Small interfering RNA-mediated CXCR1 or CXCR2 knock-down inhibits melanoma tumor growth and invasion

Small interfering RNA-mediated CXCR1 or CXCR2knock-down inhibits melanoma tumor growth and invasion

Seema Singh, Anguraj Sadanandam, Michelle L. Varney, Kalyan C. Nannuru and Rakesh K. Singh

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE

CXCR1 and CXCR2 are receptors for CXCL-8 and are differentially expressed on melanoma and endothelial cells. In this study,

we determined the functional role of these receptors in melanoma progression. We stably knock-down the expression of

CXCR1 and/or CXCR2 in A375-SM (SM; high metastatic) human melanoma cells by short-hairpin RNA transfection. Cell

proliferation, migration, invasion, ERK phosphorlyation and cytoskeletal rearrangements were carried out in vitro. In vivo

growth was evaluated using murine subcutaneous xenograft model. Our data demonstrate that knock-down of CXCR1 and/or

CXCR2 expression, inhibited melanoma cell proliferation, survival, migration and invasive potential in vitro. Moreover, we also

observed inhibition of ERK phosphorylation and cytoskeltal rearrangement in SM-shCXCR1, SM-shCXCR2 and SM-shCXCR1/2

cells. Furthermore, when SM-shCXCR1 or SM-shCXCR2 cells implanted in nude mice, tumor growth, proliferation and

microvessel density was significantly inhibited as compared to SM-control cells. In addition, we observed a significant

increase in melanoma cell apoptosis in SM-shCXCR1 and SM-shCXCR2 tumors compared to SM-control tumors. Together,

these data demonstrate that CXCR1 and CXCR2 expression play a critical role in human melanoma tumor progression

and, functional blockade of CXCR1 and CXCR2 could be potentially used for future therapeutic intervention in malignant

melanoma.

Human melanoma, is the fifth most common cancer in theUnited States and there will be 8,420 deaths from the diseasein 2008.1 Metastatic melanoma is difficult to treat due to lackof effective treatment modalities. Therefore, improvement inthe therapy of metastatic melanoma now depends on improv-ing our understanding of the complex molecular mechanismsgoverning the progression and aggressiveness of the disease.

Over the last 2 decades, it has been increasingly recog-nized that chemokines play an important role in tumor biol-ogy.2–5 Chemokines bind particular G-protein-coupled recep-tors and are known to mediate leukocyte trafficking,inflammation, angiogenesis and tumorigenesis.6–8 Interest-ingly, CXCL-8, a member of the CXC chemokine family hasbeen reported to induce migration, stimulate angiogenesisand promote tumor cell growth in melanoma and othermalignancies.9–18 The biological effects of CXCL-8 are medi-ated through 2 high affinity receptors: type A CXCL-8 recep-

tor (IL-8RA/IL-8RI or CXCR1) and type B CXCL-8 receptor(IL-8RB/IL-8RII or CXCR2).19,20 Although CXCR2 bindswith high affinity to CXCL-8 and other CXC chemokinessuch as CXCL-6, CXCL-5, CXCL-7 and CXCL-1, CXCR1 isless promiscuous and binds only to CXCL-8.12,21,22 CXCR1and CXCR2 share a high degree of sequence similarity(75.8% in amino acid sequence), but differ within the extrac-ellular and intracellular loops and the NH2- and COOH-ter-minal domains.23 We have previously shown that CXCR1and CXCR2 are differentially expressed on melanoma andendothelial cells.9,24,25 Several studies have implicated CXCR1and CXCR2 as important players in tumor progression.3,26,27

Our previous studies demonstrated that CXCR1 and CXCR2are expressed on melanoma and endothelial cells.9,24,25 Weand others have also shown that neutralization of CXCR1 orCXCR2 using small molecule antagonists affects cell prolifer-ation and migration, indicating the involvement of thesereceptors in altered cellular responses.25,28,29 Thus, involve-ment of CXCR1 and CXCR2 and their ligand CXCL-8 in dif-ferent cell process makes this ligand-receptors axis of particu-lar interest in investigating its functional role in melanomaprogression.

To examine more directly the role of CXCR1 and CXCR2in melanoma progression, we employed a gene knock-downstrategy to inhibit CXCR1 or CXCR2 expression to modulatecellular phenotypes associated with melanoma growth andinvasion. Our results clearly show that downregulation ofCXCR1 or CXCR2 inhibited melanoma tumor growth byincreasing apoptosis and decreasing cell proliferation, migra-tion and invasion.

Key words: chemokines, CXCR1, CXCR2, tumor growth, melanoma

Grant sponsor: National Cancer Institute; Grant numbers:

CA72781, P30CA036727 (Cancer Center Support Grant); Grant

sponsors: National Institutes of Health, Nebraska Research

Initiative Cancer Glycobiology Program

DOI: 10.1002/ijc.24714

History: Received 8 Jan 2009; Accepted 23 Jun 2009; Online 7 Jul

2009

Correspondence to: Rakesh K. Singh, Department of Pathology and

Microbiology, University of Nebraska Medical Center, 985900

Nebraska Medical Center, Omaha, NE 68198-5900, USA,

Fax: þ402-559-5900, E-mail: [email protected]

Can

cerCellBiology

Int. J. Cancer: 126, 328–336 (2010) VC 2009 UICC

International Journal of Cancer

IJC

Material and MethodsCell culture

The human melanoma cell line A375-SM (SM; highly meta-static) was maintained in culture as an adherent monolayerin Dulbecco’s Modified Eagle Medium (MediaTech, Herndon,VA). Culture medium was supplemented with 5% fetal bo-vine serum, 1% L-glutamine (MediaTech), 1% vitamin solu-tion (MediaTech) and gentamycin (Invitrogen, Carlsbad,CA). Cells were grown at 37�C with 5% CO2 in humidifiedatmosphere.

