ANTICANCER RESEARCH 32: 2759-2768 (2012)

SIVmac239-Nef Down-regulates Cell Surface Expression of CXCR4 in Tumor Cells and Inhibits Proliferation, Migration and Angiogenesis

CHENGZHONG CAI1, FIONA R. RODEPETER1, ANNETTE ROSSMANN1, AFSHIN TEYMOORTASH1, JIN-SEOK LEE1, KARL QUINT2, PIETRO DI FAZIO2, MATTHIAS OCKER2, JOCHEN A. WERNER1 and ROBERT MANDIC1

1Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Marburg Campus, Marburg, Germany; 2Institute for Surgical Research, Philipps University Marburg, Marburg, Germany

Abstract. Aim: To evaluate if the lentiviral accessory stromal cell-derived factor-1α (SDF-1α). CXCR4 Nef can down-regulate the C-X-C has a short, acidic extracellular N-terminal end, as well as type 4 (CXCR4) in tumor cells and affect tumor cell seven helical transmembrane domains with three intracellular proliferation, migration and angiogenesis. Materials and hydrophilic loops (1). As typically observed for G-protein- Methods: HeLa-ACC cells, which according to genotype coupled receptors, the intracellular C-terminus of CXCR4 analysis are virtually identical to the cervical cancer-derived interacts with G and thereby enables transmembrane HeLa cell line, were transfected with Nef from SIVmac239 and cell signaling after SDF-1α binding. CXCR4/SDF-1α expression levels of cell surface CXCR4 were monitored by activates numerous intracellular signaling pathways, flow cytometry. Real-time proliferation and migration of cells influencing cell adhesion, , migration, was measured with the xCELLigence system or with the in proliferation and apoptosis (2-5). vitro scratch assay. In vitro tube formation was deployed to CXCR4 is also known as a co-receptor for human and assess the effect of Nef on angiogenesis. Results: Cell surface simian viruses (HIV/SIV) during viral down-regulation of CXCR4 was observed in HeLa-ACC cells entry (6-8) and, together with its ligand SDF-1α, plays a after Nef transfection, as well as in the monkey kidney- central role in homing and bone derived COS-7 cell line after co-transfection of CXCR4 and marrow release. Knockout of CXCR4 or SDF-1α in Nef. Proliferation, as well as migration, of Nef-transfected transgenic mice results in embryonic lethality and HeLa-ACC cells appeared to be significantly reduced. In vitro underscores its critical role in the development of the tube formation was markedly lowered after Nef transfection, nervous, cardiovascular and hematopoietic systems (9, 10). and CXCR4 knockdown with siRNA. Conclusion: SIV-Nef CXCR4 is overexpressed in a wide variety of human cancer could serve as an interesting tool to study the biological types, such as lung, pancreatic, breast, prostate and kidney behavior of CXCR4-expressing tumor cells and could be cancer (11-16). It is not surprising that more and more helpful in the discovery of new therapeutic approaches for evidence indicate CXCR4/SDF-1α as playing an important the treatment of CXCR4-positive tumors. role in tumor development, since it not only promotes cancer cell proliferation and migration, but also enhances metastasis The C-X-C chemokine receptor type 4 (CXCR4), also known and tumor angiogenesis (17, 18). as fusin or CD184, is a specific receptor for the CXC Negative factor (Nef) is an accessory protein expressed by primate lentiviruses, such as HIV-1 and -2 and SIV. Nef is also a key factor for viral infection and replication and can down-modulate the cell surface receptors CD4, CCR5 and Correspondence to: Robert Mandic, MD, Department of CXCR4 that are required for HIV/SIV entry aiming to Otorhinolaryngology, Head and Neck Surgery, University Hospital prevent a lethal viral superinfection (19). It can also down- Giessen and Marburg, Marburg Campus, Baldingerstrasse, D-35033 regulate major histocompatibility (MHC) class I molecules Marburg, Germany. Tel: +49 64215861400, Fax: +49 64215862421, e-mail: [email protected] due to retention in the Golgi apparatus, which promotes immune-escape of infected cells. Nef proteins from different

Key Words: CXCR4, SDF-1α, SIVmac239, Nef, cancer, COS-7 cells, viral strains exhibit large differences in their ability to down- HeLa-ACC cells, migration, angiogenesis. regulate CXCR4. For example, Hrecka et al. (20) observed a

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Table I. Short tandem repeat analysis.

