HCRP-1 regulates cell migration, invasion and angiogenesis via Src/

FAK signaling in human prostate cancerFeifei Chen1*, Jianqiang Wu1*, Jingwei Teng1, Wang Li3, Junnian Zheng1,2,Jin Bai1

1, Cancer Institute, Xuzhou Medical University, Xuzhou, Jiangsu, China; 2, Jiangsu

Center for the Collaboration and Innovation of Cancer Biotherapy, Cancer Institute,

Xuzhou Medical College, Xuzhou, Jiangsu, China; 3, The Affiliated Hospital of

Xuzhou Medical College, Xuzhou, Jiangsu, China.

*These authors contributed equally to this work

Name: Jin Bai

Address: Jiangsu Center for the Collaboration and Innovation of Cancer Biotherapy,

Cancer Institute, Xuzhou Medical College, 84 West Huai-hai Road,

Xuzhou, Jiangsu, P.R.China

Tel: +86-0516-85582530;*E-mail: bj@xzhmu.edu.cn

Name: Junnian Zheng

Address: Jiangsu Center for the Collaboration and Innovation of Cancer Biotherapy,

Cancer Institute, Xuzhou Medical College, 84 West Huai-hai Road,

Xuzhou, Jiangsu, P.R.China

Tel: +86-0516-85582530;* E-mail: jnzheng@xzhmu.edu.cn

Running title: HCRP-1 in human prostate cancer

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AbstractProstate cancer (PCa) is the third leading malignancy engendering mortality among

men globally. The present study aimed at determining the expression of hepatocellular

carcinoma-related protein-1 (HCRP-1) in PCa, to explore its potential role in prostate

tumorigenesis in vitro and in vivo. We evaluated HCRP-1 protein with

immunohistochemistry (IHC) technology and found HCRP-1 expression was

significantly low in PCa tissues (PCTs); In addition, the decreased HCRP-1 was

significantly associated with TNM (tumor node metastasis) stage, advanced histology

grade and gleason score. Transwell, tube formation, Western blot and co-

immunoprecipitation (Co-IP) assays were utilized to determine the role of down-

regulating HCRP-1 in PCa cell migration, invasion and angiogenesis. Meanwhile, we

found HCRP-1 depletion induced Src and focal adhesion kinase (FAK)

phosphorylation, which could be reversed by Src inhibitor PP2 or FAK inhibitor.

Furthermore, down-regulated HCRP-1 evidently induced lung metastasis of PCa cells

in xenograft mode. Taken together, our study indicates HCRP-1 plays an important

role in PCa metastasis. HCRP-1 may serve as a potential therapeutic target for PCa.

KeywordsHCRP-1; prostate cancer; migration; invasion; angiogenesis

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IntroductionProstate cancer (PCa) remains the third leading malignancy engendering mortality

among men globally. Metastasis is the leading cause for the death of PCa patients, the

5-year survival rate is merely 29%[1].During metastasis process, cancer cells invade

into neighboring tissue or lymphatic circulation, blood circulation and

survive to form distant metastases[2].To better understand the mechanistic basis of

cancer metastasis, identifying the critical molecules involved in metastasis is

necessary.

HCRP-1 is identified as a member of the human endosomal sorting complex

required for transport-1 (ESCRT-I) families. It can bind and sort of ubiquitinated

receptors to intraluminal vesicles of MVBs (multivesicular bodies), and then degrade

them in lysosomes[3, 4]. HCRP-1 has been implicated in cancer progression. HCRP-1

was firstly found significantly lost in hepatocellular cell carcinoma (HCC) tissues but

abundant in normal liver tissues[5]. Loss of HCRP-1 is associated with the activation

of EGFR (epidermal growth factor receptor), which exerts an influence on EGFR

prognosis. Down-regulated HCRP-1 leads to invasive phenotype of ovarian cells and

tumor growth in vivo[6]. In addition, it is proved to be of adverse prognostic

significance that oropharyngeal squamous cell carcinoma (OOSCC) patients with less

HCRP-1 expression received preoperative chemoradiotherapy[7]. Our previous study

showed that HCRP-1 was decreased in colorectal cancer (CRC). Reduced HCRP-1

expression caused CRC cancer cells anoikis resistance, and surviving cells formed

metastasis and resulted in poor prognosis in CRC[8]. These studies indicate that

HCRP-1 acts as a tumor suppressor in cancer progression.

