Published OnlineFirst February 14, 2017; DOI: 10.1158/1078-0432.CCR-16-2530 Biology of Human Tumors Clinical Cancer Research MUC16 Regulates TSPYL5 for Lung Cancer Cell Growth and Chemoresistance by Suppressing p53 Imayavaramban Lakshmanan1, Shereen Salfity2, Parthasarathy Seshacharyulu1, Satyanarayana Rachagani1, Abigail Thomas2, Srustidhar Das1, Prabin D. Majhi1, Rama Krishna Nimmakayala1, Raghupathy Vengoji1, Subodh M. Lele3, Moorthy P. Ponnusamy1,4, Surinder K. Batra1,4, and Apar Kishor Ganti2,5 Abstract Purpose: MUC16, a tumor biomarker and cell surface–associ- (P < 0.001). Transcriptome analysis of MUC16 KD showed ated mucin, is overexpressed in various cancers; however, its role adownregulation(P ¼ 0.005) of TSPYL5 gene, which encodes in lung cancer pathogenesis is unknown. Here, we have explored for a testis-specific Y-like protein. Rescue studies via over- the mechanistic role of MUC16 in lung cancer. expression of MUC16-Cter in MUC16 KD cells showed acti- Experimental Design: To identify the functional role of vation of signaling proteins, such as JAK2 (Y1007/1008), MUC16, stable knockdown was carried in lung cancer cells STAT3 (Y705), and glucocorticoid receptor (GR), which con- with two different shRNAs. Clinical significance of MUC16 was stitutes an important axis for the regulation of TSPYL5 for evaluated in lung cancer patient tissues using IHC. We have oncogenic process. Further, inhibition of STAT3 (Y705) led to generated genetically engineered mouse model (KrasG12D; AdCre) decreased GR and TSPYL5, suggesting that MUC16 regulates to evaluate the preclinical significance of MUC16. TSPYL5 through the JAK2/STAT3/GR axis. Also, MUC16 over- Results: MUC16 was overexpressed (P ¼ 0.03) in lung expression induced cisplatin and gemcitabine resistance by cancer as compared with normal tissues. MUC16 knockdown downregulation of p53. (KD) in lung cancer cell lines decreased the in vitro growth rate Conclusions: Our findings indicate a significant role of (P < 0.05), migration (P < 0.001), and in vivo tumor growth MUC16 in tumorigenesis and metastasis of lung cancer cells (P ¼ 0.007), whereas overexpression of MUC16-carboxyl possibly via regulation of TSPYL5 through the JAK2/STAT3/GR terminal (MUC16-Cter) resulted in increased growth rate axis. Clin Cancer Res; 23(14); 3906–17. Ó2017 AACR. Introduction domain with potential phosphorylation sites (3, 4). The N-ter- minal portion of MUC16 interacts with mesothelin that facilitates MUC16 mucin is a large-molecular-weight (20 to 25 mD) the peritoneal metastasis of ovarian cancer cells (5, 6). The SEA glycoprotein with 22,152 amino acid (aa) residues in its protein domain of the tandem repeat region is responsible for the cleavage sequence (1–3). MUC16 is a type I transmembrane protein that process (7), whereas carboxyl-terminal region contains 32 aa has three major domains: highly O-glycosylated N-terminal comprising of three tyrosine, two threonine, and one serine domain, repetitive sea urchin sperm, enterokinase and agrin residues, which serve as potential phosphorylation sites for intra- (SEA) containing tandem repeat domain, and a cytoplasmic cellular signaling (8, 9). MUC16 is overexpressed and associated with poor prog- nosis in ovarian (10), breast (11), and pancreatic cancer 1Department of Biochemistry and Molecular Biology, University of Nebraska (12). MUC16 is elevated in patients with multiple brain Medical Center, Omaha, Nebraska. 2Department of Internal Medicine, University – 3 metastases from non smallcelllungcancer(NSCLC),andit of Nebraska Medical Center, Omaha, Nebraska. Department of Pathology and is associated with poor prognosis (13). Another study has Microbiology, University of Nebraska Medical Center, Omaha, Nebraska. 4Eppley Institute for Research in Cancer and Allied Diseases Fred & Pamela Buffett shown that MUC16 is elevated in stage I NSCLC patients' Cancer Center University of Nebraska Medical Center, Omaha, Nebraska. serum samples and demonstrated that MUC16 could be a 5Department of Internal Medicine, VA Nebraska-Western Iowa Health Care useful biomarker for patients with lung cancer (14). Recent System and University of Nebraska Medical Center, Omaha, Nebraska. studies have reported that MUC16 is an extremely highly Note: Supplementary data for this article are available at Clinical Cancer mutated gene, in various cancers including lung cancer (15). Research Online (http://clincancerres.aacrjournals.org/). MUC16 has been shown to be associated with enhanced Corresponding Authors: Apar Kishor Ganti, University of Nebraska Medical cancer cell growth and metastasis (4, 8). During this process, Center, 987680 Nebraska Medical Center, Omaha, NE 68198-7680. Phone: 402- MUC16 interacts with various proteins