Generation of shRNA-expression plasmids

Silencing of gene expression was achieved using short-hairpinRNA (shRNA) technology. One shRNAs targeting CXCR1(1sh-50CCC GCG TCA CTT GGT CAA GTT TGT), one tar-geting CXCR2 (2sh-CCC CAA TAC AGC AAA CTG GCGGAT), one targeting both CXCR1/2 (1/2sh-CCC CTT CTATAG TGG CAT CCT GCT) and scrambled (control) weregenerated using a CXCR1 or CXCR2 specific sequence withBglII and HindIII overhangs to allow for cloning into thepSuper.neo vector (Oligoengine, Seattle, WA). A375-SM cellswere stably transfected with pSuper.neo/scrambled (SM-con-trol), pSuper.neo/shCXCR1 (SM-shCXCR1), pSuper.neo/shCXCR2 (SM-shCXCR2) or pSuper.neo/shCXCR1/2 (SM-shCXCR1/2) plasmid using Lipofectamine reagent (Invitro-gen) following the manufacturer’s protocol. G418-sulphate-resistant colonies were isolated and maintained in mediumsupplemented with 1,000 lg/ml of G418-sulphate (Invitro-gen). To avoid clone specific effects, pooled cultures wereused for all experiments.

Gene expression analysis

Analysis of gene expression was performed using quantitativeRT-PCR as described.24 Briefly, cDNA was synthesized from5 lg total RNA using SuperScriptTM II Reverse Transcriptase(Invitrogen) and oligo(dT) primer. Two micro liter of firststrand cDNA (1:10 dilution) was amplified. The followingprimer sequences were used: CXCR1, 50-TGG GAA ATGACA CAG CAA AA-30 (forward) and 30,AGT GTA CGCAGG GTG AAT CC-30 (reverse), CXCR2, 50-ACT TTT CCGAAG GAC CGT CT-30 (forward) and 50-GTA ACA GCATCC GCC AGT TT-30 (reverse). For internal control, glycer-aldehydes 3-phosphate dehydrogenase (GAPDH), 50-CGCATT TGG TCG TAT TGG G-30 (forward), and 50-TGA TTTTGG AGG GAT CTC GC-30 (reverse) was used. Amplifiedproducts were resolved through a 1.5% agarose gel containingethidium bromide and analyzed using an Alpha Imager geldocumentation system (AlphaInnotech, San Leandro, CA).

Tumor growth analysis

Female athymic nude (6- to 8-week-old) were purchasedfrom the National Cancer Institute and maintained underspecific pathogen-free conditions. All procedures performedwere in accordance with institutional guidelines and

approved by the University of Nebraska Medical CenterInstitutional Animal Care and Use Committee. To specificallydetermine the role of CXCR1 or CXCR2 in tumor develop-ment SM-control, SM-shCXCR1 or SM-shCXCR2 cells (1 �106 in 0.1 ml of HBSS) were injected subcutaneously (s.c.)into the right flank region of nude mice. Tumor growth wasmeasured twice weekly and mice were sacrificed on Day 45after tumor implantation. Tumor volume was calculatedusing the formula p/6 � (smaller diameter)2 � (larger diam-eter). Tumors recovered from mice were fixed in zinc fixative,embedded in paraffin and processed for histopathologicalevaluation and immunohistochemistry.

Immunohistochemistry

Immunohistochemical analysis was performed as previouslydescribed.30 In brief, 6-lm-thick tumor sections were depar-affinized by EZ-Dewax (Biogenex, SanRoman, CA) andblocked for 30 min. Tumor sections were incubated overnightin a humid chamber with the following primary antibodies:anti-PCNA (1:40; Santa Cruz Biotechnology, Santa Cruz, CA)or mouse biotinylated anti-GS-IB4 (isolectin from Griffoniasimplicifolia; 1:50; Vector Laboratories, Burlingame, CA). Forconfirming the downregulation of CXCR1 and/or CXCR2,cells (10,000 cells) were seeded overnight on coverslips, fixedin ice cold 4% formaldehyde, blocked and incubated with fol-lowing primary antibodies: mouse monoclonal anti-CXCR1(1:100; R&D systems, Minneapolis, MN) and mouse mono-clonal anti-CXCR2 (1:50; R&D systems). Corresponding bio-tinylated secondary antibody was used (except for GS-IB4) atroom temperature. Immunoreactivity was detected using theABC Elite kit and DAB substrate (Vector Laboratories) perthe manufacturer’s instructions. A reddish brown precipitatein the cytoplasm indicated a positive reaction. Negative con-trols had all reagents included except the primary antibody.The number of microvessels was quantitated microscopicallywith a 5 � 5 reticle grid (Klarmann Rulings, Litchfield, NH)using 400� objective (250-lm total area).

