Comparative Proteomics Analysis Identifies Cdc42-Cdc42BPA

Subscriber access provided by READING UNIV

Article

Comparative proteomics analysis identifies Cdc42-Cdc42BPA signaling as prognostic biomarker and therapeutic target for colon cancer invasion Hui-Fang Hu, Wen Wen Xu, Yang Wang, Can-Can Zheng, Wei-Xia Zhang, Bin Li, and Qing-Yu He J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00550 • Publication Date (Web): 26 Oct 2017 Downloaded from http://pubs.acs.org on October 28, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research

Comparative proteomics analysis identifies Cdc42-Cdc42BPA signaling as prognostic biomarker and therapeutic target for colon cancer invasion

Hui-Fang Hu1, Wen Wen Xu2, Yang Wang1, Can-Can Zheng1, Wei-Xia Zhang1, Bin Li1,*, Qing-Yu He1,* 1

Key Laboratory of Functional Protein Research of Guangdong Higher Education

Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China. 2Institute of Biomedicine, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou 510632, China.

Correspondence to: Prof. Qing-Yu He, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China; Phone/Fax: 86-20-85227039; Email: [email protected] Dr. Bin Li, Institute of Life and Health Engineering, College of Life Science and Technology,

Jinan

University,

Guangzhou

510632,

China;

Phone/Fax:

86-20-85224372; Email: [email protected]

Keywords: SILAC proteomics，Cdc42, Cdc42BPA, colon cancer invasion, prognostic 1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

biomarker, targeted therapy

Conflict of interest: The authors declare no conflict of interest.

2


Page 2 of 38

Page 3 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Abstract Background Metastasis is one of the major causes of treatment failure in the patients with colon cancer. The aim of our study is to find key proteins and pathways that drive invasion and metastasis in colon cancer. Methods Eight rounds of selection of cancer cells invading through matrigel-coated chamber was performed to obtain highly invasive colon cancer sublines HCT116-I8 and RKO-I8. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) technology was used to identify the differently expressed proteins and the proteomics data

was

analyzed

by

ingenuity

pathway

analysis

(IPA).

PAK1-PBD

immunoprecipitation combined with Western blot were carried out to determine Cdc42 activity, and qRT-PCR and Western blot were used to determine gene expression. The functional role of Cdc42BPA and Cdc42 pathway in colon cancer invasion was studied by loss-of-function experiments including pharmacological blockade, siRNA knockdown, chamber invasion, and WST-1 assays. Human colon cancer tissue microarray was analyzed by immunohistochemistry for overexpression of Cdc42BPA and its correlation with clinicopathological parameters and patient survival outcomes. Results HCT116-I8 and RKO-I8 cells showed significantly stronger invasive potential as well as decreased E-cadherin and increased vimentin expressions compared with parental 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

cells. The differently expressed proteins in I8 cells compared with parental cells were identified. Bioinformatics analysis of proteomics data suggested that Cdc42BPA protein and Cdc42 signaling pathway are important for colon cancer invasion, which was confirmed by experimental data showing upregulation of Cdc42BPA and higher expression of active GTP-bound form of Cdc42 in HCT116-I8 and RKO-I8 cells. Functionally, pharmacological and genetic blockade of Cdc42BPA and Cdc42 signaling markedly suppressed colon cancer cell invasion and reversed epithelial mesenchymal transition (EMT) process. Furthermore, compared with adjacent normal tissues, Cdc42BPA expression was significantly higher in colon cancer tissues, and further upregulated in metastatic tumors in lymph nodes. More importantly, Cdc42BPA expression was correlated with metastasis and poor survival of the patients with colon cancer. Conclusions This study provides the first evidence that Cdc42BPA and Cdc42 signaling are important for colon cancer invasion, Cdc42BPA has potential implications for colon cancer prognosis and treatment.

4


Page 4 of 38

Page 5 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Background Colon cancer is the third most common cancer worldwide and leads to about 700,000 deaths every year 1. About 60% of patients have metastasis at initial diagnosis, which is one of the primary factors account for the poor prognosis of this lethal disease. Cancer metastasis is a multi-stage, complex and continuous process, which involves several important events and are contingent on the cancer cells acquiring increased cell motility, invasiveness, and the epithelial-stromal transition (EMT) 2. Identification of critical regulators and signaling pathways involved in invasion and metastasis will improve our understanding on tumor metastasis and facilitate the development of new treatment strategies. Although functions of some genes have been reported in colon cancer progression, there is as yet little information on the systematic identification of important regulators in colon cancer invasion and metastasis. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) technology is a powerful proteomics strategy to identify and quantify the differently expressed proteins among different samples

3, 4

.

In this study, we established highly invasive colon cancer sublines by serial selection of cancer cells invading through matrigel-coated invasion chamber, and performed SILAC proteomics to identify the important proteins that drive colon cancer invasion and metastasis. Among the upregulated proteins identified by SILAC proteomics in the I8 cells, Cdc42 binding protein kinase alpha (Cdc42BPA/MRCKα), a member of the serine/threonine protein kinase family, drew our strong interest. Previous studies 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 38

suggested that Cdc42BPA may mediates the Cdc42-induced peripheral actin formation to promote cytoskeletal reorganization 5. However, the biological function and clinical significance of Cdc42BPA in colon cancer are unclear. Loss-of-function experiment and invasion assay were performed in this study to examine the role of Cdc42BPA in regulation of colon cancer invasion. In addition, the Cdc42BPA expression in a human colon cancer tissue microarray was analyzed by immunohistochemistry to evaluate the potential of Cdc42BPA as diagnostic or prognostic biomarker in colon cancer. Previous studies reported that Cdc42BPA may function as a downstream effector of Cdc42 signaling pathway

6, 7

, which is activated in many cancers

8-11

. The Cdc42

downstream effectors Wiskott-Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous (WAVE) proteins can cause Arp2/3 complex-dependent actin polymerization, which plays an important role in regulation of cytoskeleton, cell movement and cancer metastasis 12. Blockade of Cdc42 signaling may be a potential therapeutic strategy in cancer treatment. However, the functional role of Cdc42 signaling in colon cancer invasion remains to be elucidated. In this study, ingenuity pathway analysis (IPA) comparison of the protein profiles of the highly invasive cells and parental cells suggested that the small GTPases Cdc42 signaling pathway has a crucial role in colon cancer invasion and metastasis. In addition, the effects of Cdc42 inhibitors on repressing the invasive potential of colon cancer cells have not been reported and warrant further investigation. ML141, an effective and selective inhibitor of Cdc42 13-15, was used here to test the effects of Cdc42 blockade on invasion ability 6


Page 7 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and expression levels of EMT markers in colon cancer cells.

