An TRIM59‐CDK6 axis regulates growth and metastasis of lung cancer

Abstract Lung cancer (LC) is a devastating malignancy with no effective treatments, due to its complex genomic profile. Using bioinformatics analysis and immunohistochemical of lung carcinoma tissues, we show that TRIM59 as a critical oncoprotein relating to LC proliferation and metastasis. In this study, high TRIM59 expression was significantly correlated with lymph node metastasis, distant metastasis, and tumour stage. Furthermore, up‐regulation of TRIM59 expression correlated with poorer outcomes in LC patients. Mechanistically, TRIM59 play a key role in promoting LC growth and metastasis through regulation of extracellular‐signal regulated protein kinase (ERK) signalling pathway and epithelial‐to‐mesenchymal transition (EMT)‐markers, as validated by loss‐of‐function studies. In‐depth bioinformatics analysis showed that there is preliminary evidence of co‐expression of TRIM59 and cyclin dependent kinase 6 (CDK6) in LC. Notably, CDK6 expression significantly decreased when TRIM59 was knocked down in the LC cells. In contrast, exogenous up‐regulation of TRIM59 expression also induced significant increases in the expression of CDK6. Moreover, the expression of CDK6 was also inhibited by the ERK signalling inhibitor, U0126. The results of both loss‐ and gain‐of‐function studies showed that TRIM59 could regulate the expression of CDK6. Collectively, these data provide evidence that TRIM59 is involved in lung carcinoma growth and progression possibly through the induction of CDK6 expression and EMT process by activation of ERK pathway.

Thus, the need to identify potential therapeutic targets to improve LC treatment is urgent.
The tripartite motif (TRIM) family proteins, contain three highly conserved domains, which consist of a common N-terminal Really Interesting New Gene finger domain, one or two B-box motifs and a coiled-coil region. 3,4 TRIM proteins are thought to be important regulators of carcinogenesis, which are involved in several biological processes, such as cell growth, development, and cellular differentiation and alteration of these proteins can affect transcriptional regulation, cell proliferation, and apoptosis. 5 Previous studies have suggested that TRIM59 were increased significantly in tumour tissues compared with noncancerous tissues from glioblastoma and gastric tumours, where increased levels were associated with advanced tumour stage and shorter patient survival times. 6,7 Up-regulation of the TRIM59 gene promotes gastric carcinogenesis via promoting the p53 ubiquitination and degradation. 6 In addition, TRIM59 acts as a key oncogene by promoting EGFR/STAT3 signalling activation and mediates the development of proliferation and migration. 7 Increasing evidence showed that TRIM59 has been implicated in mediating tumour progression. However, the mechanisms regarding how it facilitates tumourigenesis have not been elucidated.
Notably, silencing of TRIM59 significantly inhibited the proliferation and migration of non-small cell lung cancer cells cell lines by arresting cell cycle in G2 phase. 8 Cell cycle is gaining great attention in cancer research for their potential to regulate essentially every hallmark of tumour development. Yet the molecular mechanisms regulating and coordinating cell cycle remain. Bioinformatics analysis shows that expression of TRIM59 positively correlates with cyclindependent kinases 6 (CDK6) in LC cell and tissue. The CDK 6 control the G1 phase of the cell cycle, which has a central role in cell proliferation and in tumourigenesis. 9,10 It is widely accepted that CDK6 is a leading gene relating to tumour growth and metastasis. 11 Therefore, further investigation of the above association of TRIM59 and CDK6 is of pivotal importance.
In this study, we investigated the correlation of TRIM59 expression and the clinicopathological parameters in lung carcinoma and determined whether TRIM59 was involved in the epithelial-mesenchymal transition (EMT) process of LC by activation of the extracellular-signal regulated protein kinase (ERK) pathway. Furthermore, we further investigated the mechanisms that TRIM59 positively regulates CDK6 expression by activation of ERK pathway, which contribute to cancer cell growth and invasion.

