p62 functions as an oncogene in colorectal cancer through inhibiting apoptosis and promoting cell proliferation by interacting with the vitamin D receptor

Abstract Objectives The role of p62 in cancer is controversial. Evidence has shown that p62 is upregulated in different cancers and promotes tumour growth, such as in liver cancer and lung cancer. However, a recent study showed that the downregulation of p62 in hepatic stellate cells (HSCs) promotes hepatocellular carcinoma (HCC) development. How p62 is regulated in colorectal cancer (CRC) remains largely unknown. In this study, we aimed to investigate the roles and molecular mechanisms of p62 in CRC. Materials and Methods The expression levels of p62 in CRC tissues and adjacent non‐tumour tissues were determined by immunohistochemistry (IHC). Stable p62‐overexpression HCT116 cells and p62‐knockdown SW480 cells were established with lentiviral vectors. The role of p62 in CRC was investigated in in vitro and in vivo functional studies. The relationship between p62 and the vitamin D receptor (VDR) was investigated by coimmunoprecipitation (Co‐IP) assays. Results p62 was significantly upregulated in CRC, and a high p62 level was an independent risk factor for a poor prognosis in CRC patients. p62 promoted CRC migration and invasion by inhibiting apoptosis and promoting cell proliferation in vitro, and p62 aggravated tumour growth and metastasis in vivo. Co‐IP assays indicated that p62 interacts with the VDR and may target the NRF2‐NQO1 axis. Conclusions Our study suggested that p62 functions as an oncogene in CRC through inhibiting apoptosis and promoting cell proliferation by interacting with the VDR.


| INTRODUC TI ON
Colorectal cancer (CRC) is one of the deadliest cancers worldwide and develops by the accumulation of genetic and environmental factors. 1 In 2012, the International Agency for Research on Cancer (IARC) has reported that CRC is the third most common cancer in men (10.0% of global incident cancer cases in men) and the second most common cancer in women (9.2% of global incident cancer cases in women). The mortality of CRC is the third highest in all cancers, bringing a great burden to human beings, 2 but the pathogenesis of CRC remains unclear.
Protein p62 is a classic autophagy receptor and encodes the gene SQSTM1. It has four structural domains, including LIR (LC3-interacting region), PB1 (Phox/Bem 1p), TB (TRAF6-binding) and UBA (ubiquitin-associated) domains. The LIR domain is combined with the autophagy regulator ATG8/LC3. 3 Autophagy can be activated by metabolism in an emergency. When cancer cells are unable to obtain extracellular metabolites and energy sources, autophagy may promote tumour development. 4,5 As a ubiquitin-binding protein, p62 plays an important role in response to DNA damage.
Autophagy-deficiency-induced p62 accumulation can inhibit DNA double-strand break (DSB)-induced histone and chromatin ubiquitination. 6 The role of p62 in cancer is controversial. Evidence has shown that p62 is upregulated in different cancers and promotes tumour growth, such as in liver cancer 7 and lung cancer. 8 The accumulation of p62 enhanced endoplasmic reticulum (ER) stress, mitochondrial dysfunction, reactive oxygen species (ROS) production and genome damage. The inhibition of p62 can suppress tumorigenesis. 6 However, a recent report from Duran et al adds a series of interesting twists to the role of p62 in cancer. They found that p62 inhibits the activity of hepatic stellate cells (HSCs) by promoting the formation of the vitamin D receptor (VDR)-retinoid X receptor (RXR) heterodimerization and that the downregulation of p62 in HSCs promotes HCC development by reducing the interaction of VDR-RXR. 9 Vitamin D is widely known for its role in calcium and phosphate metabolism. In the mid-1970s, scientists found that a nuclear receptor, VDR, was present in numerous tissues. It is not related to calcium and bone but associated with a large number of other diseases. 10 Enhanced VDR signalling can reduce β cell failure and inhibit type 2 diabetes. 11 VDR can reduce myocardial ischaemia/ reperfusion injury by reducing oxidative stress and inhibiting apoptosis-mediated cell death. 12 Morphine-induced T-cell apoptosis is mediated through the downregulation of VDR and the activation of renin-angiotensin system (RAS). 13 In genistein, VDR can mediate the proliferation of MC3T3-E1 cells but is not affected by the oestrogen receptor. 14 Vitamin D is also important against bacterial infections. Bacteria can inhibit the expression of VDR and leucine-leucine-37 (LL-37) to avoid antibacterial defence. Vitamin D supplementation can upregulate the expression of VDR and LL-37 of macrophages and enhance the immunological defence against spontaneous peritonitis in patients with cirrhosis. 15 A recent study examined the status of LL-37, tumour necrosis factor (TNF) and vitamin D in tuberculosis (TB) patients. They find that the mean levels of LL-37 and TNF were higher in the TB group compared with healthy controls. However, there was no correlation between levels of LL-37, TNF and vitamin D in patients with TB. 16 In tumours, high VDR expression in CRC stromal fibroblasts is associated with better overall survival. 17 In addition, Vitamin D deficiency resists proliferation and promotes apoptosis though the downregulation of Wnt/β-catenin signalling in human breast and prostate cancers. 18 In this study, we aimed to investigate the roles and molecular mechanisms of p62 in CRC. We found that p62 is significantly upregulated in CRC, and p62 functions as an oncogene in CRC through inhibiting apoptosis and promoting cell proliferation by interacting with the VDR.

