated; bottom, representative images (scale bar, 40 mm). E–H, Inhibition of miR-489 by anti-miR-489 overrode the effect of KRAS knockdown on wound-healing (G) and Transwell cell migration (H) but only slightly altered KRAS knockdown–reduced cell proliferation (E) and anchorage-independent growth (F) in PANC-1 cells. The cells were transfected with KRAS siRNA (KRAS-siR) or cotransfected with KRAS-siR and anti-miR-489, with scrambled RNA (scr siR) and control RNA (ctrl RNA) serving as negative controls. The mean SD of three separate experiments was plotted. Statistics: Student t test, , P < 0.05; , P < 0.01; , P < 0.001. n.s., not significant.

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Figure 2. miR-489 is downregulated by the KRAS/NF-kB/YY1 axis in PDAC cells. A, Top, schematic representation of predicted YY1- and ETS1-binding sites within the human MIR489 promoter. Bottom, construction of the wild-type MIR489 promoter reporter (WT PMIR489 reporter) and the YY1 binding site–deleted mutant reporter (mut PMIR489 reporter). B, Top, YY1- and ETS1-binding sites within the human MIR489 promoter. Bottom, ChIP analyses of YY1 or ETS1 binding to the MIR489 promoter using antibodies against YY1 or ETS1, respectively. C and D, Effects of YY1 or ETS1 knockdown (C) or overexpression (D) on miR-489 expression through qRT-PCR analyses of miR-489 expression (left) and Western blot analyses of YY1 or ETS1 protein expression (right) in PANC-1 cells. E, Modulation of the PMIR489 promoter activity by YY1 knockdown (top) or overexpression (bottom) in PANC-1 cells. F, Regulation of KRAS signaling on YY1 expression in PANC-1 and BxPC-3 cells. Top, qRT-PCR analyses of YY1 mRNA; bottom, Western blot analyses of YY1 protein. G, Effect of the KRAS/NF-kB/YY1 axis on miR-489 expression in PANC-1 cells. Top, qRT-PCR analyses of miR-489; bottom, Western blot analyses of p65 and YY1 protein. H, Modulation of PMIR489 promoter activity by the KRAS/NF-kB/YY1 axis in PANC-1 cells. The mean SD of three separate experiments was plotted. Statistics: Student t test, , P < 0.05; , P < 0.01; , P < 0.001. n.s., not signiﬁcant.

3 cells (Supplementary Fig. S2, middle). These results together control pSIF-H1 pseudovirus–infected cells but a dramatic reduc- demonstrate that miR-489 does not affect the proliferation of tion in the luciferase signal in the lungs of mice injected with miR- PDAC cells. We further examined the effect of miR-489 on PDAC 489–overexpressing cells (Fig. 3D, top), suggesting that miR-489 cell migration and metastasis. In vitro studies showed that miR- inhibits the lung metastasis of PDAC cells. Hematoxylin and eosin 489 overexpression in PANC-1 cells significantly reduced cell (H&E) staining of the lung sections confirmed that miR-489– migration in both wound-healing (Fig. 3A, top) and Transwell overexpressing cells colonized the lung much less efficiently than migration assays (Fig. 3B, top) and markedly suppressed the the control populations (Fig. 3E, top). We next injected pSIF-miR- ability of these cells to spread out and form colonies in a 3- 489 pseudovirus–infected PANC-1-luc cells into mouse spleens dimensional (3D) cell culture assay (Fig. 3C, top). To determine and examined liver metastasis. Compared with controls, miR-489 the function of miR-489 on PDAC metastasis in vivo, we infected overexpression substantially reduced the liver metastatic coloni- firefly luciferase–labeled PANC-1 (PANC-1-luc) cells with pSIF- zation of PDAC cells (Fig. 3F, top) and resulted in fewer metastatic miR-489 pseudovirus and performed tail vein xenografts. We liver nodules (Fig. 3G, top). In a reciprocal experiment, inhibition observed strong luciferase foci in the lungs of mice injected with of miR-489 by anti-miR-489 in BxPC-3-luc (firefly luciferase–