In this study, we investigated the relationship between HCRP-1 expression and

clinicopathologic features by PCa tissue microarray (TMA). Furthermore, we showed

a role for HCRP-1 in regulation of migration, invasion and angiogenesis in human

PCa cells. Lastly, our data provided a profound understanding of novel HCRP-1 -

Src/FAK signaling in promoting PCa migration, invasion and angiogenesis.

Material and methodsPatients and samples

A PCa TMA was purchased from Shanghai OUTDO Biotech CO., LTD. (China).

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Tumors were staged and graded based on 2010 revised TNM system and WHO

criteria, respectively[9].The clinicopathological characteristics of TMA are described

in Table 1.Moreover, 90 cases of PCTs were provided.

Antibodies and Reagents

Rabbit monoclonal antibodies to EGFR, p-EGFR (Tyr1173), FAK, p-FAK (Tyr397),

Src, p-Src (Tyr416) and CD31 were purchased from Cell Signaling Technology

(Beverly, MA, USA). Antibody HCRP-1 was from Proteintech (Wuhan, China).

Antibodies against GAPDH were from Santa Cruze (CA, USA). The p-FAK inhibitor,

FAK inhibitor 14 and Src inhibitor, PP2 were from Abcam (Cambridge, MA,

USA).Cycloheximide (CHX) was ordered from Sigma (Shanghai, China).

Immunohistochemistry

The TMA slide or tissues were incubated with primary antibodies overnight at 4°C,

and visualization with diaminobenzidine (DAB; sigma, Shanghai, China). The image

was collected by light microscopy (Olympus BX-51 light microscope). The evaluation

of HCRP-1 in TMA was performed as previously described[8].

Cell Culture

Human PCa cell lines PC3 and LnCap were cultured in RPMI1640 medium enriched

with 10% fetal calf serum (Invitrogen, Shanghai, China). Cells were cultured in a

37 humidified incubator with 95% air, 5% CO℃ 2.

Transfection and PC3 stable cell line generation

HCRP-1 siRNA were ordered from GenePharma (Shanghai, China). The sequences

are as follows: HCRP-1,5′-GACACUGUUUCUUCUUCAACA-3′. Transient

transfections were performed using silentFect Lipid Reagent (Bio-Rad, Hercules, CA,

USA) following the manufacturer’s instructions. GFP-lentivirus-packing HCRP-1

shRNA vector (IBS, Shanghai, China) infectedPC3 cells for two days, and then

screened out with 2 μg/ml puromycin (Sigma, Shanghai, China) for 14 days

Angiogenesis assay

The medium from transfected PC3 and LnCap cells was collected and used as a

conditioned medium. 2×104 human umbilical vein endothelial cells in 100 μl

conditioned medium were seeded in the Matrigel pre-coated 48-well plate. Three

randomly chosen fields were selected to photograph the number of capillary-like tubes

using a Nikon inverted microscope (Nikon, Japan).

Cell migration and invasion assay

Cell migration and invasion assay were performed as described previously[10]. 4

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Modified two chamber plates with a pore size of 8 μm were employed. The transwell

filter that inserts without Matrigel (BD Biosciences) coating were used for migration

assay. The transwell filter that coated with Matrigel (BD Biosciences) were used for

invasion assay.

Western blot analysis

After transfection, cells were harvested and cell extracts were separated on SDS-

polyacrylamide gel. The proteins were blotted on nitrocellulose membrane and

incubated with appropriate primary antibodies overnight at 4°C. Secondary antibodies

included horseradish peroxidase (HRP)-goat anti-mouse and HRP-goat anti-rabbit

(ZSGB-BIO). Protein bands were detected by Tanon5200 automatic

chemiluminescence imaging analysis system using ECL reagent (Tanon, shanghai,

China).

