EnhancedExpressionof Rab27A GenebyBreast … · intimate members of the Rab family, Rab3A, Rab7,...

Enhanced Expression of Rab27A Gene by BreastCancer Cells Promoting Invasiveness and theMetastasis Potential by Secretion ofInsulin-Like Growth Factor-II

Jin-Song Wang,1 Fu-Bin Wang,2 Qiang-Ge Zhang,2 Zhen-Zhou Shen,1

and Zhi-Ming Shao1

1Breast Cancer Institute, Cancer Hospital, Department of Oncology, Shanghai Medical College,Institutes of Biomedical Science, Fudan University and 2Shanghai Institutes forBiological Sciences, Chinese Academy of Sciences, Shanghai, China

AbstractIn addition to the functions of transporting melanosome

in melanocytes and releasing contents of lytic

granules in CTLs, Rab27A was recently shown to be

involved in exocytosis of insulin and chromaffin

granules in endocrine cells; it was also reported to be

expressed in an exceptionally broad range of specialized

secretory cells. As autocrine and paracrine cytokines

are essential for invasion and metastasis in some solid

tumors, blocking them may be an effective strategy to

prevent tumor dissemination. In the present study,

we show that Rab27A is associated with invasive and

metastatic potentials of human breast cancer cells.

The overexpression of Rab27A protein redistributed the

cell cycle and increased the invasive and metastatic

abilities in breast cancer cells both in vitro and in vivo.

We also certified that Rab27A conferred the invasive

and metastatic phenotypes on breast cancer cells by

promoting the secretion of insulin-like growth factor-II

(IGF-II), which regulates the expression of p16, vascular

endothelial growth factor, matrix metalloproteinase-9,

cathepsin D, cyclin D1, and urokinase-type plasminogen

activator. These data provide functional evidence that

Rab27A acts as a novel mediator of invasion and

metastasis promotion in human breast cancer cells,

at least in part, through regulating the secretion

of IGF-II, suggesting that synergistic suppression of

Rab27A and IGF-II activities holds a promise for

preventing breast cancer invasion and metastasis.

(Mol Cancer Res 2008;6(3):372–82)

IntroductionInvasion and metastasis, which represent the devastating

attributes of cancer cells, are continuing therapeutic challenges

and common causes of death for patients with cancer (1).

However, it is clear that they are such complex processes involv-

ing several strategies, including mesenchymal movement,

amoeboid locomotion, and migration through tissues, and so

on, that the molecular mechanisms are still poorly understood

(2, 3). Thus, an improved understanding of the molecular

basis underlying cancer invasion and metastasis is essential to

develop novel and more effective molecular targets for therapy.

In breast cancer, which is the leading malignancy in women

(4), cytokines and their receptors perform significant functions

on proliferation, invasion, metastasis, and resistance to drugs

(5). The insulin-like growth factor system (IGF), which

comprises a complex network of ligands (IGF-I and IGF-II),

cognate receptors (type I, type II IGF receptors, and insulin

receptor), IGF-binding proteins, and IGF-binding protein

proteases, plays multiple roles in normal mammary gland and

breast cancer (6-10). With the studies of cytokines, some

proteins are found to collaborate with special cytokines in

cancer cells. Rab proteins, members of the superfamily of Ras-

related small GTPases, have distinct subcellular localization

and are believed to control cellular events ranging from secre-

tion and endocytosis to signal transduction and development

(11-15). A recent genome analysis revealed that the Rab family

is composed of 60 members in Homo sapiens (16). Rab27A is

a unique member in Rab family for its specific implication in

human genetic diseases (15). Loss-of-function mutations in

human Rab27A gene result in Griscelli syndrome, a rare

autosomal disorder characterized by combination of partial

cutaneous albinism and severe immunodeficiency (17, 18).

Its clinical picture seems to be a manifestation of defects in

two specialized lysosome-related organelles, due to failure of

distributing melanosome in melanocytes and releasing the

contents of lytic granules in CTL (19). A mouse mutant,

ashen (Rab27aash), has mutations in the Rab27A gene and

exhibits the same phenotypic features as Griscelli syndrome

patients (20-22). Provance et al. (23) have revealed the

profound functions of Rab27A on the two lysosome-related

organelles in normal and Griscelli syndrome or ashen cells.

