Cancer Letters

Cancer Letters

Volume 506, 28 May 2021, Pages 67-82
Cancer Letters

LACTB suppresses melanoma progression by attenuating PP1A and YAP interaction

https://doi.org/10.1016/j.canlet.2021.02.022Get rights and content

Highlights

  • LACTB suppresses the malignant phenotypes of melanoma both in vitro and in vivo.

  • LACTB inhibits YAP activity by increasing its phosphorylation at S127.

  • LACTB attenuates the interaction of PP1A with YAP, leading to decreased dephosphorylation of YAP.

  • SOX10 binds to the promoter of LACTB and negatively regulates its expression.

Abstract

Very limited progress has been made in the management of advanced melanoma, especially melanoma of uveal origin. Lactamase β (LACTB) is a novel tumor suppressor; however, its biological function in melanoma remains unknown. Herein we demonstrated markedly lower LACTB expression levels in melanoma tissues and cell lines. Overexpression of LACTB suppressed the proliferation, migration and invasion of melanoma cells in vitro. Mechanistically, LACTB inhibited the activity of yes-associated protein (YAP). We showed that the level of phospho-YAP (Serine 127) was increased upon LACTB overexpression, which prevented the translocation of YAP to the nucleus. Further, LACTB could directly bind to PP1A and attenuate the interaction between PP1A and YAP, resulting in decreased YAP dephosphorylation and inactivation in a LATS1-independent manner. Additionally, transfection of phosphorylation-defective YAP mutants reversed LACTB-induced tumor suppression. Upstream, we demonstrated that SOX10 binds to the LACTB promoter and negatively regulates its transcription. Overexpression of LACTB also suppressed the tumorigenicity and lung metastasis of MUM2B uveal melanoma cells in vivo. Taken together, our findings indicate a novel SOX10/LACTB/PP1A signaling cascade that renders YAP inactive and modulates melanoma progression, offering a new therapeutic target for melanoma treatment.

Introduction

Melanoma is a malignancy that originates in melanocytes [1]. This cancer can affect many sites of the human body including the skin, uveal tract, conjunctiva, rectum and mucosal surfaces [2,3]. Over the past decades, the development of new, targeted therapeutic agents and advancement of immunotherapy have achieved prolonged survival in melanoma patients [4,5]. However, due to frequent metastasis to distant organs, such as the lung, brain and liver, melanoma can still be life-threatening [6]. For example, approximately half of uveal melanoma patients develop liver metastasis and die within a year [7]. To date, once metastasis occurs, there are limited effective systemic therapies to save patients’ lives. Therefore, there is an urgent need to discover new targets for melanoma treatment.

Lactamase β (LACTB) is the only penicillin-binding protein homolog of mammals that is widely expressed in the liver, heart, skeletal muscle and many other tissues [8]. LACTB was reported to be localized to the mitochondrial intermembrane space and participate in submitochondrial organization and cell metabolic processes [9]. In 2017, LACTB was first demonstrated to suppress the proliferation and differentiation of breast cancer cells by modulating lipid metabolism [10]. Successively, LACTB was also found to exhibit tumor repression function in colorectal cancer by attenuating MDM2-mediated degradation of P53 [11]. In addition, it inhibited the proliferation of glioma cells and hepatocellular carcinoma cells [12,13]. However, whether LACTB plays a crucial role in the development of melanoma requires more exploration.

The Hippo pathway has long been acknowledged to regulate organ size and tissue homeostasis [14]. In the process of cancer development, Hippo pathway is also intensely involved [15]. The major molecular components of the Hippo signaling pathway include mammalian sterile 20-like 1/2 (MST1/2), large tumor suppressor homolog 1/2 (LATS1/2), yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) and TEA domain transcription factors (TEADs). Among these molecules, YAP is a transcriptional coactivator that regulates gene transcription by interacting with TEADs. The essential role of YAP in cancer initiation and progression is well known [16]. Sustained YAP activation facilitates cell proliferation, survival, migration and invasion in various kinds of human cancers [17]. Specifically, in cutaneous melanoma increased YAP activity promotes metastasis while YAP drives uveal melanoma initiation and progression [[18], [19], [20]]. LATS1/2 acts upstream of YAP. Phosphorylation of YAP at serine 127 (S127) by LATS1/2 causes its cytosolic retention and restricts its activity [21]. The Hippo pathway is regulated by various signals including soluble factors such as GPCRs and metabolic status [22]. Many other proteins such as phosphatases (PP1, PP2A and PTPN14), also impact the activity of different components of the Hippo pathway by dephosphorylating them [23]. Previous studies reported that the catalytic subunit alpha of protein phosphatase-1 (PP1A) interacts with and dephosphorylates YAP/TAZ as well as LATS1 [[24], [25], [26]]. However, the positive regulation of YAP by PP1A in melanoma has seldomly been reported.