Co-immunoprecipitation

PC3 cells were lysed and the cell lysates were precleared with mouse normal IgG and

protein G-agarose for 30 min. Then, HCRP-1 and HCRP-1-specific IgGs were

incubated with the precleared mixture overnight. Then, the immunoprecipitates were

washed with cold lysis buffer for five times and applied to Western blotting assay.

In vivo experimental metastasis models

Each group consists of eight male nude mice. 1.0×106 shHCRP-1 stably expressed

PC3 cells were injected with mice via tail vein. 8 weeks post-implantation, mice were

euthanized and lung tissues were fixed in 10% formaldehyde. HE stained was applied

for metastatic nodules. Bioluminescence imaging of the mice were analyzed using

Xenogen IVIS200 imaging system (Xenogen Corp.). Animal experiments were

performed following protocols approved by the Committee of Xuzhou Medical

University for the Use and Care of Animals.

Subcutaneous in vivo experiments

To explore tube formation in vivo, male nude mice were injected with 1.0×106

shHCRP-1 stably expressed PC3 cells in 100 μl PBS/Matrigel (50:50) subcutaneously.

The body weight was monitored and tumor volume was calculated by vernier caliper

every day. 8 weeks after implantation, mice were euthanized and tumors were

surgically removed and fixed in 10% formaldehyde for histology.

Statistical analysis

We used SPSS version 17.0 for Windows (SPSS Inc., Chicago, IL) for all analyses.

Data are presented as means ± SD. Dunnett’s t test (two-factor) was employed to 5

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evaluate differences within treatment groups. HCRP-1 staining in PCa TMA and

clinicopathologic parameters were evaluated by χ2 test. A P-value equal to or <0.05

was considered statistically significant.

ResultsThe expression of HCRP-1 is reduced in human PCa

To determine HCRP-1 expression in human PCa, we used TMA slide containing PCTs

and paired NTs for immunohistochemistry staining. Data showed that HCRP-1 was

mainly localized in the cytoplasmic (Fig. 1A). In NTs, strong cytoplasmic staining

was observed in normal prostate epithelia. 76.7% (69 of 90 cases) patients showed

positive HCRP-1 staining. In PCTs, strong expression of HCRP-1 was recorded in

42.2% (38 of 90 cases) (Fig. 1B). The expression of HCRP-1 was significant reduced

in the PCTs when compared with NTs (P＜0.001, χ2 test).

HCRP-1 expression with clinicopathological parameters

We next investigated the correlation between HCRP-1 staining and

clinicopathological features in PCa (Table 1). We found that loss of HCRP-1 was

significantly correlated with histological grade when compared I-II with III-IV

(P<0.05, Fig. 1C). Since TNM stage is a critical prognostic marker for cancer

patients, we examined whether reduced HCRP-1 expression correlates with TNM

stage. Data displayed that HCRP-1 expression was dramatically reduced in late TNM

stages (III-IV) when compared with early stages (I-II) (P<0.05, Fig. 1D). Moreover,

decreased HCRP-1 expression was significantly decreased in high gleason score (＞7)

(P<0.05, Fig. 1E). However, there is no significant correlation between HCRP-1

expression andage.

Loss of HCRP-1 promotes PCa cells migration, invasion and angiogenesis

To investigate the effect of HCRP-1 on cell phenotypes, we transiently transfected

PC3 and LnCap cells with control siRNA and HCRP-1 siRNA to inhibit HCRP-1

protein levels (Fig. 2A and B). We then assessed whether alteration of HCRP-1

affected the malignant characteristics of the prostate cancer cell lines. We first

detected the migration ability of cancer cells and data showed that the thecapacity of

cell migration was drastically promoted after knockdown of HCRP-1 expression (Fig.

