MICAL-L2 Is Essential for c-Myc Deubiquitination and Stability in Non-small Cell Lung Cancer Cells

Objectives: MICAL-L2, a member of the molecules interacting with the CasL (MICAL) family, was reported to be highly expressed in several types of cancers, however, the roles of MICAL-L2 in NSCLC pathogenesis remain to be explored. This study is designed to clarify the mechanisms by which MICAL-L2 participates in NSCLC cell proliferation. Materials and Methods: The expression levels of MICAL-L2 in human lung cancer samples were assessed by immunohistochemical staining. Cells were transfected with siRNA or plasmids to regulate MICAL-L2 expression. Cell proliferation was measured by EdU staining and CCK-8 assays. MICAL-L2 and phosphorylated/total c-Myc expression were examined by Western blotting analysis. Interaction between MICAL-L2 and c-Myc was assessed by immunofluorescence staining, Western blotting and co-immunoprecipitation assays. Western blotting, polyubiquitylation detection and protein stability assays were used to assess whether MICAL-L2 exerts its oncogenic effect via c-Myc. Results: We found that MICAL-L2 was highly expressed in human NSCLC. While overexpressing MICAL-L2 increased NSCLC cell proliferation, MICAL-L2 depletion decreased the proliferation of NSCLC cells, an effect that was linked to cell cycle arrest. MICAL-L2 physically interacted with the c-Myc protein and functioned to maintain nuclear c-Myc levels and prolonged its half-life. Knockdown of MICAL-L2 expression led to decreased c-Myc protein stability through accelerating polyubiquitylation of c-Myc and gave rise to c-Myc degradation. We further found that MICAL-L2 deubiquitinated c-Myc and blocked its degradation, presumably by inhibiting c-Myc phosphorylation at threonine residue 58. Conclusions: These results indicate that MICAL-L2 is a key regulator of c-Myc deubiquitination and stability in the nucleus, and this activity may be involved in promoting NSCLC cell proliferation.


