Molecular Cancer Research Landon Prizes for Basic and Translational Cancer Research Bridging the Lab and the Clinic in Cancer Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
Molecular Cancer Research 5, 737-748, July 1, 2007. Published Online First July 2, 2007;
doi: 10.1158/1541-7786.MCR-07-0032
© 2007 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
1541-7786.MCR-07-0032v1
5/7/737    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by MacManus, C. F.
Right arrow Articles by Waugh, D. J.J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by MacManus, C. F.
Right arrow Articles by Waugh, D. J.J.

Signaling and Regulation

Interleukin-8 Signaling Promotes Translational Regulation of Cyclin D in Androgen-Independent Prostate Cancer Cells

Christopher F. MacManus, Johanna Pettigrew, Angela Seaton, Catherine Wilson, Pamela J. Maxwell, Silvia Berlingeri, Colin Purcell, Maryalice McGurk, Patrick G. Johnston and David J.J. Waugh

Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast City Hospital, Belfast, Northern Ireland

Requests for reprints: David J.J. Waugh, Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast City Hospital, University Floor, Lisburn Road, Belfast BT9 7AB, Northern Ireland. Phone: 44-2890-263911; Fax: 44-2890-263744. E-mail: d.waugh{at}qub.ac.uk


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We have shown previously that interleukin-8 (IL-8) and IL-8 receptor expression is elevated in tumor cells of human prostate biopsy tissue and correlates with increased cyclin D1 expression. Using PC3 and DU145 cell lines, we sought to determine whether IL-8 signaling regulated cyclin D1 expression in androgen-independent prostate cancer (AIPC) cells and to characterize the signaling pathways underpinning this response and that of IL-8–promoted proliferation. Administration of recombinant human IL-8 induced a rapid, time-dependent increase in cyclin D1 expression in AIPC cells, a response attenuated by the translation inhibitor cycloheximide but not by the RNA synthesis inhibitor, actinomycin D. Suppression of endogenous IL-8 signaling using neutralizing antibodies to IL-8 or its receptors also attenuated basal cyclin D1 expression in AIPC cells. Immunoblotting using phospho-specific antibodies confirmed that recombinant human IL-8 induced rapid time-dependent phosphorylation of Akt and the mammalian target of rapamycin substrate proteins, 4E-BP1 and ribosomal S6 kinase, resulting in a downstream phosphorylation of the ribosomal S6 protein (rS6). LY294002 and rapamycin each abrogated the IL-8–promoted phosphorylation of rS6 and attenuated the rate of AIPC cell proliferation. Our results indicate that IL-8 signaling (a) regulates cyclin D1 expression at the level of translation, (b) regulates the activation of proteins associated with the translation of capped and 5'-oligopyrimidine tract transcripts, and (c) activates signal transduction pathways underpinning AIPC cell proliferation. This study provides a molecular basis to support the correlation of IL-8 expression with that of cyclin D1 in human prostate cancer and suggests a mechanism by which this chemokine promotes cell proliferation. (Mol Cancer Res 2007;5(7):737–48)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Elevated expression of the CXC chemokine, interleukin-8 (IL-8), has been correlated with the increased metastatic potential of melanoma (1), gastric (2), renal (3), ovarian (4-6), pancreatic (7-9), breast (10-13), and colorectal cancers (14, 15). Furthermore, IL-8 expression is associated with the disease progression of urogenital cancers, including transitional cell carcinoma of the bladder (16-19) and prostate cancer (20-24).

We previously showed elevated expression of IL-8 and each of its receptors, CXCR1 and CXCR2, in neoplastic cells of human prostate biopsy sections, suggesting that prostate cancer cells are subject to a continuous autocrine stimulus by this chemokine (25). The expression of IL-8, CXCR1, and CXCR2 in neoplastic prostate cells correlated with the expression of two markers of cell proliferation (cyclin D1 and Ki67) and microvessel density (CD34 staining). Furthermore, we showed both increased proliferation of the androgen-independent prostate cancer (AIPC) cell line PC3 in response to addition of exogenous IL-8 or inhibition of growth following antibody-mediated inhibition of endogenous IL-8 signaling in these cells.