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Figure 3. miR-489 inhibits the migration and metastasis of PDAC cells. A–C, miR-489 inhibited the migration of PDAC cells in vitro. PANC-1 and BxPC-3 cells were transfected with pSIF-miR-489 or the control vector pSIF-H1 (top) and anti-miR-489 or the control RNA (ctrl RNA; bottom), and the assays were performed 24 hours posttransfection. A, Wound-healing assays. Left, representative images at 0 and 16 hours after wounding (scale bars, 100 mm); right, quantification of wound closure 16 hours after wounding. B, Transwell migration assays. Left, representative images (scale bars, 40 mm); right, quantification of migrating cell numbers 24 hours after the cells were plated. C, 3D cell culture assays, with images showing spheres on day 19 after the cells were grown in 3D cell culture (scale bars, 300 mm). The inset shows an amplified view of individual pSIF-H1–transfected PANC-1 cells (top) or anti-miR-489–transfected BxPC-3 cells (bottom) invading the surrounding Matrigel (scale bars, 50 mm). D and E, miR-489 inhibited lung metastatic colonization of PDAC cells in NOD/SCID mice. D, Bioluminescence imaging of NOD/SCID mice on day 50 after tail vein injection of pSIF-miR-489 pseudovirus–infected PANC-1-luc cells (top) or anti-miR-489–transfected BxPC-3-luc cells (bottom). Left, representative images; right, bioluminescence quantification of lung metastasis (mean SD, n ¼ 5 mice in each group). E, H&E staining of the lungs (left; scale bars, 2 mm) and the number of metastatic nodules (right) in mice on day 50 after tail vein injection of the indicated PDAC cells. Data on the right are presented as mean SD (n ¼ 5 mice in each group). F and G, miR-489 inhibited liver metastatic colonization of PDAC cells in NOD/SCID mice. F, Bioluminescence imaging of NOD/SCID mice on day 60 after splenic injection of pSIF-miR-489 pseudovirus–infected PANC-1-luc cells (top) or anti-miR-489– transfected BxPC-3-luc cells (bottom). Left, representative images; right, bioluminescence quantification of liver metastasis (mean SD, n ¼ 4 mice in each group). G, Brightfield imaging of the livers (left; scale bars, 5 mm) and the number of visible liver metastases (right) in mice on day 60 after splenic injection of the indicated PDAC cells. Data on the right are presented as mean SD (n ¼ 4 mice in each group). All data are mean SD. Statistics: Student t test, , P < 0.05; , P < 0.01; , P < 0.001.

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labeled BxPC-3) cells, which have a higher endogenous miR-489 decreased the luciferase activity of the wild-type reporters but expression (Supplementary Fig. S1A, right), led to a significant barely affected that of the mutant reporters (Fig. 4B), suggesting increase in cell migration in wound healing (Fig. 3A, bottom) that ADAM9 and MMP7 are targets of miR-489. To further cor- and Transwell migration (Fig. 3B, bottom), cell invasion in 3D roborate this conclusion, we transfected pSIF-miR-489 into PANC- cell culture (Fig. 3C, bottom), and lung and liver metastasis 1 cells and found that both ADAM9 and MMP7 proteins were (Fig. 3D–G, bottom). These results together indicate that miR- greatly reduced in pSIF-miR-489–transfected cells (Fig. 4C, left, 489 inhibits the migration and metastasis of PDAC cells, lane 3). qRT-PCR analyses showed that their mRNA levels were supporting that miR-489 is a critical regulator of PDAC also significantly reduced in PANC-1 cells transfected with pSIF- metastasis. miR-489 (Fig. 4C, right). In contrast, inhibition of miR-489 by anti-miR-489 in BxPC-3 cells led to enhanced ADAM9 and MMP7 ADAM9 and MMP7 are novel targets of miR-489 expression (Fig. 4D). These results together confirmed ADAM9 and To dissect the molecular mechanism of the inhibitory effect of MMP7 as authentic targets of miR-489. In addition, depleting YY1 miR-489 on PDAC metastasis, we used miRNA target prediction or KRAS proteins in PANC-1 cells significantly reduced ADAM9 tools (29) to search for potential target genes of miR-489. We and MMP7 expression, whereas inhibition of miR-489 by anti- found that ADAM9 and MMP7, 2 metalloproteinase genes that miR-489 effectively increased their expression in these cells (Sup- have been implicated as being crucial to PDAC progression (30, plementary Fig. S3). This result is consistent with our above finding 31), were predicted to be targets of miR-489 (Fig. 4A, top). To that the KRAS/NF-kB/YY1 axis is required for repressing MIR489 experimentally test whether miR-489 targets ADAM9 and MMP7, (Fig. 2), further supporting the notion that ADAM9 and MMP7 are we constructed luciferase reporters by cloning the wild-type 30- direct targets of miR-489 in human PDAC cells. untranslated regions (UTR) of ADAM9 and MMP7 or their mutant versions (with deletion of the 7-bp sequence complementary to The miR-489:ADAM9/MMP7 regulatory axis is functionally the 50 sequence of miR-489) downstream of the firefly luciferase important for migration and metastasis of PDAC cells cDNA in the pmirGLO vector (Fig. 4A, bottom). We found that As miR-489 inhibits the migration and metastasis of PDAC cotransfection of pSIF-miR-489 into 293T cells substantially cells (Fig. 3), we then examined whether miR-489 exerts its

