Journal of Receptors and Signal Transduction

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/irst20

Inhibition of CDC42 reduces macrophage recruitment and suppresses lung tumorigenesis in vivo Bo Zhang , Jian Zhang , Lilong Xia , Jing Luo , Lei Zhang , Yanhui Xu , Xinhai Zhu & Guoping Chen To cite this article: Bo Zhang , Jian Zhang , Lilong Xia , Jing Luo , Lei Zhang , Yanhui Xu , Xinhai Zhu & Guoping Chen (2020): Inhibition of CDC42 reduces macrophage recruitment and suppresses lung tumorigenesis in vivo, Journal of Receptors and Signal Transduction, DOI: 10.1080/10799893.2020.1828916 To link to this article: https://doi.org/10.1080/10799893.2020.1828916

Published online: 01 Oct 2020.

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JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION https://doi.org/10.1080/10799893.2020.1828916

ORIGINAL ARTICLE

Inhibition of CDC42 reduces macrophage recruitment and suppresses lung tumorigenesis in vivo Bo Zhanga, Jian Zhanga, Lilong Xiab

, Jing Luob, Lei Zhangb, Yanhui Xub, Xinhai Zhub and Guoping Chenb

a

Department of Thoracic Surgery, Taizhou Hospital Affiliated to Wenzhou Medical University, Taizhou, China; bDepartment of Thoracic Surgery, Zhejiang Hospital, Hangzhou, China ABSTRACT

ARTICLE HISTORY

Background: Cell division control (CDC) 42 has been involved in the regulation of diverse cancers. Macrophage recruitment plays an important role in the pathogenesis and development of tumor. However, it remains unclear whether CDC42 contributes to macrophage recruitment and lung tumorigenesis in vivo. Methods: Small interference RNA (siRNA) was used to knock down CDC42 in the Lewis lung carcinoma (LLC)1. The invasion capability of CDC42 knockdown LLC1 cells was evaluated. LLC1 cells with CDC42 targeted small hairpin RNA (shRNA) were inoculated into C57BL/6 mice to establish the tumorbearing animal model Tumor size and metastasis related proteins were measured. In addition, the invasion of macrophages in the tumor site as well as macrophage chemokine were also determined in the model. Results: The capacity of invasion and metastasis of LLC1 cells significantly decreased when CDC42 was knocked down. When inoculated with CDC42 knockdown LLC1 cells in vivo, the tumor size and metastasis related proteins levels both decreased. The invasion capacity of macrophages and the associated macrophage chemokine were also significantly down-regulated. Conclusion: Our data suggest that the inhibition of CDC42 expression in lung cancer cells can significantly prevent the pathogenesis and development of tumor in an allograft tumor model in vivo, which might provide a novel therapeutic target and potential strategy for lung cancer treatment in the future.

Received 28 August 2020 Revised 21 September 2020 Accepted 23 September 2020

Introduction Cell division control (CDC) 42, a member of the Rho family of GTPases, is a protein involved in the regulation of actin cytoskeleton rearrangement and cell cycle [1]. CDC42 was found to be overexpressed in a variety of cancer cells [2], which was associated with the malignant degree of tumor [3] while negatively correlated with the overall survival of patients [4]. Further studies demonstrated that CDC42 could also regulate cell migration [5,6] and proliferation significantly [7,8]. The carcinogenic effect of CDC42 has been extensively investigated in colorectal cancer [3,8]. Although it has been previously reported that CDC42 expressed increased abnormally in non-small cell lung cancer [9], the mechanism of CDC42 regulating the development of lung cancer is still unknown. Macrophage, a component of innate immune system, is one of the most abundant immune cells in the microenvironment of solid tumors [10]. Macrophages are an important part of leukocytes involved in the maintenance of homeostasis, clearance of apoptotic cells, activation of antimicrobial defense against invasive organisms [11], and adaptive T cells [12]. However, macrophages have been found to be recruited to the tumor site during the development and formation of tumor microenvironment [13]. Macrophages at the CONTACT Lilong Xia Zhejiang, China

[email protected]

KEYWORDS

CDC42 knockdown; macrophage invasion; lung tumorigenesis; allograft tumor; Small interference RNA (siRNA)

tumor sites are polarized to a protumoral phenotype, with the features of suppressing T cell responses [14], promoting angiogenesis and stimulating the extravasation of tumor cell [10]. Therefore, the recruitment of macrophages plays an important role in tumorigenesis and metastases. In this study, we verified whether CDC42 contributed to the development of lung cancer in vitro and in vivo. In the LLC1 lung cancer cell line, knockdown of CDC42 significantly inhibited the growth and invasion of LLC1. In addition, we found that CDC42 is essential for the development of lung cancer in vivo. Moreover, the number of recruited macrophages was significant decreased in the tumor site of mice which were inoculated with CDC42 knockdown LLC1. In conclusion, we demonstrated the critical role of CDC42 in the development of lung cancer by recruiting macrophages.

