1 INTRODUCTION

Colorectal cancer (CRC) is the third most frequent cancer worldwide and ranks second in cancer-related mortality.1 Metastasis is still the most incomprehensible part of cancer progression, accounting for up to 90% of cancer-related deaths.2 Nuclear β-catenin accumulation is a hallmark of the activation of Wnt/β-catenin pathway.3 Particularly, it was reported that nuclear β-catenin accumulation occurs in the invasive fronts of primary tumors, suggesting that activation of Wnt/β-catenin pathway closely correlates with tumor cell invasion and metastasis.4

The product of the ASPM gene localizes to centrosomes, spindle poles, and the midbody.5 In addition to its role in embryonic development, ASPM is highly expressed in many tumor cell lines.6, 7 In human glioblastoma, ASPM promotes glioblastoma cell growth by regulating G1 restriction point progression and Wnt/β-catenin signaling.8 High expression of ASPM is positively associated with poor prognosis in bladder cancer.9 ASPM expression is incrementally upregulated in metastatic prostate cancer and promotes prostate cancer stemness by augmenting Wnt/Dvl-3/β-catenin signaling.10 In colonic adenocarcinoma, ASPM-positive expression is correlated with shorter disease-free survival (DFS) time in patients.11 Although several studies have focused on various fundamental cellular processes of ASPM and its crucial mechanism in tumorigenesis, the exact role of ASPM in CRC remains largely unknown.

In this study, we found that silencing of ASPM impairs the migration, invasion and EMT abilities of CRC cells. Furthermore, we identified that ASPM promotes the metastatic traits of CRC cells by activating the β-catenin signaling pathway.

2 MATERIALS AND METHODS 2.1 Cell cultures

All cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were free of mycoplasma and authenticated by genetic profiling using polymorphic short tandem repeat loci. SW480, HCT116, RKO, LOVO, and HEK-293T cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, Massachusetts) supplemented with 10% FBS (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific). HCT116 cells were maintained in McCoy's 5A Medium (Gibco, Waltham, Massachusetts) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were cultured at 37°C in a humidified incubator containing 5% CO2.

2.2 Transfection of shRNA and plasmid construction

The ASPM shRNA knockdown construct was subcloned into the pLKO.1 puro cloning vector (Addgene, Watertown, Massachusetts), and the shRNA sequence was 5′-CCGGCCAAAGTTGTTGACCGTATTTCTCGAGAAATACGGTCAACAACTTTGGTTTTTG-3′. The shRNA plasmid-targeted scrambled sequence was used as a negative control (sh-NC). HEK-293T cells were cultured overnight to reach 80% confluence. Then, 5 μg of pLKO.1 sh-NC or pLKO.1 sh-ASPM plasmids were introduced into cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific). After incubation for 48 h, the culture medium containing lentiviral particles was collected. HCT116 or RKO cells were infected for 24 h with lentiviral particles and treated with 1 μg/mL puromycin to establish stable cells. β-Catenin siRNA was purchased from OriGene (Beijing, China). To construct overexpressing exogenous ASPM cell lines, PCR production of ASPM was cloned into the expression vector pcDNA3.1 (Invitrogen) and then transfected into HCT116 or RKO cell lines for 48 h according to the manufacturer's instructions.

2.3 Cell proliferation assay

HCT116 and RKO cells proliferation was measured by using the Cell Counting Kit-8 (Biosharp Co., Ltd, China) assay. The 96-well plates were seeded with 2 × 103 cells/well. CCK-8 solution (10 μL) was added to each well at 0, 1, 3, and 5 days after cell transfection. After incubation for 3 h, the absorbance value per well was determined with an ultraviolet spectrophotometer at 450 nm.

2.4 Transwell assay

Cells cultured in 200 μL medium without FBS were seeded into the upper chamber of matrigel-coated (8 μm pore size chamber inserts; Corning, New York) membrane filters. The lower chamber was filled with 500 μL medium with 10% FBS. After 48 h, cells invaded through the membrane were fixed with 4% formalin for 30 min, and stained with 0.1% crystal violet.

2.5 Wound healing assay

HCT116 or RKO cells (2 × 103) were seeded in six-well plates to 95% confluence. A linear wound was scratched with a 200 μL sterile pipette tip across the monolayers. The wounded monolayers were photographed at 0 or 24 h after scratching.

2.6 Quantitative real-time PCR

Total RNA was isolated using the TRIzol kit (Invitrogen) and transcribed to cDNA using the Prime-Script RT kit (Takara, China). Quantitative real-time PCR (qPCR) was performed in 96-well plates using the StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, California). After immediate reverse transcription, cDNA was amplified using SYBR Green PCR Master Mix (Takara, Japan). GAPDH was used as the control. The primer sequences are shown in Table 1. Relative mRNA expression levels were analyzed using the 2−ΔΔCT method.