Apoptotic cells in tumor samples were identified by termi-nal deoxyribonucleotidyl transferase dUTP nick end fluores-cein labeling (TUNEL) assay according to the manufacturer’sinstructions (In situ Cell Death Detection Kit, Fluorescein;Roche, Indianapolis, IN). The number of apoptotic cells wasevaluated by counting the positive (brown-stained) cells in 10arbitrarily selected fields at 200� magnification in a double-blinded manner and expressed as average number of cells perfield view.

Cell proliferation and apoptosis assay

Cells were seeded in 96-well plates at low density (1,000cells/well). Following overnight adherence, cells were incu-bated with media alone or medium containing different se-rum concentration, with or without CXCL-8 (10 ng/ml) for72 hr. Cell proliferation was determined by MTT assay.31,32

Apoptosis was measured using the CaspACE FITC-VAD-FMK in situ marker (Promega, Madison, WI). Cells

Can

cerCellBiology

Singh et al. 329


(1 � 106) were grown in serum-free medium for 24 hr. Apo-ptosis was detected by staining the cells with CaspACEFITC-VAD-FMK solution in PBS for 30 min at 37�C. Thebound marker was localized by fluorescent detection using aconfocal microscope.

Cell motility and invasion assay

To investigate the effect of silencing CXCR1 or CXCR2expression on cell migration, cells (1 � 106 cells/well) inserum-free media were plated in the top chamber of non-coated polyethylene terephthalate membranes (6-well insert;8-lm pore size; Becton Dickinson, Franklin Lakes, NJ). Forinvasion, cells (10,000 cells/wells) were plated onto Matrigel-coated transwell chambers (24-well insert; 8-lm pore size;Corning Costar Corp., Cambridge, MA) in serum-free media.The bottom chamber contained 1.0-ml serum-free mediawith or without CXCL-8 (10 ng/ml). The cells were incu-bated for 24 hr at 37�C and cells that did not pass throughthe membrane pores were removed. Migrated cells werestained using Hema 3 kit (Fisher Scientific Company L.L.C.,Kalamazoo, MI) as per the manufacturer’s instructions. Cellswere counted in 10 random fields (200�) and expressed asthe average number of cells per field of view. The data arerepresented as the average of 3 independent experiments.

F-actin immunostaining

Cells were grown at low density (10,000 cells) overnight oncoverslips, fixed in ice cold 4% formaldehyde, permeabilizedin 0.3% Triton X-100 and stained with Texas Red-phalloidin(Molecular Probes, Eugene, OR) for 30 min at room temper-ature. Cells were further washed with PBS-T (PBS containing0.1% Tween 20) and mounted with antifade vectashieldmounting medium (Vector Laboratories). The stained cellswere analyzed using a confocal microscope (UNMC corefacility).

Western blot analysis

Cells were processed for protein extraction and Western blot-ting using standard procedures. Briefly, the cells were washedtwice with PBS and scraped in Triton X-100 buffer [1% Tri-ton X-100, 50 mmol/l TBS (pH 7.4), 10 mmol/l EDTA withprotease inhibitors (Roche Diagnostics, Mannheim, Germany)and phosphatase inhibitors (5 mM NaF and 5 mM Na3VO4;Sigma Chemicals, St. Louis, MO)]. Cell lysates were passedthrough the needle syringe to facilitate the disruption of thecell membranes and were centrifuged at 14,000 rpm for 20min at 4�C, and supernatant were collected. The proteins (50lg) were resolved by electrophoresis on 8% SDS-PAGE.Resolved proteins were transferred onto polyvinylidene diflu-oride membrane and subjected to standard immunodetectionprocedure using specific antibodies: anti-CXCR1 and anti-CXCR2 (1:50, mouse monoclonal, R&D systems); anti-pERK1/2, anti-ERK1/2 and anti-GAPDH (1:1,000, rabbit pol-yclonal, Cell Signaling Technology). Secondary antibodiesconsisted of horseradish peroxidase-conjugated (Santa Cruz

Biotechnology) and were used at 1:2,000 dilution. All theblots were processed with ECL Plus Western blotting detec-tion kit (GE Healthcare, Piscataway, NJ), and the signal wasdetected by a Typhoon 9410 Variable Mode Imager.

Statistical analysis

Differences between the groups were compared using theunpaired 2-tailed t-test using SPSS software (SPSS, Chicago,IL). In vivo analysis was done using the Mann–WhitneyU-test. All the values were expressed as mean 6 SEM. A pvalue of equal or less than 0.05 was considered statisticallysignificant.

ResultsKnock-down of CXCR1 and CXCR2 resulted in reduced

tumor growth and angiogenesis

To knock-down CXCR1 or CXCR2 expression, plasmids con-taining shRNA sequences were transfected into SM mela-noma cells (SM-shCXCR1, SM-shCXCR2 or SM-shCXCR1/2)along with SM-control. Expression of CXCR1 and CXCR2was determined in pooled sublines by RT-PCR (Fig. 1a) andimmunocytochemical analyses (Fig. 1b). To specifically deter-mine the role of CXCR1 or CXCR2 in tumor development,SM-shCXCR1, SM-shCXCR2 or SM-control cells wereinjected into the right flank region of nude mice (n ¼6/group). Seven weeks post-tumor injection, tumor volumeswere measured. Subcutaneous tumors formed from SM-shCXCR1 (2.1-fold) or SM-shCXCR2 (2.2-fold) cells weresignificantly smaller compared with the SM-control cells(SM-shCXCR1, 407.1 6 133.7 mm3; SM-shCXCR2, 393.2 699.0 mm3; SM-control, 864.6 6 78.7 mm3 Fig. 2a). All of themice injected with SM-shCXCR1, SM-shCXCR2 or SM-con-trol developed tumors.