Source Cell line D5 D13 D7 D16 vWA TH01 TPOX CSF1 Amel

DSMZ HeLa 11,12 12, 13.3 8,12 9,10 16,18 7 8,12 9,10 × Phuchareon et al. (23) ACC2/Sa 11,12 12, 13.3 8,12 9,10 16,17,18 7 8,12 9,10 × Our study ACC3/HeLa-ACC3 11,12 12, 13.3 8,12 9,10 16,17,18 7 8,12 9,10 ×

D5, D13, D7, D16, vWA, TH01, TPOX, CSF1 contain highly polymorphic regions in the that are used for genotyping. In addition amelogenin (Amel) is used for sex determination. (23) DSMZ (Deutsche Sammlung für Mikroorganismen und Zellkulturen) – German Biological Resource Centre Human and Animal Cell Lines.

strong down-regulating effect of Nef from SIVmac239 and Construction of recombinant plasmids and transfection into cell lines. HIV-2 on cell surface CXCR4 expression and SDF-1α- Full-length wild-type SIVmac239 Nef DNA (GenBank: M33262.1) and dependent migration. In contrast, HIV-1 Nef did its functionally deficient mutant Nef-M8 (Y28A-Y39A-DE184/185AA- LM194/195AA-DD204/205AA) were amplified by polymerase chain not exhibit a comparable effect. However, Venzke et al. did reaction (PCR) using previously published Nef constructs as a template not observe such differences between HIV and SIV Nef (19). (28). Full-length CXCR4 (NCBI Reference Sequence: NM_003467.2) Since down-regulation of CXCR4 by Nef was DNA was obtained by RT-PCR (Transcriptor First Strand cDNA demonstrated in Jurkat T-cells, it is of interest to evaluate if Synthesis Kit; Roche Applied Science, Basel, Switzerland. REDTaq™ Nef also affects expression of this receptor in CXCR4- ReadyMix™ PCR reaction Mix; Sigma-Aldrich Corp. St. Louis, MO, positive cells derived from solid tumors, and subsequently USA) from total RNA (RNeasy Mini Kit; Qiagen) from HUVECs, influence tumor-promoting features, such as proliferation, which are known to highly express CXCR4. PCR was performed for 40 cycles (95˚C, 30 s; 56˚C, 1 min; 72˚C, 1 min) with primers containing migration and angiogenesis. an EcoRI (Nef: 5’-AAACTTAAGCTTGCCACCATGGGTGGAG CTATTT-3’; CXCR4: 5’-AAACTTAAGCTTGCCACCATGTCCA Materials and Methods TTCCT-3’) or HindIII (Nef: 5’-TCTGCAGAATTCTCAGCGAG TTTCCTTC-3’; CXCR4: 5’-GGCTAGGAATTCCATCTGTGTTAG-3’) Short tandem repeat (STR) analysis and cell culture. The cell line restriction site. PCR products were gel-purified (QIAquick Gel initially designated as ACC3 (21) that was used in our study was Extraction Kit; Qiagen), double-digested with HindIII and EcoRI (New shown to exhibit high CXCR4 expression levels (22). Recently it England Biolabs GmbH, Frankfurt, Germany) and subcloned into the was reported (23, 24) that this cell line was cross-contaminated with pcDNA3.1(+) mammalian expression vector (Invitrogen Corporation, the cervical cancer-derived HeLa cell line in different laboratories Carlsbad, CA, USA). The recombinant constructs, pcDNA3.1(+)- world wide. In order to verify the identity of the cell line used in SIVmac239Nef-WT (Nef-WT), pcDNA3.1(+)-SIVmac239Nef-M8 (Nef- our study, DNA was isolated from ACC3 cells using the Blood and M8) and pcDNA3.1(+)-CXCR4 were confirmed by sequence analysis Cell Culture DNA Mini Kit from Qiagen (Hilden, Germany) and (4base lab GmbH, Reutlingen, Germany). Purified plasmid DNA sent for STR analysis (German Biological Resource Centre Human (QIAprep Spin Midiprep Kit; Qiagen), CXCR4 siRNA and scrambled and Animal Cell Lines, DSMZ, Braunschweig, Germany). The data RNA (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used revealed that the ACC3 cell line was most closely related to HeLa to transfect (Lipofectamin™2000, Lipofectamin™RNAiMAX; cells as previously reported by Phuchareon et al. (23) (Table Ⅰ). To Invitrogen Corporation. TranspassTM HUVEC Transfection Reagent; distinguish the cells used in our studies from regular HeLa cells, New England Biolabs GmbH, Ipswich, MA, USA) COS-7, HeLa-ACC although according to the STR analysis no significant difference and HUVECs according to the manufacturers’ protocol. should be expected, we designated the cells as HeLa-ACC during the course of our study. Since HeLa cells exhibit high cell surface Western blot and antibodies. Forty-eight hours after transfection, expression levels of CXCR4 and have often been used in research to cells were harvested and lysed as described earlier (29). Thirty-five evaluate the role of CXCR4 during the infection with HIV (25, 26), micrograms of total protein was separated in a 10% polyacrylamide HeLa-ACC cells should also be suitable to evaluate the effect of gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis SIVmac239-Nef on tumor cell proliferation, migration and (SDS-PAGE) and then transferred to a nitrocellulose membrane. angiogenesis. Membranes were blocked in 3% milk/phosphate buffered saline COS-7 (27) and HeLa-ACC cells were cultured in Dulbecco’s (PBS) (1 h; room temperature (RT)) and incubated (overnight, 4˚C) modified Eagle’s medium (DMEM) in the presence of 10% fetal with primary antibodies directed against SIV-Nef and human calf serum, 1% L-glutamine, 100 U/ml penicillin and 100 μg/ml CXCR4 (SIV-Nef, sc-65911; Santa Cruz Biotechnology Inc.. streptomycin. Human umbilical vein endothelial cells (HUVECs; C- CXCR4 antibody, ab2074; Abcam PLC, Cambridge, UK). 003-5C; Life Technologies Corporation, Carlsbad, CA, USA) were Membranes were washed three times in 3% milk/PBS and incubated cultured in EGM-2 medium supplemented with growth factors with horseradish peroxidase (HRP)-linked anti-rabbit or anti-mouse (Lonza Group Ltd., Basel, Switzerland). The culture medium was secondary antibodies (Santa Cruz Biotechnology Inc.) for 1 h at RT. replaced every three days and the cells were passaged after reaching After washing away excessive antibody, bands were visualized with 80% confluence. the enhanced chemiluminescence (ECL) method on x-ray film.