2C and D). The results from the cell invasion assay were corresponded with the cell

migration assay(Fig. 2E and F). Moreover, we evaluated the effect of decreased

HCRP-1 expression on angiogenic potential of human PCa cells. As shown in Figure 6

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2G and H, the number of HUVECs formed tubular structures was obviously increased

in conditioned medium from HCRP-1 knockdown cancer cells.

HCRP-1 regulates Src/FAK signaling pathway in PCa cells

HCRP-1 mediates the internalization and degradation of EGFR. We detected the half-

life of EGFR to clarify whether the stability of EGFR exerted influence oncells

metastasis phenotypes in HCRP-1 knockdown PCa cells. We observed that the

stability was sustained in both two cell lines (Fig. 3A, B). Ineffective receptor sorting

could lead to phosphorylated EGFR receptor accumulation in cancer cells. We

explored that whether HCRP-1 might regulate EGFR activation. Our data showed that

phosphorylation of EGFR remains unchanged (Fig. 3C). It has been well studied that

Src/FAK signaling plays an important role in cell migration, invasion and

angiogenesis. Therefore, we detected the phosphorylation of Src and FAK in cells

transfected with control siRNA and HCRP-1 siRNA. The results showed that

knockdown of HCRP-1 expression induced Src and FAK phosphorylation (Fig. 3C).

We further performed immunoprecipitation experiments in PC3 cells to investigate the

interaction of HCRP-1 and Src. The results showed that HCRP-1 or Src

immunoprecipitates were associated with endogenous Src or HCRP-1 protein (Fig.

3D), confirming the specific association between HCRP-1 and Src. There results

suggest that HCRP-1 may regulate Src/FAK signaling pathway via interaction of

HCRP-1 and Src.

Inhibition of HCRP-1 promotes cells migration, invasion and angiogenesis

through Src/FAK signaling pathway

To further investigate whether HCRP-1 regulates cells migration, invasion and

angiogenesis through Src/FAK signaling pathway, we firstly added the Src-specific

inhibitor, PP2 (5 μM), into PCa cells that was transfected with HCRP-1 siRNA.

Western blot data indicated that PP2 obviously suppressed Src and FAK

phosphorylation in HCRP-1-knockdown cells (Fig. 4A). The migration and invasion

cells were obviously decreased in HCRP-1-deficient cells treated with PP2 (Fig. 4B).

In addition, the angiogenesis of PC cells was determined by endothelial cell tube

formation assay. The number of HUVECs formed tubular structures was obviously

increased in conditioned medium from HCRP-1 knockdown cancer cells. However,

this induction was suppressed by Src-specific inhibitor PP2 (Fig. 4C).

To further explore the functions of Src/FAK signaling pathway in regulating cell

migration, invasion and angiogenesis by HCRP-1, we also employed FAK-specific 7

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inhibitor 14 (100 mM) to inhibit FAK phosphorylation. Our data testified to that FAK-

specific inhibitor 14 lead to the reduction of FAK phosphorylation which was induced

by knockdown of HCRP-1 (Fig. 5A). The migration and invasion abilities were

suppressed by treatment of PC3 and LnCap cells with FAK-specific inhibitor 14

(Fig.5B). Subsequently, we also found that angiogenesis ability was inhibited by

FAK-specific inhibitor 14 (Fig.5C). These data suggest that Src/FAK signaling is

involved in the promotion of PCa cancer cells migration, invasion and angiogenesis

induced by depletion of HCRP-1.

Depletion of HCRP-1promoted prostate cancer tumorigenesis, angiogenesis and

metastasis in vivo

To evaluate a role for HCRP-1 in tumor metastasis in vivo, an experimental metastasis

assay was performed with luciferase-expressing PC3 cells infected with scrambled

control or shRNA against HCRP-1. Two months later, a statistical difference was

noticed that PCa cells with the HCRP-1-knockdown had a higher signal in the lungs

compared with the scrambled control (Fig. 6A). Then, the mice were executed to

analyze the lung metastasis status. As shown in Fig. 6B-D, more multiple large

metastatic foci in lung were observed in the samples containing PC3-shHCRP-1 cells.