Figure 4. ADAM9 and MMP7 are novel targets of miR-489. A, ADAM9 and MMP7 were predicted to be miR-489 targets. Top, predicted miR-489 regulatory elements (seed sequences in upper case) in ADAM9 and MMP7 30UTRs. Bottom, sequences of the wild-type (pmirGLO-ADAM9-30UTR, pmirGLO-MMP7-30UTR) and the mutated (pmirGLO-ADAM9-30UTR mut, pmirGLO-MMP7-30UTR mut) 30UTR luciferase reporters. B, Luciferase reporter assays for the ADAM9-30UTR (top) and MMP7-30UTR (bottom) reporters in 293T cells, indicating that miR-489 targets ADAM9 and MMP7. C and D, ADAM9 and MMP7 were repressed by miR-489 in PDAC cells. C, Effect of miR-489 overexpression on ADAM9 and MMP7 expression in PANC-1 cells. D, Effect of miR-489 inhibition on ADAM9 and MMP7 expression in BxPC-3 cells. Left, Western blot analyses of ADAM9 and MMP7 protein expression. Right, qRT-PCR analyses of ADAM9 and MMP7 expression. The mean SD of three separate experiments was plotted. Statistics: Student t test, , P < 0.01; , P < 0.001. n.s., not signiﬁcant.

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Figure 5. The miR-489:ADAM9/MMP7 axis is functionally important for regulating migration and metastasis in PDAC cells. A–C, ADAM9 and MMP7 overexpression rescued the migration of PDAC cells in vitro. BxPC-3 cells were cotransfected with pSIF-miR-489 and pCMV-myc-ADAM9 (myc-ADAM9), pCMV-myc-MMP7 (myc-MMP7), or their control vector pCMV-myc, and the assays were performed 24 hours posttransfection. A, Wound-healing assays. Left, representative images at 0 and 16 hours after wounding (scale bar, 100 mm); right, quantification of wound closure 16 hours after wounding. B, Transwell migration assays. Left, representative images (scale bar, 40 mm); right, quantification of migrating cell numbers 24 hours after the cells were plated. C, 3D cell culture assays, with images showing spheres on day 19 after the cells were grown in 3D cell culture (scale bar, 300 mm). The inset shows an amplified view of pCMV-myc-ADAM9- or pCMV-myc-MMP7– transfected BxPC-3 cells invading the surrounding Matrigel (scale bar, 150 mm). D and E, ADAM9 and MMP7 overexpression rescued lung metastatic colonization of PDAC cells in NOD/SCID mice. D, Bioluminescence imaging of NOD/SCID mice on day 50 after tail vein injection of BxPC-3-luc cells cotransfected with pSIF-miR-489 and pCMV-myc-ADAM9, pCMV-myc-MMP7, or the control vector pCMV-myc. Left, representative images; right, bioluminescence quantification of lung metastasis (mean SD; n ¼ 5 mice in each group). E, H&E staining of the lungs (left; scale bar, 2 mm) and the number of metastatic nodules in mice on day 50 after tail vein injection of the indicated PDAC cells. Data in the right are presented as mean SD (n ¼ 5 mice in each group). F and G, ADAM9 and MMP7 overexpression rescued liver metastatic colonization of PDAC cells in NOD/SCID mice. F, Bioluminescence imaging of NOD/SCID mice on day 60 after splenic injection of BxPC-3-luc cells cotransfected with pSIF-miR-489 and pCMV-myc-ADAM9, pCMV-myc-MMP7, or the control vector pCMV-myc. Left, representative images; right, bioluminescence quantification of liver metastasis (mean SD; n ¼ 4 mice in each group). G, Brightfield imaging of the livers (left; scale bar, 5 mm) and the number of visible liver metastases (right) in mice on day 60 after splenic injection of the indicated PDAC cells. Data on the right are presented as mean SD (n ¼ 4 mice in each group). All data are mean SD. Statistics: Student t test, , P < 0.05; , P < 0.01; , P < 0.001.