2760 Cai et al: Effect of SIV-Nef on Cancer Cells

Flow cytometry. HeLa-ACC and COS-7 cells were washed in PBS/0.5% In vitro tube formation assay. In order to evaluate the effect of bovine serum albumine (BSA) and incubated with a phycoerythrin SIVmac239-Nef on angiogenesis, endothelial tube formation was (PE)-conjugated CXCR4 antibody (PE Mouse Anti-Human CD184; evaluated using an in vitro angiogenesis (Cell Biolabs, Inc. San BD Biosciences, San Jose, CA, USA). IgG2α was used as an isotype Diego, CA, USA). A total of 2×104 HUVECs transfected with Nef, control. Cell surface fluorescence was monitored by flow cytometry Nef-M8, vector only, CXCR4 siRNA, or scrambled RNA were (BD LSR II; BD Biosciences) and data analysis was performed using seeded into a 96-well plate precoated with 50 μl extracellular matrix FlowJo software (Tree Star, Inc., Ashland, OR, USA). (ECM) gel and incubated at 37˚C for 6 h. Endothelial tube formation was documented by microscopy.

Immunocytochemistry. HeLa-ACC cells that were grown on coverslips were transfected with Nef or control vector and incubated Statistical analysis. Nonparametric tests were used for data analysis. for 48 h. Cells were washed in PBS, fixed for 30 min in cold The Mann-Whitney test was implemented to evaluate the statistical (–20˚C) methanol and incubated in staining buffer significance of the proliferation rates between SIV-Nef, -Nef-M8 (3%BSA/0.3%NP40/PBS) for 30 min. Then anti-CXCR4 antibodies and vector only-transfected HeLa-ACC cells. A probability less than were added and incubation was continued for two more hours. 0.05 (p<0.05) was considered statistically significant. Subsequently, cells were washed in staining buffer and secondary Texas Red (TR)-conjugated goat anti-mouse IgG (for CXCR4) was Results added for another hour at RT. Subcellular localization was evaluated by confocal laser scanning microscopy (Fluoview; Olympus Down-regulation of CXCR4 surface expression after co- Deutschland GmbH, Hamburg, Germany). transfection of SIVmac239-Nef and CXCR4 in COS-7 cells. Mammalian expression vectors containing the from In vitro scratch assay. HeLa-ACC cells transfected with Nef, Nef-M8 Nef, Nef-M8 or CXCR4 were generated as described above. or vector only were seeded into 6-well plates and cell growth was allowed to continue until confluence was reached. The cell COS-7 cells were deployed to assess the expression levels of monolayer was scratched with a 10 μl pipette tip and dislodged cells Nef, Nef-M8 and CXCR4 and to evaluate the influence of were washed away with PBS. Cell incubation was continued under Nef on CXCR4 in this well-established cell system. Only standard conditions and the degree of cell migration into the scraped little amounts of endogenous CXCR4 protein were detected area was documented every 24 h (30). in COS-7 cells (Figure 1A). COS-7 cells were co-transfected with CXCR4 and Nef, Cell proliferation and migration. Real-time cell proliferation was Nef-M8 or pcDNA3.1(+) (vector only control) (Figure 1A). monitored with the xCELLigence system (Roche, Mannheim, Flow cytometry analysis revealed a reduction in CXCR4 Germany). Cells were suspended in 150 μl DMEM and added into a 96-well microtiter plate specifically designed to measure cellular surface fluorescence of COS-7 cells after co-transfection of impedance (E-Plate; Roche). The measured impedance, which is Nef but not of Nef-M8 or vector only, pointing to a Nef- dependent on the level of confluence, is expressed as an arbitrary dependent down-regulation of the receptor from the cell unit called the Cell Index. The Cell Index at each time point is surface in this cell line (Figure 1B). defined as (Rn-Rb)/(15Ω), where Rn is the cell-electrode impedance of the well when it contains cells and Rb is the SIV -Nef down-regulates expression of endogenous background impedance of the well containing medium alone. A mac239 3 CXCR4 in tumor cells. Tumor cells, such as HeLa-ACC, total of 5×10 HeLa-ACC cells transfected with Nef, Nef-M8, vector only, CXCR4-specific siRNA, or scrambled RNA were overexpress CXCR4. Immunocytochemical analyses seeded on the E-plate. The proliferation of the cells was monitored identified CXCR4 in the cytoplasm, as well as at the plasma every 15 min. SDF-1α was added to a final concentration of 100 membrane (Figure 2A). Substantial endogenous expression ng/ml 24 h after cell seeding and proliferation was monitored of CXCR4 was also documented in western blots of vector continuously for 72 more hours. The cell monolayer was scratched only (control)-transfected HeLa-ACC cells (Figure 2B). once the impedance reached the maximum in the same way as HeLa- cells were further transfected with Nef or Nef-M8 described for the in vitro scratch assay and the Cell Index was ACC and the expression of Nef was determined by western blot monitored as described above. analysis. There was no visible change in total CXCR4 Evaluation of phospho-extracellular signal-regulated kinases (ERK), protein expression levels after transfection with Nef (Figure phospho- signal transducer and activator of transcription (STAT3) 2B). However, Flow cytometry analysis revealed a reduction and caspase-3 in HeLa-ACC cells. Twenty-four hours after of cell surface CXCR4 in HeLa-ACC cells after transfection transfection of Nef or vector- only control, HeLa-ACC cells were with Nef-WT, similarly as previously observed in COS-7 treated with SDF-1α (100 ng/ml). Cells were lysed at time points cells co-transfected with Nef and CXCR4 (Figure 2C). No 0, 5, 10 and 30 min. p-STAT3 (Santa Cruz Biotechnology Inc.), significant procaspase-3 cleavage was observed after Nef phosphorylated ERK1/2 (Sigma, Saint Louis, MO, USA) and transfection (Figure 2B). caspase-3 (Santa Cruz Biotechnology Inc.) were evaluated by western blot analysis. Beta-actin (Sigma) was used as control. Fourty-eight hours after transfection with Nef, Nef-M8 or vector SIVmac239-Nef reduces proliferation and migration of tumor only, HeLa-ACC cells were lysed and protein expression was cells. The xCELLigence system was deployed to measure evaluated by western blot analysis. real-time cell proliferation. Nef, Nef-M8, vector only,