INTRODUCTION
Lung cancer is the most common type of malignant tumor and one of the malignancies with the fastest increase in morbidity and mortality worldwide (Torre et al., 2016). Non-small cell lung cancer (NSCLC) accounts for more than 80% of all lung cancer cases. Approximately 75% of NSCLC patients are diagnosed in the middle and advanced stages of the disease, and the 5year survival rate is very low. c-Myc, an oncogene-encoded protein, is highly expressed in NSCLC and plays a key role in its carcinogenesis (Wu et al., 2015;Li et al., 2019). Consequently, understanding the mechanisms that control the level and activity of c-Myc in NSCLC is important to support the development of novel therapeutic interventions.
c-Myc is a multifunctional transcription factor with roles in various cellular processes, including metabolism and growth (Dang, 2012). Normally, dysregulated c-Myc expression, rather than the expression of a mutated form of the protein, is responsible for its oncogenic effects. Its expression could be influenced at the transcriptional level by c-Myc gene amplification. Protein stability is also an important mechanism underlying the regulation of c-Myc protein content owing to its short half-life in proliferating cells. c-Myc has been identified to undergo phosphorylation modification by the proteasome, which triggers c-Myc ubiquitylation and degradation (Hann, 2006;Farrell and Sears, 2014). Meanwhile, the ubiquitination of c-Myc can be reversed by deubiquitinating enzymes such as USP28 and USP36 (Popov et al., 2007;Sun et al., 2015). The degradation of c-Myc has recently also been found to be regulated through a ubiquitin-independent pathway, i.e., autophagic lysosomal degradation (Liu et al., 2018;Murai et al., 2018). Generally, c-Myc is regulated and exerts its oncogenic activity in the nucleus. However, whether c-Myc is deubiquitinated in the nucleus in NSCLC cells remains to be determined.
Remodeling of the actin cytoskeleton, a molecular framework that provides physical support for cell structure and proliferation, depends on the spatial regulation of an upstream signaling network (Giridharan and Caplan, 2014;Roy and Burkhardt, 2018;Hohmann and Dehghani, 2019;Phuyal and Farhan, 2019). Molecules interacting with CasL (MICALs) comprise a family of flavoprotein monooxygenases that interact with F-actin and participate in a multitude of activities related to cytoskeleton dynamics. MICAL-L2, a member of the MICAL family, is a recently identified Rab13-binding protein and coordinates with Rab13 to promote neurite outgrowth (Rahajeng et al., 2010;Sakane et al., 2010). MICAL-L2 has been found to be highly expressed in multiple types of cancer, including ovarian, gastric, and breast cancers (Ioannou et al., 2015;Zhu et al., 2015;Min et al., 2019). Recent studies have shown that MICAL-L2 localization to the leading edge of migrating cells is necessary for the metastatic behavior of cancer cells (Ioannou et al., 2015;Sakane et al., 2018). Meanwhile, the silencing of MICAL-L2 was reported to inhibit both the nuclear translocation of beta-catenin and ovarian cancer cell proliferation (Zhu et al., 2015). We have also recently shown that MICAL-L2 attenuates lysosomemediated EGFR degradation and enhances the migratory ability of gastric cancer cells, suggesting that MICAL-L2 functions to prevent the degradation of its client proteins and may play an oncogenic role in gastric cancer (Min et al., 2019). Although MICAL-1, another member of the MICAL family, was reported to exert its effect on breast cancer cell proliferation via ROS-sensitive PI3K/AKT/ERK signaling , the function of MICAL-L2 in NSCLC cell proliferation and progression is largely unknown.
Interestingly, proteomic profiling has demonstrated that MICAL-L2 interacts with c-Myc (Agrawal et al., 2010). Strong MICAL-L2 staining has also been observed in the nuclei of ovarian cancer tissues (Zhu et al., 2015). Consequently, we hypothesized that MICAL-L2 can promote NSCLC cell proliferation through binding to c-Myc and may help attenuate c-Myc degradation. In the present study, we found that MICAL-L2 was highly expressed in NSCLC tissues and cells, and its expression was associated with cell proliferation. We further found that MICAL-L2 acts as a positive regulator of c-Myc protein stabilization by preventing its ubiquitin-dependent degradation in the nucleus. These findings indicate that MICAL-L2 promotes NSCLC cell proliferation by maintaining c-Myc content.

Ethics Statement
All immunohistochemistry assays with human tumor specimens were conducted under the institutional guidelines of Jiangsu Province.

Cell Culture
The human NSCLC cell lines A549, NCI-H1299, NCI-H292, and PC9; the normal human fibroblast cell line MRC5; and the human bronchial epithelial cell line BEAS-2B were obtained from the Cell Biology Institute of the Chinese Academy of Science (Shanghai, China). All the cells were maintained in Dulbecco's modified Eagle's medium (DMEM, high glucose) or RPMI 1640 (Hyclone, Thermo Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 • C in a humidified incubator with 5% CO 2 . Cells were grown on plastic dishes for RNA isolation and protein extraction and coverslips for fluorescence staining.

Plasmids and siRNAs
Full-length MICALL2 was PCR-amplified from the pCMV-SPORT6-MICAL-L2 plasmid (YouBio, Hunan, China) and cloned into the pCMV-C-HA or pEGFP-N1 vector (Beyotime, Nantong, China) as previously described (Min et al., 2019). For plasmid construction, the cDNA of the c-Myc gene was amplified by PCR from NCI-H1299 cells and inserted into the pCMV-N-Flag vector (Beyotime). All the constructs were verified by sequencing. When the cells had reached ∼80% confluence, they were transfected with the relevant plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Cell Counting Kit-8 Assay
Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay. Briefly, cells were seeded in a 96-well plate and then transfected with siRNA or plasmids. After culturing for the indicated times, the culture medium was adjusted to 90 µL per well, and 10 µL of the CCK-8 solution (Selleck Chemicals) was added to each well for 1 h. The OD of each sample was measured at 450 nm using a microplate reader (Bio-Tek, Elx800, VT. USA). Each group had five replicates.