Methods Cell culture and drugs Human colon cancer cell lines HCT116 and RKO (ATCC, Rockville, MD, USA) were maintained in RPMI 1640 and DMEM (Thermo Fisher Scientific, San Jose, CA, USA), respectively, supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) at 37°C in 5% CO2. ML141 was bought from Selleck Chemicals (Huston, TX, USA) and dissolved in dimethyl sulfoxide (DMSO).

Establishment of highly invasive colon cancer sublines The method was performed as described previously16. Colon cancer cell lines HCT116 and RKO (3 x 105 cells) were seeded into the upper compartment of 8 µm pore size invasion chamber coated with matrigel (BD Biosciences, Bedford, MA, USA) and incubated for 48 h. The cells that invaded to the bottom of the chamber were digested with trypsin. The cells were cultured until the cells were adequate for next round of invasion selection, and then the cells were applied into a new invasion chamber. The invasion selection was repeated eight rounds to finally get the highly invasive cells HCT116-I8 and RKO-I8.

SILAC labeling, protein digestion and mass spectrometry (MS) analyses 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 38

HCT116-I8 and HCT116 cells were cultured for at least seven cell doublings in SILAC RPMI 1640 media (Thermo Fisher Scientific), supplemented with 10% dialytic fetal bovine serum (Thermo Fisher Scientific) and either ‘‘light media” (Arg0, Lys0) or ‘‘heavy media” (Arg10, Lys8; Cambridge Isotope Laboratories, Andover, MA, USA), respectively. The lysates of ‘‘heavy”-labeled HCT116-I8 and ‘‘light”-labeled HCT116 cells were mixed with equal amounts and MS analysis were performed as described previously

17

. Protein digestion

18

. The peptide mixtures

were analyzed in the LTQ-Orbitrap mass spectrometer (LTQ MS; Thermo Electron) according to the manufacturer’s instructions. MS parameters: capillary temperature, 120°C; spray voltage, 2.30 kV; scan range, 350–1500 m/z; resolution, 35 K fwhm (full width at half-maximum) at 400 m/z; MS/MS CID-scans, 10 most intense precursor ions; dynamic exclusion, applied; repeat, 1; and duration, 90 s. Finally, proteins identification and quantitation were performed by MaxQuant software (v. 1.5.2.8) against the Uniprot-Swiss Human database (2017_08 Release, 20237 entries). Protein and peptide FDRs were set to 1%, and the normalized ratio of ‘‘heavy” versus ‘‘light” was calculated by MaxQuant. Other parameters used were: (a) variable modifications, methionine oxidation, protein N-acetylation, Gln→pyro-Glu; (b) fixed modifications, cysteine carbamidomethylation; (c) database: target-decoy human MaxQuant; (d) heavy labels: R10K8; (e) MS/MS tolerance: FTMS-20 ppm, ITMS-0.5 Da; (f) minimum peptide length, 7; (g) maximum missed cleavages, 2; (h) maximum of labeled amino acids, 3. All MS data are available in ProteomeXchange consortium (identifier:

PXD007931).

Username: [email protected]. 8


Password:

Page 9 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


gmwt6TEy.

Bioinformatics pathway analysis Proteins upregulated or downregulated (fold change ≥ 1.5 or ≤ 0.67，P < 0.01) in three biological replicates were defined as differently expressed proteins. As described previously with minor modifications19,

20

, differently expressed proteins were

analyzed by Ingenuity Pathway Analysis (IPA) program.

siRNA and transfection The siRNA against Cdc42BPA (si-Cdc42BPA) was purchased from TranSheep Bio (Shanghai, China) and the target sequences were 5’-GAGTGAACTTGATAAGCTT-3’ for si-Cdc42BPA#1 and 5’-GGGCCGAAGATCTAGACAA-3’ for si-Cdc42BPA#2. For transfection, Lipofectamine 3000 reagent (Thermo Fisher Scientific) was used according to the manufacturer’s recommendations 21.

qRT-PCR The qRT-PCR was performed as described previously

22, 23

. In brief, total RNA was

extracted with Trizol reagent according to the manufacturer's instructions (Thermo Fisher Scientific). Reverse transcription was conducted using PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). The mRNA expression levels of Cdc42BPA and of GAPDH as internal control were detected by quantitative real-time PCR using SYBR® Premix Ex Taq™ II (Takara) on a Bio-Rad Mini Option real-time 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

PCR

system

(Bio-rad,

Hercules,

CA,

USA).

The

Page 10 of 38

primers

used

were:

5'-GTAATGTAAAGGGTGGAAAGGC-3' (forward) and 5'-GATGGAAGGGATCAGAGGTG-3' (reverse) for Cdc42BPA; 5'-GAAGGTGAAGGTCGGAGTC-3' (forward) and 5'-AAGATGGTGATGGGATTTC-3' (reverse) for GAPDH.

Western blot Details on preparation of cell lysates and Western blot were described previously 24. Cells were lysed in lysis buffer (Cell Signaling Technology, Beverly, MA, USA) according to the manufacturer’s instructions, and the protein concentration was determined with a BCA kit (Thermo Fisher Scientific). The proteins samples were loaded into SDS-polyacrylamide gel for electrophoresis and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% milk for 1 h, the membranes were probed with diluted primary antibody for 1-2 h at room temperature, then washed with Tris-Buffered Saline Tween-20 (TBST) and incubated with corresponding secondary antibody for 1 h at room temperature. The reaction was visualized using ECL (Bio-Rad) and detected by exposure to autoradiographic film. The primary antibodies used included Cdc42BPA (Abcam, Cambridge, UK), E-cadherin (BD Biosciences), vimentin (Santa Cruz Biotechnology, Santa Cruz, CA, USA, and actin (Proteintech, Chicago, IL, USA).

