Cul4A Modulates Invasion and Metastasis of Lung Cancer through Regulation of ANXA10

Cullin 4A (Cul4A) is overexpressed in a number of cancers and has been established as an oncogene. This study aimed to elucidate the role of Cul4A in lung cancer invasion and metastasis. We observed that Cul4A was overexpressed in non-small cell lung cancer (NSCLC) tissues and the overexpression of Cul4A was associated with poor prognosis after surgical resection and it also decreased the expression of the tumor suppressor protein annexin A10 (ANXA10). The knockdown of Cul4A was associated with the upregulation of ANXA10, and the forced expression of Cul4A was associated with the downregulation of ANXA10 in lung cancer cells. Further studies showed that the knockdown of Cul4A inhibited the invasion and metastasis of lung cancer cells, which was reversed by the further knockdown of ANXA10. In addition, the knockdown of Cul4A inhibited lung tumor metastasis in mouse tail vein injection xenograft models. Notably, Cul4A regulated the degradation of ANXA10 through its interaction with ANXA10 and ubiquitination in lung cancer cells. Our findings suggest that Cul4A is a prognostic marker in NSCLC patients, and Cul4A plays important roles in lung cancer invasion and metastasis through the regulation of the ANXA10 tumor suppressor.


Cell Invasion Assay
The invasion assays were performed in 24-well (6.5 mm diameter) cell-culture inserts (8.0 mm pore size, Corning, Tewksbury, MA, USA) coated with an indicator layer of growth factor reduced Matrigel (BD Transduction Laboratories, San Jose, CA, USA). The cells were plated in the upper well in 0.2% serum and then incubated with 5% FBS and 100 ng/mL fibronectin in the lower chambers. After 24 h, cells in the upper chamber were removed with a cotton swab. Cells that migrated into the lower chamber were fixed in 4% PFA and then stained with 0.5% crystal violet. Filters were photographed and the total number of cells was quantified.

Western Bot Analysis
Whole protein was extracted using mammalian protein extraction reagent (M-PER) from the cell lines. The proteins were digested using the Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego, CA, USA) and complete protease inhibitor cocktails (Roche, Lewes, UK) according to the manufacturer's protocols. The digested proteins were separated on 4-15% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). The following primary antibodies were used: Cul4A (Abcam, Cambridge, MA, USA), ANXA10 (GeneTex), and β-actin (Sigma, St. Louis, MO, USA). After incubation with indicated secondary antibodies, the membranes were washed thoroughly and an enhanced chemiluminescence (ECL) blotting analysis system (GE Healthcare Life Sciences, Piscataway, NJ, USA) was used for antigen-antibody detection. The relative intensities of protein bands were analyzed by densitometry using ImageJ 1.46r software (National Institutes of Health, Bethesda, MD, USA).

Transfection with Small Interfering RNA (siRNA) and Vectors
Pre-designed and validated Cul4A (Dharmacon, Lafayette, CO, USA), ANXA10 (Santa Cruz, Santa Cruz, CA, USA), and universal negative control siRNAs were transfected (final concentration = 50 nM) in cells grown to 80% confluence on 6-well plates using an antibiotic-free media and Lipofectamine™ RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. At 96 h after transfection, the cells were treated with gemcitabine for 72 h followed by counting for viable cells. The pCMV6-ANXA10-GFP (OriGene, Rockville, MD, USA) and empty pCDNA3 (Invitrogen, Carlsbad, CA, USA) vectors were transfected with OmniFect™ transfection reagent (TransOMIC, Huntsville, AL, USA), following the manufacturer's instructions. Cells were plated in 6-well plates in antibiotic-free media and then transfection was performed with cells at 80% confluence, with a final concentration of 0.5 µg for each vector.

Protein Degradation Assay
Protein degradation assay was used to evaluate the effects of Cul4A on the decay of ANXA10 in lung cancer cells. Cells, transfected with Cul4A siRNA and Cul4A vector, were plated on 6 cm culture dishes. At 80% confluence, the cells were exposed to 100 mg/mL of cycloheximide. Then, the cells were harvested at the indicated time points. Total cellular proteins were extracted and analyzed by western blot analysis using β-actin as a loading control.

