Rock2 stabilizes β-catenin to promote tumor invasion and metastasis in colorectal cancer

https://doi.org/10.1016/j.bbrc.2015.10.103Get rights and content

Highlights

  • Rock2 is overexpressed in CRC.

  • High expression of Rock2 is associated with the progression and outcome of CRC.

  • Rock2 promotes the invasion and metastasis of CRC via the β-catenin/TCF4 pathway.

  • Rock2 stabilizes β-catenin by inhibiting its ubiquitination and degradation.

Abstract

Rho-associated coiled-coil-containing protein kinase 2 (Rock2) is an effector for the small GTPase Rho and plays an important role in tumor progression and metastasis. However, the effect of Rock2 in colorectal cancer (CRC) still remains unclear. In this study, we found that Rock2 expression was markedly increased in clinical CRC tissues compared with adjacent non-cancerous tissues. High expression of Rock2 was correlated with tumor metastasis and poor prognosis in CRC. In addition, the knockdown of Rock2 suppressed the invasion and metastasis of CRC cells both in vitro and in vivo. Furthermore, we found that the β-catenin/TCF4 pathway contributed to the effects of Rock2 in CRC cells, and Rock2 stabilized β-catenin by preventing its ubiquitination and degradation. Taken together, this novel pathway for β-catenin control plays a biologically relevant role in CRC metastasis.

Introduction

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death worldwide. Although surgical resection is regarded as the standard curative treatment for CRC, the metastasis and recurrence rates after radical resection of CRC remain high [1]. Thus, it is important to investigate the molecular mechanisms involved in CRC progression and metastasis.

Rho-associated coiled-coil forming protein kinase 2 (Rock2) belongs to a family of serine/threonine kinases that regulate important cellular processes, such as cell morphology, shape, adhesion and migration [2], [3], [4]. Recently, emerging evidence has linked the biological function of Rock2 to tumor metastasis. For example, increased Rock2 expression has been reported in several types of cancers, including hepatocellular carcinoma (HCC), human laryngeal squamous cell carcinoma and lung cancer, and this overexpression is associated with metastasis and shorter survival of cancer patients [5], [6], [7]. However, the expression of Rock2 in CRC and its specific mechanism of action in the process of CRC invasion and metastasis remain unclear.

The protein β-catenin, a key mediator of the β-catenin/TCF4 signaling pathway [8], plays an important role in CRC carcinogenesis and tumor metastasis [9], [10]. It is well known that β-catenin is targeted to ubiquitin-proteasome-mediated degradation [11]. In addition, we previously demonstrated that Rock2 can modify the ubiquitin degradation of MMP2 and CDC25A [12], [13]. Thus, we presumed that Rock2 might also regulate β-catenin by affecting β-catenin ubiquitination degradation.

In this study, we first demonstrated that Rock2 is upregulated in CRC tissues compared with non-tumor tissues and that the overexpression of Rock2 is associated with metastasis and poor survival of CRC patients. In addition, we also provided the first evidence that Rock2 promotes the migration and invasion of CRC cells via the β-catenin/TCF4 pathway. Further investigations indicated that Rock2 can stabilize β-catenin expression by modulating the ubiquitination and degradation of β-catenin.

Section snippets

Samples

Human CRC specimens were collected from 116 patients who underwent CRC resection at the Second Affiliated Hospital of Nanchang University between January 2008 and December 2013. The clinicopathologic characteristics of CRC patients are described in Supplemental Table S1. Informed consent was obtained from each patient, and the study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University.

Cell culture, plasmids and reagents

The human CRC cell lines SW480, SW620, LOVO, DLD-1 and

Rock2 expression is associated with the progression and outcome of CRC

To explore the expression and significance of Rock2 in CRC tissues, we first examined Rock2 expression in 116 CRC tissue samples and the corresponding adjacent tissues by qRT-PCR, immunohistochemistry (IHC) and western blotting. The qRT-PCR experiments revealed that the average fold change of Rock2 mRNA expression in tumor tissues was significantly higher than in paired nontumor tissues (Fig. 1A). The IHC results showed that the Rock2 protein was highly expressed in 62.07% (72/116) of the CRC

Discussion

Rock2 is among the immediate downstream effectors of Rho, which play important roles in many physiological functions, including neurite growth retardation, postnatal development, and smooth muscle contractions [4]. In recent years, studies have shown that Rock2 is highly expressed in a variety of malignant tumors, where it plays an important role in tumor invasion and metastasis. For example, the upregulated expression of Rock2 could promote HCC invasion and metastasis [5]. Additionally, in

Acknowledgments

We thank Elsevier's English Language Editing service for editing the manuscript. This study was supported by grants from the National Natural Science Foundation of China (no. 81360325), the Project of Jiangxi Provincial Department of Science and Technology (nos.20132BDH80031 and 20142BAB215037) and the Project of Jiangxi Provincial Department of Education (no.KJLD12053).

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    Increased ROCK2 expression was also found in gastrointestinal tumors compared to adjacent non-cancer tissues, being not only correlated with tumor grade, infiltration depth, lymph node invasion and Ki-67 index, but also, projected poor prognosis [70,76,77]. Particularly, overexpression of ROCK2 in colon cancer is a critical mediator of invasion through its modulation of matrix metalloproteinase 2 (MMP-2) and -13 (MMP-13) at the sites of invadopodia [78], being directly correlated with tumor metastasis and poor prognosis in this tumor [79]. A more recent study in oral squamous cell carcinomas revealed advanced clinical stage and increased density of cancer-associated fibroblasts in samples with high ROCK2 expression, suggesting that this kinase might be important for tumor progression [80].

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    Finally, expression of the corresponding protein was observed via chemiluminescence. qRT-PCR assays were performed as previously described [24,25]. Primer sequences were as follows: ROCK2 (forward: 5′-GGTATCTGTACATGGTAATGG-3′; reverse: 5′-GGAGTGTATTGCATCCAGAG-3′), PFKFB3 (forward: 5′-GATGCCCTTCAGGAAAGCCT-3′; reverse: 5′-TTGAACACTTTTGTGGGGACGC-3′), and GAPDH (forward: 5′-CCTGCCGGTGACTAACCCTG-3′; reverse: 5′-AGTTAAAAGCAGCCCTGGTG-3′).

  • Rock2 promotes RCC proliferation by decreasing SCARA5 expression through β-catenin/TCF4 signaling

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    Our previous work as well as other studies provided evidence that Rock2 is frequently overexpressed in hepatocellular carcinoma (HCC), colorectal cancer (CRC), melanoma and so on [10–13]. Additionally, Rock2 overexpression is associated with cancer progression and poor prognosis [11,14]. However, the expression of Rock2 in RCC and the specific mechanism of its effect on RCC proliferation remain unclear.

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1

These authors contributed equally to this work.

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