TABLE 1. The sequences of genes Genes Primers ASPM F: 5′-GGCCCTAGACAACCCTAACGA-3′ R: 5′-AGCTTGGTGTTTCAGAACATCA-3′ β-catenin F: 5′-GCGCCATTTTAAGCCTCTCG-3′ R: 5′-AAATACCCTCAGGGGAACAGG-3′ GAPDH F: 5′-GACTCATGACCACAGTCCATGC-3′ R: 5′-AGAGGCAGGGATGATGTTCTG-3′ Abbreviations: F, forward; R, reverse. 2.7 Immunohistochemistry

Forty pairs of human CRC specimens and adjacent normal mucosa tissue were collected from patients undergoing standard surgical procedures at the First People's Hospital of Lianyungang. Written consent was obtained from all the participants involved in the study. Immunohistochemistry (IHC) analysis was performed, and final staining scores were determined as previously described.12 The staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). The staining area was scored as 0 (<10% positive staining), 1 (10%–25% positive staining), 2 (25%–50% positive staining), 3 (50%–75% positive staining), and 4 (>75% positive staining). The final staining scores were determined by the formula: overall score = percentage score × intensity score. An overall score of ≤3 was defined as negative, >3 to ≤6 as weakly positive, and >6 as strong positive. The experimental protocols were approved by the Institutional Review Committees of the First People's Hospital of Lianyungang (approval number: 20191009).

2.8 Western blot

Total proteins were extracted from cells using RIPA Lysis Buffer. Nuclear/cytoplasmic fractionation was performed with a Nuclei Isolation Kit (KeyGEN BioTECH) according to the manufacturer's protocols. Samples were loaded on 10% SDS-PAGE. After electrophoresis, the proteins were transferred from gels to PVDF membranes. The membranes were incubated with the primary antibodies overnight at 4°C. The primary antibodies included anti-ASPM (Proteintech, China), anti-E-cadherin, anti-N-cadherin, anti-β-catenin, anti-Lamin B1, or anti-β-actin (Cell Signaling Technology). After washing, the membranes were then incubated with HRP-conjugated secondary antibody for 2 h. The immunoreactive bands were visualized by the ECL Plus system (Tanon, China). Lamin B1 was used as the nuclear fraction control.

2.9 Co-immunoprecipitation (Co-IP)

Total protein was extracted from HCT116 or RKO cells using RIPA buffer. The cell lysates were centrifuged, the supernatant removed, and the pellet was discarded. Protein lysates were precleared by incubation with 20 μL of protein A agarose (Abcam, Cambridge, UK) at 4°C for 1 h. Following brief centrifugation, the supernatant was transferred to a new tube. The cell lysate was incubated with either anti-ASPM (Proteintech, China) or IgG antibody (Santa Cruz Biotechnology, Santa Cruz, California) and protein A agarose for overnight incubation at 4°C. Then, the supernatant was removed, and the protein A beads were washed in lysis buffer. Finally, the precipitated proteins were denatured and evaluated by western blotting using anti-ASPM (Proteintech, China) or anti-β-catenin antibody.

2.10 Luciferase reporter assay

The TOP-flash/FOP-flash luciferase reporter system (Genomeditech, Shanghai, China) was used to measure β-catenin-driven TCF/LEF transcriptional activation. HCT116 or RKO cells were transfected with TOP-Flash or FOP-Flash luciferase reporter plasmids according to the manufacturer's protocol. After 24 h, firefly and Renilla luciferase activities were analyzed using the Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin). The firefly luciferase activity level was normalized to the Renilla luciferase activity level. For analysis of the β-catenin promoter, the full-length β-catenin promoter was cloned into the pGL3-Basic vector (Promega) to generate β-catenin promoter reporter plasmids. Plasmids containing firefly luciferase reporters and pTK-RL plasmids were cotransfected into HCT116 or RKO cells, and the activities of both firefly and Renilla luciferase reporters were determined 48 h after transfection using the Dual-Luciferase Assay Kit (Promega). Promoter activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity.

2.11 Xenograft metastasis assay

We constructed an experimental metastatic model by tail vein injection of 2 × 106 sh-NC or sh-ASPM transfected HCT116 cells into BALB/C-nu/nu mice (n = 3 in each group, Shanghai Laboratory Animal Center, China) via the tail vein. After 4 weeks, the mice were euthanized by cervical dislocation. Lung tissue samples were embedded in paraffin and stained with hematoxylin and eosin (H&E). All procedures were approved by the Committee for Animal Research of the First People's Hospital of Lianyungang (approval number: 20191009) and complied with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1985).