| Cell cultures
The human LC cell lines H441, SPC, H292, A549, H1299, and H460 were purchased from the Chinese Academy of Science Cell Bank (Shanghai, China). Cell lines were routinely checked for contamination by Mycoplasma, using Hoescht staining, and were authenticated by DNA-Fingerprinting and isoenzyme analyses. Both cell lines were maintained in RPMI1640 (Gibco, USA) supplemented with 10% foetal bovine serum (Gibco) and 1% penicillin/streptomycin (Corning, Lowell, MA, USA) at 37°C in a humidified incubator with 5% CO 2 .

| Immunohistochemistry
The incubated slides were then deparaffinized in xylene and rehydrated with graded alcohol. Next, antigens were retrieved using citrate buffer (pH 6.0). The samples were covered with 10% normal goat serum in phosphate buffered saline (PBS) for approximately 10 minutes at room temperature and then incubated with anti-TRIM59 (Catalog no. PA5-38726; Invitrogen) at 4°C overnight. For immunohistochemical detection an HRP-Polymer Detection Kit (Abcam) followed by a DAB Substrate Kit (Abcam) were used, and slides were subsequently counterstained with haematoxylin. The percentage of TRIM59-positive cells was scored in five groups: 0 (0% to ≤25%), 1 (25% to ≤50%), 2 (50% to ≤75%), and 3 (>75%). The 0 and 1 groups were defined as low expression, while 2 and 3 groups were defined as high expression.

| RNA extraction and real-time PCR
Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. For first-strand cDNA synthesis, 500 ng of total RNA was retrotranscribed using Prime Script RT GENG ET AL.

| Bioinformatics analysis
The expression levels of genes were investigated in paired LC tissue samples based on Oncomine datasets (https://www.oncomine.org/). Co-expression data were from Oncomine datasets. We used the following filters: gene "TRIM59" Analysis Type: "Co-expression analysis" Cancer Type: "LC."

| Construction of H1299 and A549 TRIM59knockdown cells
Human TRIM59 shRNAs (Sigma) were used to knock down TRIM59 according to the protocols provided by the manufacturer. Cells were seeded on six-well plates and transfected the next day with TRIM59 or control shRNAs. The cells were collected at the indicated time points and were subjected to western blot evaluations. Western blot evaluations using anti-TRIM59 antibody demonstrated that these shRNA had over 90% of silencing efficacies for TRIM59.

| Wound closure assay
Cells were allowed to form a confluent monolayer in a 24-well plate.
The wound was created by scraping a conventional pipette tip across the monolayer. Cells were washed with PBS, cultured in serum-and antibiotic-free media at 37°C, and photographed at 0 and 24 hours.

| Tumour xenograft transplantation assay
Subcutaneous xenografts were created in the flank regions of 4-weekold female BALB/c nude mice. A total of 5 × 10 6 A549 cells stably expressing scrambled shRNA or shTRIM59 were implanted. In addition, a total of 5 × 10 6 H460 cells stably transfected with pCMV3-GFP-TRIM59 or pCMV3-GFP-N1 (Vector) were independently injected subcutaneously into nude mice. Monitoring of tumour nodules was performed and tumour volumes were estimated with the following formula: Volume = width × length × (width + length)/2. The mice were euthanized on day 30 or 28 and tumours were taken. The animal studies were approved by the Wannan Medical College Ethics Review Board.

| Statistical analysis
All calculations were performed with the SPSS 20.0 software program (SPSS Inc, Chicago, IL, USA). The relationship of TRIM59 expression and clinicopathological parameters was evaluated with chi-squared test. The Kaplan-Meier method was used to analyse patient survival.
Student's t tests were used to evaluate continuous variables between subgroups. Statistical significance was defined as P < 0.05.