Immunohistochemistry staining of metastatic nodules in lungs showed that p-Src and

p-FAK expression in the HCRP-1-knockdown group was high compared with the

control group (Fig. 6E). Next, we employed xenograft models using nude mice

subcutaneously transplanted with PC3 cells that were mock treated or depleted of

HCRP-1 to evaluate the effect of HCRP-1 on tumor growth in vivo. As shown in Fig.

6F and G, HCRP-1 knockdown cells formed bigger tumors than the control. Also, the

expression levels of HCRP-1, p-Src, p-FAK and CD31 were high in the HCRP-1

knockdown subcutaneous tumors (Fig. 6H). Next, These findings provided

compelling evidence for the ability of HCRP-1 to confer tumorigenesis and metastasis

in vivo.

DiscussionIn our work, we provide a set of data suggesting critical roles for HCRP-1 in PCa

angiogenesis and metastasis. First, it was found that in human PCa patients, low

HCRP-1 expression correlates/is associated with/related to clinicopathologic

parameters, revealing HCRP-1 as a novel candidate tumor-suppressor gene in PCa

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(Fig. 1). Second, evidence was presented, demonstrating a supporting role for HCRP-

1 in prostate cancer cell migration, invasion and angiogenesis (Fig. 2). Third, HCRP-1

was shown to regulate cell migration, invasion and angiogenesis through interacting

with Src/FAK signaling (Fig. 3-5). Finally, the results of mouse xenograft experiments

revealed a role for HCRP-1 in promoting prostate cancer angiogenesis and metastasis

in vivo (Fig. 6).

The vital role for HCRP-1 expression in cancer development has been reported

in several malignancies, such as ovarian cancer, HCC and breast cancer[5, 6, 11]. Low

HCRP-1 mRNA expression was independently associated with shorter disease-free

survival in HCC and breast cancer[5]. We previous found that RCC patients were

significantly correlated with decreased overall survival only in tumors with low or

missing HCRP-1 expression[10]. In PCTs, we identified decreased expression of

HCRP-1 protein in cancer tissues. Moreover, we explored the correlation between

HCRP-1 staining and clinicopathological features. Our results displayed that loss

expression of HCRP-1 occurred in most of PCa patients, and this reduced expression

had significantly correlation with histological grade, TNM stage and Gleason score.

Collectively, these observations suggest that HCRP-1 have a potential effect on PCa

progression and metastasis.

Distant metastasis has been reported as the main cause of death in PCa

patients[12]. A critical step in cancer metastasis is the migration and invasion of

cancer cells[13]. Since the invasiveness of cancer cells depends on increased

migratory and invasive properties, we explored the potential role of HCRP-1 in

suppressing migration and invasion of PCa cells. Knockdown of HCRP-1 protein lead

to significant induction and alteration of PCa cells migratory and invasive properties.

Similarly, ablation of HCRP-1 obviously promoted tumor progression and

metastasisin vivo.

Solid tumors growth and metastasis depends on angiogenesis, including PCa,

which is the progression of forming new blood vessels from the existing network of

capillaries[14]. Targeting angiogenesis is effective in cancer therapy not only because

angiogenesis supplies oxygen and nutrients for the survival of cancer cells, but also

provides the way for metastatic spread of these cancer cells[15]. Here, we showed that

inhibition of HCRP-1 expression promoted tube formation of HUVECs induced by

prostate cancer cells. In vivo, knockdown of HCRP-1 promoted CD31 expression in

prostate tumors. These data revealed that angiogenesis serve as a crucial role in 9

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promoting cancer metastasis by knockdown of HCRP-1.

In order to further elucidate the mechanisms involved in HCRP-1-induced

prostate cancer cell migration, invasion and angiogenesis, PC3 and LnCap cells were

transfected with siRNA for HCRP-1, phosphorylation of Src and FAK was detected.