Melanocyte and CTL are only a subset of cell types expressing

Rab27A, according to previous studies which indicated that

Received 4/8/07; revised 8/21/07; accepted 8/28/07.Grant support: National Natural Science Foundation of China grants 30371580and 30572109 (Z-M. Shao), and Shanghai Science and Technology Committeegrants 03J14019, 06DJ14004, and 06DZ19504 (Z-M. Shao).The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).Requests for reprints: Zhi-Min Shao, Department of Breast Surgery, BreastCancer Institute, Cancer Hospital/Cancer Institute, Fudan University, 270 DongAn Road, Shanghai 200032, P.R. China. Phone: 86-1361-1709888; Fax: 86-21-64434556. E-mail: [email protected] D 2008 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-07-0162

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Rab27A protein is widely expressed in specialized secretory

cells, including exocrine (particularly in mucin- and zymogen-

secreting cells), endocrine, ovarian, and hematopoietic cells,

most of which undergo regulated exocytosis (24). Recent

investigation revealed the roles of Rab27A in exocytosis of

insulin and chromaffin granules in endocrine cells (25-27).

Consistently, three families of Rab27A effectors have been

recognized and thoroughly investigated (28).

In the present study, we systematically up-regulated and

down-regulated the expression of Rab27A protein and tried to

explore the roles of Rab27A in invasion and metastasis in breast

cancer cells. We showed that Rab27A is critical to invasive and

metastatic phenotypes of breast cancer cells by regulating the

secretion of IGF-II, which modulates the expression of vas-

cular endothelial growth factor (VEGF), cathepsin D, cyclin

D1, urokinase-type plasminogen activator (uPA), matrix metal-

loproteinase-9 (MMP-9), and p16.

ResultsExpression Analyses of Rab27A in Breast Cancer Cells

To investigate whether Rab27A is involved in invasion and

metastasis in breast cancer cells, we analyzed Rab27A

expression using reverse transcription-PCR (RT-PCR) and

Western blot in a panel of breast cancer cell lines with different

invasive and metastatic potentials comprising MCF-7, MDA-

MB-231, MDA-MB-435, and MDA-MB-435HM (29). We

found that Rab27A mRNA (Fig. 1A) but not other functionally

intimate members of the Rab family, Rab3A, Rab7, Rab33A,

and Rab37 (data not shown), increased as invasive and

metastatic ability increased. Semiquantitative real-time RT-

PCR analyses revealed that the levels of Rab27A mRNA in

MDA-MB-231, MDA-MB-435, and MDA-MB-435HM were

elevated 2.1-, 3.4-, and 6.9-fold, respectively, compared with

that in MCF-7, which has the weakest invasive and metastatic

potential (refs. 29, 30; Fig. 1B; P < 0.05 or P < 0.01). Western

blot also showed that breast cancer cell lines differentially

expressed the Rab27A protein, which was 2.83-, 4.9-, and

9.19-fold in MDA-MB-231, MDA-MB-435, and MDA-

MB-435HM, respectively, compared with that in MCF-7 as

determined by densitometric analyses (Fig. 1C). These data

suggested that invasion and metastasis in breast cancer cells

were likely to correlate with endogenous Rab27A expression.

Rab27B, the unique homologue of Rab27A, was not expressed

in all the four breast cancer cell lines (data not shown).

Subcellular Localization of Rab27A in Breast CancerCells

To characterize the localization of endogenous Rab27A in

breast cancer cells, we did immunofluorescence and confocal

microscopy on human breast cancer cells with antibody specific

to Rab27A. As shown in Fig. 2, Rab27A was found to be

targeted to diffuse in cytoplasm in three breast cancer cell lines

and particularly concentrated in the perinuclear area in MDA-

MB-231 and MDA-MB-435 cells. This perinuclear localization

of a dot-like pattern is not similar to that of the ring-like pattern

in parietal cells of the stomach (24). Meanwhile, we did not find

any specific relationship in location between Rab27A and

cytoskeletons (data not shown). It is likely that in breast cancer

cells, Rab27A participates in secretion but does not link to

cytoskeletons by its effectors, which was reported by Kuroda

and Fukuda (31).

Rab27A Overexpression Redistributed Cell Cycle andEnhanced Invasive Potential of Breast Cancer CellsIn vitro

Rab27A expression may affect the invasive and metastatic

phenotypes of breast cancer cells. To investigate this possibility,

the Rab27A eukaryotic expression vector pcDNA3.1(+)-

Rab27A was constructed and transfected into MDA-MB-231

and MDA-MB-435 cells and generated stable transfectants. For

each cell line, two of the Rab27A transfectants, mock-

transfected and parental cells in which Rab27A expression

was detected by RT-PCR (data not shown) and Western blot

(Fig. 3), were used for further studies.

FIGURE 1. Expression of Rab27A in breast cancer cell lines. A.Expression of Rab27A mRNA in four human breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-435, and MDA-MB-435HM detected by RT-PCR. B. Comparison of the relative levels of Rab27A mRNA in fourhuman breast cancer cell lines by semiquantitative real-time RT-PCR.Bars, SD. The quantification of Rab27A mRNA in MCF-7 is given anarbitrary value of 1.C. Differential expressions of Rab27A protein in breastcancer cell lines. Relative amount of Rab27A protein (fold F SD) bydensitometric analyses is indicated below the blots. The quantification ofRab27A protein in MCF-7 is given an arbitrary value of 1.