In Vivo Ubiquitination Assay
The 293T cells were co-transfected with a combination of pBabe-Cul4A-myc-his and pCMV6-ANXA10-GFP (OriGene) with or without pRK5-HA-Ubiquitin-WT (Addgene, Cambridge, MA, USA). All the cells were treated with 10 µg/mL of MG132 for 24 h prior to lysis. Anti-GFP antibody was used for immunoprecipitation. Anti-HA tag antibody (Cell Signaling, Danvers, MA, USA) was used for the western blot analysis.

Tail Vein Injection Mouse Xenograft Models
Tail vein injection was used to establish a lung metastasis xenograft model for the Cul4A knockdown and metastasis. Approval from the Institutional Animal Care and Use Committee (IACUC) was obtained for the experiments (IACUC No. 2014121206). Female Balb/c athymic nude mice (5-6 weeks old) were housed under specific pathogen-free conditions. Cells were cultured in RPMI media, then suspended at a concentration of 1 × 10 6 cells/100 µL, and 100 µL of this suspension was injected into the tail vein of the mice. Fluorescence molecular tomography (FMT) (PerkinElmer, Waltham, MA, USA) imaging was performed 6 weeks after injection of the lung cancer cells. ProSense680 (PerkinElmer) was injected into the tail vein and then FMT imaging was performed 48 h later. The mice were euthanized at 8 weeks, and their lungs were harvested for further analysis.

Statistical Analysis
Data are presented as mean values ± standard error of deviation (SD). Student's t-test was used for comparing the means, unless otherwise mentioned. Statistical analysis was carried out using MedCalc version 15 (MedCalc Software, Ostend, Belgium). A p value < 0.05 was considered statistically significant. All the statistical tests were two-tailed.

Cul4A Is Upregulated in the NSCLC Tissues
First, we examined the Cul4A protein expression in 73 primary NSCLC tissues. An increased expression of Cul4A was observed in 59 (80.8%) tumor tissues compared to the paired normal tissues ( Figure 1A). Receiver operating characteristic (ROC) curves and the Youden index were used to determine the optimal cutoff value of Cul4A IRS for disease recurrence after surgical resection of NSCLC lung cancer ( Figure S1). High expression of Cul4A levels, which was defined by an IRS score greater than 6, were detected in 12 of the 73 (16.4%) NSCLC tissue specimens that were analyzed, and the high expression was associated with a significantly decreased disease-free survival (DFS) after surgical resection of the lung cancer ( Figure 1B,C). The expression of ANXA10 was also examined and a significantly negative correlation of the expression of Cul4A and ANXA10 was determined using the Spearman rank correlation test ( Figure 1D,E). ProSense680 (PerkinElmer) was injected into the tail vein and then FMT imaging was performed 48 h later. The mice were euthanized at 8 weeks, and their lungs were harvested for further analysis.

Statistical Analysis
Data are presented as mean values ± standard error of deviation (SD). Student's t-test was used for comparing the means, unless otherwise mentioned. Statistical analysis was carried out using MedCalc version 15 (MedCalc Software, Ostend, Belgium). A p value < 0.05 was considered statistically significant. All the statistical tests were two-tailed.

Cul4A is Upregulated in the NSCLC Tissues.
First, we examined the Cul4A protein expression in 73 primary NSCLC tissues. An increased expression of Cul4A was observed in 59 (80.8%) tumor tissues compared to the paired normal tissues ( Figure 1A). Receiver operating characteristic (ROC) curves and the Youden index were used to determine the optimal cutoff value of Cul4A IRS for disease recurrence after surgical resection of NSCLC lung cancer ( Figure S1). High expression of Cul4A levels, which was defined by an IRS score greater than 6, were detected in 12 of the 73 (16.4%) NSCLC tissue specimens that were analyzed, and the high expression was associated with a significantly decreased disease-free survival (DFS) after surgical resection of the lung cancer ( Figure 1B,C). The expression of ANXA10 was also examined and a significantly negative correlation of the expression of Cul4A and ANXA10 was determined using the Spearman rank correlation test ( Figure 1D,E).