Ethynyl-2-deoxyuridine (EdU) Incorporation Assays
Cell proliferation was further measured using an EdU staining kit (Ribobio). In brief, cells were seeded into a 96-well plate and 0.2 µL of EdU was added to the medium for 2 h. The cells were then washed with PBS, fixed in formaldehyde for 30 min, incubated with glycine, and washed with PBS containing 0.5% Triton X-100. The cells were stained with Apollo and then counterstained with Hoechst 33342. The number of EdU-labeled cells was determined using a fluorescence microscope (Carl Zeiss Meditec, Jena, Germany) and normalized to the total number of Hoechst 33342-stained cells.

Flow Cytometry
Cell cycle analysis was conducted by flow cytometry. Briefly, at 48 h post-transfection, cells were harvested and suspended in 75% ice-cold ethanol at a concentration of 1.5 × 10 6 cells/mL. RNase and propidium iodide staining solution were then added to the cell suspension and the mixture was incubated for 30 min at 37 • C in the dark. The cell cycle distribution of the stained cells was analyzed by flow cytometry.

Western Blotting
Cells were homogenized in lysis buffer (Beyotime) and protein concentrations were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of total protein were separated by SDS-PAGE and transferred to pure nitrocellulose membranes. The bands were visualized using enhanced chemiluminescence (Millipore, Billerica, MA, USA). Digital images of the positive bands were analyzed with Quantity One software (Bio-Rad, Hercules, CA, USA). Antibodies targeting the following proteins were used: GAPDH (G9545, Sigma-Aldrich), MICAL-L2 (PA5-24826, Thermo Fisher Scientific), c-Myc (#13877, Cell Signaling Technology, Danvers, MA, USA), cyclin-D1 (#2978, Cell Signaling Technology),

Gene
Sequence

Real-Time Quantitative PCR
Total RNA was extracted using Trizol reagent (Invitrogen) and reverse transcription was performed using HiScript R Q RT SuperMix (Vazyme, Nanjing, China) according to the manufacturer's instructions. qPCR was performed using AceQ R qPCR SYBR R Green Master Mix (High ROX Premixed) (Vazyme) in an ABI StepOne TM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). mRNA expression levels were quantified using the 2 CT method (Applied Biosystems). The sequences of the primers used are listed in Table 1.

Protein Stability Assays
In brief, cells were incubated in the presence of cycloheximide (CHX) for 0, 30, 60, 90, 120 min. Then, the cells were collected and lysed, and the lysates were separated by SDS-PAGE for western blotting analysis of protein abundance at each time point.

Immunofluorescence
The cells used for immunostaining were fixed in 4% paraformaldehyde for 15 min, permeabilized in 0.2% Triton X-100, and blocked with 1% BSA for 1 h at room temperature. The cells were then incubated with antibodies against MICAL-L2 (sc-376675, Santa Cruz Biotechnology) and c-Myc (sc-764x; Santa Cruz Biotechnology) at 4 • C overnight followed by incubation with species-matched Alexa-or TRITC-conjugated secondary antibodies for 1 h at room temperature in a moist chamber. Cell nuclei were counterstained with DAPI (Southern Biotech, Birmingham, AL, USA). Images were acquired using an Olympus BX43 microscope (Olympus, Tokyo, Japan) coupled to an Olympus DP74 digital camera.

Co-immunoprecipitation (Co-IP) Assay
Co-IP was conducted as previously described (Deng et al., 2016). In brief, cellular proteins were prepared and incubated with antibody at 4 • C overnight. Antibody-protein complexes were precipitated using protein A + G agarose beads (Beyotime). The agarose-associated antibody-protein complexes were then dissolved in SDS-PAGE and the bound proteins were probed with antibodies as indicated in the figure legends.

Polyubiquitination Assays
Polyubiquitination assays were conducted using Co-IP. Briefly, cells were transfected with HA-ubiquitin and the relevant plasmids. Cell lysates were harvested at 48 h post-transfection; 80% of each cell lysate was used for Co-IP and the rest was subjected to Western blotting under denaturing conditions. The bound proteins were analyzed using Western blotting.

