USP46 Inhibits Cell Proliferation in Lung Cancer Through PHLPP1/AKT Pathway

Hindawi BioMed Research International Volume 2020, Article ID 2509529, 10 pages https://doi.org/10.1155/2020/2509529 Research Article USP46 Inhibits Cell Proliferation in Lung Cancer through PHLPP1/AKT Pathway Wei Wang,1,2 Meng Chen,1,2,3 Hailing Xu,1,4 Dongqing Lv,1,4 Suna Zhou,1,2 and Haihua Yang 1,2,3 1Laboratory of Cellular and Molecular Radiation Oncology, Radiation Oncology Institute of Enze Medical Health Academy, Affiliated Taizhou Hospital of Wenzhou Medical University, Taizhou, 317000 Zhejiang Province, China 2Department of Radiation Oncology, Affiliated Taizhou Hospital of Wenzhou Medical University, Taizhou, 317000 Zhejiang Province, China 3School of Medicine, Shaoxing University, Shaoxing City, 312000 Zhejiang Province, China 4Department of Pulmonary Medicine, Enze Hospital, Affiliated Taizhou Hospital of Wenzhou Medical University, Taizhou, 317000 Zhejiang Province, China Correspondence should be addressed to Haihua Yang; [email protected] Received 24 April 2020; Revised 3 August 2020; Accepted 10 September 2020; Published 24 September 2020 Academic Editor: Michael Linnebacher Copyright © 2020 Wei Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Previous studies have shown that ubiquitin-specific protease 46 (USP46) is a tumor suppressor in colon cancer and renal cell carcinoma. However, its specific role in other cancers is still poorly understood. This study is aimed at investigating the role of USP46 in lung cancer tumorigenesis and identifying its underlying mechanisms. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB) were used to measure the expression levels of USP46 and PHLPP1 in lung cancer tissue and adjacent normal tissue from patients with lung cancer. We examined the ability of USP46 to regulate cell proliferation in lung cancer cells via cell proliferation assay, radiation assay, genetic overexpression and knockdown, and chemical inhibition of relevant genes. We investigated the underlying mechanisms in multiple lung cancer cell line models by coimmunoprecipitation and ubiquitination assays. In this study, we identified a strong downregulation of the expressions of USP46 and PHLPP1 in lung cancer tissues relative to normal adjacent tissues. USP46 was further shown to inhibit lung cancer cell proliferation under conditions of normal growth and during radiation-induced DNA damage by antagonizing the ubiquitination of PHLPP1 resulting in the inhibition of AKT signaling. Exposure to radiation and AKT inhibition significantly reversed the effect of USP46 siRNA on lung cancer cell proliferation. USP46 is downregulated in lung cancer and suppresses the proliferation of lung cancer cells by inhibiting the PHLPP1/AKT pathway. AKT inhibition slows the proliferation of lung cancer cells that have been downregulated by USP46 and exposed to radiation. This suggests a potential therapeutic avenue for USP46- downregulated lung cancer through a combination of radiation and AKT inhibitor treatment. 1. Introduction Protein posttranslational modifications play important roles in regulating numerous biological processes, such as Lung cancer is the most deadly form of cancer for the sexes the cell cycle, DNA damage repair, and apoptosis [3]. Ubi- and has one of the worst survival rates [1]. Every year, about quitination is one of the most ubiquitous posttranslational 1.6 million people are estimated to die from lung cancer [2]. modifications and is dynamically regulated by ubiquitin Recent advancements in cancer therapies have improved the ligases and deubiquitinase [4]. Ubiquitin-specific protease survival rate of patients with lung cancer; however, there are 46 (USP46) is a member of the cysteine protease family still unmet needs in terms of therapies. A key step toward which functions as a deubiquitinase and has been associated developing effective therapies against lung cancer is elucidat- with neurological disorders [5] and behavioral abnormalities ing the molecular mechanisms underlying tumorigenesis. [6]. The vital role of USP46 in regulating neuronal signaling 2 BioMed Research International may be the most probable cause of these phenotypes. For LY294002 (25 μmol/L; S1105, Selleck, USA) was dissolved example, USP46 has been shown to regulate glutamate recep- in DMSO (D2650, Sigma, USA) and added to the cell culture. tor (GLR) function by regulating the abundance of GLR-1 [7]. Likewise, USP46 has been shown to deubiquitinate 2.3. Quantitative Real-Time Polymerase Chain Reaction alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (qRT-PCR). RNAs were isolated using a TRIzol reagent (Invi- receptors (AMPARs) leading to the stabilization of the recep- trogen, Waltham, USA). cDNA was synthesized