Knockdown of Cul4A Is Associated with the Upregulation of ANXA10 in Lung Cancer Cells
We further explored the expression of the cancer metastasis suppressor ANXA10 after knocking down Cul4A. We observed an increase in the ANXA10 protein level in Cul4A knockdown lung cancer cells using western blotting ( Figure 2A). The expression of ANXA10 mRNA was further evaluated by RT-PCR in the Cul4A knockdown H460 and A549 lung cancer cells. Remarkably, no obvious change in the ANXA10 mRNA levels was observed in the Cul4A shRNA transfected groups of lung cancer cells compared to the cells transfected with the empty vector ( Figure 2B). Cul4A overexpression in the H460 lung cancer cells resulted in lowered protein levels of ANXA10 ( Figure  2C). Similar to the results observed with transient siRNA transfection, H460 and A549 lung cancer cells were stably transfected with Cul4A shRNA using retroviral transduction, which resulted in the knockdown of Cul4A, and showed increased ANXA10 protein levels ( Figure 2D).

Knockdown of Cul4A Is Associated with the Upregulation of ANXA10 in Lung Cancer Cells
We further explored the expression of the cancer metastasis suppressor ANXA10 after knocking down Cul4A. We observed an increase in the ANXA10 protein level in Cul4A knockdown lung cancer cells using western blotting ( Figure 2A). The expression of ANXA10 mRNA was further evaluated by RT-PCR in the Cul4A knockdown H460 and A549 lung cancer cells. Remarkably, no obvious change in the ANXA10 mRNA levels was observed in the Cul4A shRNA transfected groups of lung cancer cells compared to the cells transfected with the empty vector ( Figure 2B). Cul4A overexpression in the H460 lung cancer cells resulted in lowered protein levels of ANXA10 ( Figure 2C). Similar to the results observed with transient siRNA transfection, H460 and A549 lung cancer cells were stably transfected with Cul4A shRNA using retroviral transduction, which resulted in the knockdown of Cul4A, and showed increased ANXA10 protein levels ( Figure 2D).

Knockdown of Cul4A Is Associated with the Upregulation of ANXA10 in Lung Cancer Cells
We further explored the expression of the cancer metastasis suppressor ANXA10 after knocking down Cul4A. We observed an increase in the ANXA10 protein level in Cul4A knockdown lung cancer cells using western blotting ( Figure 2A). The expression of ANXA10 mRNA was further evaluated by RT-PCR in the Cul4A knockdown H460 and A549 lung cancer cells. Remarkably, no obvious change in the ANXA10 mRNA levels was observed in the Cul4A shRNA transfected groups of lung cancer cells compared to the cells transfected with the empty vector ( Figure 2B). Cul4A overexpression in the H460 lung cancer cells resulted in lowered protein levels of ANXA10 ( Figure  2C). Similar to the results observed with transient siRNA transfection, H460 and A549 lung cancer cells were stably transfected with Cul4A shRNA using retroviral transduction, which resulted in the knockdown of Cul4A, and showed increased ANXA10 protein levels ( Figure 2D).

Knockdown of Cul4A Represses Metastasis and Invasion in Lung Cancer Cells
Effects of Cul4A knockdown on metastasis and invasion of Cul4A shRNA transfected H460 and A549 stable lung cancer cells were evaluated using cell migration and invasion assays. We observed that the knockdown of Cul4A significantly repressed metastasis ( Figure 3A-D) and invasion ( Figure  3E-H) of lung cancer cells.

Knockdown of Cul4A Represses Metastasis and Invasion in Lung Cancer Cells
Effects of Cul4A knockdown on metastasis and invasion of Cul4A shRNA transfected H460 and A549 stable lung cancer cells were evaluated using cell migration and invasion assays. We observed that the knockdown of Cul4A significantly repressed metastasis ( Figure

Knockdown of Cul4A Represses Metastasis and Invasion in Lung Cancer Cells
Effects of Cul4A knockdown on metastasis and invasion of Cul4A shRNA transfected H460 and A549 stable lung cancer cells were evaluated using cell migration and invasion assays. We observed that the knockdown of Cul4A significantly repressed metastasis ( Figure 3A-D) and invasion ( Figure  3E-H) of lung cancer cells.