One possible mechanism for decreased tumor growth isattenuated neovascularization. To determine whether down-regulation of CXCR1 and CXCR2 could potentially disruptthe neovascularization, we examined vascularity in tumors.Staining on SM-shCXCR1 and SM-shCXCR2 tumor sectionsshowed a 3.1- to 3.3-fold decrease in the number of bloodvessels as compared to control tumors (Fig. 2b).

Inhibition of CXCR1 and CXCR2 expression decreased the

proliferation and survival both in vitro and in vivo

Chemokines are potent regulators of proliferation and sur-vival. Hence, it was of interest to address whether knock-down of CXCR1 or CXCR2 may affect melanoma cell prolif-eration. The average number of PCNA-positive cells by im-munohistochemical staining showed a 2.2-fold (p < 0.05)decrease for SM-shCXCR1 and 2.3-fold (p < 0.05) decreasefor SM-shCXCR2 tumors compared with SM-control tumors(Fig. 3a). Additionally, SM-shCXCR1 and SM-shCXCR2tumors had significantly increased numbers (2.1- to 2.2-fold,p < 0.05) of TUNEL-positive cells as compared with SM-control tumors (Fig. 3b).

Can

cerCellBiology

330 CXCL-8 receptors in human melanoma


Figure 1. CXCR1 and/or CXCR2 knock-down in A375SM melanoma cells. Cells were stably transfected with SM-control, SM-shCXCR1, SM-

shCXCR2 or SM-shCXCR1/2 plasmid construct containing scrambled, CXCR1, CXCR2 or CXCR1/2 shRNA. (a) RT-PCR shows decreased

expression of CXCR1 or CXCR2 mRNA in pooled sublines. GAPDH was used as a loading control. (b) Immunocytochemistry showing

decreased expression of CXCR1 or CXCR2 in SM-shCXCR1, SM-shCXCR2 and SM-shCXCR1/2 cells. Pictures are representative of at least 3

experiments. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 2. Knock-down of CXCR1 or CXCR2 reduces melanoma tumor growth and decreases microvessel density in vivo. Melanoma cells (SM-

shCXCR1, SM-shCXCR2 or SM-control) were subcutaneously (s.c.) injected into the right flank region of nude mice. Tumor volume was

measured twice weekly with a caliper and was calculated by using the formula p/6 � (smaller diameter)2 � (larger diameter). (a) Tumor

volume from Day 0 to Day 45 (n ¼ 6). Growth of SM-shCXCR1 and SM-shCXCR2 were significantly reduced (p < 0.05) compared with SM-

control group. (b) Immunohistochemical staining for microvessels with anti-GS-IB4. The representative pictures are shown at 200�. (c) The

values are average number of microvessels 6 SEM. Microvessel density was quantitated microscopically with a 5 � 5 reticle grid at 400�magnification. The values are mean 6 standard error of mean (SEM). *Significantly different from SM-control (p < 0.05). [Color figure can

be viewed in the online issue, which is available at www.interscience.wiley.com.]


Similarly, in an in vitro assay (in the absence of serum),the presence of CXCL-8 did not enhance cell proliferation inSM-shCXCR1, SM-shCXCR2 or SM-shCXCR1/2 melanomacells as compared to SM-control cells. SM-shCXCR1, SM-shCXCR2 and SM-shCXCR1/2 cells showed a 28–35%decrease in proliferation (Fig. 3c). The in vitro apoptosisassay (Caspase FITC) of SM-shCXCR1, SM-shCXCR2 andSM-shCXCR1/2 cells showed an increase of �1.3- to 1.7-folds (Fig. 3d), which supports the above findings. Taken to-gether, our data suggest a role for CXCR1 and CXCR2 inmelanoma cell proliferation and survival.

Reduced CXCR1 and CXCR2 expression also inhibited

migration and invasiveness of SM cells

Overall growth of the tumor is determined by the interactionof tumor cells with the host microenvironment. Our recentstudy has suggested that CXCR1 and CXCR2 signaling notonly helps in the migration of melanoma cells and angiogen-esis, but may also help in the motility and invasion of mela-noma cells (unpublished). Therefore, we investigated whetherdownregulation of CXCR1 or CXCR2 would affect tumor cellchemotaxis and invasion. Our data demonstrated a significant(p < 0.05) inhibition in CXCL-8-induced chemotaxis of SM-shCXCR1 (4.0-fold), SM-shCXCR2 (5.0-fold) and SM-shCXCR1/2 (5.0-fold) cells as compared to SM-control cells(Figs. 4a-upper panel and 4b-left panel). Similarly, the num-

ber of invading cells decreased by 1.8- to 2.0-fold as com-pared with control cells in an in vitro Matrigel invasion assay(Figs. 4a-lower panel and 4b-right panel).