| Cell lines and cell culture
Human CRC cell lines HT29, HCT116, SW480, SW620, Caco2 and DLD-1 and the normal human colon cell line HCoEpiC were cultured in high glucose DMEM containing 10% foetal bovine serum (FBS; Gemini, Calabasas, CA, USA). All cells were maintained in a humidified incubator in 5% CO 2 at 37°C.

| Lentiviral transduction
The hU6-MCS-CMV-EGFP knockdown lentiviral vector for SQSTM1, the Ubi-MCS-3FLAG-SV40-puromycin overexpressing lentiviral vector for SQSTM1 and the control lentiviral vector were obtained from Genechem Co., Ltd. (Shanghai, China). The SW480 p62-knockdown cells using lentivirus infection and the efficiency of transduction were controlled by GFP fluorescence. A stable HCT116 p62-overexpression cells was established using lentivirus infection and selected with 2 ng/mL puromycin.

| Cell viability analysis
Cell viability was detected using the CCK-8 assay. Cells were seeded in 96-well plates at a density of 5 × 10 3 cells in 100 μL of medium and cultured for 1-4 days. Then, CCK-8 reagent was added to each well. After an hour reaction at 37°C, the absorbance of the density of each well was read at a wavelength of 450 nm with a microplate reader (Thermo, Waltham, MA, USA).

| Wound healing
Cells were seeded into 6-well plates. After confluence, cells were scratched using a 1 mm wide tip and cultured in serum-free DMEM.
Images were captured with a microscope at 100× magnification at 0, 24 and 48 hours. Wound spacing was calculated and analysed.

| Colony formation
One thousand cells were seeded into 6-well plates and incubated at 37°C for 14 days. Then, cells were fixed in 100% methanol and stained with 0.5% crystal violet, and colonies were counted.

| Mouse xenograft and metastasis models
Male athymic nude mice (BALB/c, 5 weeks old) were purchased from the Xi'an Jiaotong University Medical Laboratory Animal Center. All experiments were approved by Xi'an Jiaotong University. For xenograft models, five nude mice in each group were subcutaneously injected with 1 × 10 6 cells. After a month, the mice were sacrificed, and the tumours were weighed. For metastasis models, each group of mice was injected with 1 × 10 6 cells in the tail vein and sacrificed after 2 months. Lung tissue was removed for HE staining.