An increased translation of proteins that regulate cell growth and cell cycle progression is fundamental to the increased proliferation rates of malignant tumor cells (26). Overexpression of the translation initiation factor eIF4E has been shown to alter cellular morphology, induce cell transformation, and potentiate cell proliferation, tumorigenesis, and metastasis, mediated through selective translation of mRNAs that encode oncoproteins involved in cell cycle progression (e.g., cyclin D1), cell survival (e.g., Akt), and angiogenesis (e.g., vascular endothelial growth factor and matrix metalloproteinase-9; ref. 27). Protein translation is controlled by the integration of numerous signals. Activation of the translational machinery has been shown in response to signals emanating from the Ras-, phosphatidylinositol 3-kinase (PI3K)–, mitogen-activated protein kinase (MAPK)–, and mammalian target of rapamycin (mTOR)–stimulated pathways (28). The latter of these signaling kinases, mTOR, is central to the regulation of protein translation through its concerted effects on the ribosomal protein S6 kinases, the eukaryotic translation initiation factors (eIF), and the eukaryotic translation initiation factor binding proteins (4E-BP). The subsequent activation of the 48S ribosomal complex regulates the translation of mRNA transcripts with highly ordered 5'-oligopyrimidine tracts, whereas the activation of the eIF4F complex leads to translation of mRNA transcripts with highly structured, capped 5'-regions.

The D-type cyclins, D1, D2, and D3, play a critical role in regulating the commitment of cells to enter the cell cycle. Cyclin D associates with the cyclin-dependent kinases 4 and 6, forming protein complexes that underpin the progression from the early to mid-G1 phase of the cell cycle. These cyclin D/cyclin-dependent kinase 4/6 complexes phosphorylate the retinoblastoma protein, inactivating its functional activity to complex with E2F and repress the activity of this transcription factor. The subsequent release of E2F leads to transcriptional induction of genes required for progression from G1 to S phase, including that of cyclin E. Therefore, the expression of cyclin D1 is a key determinant of E2F-mediated transcriptional activity and cell proliferation. Consequently, cyclin D1 expression is tightly regulated to prevent unwarranted entry of cells into the cell cycle.

In this study, we show that IL-8 signaling regulates the translation of cyclin D1, potentiating its expression in cell-based models of AIPC. This observation is further supported by characterization of IL-8 signaling in AIPC cells confirming an activation of PI3K and mTOR signaling pathways and a phosphorylation-mediated downstream regulation of proteins implicated in cap-dependent and ribosome-mediated regulation of protein translation. Our observations enhance our understanding of signaling pathways modulated by IL-8 in AIPC cells, provide a molecular basis to explain our prior correlation of IL-8 expression with cyclin D1 expression in human prostate biopsy tissue, and suggest a mechanistic basis by which this chemokine regulates cell proliferation (25).


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
IL-8 Signaling Increases Cyclin D1 Expression in Prostate Cancer Cells
Our prior studies on prostate biopsy tissue identified a statistically significant correlation between IL-8 and cyclin D1 expression in prostate cancer cells and showed that both endogenous IL-8 and exogenously administered recombinant human IL-8 (rh-IL-8) were linked to AIPC cell proliferation (25). The primary objective of this study was to provide evidence that IL-8 signaling regulates cyclin D1 expression in AIPC cells. Our current experiments were conducted on two AIPC cell lines, PC3 and DU145, each of which was shown to express CXCR1 and CXCR2 receptors by immunoprecipitation-immunoblotting (Fig. 1A ) and flow cytometry analysis (data not shown), consistent with our prior characterization of their expression in epithelial-derived prostate cancer cells.


