B7-H3 promotes the migration and invasion of colorectal cancer cells via regulating the actin cytoskeleton and RhoA/ROCK1/LIMK1 signaling pathway

doi:10.21203/rs.3.rs-3617952/v1

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B7-H3 promotes the migration and invasion of colorectal cancer cells via regulating the actin cytoskeleton and RhoA/ROCK1/LIMK1 signaling pathway

https://doi.org/10.21203/rs.3.rs-3617952/v1

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Aberrant expression of B7 homolog 3 protein (B7-H3) has been detected in various cancers including colorectal cancer (CRC) and implicated in modulating multiple biological functions of CRC cells. However, its role in CRC metastasis has not yet been determined. In this study, we demonstrated that B7-H3 was highly expressed in CRC tissues and positively associated with poor prognosis of CRC patients. B7-H3 knockdown significantly inhibited the migration and invasion of CRC cells. B7-H3 overexpression had the opposite effect. Moreover, we determined that B7-H3 could regulate actin cytoskeleton and the RhoA/ROCK1/LIMK1 pathway. Importantly, the BDP5290, an inhibitor of the RhoA/ROCK1/LIMK1 axis, reversed the effects of B7-H3 overexpression on actin filament accumulating, migration, and invasion of CRC cells. In sum, our study concluded that B7-H3 facilitated CRC cell actin filament accumulating, migration, and invasion through the RhoA/ROCK1/LIMK1 axis.

Colorectal cancer

B7-H3

migration

invasion

F-actin

LIMK1

Worldwide, colorectal cancer (CRC) is the third most prevalent and second most lethal form of cancer, posing a huge threat and burden to society[1]. In recent years, advances in surgery, chemotherapy, and immunotherapy have achieved improved results, but relapse and metastasis still occur in most patients with CRC [2–4]. Moreover, metastasis is the main reason for the death of cancer patients. Therefore, it is necessary to elucidate the molecular mechanisms of metastasis of CRC.

B7 homolog 3 protein (B7-H3), also known as CD276, belongs to the B7 superfamily of immune checkpoint molecules. A number of clinical studies showed that B7-H3 is lowly expressed in most normal tissues but is highly expressed in tissues of multiple cancers, including breast, lung, ovarian, gastric, and CRC[5]. Furthermore, the high expression of B7-H3 was positively associated with TNM stage and prognosis in many cancers[6]. B7-H3 has been demonstrated to be associated with a variety of biological functions of cancers[6, 7]. For example, overexpression of B7-H3 promoted angiogenesis in CRC through activating the NF-κB/VEGFA pathway [8]. In clear cell renal cell carcinoma (ccRCC), B7-H3 could bind to exogenous Fibronectin, activate kinases [namely, phosphorylated (p)-phosphoinositide 3-kinase, p-protein kinase B, p-p38 and p-extracellular regulated protein kinase], and promote the migration and invasion of ccRCC cells [9]. Tingting Yu et al. noted that B7-H3 overexpression markedly elevated the ability of proliferation, epithelial-mesenchymal transition, invasion and migration of lung cancer cells [10]. However, the precise roles and molecular mechanisms of B7-H3 in modulating migration and invasion of CRC cells remain unclear.

In this study, we observed that B7-H3 was highly expressed in tumor tissues of CRC and positively poor prognosis of patients with CRC. The expression of B7-H3 was positively related to LIMK1 in CRC tissues. B7-H3 knockdown significantly inhibited the migration and invasion of CRC cells. B7-H3 overexpression had the opposite effect. Moreover, B7-H3 promoted the migration and invasion of CRC cells through modulating actin cytoskeleton remodeling.

CRC patients and specimens

Tissue microarray (TMA) chips containing 87 adjacent normal tissues and 94 CRC tissue samples were purchased from Shanghai Biochip Co., Ltd. (Shanghai, China). The follow-up data was also obtained. Table 1 shows the clinical data of each patient with CRC. Our experiments based on CRC specimens were approved by the Ethics Review Board of the First Affiliated Hospital of Soochow University.

Table 1

Clinical characteristics of patients
CRC patients	Number
NO. of patients	94
Gender
Male	46
Female	48
Age (years)
Median	65
Range	29–82
Death
Yes	65
No	29
AJCC stage
I-II	56
III-IV	38

Immunohistochemistry

We performed the IHC assay as previously described [11]. Briefly, The CRC tissues in the TMA chip were subjected to deparaffinization, antigen retrieval, blockage, and incubation with rabbit anti-human B7-H3 antibody (Proteintech, Wuhan, China) or rabbit anti-human LIMK1 antibody (Proteintech) at 4°C overnight. Then, these tissues were incubated with the corresponding HRP-labeled secondary antibody (Genetech, China), this was followed by visualization by DAB staining (brown) and hematoxylin counterstaining (purple). The score of IHC was judged using the H-score semiquantitative scoring system[11].