| Immunohistochemistry (IHC)
Paraffin-embedded slides and tissue microarrays were dewaxed with dimethylbenzene (I and II) and gradient alcohol (100%, 95%, 85% and 75%). The antigen retrieval was performed by microwaving sections in citrate buffer for 2 minutes. Slides were incubated with 3% hydrogen peroxide (H 2 O 2 ) for 20 minutes and then incubated with goat serum for 30 minutes at room temperature. Without washing, primary antibodies SQSTM1/p62 (1:1000; ab109012; Abcam), Ki67 (1:50; ab8191; Abcam) and caspase-7 p20 (1:50; SC-28295; Santa Cruz Biotechnology, Santa Cruz, DE, USA) were applied at 4°C overnight. Biotinylated anti-IgG was added and incubated at room temperature for 1 hour. After incubating with streptomycin-HRP for 30 minutes, the slides were stained with DAB and then counterstained with haematoxylin. After washing with flowing water, the slides were dehydrated with gradient alcohol and dimethylbenzene. Finally, the slides were sealed with neutral balsam and coverslips.

| Evaluation of immunostaining intensity
Tissue microarrays were scored independently by two pathologists who were blinded to the clinicopathologic features and outcomes of the patients. The staining was evaluated under low magnification (40×), then selected five views under high magnification (200×) for analysis.

| Statistical analysis
graphpad prism 5 (La Jolla, CA, USA) was used for all statistical analyses. The comparisons between two groups were performed using Student's t test. Before using Student's t test, we performed a variance homogeneity test and normality test for the variable data. The overall survival rates were estimated using Kaplan-Meier analyses and log-rank tests. The chi-squared test was used to determine whether there was a significant difference in the distribution of p62 samples among the different categories. A Cox regression model was used for univariate and multivariate analyses to calculate the hazard ratio and its 95% confidence interval. Data are presented as the mean ± SD. All statistical tests were two-sided, and values of P < 0.05 were considered significantly different.
F I G U R E 1 p62 is upregulated in CRC tissues and indicates poor prognosis in human CRC. A, SQSTM1/p62 mRNA expression levels are upregulated in CRC tissues compared to normal controls in the TCGA dataset (Student's t test, P < 0.001). B, Relative mRNA expression of p62 in primary CRC tissues and paired adjacent non-tumour tissues (Non) (n = 22), (Student's t test, P < 0.001). C, Western blot analysis of p62 in 6 representative paired adjacent non-tumour tissues (N) and CRC tissues (T). Bars represent the standard error of the mean ± SD from three independent experiments (Student's t test, **P < 0.01). D, Representative images of IHC staining of p62 in 92 CRC tissues and 71 adjacent non-tumour tissues. The high (right) and low (left) expression levels of p62 were evaluated semi-quantitatively by the staining intensity (high score: 7-12; low score: 0-6). E, Chi-square analysis of the p62 levels in 92 CRC tissues and 71 adjacent non-tumour tissues (P < 0.001). F, Kaplan-Meier curve depicting the overall survival of the CRC patients (n = 92). The curves were stratified based on the p62 level (log-rank test, P = 0.01)

| p62 is upregulated in CRC tissues and indicates poor prognosis in human CRC
We first investigated the involvement of p62 in CRC patients from the TCGA dataset and identified that SQSTM1/p62 mRNA expression was significantly increased in CRC tissues compared to normal controls ( Figure 1A, P < 0.001). We also examined the expression  (Table 1, P < 0.05). In addition, the patients with higher expression of p62 had shorter overall survival than those with lower expression of p62, indicating that the p62 level was an independent risk factor for a poor prognosis in CRC patients ( Figure 1F, P = 0.01 and Table 2, P < 0.05).

| p62 shows tumour-promoting ability in CRC cells
The upregulation of p62 in clinical CRC tissues suggested the poten-    Figure 2D, P < 0.05) and the reduced colony formation ability in p62-knockdown SW480 cells compared with that of the SW480-EV cells ( Figure 2D, P < 0.001). These data collectively suggest that p62 plays a tumour-promoting role in CRC cells. In addition, the effect of p62 on cell apoptosis was evaluated.
The knockdown of p62 in SW480 cells induced early apoptosis compared to the control cells ( Figure 2E, P < 0.001). The overexpression of p62 in HCT116 cells suppressed early apoptosis compared to the control cells, but no significant difference was found ( Figure S1, P > 0.05). The effect of p62 on cell apoptosis was further examined by the reduced protein expression of cleaved-caspase-7(cleaved-cas7) and cleaved-PARP1 in p62-overexpression HCT116 cells and by the increased expression of these proteins in p62-knockdown SW480 cells ( Figure 2F, P < 0.01). These results indicated that the oncogenic effect of p62 is also associated with the suppression of cell apoptosis.