Cell Migration and Invasion are Restored by Knockdown of ANXA10 in Cul4A Knockdown Lung Cancer Cells
To evaluate the effect of ANXA10 on the cell migration and invasion of lung cancer cells, ANXA10 knockdown assay using ANXA10 siRNA was performed in Cul4A knockdown H460 and

Cell Migration and Invasion Are Restored by Knockdown of ANXA10 in Cul4A Knockdown Lung Cancer Cells
To evaluate the effect of ANXA10 on the cell migration and invasion of lung cancer cells, ANXA10 knockdown assay using ANXA10 siRNA was performed in Cul4A knockdown H460 and A549 stable lung cancer cells ( Figure 4A). Both cell migration ( Figure 4B-E) and invasion ( Figure 4F-I) were restored following the knockdown of ANXA10 in Cul4A knockdown H460 and A549 lung cancer stable cells. A549 stable lung cancer cells ( Figure 4A). Both cell migration ( Figure 4B-E) and invasion ( Figure 4F-I) were restored following the knockdown of ANXA10 in Cul4A knockdown H460 and A549 lung cancer stable cells.

Knockdown of Cul4A Represses Metastasis of Lung Cancer Tumors in Tail Vein Injection Mouse Models
Tail vein injection models were established using H460 and A549 cells that were stably transfected with Cul4A shRNA to confirm the effect of Cul4A on lung cancer metastasis. A significant reduction in lung metastasis was observed in the Cul4A knockdown groups of H460 using FMT imaging ( Figure 5A,B) and A549 ( Figure 5D,E) lung cancer cells.
An increased expression of ANXA10 was also observed in the Cul4A knockdown H460 ( Figure 5C) and A549 cells compared to the empty virus transfected lung cancer cells ( Figure 5F).

Knockdown of Cul4A Represses Metastasis of Lung Cancer Tumors in Tail Vein Injection Mouse Models
Tail vein injection models were established using H460 and A549 cells that were stably transfected with Cul4A shRNA to confirm the effect of Cul4A on lung cancer metastasis. A significant reduction in lung metastasis was observed in the Cul4A knockdown groups of H460 using FMT imaging ( Figure 5A,B) and A549 ( Figure 5D,E) lung cancer cells.
An increased expression of ANXA10 was also observed in the Cul4A knockdown H460 ( Figure  5C) and A549 cells compared to the empty virus transfected lung cancer cells ( Figure 5F).

ANXA10 Expression Is Regulated by Protein Degradation
Protein degradation assay was used to evaluate the stability of ANXA10 protein in Cul4A knockdown and overexpressed H460 lung cancer cells. After treated with cycloheximide for the indicated periods, protein degradation was dramatically increased in the Cul4A overexpressed lung cancer cells ( Figure 6A), while Cul4A knockdown lung cancer cells showed a marked reduction in protein degradation ( Figure 6B). The NEDD8 inhibitor specifically blocks the NEDDylation and subsequent function of Cul4A [30]. Protein degradation assay was also performed in H460 lung cancer cells after the treatment of MLN4924 (Sigma, St. Louis, MO, USA), a NEDD8 inhibitor. Decreased protein degradation was observed in the MLN4924 treated lung cancer cells ( Figure 6F).
Protein degradation assay was used to evaluate the stability of ANXA10 protein in Cul4A knockdown and overexpressed H460 lung cancer cells. After treated with cycloheximide for the indicated periods, protein degradation was dramatically increased in the Cul4A overexpressed lung cancer cells ( Figure 6A), while Cul4A knockdown lung cancer cells showed a marked reduction in protein degradation ( Figure 6B). The NEDD8 inhibitor specifically blocks the NEDDylation and subsequent function of Cul4A [30]. Protein degradation assay was also performed in H460 lung cancer cells after the treatment of MLN4924 (Sigma, St. Louis, MO, USA), a NEDD8 inhibitor. Decreased protein degradation was observed in the MLN4924 treated lung cancer cells ( Figure 6F). In reciprocal immunoprecipitation and in vivo ubiquitination assays, the cells were treated with 10 µg/mL of MG132 for 24 h prior to being lysed. Anti-GFP antibody was used for the immunoprecipitation. Anti-HA antibody was used for the western blot (WB) analysis. (F) Protein degradation assay for ANXA10 in H460 lung cancer cells treated with DMSO and 1 µM of MLN4924, a NEDD8 inhibitor, for 24 h. H460 cells were incubated with 100 µg/mL cycloheximide (CHX) for the indicated time periods. The expression of ANXA10 was quantified by densitometry normalized to actin, and using o hour groups as the control.