Immunohistochemistry
Lung adenocarcinoma (LUAD) tissue microarrays were purchased from Outdo Biotech (Shanghai, China). Thirty LUAD case samples and their corresponding paracancerous tissues were used in this study. Briefly, the microarray tissues were deparaffinized, rehydrated, blocked with 3% BSA, and incubated with antibodies against MICAL-L2 (PA5-24826, Thermo Fisher Scientific) and c-Myc (bs4963R, Biosynthesis Biotechnology Co., Ltd). After 1 h of incubation with biotinylated secondary antibodies and subsequent DAB solution staining, the samples were observed under a microscope, counterstained with hematoxylin, and then coverslipped with neutral gum. MICAL-L2 and c-Myc immunoreactivity was semi-quantified using the immunoreactive score (IRS).

Statistical Analysis
All data were expressed as means ± S.E.M. Statistical analysis was performed using SPSS version 19.0 (SPSS, Chicago, IL, USA). The Student's t-test was used for comparisons between two groups.

The Expression and Relationship of MICAL-L2 and c-Myc in Human LUAD Samples
The expression of MICAL-L2 and c-Myc was evaluated in a tissue microarray comprising 30 paired LUAD cases. We found that MICAL-L2 was highly expressed in LUAD tissues when compared with that in matched paracancerous tissues (P < 0.001). LUAD tissues also exhibited strong MICAL-L2 staining in the nuclei of most tumor cells and the cytoplasm of some tumor cells (Figures 1A,B). c-Myc has been implicated in cancer progression and growth. The expression pattern of c-Myc was similar to that of MICAL-L2 ( Figure 1C). Analysis of The Cancer Genome Atlas (TCGA) lung cancer dataset indicated that the expression of MICAL-L2 was significantly higher in cancer samples than in paracancerous tissues ( Figure 1D). Next, we analyzed the expression of MICAL-L2 in a set of 4 NSCLC cell lines by immunoblotting, and found that MICAL-L2 expression was higher in most NSCLC cell lines compared with the non-malignant bronchial epithelial cell line BEAS-2B and the normal human fibroblast cell line MRC5 (Figures 1E,F).
Overall, the clinical data indicated that MICAL-L2 expression was upregulated in lung cancer.

MICAL-L2 Deficiency Downregulated c-Myc Expression in NSCLC Cells
We subsequently explored whether MICAL-L2 plays a key role in c-Myc expression in NSCLC cells. We first knocked down MICAL-L2 expression in A549 cells using siRNA and determined the knockdown efficiency by Western blotting. As shown in Figure 2A, both siMICAL-L2 #2 and #3 significantly downregulated the expression of MICAL-L2. Moreover, siMICAL-L2 also suppressed the expression of c-Myc in these cells, and a similar result was found for c-Myc expression in MICAL-L2-depleted H1299 cells ( Figure 2B). Immunofluorescence staining showed that, in A549 cells transfected with siMICAL-L2 (#2 and #3), c-Myc was mainly detected in the nucleus and its fluorescence was decreased in MICAL-L2-knockdown A549 cells when compared with that in control cells (Figure 2C and Supplementary Figure 1A). No significant translocation of c-Myc from the nucleus to the cytoplasm was detected in these cells (Supplementary Figure 2).

MICAL-L2 Positively Regulates NSCLC Cell Proliferation
c-Myc expression is known to enhance the proliferation rates of various types of cells, including lung cancer cells. We noticed that after ectopic expression of MICAL-L2, both c-Myc expression (Figures 3A,B) and the proliferative potential of PC9 cells were increased ( Figure 3C). EdU and CCK-8 incorporation assays also indicated that the proliferative ability of MICAL-L2-overexpressing PC9 cells was increased compared with that in the control group (Figures 3C,D). Immunofluorescence analysis further showed that c-Myc expression was markedly increased in PC9 cells overexpressing MICAL-L2 (Figure 3E and Supplementary Figure 1B). In contrast, pretreatment with MICAL-L2 siRNA significantly decreased the proliferative capacity of A549 and H1299 cells (Figures 4A-D). Flow cytometric analysis showed that knocking down MICAL-L2 in A549 and H1299 cells increased the number of cells in the G0/G1-phase of the cell cycle and decreased that in the S-phase. This implied that MICAL-L2 positively regulates the proliferative phase of NSCLC cells, particularly the G1 to S-phase transition of the cell cycle (Figures 4E,F).