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Figure 1. SIVmac239-Nef down-regulates cell surface expression of the C-X-C chemokine receptor type 4 (CXCR4) in COS-7 cells. A: Western blot analysis demonstrates co-expression of CXCR4 and wild-type Nef (Nef-WT) or Nef-M8 in COS-7 cells after co-transfection of the respective two constructs. Cells transfected with CXCR4 and with empty vector (control) express CXCR4 but exhibit no Nef signal. No significant change is noted in the total CXCR4 protein level after transfection with Nef. Actin was used as an internal control. B: Flow cytometry analysis demonstrates a reduction of CXCR4 from the cell surface in cells co-transfected with CXCR4 and Nef compared to cells co-transfected with CXCR4 and Nef-M8 or with empty vector (control).

CXCR4 siRNA and scrambled RNA transfected HeLa-ACC of Nef and Nef-M8 to the vector control (relative cells were seeded into specialized 96-well culture plates and proliferation rate) was plotted to highlight potential effects cellular impedance was measured continuously as described of Nef (Figure 3B). Nef-transfected HeLa-ACC cells exhibited in the Materials and Methods. Control cells treated with a markedly reduced proliferation rate in both untreated and SDF-1α exhibited an enhanced proliferation rate compared SDF-1α-treated groups, whereas Nef-M8-transfected cells to untreated cells (Figure 3A). The proliferation rate of the exhibited a similar proliferation rate to the one of the vector control was taken as baseline (1.0) and the proliferation ratio only-transfected cells, pointing to a specific effect of the

2762 Cai et al: Effect of SIV-Nef on Cancer Cells

Figure 2. Nef from SIVmac239 down-regulates endogenous CXCR4 in HeLa-ACC cells. A: Immunocytochemical analysis of HeLa-ACC cells. HeLa-ACC cells stain positively for endogenous CXCR4, which is found at the plasma membrane, as well as in the cytoplasm and nucleus. B: Western blot analysis reveals that all cells, transfected and untransfected, are positive for endogenous CXCR4, whereas only the Nef-transfected cells express the viral protein. No cleaved procaspase-3 was found in Nef-transfected cells. Actin was used as an internal control. C: Flow cytometry analysis demonstrates cell surface down-regulation of CXCR4 in wild-type Nef (Nef-WT)-transfected HeLa-ACC cells.

viral protein on HeLa-ACC cell proliferation. CXCR4 siRNA- normalized for better comparison. After scratching the cell transfected HeLa-ACC cells exhibited a pronounced inhibition layer, the subsequent rise in impedance was the lowest in the of the proliferation rate compared to those transfected with Nef-transfected HeLa-ACC cells compared with those scrambled RNA (as baseline 1.0) (Figure 3C) as similarly transfected with Nef-M8 or vector only, pointing to Nef as an observed for the wild-type Nef (Figure 3B). inhibitor of HeLa-ACC cell migration (Figure 4A). The short An in vitro scratch assay was performed to evaluate the time required for impedance to rise again after scratching the influence of Nef on cellular migration. Cell numbers were cell layer indicates that migration rather than proliferation is

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Figure 3. Nef from SIVmac239 reduces proliferation of HeLa-ACC cells. A: HeLa-ACC cells (vector control) exhibits enhanced proliferation after addition of 100 ng/ml stromal cell derived factor-1α (SDF-1α). B: Proliferation rate of wild-type Nef (Nef-WT) and Nef-M8-transfected HeLa-ACC cells relative to the vector- only transfected control cells, normalized to 1.0, reveals a significant (p=0.02 at 48 h) inhibition of proliferation in the Nef-WT-transfected cells. This effect was even more pronounced in the SDF-1α-treated cells (p=0.01 at 48 h). C: CXCR4 siRNA-transfected HeLa- ACC cells exhibited a reduced proliferation rate comparable to the one of wild-type Nef-transfected cells. (baseline=scrambled RNA).