Knock-down of CXCR1 and CXCR2 affects CXCL-8-induced

actin reorganization and phosphorylation of ERK1/2 in

melanoma cells

Actin polymerization stimulates motility of cells, therefore weexamined the effect of downregulation of CXCR1 or CXCR2on actin polymerization of melanoma cells. The staining ofSM-shCXCR1, SM-shCXCR2 and SM-shCXCR1/2 cellsshowed a reduction in lamellipodial structures as comparedto control cells (Fig. 5b). Thus, the decrease in actin polymer-ization reduced the motility and in turn, the invasiveness ofthe melanoma cells. CXCR1 or CXCR2-associated changes ingene expression indicate their important role in cell signaling.The ERK1/2 MAPK pathway is constitutively activated inmost melanomas, and plays a major role in mediating theirsurvival and proliferation.33 Therefore, we investigated theeffect of downregulation of CXCR1 or CXCR2 on ERK1/2MAP kinase phosphorylation. Figure 5a showed that the lev-els of ERK1/2 phosphorylation were reduced in SM-shCXCR1, SM-shCXCR2 and SM-shCXCR1/2 cells as com-pared to SM-control cells. Thus, suggesting the involvementof CXCR1 and CXCR2 mediated signaling in melanoma cells.

Figure 3. Reduced melanoma cell proliferation and apoptosis both in vivo and in vitro. Immunohistochemical staining of PCNA (a) and

TUNEL (b) in the tumor section. Proliferating and apoptotic cells were counted in 10 arbitrarily selected fields (200�) in a double-blinded

manner. (c) In vitro cellular proliferation was determined at 72 hr by MTT assay (left panel). The values are mean percent inhibition of

proliferation 6 SEM. (d) The frequency of CaspACE-positive cells was determined by counting in 10 fields (200�) for each treatment. The

values are expressed as average number of cells per field view (right panel). *Significantly different from SM-controls (p < 0.05).

Can

cerCellBiology



DiscussionDespite extensive studies on melanoma, the molecular mech-anisms of melanoma tumor growth are still not fully under-stood. Numerous studies have shown that chemokines andchemokine receptors exert a variety of biological functions,including regulation of proliferation, angiogenesis and sur-vival.34,35 In addition, functional chemokine receptors possi-bly play pivotal role in tumor growth and metastasis of vari-ous cancer. Therefore, the expression of chemokines andchemokine receptors give an advantage to tumor cells and

may grant them the enhanced ability to proliferate and dis-seminate. Earlier reports from our group suggest that modu-lation of CXCL-8 expression in melanoma cells influence thetumor growth and metastasis.32,36 Modulation of chemokinereceptors on tumor cells has not been extensivelyinvestigated.

Prompted by initial observations, an important questionaddressed in this study deals with the functional significanceof CXCR1 or CXCR2 expression in turn melanoma tumorgrowth. All the mice injected subcutaneously with

Figure 4. Downregulation of CXCR1 and/or CXCR2 reduces cell motility and invasion. (a) Cells were seeded on noncoated or Matrigel-coated

membranes for motility (upper panel) and invasion (lower panel) assays overnight. Migrated cells were stained and photographed at 200�magnification. (b) Migrated (left panel) and invaded (right panel) cells were counted in 10 random fields (200�) and expressed as the

average number of cells per field of view 6 SEM. This is a representative of 3 experiments done in triplicate. *Significantly different from

SM-control cells (p < 0.05).

Figure 5. Effect of CXCR1 and/or CXCR2 downregulation on actin reorganization and ERK1/2 phosphorylation. (a) Cell lysates (50 ug) were

fractionated by SDS-PAGE and subjected to Western blotting using pERK1/2, ERK1/2 and GAPDH antibody. (b) Cells were grown on

coverslips and stained with Texas Red phalloidin. Decreased lamellipodial structures were observed in SM-shCXCR1, SM-shCXCR2 and SM-

shCXCR1/2 cells compared with the SM-control cells.Can

cerCellBiology

Singh et al. 333


SM-shCXCR1 or SM-shCXCR2 cells developed significantlysmaller tumors as compared to vector control, demonstratingsignificance of CXCR1 or CXCR2-dependent signaling. Ear-lier reports demonstrate that blockade of CXCL-8 activity byneutralizing anti-CXCL-8 antibodies significantly inhibitedthe growth and metastasis of human melanoma cells in nudemice by suppressing angiogenesis and invasion.37 Our recentdata suggest that inhibition of CXCR1 and CXCR2 signalingusing small molecule antagonists inhibited melanoma growthand invasion.38 In support, our present study showed thatknock-down of both the receptors regulated primary tumorgrowth by decreasing cell proliferation, angiogenesis and sur-vival. In addition, we also observed decreased chemotaxisand invasion of CXCR1 or CXCR2 knock-down SM cells.Hence, the results of our study suggest an important associa-tion between the expression of the CXCR1 or CXCR2 recep-tors and melanoma growth and invasion.

Increased cell proliferation and decreased cell death play apivotal role in tumor progression. Any decreased tumorgrowth of SM-shCXCR1 and SM-shCXCR2 cells may be dueto their decreased response to CXCL-8-induced cell prolifera-tion, motility, survival or invasion. The present in vitro datareveal that downregulation of CXCR1 or CXCR2 results in asignificant decrease in proliferation, especially under serumstarvation. Our results are in agreement with our previousobservation that neutralization of CXCR1 and CXCR2 inhib-ited the proliferation of melanoma cells expressing CXCL-8,25

thus further strengthening that CXCL-8 functions in an auto-crine manner by binding to CXCR1 and/or CXCR2.