2.12 Statistical analysis

Data are expressed as mean ± SD. Data were analyzed using GraphPad Prism version 7. Statistical differences between groups were evaluated using two-tailed t-tests. Pearson correlation analysis was performed to determine the correlation between ASPM and β-catenin levels. Statistical significance was set at P < 0.05.

3 RESULTS 3.1 High expression of ASPM positive correlates with highly aggressive phenotypes of CRC cells

First, we analyzed the endogenous levels of ASPM in the four CRC cell lines (Figure 1A,B). HCT116 and RKO cells expressing higher levels of ASPM were chosen to investigate the significance of ASPM in CRC by wound healing and transwell invasion assays. As shown in Figure 1C,D, the migration ability of HCT116, RKO, SW480, and LOVO cells weakened in the following sequence (HCT116 > RKO > SW480 > LOVO). The transwell invasion assay showed that the invasive ability of HCT116 cells was stronger than that of RKO and SW480 cells (Figure 1E,F). These results indicated that the expression level of ASPM might be positively correlated with the metastatic ability of CRC cells.

High expression of ASPM positive correlates with highly aggressive phenotypes of CRC cells. (A) qPCR analysis of ASPM in four CRC cell lines. (B) Western blot analysis of ASPM in four CRC cell lines. (C) Wound healing assays to access the migration abilities of HCT116, RKO, SW480, and LOVO. (D) The quantitative analysis of wound healing rates. (E) Transwell invasion assays to evaluate the invasion ability of cells. (F) The quantitative analysis of cells across the transwell membrane

3.2 ASPM deficiency inhibits colon cancer cell migration and invasion

Next, shRNA knocked down endogenous of ASPM was applied into HCT116 and RKO cells. The protein levels of ASPM were verified by western blotting (Figure 2A). Cell proliferation was evaluated in cells with or without ASPM downregulation, and we observed that silencing of ASPM had no inhibitory effect on CRC cell proliferation when compared with the negative control (sh-NC) group (Figure 2B,C). Additional migration and transwell invasion assays showed that loss of ASPM substantially reduced both cell motility (Figure 2D,E) and invasion ability (Figure 2F,G). To further evaluate the role of ASPM in CRC cell EMT process, we detected the protein expressions of EMT markers. Comparing with sh-NC treated cells, knocking down ASPM resulted in increasing E-cadherin protein level and decreasing N-cadherin protein level (Figure 2H,I). A pulmonary metastasis model was established to investigate the metastatic ability of HCT116 cells in vivo. As shown in Figure 2J,K, lower ASPM expression significantly reduced the number of metastatic nodules. Collectively, these results indicated that CRC cells deficient in ASPM exhibit low metastatic ability.

ASPM deficiency weakens the migration and invasion of CRC cells. (A) Western blot analysis to access the efficiency of sh-ASPM transfection in HCT116 and RKO cells. sh-NC: Negative control shRNA, sh-ASPM: ASPM shRNA. (B,C) Cell proliferation in cells after sh-ASPM transfection was determined by CCK-8 assay. (D) Wound healing assays to access the effects of ASPM deficiency on the migration abilities of HCT116 and RKO cells. (E) The quantitative analysis of wound healing rates. (F) Transwell invasion assays to evaluate the effects of ASPM deficiency on the invasion abilities of HCT116 and RKO cells. (G) The quantitative analysis of cells across the transwell membrane. (H,I) Western blot to determine the protein levels of epithelial marker (E-cadherin) and mesenchymal marker (N-cadherin) in HCT116 and RKO cells. (J) The metastatic nodules in the lung were determined by H&E staining. (K) The quantitative of data of lung metastasis by counting the micrometastatic nodules per mice. **P < 0.05 compared with sh-NC

3.3 The relationship of ASPM and β-catenin in CRC

Forty pairs of CRC tissues and adjacent normal mucosa tissues were examined by IHC analysis using anti-ASPM or anti-β-catenin antibodies (Figure 3A). The ASPM and β-catenin scores in the CRC group were higher than those in the adjacent tissue group (Figure 3B,C). The ASPM and β-catenin scores of the metastasis subgroup were also higher than those of the non-metastasis subgroup (Figure 3D,E). Interestingly, we found that ASPM was positively correlated with β-catenin in CRC tissues (P < 0.01) (Figure 3F). In the metastasis subgroup, there was a strong correlation between ASPM and β-catenin levels (P < 0.01) (Figure 3G). Therefore, these results indicated that high levels of both ASPM and β-catenin play a critical role in the metastasis of CRC.