Boyden chamber invasion assay Cell invasion assay was performed with the use of 8 µm pore size invasion chamber 10


Page 11 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


coated with matrigel (BD Biosciences) 25. The cells suspended in serum-free medium were seeded to the upper chamber, and the lower compartment was filled with complete medium. After 24 h, the invaded cells adhering to the bottom surface of the chamber membrane were fixed with methanol and then stained with crystal violet (0.2%). Images of three different fields were captured from each membrane, and the number of invaded cells was counted.

WST-1 cell viability assay Cell viability was measured using a WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime Biotechnology, Shanghai, China) 21. The cells were seeded into 96-well plates, and WST-1 was added at the end of experiment. The cells were incubated for 2 h at 37ºC, and absorbance at 450 nm was measured on an automated microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA).

Cdc42 activity assay Details on sample preparation were described previously 26. Cdc42 Activation Assay Kit (Cell Biolabs, San Diego, CA, USA) was used to determine Cdc42 activity according to the manufacturer’s instructions. In brief, PAK1 PBD Agarose beads were used to selectively isolate and pull-down the active form of Cdc42 from cell lysates, and the precipitated GTP-Cdc42 was detected by Western blot analysis using Cdc42 specific monoclonal antibody.

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Tissue microarray and immunohistochemistry A human colon cancer tissue microarray (TMA) containing 100 cases of colon cancer and 80 cases of corresponding adjacent normal tissue (Shanghai Outdo Biotech, Shanghai, China) was used to determine the correlation of Cdc42BPA expression with clinicopathological parameters. Another TMA containing 33 pairs of primary colon cancer and matched lymph nodes with metastatic colon cancer (Biomax, Rockvile, MD) was included to compare Cdc42BPA expression in primary tumors with that of metastasized tumors of colon cancer. Immunohistochemistry was performed as described previously

16, 27

. In brief, paraffin-embedded TMAs were deparaffinized in

xylene, rehydrated in a graded series of ethanol solutions, and subjected to antigen retrieval in citrate buffer. After blocking with normal serum, the slides were incubated overnight with Cdc42BPA antibody, washed with phosphate buffered saline, and then incubated with corresponding biotinylated secondary antibody. After incubation with peroxidase-conjugated avidin-biotin complex, immunostaining was visualized using 3, 3’-diaminobenzidine (Dako, Mississauga, ON, Canada) as chromogen, and then the slides were counterstained with hematoxylin. A scale of 1 to 4, representing negative (1), weak (2), moderate (3) and strong (4) staining, respectively, was used to grade the intensity of staining. The stained sections were reviewed independently by two observers. Specimens assigned scores of 1 to 2 were considered low expression, whereas those with scores 3 to 4 were regarded as having high expression.

Statistical analysis 12


Page 12 of 38

Page 13 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


All assays were performed in triplicate on three independent experiments. The data were expressed as the mean ± SD and analyzed by t-test using GraphPad Prism software (version 5.00, San Diego, CA, USA). The correlation between Cdc42BPA expression and clinicopathological parameters was assessed using Fisher exact test. The association between Cdc42BPA expression and patient survival was plotted using the Kaplan-Meier method, and statistical difference was compared using the log-rank test. P < 0.05 was considered statistically significant.

Results HCT116-I8 and RKO-I8 sublines were highly invasive and showed increased EMT Through eight rounds of selection of the HCT116 and RKO cells invading through invasion chamber (Figure 1A), we obtained highly invasive colon cancer sublines designated HCT116-I8 and RKO-I8 (Figure 1B). The expression levels of EMT markers were analyzed and the I8 cells showed decreased E-cadherin and increased vimentin expression compared with their respective parental colon cancer cell lines (Figure 1C), although there is no difference in morphology (Figure 1D). The comparable proliferation rates of I8 cells and parental cells indicated that higher invasive potential of I8 cells was not due to increase in proliferation (Figure 1E).

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

SILAC proteomics suggests the upregulation of Cdc42BPA in highly invasive colon cancer cells The cell lysates of “heavy” labeled HCT116-I8 and “light” labeled HCT116 were mixed for mass spectrometry (MS) analysis. Schematic diagram of SILAC proteomics was shown (Figure 2A). With stringent criteria, a venn diagram showed that 3161 proteins with quantitative information were identified in the three replicated experiments (Figure 2B) (Supplementary Table S1 and S2). For each experiment, the identified proteins were assembled with a minimum of two peptides with a false discovery rate (FDR) of 1% (minimal peptide length: seven amino acids). The ratio of heavy/light was automatically calculated and normalized by MaxQuant program. A total of 128 proteins were identified to be upregulated or downregulated (fold change ≥ 1.5 or ≤ 0.67, P < 0.01) in HCT116-I8 cells (Supplementary Table S3). For example, FHL2, which has been reported to promote invasive potential and EMT in colon cancer28, was expressed at a level 3.8 fold higher in HCT116-I8 cells. NDRG1, a known suppressor of colon cancer invasion and metastasis 29, was found to be 2.61 fold lower in the I8 cells (Supplementary Table S3). These data suggested that the highly invasive sublines could be used as a cell model for identifying novel regulators of cancer invasion and metastasis. In addition, ID Associated Network Function analysis and Molecular and Cellular Function analysis showed that the dysregulated proteins in I8 cells were mainly related to cellular movement and could provide important clues for mechanistic study of cancer cell invasion (Figure 2C and D), which proved that our mass spectrometry data was reliable. Among the upregulated 14


Page 14 of 38

Page 15 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


proteins in HCT116-I8 cells, Cdc42BPA (fold change = 2.1, P < 0.01) drew out strong interest, because its function and clinical significance in colon cancer remains unknown.