Figure 1
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 1. IL-8 signaling regulates cyclin D1 expression in AIPC cells. A. Characterization of CXCR1 (left) and CXCR2 expression (right) in PC3 and DU145 prostate cancer cells by immunoprecipitation (IP)-immunoblotting. B. Immunoblot showing a transient time-dependent increase in cyclin D1 expression in PC3 cells (left) and DU145 cells (right) following stimulation with 3 nmol/L rh-IL-8. Blots are representative of three independent experiments. C. Left, immunoblot showing the concentration-dependent increase in cyclin D1 expression in PC3 cells in response to stimulation with 1 and 10 nmol/L rh-IL-8 for 10 min. Right, immunoblot showing the effect of abrogating constitutive IL-8 signaling in PC3 cells on cyclin D1 expression. Cells were electroporated with anti-IL-8 (2 µg/mL), anti-CXCR1 (4 µg/mL), or anti-CXCR2 (4 µg/mL) antibodies or an IgG-negative control antibody. D. Left, effect of 1 µg/mL actinomycin D (ActD) or 1 µg/mL cycloheximide (CHX) on the IL-8–stimulated cyclin D1 expression in PC3 cells. Cells were treated with these inhibitors for 3 h before stimulation with 3 nmol/L rh-IL-8 for 20 min. Treatment with actinomycin D had no effect on the IL-8–promoted increase in cyclin D1 expression, whereas basal and IL-8–promoted expression of cyclin D1 was attenuated by treatment with cycloheximide. Blots (A-D) are representative of three experiments and equivalent loading of protein samples was verified on all blots by detection of GAPDH expression. Right, the relative cyclin D1 mRNA transcript levels in PC3 cells in response to stimulation with 3 nmol/L rh-IL-8 over a 45-min time course. Columns, mean values calculated by analysis of four independent experiments; bars, SE.

 
Immunoblotting experiments confirmed that stimulation of AIPC cells with rh-IL-8 resulted in an increased cyclin D1 expression. Increased expression of cyclin D1 in PC3 cells was detectable as early as 2 min after stimulation with 3 nmol/L rh-IL-8 and remained elevated out to 60 min (Fig. 1B, left). A transient increase in cyclin D1 expression was also detected in DU145 cells following stimulation with 3 nmol/L rh-IL-8 (Fig. 1B, right). In addition to showing a time-dependent relationship, we confirmed the specificity of this response by further showing a concentration dependence to IL-8–promoted cyclin D1 expression, shown for PC3 cells in response to increasing concentrations of rh-IL-8 for 10 min (Fig. 1C, left). We also examined whether blockade of constitutive IL-8 signaling in PC3 cells, conducted by either targeting the constitutive IL-8 expression of IL-8 or inhibiting each of its receptors with neutralizing antibodies, would diminish the basal expression of cyclin D1 observed in PC3 cells. Administration of neutralizing antibodies against IL-8, CXCR1, and CXCR2 (at concentrations shown previously to block cell proliferation; ref. 25) each attenuated the basal expression of cyclin D1 detected in PC3 cells (Fig. 1C, right).

The rapidity with which IL-8 signaling potentiated cyclin D1 expression in AIPC cells suggested to us that this response was consistent with chemokine-promoted regulation of protein translation as opposed to transcriptional regulation of the cyclin D1 gene. To test this hypothesis, AIPC cells were stimulated with 3 nmol/L rh-IL-8 in the absence or presence of either the RNA synthesis inhibitor actinomycin D or the translation inhibitor cycloheximide. Pretreatment with actinomycin D had no effect on the ability of rh-IL-8 signaling to transiently increase cyclin D1 expression in these cells (Fig. 1D, left). However, treatment with cycloheximide abolished both the basal expression and the IL-8–promoted increase in cyclin D1 expression in PC3 cells. Quantitative PCR also confirmed that rh-IL-8–induced signaling did not increase cyclin D1 mRNA transcript levels over the same time course in which we observed increased cyclin D1 protein expression (Fig. 1D, right).

IL-8 Signaling Promotes Activation of PI3K/Akt Signaling in AIPC Cells
The secondary objective of this study was to identify the molecular basis by which IL-8 signaling may regulate cyclin D1 expression and promote cell proliferation. In a series of further immunoblotting experiments, we sought to show that IL-8 induced the activation of signaling pathways in AIPC cells known to be associated with the regulation of the translation machinery and/or regulation of cell proliferation.

