Ling Zhi-8 reduces lung cancer mobility and metastasis through disruption of focal adhesion and induction of MDM2-mediated Slug degradation

Highlights

• Ling Zhi-8 reduces lung cancer metastasis and mobility.

• Ling Zhi-8 disrupts the focal adhesion and inhibits FAK activity.

• Ling Zhi-8 promotes MDM2-mediated ubiquitination of Slug degradation.

• Ling Zhi-8 potentially serves as an antitumor agent.

Abstract

We recently reported that recombinant Ling Zhi-8 (rLZ-8), a medicinal mushroom Ganoderma lucidum recombinant protein, effectively prevents lung cancer cells proliferation in vivo mice model. In our current study, we demonstrated that rLZ-8 suppressed tumor metastasis and increased the survival rate in Lewis lung carcinoma cell-bearing mice. The epithelial to mesenchymal transition (EMT) process is regarded as the critical event in tumor metastasis. Herein, we showed that rLZ-8 effectively induced changes in EMT by interfering with cell adhesion and focal adhesion kinase (FAK) functions in lung cancer cells. Slug, a transcription factor, represses E-cadherin transcription and is regarded as a critical event in EMT and tumor metastasis. Functional studies revealed that downregulation of Slug as a result of rLZ-8-induced FAK inactivation enhanced E-cadherin expression and repressed cancer cell mobility. Moreover, we found that rLZ-8 enhanced the ubiquitination proteasome pathway (UPP)-mediated degradation of Slug in CL1-5 cells. Mechanistically, we demonstrated that rLZ-8 promoted the interaction between MDM2 and Slug, resulting in Slug degradation; however, MDM2-shRNA abolished rLZ-8-enhanced Slug degradation. This study is the first to determine anti-metastatic activity of rLZ-8 and its potential mechanism, with how the regulation of EMT and cell mobility is via the negative modulation of FAK, and thereby leading to the ubiquitination and degradation of Slug. Our findings suggest that the targets of FAK play a key role in metastasis. Moreover, rLZ-8 may be useful as a chemotherapeutic agent for treating lung cancer.

Introduction

Ling-Zhi 8 (LZ-8) from Ganoderma lucidum is a protein that consists of 110 amino acid residues which share the similarities with the variable region of the immunoglobulin heavy chain [1], [2], [3]. Our previous study demonstrated that LZ-8 effectively prevents lung cancer cell proliferation via the mediation of the ribosomal protein S7-MDM2-p53 pathway [4]; however, the anti-metastasis mechanism of LZ-8 remains unclear.

Lung cancer is well-known with its highest mortality rate of all cancers and non-small-cell lung cancer (NSCLC) appears as the predominant type. Generally, patients with metastatic disease have lower survival rate [5], [6]. Therefore, inhibiting the metastasis represents the ultimate goal of treatment. The epithelial-to-mesenchymal transition (EMT) plays a key role in tumor metastasis [6], [7]. It is given that the EMT often refers to the process whereby cells change their morphology and develop with enhanced motility. Specifically, the EMT is characterized by a high correlation between a gain of mesenchymal-like markers, such as N-cadherin or vimentin, and a loss of epithelial-like cell junction proteins such as E-cadherin [8]. EMT regulators, for example Snail and Slug, mediate the transcriptional repression of E-cadherin and induce tumor cell metastasis [9], [10], [11].

Focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, involves in the regulation of cell motility, progression, metastasis, and survival [12], [13]. FAK expression and activity are upregulated in certain cancers, such as lung, breast, and head and neck cancers, and are correlated with malignant and metastatic disease [13], [14]. FAK is activated when cells bind to extracellular matrix proteins, otherwise they can be stimulated at the presence of growth factors, for instance, the activated FAK forms a complex with Src. Interestingly, growing evidence suggests that FAK/Src play a critical role in both the EMT promotion and the tumorigenic functions of intracellular signaling pathways, such as the MAPK-ERK pathway and the PI3K-Akt cascade [15], [16], [17]. The activation of these pathways is followed by the induction of repressors of E-cadherin, the subsequent downregulation of E-cadherin, the enhancement of tumor cell migration/invasion, and ultimately metastasis. However, much remains to be elucidated regarding the relationship between FAK and the EMT.

Overexpression of Slug in NSCLC patients is significantly associated with a lower overall survival rate [18]. Upregulated expression of the SNAI genes (Snail and Slug) has been detected upon the activation of several intracellular signaling pathways, such as the ERK, Akt, and Smad pathways [19]. Moreover, it has been shown that FAK-dependent regulation of Snail expression via PI3K and MAPK signaling pathways [20]. These findings suggest that pharmacological inhibition of FAK signaling pathways may prevent loss of E-cadherin function which is associated with the acquisition of invasive motility. However, the molecular basis of the linkage between the involvement of FAK and the stability of Slug in tumorigenesis remains unknown. Snail and Slug are degraded by ubiquitination via the proteasome pathway [18], [21], [22], resulting in E-cadherin expression. Therefore, we propose that rLZ-8 inhibits FAK signaling pathways, and thereby resulting in the decreased expression of Slug, concomitantly giving effect on the initiation of EMT and cell mobility in lung cancer cells.

In our current study, we explored the inhibitory effects of rLZ-8 on FAK functions as well as its capability to reduce the lung tumorigenesis and metastasis in both the human NSCLC cells and LLC1 xenograft mouse model. Our novel findings have suggested that rLZ-8 suppresses EMT via the inhibition of FAK-mediated signaling and it concurrently enhances the MDM2-mediated ubiquitination-dependent Slug degradation. Here, this report has supported our hypothesis and it will be the first report to reveal a novel function of the rLZ-8 in the prevention of lung cancer cell progression and metastasis.