Cdc42BPA promotes colon cancer cell invasion We aim to explore whether Cdc42BPA is responsible for the high invasiveness of I8 cells. Firstly, qRT-PCR and Western blot were performed to confirm the proteomics data, and the results showed that Cdc42BPA was upregulated at mRNA and protein levels in HCT116-I8 and RKO-I8 cells (Figure 3A and B). Secondly, we carried out loss-of-function experiment with different siRNAs against Cdc42BPA. The HCT116-I8 and RKO-I8 cells transfected with different concentrations of siRNAs were compared for their invasion ability. The results showed that knockdown of CDC42BPA significantly reduced invasive potential of I8 cells in a dose-dependent manner (Figure 3C and D). Moreover, the increased E-cadherin expression and decreased vimentin expression in the I8 cells with Cdc42BPA knockdown indicated the crucial role of Cdc42BPA in EMT and invasion (Figure 3E). We also found that knockdown of Cdc42BPA did not exert any effect on proliferation rates of HCT116-I8 and RKO-I8 cells, eliminating the possibility that the weaker invasion ability of Cdc42BPA-knockdown I8 cells was due to decreased proliferation (Figure 3F). Collectively, these data demonstrated that Cdc42BPA may be a positive regulator of cancer cell invasion and could be a therapeutic target for cancer treatment.

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Dysregulation of Cdc42BPA expression is correlated with metastasis and poor survival in colon cancer To study the clinical significance of Cdc42BPA in human cancer, we determined Cdc42BPA expression level by immunohistochemistry using a tissue microarray containing 100 cases of primary colon cancer tissues and 80 cases of adjacent normal tissues. As shown in Figure 4A-C, the majority of the tumor cases had stronger Cdc42BPA cytoplasmic staining than corresponding normal tissues. Notably, 49.5% (49/99) of colon cancer tissues, but only 22.5% (18/80) of normal tissues, showed moderate or strong Cdc42BPA expression. In contrast, only 50.5% (50/99) of the tumor tissues had absent or weak Cdc42BPA expression versus 77.5% (62/80) in normal tissues. We found that high Cdc42BPA expression was significantly associated with lymph node metastasis (P = 0.002) and distant metastasis (P = 0.026) (Table 1). More importantly, Kaplan-Meier survival analysis indicated that patients with high tumor Cdc42BPA expression had markedly shorter survival (median survival = 15.0 months) than the patients with low tumor Cdc42BPA expression (median survival = 25.5 months). Log-rank analysis showed that high Cdc42BPA expression was significantly correlated with shorter survival (log-rank = 6.447, P = 0.011; Figure 4D). Furthermore, Cdc42BPA expression was examined in a tissue microarray containing 33 pairs of primary colon cancer tissues and the matched metastatic tumor tissues in lymph nodes. The data showed that majority of the metastatic tumors had higher Cdc42BPA expression than the corresponding primary tumors (Figure 4E and F), corroborating our findings above that high Cdc42BPA 16


Page 16 of 38

Page 17 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


expression is correlated with metastasis and poor prognosis. Taken together, our results strongly supported the rationale of Cdc42BPA as a biomarker for colon cancer diagnosis and prognosis.

Inhibition of Cdc42 signaling pathway suppresses colon cancer cell invasion Since Cdc42BPA is a downstream regulator of Cdc42 signaling and IPA analysis of the proteins significantly changed in I8 cells showed that Cdc42 signaling pathway were significantly associated with colon cancer cell invasion (Supplementary Table S4), we next investigated the significance of Cdc42 signaling pathway in the invasive potential of I8 cells. As shown in Figure 5A, Cdc42 activity was determined and significantly higher expression of Cdc42-GTP, the active form of Cdc42, was detected in the immunoprecipitates of PAK1 PBD agarose beads in HCT116-I8 and RKO-I8 cells compared with the parental cells, indicating that highly invasive colon cancer cells have higher Cdc42 activity. To examine the functional role of Cdc42 signaling pathway in colon cancer cell invasion, HCT116-I8 and RKO-I8 cells were treated with different concentrations of ML141, a specific Cdc42 pathway inhibitor, and cancer cell invasion was compared. We found that ML141 treatment markedly repressed invasive potential of I8 cells in a dose-dependent manner (Figure 5B and C). The comparable proliferation rates of the I8 cells treated with ML141 or vehicle control indicated that the inhibitory effect of ML141 on invasive potential of I8 cells was not due to any effect on proliferation (Figure 5D). In addition, the increased E-cadherin expression and decreased vimentin expression in the ML141-treated cells 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

suggested that blockade of Cdc42 signaling pathway suppressed colon cancer cell invasion through reversal of EMT (Figure 5E).

Discussion Cancer metastases, compared with the primary cancer, account for as high as 90% death of cancer patients

30, 31

. EMT, the biological process by which polarized

epithelial cells lose their adhesion property and obtain mesenchymal cell phenotypes, is closely related to tumorigenesis and metastasis

32-35

. EMT and invasion, as the

initial steps, are important for tumor metastasis, but the mechanism involved is unclear. The highly invasive colon cancer sublines we established in this study may be not only an ideal cell model to identify the functional proteins and signaling pathways responsible for cancer invasion, but also be valuable tool for evaluating novel therapeutic agents which can block tumor metastasis. With the rapid development of protein profiling technologies and improvement of bioinformatics analytical strategies, comparative quantitative proteomics provides a high-throughput platform for investigation of the mechanisms involved in colon cancer invasion and metastasis, which could benefit the development of useful diagnostic and prognostic biomarkers as well as potential therapeutic targets for cancer treatment. SILAC, a relatively mature quantitative proteomics strategy based on mass spectrometry, has been widely used in identification of cancer biomarkers 3, 4, 36

and mechanistic study of anticancer drug 21, 22. In this study, SILAC combined with 18


Page 18 of 38

Page 19 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mass spectrometry were used to search for the differently expressed proteins in HCT116-I8 cells, and the potential pathways involved were predicted by bioinformatics according to the proteomics data. The rationale of use of SILAC proteomics in identification of genetic alterations in highly invasive cancer sublines was confirmed by the enrichment of cellular movement-associated network in the analyzed data (Figure 2). Therefore, our proteomics data is a valuable database for the investigation on the molecular mechanisms of cancer invasion and metastasis. Cdc42BPA can regulate actin cytoskeletal reorganization via phosphorylation of PPP1R12C and MYL9/MLC2 and interacts with adaptor protein LRAP35a to form a tripartite complex with a myosin II-like myosin heavy chain MYO18A

37, 38

. ROCK

and MRCK (Cdc42BPA and MRCKβ) are known to cooperatively regulate the processes required for cancer cell invasion including the formation of stress fibers, cytoskeleton remodeling and establishment front–rear polarity by transducing extracellular stimuli through phosphorylation of multiple intracellular targets

39-41

.