We found that knockdown of HCRP-1 induced phosphorylation of Src and FAK. Src

is a non-receptor tyrosine kinase, which is induced by various kinds of cellular signal

molecules and have a great effect in regulating numerous processes, including cell

growth, differentiation, adhesion and the migration signaling pathway[16, 17]. As an

other non-receptor protein tyrosine kinase, FAK is widely expressed and regulats

several biological processes, including cell adhesion, angiogenesis and migration[18,

19]. At the sites of cell-substratum focal adhesions, FAK can combine withSrc to form

a dual-kinase complex[20], which coordinate cell behavior through regulating

downstream pathways and molecules, including AKT, p38 and ERK kinase[21, 22].

Autophosphorylation of Src/FAK complexes is a critical event in cancer progression

and metastasis[23]. Src/FAK pathway kinases in solid tumors has been viewed as an

attractive target in cancer therapy. Inhibition of Src family kinase exerts effects on the

tumor microenvironment [24, 25]. In our present study, the Src-specific inhibitor PP2

were used to abrogate the activation of Src, and the results showed that FAK activity

and cells migration, invasion and angiogenesis were suppressed by PP2 in PC3 and

LnCap cells. Additionally, FAK inhibitor 14 could inhibit the phosphorylation levels

of Src and FAK. Combined with our results, we suggested that HCRP-1 knockdown

might induce cell migration, invasion and angiogenesis by deregulating Src/FAK-

associated signaling pathways in prostate cancer cells.

HCRP-1 is involved in binding and sorting of EGFR and the consequent

degradation with lysosomes[4]. Src/FAK and EGFR converge on common

downstream pathways[26], which exhibit antitumor activity in patients with advanced

NSCLC co-treated with inhibitors of both signaling pathways[27]. We detected the

half-life of EGFR to elucidate whether EGFR stability exerted influence on Src/FAK

pathway in HCRP ablation cells. The stability was sustained in both WT and HCRP-1

knockdown cancer cells. Next, we investigated the phosphorylation of EGFR and our

data showed that reduced HCRP-1 expression did not change EGFR activity. We

assumed that HCRP-1 could regulate Src/FAK signaling through other mechanism. As

the ESCRT-I subunit, Tsg101 (tumor susceptibility gene101) deletion impaired Src-

dependent activation of STAT3 and FAK, resulted in cell migration suppression[28]. 10

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Moreover, as other ESCRT-I subunit, Alix binds to Src at two points via SH2 (Src

homology 2) and SH3 (Src homology 3) domains. Alix that binds to Src are typically

phosphorylated by Src[29]. Therefore, we investigated the endogenous interaction of

HCRP-1 and Src. We demonstrated that endogenous Src and HCRP-1 in untransfected

cells were associated, suggests that the interaction between Src and HCRP-1 has

physiological relevance. We supposed that the interaction of HCRP-1 and Src

contributed to the regulation of Src activity.

In conclusion, we have established a vital role for HCRP-1 in PCa and our clinical

analyses point the prospect of HCRP-1 as a potential molecular therapeutic target for

highly aggressive prostate cancer. We also revealed that HCRP-1 regulated prostate

cancer cell migration, invasion and angiogenesis. In mechanism, HCRP-1 may

regulate Src/FAK signaling pathway through interaction with Src. Although more

details still need to explore the functions of HCRP-1 in a physiologic condition, our

data uncover the importance of HCRP-1 in PCa progression and metastasis.

AcknowledgmentsThis work was supported by the Postgraduate Research & Practice Innovation

Program of Jiangsu Province (KYCX19_2213). We thank all the staff of Jiangsu

Cancer Biotherapy Institute in Xuzhou Medical University for technical support.

Competing interestsThe authors have declared no conflicts of interests.