Because proliferation is important for tumor growth andmetastasis, decreased proliferation could influence the apopto-sis, migration and invasion of CXCR1 and CXCR2 downregu-lated melanoma cells. Interestingly, the numbers of cellsmigrated and invaded were significantly less than control cells.Cell motility and invasiveness are associated with actin fila-ments that are organized on the lamellipodia.39 In addition, ithas also been shown that CXCR1 and CXCR2 mediate cytos-keletal reorganization of microvascular endothelial cells.40

Therefore, we next chose to focus on the effect of downregula-tion of CXCR1 or CXCR2 on actin cytoskeleton organization.Staining of SM-shCXCR1 and SM-shCXCR2 cells showed thepresence of fewer lamellipodial structures as compared to con-trol cells. This suggests that downregulation of CXCR1 orCXCR2 reduces actin reorganization, thereby effecting the mo-tility and, in turn, the invasiveness of melanoma cells. The mo-lecular basis of CXCR1 or CXCR2 induced changes in actinorganization is yet to be investigated.

Another important finding of this study was the enhancedapoptosis of SM-shCXCR1 and SM-shCXCR2 cells. An apo-ptotic property of these cells was observed due to thedecrease in the number of proliferating cells both in vitroand in tumor tissues. Additionally, number of TUNEL-posi-tive cells in tumors from mice injected with SM-shCXCR1 orSM-shCXCR2 cells was higher as compared to SM-controlinjected mice. Our results are consistent with the recent

report where CXCL-8 mediated chemotaxis of melanomacells is shown to be mediated mainly through CXCR1.41

However, our in vitro study did not show any significant dif-ference in CXCL-8 mediated apoptosis, motility and invasionbetween CXCR1 and/or CXCR2 downregulated melanomacells, suggesting the role of both the receptors.

ERK1/2 phosphorylation is an important signaling path-way involved in the control of growth signals, cell survivaland invasion.42 Our recent (unpublished) observation alongwith other studies support the idea that stimulation ofCXCR1 and CXCR2 leads to the activation of MAP kinase.43

Therefore, from our study it is evident that signaling medi-ated by the CXCR1 or CXCR2 can lead to survival and pro-liferation of cells.

Angiogenesis is another essential step for tumor growthand metastasis and expression of CXCR1, CXCR2 and VEGFcan provide a positive feedback loop.44 Our immunohisto-chemistry results support that primary tumor vasculature con-tributed to in vivo differences between SM-shCXCR1, SM-shCXCR2 and control tumors. These results are in agreementwith another report where abrogation of CXCL-8-CXCR2within the tumor markedly inhibited tumor-associated angio-genesis and growth.45 Similarly, attenuation of CXCR2 activityhas been shown to inhibit angiogenesis and tumor growth innude mice.46 In the murine system, CXCL-8 does not exist.The most likely functional murine homolog to human CXCL-8 is murine CXCL1 (KC/Gro1) and CXCL2 (MIP-2/Gro2).4,47

It has been shown that these murine homologs contribute totumor growth, angiogenesis and progression.4,47,48

The role of CXCR2-ligands and CXCR2-dependent signal-ing during tumor progression is complex. A recent reportdemonstrates that CXCR2 signaling plays distinct role duringearly tumorigenesis and late tumor progression.49 These find-ings suggest that CXCL-8 and CXCL-1 signaling throughCXCR2 might limit tumor growth by reinforcing senescenceearly in tumorigenesis.50 During tumorigenesis and progres-sion, CXCR2 and its ligand CXCL-8 and CXCL1 are aber-rantly expressed and CXCR2-dependent signaling tipped to-ward supporting phenotypes associated with progression andinvasion.4,50 Our present data demonstrate that loss ofCXCR2 in malignant melanoma cells inhibits cell prolifera-tion, survival and invasiveness.

In conclusion, our data provide direct evidence for therole of CXCR1 and CXCR2 in melanoma progression.Knock-down of CXCR1 or CXCR2 modulates cellular pheno-types associated with melanoma tumor growth and angiogen-esis, thus indicating CXCR1 and CXCR2 as targets for futuretherapeutic interventions.

AcknowledgementsThis work was supported in part by Cancer Center Support Grant fromNational Cancer Institute, National Institutes of Health and NebraskaResearch Initiative Cancer Glycobiology Program (R.K.S.). The authorsthank Dr. Ajay P. Singh, University of Nebraska Medical Center, NE; forcareful reading of this manuscript.

Can

cerCellBiology



References

1. Jemal A, Siegel R, Ward E, Hao Y, Xu J,Murray T, Thun MJ. Cancer statistics,2008. CA Cancer J Clin 2008;58:71–96.

2. Homey B, Muller A, Zlotnik A.Chemokines: agents for theimmunotherapy of cancer? Nat RevImmunol 2002;2:175–84.

3. Murphy C, McGurk M, Pettigrew J,Santinelli A, Mazzucchelli R, Johnston PG,Montironi R, Waugh DJ. Nonapical andcytoplasmic expression of interleukin-8,CXCR1, and CXCR2 correlates with cellproliferation and microvessel density inprostate cancer. Clin Cancer Res 2005;11:4117–27.