However, the function of MRCK in cancer was rarely reported compared than ROCK, and in particular, the clinical significance and biological function of Cdc42BPA in colon cancer are unknown. Our results showed that Cdc42BPA overexpression in colon cancer is significantly associated with metastasis and poor survival (Figure 4). In addition, expression level of Cdc42BPA was found to be further higher in metastatic colon cancer tissues in lymph nodes compared with the primary tumors. To our knowledge, this study provide the first evidence indicating that Cdc42BPA is a potential biomarker for colon cancer diagnosis and prognosis, and may be a potential 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

therapeutic

target

in

the

treatment

Page 20 of 38

of

human

cancer.

A recent screening of small molecule chemical library resulted in the identification of specific MRCK inhibitors, further contributed to the future implication of Cdc42BPA blockade in cancer therapy 6. Rho family of GTPases, including Cdc42, is involved in actin and microtubule dynamics, which controls cellular processes such as cell migration 42. It is known that Cdc42 cycles between an active-GTP bound state and an inactive GDP-bound state and regulates the polymerization of the actin cytoskeleton

43-45

. Our study highlights

the important role of Cdc42 pathway in colon cancer invasion. IPA analysis of proteomics data suggested dysregulation of Cdc42 signaling in the highly invasive colon cancer cells (Supplementary Table S4), which was supported by our experimental data showing that Cdc42 activity was significantly higher in the HCT116-I8 and RKO-I8 cells. Cdc42 is frequently overexpressed in human cancer and plays an important role in cell movement and apoptosis

46, 47

. It has reported that

Cdc42 signaling promoted cancer invasion and was closely related to cancer metastasis

10, 11, 48, 49

. More importantly, we found that pharmacologic blockade of

Cdc42 pathway with ML141 significantly suppressed colon cancer invasion, accompanied with increased E-cadherin and decreased vimentin expression. There is increasing recognition that conventional treatment alone is insufficient in cancer therapy, especially for the cancer patients with metastasis, and there is urgent need to develop new therapeutic agents. Our findings here may provide potential therapeutic strategy for treatment of cancer metastasis. 20


Page 21 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Conclusions In summary, this study supports the importance of Cdc42-Cdc42BPA signaling in cancer invasion, and supports its potential application as therapeutic targets in colon cancer. Overexpression of Cdc42BPA and its correlation with patient outcome suggests its potential as diagnostic and prognostic biomarker in colon cancer.

Acknowledgement This work was supported by the National Key Research and Development Program of China (No. 2017YFA0505100), the National Natural Science Foundation of China (31570828,

81672953),

Guangdong

Natural

Science

Research

Grant

(2016A030313838), and the Fundamental Research Funds for the Central Universities (21617434). We thank Mr. Xing-Feng Yin and Mr. Zheng-Hua Sun for their technical assistance on mass spectrometry.

References 1.

Fotiadis, C. I.; Stoidis, C. N.; Spyropoulos, B. G.; Zografos, E. D., Role of

probiotics, prebiotics and synbiotics in chemoprevention for colorectal cancer. World journal of gastroenterology 2008, 14, (42), 6453-7. 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.

Valastyan, S.; Weinberg, R. A., Tumor metastasis: molecular insights and

evolving paradigms. Cell 2011, 147, (2), 275-92. 3.

Yan, G. R.; Tan, Z.; Wang, Y.; Xu, M. L.; Yu, G.; Li, Y.; He, Q. Y., Quantitative

proteomics characterization on the antitumor effects of isodeoxyelephantopin against nasopharyngeal carcinoma. Proteomics 2013, 13, (21), 3222-32. 4.

Yan, G. R.; Yin, X. F.; Xiao, C. L.; Tan, Z. L.; Xu, S. H.; He, Q. Y., Identification

of novel signaling components in genistein-regulated signaling pathways by quantitative phosphoproteomics. Journal of proteomics 2011, 75, (2), 695-707. 5.

Wilkinson, S.; Paterson, H. F.; Marshall, C. J., Cdc42-MRCK and Rho-ROCK

signalling cooperate in myosin phosphorylation and cell invasion. Nature cell biology 2005, 7, (3), 255-61. 6. Unbekandt, M.; Croft, D. R.; Crighton, D.; Mezna, M.; McArthur, D.; McConnell, P.; Schuttelkopf, A. W.; Belshaw, S.; Pannifer, A.; Sime, M.; Bower, J.; Drysdale, M.; Olson, M. F., A novel small-molecule MRCK inhibitor blocks cancer cell invasion. Cell communication and signaling : CCS 2014, 12, 54. 7.

Unbekandt, M.; Olson, M. F., The actin-myosin regulatory MRCK kinases:

regulation, biological functions and associations with human cancer. Journal of molecular medicine (Berlin, Germany) 2014, 92, (3), 217-25. 8.

Chang, J. S.; Su, C. Y.; Yu, W. H.; Lee, W. J.; Liu, Y. P.; Lai, T. C.; Jan, Y. H.;

Yang, Y. F.; Shen, C. N.; Shew, J. Y.; Lu, J.; Yang, C. J.; Huang, M. S.; Lu, P. J.; Lin, Y. F.; Kuo, M. L.; Hua, K. T.; Hsiao, M., GIT1 promotes lung cancer cell metastasis through modulating Rac1/Cdc42 activity and is associated with poor prognosis. 22


Page 22 of 38

Page 23 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Oncotarget 2015, 6, (34), 36278-91. 9.

