Cancer Letters

Cancer Letters

Volume 358, Issue 1, 1 March 2015, Pages 76-84
Cancer Letters

Original Articles
Semaphorin-3F suppresses the stemness of colorectal cancer cells by inactivating Rac1

https://doi.org/10.1016/j.canlet.2014.12.040Get rights and content

Highlights

  • Low level of SEMA3F in CRC is associated with poor prognosis of patients.

  • SEMA3F inhibits the capability of cell self-renewal and clone formation of cancer cells in vitro and tumorigenesis in vivo.

  • Rac1 activation plays a crucial role in stemness inhibited by SEMA3F.

  • Combination of SEMA3F and GTP-Rac1 is an independent prognosticator of overall survival of patients.

Abstract

Tumor cell stemness has been recognized as a key contributor to tumor initiation, progression and recurrence. Our previous studies have found that semaphorin-3F (SEMA3F), an axon guidance molecule in the development of central nervous system, inhibited the growth and metastasis of colorectal cancer (CRC). However, a possible role for SEMA3F in regulating cancer cell stemness remains unknown. Here, we report a novel mechanism of the acquirement of stemness of CRC cells regulated by SEMA3F. Knockdown of SEMA3F significantly promoted the self-renewal and tumorigenicity of CRC cells, and increased the expression of stemness-associated genes, while overexpressing SEMA3F reduced the stemness of CRC cells. Mechanistically, GTP-Rac1 was involved in SEMA3F mediated regulation of CRC cell stemness by targeting the Wnt/β-catenin pathway. Clinically, GTP-Rac1 expression was inversely correlated with SEMA3F levels in CRC samples and patients with SEMA3Flow/GTP-Rac1high CRC showed poorer prognosis. Our findings demonstrate the ability of SEMA3F to inhibit the stemness of human CRC cells by suppressing Rac1 activation, which suggests a novel therapeutic approach for CRC.

Introduction

Colorectal cancer (CRC) is one of the most common malignancies around the world [1]. Semaphorin-3F (SEMA3F) is a member of axon guidance factors, which plays an important role in the process of neuron axon guidance. Emerging evidence has indicated that some molecules in the family of axon guidance factors, such as Netrin-1 and Slit-2, elicit multiple signaling events in normal stem cells, including mesenchymal stem cell apoptosis and motility [2], [3], hematopoietic stem cell migration [4] and intestinal stem cell proliferation [5]. SEMA3F, characterized as a tumor suppressor, has also been found to be aberrantly expressed and inhibit the progression of many cancers [6], [7]. We have revealed that SEMA3F, which suppresses tumor cell proliferation and migration, is often absent in CRC [8]. However, the underlying mechanisms by which SEMA3F interferes with the progression of CRC remains unknown.

An increasing body of evidence reveals that a small subpopulation of cancer cells is responsible for the initiation and progression of malignancies including CRC [9]. These cells are named as “cancer stem cells (CSCs)” because of their stem-like properties (stemness) including self-renewal, multi-differentiation capability and tumorigenicity potential [10], [11]. Tumorigenesis in immunodeficient mice is regarded as a golden standard for identification of CSCs. So far, some therapeutic strategies against CSCs have shown promising perspective in controlling cancer [12]. However, non-CSCs are most likely to be transformed into CSCs under the pressure of genetic alterations or microenvironmental cues [13], [14], [15]. Therefore, investigation into the regulatory mechanisms governing the stemness of CSCs is crucial for understanding cancer progression and developing new anti-cancer strategies.

We investigated the effect of SEMA3F on the stemness of CRC cells and report the identification of that for key pathways responsible for SEMA3F-mediated CRC stemness. We also determined the expression and the prognostic significance of SEMA3F in CRC specimens.

Section snippets

Tissue specimens and cell culture

Surgical specimens were obtained at operation from 187 patients with colorectal carcinoma (Southwest Hospital, Third Military Medical University) with written consent during the period from 2002 to 2008. The patients received neither chemotherapy nor radiotherapy before surgery. Histological diagnoses were made independently by at least two experienced senior pathologists. All human specimens used in the experiments were approved by Ethics committee of the Third Military Medical University. The

High level of SEMA3F in CRC is associated with longer survival of patients

To investigate the prognostic significance of SEMA3F expression in human CRC, IHC was conducted on tumor sections from 187 patients. As shown in Fig. 1A, SEMA3F expression was observed in the cytoplasm of glandular cells of normal colorectal mucosa as well as in early stage CRC cells. In cancer cells with advanced TNM, the expression of SEMA3F was low or absent (Fig. 1A). The correlation between the level of SEMA3F in tumor and the clinicopathological features of CRC was categorized in Table 1,

Discussion

Emerging evidence indicates that the stemness of cancer cells is responsible for tumor initiation, progression and recurrence [20]. Here, we revealed for the first time a role for SEMA3F in the regulation of stemness of CRC cell. SEMA3F over-expression in CRC reduced LGR5+ CSCs subpopulation and stemness-associated molecules, suggesting that SEMA3F may act as a stemness regulator in CRC. Our study further indicated Rac1 as a critical molecule in the regulation of stemness by SEMA3F. These

Conflict of interest

The authors declare that they have no conflict of interests.

Acknowledgements

This project was supported by grants from the National Basic Research Program of China (973 Program, No. 2010CB529403), the National Natural Science Fund for Youth (No. 81101607 and 81201951). We thank Ms. Qing-hua Ma and Mr. Zeng-tian Sun for their assistance in FACS techniques, and Mrs. Wei Sun and Miss Li-ting Wang (Central Laboratory, Third Military Medical University, Chongqing, China) for their technical assistance in immunofluorescence analysis.

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    These authors contributed equally to the study.

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