ASPM is positively correlated with β-catenin in CRC tissues. (A) Representative images of staining with ASPM or β-catenin antibody in CRC tissue showed strong, moderate, and weak expression, respectively (scale bar: 100 μm). (B,C) Statistical analysis of ASPM staining and β-catenin staining between CRC tissues and adjacent normal mucosa tissues. **P < 0.05 compared with adjacent. (D,E) Statistical analysis of ASPM staining and β-catenin staining between metastasis and no-metastasis CRC tissues. **P < 0.05 compared with no metastasis subgroup. (F) Pearson correlative analysis of staining scores for ASPM and β-catenin. (G) Pearson correlative analysis of staining scores for ASPM and β-catenin in the metastasis subgroup

3.4 ASPM upregulates β-catenin transcription and nuclear translocation

To further explore whether ASPM regulates the β-catenin signaling pathway, we used TOP-flash and FOP-flash luciferase reporter plasmids. As shown in Figure 4A, ASPM knockdown decreased TCF/LEF transcriptional activation in RKO and HCT116 cells. Interestingly, we found that ASPM could interact with endogenous β-catenin, as confirmed by the Co-IP assay (Figure 4B). Subsequently, inhibition of ASPM downregulated the protein expression of total cellular β-catenin (Figure 4C). Subcellular fractionation revealed that overexpression of ASPM resulted in enhanced nuclear accumulation of β-catenin (Figure 4D). However, there were only marginal differences in the cytoplasmic levels of β-catenin in ASPM-overexpressing CRC cells. As ASPM facilitates nuclear entry of β-catenin, we next hypothesized whether it is a binding partner of β-catenin that would concurrently translocate to the nucleus. Nuclear extracts were prepared and subjected to immunoblotting, and nuclear translocation of ASPM was undetected (Figure 4D). To probe the mechanisms underlying the correlation between ASPM and β-catenin levels in CRC, we tested the effects of ASPM on β-catenin transcription. Indeed, β-catenin mRNA levels were significantly increased in HCT116 and RKO cells upon ASPM overexpression, while they were reduced after ASPM knockdown (Figure 4E). Moreover, the promoter luciferase reporter assay showed that ASPM could activate the full-length β-catenin promoter (Figure 4F). Meanwhile, ASPM did not affect the stability of β-catenin in the presence of the protein synthesis inhibitor cycloheximide (data not shown). Altogether, these observations imply that ASPM upregulates β-catenin transcription in CRC cells, most likely by acting on its promoter.

ASPM promotes CRC cells migration and invasion via promoting β-catenin translocation to nucleus. (A) Luciferase reporter assay using TOP-flash and FOP-flash vectors were used to evaluate the β-catenin-dependent Wnt/β-catenin signal. **P < 0.05 compared with sh-NC. (B) The enrichment of β-catenin was obtained in anti-ASPM group by comparison with matched IgG in Co-IP assay. (C) The expression of total-β-catenin was determined by western blot. (D) Cells transfected with empty vector or ASPM overexpression plasmid for 48 h. Part of the cells was used to extract nuclear and cytosolic fractions. Cell lysates were immunoprecipitated with anti-ASPM or anti-β-catenin. (E) The relative mRNA levels of PRDX1 in ASPM overexpressing or silenced CRC cell lines were determined by qPCR. (F) Luciferase activity of β-catenin promoter in CRC cells was raised by ASPM plasmid. **P < 0.05 compared with vector

3.5 β-Catenin is required for ASPM-mediated CRC cell migration and invasion

Aberrant activation of the canonical Wnt/β-catenin signaling pathway is often observed during the initiation and progression of cancer. In line with other reports, we demonstrated that silencing of β-catenin using si-β-catenin in HCT116 or RKO cells significantly blocked the ability of ASPM to promote cell migration and invasion (Figure 5A–C).13, 14 Similarly, western blot assay suggested that β-catenin deletion reversed E-cadherin and N-cadherin expressions in ASPM overexpressing HCT116 or RKO cells (Figure 5D). Based on these data, we concluded that that β-catenin is involved in ASPM-mediated CRC cell migration, invasion, and EMT process (Figure 5E).