Promoting Breast Cancer Invasiveness and Metastasis

Mol Cancer Res 2008;6(3). March 2008

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To investigate whether Rab27A expression could affect

breast cancer cell proliferation, we did flow cytometry analyses.

The results showed that the percentage of cells in the S phase

was dramatically increased from 40% to 70% and that in the G0

and G1 phase was accordingly decreased from f50% to 20%

(P < 0.01) with Rab27A transfection, suggesting that Rab27A

overexpression in breast cancer cells redistributes cell cycle and

causes more cells to divide (data not shown).

To validate the effects of Rab27A on the invasive potential

of breast cancer cells, we did in vitro invasion assay to quantify

the invasion ability. We found that all cell lines tested were

invasive through Matrigel after 12 hours; however, Rab27A

transfectants of both MDA-MB-231 and MDA-MB-435 were

highly invasive compared with parental or mock-transfected

cells after 24 and 48 hours by culturing in Transwell (P < 0.01;

data not shown). These results indicated that the invasive

potential of breast cancer cells in vitro has close correlation

with the level of Rab27A expression.

Rab27A Overexpression Redistributed Cell Cycle andEnhanced Invasive Potential of Breast Cancer CellsIn vitro by Up-Regulating VEGF, uPA, Cathepsin D,Cyclin D1, and MMP-9 and Down-Regulating p16

The progression of cancer is usually accompanied by the genetic

underpinnings of cancer invasion and metastasis (32). To further

investigate the molecular basis underlying the Rab27A-mediated

invasive phenotype of breast cancer cells in vitro, we focused our

attention on several extensively recognized invasive- and meta-

static-associated genes. Semiquantitative real-time RT-PCR and

Western blot analyses showed that Rab27A overexpression was

constitutive with up-regulation of VEGF, cathepsin D, cyclin D1,

uPA, and MMP-9, and with down-regulation of p16 in MDA-MB-

231 and MDA-MB-435 cells (Fig. 4A-C; P < 0.05 or P < 0.01). In

contrast, no significant difference in the expression of p21, trans-

forming growth factor-a, cyclinA, cyclin E,MMP-1,MMP-2, tissue

inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, uPA receptor,

and basic fibroblast growth factor was found (data not shown).

FIGURE 2. Subcellular localizationof Rab27A in MCF-7, MDA-MB-231,and MDA-MB-435 cells as detected byimmunofluorescence confocal micros-copy. Note that in MDA-MB-231 andMDA-MB-435 cells, Rab27A is particu-larly concentrated in the perinucleusother than diffusing in the cytoplasmlike MCF-7 cells.

FIGURE 3. Rab27A proteindetected by Western blot is shownin Rab27A stable transfectants,mock-transfected and parental cellsof the MDA-MB-231 and MDA-MB-435 cell lines.

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At the same time, overexpressed Rab27A concomitantly

increased the amount of vesicles in MDA-MB-231 and MDA-

MB-435 cells (figure not shown), indicating that Rab27Amay be

a decisive regulator of vesicle formation in breast cancer cells.

Reduced Rab27A Expression Attenuated the InvasivePotential of Breast Cancer Cells In vitro by Down-Regulating VEGF, uPA, Cathepsin D, Cyclin D1, andMMP-9 and Up-Regulating p16

To determine whether Rab27A expression is critical to the

invasive phenotype of breast cancer cells in vitro, we constructed

RNA interference (RNAi) vectors targeting the Rab27A gene.

First, we certified that the two fragments of RNAi effectively

reduced the exogenous and endogenous Rab27A detected by

Western blot in 293T cells (P < 0.01; data not shown). Then, we

applied them on MDA-MB-231 and MDA-MB-435 parental

cells. Approximately 60% to 65% of reductions in Rab27A

mRNA and protein by 48 to 72 h after transfection were observed

in RNAi-targeted MDA-MB-231 and MDA-MB-435 cells com-

pared with parental and nonsilencing control (data not shown).

Reduced Rab27A expression resulted in a significant decrease in

the invasive potential of MDA-MB-231 and MDA-MB-435

parental cells in vitro (data not shown). With Rab27A RNAi, the

expressions of VEGF, cathepsin D, cyclin D1, uPA, and MMP-9

in both mRNA and protein were down-regulated and those of p16

was up-regulated (Fig. 5A-C; P < 0.05 or P < 0.01). These results

further verified that Rab27A affects the invasive potential in

breast cancer cells in vitro by modulating the expression of

VEGF, cathepsin D, cyclin D1, uPA, MMP-9, and p16.

Reduced Rab27A expression by RNAi also eliminated the

concentrated perinuclear localization in MDA-MB-231 and

MDA-MB-435 cell lines (data not shown).