Chrysanthou, E.; Gorringe, K. L.; Joseph, C.; Craze, M.; Nolan, C. C.;

Diez-Rodriguez, M.; Green, A. R.; Rakha, E. A.; Ellis, I. O.; Mukherjee, A., Phenotypic characterisation of breast cancer: the role of CDC42. Breast cancer research and treatment 2017, 164, (2), 317-325. 10. Stengel, K.; Zheng, Y., Cdc42 in oncogenic transformation, invasion, and tumorigenesis. Cellular signalling 2011, 23, (9), 1415-23. 11. Aguilar, B. J.; Zhou, H.; Lu, Q., Cdc42 Signaling Pathway Inhibition as a Therapeutic Target in Ras-Related Cancers. Current medicinal chemistry 2017. doi: 10.2174/0929867324666170602082956. [Epub ahead of print] 12. Zhang, J.; Ma, R.; Li, L.; Wang, L.; Hou, X.; Han, L.; Ge, J.; Li, M.; Wang, Q., Intersectin 2 controls actin cap formation and meiotic division in mouse oocytes through the Cdc42 pathway. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2017, 31, (10), 4277-4285. 13. Surviladze, Z.; Waller, A.; Strouse, J. J.; Bologa, C.; Ursu, O.; Salas, V.; Parkinson, J. F.; Phillips, G. K.; Romero, E.; Wandinger-Ness, A.; Sklar, L. A.; Schroeder, C.; Simpson, D.; Noth, J.; Wang, J.; Golden, J.; Aube, J., A Potent and Selective Inhibitor of Cdc42 GTPase. In Probe Reports from the NIH Molecular Libraries Program, National Center for Biotechnology Information (US): Bethesda (MD), 2010. 2010-. 2010 Feb 27 [updated 2010 Dec 16]. 14. Chen, H. Y.; Yang, Y. M.; Stevens, B. M.; Noble, M., Inhibition of 23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

redox/Fyn/c-Cbl pathway function by Cdc42 controls tumour initiation capacity and tamoxifen sensitivity in basal-like breast cancer cells. EMBO molecular medicine 2013, 5, (5), 723-36. 15. Kumar, A.; Al-Sammarraie, N.; DiPette, D. J.; Singh, U. S., Metformin impairs Rho GTPase signaling to induce apoptosis in neuroblastoma cells and inhibits growth of tumors in the xenograft mouse model of neuroblastoma. Oncotarget 2014, 5, (22), 11709-22. 16. Li, B.; Xu, W. W.; Lam, A. K. Y.; Wang, Y.; Hu, H. F.; Guan, X. Y.; Qin, Y. R.; Saremi, N.; Tsao, S. W.; He, Q. Y.; Cheung, A. L. M., Significance of PI3K/AKT signaling pathway in metastasis of esophageal squamous cell carcinoma and its potential as a target for anti-metastasis therapy. Oncotarget 2017, 8, (24), 38755-38766. 17. Wisniewski, J. R.; Zougman, A.; Mann, M., Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. Journal of proteome research 2009, 8, (12), 5674-8. 18. Wang, Y.; Huang, Z. H.; Li, Y. J.; He, G. W.; Yu, R. Y.; Yang, J.; Liu, W. T.; Li, B.; He, Q. Y., Dynamic quantitative proteomics characterization of TNF-alpha-induced necroptosis. Apoptosis : an international journal on programmed cell death 2016, 21, (12), 1438-1446. 19. Li, L. P.; Lu, C. H.; Chen, Z. P.; Ge, F.; Wang, T.; Wang, W.; Xiao, C. L.; Yin, X. F.; Liu, L.; He, J. X.; He, Q. Y., Subcellular proteomics revealed the epithelial-mesenchymal transition phenotype in lung cancer. Proteomics 2011, 11, (3), 24


Page 24 of 38

Page 25 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


429-39. 20. Wang, T.; Cui, Y.; Jin, J.; Guo, J.; Wang, G.; Yin, X.; He, Q. Y.; Zhang, G., Translating mRNAs strongly correlate to proteins in a multivariate manner and their translation ratios are phenotype specific. Nucleic acids research 2013, 41, (9), 4743-54. 21. Wang, Y.; Yu, R. Y.; Zhang, J.; Zhang, W. X.; Huang, Z. H.; Hu, H. F.; Li, Y. L.; Li, B.; He, Q. Y., Inhibition of Nrf2 enhances the anticancer effect of 6-O-angeloylenolin in lung adenocarcinoma. Biochemical pharmacology 2017, 129, 43-53. 22. Wang, Y.; Zhang, J.; Huang, Z. H.; Huang, X. H.; Zheng, W. B.; Yin, X. F.; Li, Y. L.; Li, B.; He, Q. Y., Isodeoxyelephantopin induces protective autophagy in lung cancer cells via Nrf2-p62-keap1 feedback loop. Cell death & disease 2017, 8, (6), e2876. 23. Xu, W. W.; Li, B.; Guan, X. Y.; Chung, S. K.; Wang, Y.; Yip, Y. L.; Law, S. Y.; Chan, K. T.; Lee, N. P.; Chan, K. W.; Xu, L. Y.; Li, E. M.; Tsao, S. W.; He, Q. Y., Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression. 2017, 8, 14399. 24. Li, B.; Xu, W. W.; Guan, X. Y.; Qin, Y. R.; Law, S.; Lee, N. P.; Chan, K. T.; Tam, P. Y.; Li, Y. Y.; Chan, K. W.; Yuen, H. F.; Tsao, S. W.; He, Q. Y.; Cheung, A. L., Competitive Binding Between Id1 and E2F1 to Cdc20 Regulates E2F1 Degradation and