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19. Cox BD, Natarajan M, Stettner MR, Gladson CL. New concepts regarding focal adhesion kinase promotion of cell migration and proliferation. J Cell Biochem. 2006; 99: 35-52.20. Mitra SK, Schlaepfer DD. Integrin-regulated FAK-Src signaling in normal and cancer cells. Curr Opin Cell Biol. 2006; 18: 516-23.21. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell. 2002; 110: 673-87.22. Guo W, Giancotti FG. Integrin signalling during tumour progression. Nat Rev Mol Cell Biol. 2004; 5: 816-26.23. Liang Y, Yi L, Liu P, Jiang L, Wang H, Hu A, et al. CX3CL1 involves in breast cancer metastasizing to the spine via the Src/FAK signaling pathway. J Cancer. 2018; 9: 3603-12.24. Wu Y, He L, Zhang L, Chen J, Yi Z, Zhang J, et al. Anacardic acid (6-pentadecylsalicylic acid) inhibits tumor angiogenesis by targeting Src/FAK/Rho GTPases signaling pathway. J Pharmacol Exp Ther. 2011; 339: 403-11.25. Vultur A, Buettner R, Kowolik C, Liang W, Smith D, Boschelli F, et al. SKI-606 (bosutinib), a novel Src kinase inhibitor, suppresses migration and invasion of human breast cancer cells. Mol Cancer Ther. 2008; 7: 1185-94.26. Leung EL, Tam IY, Tin VP, Chua DT, Sihoe AD, Cheng LC, et al. SRC promotes survival and invasion of lung cancers with epidermal growth factor receptor abnormalities and is a potential candidate for molecular-targeted therapy. Mol Cancer Res. 2009; 7: 923-32.27. Haura EB, Tanvetyanon T, Chiappori A, Williams C, Simon G, Antonia S, et al. Phase I/II study of the Src inhibitor dasatinib in combination with erlotinib in advanced non-small-cell lung cancer. J Clin Oncol. 2010; 28: 1387-94.28. Tu C, Ortega-Cava CF, Winograd P, Stanton MJ, Reddi AL, Dodge I, et al. Endosomal-sorting complexes required for transport (ESCRT) pathway-dependent endosomal traffic regulates the localization of active Src at focal adhesions. Proc Natl Acad Sci U S A. 2010; 107: 16107-12.29. Schmidt MHH, Dikic I, Bogler O. Src phosphorylation of Alix/AIP1 modulates its interaction with binding partners and antagonizes its activities. J Biol Chem. 2005; 280: 3414-25.

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Figure legends

Figure 1. Correlation between HCRP-1 expression and clinicopathologic

parameters in prostate cancer.

A. Representative immunohistochemical photographs were taken at different

magnifications in NTs and PCTs (Top panel ×100, bottom panel ×400). B. Compared

with that in the tumor adjacent normal prostate tissue, the overall expression level of

HCRP-1 in the prostate cancer tissues was significantly lower (P<0.001, χ2 test). C.

Decreased HCRP-1 expression was correlated with histological grade (P<0.05, χ2 test,

comparing I-II versus III-IV). D. Decreased HCRP-1 expression was correlated with

TNM stage (P<0.05, χ2 test, comparing T1-2 versus T3-4). E. Decreased HCRP-1

expression was correlated with Gleason score (P<0.05, χ2 test, comparing≤7 versus ＞7).

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Figure 2. Effect of the reduction in HCRP-1 expression on the abilities of cell

migration, invasion and angiogenesis in vitro.

A,B. Forty-eight hours after transfection, the expression of HCRP-1 in the PC3 and

LnCap cells was evaluated by western blotting GAPDH was used as an internal

control. C-F. Cell migration and matrigel cell invasion assays were performed in PC3

and LnCap cells after transfection, respectively. Representative fields of migrating or

invading cells on the membrane (magnification, ×200). G,H. Representative pictures

were taken in situ for tube formation in the supernatant of PC3 and LnCap cells

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transduced with siRNA foe HCRP-1 or control. The data are presented as mean±SD

for triplicate determinations. *** P<0.001.

Figure 3. Endogenous interaction of HCRP-1 and Src contributes to Src/FAK

signaling pathway.