Material and methods LLC1 cell line and transfection LLC1 cells were purchased from ATCC. Cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal

Department of Thoracic Surgery, Zhejiang Hospital, No. 1229 Gudun Road, Xihu District, Hangzhou,

ß 2020 Informa UK Limited, trading as Taylor & Francis Group

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B. ZHANG ET AL.

bovine serum (Sigma, St. Louis, MO) and 1% penicillin and streptomycin at 37  C with 5% CO2 in a humidified incubator. Cells were transfected with duplexes of small interfering RNA (siRNA) or small hairpin RNA (shRNA) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA) for transient and stable knockdown of LLC1 cells, respectively. The duplexes of siRNA and shRNA were purchased from Santa Cruz Biotechnology (Dallas, TX). Briefly, Lipofectamine 3000 and 100 ng of siRNA (or shRNA) were diluted with Opti-MEM, respectively, and then mixed together. The mixed siRNA (or shRNA) and lipofectamine in Opti-MEM were incubated at room temperature for 15 min before addition into the well with confluent cells. The cells were incubated with lipofectamine wrapped siRNA (or shRNA) for 36 h before selection of single cell colony in 96well plates.

Wound healing assay A scratch across the center of the well was made on the LLC1 monolayer cells using 1 ml pipette tip. The detached cells were washed with medium twice gently. Fresh medium was added and cells were cultured for additional 24 h before photos were taken on a microscope to evaluate the wound healing process.

Crystal violet staining Cells subjected to crystal violet staining were washed with cold PBS, and fixed with ice-cold methanol for 10 min. Then cells were stained with 0.5% crystal violet solution in 25% methanol for 10 min. Cells were washed with water several times to remove the dye reagent residue prior to microscopic observation.

RNA extraction, cDNA synthesis and quantitative reverse transcript PCR (qRT-PCR) Total RNA was isolated using Trizol following manufacture’s instruction. 1 mg of total RNA was used to synthesize cDNA using a cDNA synthesis kit (Thermo Fisher Scientific). Specific primers were used in qRT-PCR as in Table 1 [15–17]. The PCR conditions are as follows: 95  C for 5 min, and 40 cycles of 95  C 20 s and 60  C 30 s, followed by 72  C 3 min.

Western blot Total protein was extracted using RIPA buffer and protein concentration was determined using Bradford assay. Equal

amount of protein (20 mg) was loaded into each well for western blot assay. The proteins were run at constant 120 V for around 45 min before transferred onto PVDF membrane. The transfer was done at constant 100 V for 50 min. The PVDF membrane was incubated with primary antibody (1:1000 dilution) overnight at 4  C before washed with PBS þ 0.1% Tween 20. The membrane was then incubated with a secondary antibody (1:5000 dilution) at room temperature for 1 h prior to exposure. The antibodies included: Integrin b-1 (ab30394, Abcam, Cambridge, United Kingdom), MMP2 (4022, Cell Signaling, Danvers,Massachusetts), MMP9 (ab38898, Abcam, Cambridge, United Kingdom), MMP14 (ab53712, Abcam, Cambridge, United Kingdom) and GAPDH (G8795, Millipore-Sigma, St. Louis, Missouri).

Transwell assay Cell migration assays were performed using 24-well transwell chambers (8 mm pore size, BD Biosciences). Approximate 1  105 cells were resuspended in 100 lL serum-free medium and added into the top chambers, which was coated with Matrigel matrix (Corning, Corning, NY). The culture medium collected from siRNA scramble control (siNC) and si-CDC42 LLC1 cells were added into separate bottom chambers. After incubation at 37  C for 24 h, the cells that did not migrate through the pores were removed with a cotton-tipped applicator, and cells on the lower surface of the membrane were stained with crystal violet.

ELISA assay ELISA assays were performed using commercial kits according to the manufacturer’s instruction. CSF-1, MCP-1, CCL5, CXCL1, CXCL2, SDF-1 mouse ELISA kits were purchased from R&D system (Minneapolis, MN).