Thymidylate

Synthase

Expression

to

Promote

Esophageal

Cancer

Chemoresistance. Clinical cancer research : an official journal of the American 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Association for Cancer Research 2016, 22, (5), 1243-55. 25. Li, B.; Xu, W. W.; Han, L.; Chan, K. T.; Tsao, S. W.; Lee, N. P. Y.; Law, S.; Xu, L. Y.; Li, E. M.; Chan, K. W.; Qin, Y. R.; Guan, X. Y.; He, Q. Y.; Cheung, A. L. M., MicroRNA-377 suppresses initiation and progression of esophageal cancer by inhibiting CD133 and VEGF. Oncogene 2017, 36, (28), 3986-4000. 26. Yiin, J. J.; Hu, B.; Jarzynka, M. J.; Feng, H.; Liu, K. W.; Wu, J. Y.; Ma, H. I.; Cheng, S. Y., Slit2 inhibits glioma cell invasion in the brain by suppression of Cdc42 activity. Neuro-Oncology 2009, 11, (6), 779-89. 27. Xu, W. W.; Li, B.; Lam, A. K.; Tsao, S. W.; Law, S. Y.; Chan, K. W.; Yuan, Q. J.; Cheung, A. L., Targeting VEGFR1- and VEGFR2-expressing non-tumor cells is essential for esophageal cancer therapy. Oncotarget 2015, 6, (3), 1790-805. 28. Zhang, W.; Jiang, B.; Guo, Z.; Sardet, C.; Zou, B.; Lam, C. S.; Li, J.; He, M.; Lan, H. Y.; Pang, R.; Hung, I. F.; Tan, V. P.; Wang, J.; Wong, B. C., Four-and-a-half LIM protein 2 promotes invasive potential and epithelial-mesenchymal transition in colon cancer. Carcinogenesis 2010, 31, (7), 1220-9. 29. Jin, R.; Liu, W.; Menezes, S.; Yue, F.; Zheng, M.; Kovacevic, Z.; Richardson, D. R., The metastasis suppressor NDRG1 modulates the phosphorylation and nuclear translocation of beta-catenin through mechanisms involving FRAT1 and PAK4. Journal of cell science 2014, 127, (Pt 14), 3116-30. 30. Gupta, G. P.; Massague, J., Cancer metastasis: building a framework. Cell 2006, 127, (4), 679-95. 31. Steeg, P. S., Tumor metastasis: mechanistic insights and clinical challenges. 26


Page 26 of 38

Page 27 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Nature medicine 2006, 12, (8), 895-904. 32. Soltermann, A., [Epithelial-mesenchymal transition in non-small cell lung cancer]. Der Pathologe 2012, 33 Suppl 2, 311-7. 33. Blick, T.; Hugo, H.; Widodo, E.; Waltham, M.; Pinto, C.; Mani, S. A.; Weinberg, R. A.; Neve, R. M.; Lenburg, M. E.; Thompson, E. W., Epithelial mesenchymal transition traits in human breast cancer cell lines parallel the CD44(hi/)CD24 (lo/-) stem cell phenotype in human breast cancer. Journal of mammary gland biology and neoplasia 2010, 15, (2), 235-52. 34. Pinto, C. A.; Widodo, E.; Waltham, M.; Thompson, E. W., Breast cancer stem cells and epithelial mesenchymal plasticity - Implications for chemoresistance. Cancer letters 2013, 341, (1), 56-62. 35. Savagner, P., Epithelial-mesenchymal transitions: from cell plasticity to concept elasticity. Current topics in developmental biology 2015, 112, 273-300. 36. Yan, G. R.; Xu, S. H.; Tan, Z. L.; Liu, L.; He, Q. Y., Global identification of miR-373-regulated genes in breast cancer by quantitative proteomics. Proteomics 2011, 11, (5), 912-20. 37. Tan, I.; Yong, J.; Dong, J. M.; Lim, L.; Leung, T., A tripartite complex containing MRCK modulates lamellar actomyosin retrograde flow. Cell 2008, 135, (1), 123-36. 38. Lee, I. C.; Leung, T.; Tan, I., Adaptor protein LRAP25 mediates myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) regulation of LIMK1 protein in lamellipodial F-actin dynamics. The Journal of biological chemistry 2014, 289, (39), 26989-7003. 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

39. Amano, M.; Nakayama, M.; Kaibuchi, K., Rho-kinase/ROCK: A key regulator of the cytoskeleton and cell polarity. Cytoskeleton (Hoboken, N.J.) 2010, 67, (9), 545-54. 40. Leung, T.; Chen, X. Q.; Manser, E.; Lim, L., The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Molecular and cellular biology 1996, 16, (10), 5313-27. 41. Katoh, K.; Kano, Y.; Ookawara, S., Rho-kinase dependent organization of stress fibers and focal adhesions in cultured fibroblasts. Genes to cells : devoted to molecular & cellular mechanisms 2007, 12, (5), 623-38. 42. Kim, Y.; Ha, C. M.; Chang, S., SNX26, a GTPase-activating protein for Cdc42, interacts with PSD-95 protein and is involved in activity-dependent dendritic spine formation in mature neurons. The Journal of biological chemistry 2013, 288, (41), 29453-66. 43. Benitah, S. A.; Valeron, P. F.; van Aelst, L.; Marshall, C. J.; Lacal, J. C., Rho GTPases in human cancer: an unresolved link to upstream and downstream transcriptional regulation. Biochimica et biophysica acta 2004, 1705, (2), 121-32. 44. Ellis, S.; Mellor, H., Regulation of endocytic traffic by rho family GTPases. Trends in cell biology 2000, 10, (3), 85-8. 45. Symons, M.; Rusk, N., Control of vesicular trafficking by Rho GTPases. Current biology : CB 2003, 13, (10), R409-18. 46. Sinha, S.; Yang, W., Cellular signaling for activation of Rho GTPase Cdc42. Cellular signalling 2008, 20, (11), 1927-34. 47. Xu, Y.; Qi, Y.; Luo, J.; Yang, J.; Xie, Q.; Deng, C.; Su, N.; Wei, W.; Shi, D.; Xu, 28