inhibitory effects on PDAC metastasis by targeting ADAM9 2-(4-chlorine-3-triﬂuoromethyl phenyl)-sulfonamido-4-phe- and MMP7. Intriguingly, either ADAM9 or MMP7 knockdown nylbutyric acid (SCTPSPA; ref. 34) substantially suppressed by shRNA in PANC-1 cells barely altered cell proliferation and the lung metastasis of PANC-1-luc cells (Supplementary Fig. anchorage-independent growth (Supplementary Fig. S4A and S4J and S4K). These results indicate that inhibition of ADAM9 S4B) but robustly decreased cell migration in wound-healing, and MMP7 recapitulates the inhibitory effect of miR-489 on Transwell migration, and 3D cell culture migration assays the migration and metastasis of PDAC cells. To verify the (Supplementary Fig. S4C–S4E) and signiﬁcantly suppressed functional role of the miR-489:ADAM9/MMP7 regulatory axis both lung and liver metastases in NOD/SCID mice (Supple- in PDAC migration and metastasis, we constructed miR-489– mentary Fig. S4F–S4I). Interestingly, we found that either resistant expression vectors (pCMV-myc-ADAM9 and pCMV- ADAM9 inhibitor SI-27 (32, 33) or MMP7 inhibitor sulfur- myc-MMP7, without their 30UTRs) for ectopic expression of

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Figure 6. Comparison of miR-489 expression and YY1, ADAM9,andMMP7 expression in human PDAC specimens. A, miR-489 expression was further reduced in human PDAC specimens from metastatic patients, whereas expression of YY1, ADAM9, and MMP7 was enhanced. qRT-PCR analyses of miR-489 and YY1, ADAM9,and MMP7 mRNA expression in 30 primary tumor and paired adjacent normal tissue specimens from nonmetastatic or metastatic patients. B, Pearson correlation analyses of miR-489 and YY1 mRNA (left), ADAM9 mRNA (middle), or MMP7 mRNA (right) in 60 human PDAC specimens. C, Immunohistochemical staining of YY1, ADAM9, and MMP7 (brown) and in situ hybridization of miR-489 (blue) in human PDAC sections with wild-type KRAS (left, n ¼ 10) or mutant KRAS (right, n ¼ 10). Sections were counterstained with Mayer hematoxylin (blue) for immunohistochemical staining or nuclear fast red sodium salt (light red) for in situ hybridization analyses. Four of 10 stained tumor specimens in each group are shown as representative images. Scale bars, 50 mm. D, Model of miR-489 as a key regulatory node linking oncogenic KRAS mutations to PDAC metastasis. All data are mean SD. Statistics: Student t test, , P < 0.05; , P < 0.01; , P < 0.001.