responsible for this effect. Furthermore, microscopical analysis Influence of SIVmac239-Nef on SDF-1/CXCR4 signaling in of HeLa-ACC cells revealed a difference in the rate of cells tumor cells. To determine whether SIV-Nef has an effect on moving into the cell-free region (Figure 4B). Nef-transfected MAPK/ERK signaling, HeLa-ACC cells were treated with cells displayed the lowest migratory ability compared with SDF-1α and lysed at the indicated time points as described those transfected with Nef-M8 or vector control, further in the Materials and Methods. Phospho-ERK1/2 and pointing to an inhibitory effect of Nef on cell migration. phospho-STAT3 were detected by western blot (Figure 4C).

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Figure 4. Effect of Nef on migration, signaling and angiogenesis. A and B: Nef from SIVmac239 inhibits migration of HeLa-ACC cells in an in vitro scratch assay. A: The xCELLigence system reveals a quick increase in electric impedance after scratching the cell layer of Nef-M8- and vector- only but not wild-type Nef (Nef-WT)- transfected HeLa-ACC cells. B: Similarly, microscopical analysis shows reduced migration of Nef-WT-transfected HeLa-ACC cells into the scratched cell-free area. C: Stromal cell derived factor-1α (SDF-1α)-treatment of HeLa-ACC cells reduces phospho-signal- transducer and activator of transcription-3 (STAT3) and phospho-extracellular signal-regulated kinase 1/2 (ERK1/2) levels. This effect appears to be more pronounced in the Nef-WT-transfected cells since the phospho-protein levels dropped as early as 10 min after SDF-1α addition. D: Nef inhibits angiogenesis. Nef-WT-transfected human umbilical vein endothelial cells lost most of their in vitro tube formation ability compared with control cells. A similar effect was observed after CXCR4 knockdown with siRNA.

No effect on procaspase-3 cleavage was observed. SIV-Nef RNA-transfected HUVECs, pointing to an inhibitory effect appeared to induce a more enhanced decline in phospho- of SIVmac239-Nef on angiogenesis that is comparable to ERK1/2 and phospho-STAT3 expression after treatment with CXCR4 inhibition. SDF-1α than did the negative control (vector only), however, this effect was not pronounced. Discussion

SIVmac239-Nef inhibits in vitro angiogenesis. HUVECs were Nef is a 27 kDa, N-terminal myristoylated HIV/SIV transfected with Nef, Nef-M8, CXCR4 siRNA or scrambled accessory protein. During infection with HIV/SIV, Nef RNA. In vitro tube formation abilities were compared using down-regulates CD4, the major receptor for virus entry, as light microscopy after 6 hours (Figure 4D). Nef- and CXCR4 well as its two co-receptors CXCR4 and CCR5, to prevent siRNA-transfected HUVECs exhibited reduced tube lethal viral superinfection of the infected cell (19, 20, 28, 31- formation ability compared with Nef-M8- and scrambled 33). Nef was also found to down-regulate MHC class I