MICAL-L2 Maintains c-Myc Levels by Attenuating c-Myc Degradation
To investigate the mechanism involved in the MICAL-L2dependent regulation of c-Myc expression, the mRNA levels of c-Myc in MICAL-L2-knockdown or overexpressing A549, H1299, and PC9 cells were analyzed by RT-qPCR. Despite a marked increase or decrease in MICAL-L2 expression, c-Myc mRNA levels were not significantly altered (Figures 5A-C). This suggested that MICAL-L2 might positively regulate c-Myc expression through different mechanisms, such as by disrupting its degradation. Consequently, we then knocked down MICAL-L2 expression in both A549 and H1299 cells by siRNA and examined c-Myc protein expression after treating the cells with CHX, a translation blocker. The results showed that knockdown of MICAL-L2 promoted c-Myc degradation relative to control cells (Figures 5D,E). The above results suggested that MICAL-L2 may help maintain c-Myc stability by reducing its degradation. The results further showed that MICAL-L2 depletion reduced the cyclin-D1 content, whereas ectopic c-Myc expression markedly upregulated cyclin-D1 levels in MICAL-L2depleted cells (Supplementary Figure 3).
To further confirm that the inhibitory effects on proliferation exerted by MICAL-L2 knockdown were mediated through a reduction in c-Myc protein levels, we then examined the expression of cell cycle-related genes in siMICAL-L2-transfected cells by qPCR. The results showed that the depletion of MICAL-L2 led to a significant decrease in the mRNA and protein levels of CCND1, CDK2, and CDK4 when compared with controls (Figures 6A,B,D). Meanwhile, as shown in Figures 6C,E, MICAL-L2 overexpression led to a marked increase in the mRNA and protein levels of CCND1, CDK2, and CDK4. In addition to their roles in regulating the G1 to S-phase transition of the cell cycle, these genes are also key c-Myc targets.

MICAL-L2 Regulates c-Myc Stability via Suppressing Ubiquitin-Mediated c-Myc Degradation
c-Myc degradation is known to be regulated via multiple pathways. To determine which pathway is involved in the MICAL-L2-mediated degradation of c-Myc, we treated cells with various inhibitors of known degradation pathways, namely, AICAR (macroautophagy inhibitor), chloroquine (lysosomal proteolysis inhibitor), MG-132 and Velcade (proteasome inhibitor). The results showed that only the proteasome inhibitor MG-132 and Velcade could block MICAL-L2 knockdown-induced c-Myc degradation (Figures 7A-D), suggesting that MICAL-L2 may inhibit c-Myc degradation via the proteasome pathway.
To further uncover the potential mechanism underlying c-Myc proteasomal degradation, we examined c-Myc ubiquitylation levels in MICAL-L2-depleted cells. As shown in Figure 7E, c-Myc polyubiquitylation levels were increased in H1299 cells transfected with siMICAL-L2. In contrast, c-Myc polyubiquitylation was significantly reduced following the ectopic expression of MICAL-L2 in H1299 cells ( Figure 7F). Our data suggested that MICAL-L2 regulates c-Myc ubiquitylation in NSCLC cells. c-Myc stability was reported to be highly correlated with low levels of threonine 58 (T58) phosphorylation. Additionally, pT58-c-Myc can be recognized by the E3 ubiquitin ligase complex SCF Fbw7 , leading to c-Myc degradation through the proteasomal system (Yada et al., 2004). Consequently, we then evaluated the role of MICAL-L2 in modulating pT58c-Myc expression using Western blotting. The pT58-c-Myc level was decreased in PC9 cells overexpressing MICAL-L2, but was increased in MICAL-L2-depleted A549 and H1299 cells ( Figure 7G). The above results suggested that MICAL-L2 maintained c-Myc protein levels possibly through repressing polyubiquitylation-mediated c-Myc degradation.