FIGURE 4. Western blotanalyses of the expression ofp16 and VEGF (A), cathepsin Dand cyclin D1 (B), and uPA andMMP-9 (C) in Rab27A trans-fectants and control cells.



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Rab27A Overexpression Promoted Orthotopic TumorGrowth and Spontaneous Pulmonary Metastasis In vivo

To examine the effects of Rab27A overexpression on tumor

growth and metastasis, we did the in vivo experiments using an

orthotopic xenograft tumor model in the athymic mice. The

results showed that Rab27A transfectants double the volume of

primary tumor compared with mock-transfected or parental

cells at the end of the in vivo assays (P < 0.05; data not shown).

We also found Rab27A up-regulation induced much more

hemorrhage and necrosis in primary tumors (data not shown).

To study metastatic potential in vivo , the lungs of mice in both

MDA-MB-231 and MDA-MB-435 groups were physically

examined at autopsy and then subjected to microscopic

examination for morphologic evidence of tumor cells by light

microscopy on H&E-stained paraffin sections. We found that

Rab27A overexpression dramatically increased the pulmo-

nary metastasis in both MDA-MB-435 and MDA-MB-231 cells

(P < 0.01; data not shown). Furthermore, we compared

Rab27A expression both in the mRNA and protein levels in

primary tumors and in pulmonary metastases. The results

showed that the latter seems to express much more Rab27A

than the former, especially in Rab27A-transfected groups

(Fig. 6; P < 0.05 or P < 0.01), which is another evidence

that a high level of Rab27A is necessary for acquisition of

invasive and metastatic potentials of breast cancer cells in vivo .

Additionally, similar effects of Rab27A on p16, VEGF, uPA,

FIGURE 5. Reduced Rab27Aexpression by RNAi results indown-regulation of VEGF, cathep-sin D, cyclin D1, uPA, and MMP-9, and up-regulation of p16. A toC. Western blot analyses of theexpression of p16 and VEGF (A),cathepsin D and cyclin D1 (B),and uPA and MMP-9 (C) inparental cells, negative controls,and microRNA-targeted parentalcells. Relative expressions (fold FSD) of genes are indicated belowthe Western blotsd.

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cathepsin D, cyclin D1, and MMP-9 expression noted in the

in vitro studies were also observed in in vivo studies (data not

shown). With Rab27A RNAi, the volume of primary tumor

and the number of pulmonary metastasis were accordingly

decreased (data not shown).

Rab27A Promoted the Secretion but not the Expressionof IGF-II in Breast Cancer Cells In vitro

Rab27A is not found as a transcription factor. We speculate

that Rab27A is involved in transporting or releasing proteins

that can regulate the expression of other genes. To gain insight

into the molecular basis, we first examined the expression of

IGF, which has the homologous structure to insulin in human

breast cancer cells. RT-PCR showed that the four breast cancer

cell lines coincidentally express insulin receptor, IGF-II, type I

and type II IGF receptors, but not IGF-I (Supplementary

Data G). We then investigated the relationship between the

expression of IGF-II and Rab27A and showed that IGF-II

mRNA was not changed by Rab27A up-regulation or down-

regulation in breast cancer cells (data not show). Finally, we

detected IGF-II protein in cells by Western blot and in

conditioned medium by ELISA at different time points.

Interestingly, IGF-II protein in conditioned medium of either

MDA-MB-231 or MDA-MB-435 was increased by Rab27A

overexpression after 12 hours, and these disparities reached

their peaks after 48 hours (P < 0.05 or P < 0.01; data not

shown). In contrast, IGF-II protein detected by Western blot in

both MDA-MB-231 and MDA-MB-435 cells was down-

regulated by Rab27A overexpression accordingly after

24 hours but not after 12 hours (Fig. 7A and B; P < 0.05 or

P < 0.01). These results suggested that Rab27A contributes

to secretion but not expression of IGF-II in breast cancer cells.

Whether IGF-II plays a key role in Rab27A pathway, we try

to block the IGF-II signaling pathway in MDA-MB-231 and

MDA-MB-435 cells and their Rab27A transfectants using IGF-

IR inhibitor NVP-ADW742 (Novartis Pharma AG). Western

blot showed that NVP-ADW742 efficiently decreased the

expression of total Akt and especially phosphorylated Akt,

which is the main molecule downstream of IGF-IR signaling

pathway (Fig. 7C). With inhibition of IGF-IR, MDA-MB-231

and MDA-MB-435 cells and their Rab27A transfectants

displayed decreased invasive potential in vitro (P < 0.05 or

P < 0.01; data not shown). We also found that the inhibition of

IGF-IR attenuated the effects of Rab27A on the expression of

MMP-9, VEGF, cyclin D1, cathepsin D, uPA, and p16 to the

levels in their parental cells (data not shown). We did coim-

munostaining of Rab27A and IGF-II by immunofluorescence

and confocal microscopy on MDA-MB-231 and MDA-MB-

435 and found that Rab27A colocalized with IGF-II within

these cells (data not shown).