β-Catenin mediates the function of ASPM to promote the migration and invasion of CRC cells. (A) The expression of β-catenin was detected by western blot analysis when β-catenin was knocked down in CRC cells transduced with ASPM plasmid. (B) The migration abilities were assessed by wound healing assay when β-catenin was knocked down in CRC cells transduced with ASPM plasmid. (C) The invasion abilities were assessed by transwell assay when β-catenin was knocked down in CRC cells transduced with ASPM plasmid. **P < 0.01 compared with vector, ##P < 0.01 compared with ASPM. (D) The expressions of N-cadherin and E-cadherin were detected by western blot analysis when β-catenin was knocked down in CRC cells transduced with ASPM plasmid. (E) Schematic representation of ASPM and β-catenin implicated in CRC cells migration and invasion. ASPM binds with the promoter region of β-catenin and subsequently promotes β-catenin translocation through cellular cytoplasm to nucleus

4 DISCUSSION

Tumor invasion and metastasis are regarded as the most important features of malignant tumors and are complex and multistep processes.15 CRC cells exert distant invasive potential and metastatic ability. These characteristics have been considered to be responsible for at least 90% of colon cancer-associated mortality.16 Overexpression of ASPM has been reported in breast, prostate, bladder, and colon cancers9-11, 17 and several studies have shown that knockdown of ASPM inhibits cancer cell proliferation, migration, and invasion in vitro and reduces the size of some solid tumors in vivo.18-20 ASPM expression is more intense in stage III and IV than II and I stage CRC patients, and positively correlated with lymph node metastasis.11 However, there are few reports on the specific role of ASPM in CRC metastasis.

In this study, we discovered that ASPM was highly overexpressed in CRC samples compared with that in control samples. Enhanced ASPM expression was significantly associated with aggressive CRC cell traits. We illustrated that ASPM is imperative for the migration and invasion of CRC cells. ASPM knockdown distinctly depressed cell migration and invasion in wound healing and transwell assays. Besides, we found that inhibition of ASPM resulted in decrease of N-cadherin and increase of E-cadherin, which caused a suppression of the EMT process. Similarly, the experimental lung metastasis model also indicated that silencing of ASPM weakened the metastatic ability of CRC cells in vivo. Hence, these results suggested that ASPM may act as a pro-oncogene in CRC development.

The canonical Wnt/β-catenin pathway plays a critical role in the metastasis and invasion of cancer cells.21 Cytoplasmic β-catenin is normally short-lived and is rapidly targeted for degradation. Stabilization of cytoplasmic β-catenin leads to the accumulation of translocated β-catenin in the nucleus, and complex formation of the latter with the TCF/LEF transcription factor in turn regulates the expression of Wnt/β-catenin target genes. Importantly, we demonstrated for the first time that ASPM can directly regulate β-catenin transcription in CRC cells, thereby affecting its expression and thereby regulating the accumulation of translocated β-catenin in the nucleus. Pearson correlation analysis using CRC tissues further supports the direct relationship between ASPM and β-catenin. Finally, the present study reveals that ASPM promotes CRC cell migration and invasion, which is mediated by the transcriptional activation of β-catenin. In the cytoplasm fraction, β-catenin is phosphorylated by a large multiprotein complex termed as the “APC/Axin/GSK-3 complex” and then degraded by the proteasome. In prostate cancer, ASPM also directly interacts with disheveled-3 (Dvl-3), a cardinal upstream regulator of canonical Wnt signaling, and inhibits its proteasome-dependent degradation, thereby increasing its protein stability and enabling the Wnt-induced β-catenin transcriptional activity.22 Although, we demonstrated that ASPM had no effect on β-catenin protein synthesis by using the protein synthesis inhibitor cycloheximide, the effect of ASPM on the intracellular degradation pathway needs to be further explored.

ASPM facilitates the growth of lung squamous cell carcinoma (LSCC) cells by regulating CDK4.5 Surprisingly, ASPM depletion did not affect CRC cell growth, and the underlying mechanism requires further investigation. Additionally, the aberrant expression of ASPM affects CRC clinicopathological staging, and prognosis significance would be needed to investigate further based on a clinical correlative study in a large patent cohort. Despite the finding that ASPM binds with the promoter region of β-catenin and facilitates β-catenin entry into the nucleus, we were unable to clearly map the exact binding domain between ASPM and β-catenin in this study, which will be studied in further experiments. After defined the binding domain between ASPM and the promoter of β-catenin, we need to mutate the binding sites and identify the binding sites play an important role in maintaining the binding of ASPM and β-catenin.

Altogether, we provide evidence that ASPM is involved in CRC cell aggressiveness by assisting β-catenin translocation from the cytoplasm to the nucleus. Therefore, the ASPM-β-catenin signaling pathway may be a novel and suitable target for clinical intervention in CRC metastasis.

ACKNOWLEDGMENT

We would like to thank Editage (www.editage.cn) for English language editing.

CONFLICT OF INTEREST

The authors declared that they have no conflicts of interest to this work.