MICAL-L2 Interacts With c-Myc
The above results indicated that MICAL-L2 regulates c-Myc expression at the posttranscriptional level. We then further explored whether MICAL-L2 and c-Myc colocalize. The results of the immunofluorescence assay indicated that they were located in the same part of the cell (Figure 8A). The interaction between endogenous MICAL-L2 and c-Myc was also confirmed by coimmunoprecipitation assays using H1299 cells ( Figure 8B). Moreover, Flag-tagged c-Myc pulled down HA-MICAL-L2 after the co-transfection of the two plasmids into  The protein levels of c-Myc were examined in A549 and H1299 cells transfected with control siRNA or siMICAL-L2 and treated with cycloheximide (CHX, 10 µg/mL) for the indicated times. *P < 0.05, **P < 0.01, ***P < 0.001 relative to cells expressing control siRNA. (Figure 8C), while HA-MICAL-L2 could also pull down Flag-tagged c-Myc after co-transfection ( Figure 8D). These results suggested that MICAL-L2 directly binds to c-Myc.

Cos-7 cells
We subsequently divided c-Myc and MICAL-L2 into fragments based on their known domains (Figures 8Ea,Fa) and expressed them individually in Cos-7 cells. The results indicated that the central region of c-Myc (amino acids [aa] 145-354) was crucial for its interaction with MICAL-L2 (Figure 8Eb), and the CH+LIM domain region of MICAL-L2 (aa 1-248) was required for its interaction with c-Myc (Figure 8Fb). We noticed that MICAL-L2 construct #3, which contains the CH+LIM domains, did not interact with c-Myc. The CC region of MICAL1 can bind to its LIM domain, thereby mediating its autoinhibition. Because construct #3 included regions other than the CH+LIM domains, it was possible that these other regions might inhibit the function of the CH+LIM domain region and prevent its binding to c-Myc. Notably, construct #2 of c-Myc (aa 355-439) was a smallsized fragment that could not be detected by Western blotting. However, as shown in Figure 8Ec, this construct was successfully expressed in cells. immunofluorescence analysis indicated that construct #2 of c-Myc and MICAL-L2 did not colocalize.
Overall, our clinical and in vitro data supported that MICAL-L2 may promote NSCLC cell proliferation via the c-Myc pathway (Figure 9).