Flow cytometry Cell suspension from tumor tissue was generated as previously described with little modification [18]. Collagenase I combined with DNase I was used to digest peptide bonds in collagen in the extracelluar structure of tumor tissue. Chopped tumor tissue was incubated with 1 mg/mL of collagenase I and 0.25 mg/mL of DNase I in the LLC1 culture medium at 37  C for 1–2 h with occasional shaking. Cells were then filtered through 10 mm strainer to eliminate debris and obtain single cell suspension. F4/80 antibody conjugated with PE color was purchased from Biolegend (San Diego, CA).

Table 1. List of primer sequences for real time PCR. Name Integrin b-1 MMP2 MMP9 MMP14 GAPDH

Forward AACGAGGTCATGGTTCATGTTG CAGGGAATGAGTACTGGGTCTATT AATCTCTTCTAGAGACTGGGAAGG AG GTACTACCGGTTCAATGAAGAAT AGAGAGAGGCCCTCAGTTGCT

Reverse AACGAGGTCATGGTTCATGTTG ACTCCAGTTAAAGGCAGCATCTAC AGCTGATTGACTAAAGTAGCTGGA GGATACCCTGGCTCTACCTTC TTGTGAGGGAGATGCTCAGTGT

JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION

Immunohistochemistry Tumor tissue was prepared in formalin-fixed and paraffinembedded sections. Deparaffinization, antigen retrieval and blocking incubation were performed prior to F4/80 antibody staining. HRP-conjugated secondary antibody and DAB substrate were used to visualize the primary antibody F4/80 stain.

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transplanted into C57BL/6 mice (shRNA-CDC42) to evaluate the growth of lung tumors. The volume and weight of the tumor in shRNA-CDC42 mice group were significantly decreased than LLC1 cells transplanted group (Sh-Ctrl) (Figure 3(A)). Consistent with in vitro results, metastasisrelated proteins (i.e. Integrin b-1, MMP2, MMP9 and MMP14) at both mRNA (Figure 3(B)) and protein (Figure 3(C)) levels were also found to be significantly reduced in shRNA-CDC42 mice group.

Establishment of allograft tumor model Male pathogen-free C57BL/6 mice were purchased from Shanghai Laboratory Animal Center (Shanghai, China). The mice were maintained in an air-conditioned room with free accessibility to water and chow food. LLC1 control cells and CDC42 knockdown LLC1 cells were both mixed with Matrigel (Corning, Corning. NY) at 1:1 ratio and injected subcutaneously into two groups of C57BL/6 mice (N ¼ 5/group) at a dose of 2  106, respectively. Allograft tumors were evaluated 4–5 weeks after injection. All the experiments were approved by the Ethics Committee of Zhejiang Hospital.

Results Knockdown of CDC42 inhibited LLC1 migration and invasion LLC1 lung cancer cell line was used to investigate the effect of CDC42 on cell migration and invasion. Three duplexes of siRNA targeting CDC42 were used to eliminate the potential off-target effects. The efficiency of CDC42 suppression was demonstrated at RNA level by qRT-PCR (Figure 1(A)). The suppression of CDC42 protein level was measured by western blotting (Figure 1(B)) and immunofluorescence (Figure 1(C)). The results revealed that significant reduction of CDC42 mRNA and protein levels were observed with treatment of duplexes of siRNA. #3 cell colony was used in the following experiments due to its high efficiency of CDC42 suppression. Next, the effect of CDC42 on the migration of LLC1 cells was evaluated in a wound healing experiment. After 24 h of recovery, LLC1 cells with decreased CDC42 expression demonstrated a significantly diminished capability of migration, as shown by larger wound width (Figure 1(D)). A decreased migration and invasion capability of LLC1 cells with decreased CDC42 expression was also observed in the transwell assay, as demonstrated by less cells passing through the chamber membrane (Figure 1(E)). Finally, it was found that both mRNA and protein expression levels of some several important metastasis-related proteins (i.e. Integrin b-1, MMP2, MMP9 and MMP14) decreased (Figure 2(A,B)).

Knockdown of CDC42 inhibited tumor growth and decreased metastasis-related protein levels Since CDC42 played an important role in the migration and invasion of LLC1 cells, we speculate that knockdown of CDC42 can also hinder tumor development in vivo. To verify our speculation, a stable CDC42 knockout LLC1 cell line was

Knockdown of CDC42 inhibited macrophage chemotaxis in vitro It has been recently reported that the recruitment of macrophages plays a key role in cancer progression and metastasis [11]. In this study, we examined macrophage chemotaxis in a transwell assay. Figure 4(A) displayed that the migrated macrophages were significantly reduced in the presence of siCDC42 cell culture medium (Figure 4(A)). In addition, a series of macrophage chemotactic factors were also determined in siNC and si-CDC42 LLC1 cells. Significant reduction in the content of macrophage chemotactic factors was also significantly reduced (p

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