Cell lines and culture conditions

HCT116 and RKO cell lines (ATCC, Manassas, VA, USA) were maintained at 37°C 5% CO₂ and cultured in DMEM (Biological Industries, BeitHaemek, Israel) supplemented with 10% fetal bovine serum (FBS, Biological Industries) and 1% penicillin-streptomycin (Beyotime, Shanghai, China).

Infection of lentivirus in CRC cells

Lentivirus vectors carrying human 4IgB7-H3 cDNA and B7-H3 short hairpin RNA (shRNA) were purchased from Shanghai Genechem Co., Ltd. (Shanghai, China). An empty backbone vector was used as a control. For infection, CRC cells were infected with lentiviral particles at an MOI of 30, and the overexpression or interference efficiency was verified by western blot.

Western blot analysis

We performed western blot as described in our previous studies[12]. The protein bands were incubated overnight with the following primary antibodies: rabbit anti-human B7-H3 antibody (Proteintech), rabbit anti-human RhoA antibody (Proteintech), rabbit anti-human ROCK1 antibody (Proteintech), rabbit anti-human LIMK1 antibody (Proteintech) and mouse anti-human LIMK1 antibody (Proteintech).

Wound healing assay

We used a 20 µl pipette tip to draw a straight line in the center of CRC cells in 6-well plates. After the line was drawn, the cells were left standing for 30 min, and the time was recorded. The scratch distance was observed and photographed at 0 h and 24 h.

Migration and invasion assays

To investigate the effects of B7-H3 on CRC cell migration and invasion, we performed transwell experiments. Cells in each group (5×10⁴) were cultured in a medium without FBS and added to the upper chamber in a 24-well plate (BD Biosciences, NJ, USA). The lower chamber contained medium with 20% FBS. For invasion assay, Matrigel (200 µg/ml, Corning, Shanghai, China) was used to coat the upper chamber. After culturing for 24 h, the cells on the filter membranes were fixed using 4% paraformaldehyde and stained using 0.1% crystal violet.

F-actin immunofluorescence staining

Cells in each group (1×10⁴) were cultured in a 24-well plate and incubated overnight. Then, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton, and then blocked with 5% FBS. F-actin staining was used with an actin-Tracker Red-555 kit (Beyotime) according to the manufacturer’s instructions. Nuclei were stained with DAPI.

Statistical analysis

Student’s t-test was used for statistically significant differences between groups. For clinical data, the SPSS software (IBM Corporation, Armonk, NY, USA) was used. Pearson correlation analysis was used to analyze the correlation assay. Survival analysis was performed by using the Kaplan–Meier method. A P-value of < 0.05 was considered statistically significant in all cases.

B7-H3 is highly expressed in CRC tissues and positively associated with lymph node metastasis and poor prognosis.

We first performed an IHC assay to investigate the expression of B7-H3 in 90 pairs of CRC cancer tissues and adjacent normal tissues. As shown in Fig. 1A, the protein expression of B7-H3 in CRC tissues was much higher than that in adjacent normal tissues. Furthermore, the B7-H3 expression was higher in patients at AJCC stage III-IV compared with patients at AJCC stage I-II (Fig. 1B). To explore the relationship between B7-H3 expression and the prognosis of CRC patients, the patients were divided into the B7-H3 low expression group (IHC score ≤ 6) and the B7-H3 high expression group (IHC scores > 6). Kaplan-Meier analysis showed that the survival time of patients in the B7-H3 high-expression group was significantly lower than in the B7-H3 low-expression group (Fig. 1C).

B7-H3 promoted CRC cell migration and invasion

Then, we explored the effects of B7-H3 on the migration and invasion of CRC cells. Stable B7-H3 knockdown CRC cell lines were established using a B7-H3 shRNA[12]. As shown in Fig. 2A, the B7-H3 protein levels were obviously decreased in B7-H3 knockdown HCT116 and RKO cells compared with the control cells. Subsequently, Transwell and wound healing assays were used to measure the migration and invasion of B7-H3 knockdown CRC cells. Results showed that silencing of B7-H3 led to a decrease in the capacity for migration and invasion of HCT116 and RKO cells. Importantly, B7-H3 knockdown obviously inhibited the lung metastasis of HCT116 cells in vivo (Fig. 2).