2765 ANTICANCER RESEARCH 32: 2759-2768 (2012) molecules from the cell surface of infected cells, reduce factors, such as STAT3, leading to changes in MHC class II levels in antigen-presenting cells and CD8 expression and cell cycle progression. No signs of levels in cytotoxic T- (33-37), thereby promoting apoptosis, such as cleavage of procaspase-3, were observed immune escape of the virus (38, 39). in any of the Nef-transfected cells, indicating that the CXCR4 is known not only as a co-receptor for HIV/SIV inhibitory effect of SIV-Nef exerted on HeLa-ACC cells is entry, but is also involved in malignant transformation, not necessarily related to the activation of programmed cell proliferation, migration and metastasis, where it plays an death (Figure 2B and 4C). However, Nef affects important role in directing metastatic CXCR4+ cells to intracellular signals through complex mechanisms which organs that express high SDF-1α levels (22, 40-43). are not fully understood. STAT3 can also be phosphorylated Therefore, targeting the CXCR4 receptor, e.g. with CXCR4- or activated from a crosstalk signaling pathway via the inhibiting peptides such as ALX40-4C, is used as a mammalian target of rapamycin and Janus kinases. ERK therapeutic approach to prevent HIV/SIV infection, as well was induced and PI3K inhibited by HIV-Nef in Jurkat T- as tumor metastasis (44, 45). cells and primary peripheral CD4+ T-lymphocytes (49). In the present study, wild-type Nef from SIVmac239, which Wild-type but not mutated HIV-Nef was previously found was previously shown to exhibit a potent down-regulating to induce apoptosis in Jurkat T-cells (50). Subsequent effect on CXCR4 in Jurkat T-cells, was used to transfect mapping of HIV-Nef revealed two specific regions (motifs) HeLa-ACC cells to study if this viral protein can affect that exert apoptosis, with motif 1 (M1) being the most CXCR4 levels and related tumor relevant parameters, such powerful (51). Interestingly, in a recent study, this group as proliferation and migration. used an HIV-Nef peptide (Nef-M1) representing the pro- In HeLa-ACC cells, CXCR4 was found located at the apoptotic motif 1 to treat four colorectal cancer cell lines, as plasma membrane, as well as in the cytoplasm and the well as colorectal xenografts, in SCID mice. Here, they nucleus (Figure 2A). This is in accordance with previous observed a potent antitumor effect and concluded that the reports that found a correlation of cytoplasmic and nuclear interaction of the Nef-M1 peptide with CXCR4 is CXCR4 expression with lymph node metastasis and responsible for the observed effect (52). However, there are reduced overall survival in breast cancer and non-small no obvious sequence similarities between these two HIV-Nef cell lung cancer (46, 47). One explanation for the observed apoptosis-inducing motifs and SIVmac239-Nef. high level of intracellular CXCR4 could be a concomitant Taken together, it was demonstrated that Nef from expression of SDF-1α, since autocrine secretion of this SIVmac239 can down-regulate expression of plasma factor could result in enhanced endocytosis of the receptor, membrane-associated CXCR4 in the CXCR4+ tumor cell with subsequent cytoplasmic and nuclear localization. line HeLa-ACC and that expression of the viral protein Indeed, the co-expression of CXCR4 and SDF-1α was resulted in reduction of cell proliferation, migration and found to predict lymph node metastasis in colorectal angiogenesis. SIV-Nef could therefore serve as an cancer (48). interesting experimental tool for the study of CXCR4- Transfection of HeLa-ACC cells with Nef resulted in expressing tumors and could potentially help to pinpoint down-regulation of CXCR4 from the cell surface, however, new therapeutic approaches for the treatment of these without markedly influencing the total CXCR4 protein malignancies. expression. Investigating the real-time proliferation the and migration of Nef-transfected cells revealed that tumor cells Note transfected with the viral protein had significantly lower proliferation and migration rates. HUVECs transfected with A previously accepted submission of the data presented in this manuscript erroneously assuming use of a head and neck adenoid- Nef exhibited a reduction in angiogenesis in the tube cystic carcinoma cell line (ACC3), which indeed was virtually formation assay. CXCR4 with SDF-1α influence numerous identical to the cervical cancer-derived cell line HeLa (see Table I of intracellular signaling pathways, such as the this article), was retracted from Oral Oncology since this journal phosphatidylinositol 3-kinase (pI3K)/protein kinase B covers head and neck cancer only (http://dx.doi.org/10.1016/ (PKB, AKT), cyclic adenosine monophosphate j.oraloncology.2011.06.502). (cAMP)/protein kinase A (PKA), mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and Acknowledgements diacylglycerol (DAG)/Ca2+/protein kinase C (PKC) pathways to regulate cell adhesion, chemotaxis, migration, We thank Ms. Roswitha Peldszus and Ms. Maria Sadowski (Department of Otorhinolaryngology, UKGM GmbH) and Mr. proliferation and apoptosis. The MAPK/ERK pathway Thorsten Volkmann (Department of Hematology, Oncology and plays an important role in CXCR4/SDF-1α signaling. ERK Immunology, UKGM GmbH) for their excellent technical can phosphorylate and activate downstream cellular assistance. This study was supported by the Alfred und Ursula proteins and further phosphorylate nuclear transcription Kulemann Stiftung (Philipps University Marburg, Germany).

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