| p62 promotes the invasion and migration abilities in CRC cell lines
The oncogenic role of p62 in CRC was further evaluated for invasion and migration. Transwell assays showed that invasion and migration were significantly enhanced in p62-overexpression HCT116 cells ( Figure 3A, P < 0.01). Conversely, p62-knockdown SW480 cells showed the opposite effect on cell invasion and migration ( Figure 3B, P < 0.01). In addition, the monolayer scratch healing assays showed that the overexpression of p62 significantly increased the migration ability of HCT116 cells ( Figure 3C, P < 0.05), while knockdown of p62 in SW480 cells suppressed the migration capacity ( Figure 3D, P < 0.01), indicating that p62 enhances CRC invasion and migration abilities.

| p62 aggravates tumour growth and metastasis in vivo
To   in mouse tumours injected with p62-knockdown SW480 cells than those injected with sw480-EV cells. Cleaved-cas7 expression levels were higher in mouse tumours injected with p62-knockdown SW480 cells than those injected with sw480-EV cells ( Figure 4E).
The lung volume of nude mice was decreased in the mice injected with p62-knockdown SW480 cells compared with those injected with SW480-EV cells ( Figure S2). These data indicated that p62 promotes tumour metastasis in CRC.

| p62 interacts with the vitamin D receptor and may target the Nrf2-NQO1 axis through VDR in human CRC cells
To characterize the mechanism of p62 as an oncogene in CRC cells, we measured the protein expression levels of VDR in human CRC cell lines. p62 deficiency enhanced the protein levels of VDR in SW480, and p62 overexpression reduced the protein levels of VDR in HCT116 ( Figure 5A, P < 0.01). We next examined whether p62 and VDR interact. Co-IP assays showed that exogenous VDR could coprecipitate with endogenous p62, and exogenous p62 could also coprecipitate with endogenous VDR in p62-overexpression HCT116 cells ( Figure 5B). We also examined the protein levels of Nrf2 and NQO1 and found that the expression of Nrf2 and NQO1 proteins were increased in p62-overexpression HCT116 cells, while the expression levels were decreased in p62-knowdown SW480 cells ( Figure 5C, P < 0.01). Additionally, a piece of the regulatory network of p62 in CRC was elucidated ( Figure 5D).