To further verify these effects, a B7-H3 overexpression lentivirus and infected CRC cell lines were constructed. Western blot confirmed that the protein expression levels of B7-H3 were higher in stable B7-H3-overexpressing HCT116 and RKO cell lines than in control cells (Fig. 3A). As shown in Fig. 3B, B7-H3 overexpression significantly elevated the capacity for migration and invasion of HCT116 and RKO cells (Fig. 3B-D).

B7-H3 modulated actin cytoskeleton and RhoA/ROCK1/LIMK1 pathway

To investigate how B7-H3 modulates CRC cell migration and invasion, we performed the enrichment of molecular function using the differentially expressed genes (DEGs) between negative control (NC) and shB7H3 HCT116 cells, which were obtained from our previous study [12]. As shown in Fig. 4A, B7-H3 was involved in the cytoskeletal motor activity and microtubule binding, suggesting that B7-H3 may regulate CRC cell migration and invasion through the actin cytoskeleton. The results of immunofluorescent staining showed that the actin filament accumulating was significantly lower in the B7-H3 knockdown group than in the control group (Fig. 4B). B7-H3 overexpression had the opposite effect (Fig. 4C). It has been well documented that the RhoA/ROCK1/LIMK1 signaling pathway was involved in regulating actin cytoskeleton[13, 14]. We observed that depletion of B7-H3 reduced the protein expression levels of RhoA, ROCK1, and LIMK1 in CRC cells and that the overexpression of B7-H3 enhanced the expression of RhoA, ROCK1 and LIMK1 (Fig. 4C-E).

B7-H3 promoted CRC cell migration and invasion through the RhoA/ROCK1/LIMK1 pathway

To demonstrate whether B7-H3-induced migration and invasion of CRC cells were associated with the RhoA/ROCK1/LIMK1, BDP5290, an inhibitor of ROCK1, was used. As shown in Fig. 5A, the BDP5290 treatment counteracted the promotion of B7-H3 overexpression on LIMK1 expression in HCT116 and RKO cells. Moreover, the addition of BDP5290 reversed the effects of B7-H3 overexpression on CRC cell migration and invasion (Fig. 5B). Treatment with BDP5290 decreased the actin filament accumulating in B7-H3 overexpressing HCT116 and RKO cells (Fig. 5C). These results suggested that B7-H3 promoted CRC cell migration and invasion through the RhoA/ROCK1/LIMK1 pathway.

B7-H3 was positively correlation with LIMK1 expression in CRC tissue specimens

As shown in Fig. 6A, compared with adjacent normal tissues, the protein levels of LIMK1 were significantly up-regulated in CRC tissues. Moreover, the LIMK1 protein expression was higher in patients with the III-IV AJCC stage than that in patients with I-II AJCC stage (Fig. 6B). Patients with high expression of LIMK1 (IHC score＞6) predicted a shorter overall survival compared with patients with low expression of LIMK (IHC ≤ 6) (Fig. 6C). Then, we investigate the clinical correlation between B7-H3 and LIMK1 protein levels in CRC patient specimens. As shown in Fig. 6D and 6E, there was a positive correlation between B7-H3 and LIMK1 protein expression in CRC tissues. Importantly, Kaplan-Meier analysis showed that patients with simultaneous overexpression of B7-H3 and LIMK1 had the shortest overall survival time (Fig. 6F).

Deregulated expression of B7-H3 has been found in human malignant tumors including CRC. Low expression of B7-H3 was a significant prognostic biomarker of CRC associated with better survival [15]. In this study, we observed that B7-H3 was highly expressed in the cancer tissues of a cohort of CRC patients, and its high expression was positively correlated with TNM stage and lymph node metastasis. More importantly, B7-H3 high expression was associated with worse survival of CRC patients. These results suggest that B7-H3 plays an important role in the progression of CRC. A number of studies showed a multifaceted role of B7-H3 in tumor progression by regulating various cancer hallmarks such as glycolysis, angiogenesis, chemoresistance, EMT, and immune evasion[6]. For instance, conditioned medium from B7-H3 silencing CRC cells markedly suppressed the migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs) [8]. B7-H3 overexpression enhanced the resistance of CRC cells to oxaliplatin by upregulating the expression of XRCC1 via the PI3K-AKT pathway[16]. Hu et al. found that B7-H3 overexpression obviously promoted CRC cell multiplication and migration [17]. Moreover, overexpression of B7-H3 facilitated cell migration and invasion in CRC by elevating MMP9 through upregulation of the Jak2-Stat3 signaling pathway[18]. Herein, we verified that B7-H3 depletion restrained CRC cell migration and invasion, while B7-H3 overexpression worked oppositely. Therefore, we thought that B7-H3 would act as a key regulator for CRC metastasis.