Rab27A Interacts with Two of Its Effectors, SYTL4 andMelanophilin, in Breast Cancer Cells

Eleven effectors, which are divided into three groups, are

essential for Rab27A to function as a transporter (28). Our

results manifested that melanophilin and SYTL4, but not other

effectors, were expressed in the mRNA and protein levels in

breast cancer cells (Supplementary data H-K). Using immuno-

precipitation, we found that SYTL4 was coprecipitated with

Rab27A in all four breast cancer cell lines but with

melanophilin only in MCF-7 and MDA-MB-231 cells (data

not shown), which suggests that Rab27A participates in some

steps of IGF-II secretion in breast cancer cells by interacting

with the effectors of SYTL4 and/or melanophilin, which may

have the different function from the former. We presume that

the reason Rab27A bands detected by anti-FLAG and anti-

Rab27A antibodies double in Western blot is that whereas one

portion of Rab27A binds with melanophilin, the other portion

binds with SYTL4 in MCF-7 and MDA-MB-231 cell lines.

DiscussionAs the unique member of Rab protein family for its specific

implication in human genetic diseases (15), Rab27A was first

reported in 1997 (33). Because of Griscelli syndrome, the

functions of Rab27A have been investigated in detail in

melanocytes and CTLs, and recent studies showed that Rab27A

is expressed and responsible for transportation and secretion of

some cytokines in exocrine and endocrine cells (25-27).

Paracrine and autocrine cytokines were reported in breast

cancer cells (34). Although Rab27A contributes to transporta-

tion of melanosome in melanocytes (35, 36), the correlation

between Rab27A expression and the phenotypes of other

tumors still remains unknown.

In the present study, we showed that the expression of

Rab27A but not other several Rab proteins, Rab3A, Rab7,

Rab33A, and Rab37, which are functionally intimate with

FIGURE 6. Western blot revealed the differential expression of Rab27A protein (fold F SE) in primary tumor (P) and pulmonary metastases (M ) frommice bearing Rab27A transfectants, mock-transfected and their parental cells.



377



Rab27 (37), increased in human breast cancer cells as invasive

and metastatic potential increased, which indicated possible

involvement of Rab27A in invasive and metastatic phenotypes

of breast cancer cells. We found that overexpression of the

Rab27A protein resulted in a much higher percentage of cells in

the S phase. These findings were consistent with our in vivo

observations that up-regulation of Rab27A promoted the fast

growth of primary tumors. Furthermore, we also showed that

Rab27A expression positively regulated the invasive and

pulmonary metastatic potentials of MDA-MB-231 and MDA-

MB-435 cells in vitro and in vivo . All the pulmonary meta-

stases expressed much more Rab27A mRNA and protein than

the primary tumors, especially in the Rab27A transfectants,

indicating that Rab27A may have a distinct role in invasion and

metastasis of breast cancer cells.

Tolmachova et al. (24) observed that Rab27A is expressed in

a comprehensive range of classic exocrine secretory cells and it

is noticeable that the polarization of Rab27A is toward the

apical/luminal side of the cells, coinciding with the location of

secretory granules. In our results of immunofluorescence for

Rab27A, Rab27A seems to be targeted to diffuse in cytoplasm

in all three cell lines and in MDA-MB-231 and MDA-MB-435

cells particularly concentrated in the perinucleus.

The development and progression of breast cancer are also

accompanied by genetic alterations of multiple oncogenes and

tumor suppressor genes (32). In the present study, we showed

that Rab27A positively modulated the expression of VEGF,

cathepsin D, cyclin D1, uPA, and MMP-9 and negatively

modulated p16. These results were further supported by the

data obtained from RNAi targeting Rab27A in vitro .

Rab27A is not found as a transcriptional factor; thus, we

speculate that some proteins will be mediators to link Rab27A

and other genes that have effects on cellular behaviors such as

IGFs. Recently, Rab27Awas found to be involved in exocytosis

of insulin in pancreatic h cells (27, 38, 39). IGF, which has a

homologous structure to insulin, was thought to induce tumor

FIGURE 7. Rab27A is involved in IGF-II secretion. A and B. Western blot analyses of IGF-II protein affected by Rab27A up-regulation after 24, 36, and48 h in MDA-MB-231 and MDA-MB-435 breast cancer cell lines. Relative expressions (fold F SD) of IGF-II are indicated below the blots. C. Western blotanalysis to confirm blockage of the expression of total Akt and phosphorylated Akt downstream of IGF-II signaling pathway by the IGF-IR inhibitor NVP-ADW742 in MDA-MB-231, MDA-MB-435, and their Rab27A transfectants.