by using a fi tor [8]. Recent studies have identified other substrates of cDNA synthesis kit (Thermo Fisher Scienti c, Waltham, USA) following the instructions of the manufacturer. The USP46 that have important functions in cancer progression. ° thermal cycle was set as follows: 95 C for 10 min followed However, the role of USP46 in lung cancer remains unclear. ° ° USP46 had been shown to deubiquitinate the PH domain by 40 cycles of 95 C for 15 s and 60 C for 45 s. All data repre- leucine-rich repeat protein phosphatase 1 (PHLPP1) in colo- sent an average of three replicates. The primers used were as rectal cancer [9]. The PHLPP1 ubiquitination primes its deg- follows: USP46 (NM_001134223.2): forward primer: 5′ radation, and deubiquitination thus protects PHLPP1 from -AGAAGCCCAGAAAAGGATGAGG-3′, reverse primer: degradation and enhances its activity in the cell [9]. One of 5′-CAAAAGCCAGAAGCCGTGAC-3′; PHLPP1 (NM_ the key substrates of PHLPP1 is Protein Kinase B (PKB, also 194449.4): forward primer: 5′-AATGTGCCTGAGTGGG called AKT). PHLPP1 removes activating phosphorylations TATGTG-3′, reverse primer: 5′-TCATCAGAA GGTTAG in AKT proteins thus antagonizing the activity of AKT [10]. GTGGGAG-3′; and beta-actin (HQ154074.1): forward AKT family proteins are serine/threonine kinases that primer: 5′-CGTGGACATCCGCAAAGAC-3′, reverse play critical roles in cell proliferation, DNA damage repair, ′ ′ and cell survival during conditions of stress [11]. In different primer: 5 -TGCTGGGAGCCAGAGCAG-3 . types of cancers, AKT family genes are frequently mutated, 2.4. Western Blotting (WB). A radioimmunoprecipitation and inhibitors against the kinases are used in the treatment assay (RIPA) lysis buffer (JRDUN, Shanghai, China) contain- of several cancers [12]. Previously, in colorectal cancer, ing an EDTA-free protease inhibitor cocktail (Roche, Heidel- USP46 had been shown to antagonize the activity of AKT berg, Germany) was used to extract whole protein lysates, by deubiquitinating PHLPP1 [9]. Consistent with the anti- followed by protein concentration measurement by an proliferation activity of USP46, the protein was found to be enhanced BCA protein assay kit (Thermo Fisher Scientific). downregulated in colorectal cancer [9], suggesting that Twenty-five micrograms of proteins from each sample was USP46 is an important tumor suppressor gene. USP46 has run in 10% SDS-PAGE gel and transferred to a nitrocellulose equally shown a tumor suppressor activity in renal cell carci- membrane (Millipore, Billerica, USA) overnight. After block- noma with a similar mechanism to that described in colorec- ing with 5% nonfat dry milk for 1 h at room temperature, the tal cancer [13]. However, the detailed relationship between ° membranes were probed at 4 C overnight with primary anti- USP46 and the PHLPP1/AKT pathway is still less investi- bodies followed by secondary anti-mouse IgG antibody gated in lung cancer. ° (1 : 1,000; Beyotime, Shanghai, China) for 1 h at 37 C. An Herein, we investigated the potential role of USP46 in enhanced chemiluminescence system (Tanon, Shanghai, lung cancer tumorigenesis and found that the expressions China) was used to visualize protein expressions. Detailed of USP46 and PHLPP1 were downregulated in lung cancer. information of primary antibodies is as follows: anti-USP46 Mechanistically, we found that USP46 inhibits cell prolifera- (Ab88795, Abcam, UK), anti-PHLPP1 (Ab135957, Abcam, tion in lung cancer cells by inhibiting the AKT pathway, in UK), anti-p-AKT (#9271, CST, USA), anti-AKT (#9272, part by preventing DNA damage repair. CST, USA), and anti-β-actin (#4970, CST, USA). 2.5. Knockdown and Overexpression of USP46. Lentiviral 2. Materials and Methods plasmids (pLKO.1) containing three siRNAs directed to dif- 2.1. Human Tissue Samples. Thirty pairs of lung cancer tis- ferent regions of human USP46 (NM_001134223.2) and a sues and adjacent-matched normal tissues were used to negative control siRNA (siNC) were synthesized (Major, investigate mRNA and protein levels of different genes. All Shanghai, China). The USP46 overexpression plasmid was patients provided informed and written consent. The inde- constructed by inserting the full-length human USP46 cDNA pendent ethics committee of the Affiliated Taizhou Hospital sequence into a lentiviral plasmid (pLVX-puro), while the of Wenzhou Medical University approved this study, and it vector plasmid was used as a negative control (oeNC). Exper- was in accordance with the Declaration of Helsinki. iments were performed 48 h after the transit transfection of the plasmids into lung cancer cells using Lipofectamine 2000 (Thermo Fisher Scientific) following the instructions 2.2. Cell Culture. Cell

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