| D ISCUSS I ON
In previous reports, p62 accumulation is frequently observed in a variety of solid tumours. The role of p62 in multiple solid tumours F I G U R E 5 P62 interacts with the vitamin D receptor. A, Expression of the VDR was detected by Western blotting. B, Total lysates from p62-overexpression HCT116 cells were subjected to IP with VDR Ab or p62 Ab, followed by Western blotting using the indicated antibodies. C, Expression levels of Nrf2 and NQO1 were detected by Western blotting. Bars represent the standard error of the mean ± SD from three independent experiments. *Represents Student's t test *P < 0.05 and **P < 0.01. D, Proposed molecular mechanism of p62 in CRC. P62 functions as an oncogene by inhibiting apoptosis and promoting cell proliferation by interacting with the VDR, and p62 may affect the Nrf2-NQO1 axis through the VDR has been demonstrated, highlighting its oncogene effects in ROS generation and cell growth. 6 Only one study reported that p62 is a prognostic predictor associated with CRC cell proliferation in vitro. 19 However, studies about the biological effects of p62 in CRC remain unclear. In this study, we first demonstrated that p62 was an oncogene for CRC both in vivo and in vitro and that overexpressed p62 significantly inhibited apoptosis. This may be related to the interaction between p62 and VDR. We first comprehensively analysed the TCGA database and the detection of clinical samples, which proved that p62 is overexpressed in CRC and that the enrichment of p62 predicts poor survival.
Apoptosis  Figure 2E,F). This is the first study to demonstrate that p62 is associated with inhibition of apoptosis in CRC. Then, we used transwell assays and wound healing assays to demonstrate that p62 promotes the invasion and migration abilities in CRC cell lines. p62 also aggravates tumour growth and metastasis in vivo. Therefore, we reasoned that p62 was an oncogene for CRC by inhibiting apoptosis.
We also investigated the effect of p62 on cell proliferation in CRC. C-Myc is a member of the myc family, which promotes cell proliferation and inhibits differentiation. C-Myc is often overexpressed in many cancers. 24 The regulation of the cell cycle mainly depends on cyclins and cell cycle protein kinases (CDKs shown to be associated with early cancer incidence and tumour progression. Cyclin D1 responds to mitotic growth factors and activates its homologous CDK in the G1 phase. 25 Our study shows that the expression levels of c-Myc, CKD2 and cyclinD1 proteins were increased in p62-overexpression HCT116 cells, while their expression was decreased in p62-knowdown SW480 cells, demonstrating that p62 also promotes cell proliferation in CRC.
Medical standards have been improving in recent years, but vitamin D deficiency is still a major health problem in the population.
The risk factors for developing vitamin D deficiency include low sun exposure, higher geographical latitude, 28 heavy use of sunscreen 29 and a diet containing inadequate levels of vitamin D. 30 In addition to the function of maintaining bone health and metabolism, the liganded VDR functions also involve the regulation of cell proliferation and apoptosis. 31 In VDR-null mice, apoptosis of mammary epithelial cells was significantly delayed, supporting the physiological regulation of VDR in apoptosis and glandular development. 32 In pancreatic islet cells, calcitriol, which is the hormonally active metabolite of vitamin D, preventing TNF/IL-1-β/ IFN-ϒ-induced apoptosis. 33 In endometrial cancer cells, progesterone and calcitriol combination therapy increased the expression of the vitamin D receptor and inhibited cell proliferation through caspase activation and induction of G0/G1 cell cycle arrest, which is related to the downregulation of cyclins D1. 34 Vitamin D deficiency and the concomitant loss of vitamin D receptors promotes the growth of breast and prostate cancer cells. 18 VDR knockout mice showed increased sensitivity to carcinogenic challenge. 30 Calcitriol can inhibit the growth of breast cancer cell lines, especially in cells expressing ER and VDR. The VDR is a target for breast cancer therapy. 35 Angeles Duran et al reported that p62 is critical for VDR-RXR heterodimerization and inhibition of HSCs activation. The VDR signal was damaged after p62 deletion, which enhanced HSCs activation and promoted HCC. 9 Our study showed that p62 deficiency enhanced the protein levels of VDR in SW480, while p62 overexpression reduced the protein levels of VDR in HCT116. Co-IP assays showed that p62 interacts with VDR, which we speculated was related to apoptosis and cell proliferation.
Nrf2 is a basic leucine zipper protein that regulates the expression of antioxidant proteins to protect the body from oxidative damage caused by injury and inflammation. 36 High p62 expression is required to activate Nrf2 and induce c-Myc to protect HCC cells from oxidative stress-induced death. 37 Vitamin D could activate the Nrf2-Keap1 antioxidant pathway and improve renal disease in diabetic rats. 38 NQO1 is a conserved target gene of Nrf2 and can be used to monitor the activity of the Nrf2 pathway. 39 Our research found that the expression levels of Nrf2 and NQO1 proteins were increased in p62-overexpression HCT116 cells, while the expression levels were decreased in p62-knowdown SW480 cells.
In conclusion, our findings provide new mechanistic insight into the basic theory of CRC progression and suggest that the blocking of p62 may represent a potential therapeutic strategy for CRC treatment.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts to disclose.

AUTH O R ' S CO NTR I B UTI O N
Each author's contribution to the article is as follows: LD, SML and