Page 28 of 38

Page 29 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


F.; Li, X.; Xu, P., Hepatitis B Virus X Protein Stimulates Proliferation, Wound Closure and Inhibits Apoptosis of HuH-7 Cells via CDC42. International journal of molecular sciences 2017, 18, (3). doi: 10.3390/ijms18030586. 48. Feng, Y.; Feng, L.; Yu, D.; Zou, J.; Huang, Z., srGAP1 mediates the migration inhibition effect of Slit2-Robo1 in colorectal cancer. Journal of experimental & clinical cancer research : CR 2016, 35, (1), 191. 49. Cai, S.; Ye, Z.; Wang, X.; Pan, Y.; Weng, Y.; Lao, S.; Wei, H.; Li, L., Overexpression of P21-activated kinase 4 is associated with poor prognosis in non-small cell lung cancer and promotes migration and invasion. Journal of experimental & clinical cancer research : CR 2015, 34, 48.

Figure Legends Figure 1. Establishment of highly invasive colorectal cancer cell sublines. (A) Highly invasive colon cancer sublines (designated I8 cells) were established by serial selection of cancer cells invading through matrigel-coated Boyden chambers. (B) Invasion ability of HCT116-I8 and RKO-I8 cells was compared with that of corresponding parental cells using chamber invasion assay. The quantification data indicated markedly increased invasive potential of I8 cells. (C) Western blot analysis of E-cadherin and vimentin expression levels in I8 cells and parental cells. Actin was included as loading control. (D) Morphology of I8 cells and parental cells. (E) WST-1

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

assay was used to detect cell proliferation rates of I8 cells and corresponding parental cells. Bars, SD; ***, P < 0.001 compared with control cells.

Figure 2. SILAC proteomics suggests the upregulation of Cdc42BPA in highly invasive colon cancer cells (A) Experimental scheme of SILAC proteomics to identify the differently expressed proteins in HCT116-I8 cells compared with parental cells. (B) Venn diagram showing the identification of overlapped proteins in three biological replicates. (C, D) ID Associated Network Function and Molecular and Cellular Function analyses of the dysregulated proteins in I8 cells.

Figure 3. Cdc42BPA promotes colon cancer cell invasion. (A, B) The mRNA and protein abundance of Cdc42BPA in I8 cells and parental cells were measured by qRT-PCR and Western blot. (C) Knockdown of Cdc42BPA with the indicated concentrations of different siRNAs in HCT116-I8 and RKO-I8 cells. (D) The I8 cells with Cdc42BPA knockdown were subjected to invasion assay. (E) Comparison of E-cadherin and vimentin expressions in Cdc42BPA-knockdown cells and control cells. (F) Knockdown of Cdc42BPA did not affect proliferation rate of I8 cells as determined by WST-1 assay. Bars, SD; *, P < 0.05; **, P < 0.01 compared with control cells.

Figure 4. Clinical significance of Cdc42BPA in colon cancer. (A) Representative images of the different scores for Cdc42BPA staining in colon cancer. (B) 30


Page 30 of 38

Page 31 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Representative results of immunohistochemical staining for Cdc42BPA expression in paired tumor and adjacent normal tissues. (C) Cdc42BPA expression in 100 cases of tumors and 80 cases of adjacent normal tissues was analyzed. (D) Kaplan-Meier plot was used to compare overall survival of 99 patients with colon cancer based on Cdc42BPA expression. (E, F) Cdc42BPA expression in 33 cases of metastatic colon cancer in lymph nodes and matched primary tumors was detected by immunohistochemistry and analyzed.

Figure 5. Inhibition of Cdc42 signaling pathway suppresses colon cancer cell invasion. (A) Expression level of active GTP-bound form of Cdc42 in HCT116-I8 and RKO-I8 cells as well as the parental cells was compared by using PAK1-PBD immunoprecipitation and Western blot. (B, C) Chamber invasion assay was performed to determine the invasive potential of the I8 cells treated with the indicated concentrations of ML141 or vehicle control (B). Quantification of invaded cells indicated that treatment with ML141 significantly suppressed colon cancer cell invasion in a dose-dependent manner (C). (D) I8 cells treated with ML141 or DMSO control had similar proliferation rates. (E) Western blot analysis of expressions of E-cadherin and vimentin in HCT116 cells treated with ML141 compared with DMSO-treated cells. Bars, SD; *, P < 0.05; **, P < 0.01 compared with control cells.

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 38

Table 1. Correlation between Cdc42BPA expression levels and clinicopathological parameters in 99 cases of colon cancer.

Variable Age ( years)

n

LowCdc42BPA

High Cdc42BPA

≤55

15

8

7

>55

84

42

42

Female

41

19

22

Male

58

31

27

1/2/3

67

38

29

4

32

12

20

N0

52

34

18

N1

47

16

31

M0

94

50

44

M1

5

0

5

Stages I & II

72

39

33

Stages III & IV

27

11

16

P value

1.000

Gender 0.543

T-Stage 0.088

N-Stage 0.002**

M-Stage 0.026*

Pathologic stage

32


0.265

Page 33 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents graphic for TOC only

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 34 of 38

Page 35 of 38


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 36 of 38

Page 37 of 38


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

Page 38 of 38

B

Paired normal tissue

Primary tumor

Cdc42BPA staining in colon cancer tissues

1

2

3

Case#1

4

Case#2

C

D

P < 0.001

Percentage of cases

100%

Strong

80%

Moderate

60%

Negative

n = 99 P < 0.05

Weak

Cdc42BPA low

40% 20% 0%

E

Cdc42BPA high

Normal n = 80

Primary tumor

Tumor n = 100

Metastatic tumor

F

Case#1

Percentage of cases

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Case#2

P < 0.01

100%

Strong

80%

Moderate

60%

Negative

Weak

40% 20% 0%

Primary Tumor n = 33

Figure 5


Metastatic tumor n = 33

Comparative Proteomics Analysis Identifies Cdc42-Cdc42BPA

Recommend Documents