such as mesothelin 559-6210; Fax: 402-559-6520; E-mail: [email protected]; or Surinder K. Batra, (5), JAK2 (11), and Src (8), and their association facilitates Department of Biochemistry and Molecular Biology, University of Nebraska cancer cell growth and metastasis. Medical Center, Omaha, Nebraska, 68198-5870. Phone: 402-559-5455, Fax: Due to its large size, several studies have been conducted 402-559-6650; E-mail: [email protected] with a small portion of the carboxyl terminal (Cter) of MUC16 doi: 10.1158/1078-0432.CCR-16-2530 (344 aa and 114 aa; refs. 4, 8, 16). It has been reported that Ó2017 American Association for Cancer Research. MUC16-Cter has a strong oncogenic role in ovarian (8) and 3906 Clin Cancer Res; 23(14) July 15, 2017 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst February 14, 2017; DOI: 10.1158/1078-0432.CCR-16-2530 MUC16, TSPYL5, and Lung Cancer performed in accordance with the U.S. Public Health Service Translational Relevance "Guidelines for the Care and Use of Laboratory Animals" under Although MUC16 has been shown to be involved in the an approved protocol by the Institutional Animal Care and Use growth and metastasis of several cancers, its role in lung Committee of the University of Nebraska Medical Center carcinoma remains unclear. Herein, we have shown sub- (UNMC). The mouse tumor tissues were utilized for immunos- type-specific expression of MUC16 in lung adenocarcinoma. taining as described previously (24). MUC16 expression seems to increase the aggressiveness of lung cancer cells. In addition, MUC16 appears to mediate TMA and immunohistochemistry chemoresistance via expression of TSPYL5 and consequent The clinical specimen for IHC was a commercial tissues micro- inactivation of p53 (wild-type) in lung cancer. Targeting the array (TMA; LC121 and LC 814; US Biomax). The LC121 included MUC16/TSPYL5 pathway may help in decreasing the aggres- 120 cases of various histologic types of lung carcinoma [squamous siveness and metastatic potential of lung cancer cells and in cell carcinoma (n ¼ 20), large cell carcinoma (n ¼ 37), adenocar- overcoming chemoresistance, thereby improving outcomes. cinoma (n ¼ 44), and normal lung tissues (n ¼ 10)]. Similarly, LC814 included 40 cases of lung carcinoma (n ¼ 40) and meta- static lymph node carcinoma (n ¼ 40). The TMA was analyzed for MUC16 expression by IHC as described previously (24). pancreatic cancers (4). However, the mechanistic and function- al role of MUC16 in lung cancer is not well understood. Immunoblot analysis Testis-specific Y-like protein 5 TSPYL5 ( ) gene is located at chro- Western blot assay was performed as described previously (24). fi mosome 8q22 (17); it has been frequently ampli ed in breast The blots were incubated with following primary antibodies with cancer and is associated with a poor prognosis (18). Epping and respective dilutions: MUC16 (mouse, 1:1,000), MUC16 (mouse, colleagues have demonstrated that TSPYL5 interacts with ubiqui- 1:1,000), pJAK2 #8082, JAK2 #3230, pSTAT3 #9145, STAT3 fi tin-speci c protease 7 (USP7) that facilitates p53 degradation to #12640, GR #12041, pSrc #2101 (Rabbit, 1:2,000; Cell Signaling suppress the tumor-suppressor activity of p53 (17). TSPYL5 has Technology), E-cadherin (mouse, 1:1,500), and N-cadherin been shown to be involved in cancer cell growth by activating Akt (mouse, 1:1,500) antibodies were a kind gift from Dr. Keith R signaling and was found to be involved in radioresistance in lung Johnson, UNMC, Omaha, NE; CK-18 (mouse, 1:1,500; Abcam cancer cells (19). The nuclear hormone receptor family protein, #668), TSPYL5 (rabbit 1:500; Santa Cruz Biotechnology, #sc- glucocorticoid receptor (GR), is overexpressed in lung cancer and 98185), p53 (mouse 1:500; Santa Cruz Biotechnology, #sc- promotes cancer cell proliferation (20). Ligand (glucocorticoid) 126), and anti–b-actin (mouse 1:5,000; Sigma #A1978, diluted binding to GR leads to translocation of GR from cytoplasm to in 2% BSA in PBS). Similarly, immunoprecipitation assay was nucleus, where it directly binds to DNA and is involved in gene performed as described previously (22). The signals were detected regulation (21). In this study, we evaluated the role of MUC16 in with the ECL chemiluminescence Kit (Amersham Bioscience). the growth, proliferation, spread, and chemosensitivity of lung cancer. Quantitative real-time PCR, growth kinetics, transwell Materials and Methods migration, and wound-healing assay qPCR, growth kinetics, transwell migration, and wound-heal- Cell culture and transfection ing assays were performed as described previously (11, 24). H292, H1975, and A549 lung cancer cells were cultured in RPMI medium supplemented with 10% FBS and antibiotics. Phosphorylation-specific