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division, invasion, and metastasis by regulating other gene

expression (40). Our results revealed that IGF-II, type I and

type II IGF receptors, and insulin receptor, but not IGF-I, were

coincidentally expressed in four breast cancer cell lines. IGF-II

released to cell culture supernatant was positively modulated

by Rab27A expression. Accordingly, Rab27A affected IGF-II

protein on the opposite side and did not change the mRNA

of IGF-II in these cells, suggesting that Rab27A is involved

in secretion but not expression of IGF-II. We also blocked

the IGF-II signaling pathway using IGF-IR inhibitor NVP-

ADW742, which was shown to have much more potent inhi-

bitory effect against IGF-IR than insulin receptor, HER2,

platelet-derived growth factor receptor, VEGF receptor 2, etc.

(41). It was clearly shown that NVP-ADW742 efficiently

decreased the expression of total Akt and especially phosphor-

ylated Akt, which is the main molecule downstream of IGF-IR

signaling pathway. With inhibition of IGF-IR, MDA-MB-231

and MDA-MB-435 cells and their Rab27A transfectants

displayed decreased invasive potential in vitro . At the same

time, we found colocalization of Rab27A and IGF-II in MDA-

MB-231 and MDA-MB-435 cells, which suggests that Rab27A

plays a key role in IGF-II pathway.

Cyclin D1 is overexpressed in up to 50% of primary breast

cancers, in part due to amplification of the cyclin D1 gene,

CCND1 (42). Hartmann et al. (43) also documented that IGF-II

positively modulates cyclin D1 transcription induced by Sonic

hedgehog in human medulloblastoma cells. In our experiment,

the expression of cyclin D1 and a novel cell cycle–related

tumor suppressor gene, p16 , which has been highlighted as

important in resistance to oncogenic transformation in mam-

mary epithelium (44), were changed by Rab27A up-regulation

or down-regulation in breast cancer cells.

The expression of MMP-9 was also evaluated using gelatin

zymography in human non–small cell lung cancer cells treated

by IGF-II (45). MMPs are a family of zinc-dependent neutral

endopeptidases that play a key role in degrading the extra-

cellular matrix and basement membrane in various cancers

and therefore promotes metastasis and angiogenesis (42, 46).

Increased numbers of blood vessels and hence increased

angiogenic activity are essential for the growth and metastasis

of cancer (47). Other than MMPs, VEGF and basic fibroblast

growth factor are the two most potent factors that can stimulate

angiogenesis and facilitate the malignant dissemination of

cancer cells (48). Kwon et al. (49) observed that IGF-II

mediated VEGF expression by the tyrosine kinase–Src–

extracellular signal-regulated kinase 1/2 pathway and the

phosphatidylinositol 3-kinase pathway in human keratinocytes.

In addition, previous studies have shown that up-regulated

expression of uPA, uPA receptor, and cathepsin D are

associated with increased tumor cell invasion and metastasis

in several malignancies, including breast cancer (50, 51). In our

study, we found that MMP-9, VEGF, uPA, and cathepsin D

expression, which can be up-regulated by IGF-II overexpres-

sion (52, 53), were positively modulated by Rab27A in MDA-

MB-231 and MDA-MB-435 cells; however, no significantly

different expression in TIMP-1, TIMP-2, MMP-1, MMP-2,

basic fibroblast growth factor, and uPA receptor was observed.

Taken together, these results indicate that Rab27A may be a

decisive regulator of secretion of IGF-II, which regulates the

expression of cyclin D1, VEGF, MMP-9, uPA, cathepsin D, and

p16; and then affects cell cycle distribution, and the invasive

and metastatic potentials in breast cancer cells.

Eleven putative Rab27A effectors that are classified into

three distinct groups based on their structures have been

reported in human and mice (28). The first group consists of the

members of the synaptotagmin-like protein (Slp) family (Slp1/

JFC1, Slp2-a, Slp3-a, Slp4/granuphilin, and Slp5) and rabphi-

lin, all of which contain an NH2-terminal Rab27A-binding

domain (Slp homology domain) and COOH-terminal tandem

C2 domains, which are putative phospholipid binding sites

(26, 36, 54-56). The second group of Rab27A-binding proteins

that contain an NH2-terminal Rab27A-binding domain, the

same as the Slp members, but lack tandem C2 domains consists

of the members of the Slac2 family (Slac2-a/melanophilin,

Slac2-b, and Slac2-c/MyRIP) and Noc2 (36, 54-57). The final

group consists of only Munc13-4, which contains a novel

Rab27A-binding domain distinct from the Slp homology

domain and is a putative priming factor for exocytosis by cer-

tain immune cells (58, 59). Expression of several Rab27A

effectors in one secreting cell type has also been reported (25,

26, 36). In our study, we detected that melanophilin and SYTL4

are expressed in four breast cancer cell lines. However, in the

subsequent immunocoprecipitation, we confirmed that SYTL4,

which was reported to be related to the expression of ER in

breast cancer tissues (60), binds to Rab27A in all four breast

cancer cell lines but binds to melanophilin only in MCF-7 and

MDA-MB-231cells. These results revealed the complexity

of the effectors of Rab27A in vivo , which may bind to different

Rab proteins in different cell lines.