4. Singh S, Sadanandam A, Singh RK.Chemokines in tumor angiogenesis andmetastasis. Cancer Metastasis Rev 2007;26:453–67.

5. Wang JM, Deng X, Gong W, Su S.Chemokines and their role in tumorgrowth and metastasis. J Immunol Methods1998;220:1–17.

6. Richmond A, Fan GH, Dhawan P, Yang J.How do chemokine/chemokine receptoractivations affect tumorigenesis? NovartisFound Symp 2004;256:74–89; discussion89–91,106–11,266–9.

7. Rollins BJ. If it’s Tuesday, this must be aBelgian chemokine. Blood 2001;97:2193.

8. Rossi D, Zlotnik A. The biology ofchemokines and their receptors. Annu RevImmunol 2000;18:217–42.

9. Li A, Dubey S, Varney ML, Singh RK.Interleukin-8-induced proliferation,survival, and MMP production in CXCR1and CXCR2 expressing human umbilicalvein endothelial cells. Microvasc Res 2002;64:476–81.

10. Singh RK, Varney ML. IL-8 expression inmalignant melanoma: implications ingrowth and metastasis. Histol Histopathol2000;15:843–9.

11. Matsushima K, Morishita K, Yoshimura T,Lavu S, Kobayashi Y, Lew W, Appella E,Kung HF, Leonard EJ, Oppenheim JJ.Molecular cloning of a human monocyte-derived neutrophil chemotactic factor(MDNCF) and the induction of MDNCFmRNA by interleukin 1 and tumornecrosis factor. J Exp Med 1988;167:1883–93.

12. Matsushima K, Oppenheim JJ. Interleukin8 and MCAF: novel inflammatorycytokines inducible by IL 1 and TNF.Cytokine 1989;1:2–13.

13. Bar-Eli M. Role of interleukin-8 in tumorgrowth and metastasis of humanmelanoma. Pathobiology 1999;67:12–18.

14. Schadendorf D, Moller A, Algermissen B,Worm M, Sticherling M, Czarnetzki BM.IL-8 produced by human malignantmelanoma cells in vitro is an essential

autocrine growth factor. J Immunol 1993;151:2667–75.

15. Miyamoto M, Shimizu Y, Okada K, KashiiY, Higuchi K, Watanabe A. Effect ofinterleukin-8 on production of tumor-associated substances and autocrine growthof human liver and pancreatic cancer cells.Cancer Immunol Immunother 1998;47:47–57.

16. Wang JM, Taraboletti G, Matsushima K,Van DJ, Mantovani A. Induction ofhaptotactic migration of melanoma cells byneutrophil activating protein/interleukin-8.Biochem Biophys Res Commun 1990;169:165–70.

17. Youngs SJ, Ali SA, Taub DD, Rees RC.Chemokines induce migrational responsesin human breast carcinoma cell lines. Int JCancer 1997;71:257–66.

18. Koch AE, Polverini PJ, Kunkel SL, HarlowLA, DiPietro LA, Elner VM, Elner SG,Strieter RM. Interleukin-8 as amacrophage-derived mediator ofangiogenesis. Science 1992;258:1798–801.

19. Murphy PM, Tiffany HL. Cloning ofcomplementary DNA encoding afunctional human interleukin-8 receptor.Science 1991;253:1280–3.

20. Holmes WE, Lee J, Kuang WJ, Rice GC,Wood WI. Structure and functionalexpression of a human interleukin-8receptor. Science 1991;253:1278–80.

21. Oppenheim JJ, Zachariae CO, Mukaida N,Matsushima K. Properties of the novelproinflammatory supergene ‘‘intercrine’’cytokine family. Annu Rev Immunol 1991;9:617–48.

22. Miller MD, Krangel MS. Biology andbiochemistry of the chemokines: a familyof chemotactic and inflammatorycytokines. Crit Rev Immunol 1992;12:17–46.

23. Jones SA, Moser B, Thelen M. Acomparison of post-receptor signaltransduction events in Jurkat cellstransfected with either IL-8R1 or IL-8R2.Chemokine mediated activation of p42/p44MAP-kinase (ERK-2). FEBS Lett 1995;364:211–14.

24. Li A, Dubey S, Varney ML, Dave BJ, SinghRK. IL-8 directly enhanced endothelial cellsurvival, proliferation, and matrixmetalloproteinases production andregulated angiogenesis. J Immunol 2003;170:3369–76.

25. Varney ML, Li A, Dave BJ, Bucana CD,Johansson SL, Singh RK. Expression ofCXCR1 and CXCR2 receptors in malignantmelanoma with different metastaticpotential and their role in interleukin-8(CXCL-8)-mediated modulation ofmetastatic phenotype. Clin Exp Metastasis2003;20:723–31.

26. Brat DJ, Bellail AC, Van Meir EG. Therole of interleukin-8 and its receptors ingliomagenesis and tumoral angiogenesis.Neuro Oncol 2005;7:122–33.

27. Kline M, Donovan K, Wellik L, Lust C, JinW, Moon-Tasson L, Xiong Y, Witzig TE,Kumar S, Rajkumar SV, Lust JA. Cytokineand chemokine profiles in multiplemyeloma; significance of stromalinteraction and correlation of IL-8production with disease progression. LeukRes 2007;31:591–8.