Actin cytoskeleton exerts a critical role in the migration and movement of tumor cells[19–21]. For instance, leucine zipper tumor suppressor family member 3 (LZTS3) regulated CRC cell proliferation, migration, and actin cytoskeleton, which inhibited the progression of CRC[22]. A study by Guanghui Hu et al. showed that the loss of liver kinase B1 (LKB1) facilitated tumor cell metastasis in CRC via the upregulation of TRAF2 and NCK-interacting protein kinase (TNIK) and subsequently induced cytoskeleton remodeling[23]. In addition, phospholipase C Beta 1 (PLCB1) depletion resulted in a decrease in gastric cancer cell migration, and invasion by modulating actin cytoskeletal remodeling and EMT [24]. In the current study, we used RNAseq data to perform KEGG analysis and found that B7-H3 was involved in the cytoskeletal motor activity, suggesting that B7-H3 may regulate CRC cell migration and invasion through the actin cytoskeleton. Moreover, B7-H3 knockdown inhibited; while B7-H3 overexpression promoted; the actin filament accumulating in CRC cells. These data suggest that B7-H3 might regulate the actin cytoskeleton, migration, and invasion of CRC cells.

The RhoA/ROCK1/LIMK1 signaling pathway has been well documented to be involved in the regulation of several biological processes, such as actin cytoskeleton, migration, and invasion of cancer cells[25]. For example, calcium and transient receptor potential vanilloid 4 (TRPV4), a calcium channel, was involved in cytoskeleton regulation and RhoA/ROCK1/LIMK1 signaling pathway, which controlled cell metastasis of endometrial cancer cells[14]. Li Yan et al. reported that Mex-3 RNA Binding Family Member A (MEX3A) elevated cell proliferation and migration in breast cancer through the RhoA/ROCK1/LIMK1 signaling pathway [26]. Iroquois homeobox 5 (IRX5) overexpression significantly enhanced cell metastasis in CRC by blocking the RhoA/ROCK1/LIMK1 signaling pathway[27]. Herein, our western blot assay indicated that B7-H3 silencing reduced the protein expression levels of RhoA, ROCK1, and LIMK1. Furthermore, the BMS-4, an inhibitor of the RhoA/ROCK1/LIMK1 axis, reversed the effects of B7-H3 overexpression on actin filament accumulating, migration, and invasion of CRC cells. More importantly, we observed that B7-H3 expression was positively associated with LIMK1 expression in cancer tissues of CRC patients, and patients with simultaneous overexpression of B7-H3 and LIMK1 had the shortest overall survival time. These data illustrate that B7-H3-mediated RhoA/ROCK1/LIMK1 signal transduction was key for CRC cell metastasis. However, the precise molecular mechanism of B7-H3 regulating the RhoA/ROCK1/LIMK1 pathway remains unclear. Further investigations are needed.

In summary, our findings indicated that B7-H3 positively regulated actin cytoskeleton, migration and invasion of CRC cells via the RhoA/ROCK/LIMK1 pathway. Our present findings provided potential targets in CRC therapy and prevention.

Data availability

The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This work was supported by Jiangsu Provincial Medical Key Discipline (ZDXK202246); the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (20KJA310005) and Key Project of Medical Research of Jiangsu Commission of Health (ZDA2020008).

Competing Interests

The authors declare that they have no competing interests.

Author contributions

TS, QX and SX designed experiments. AZ and XZ analyzed the data, interpreted the results, and wrote the manuscript. HW and JW performed the experiments and analyzed the data. MZ and JX made suggestions during the writing. All authors read and approved the final manuscript.

Ethics Approval

This study was approved by the Ethics Review Board of the First Affiliated Hospital of Soochow University (reference number: 2021-327).

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No competing interests reported.

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Version 1

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You are reading this latest preprint version

B7-H3 promotes the migration and invasion of colorectal cancer cells via regulating the actin cytoskeleton and RhoA/ROCK1/LIMK1 signaling pathway

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

CRC patients and specimens

Immunohistochemistry

Cell lines and culture conditions

Infection of lentivirus in CRC cells

Western blot analysis

Wound healing assay

Migration and invasion assays

F-actin immunofluorescence staining

Statistical analysis

Results

B7-H3 promoted CRC cell migration and invasion

B7-H3 modulated actin cytoskeleton and RhoA/ROCK1/LIMK1 pathway

B7-H3 promoted CRC cell migration and invasion through the RhoA/ROCK1/LIMK1 pathway

B7-H3 was positively correlation with LIMK1 expression in CRC tissue specimens

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1