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. Ó2016 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, Ó2016 American Association for Cancer Research. our findings not only provide new mechanistic insights into how 100 Cancer Res; 77(1) January 1, 2017 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst October 28, 2016; DOI: 10.1158/0008-5472.CAN-16-1898 MIR489 Is a Key Regulator for PDAC Metastasis 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 Â 104)or technology), anti-p65 (10745-1-AP, Proteintech), anti-ADAM9 BxPC-3-luc cells (1 Â 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 Â 105) orBxPC-3-luc cells (5 Â 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