28. Norgauer J, Metzner B, Schraufstatter I.Expression and growth-promoting functionof the IL-8 receptor b in humanmelanoma cells. J Immunol 1996;156:1132–7.

29. Wang JM, Chertov O, Proost P, Li JJ,Menton P, Xu L, Sozzani S, Mantovani A,Gong W, Schirrmacher V, Van DJ,Oppenheim JJ. Purification andidentification of chemokines potentiallyinvolved in kidney-specific metastasis by amurine lymphoma variant: induction ofmigration and NFjB activation. Int JCancer 1998;75:900–7.

30. Varney ML, Johansson SL, Singh RK.Distinct expression of CXCL8 and itsreceptors CXCR1 and CXCR2 and theirassociation with vessel density andaggressiveness in malignant melanoma.Am J Clin Pathol 2006;125:209–16.

31. Li A, Varney ML, Singh RK. Expression ofinterleukin 8 and its receptors in humancolon carcinoma cells with differentmetastatic potentials. Clin Cancer Res 2001;7:3298–304.

32. Singh RK, Varney ML. Regulation ofinterleukin 8 expression in humanmalignant melanoma cells. Cancer Res1998;58:1532–7.

33. Hue J, Kim A, Song H, Choi I, Park H,Kim T, Lee WJ, Kang H, Cho D. IL-18enhances SCF production of melanomacells by regulating ROI and p38 MAPKactivity. Immunol Lett 2005;96:211–17.

34. Rollins BJ. Chemokines. Blood 1997;90:909–28.

35. Balkwill F. Cancer and the chemokinenetwork. Nat Rev Cancer 2004;4:540–50.

36. Singh RK, Gutman M, Radinsky R, BucanaCD, Fidler IJ. Expression of interleukin 8correlates with the metastatic potential ofhuman melanoma cells in nude mice.Cancer Res 1994;54:3242–7.

37. Huang S, Mills L, Mian B, Tellez C,McCarty M, Yang XD, Gudas JM, Bar-EliM. Fully humanized neutralizing antibodiesto interleukin-8 (ABX-IL8) inhibitangiogenesis, tumor growth, and metastasisof human melanoma. Am J Pathol 2002;161:125–34.

Can

cerCellBiology

Singh et al. 335


38. Singh S, Sadanandam A, Nannuru KC,Varney ML, Mayer-Ezell R, Bond R, SinghRK. Small-molecule antagonists for CXCR2and CXCR1 inhibit human melanomagrowth by decreasing tumor cellproliferation, survival, and angiogenesis.Clin Cancer Res 2009;15:2380–6.

39. Yamazaki D, Kurisu S, Takenawa T.Regulation of cancer cell motility throughactin reorganization. Cancer Sci 2005;96:379–86.

40. Schraufstatter IU, Chung J, Burger M. IL-8activates endothelial cell CXCR1 andCXCR2 through Rho and Rac signalingpathways. Am J Physiol Lung Cell MolPhysiol 2001;280:L1094–L1103.

41. Ramjeesingh R, Leung R, Siu CH.Interleukin-8 secreted by endothelial cellsinduces chemotaxis of melanoma cellsthrough the chemokine receptor CXCR1.FASEB J 2003;17:1292–4.

42. Smalley KS. A pivotal role for ERK in theoncogenic behaviour of malignantmelanoma? Int J Cancer 2003;104:527–32.

43. Venkatakrishnan G, Salgia R, GroopmanJE. Chemokine receptors CXCR-1/2activate mitogen-activated protein kinasevia the epidermal growth factor receptor inovarian cancer cells. J Biol Chem 2000;275:6868–75.

44. Folkman J. Angiogenesis and apoptosis.Semin Cancer Biol 2003;13:159–67.

45. Mestas J, Burdick MD, Reckamp K,Pantuck A, Figlin RA, Strieter RM. Therole of CXCR2/CXCR2 ligand biologicalaxis in renal cell carcinoma. J Immunol2005;175:5351–7.

46. Keane MP, Belperio JA, Xue YY, BurdickMD, Strieter RM. Depletion of CXCR2inhibits tumor growth and angiogenesis ina murine model of lung cancer. J Immunol2004;172:2853–60.

47. Payne AS, Cornelius LA. The role ofchemokines in melanoma tumor growthand metastasis. J Invest Dermatol 2002;118:915–22.

48. Son DS, Parl AK, Rice VM, Khabele D.Keratinocyte chemoattractant (KC)/humangrowth-regulated oncogene (GRO)chemokines and pro-inflammatorychemokine networks in mouse and humanovarian epithelial cancer cells. Cancer BiolTher 2007;6:1302–12.

49. Acosta JC, O’Loghlen A, Banito A,Guijarro MV, Augert A, Raguz S,Fumagalli M, Da Costa M, Brown C,Popov N, Takatsu Y, Melamed J, et al.Chemokine signaling via the CXCR2receptor reinforces senescence. Cell 2008;133:1006–18.

50. Acosta JC, Gil J. A role for CXCR2 insenescence, but what about in cancer?Cancer Res 2009;69:2167–70.

Can

cerCellBiology



Documents

Small interfering RNA-mediated CXCR1 or CXCR2 knock-down inhibits melanoma tumor growth and invasion