A,B. Cells were treated CHX for 0, 0.5 h, 1h, 2h after transfected with si-HCRP-1 or

control siRNA for 48 h, lysates obtained from these cells were submitted to Western

blot detection for the protein expression of EGFR. C. Activities of EGFR, Src and

FAK were determined by using Western blot analysis with antibodies specific for total

and phosphorylated forms of EGFR, Src and FAK in silencing of HCRP-1 and control

group for both PC3 and LnCap cell lines. D. Src were coprecipitated with HCRP-1 in

whole-cell lysates of PC3 cells.

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Figure 4. Inhibition of Src activation blocked HCRP-1-induced cell migration,

invasion and angiogenesis.

A. PC3 and LnCap cells were pretreated with HCRP-1 siRNA and the Src inhibitor,

PP2. The whole cell lysates were analyzed for the protein levels of p-Src, Src, p-FAK,

FAK and GAPDH by Western blot. B. Cell migration and Matrigel cell invasion were

evaluated after HCRP-1 siRNA treatment and treatment with PP2 (5 μM).

Representative fields of migrating or invading cells on the membrane (magnification,

×200). C. Tube formation was explored in the supernatant of PC3 and LnCap cells.

The data are presented as mean±SD for triplicate determinations. *P<0.05 versus

siCtrl group; #P<0.05 versus siHCRP-1 group.

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Figure 5. Inhibition of FAK activation blocked HCRP-1-induced cell migration,

invasion and angiogenesis.

A. PC3 and LnCap cells were pretreated with HCRP-1 siRNA and the FAK inhibitor,

FAK inhibitor 14(200mM). The whole cell lysates were analyzed for the protein

levels of p-Src, Src, p-FAK, FAK and GAPDH by Western blot. B. Cell migration and

Matrigel cell invasion were evaluated after HCRP-1 siRNA treatment and treatment

with FAK inhibitor 14. Representative fields of migrating or invading cells on the

membrane (magnification, ×200). C. Tube formationwas explored in the supernatant

of PC3 and LnCap cells. The data are presented as mean±SD for triplicate

determinations. *P<0.05 versus siCtrl group; #P<0.05 versus siHCRP-1 group.18

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Fig. 6 Loss of HCRP-1 induces tumor metastasis and angiogenesis in vivo.

A. PC cells were stably transfected with shHCRP-1 lentivirus. Bioluminescence

imaging of Balb/c mice were intravenously injected with PC cells stably expressed

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shHCRP-1. B. Number of metastatic nodules injected with PC cells stably transfected

with HCRP-1 lentivirus compared with the control groups. Metastatic nodules were

highlighted by arrows. C. H&E staining sections of lungs 2 months after injection of

HCRP-1KD-PC3 cell lines in BALB/c nude mouse through tail vein. D. Number of

lung metastatic nodules was counted under a dissecting microscope. E. IHC detection

of HCRP-1, p-Src and p-FAK protein expression in metastatic nodules. F,G. HCRP-

1KD-PC3 cells were subcutaneously injected into BALB/c nude mice (n = 5, for each

experimental group), and 8 weeks later, the xenograft tumors were peeled off and

weighted. The tumor weights are presented as the means ± S.D. **P<0.01. H. IHC

detection of HCRP-1, p-Src and p-FAK protein expression in xenograft tumors

formed by PC3-shCtrl/shHCRP-1 cells in BALB/c nude mice.

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Variables

HCRP-1 staining

Negative(%) Positive(%) Total P-value

Cancer cases 52（57.8） 38（42.2） 90

＜0.001Latero-cancer tissue 21（23.3） 69（76.7） 90

Age 0.805

≤ 70 years 26（50.1） 18（40.9） 44

＞70 years 26（56.5） 20（43.5） 46

Histopathologic Grade 0.014

G1-2 13（40.6） 19（59.4） 32

G3-4 39（67.2） 19（32.8） 58

Gleason score 0.045

≤ 7 14（43.8） 18（56.2） 32

＞7 38（65.5） 20（34.5） 58

TNM stage 0.022

I-II 29（49.2） 30（50.8） 59

III-IV 23（74.2） 8 （25.8） 31

Table 1. HCRP-1 staining and patients clinicopathological characteristics. *P values are

from χ2 test

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