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ADAM9 and MMP7 proteins. Cotransfection of these vectors multiple lines of evidence, including in vitro cell migration and in PANC-1 cells barely altered cell proliferation and anchor- invasion assays, in vivo xenograft experiments, and human PDAC age-independent growth (Supplementary Fig. S5) but largely specimens. rescued the impact of miR-489 on cell migration in vitro We found for the first time that the transcription factor YY1 acts as well as lung and liver metastases in NOD/SCID mice downstream of KRAS/NF-kB signaling to suppress the expression (Fig. 5), supporting the notion that targeting ADAM9 and of miR-489. YY1 is a ubiquitously distributed transcription factor MMP7 is an important mechanism of miR-489–mediated that directly binds to the promoter region of its target genes (40). suppression of cell migration and metastasis of PDAC cells. Consistently, our results showed that YY1 inhibits miR-489 Collectively, these results indicate that the miR-489:ADAM9/ expression by binding to 2 closely spaced sites that are located MMP7 axis is of functional importance in regulating PDAC near the MIR489 transcription start site and that this negative metastasis. regulation greatly attenuates the expression of miR-489, thus promoting PDAC metastasis. These findings enriched our under- The expression of miR-489 and YY1, ADAM9, and MMP7 standing of the mechanism of KRAS signaling and YY1 function in mRNAs is correlated in human PDAC specimens cancer development, especially in PDAC metastasis progression. To test whether our above findings in PDAC cells are clin- Previous studies have shown that YY1 acts as an oncogene to ically relevant, we examined the miR-489 level as well as the promote cancer development by regulating the cell cycle, cell levels of YY1, ADAM9,andMMP7 mRNAs in 30 human apoptosis, chemoresistance, inflammation, and cell invasion (40, primary PDAC tumors from nonmetastatic and metastatic 41), which is in keeping with our finding of upregulation of YY1 patients, respectively. We found that compared with paired and YY1 downregulation of miR-489 to promote PDAC cell adjacent normal tissue, the miR-489 level was reduced in metastasis. In addition, dysregulation of YY1 in PDAC and several primary tumors from nonmetastatic patients and further other types of cancers has been correlated with the poor prognosis reduced in those from metastatic patients, whereas YY1, of patients (41–44). ADAM9,andMMP7 mRNAs were all significantly upregulated The important role of miRNAs in tumorigenesisiswellestab- in nonmetastatic primary tumors and further upregulated in lished. An increasing body of evidence has demonstrated that metastatic primary tumors (Fig. 6A). Importantly, we found a miRNAs control nearly all aspects of tumorigenesis, including significant inverse correlation between miR-489 and YY1 cancer cell proliferation, apoptosis, and metastasis (11). Here, mRNA levels (Pearson R ¼0.797, P < 0.001), ADAM9 mRNA we uncovered the role of miR-489 as a critical suppressor of levels (Pearson R ¼0.804, P < 0.001), or MMP7 mRNA levels PDAC metastasis, which is consistent with the global miRNAs (Pearson R ¼0.818, P < 0.001; Fig. 6B). We further found that profilingdatafromapreviousstudyshowingastrongnegative PDAC tumors with wild-type KRAS showed greater miR-489 correlation of miR-489 expression in human PDAC tumors with expression and diminished YY1, ADAM9, and MMP7 immu- liver metastasis (45). In addition, miR-489 has been found to be nohistochemical staining (Fig. 6C, left; n ¼ 10), whereas downregulated in breast, ovarian, and non–small cell lung tumors with mutant KRAS showed a significantly lower level cancers (46–48). Of note, upregulation of miR-21 by oncogenic of miR-489 expression and more intense YY1, ADAM9, and KRAS have been shown to promote pancreatic tumor growth MMP7 staining (Fig. 6C, right, n ¼ 10; Supplementary Fig. S6). (22). Interestingly, our findings show that oncogenic KRAS Collectively, these results strongly suggest that the newly dis- signaling employs miR-489 to specifically promote metastasis. covered YY1/miR-489:ADAM9/MMP7 regulatory axis is clini- More importantly, we found that miR-489 expression was well cally relevant in human PDAC. correlated with the progression of PDAC, which is consistent with the report that miR-489 may be a useful biomarker for Discussion PDAC clinicopathologic parameters (45). Moreover, in light of the role of miR-489 in PDAC metastasis, this miRNA is also a KRAS mutations occur with a frequency of up to 90% in potential candidate for designing novel therapeutics targeting pancreatic cancer and as early as the PanIN stage (4). Mutant the metastasis of PDAC. KRAS, alone or in conjunction with other mutant genes such as We went further to show that miR-489 negatively regulates p53 or p16, drives initiation of pancreatic cancer by inducing PDAC metastasis by targeting 2 key metastasis mediators, ADAM9 acinar-to-ductal transformation and maintaining progression to and MMP7. These genes encode metalloproteinases that remodel metastasis (5, 35). Moreover, both basic and clinical studies the extracellular matrix, thereby facilitating cancer cells in forming have pointed out that many risk factors influence PDAC pro- a local or distant metastasis and are highly expressed in many gression by inducing inflammation, which accompanies cancer kinds of cancers, including PDAC (49, 50). Given the important from initiation to metastasis (36).NF-kB signaling has been role of ADAM9 and MMP7 in regulating metastasis, the regulation recognized as one of the most important pathways that link of these 2 important metalloproteinases by the KRAS/NF-kB/YY1/ inflammation to cancer, including PDAC, which confers a recip- miR-489 axis may be critical to PDAC metastasis. rocal interaction between inflammation and tumor progression In summary, our study defined a regulatory axis centered on (37, 38). The upregulation of p65, the most common NF-kB miR-489 that plays an important role in oncogenic KRAS-driven family member, greatly induces the constitutive activation of PDAC metastasis. Mechanistically, miR-489 targets 2 metallo- NF-kB signaling and promotes PDAC progression into metas- proteinase genes, ADAM9 and MMP7, to inhibit metastasis. tasis, but the key downstream molecular events still remain Oncogenic KRAS signaling activates inflammatory NF-kBsignal- largely elusive (8, 10, 39). In the present study, we established ing to upregulate YY1, the transcriptional repressor of MIR489, miR-489 as a key regulator of PDAC metastasis, acting down- thus attenuating the level of miR-489 and thereby promo- stream of KRAS/NF-kB signaling (Fig. 6D). The importance of ting tumor progression and metastasis. This study not only miR-489 in mediating PDAC metastasis was supported by identified an miRNA-based mechanism underlying oncogenic