Collectively, our results indicate that Rab27A plays a

positive regulatory role in invasion and metastasis of human

breast cancer cells. Rab27A has effects on the invasive and

metastatic potentials in breast cancer cells by modulating the

secretion of IGF-II, which regulates the expression of p16,

VEGF, uPA, cathepsin D, cyclin D1, and MMP-9. In this pro-

cess, Rab27A effectors, SYTL4, and melanophilin may be

important participants by binding to Rab27A. These findings

may lead to a new therapeutic strategy against breast cancer.

Materials and MethodsCell Lines and Animals

Human breast cancer cell lines MCF-7, MDA-MB-231,

MDA-MB-435, and MDA-MB-435HM were used in this study.

The former three cell lines were obtained from the American

Type Culture Collection and grown in complete growth medium

as recommended by the manufacturer. The last one was

established from MDA-MB-435 using an in vivo stepwise

selection scheme in our laboratory (61) and grown in the same

medium to its parental cell line MDA-MB-435. All the cultured

cells were maintained in a humidified 5% CO2 atmosphere at

37jC. Female BALB/c-nu/nu nude mice, 4 to 6 wk old, were

obtained from Shanghai Institute of Materia Medica, Chinese

Academy of Sciences (Shanghai, China), and housed in laminar-

flow cabinets under specific pathogen-free conditions with food

and water ad libitum. All experiments on mice were conducted in

accordance with the guidelines of NIH for Care and Use of

Laboratory Animals. The study protocol was also approved by

the Shanghai Medical Experimental Animal Care Committee.



379



RNA Isolation and Semiquantitative Real-time RT-PCRTotal RNA was isolated from cultured cells or tissues using

the Trizol reagent (Invitrogen) according to the manufacturer’s

instruction. The semiquantitative real-time RT-PCR using

SYBR green I to compare the relative expression of specific

gene mRNA was done as described previously (62).

Immunoblotting and CoimmunoprecipitationThe primary antibody for IGF-II was obtained from Upstate.

The primary antibodies for SYTL4 and melanophilin were

produced as described previously (63, 64). Other primary

antibodies were obtained from Santa Cruz Biotechnology

(65, 66). For immunoblotting, proteins were resolved by

SDS-PAGE and transferred to nitrocellulose membranes

(Whatman Schleicher and Schuell). Immunoblots were devel-

oped in Western Lightning Chemiluminescence Reagent Plus

(Perkin-Elmer Life and Analytical Sciences) and exposed to

X-ray films (Eastman Kodak). When necessary, blots were

stripped and reblotted with other antibodies. The level of each

protein was normalized versus the corresponding protein level

for internal a-tubulin. For immunoprecipitation, FLAG-tagged

Rab27A from 1 � 107 cells were immunoprecipitated with anti-

FLAG M2 beads (Sigma) and were eluted with 0.8 mg FLAG

peptide per milliliter in a total volume of 30 AL (67).

Construct Human Rab27A Expression Vector and StableTransfection

The entire open reading frame of the human Rab27A gene

(GeneID 5873) was amplified from 293T cells by RT-PCR

using the gene-specific primers (the upstream primer, 5¶-GCT-AAGCTTGGTGAACTACTGAGTTCTTC-3¶, with an added

HindIII site underlined; the downstream primer, 5¶-GACG-AATTCTGTCGCTTACTTGACTTCTC-3¶, with an added

EcoRI site underlined), and then subcloned in-frame into the

multiple cloning site of pcDNA3.1(+) vector. MDA-MB-231

and MDA-MB-435 cells were transfected by Lipofectamine

2000 transfection reagent (Invitrogen) according to the

manufacturer’s instruction. After selection in the presence of

1,000 Ag/mL Geneticin (G418 sulfate; Invitrogen) for 4 wk,

the pcDNA3.1(+)-Rab27A–positive and the empty vector–

positive colonies named as 231/Rab27A or 435/Rab27A and

231/vector or 435/vector were identified by RT-PCR and

Western blotting analysis as described above. To express

FLAG-tagged proteins, Rab27A cDNAs were properly inserted

into pUHD30F.