| Clinical significance of TRIM59 in LC
To identify TRIM59 aberrantly expressed in LC, we first analysed all the differentially expressed TRIM59 within the microarray dataset from the Oncomine database. The results showed that TRIM59 expression was significantly higher in LC tissues compared to adjacent noncancerous tissues ( Figure 1A Figure 1D). Therefore, our data suggested that TRIM59 overexpression is significantly associated with poor prognosis in LC patients.

| TRIM59 promotes LC growth and metastasis
The above data showed that TRIM59 positively correlates with a more aggressive tumour phenotype. Accordingly, we investigated the functions of TRIM59 in LC cell lines using in vitro assays. In order to assess the effects of TRIM59 on in vitro cell growth and invasion, we first examined the endogenous TRIM59 levels of different LC cell lines and regulated their TRIM59 expression levels via stably transfecting TRIM59-specific short hairpin (sh) RNAs in these cells. We found significantly increased TRIM59 levels in A549 and H1299 cell lines (TRIM59 high) (Figure 2A). To test the biological function of TRIM59 in LC progression, we carried out loss-of-function studies using H1299 and A549 cells as models to generate stable TRIM59-Knockdown cell lines ( Figure 2A). As shown in Figure 2B,C, Stable In addition, scratch wound assays showed that wound recovery was significantly impaired by TRIM59 knockdown in comparison with the controls ( Figure 2D). Moreover, Matrigel invasion assays verified the result, which showed that TRIM59 down-regulation also induced strong inhibition on invasion of H1299 and A549 cells ( Figure 2E). Thus, TRIM59 appears to be the critical oncoprotein in regulating tumour cell invasion. It is well-known that the epithelial-to-mesenchymal transition (EMT)-a fundamental cell-biological process that plays key roles in cancer cell invasion and metastasis. 12 This prompted us to investigate the effects of TRIM59 down-regulation on EMT-markers expression.
As expected, Western blot analysis showed that TRIM59 downregulation led to the up-regulation of E-cadherin and down-regulation of N-cadherin, Vimentin, Snail, and Slug in both H1299 and A549 cells ( Figure 3A). These experiments provide strong evidence that TRIM59 potentially promotes EMT process in LC cells. Together, these data show that TRIM59 is the major factor that promotes LC cell growth and invasion.

| TRIM59 induced ERK phosphorylation
Mitogen-activated protein kinase signalling pathway has emerged as a central feature of EMT process. 13 Furthermore, accumulating evidence indicates that ERK pathway play an critical role in LC cell growth and invasion through EMT. 14,15 The role of TRIM59 and involvement of EMT in growth and invasion led us to test this pathway. Thus, we investigated the effects of TRIM59 down-regulation on p-ERK and ERK expression, the key downstream effectors of the MAPK signalling pathway. Stable depletion of TRIM59 with shRNA in H1299 and A549 cells strongly inhibited ERK activation ( Figure 3B). To test whether TRIM59 promote cancer cell growth and invasion through regulation of the ERK signalling pathway, we further examined the effects of ERK pathway activation on tumour proliferation and invasion of H1299 and A549 cells. These experiments showed that the proliferation and invasion of H1299 and A549 cells were strongly suppressed by ERK inhibitor U0126 at 10 μmol/L ( Figure 3C,D). These data indicate that TRIM59 induced cancer cell growth and invasion possibly through regulation of the ERK signalling pathway.

| TRIM59 induced up-regulation of CDK6 expression is dependent on ERK pathway
To illustrate the molecular mechanisms of TRIM59 in governing gene expression compared with GFP-treated cells ( Figure 5C). As expected, the expression of CDK6 was significantly inhibited by ERK inhibitor in TRIM59 overexpressed cancer cells ( Figure 5C).
Taken together, these loss-and gain-of-functional studies demonstrate that TRIM59 can regulate the expression of CDK6 by activation of the ERK pathway.
T A B L E 1 Analysis of association between TRIM59 expression and clinicopathological parameters in lung cancer  Error bars indicate means ± SEM. All these experiments have been repeated three times. The significance was determined by Student t test. *P < 0.05; **P < 0.01