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KRAS–associated inflammation that promotes PDAC metastasis Acknowledgments but also provided a potential candidate molecule with which to We thank Prof. Xinyuan Liu for pGL3-basic and pmirGLO vectors and design oligonucleotide drugs targeting PDAC metastasis. Prof. Ying Yu for help in animal luminescence imaging. The authors are grateful to Dr. Yonggang Zheng for his critical reading of this article. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Grant Support This work was supported by grants from the National Natural Science Authors' Contributions Foundation of China (31325008, 91640201, 91419307, 31300656, and 31270840), Ministry of Science and Technology of China (2014CB943103, Conception and design: P. Yuan, Y. Li, W. Lou, M.-F. Liu 2014CB964802, and 2012CB910803), Science and Technology Commission of Development of methodology: P. Yuan, X.-H. He, Y.-F. Rong, W. Lou, M.-F. Liu Shanghai Municipality (13ZR1464300 and 16XD1404900), and Chinese Acad- Acquisition of data (provided animals, acquired and managed patients, emy of Sciences (KJZD-EW-L01-2, 2013KIP202, and "Strategic Priority Research provided facilities, etc.): P. Yuan, X.-H. He, Y.-F. Rong, J. Cao, Y. Hu Program" Grant XDB19010202). Analysis and interpretation of data (e.g., statistical analysis, biostatistics, The costs of publication of this article were defrayed in part by the payment of computational analysis): P. Yuan, X.-H. He, Y.-F. Rong, Y.-P. Hu, M.-F. Liu page charges. This article must therefore be hereby marked advertisement in Writing, review, and/or revision of the manuscript: P. Yuan, Y. Li, Y. Liu, D. Li, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. W. Lou, M.-F. Liu Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Liu Received July 15, 2016; revised October 15, 2016; accepted October 19, 2016; Study supervision: W. Lou, M.-F. Liu published OnlineFirst October 28, 2016.

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Correction: KRAS/NF-kB/YY1/miR-489 Signaling Axis Controls Pancreatic Cancer Metastasis Peng Yuan, Xiao-Hong He, Ye-Fei Rong, Jing Cao, Yong Li, Yun-ping Hu, Yingbin Liu, Dangsheng Li, Wenhui Lou, and Mo-Fang Liu

In the original version of this article (1), there are errors in Figs. 1D and H and 5B. Specifically, in Fig. 1D, the same migration assay image was used in the second and seventh panels, and an overlapping image was used in the third and fourth panels. In addition, the same migration assay image was used in the fifth panel of Fig. 1D and in the fifth panel of Fig. 1H. Finally, an overlapping image was used in the second and third panels of Fig. 5B. The errors have been corrected in the latest online HTML and PDF versions of the article. The authors regret these errors.

Reference 1. Yuan P, He XH, Rong YF, Cao J, Li Y, Hu YP, et al. KRAS/NF-kB/YY1/miR-489 signaling axis controls pancreatic cancer metastasis. Cancer Res 2017;77:100–11.

Published online September 15, 2020. Cancer Res 2020;80:4022 doi: 10.1158/0008-5472.CAN-20-2503 2020 American Association for Cancer Research.

AACRJournals.org | 4022 Published OnlineFirst October 28, 2016; DOI: 10.1158/0008-5472.CAN-16-1898

KRAS/NF-κB/YY1/miR-489 Signaling Axis Controls Pancreatic Cancer Metastasis

Peng Yuan, Xiao-Hong He, Ye-Fei Rong, et al.

Cancer Res 2017;77:100-111. Published OnlineFirst October 28, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-1898

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