DISCUSSION
In this paper, MICAL-L2, as a CasL interacting protein, preferentially binds to c-Myc. Notably, our report is the first to reveal such interaction in detail at the molecular level by performing binding domain analysis between MICAL-L2 and c-Myc. By interaction with c-Myc, MICAL-L2 is not only involved in this protein stability, but also participated in the subcellular c-Myc signaling pathway of cell proliferation. Furthermore, analysis of clinic specimens revealed increased expression of MICAL-L2 in lung adenocarcinoma tissues. Taken together, our results show that MICAL-L2 is a novel oncoprotein in NSCLC pathogenesis. We also reveal a characteristic of MICAL-L2 which could function as a regulator for c-Myc-mediated NSCLC cell proliferation.
Evidence from an increasing number of studies has indicated that the MICAL protein family, which includes MICAL1/2/3 and MICAL-L1/2, are important regulators of actin cytoskeleton dynamics and membrane trafficking (Terai et al., 2006;Giridharan et al., 2012;Fremont et al., 2017). We have previously reported that MICAL2 is involved in the regulation of breast cancer cell migration through the suppression of EGFR degradation . MICAL-L2, which lacks the MO domain and owns CC region compared with MICAL2 (Cai et al., 2018), is also a key member of the MICAL family and is frequently reported to serve as a cargo recognition protein (G) A549 and H1299 cells were transfected with control siRNA or siMICAL-2. Total proteins were then extracted and analyzed for the expression of phosphorylated-c-Myc (T58) by Western blotting. PC9 cells were transfected with empty vector or MICAL-L2 expression plasmids. Total proteins were then extracted and analyzed for the expression of phosphorylated-c-Myc (T58) by Western blotting. Western blotting bands corresponding to phosphorylated-c-Myc/c-Myc were quantified and normalized against GAPDH. *P < 0.05, **P < 0.01 relative to control cells. (Sun et al., 2016). MICAL-L2 is well-known as an effector protein of Rab13. Accumulating evidence has indicated that the Rab13/MICAL-L2 complex is a critical regulator of epithelial junctional assembly, cytoskeletal reorganization, and neurite outgrowth (Nakatsuji et al., 2008;Sakane et al., 2010Sakane et al., , 2012. MICAL-L2 is also involved in glucose transporter 4 trafficking as an effector of insulin-activated Rab13 (Sun et al., 2016). It is increasingly recognized that, although Rab is the main MICAL-L2 interaction partner, other proteins can also bind MICAL-L2. Of note, although proteomic profiling has shown that MICAL-L2 can interact with c-Myc (Agrawal et al., 2010), its oncogenic impact has never been explored. In MTD-1A epithelial cells, MICAL-L2 was detected both at cell-cell tight junctions and in the cytoplasm (Terai et al., 2006), while in L6 myoblasts GFP-MICAL-L2 was shown to be distributed along cytoplasmic filament-like structures. In this study, staining  for MICAL-L2 in the NSCLC cell lines A549 and H1299 was primarily localized to the cytoplasm and nucleus. The different intracellular localization of MICAL-L2 may have been due to the different cell types used. The major finding of the present study was the identification of the link between MICAL-L2 and c-Myc. Consistent with the role of MICAL2 in promoting p53 ubiquitination during colorectal cancer development , our data indicated that MICAL-L2 knockdown shortened the half-life of c-Myc and decreased c-Myc protein levels, suggesting that MICAL-L2 may promote NSCLC cell proliferation through maintaining c-Myc function.
In the present study, we found that MICAL-L2 positively regulated c-Myc at the protein level, but not at the mRNA level. MICAL-L2 knockdown increased c-Myc polyubiquitination, whereas MICAL-L2 overexpression elicited the opposite effect. These results suggested that MICAL-L2 might modulate c-Myc expression through the ubiquitin-proteasome system rather than through a transcription-dependent mechanism. We also found that MICAL-L2 blocked c-Myc polyubiquitination and degradation, presumably by inhibiting c-Myc phosphorylation at T58. As p-T58 can recruit SCF Fbw7 , a component of the SCF E3 ubiquitin ligase complex, to c-Myc (Welcker et al., 2004;Yada et al., 2004), T58 dephosphorylation may play an important role in preventing c-Myc polyubiquitination and its subsequent degradation. Moreover, we identified the domains in MICAL2 and c-Myc that are likely to be involved in their interaction. c-Myc is a multifaceted transcription factor that has been suggested to regulate the expression of up to 15% of all human genes. Recent evidence indicates that c-Myc regulation may be context-dependent; for instance, some transcription factors or cofactors were reported to determine the outcome of c-Myc-mediated transcription (Wu et al., 2017). Here, we found that the interaction between MICAL-L2 and c-Myc was related to cell proliferation, as MICAL-L2 could regulate the expression of c-Myc target genes (Dang, 1999), including CCND1, CCNE, CDK2, and CDK4, as well as the protein expression of CCND1, CDK2, and CDK4. Together, these results further support that MICAL-L2-mediated c-Myc stability is critical for NSCLC cell proliferation.
Because cell proliferation is a complex process involving multiple cellular pathways, MICAL-L2 could potentially act through several different signaling pathways to achieve the same result. Mechanistically, MICAL-L2 was shown to be significantly upregulated in ovarian cancer tissues and to activate the Wnt/beta-catenin signaling pathway (Zhu et al., 2015). Here, we have described a novel role for MICAL-L2 in preventing c-Myc ubiquitination and degradation in the nucleus. This results in the increased expression of c-Myc target genes, including cell cycle-related genes, as well as the promotion of NSCLC cell proliferation, which may finally lead to NSCLC development. Further in vivo studies are needed to elucidate how MICAL-L2 regulates NSCLC growth and whether MICAL-L2 may be a potential therapeutic target for the treatment of this cancer.