In vitro Invasion AssayIn vitro invasion assays were conducted with a Matrigel

invasion chamber (BD Labware) as previously described with

some modifications (68). Each well insert was coated with

100 AL of 1:3 dilution of Matrigel in serum-free culture

medium. Then, 750 AL of medium containing 10% fetal bovine

serum were added to the lower chamber as a chemoattractant,

and 1 � 105 cells in 500 AL of serum-free medium were added

to the top of the Matrigel layer. The cells were incubated at

37jC for 6, 12, 24, or 48 h, respectively. The cell suspension

was aspirated, and excess Matrigel was removed from the filter

using a cotton swab. Invasion ability was assessed by counting

the cells that had traveled across the filter and attached to the

bottom side of the filter. Then, the filters were fixed in 10%

formalin and stained with H&E. The cells that had invaded

through the Matrigel and reached the lower surface of the filter

were counted under a light microscope at a magnification of�200.

Flow CytometryCells at the exponential growth phase were harvested and

single-cell suspensions containing at least 1 � 106 cells were

made. The cells were treated following the standardized

protocol, and cell cycle analyses were done by flow cytometry

as described previously (69).

Immunofluorescence and Confocal MicroscopyGenerally, cells grown on glass coverslips were fixed in

cold methanol for 10 min before immunofluorescence

staining. Proper antibody or combinations were chosen for

staining. Nuclear DNA was stained with 4,6-diamidino-2-

phenylindole. Fluorescence images were captured with a cold

charge-coupled device (SPOT II; Diagnostic Instruments)

on an Olympus BX51 microscope or with a Leica TCS SP2

laser confocal microscope. For confocal microscopy with

fixed cells, optical sections were scanned at 0.1- to 0.2-Amintervals when needed. Z-stack images were then formed by

maximal projection.

RNA interferenceTo further show the role of Rab27A in progression of breast

cancer cells, we used the microRNA interference technique to

down-regulate Rab27A gene expression. The two pre-micro-

RNA sequences that targeted the human Rab27A gene from the

516-bp sequence (5¶-TGCTGTCCAGAAGCATCTCAATTGC-TGTTTTGGCCACTGACTGACAGCAATTGATGCTT-

CTGGA-3¶) and from the 611-bp sequence (5¶-TGCTGTTA-ACTGATCCGTAGAGGCATGTTTTGGCCACTGACTGA-

CATGCCTCTGGATCAGTTAA-3¶) were designed by the

Invitrogen RNAi Designer and ligated with pcDNA 6.2-GW/

EmGFP-miR-lacZ (V49350-00, Invitrogen). We transfected the

recombinant plasmids into 293T cells, which were expressed

exogenous Rab27A tagged with FLAG to verify their function

on suppression to the exogenous and endogenous expression

of Rab27A. Then, we transfected the microRNAs into MDA-

MB-231 and MDA-MB-435 cells, and their parental cells and

pcDNA 6.2-GW/EmGFP-miR-lacZ vector–only transfectants

were used as controls.

Tumorigenicity and Metastasis Assays in Nude MiceThe tumorigenicity and spontaneous metastatic potential of

the cell lines in the same orthotopic model described above

were determined by injection into the mammary fat pad as

described previously (70). The rate of primary tumor growth

was determined by plotting the means of two orthogonal

diameters of the tumors, measured twice per week. Mice were

sacrificed and autopsied at 4 wk of postinoculation for MDA-

MB-231 group and 8 wk for MDA-MB-435 group. To confirm

the presence of lung metastases, sections were cut at 50-Amintervals and H&E staining was done. In this study, we count

Wang et al.


380



metastasis in the whole lung. Two independent pathologists

calculated the number of metastasis.

Measurement of IGF-II by ELISAThe medium without serum was collected after 6, 12, 24, 36,

and 48 h for IGF-II ELISA determinations as described below.

The cells were taken through three freeze-thaw cycles and

centrifuged; the supernatant was collected for determination of

protein concentration and the cells were dissolved according to

the protocol of RT-PCR or Western blot. IGF-II levels in the cell

culture medium were measured using a Quantikine kit from

R&D Diagnostics using the procedure provided by the supplier.

Human recombinant IGF-II included in the kit was used to

construct a standard curve and to obtain the absolute values of

IGF-II protein content. The values were then normalized to the

total protein concentration in each dish.

Statistical AnalysisANOVA and Student’s t test were used to determine the

statistical significance of differences between experimental

groups, with P < 0.05 as the statistically significant level.

Graphs were created with Excel software (Microsoft Office for

Windows 2000).

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2008;6:372-382. Mol Cancer Res Jin-Song Wang, Fu-Bin Wang, Qiang-Ge Zhang, et al. by Secretion of Insulin-Like Growth Factor-IICells Promoting Invasiveness and the Metastasis Potential

Gene by Breast CancerRab27AEnhanced Expression of

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