Sex-determining region Y (SRY)-related HMG box-containing factor 10 (SOX10) is a vital transcription factor that is required for inducing the melanocyte lineage [27]. SOX10 is essential in the initiation of melanoma and is a reliable biological marker for primary and metastatic melanoma [28]. Loss of SOX10 led to cell cycle arrest of melanoma cells [29]. In addition, SOX10 promotes melanoma cell migration and invasion, further suggesting a regulatory role for SOX10 in melanoma development and patient survival [30].

In the present study, we demonstrated excessively low LACTB expression in tissue samples of melanoma and various kinds of melanoma cell lines. LACTB exerted a tumor suppressive effect on melanoma both in vitro and in vivo. In addition, we elucidated the molecular mechanism underlying the tumor suppressive effect of LACTB. LACTB overexpression prevented YAP from translocating to the nucleus by attenuating its interaction with PP1A and its consequent dephosphorylation. Upstream, we identified SOX10 as the regulatory transcription factor of LACTB. Our findings provide rational for the further study of this novel tumor suppressor as a therapeutic target for melanoma.

Section snippets

Chemicals and antibodies

Annexin V was obtained from Sigma-Aldrich (Shanghai, China). Okadaic acid was purchased from MedChemExpress (Wuhan, China). The antibodies used in this study were as follows: anti-YAP (#4912), anti-LATS1 (#9153), anti-phospho-LATS1 (Thr1079) (#8654), anti-LATS2 (#5888) and normal rabbit IgG (#2729) were from Cell Signaling Technology (MA, USA); anti-YAP1 (phospho-S127) (ab76252), anti-PP1A (ab137512), anti-H3 (ab1791) and anti-SOX10 (ab155279) were purchased from Abcam (Shanghai, China);

LACTB is markedly downregulated in melanoma and has prognostic significance

Aiming to investigate the role of LACTB in melanoma, we first detected its expression in tissue sections from melanoma patients. The results of immunofluorescent staining showed downregulated LACTB in specimens from melanoma patients compared with that in normal skin (Fig. 1A). Besides, we conducted survival analysis online at Gene Expression Profiling Interactive Analysis (GEPIA). A total of 536 melanoma patients were included in the analysis. The patients were divided into low LACTB

Discussion

In the present study, we demonstrated that LACTB was downregulated in melanoma and that lower LACTB expression correlated with shorter survival time. Our data indicated that ectopic expression of LACTB suppressed the proliferation, migration and invasion of melanoma cells. In addition, cell apoptosis and cell cycle arrest at the G2/M phase were induced upon LACTB overexpression. In vivo subcutaneous xenograft model and lung metastatic animal model also confirmed the tumor suppressive potency of

Credit author statement

Yawen Ma: Conceptualization, Methodology, Investigation, Writing-Original Draft Lihua Wang: Conceptualization, Investigation, Writing- Review& Editing Fanglin He: Visualization, Resources Jie Yang: Validation, Resources Yi Ding: Visualization Shengfang Ge: Supervision, Writing - Review& Editing Xianqun Fan: Supervision Yixiong Zhou: Investigation, Software, Writing - Review& Editing Xiaofang Xu: Investigation, Visualization, Writing - Review& Editing Renbing Jia: Writing - Review& Editing,

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by Shanghai Municipal Science and Technology Major Project (19JC1410200), Innovative research team of high-level local universities in Shanghai (SSMU-ZDCX20180401) and the National Natural Science Foundation of China (81902804).

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      Based on previous research, LACTB is considered as a tumor suppressor in breast cancer, possibly through the regulation of lipid metabolism (Keckesova et al., 2017). In addition, LACTB dysregulation in melanoma, gastric cancer, glioma cancer, and colorectal cancer patients can serve as a marker for tumor development and prognosis (Jiang et al., 2020; Li et al., 2019; Ma et al., 2021; Wang et al., 2020; Xu et al., 2020; Yang et al., 2021a, 2021b; Zeng et al., 2018). Moreover, LACTB overexpression has been shown to contribute to several cancers, including nasopharyngeal carcinoma and pancreatic adenocarcinoma (Peng et al., 2021; Xie et al., 2021).

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    1

    Yawen Ma, Lihua Wang and Fanglin He contributed equally to this work.

    2

    Present address: Department of Ophthalmology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, No. 639, Zhi Zao Ju Road, Shanghai, 200,011, China.

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