ANXA10 Is Ubiquitinated by Cul4A Through a Protein-Protein Interaction
We evaluated the role of Cul4A mediated ubiquitination in ANXA10 degradation. Co-immunoprecipitation assay was performed and the association of ANXA10 with Cul4A was observed ( Figure 6C,D). Further in vivo ubiquitination assays showed that the overexpression of Cul4A increased the ubiquitination of ANXA10 in the 293T cells ( Figure 6E).

Discussion
Cul4A has been implicated in multiple cancers, namely breast [7], mesothelioma [8], lung [9], and liver cancers [10]. In this study, we observed that the upregulated Cul4A is associated with poor prognosis in NSCLC lung cancer patients after surgery. On the other hand, the knockdown of Cul4A is associated with the decreased invasion and metastasis of lung cancer cells and tumors. The knockdown of Cul4A is associated with the increased expression of ANXA10, a tumor suppressor protein, in lung cancer cells. Further experimentation revealed that Cul4A regulates ANXA10 through ubiquitination and protein degradation in lung cancer cells. We hypothesized that Cul4A mediated degradation of ANXA10 was one of the key mechanisms in lung cancer invasion and metastasis. To our knowledge, the association of Cul4A and ANXA10 in NSCLC tissues and lung cancer cells was also reported for the first time after reviewing published papers.
A nude mouse tail vein injection metastasis model was established in our study, which showed a decreased metastatic potential in Cul4A knockdown lung cancer cells. Cul4A has been reported to promote cancer metastasis and invasion in osteosarcoma [31] cells. In breast cancer cells, Cul4A induces an epithelial-mesenchymal transition, and it promotes cancer metastasis by regulating the expression of the EMT regulatory ZEB1 gene [32]. In colorectal cancers, overexpression of Cul4A induces the epithelial-mesenchymal transition through the regulation of H3K4 trimethylation at the E-cadherin, N-cadherin, and vimentin gene promoters [33]. In gastric cancers, overexpression of Cul4A promoted gastric cancer cell proliferation and epithelial-mesenchymal transition by downregulating LATS1-Hippo-YAP signaling [34]. Our study showed that the overexpression of Cul4A was associated with the downregulation of the tumor suppressor ANXA10 in lung cancer tissues and cells, which may provide another mechanism for the role of Cul4A in lung cancer invasion and metastasis. The mechanism of lung cancer invasion and metastasis regulated by Cul4A is complex, and further studies regarding Cul4A and other metastasis suppressors are still warranted in the future.
ANXA10 is the latest identified member of the annexin family of calcium (Ca 2+ ) and phospholipid-binding proteins [35]. The downregulation of ANXA10 correlates with decreased differentiation, invasion, and tumor progression, pointing to a possible tumor suppressor role [35]. In bladder cancer, the downregulation of ANXA10 is also related to the aggressiveness of the cancer [25]. In hepatocellular carcinoma, the downregulation of ANXA10 correlates with p53 mutation and it is associated with vascular invasion, tumor progression, and poor prognosis [36]. Decreased ANXA10 has been correlated with increased invasion in a colorectal cancer cell line and with the increased proliferation and migration in a gastric cancer cell line [37]. Additionally, the upregulation of S100A4, which is considered a mediator of metastasis, has been reported to downregulate ANXA10 in a lung cancer cell line [38]. In our study, the knockdown of ANXA10 increases lung cancer cells migration and invasion. These reports together show strong evidence of the tumor suppressor and metastasis role of ANXA10 in cancer cells.
In conclusion, our results showed that Cul4A is important in lung cancer cell invasion and metastasis through the inhibition of ANXA10, a tumor suppressor. Our results add ANXA10 to the repertoire of tumor suppressor proteins that are inhibited by Cul4A in cancer, and thus, it suggests Cul4A as a potential drug target for the development of novel therapy for lung cancer in the future.

Conclusions
Our findings suggest that Cul4A is a prognostic marker in NSCLC patients after surgery of lung cancer. Cul4A also plays important roles in lung cancer invasion and metastasis partially through ubiquitin-mediated protein degradation of ANXA10 in lung cancer cells. The role of ANXA10 as a tumor and metastasis suppressor in lung cancer cells was further confirmed.