| TRIM59 promoted cancer growth and invasion
To determine whether TRIM59 governs tumour growth and invasion in vivo, we further examined the effects of TRIM59 expression on in vivo tumour growth of LC cells. We first set up subcutaneous xenograft tumour models by subcutaneously injecting shTRIM59transfected and control shRNA transfected A549 cells in nude mice.
Subcutaneous tumour growth was then monitored and compared F I G U R E 3 TRIM59 induced EMT program and ERK phosphorylation. (A) H1299 and A549 cells tranfected with shCtrl or shTRIM59 plasmid were subjected to western blotting for detection of E-cadherin, N-cadherin, Snail, Slug, and Vimentin expression. (B) Western blotting measurement of activation of ERK signaling by TRIM59 knockdown in H1299 and A549 cells. (C) DNA synthesis of H1299 and A549 cells treated with negative control and U0126 (10 μmol/L) was measured using an EdU-incorporation assay. Scale bars, 100 μm. Results are expressed as means ± SEM. Data were assessed by Student's t test. *P < 0.05; **P < 0.01. (D) In vitro invasion assays using transwell chambers in H1299 and A549 cells treated with negative control and U0126 (10 μmol/L). Scale bars, 100 μm. Values are means ± SEM from three independent experiments. Significance was determined by Student's t test between the two groups. As shown in Figure 6A,B, the growth of tumour volume by shTRIM59-transfected cells was significantly suppressed compared with control cells. Likewise, the average weight in the A549 cells with TRIM59 knockdown group were significantly lower than in the control group ( Figure 6D). In concordance with the in vitro findings, significantly fewer proliferating cells were observed F I G U R E 4 Coexpression of TRIM59 and CDK6 in lung cancer. (A, B) Co-expression data from Oncomine (www.oncomine.org). We used the following filters: gene "TRIM59" Analysis Type: "Co-expression analysis" Cancer Type: "Lung cancer." The colour changed according to a weaker (blue) or higher (red) expression in wang cell line 2 and Lee lung GENG ET AL.  Mitogen-activated protein kinase is a critical sensor of cellular proliferation and differentiation. 21 MAPK overexpression or up-regulation of its activity has been linked to cancer. 21 MAPK has emerged as a central feature of EMT program in LC. 14 Moreover, accumulating evidence indicates that ERK activation play an active role in cancer cell growth and invasion through EMT. 22,23 In this study, we observed that down-regulation of TRIM59 significantly attenuates ERK signalling in H1299 and A549 cells. In addition, the inhibition of ERK pathway activation significantly suppressed the growth and invasion in these cells. Together, these experiments identify TRIM59-mediated LC cancer cell growth and invasion is dependent on ERK activation.
Previous studies have suggested that the expression of TRIM59 is related to the cell cycle. 8 However, little is known about the TRIM59-related molecules that regulate the cell cycle or their possible roles in cancer cell invasion. The ability to sustain unscheduled proliferation is a hallmark of cancer. 24 The normal process of cell division occurs via the cell cycle, a series of highly regulated steps that are orchestrated at the molecular level by specific cyclins that act in association with CDKs. CDK4/6 play a key role in cell-cycle progression by phosphorylating and inactivating the retinoblastoma protein, a tumour suppressor that restrains G1-to S-phase progression. 25 Rapidly emerging data with selective inhibitors of CDK4/6 have validated these cell-cycle kinases as anticancer drug targets, corroborating longstanding preclinical predictions. 26,27 In this study, we found that stable depletion of In summary, our data identify TRIM59 as a key promoter of tumour progression, which uncovers that TRIM59 may become a prognostic marker and therapeutic target for lung carcinoma. Moreover, TRIM59 is involved in lung carcinoma growth and invasion through the induction of CDK6 expression and EMT progression by activation of the ERK pathway.