Activation of the PI3K pathway in AIPC cells was initially studied using two antibodies that detect specific phosphorylation on the Thr308 and Ser473 residues of the PI3K substrate protein Akt. Phosphorylation of Thr308 was detectable 2 min after stimulation with 1 nmol/L rh-IL-8 and remained elevated above basal levels for greater than 10 min in PC3 cells (Fig. 2A, left, top ). We also detected prolonged phosphorylation of the Ser473 residue of Akt in response to stimulation with 1 nmol/L rh-IL-8, being first detectable at 1 min, reaching a peak level at 10 min and returning to basal levels by 60 min (Fig. 2A, left, middle). Phosphorylation of Akt on Thr308 and Ser473 was also detected in DU145 cells in response to stimulation with rh-IL-8 (Fig. 2A, right). The magnitude of rh-IL-8–induced Ser473 phosphorylation on Akt relative to Akt expression was further analyzed by densitometry (Fig. 2B, left), which revealed that the intensity and the maintenance of phosphorylation on this residue were not as prolonged in the DU145 cells as that observed in PC3 cells. Therefore, we propose that the prolonged Akt phosphorylation response observed in the PC3 cells may reflect the absence of the phosphatase PTEN in these cells.


Figure 2
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 2. IL-8 signaling activates PI3K and MAPK signaling in AIPC cells. A. Immunoblots showing the time-dependent stimulation of PI3K activity in PC3 cells (left) and DU145 cells (right) in response to rh-IL-8 (1 nmol/L), indicated by increased phosphorylation of the substrate protein Akt on residues Thr308 (top) and the Ser473 residue (middle top). Immunoblotting was also conducted to analyze Akt expression in these samples (middle bottom). B. rh-IL-8 stimulated fold changes in the relative phospho-Akt (pAKT)/Akt ratio in PC3 and DU145 cells (left) and Akt expression detected in PC3 and DU145 cells (right), calculated by densitometry analysis of the respective immunoblots. C. Immunoblots showing the time-dependent phosphorylation of p42 Erk1 in PC3 cells (left) and DU145 cells (right) in response to 3 nmol/L rh-IL-8. Equal loading of protein in above experiments was confirmed by parallel analysis of the nonphosphorylated protein species in these samples and by reprobing the original membranes for GAPDH expression.

 
In addition to detecting increased phosphorylation of Akt, densitometry analysis of immunoblots also suggested that IL-8 signaling increased Akt expression in AIPC cells (Fig. 2B, right). This was particularly evident in PC3 cells where greater than 1.5-fold increases in Akt expression relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detectable 10 min after stimulation with rh-IL-8. Increases in Akt expression in DU145 cells were also indicated by densitometry analysis, detected at time points exceeding 30 min after stimulation with rh-IL-8 (Fig. 2B, right). Therefore, our experiments indicate that IL-8 signaling increases both the activation and the expression level of Akt in AIPC cells.

IL-8 Signaling Promotes the Activation of Phospholipase D and Atypical Protein Kinase C{zeta} in AIPC Cells
Phospholipase D (PLD) catalyzes the conversion of phosphatidylcholine to phosphatidic acid (PA) and is activated by IL-8 signaling in neutrophils (29, 30). PA is an important upstream regulator of mTOR (31), atypical protein kinase C{zeta} (PKC{zeta}; ref. 32), and PI3K activation (33). Addition of 3 nmol/L rh-IL-8 stimulated a rapid increase in PLD activity in PC3 cells, increasing basal PLD activity to a peak of 169 ± 26% (P < 0.05) of that detected in unstimulated cells within 1 min (Fig. 3A ). Further experiments were conducted to determine the contribution of PA in mediating the IL-8–promoted increase in Akt signaling. Treatment of PC3 cells with 0.3% (v/v) butan-1-ol, an inhibitor of PLD activity, attenuated but did not abrogate the rh-IL-8–promoted phosphorylation of the Thr308 residue of Akt (Fig. 3B).


Figure 3
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 3. IL-8 signaling promotes the activation of PLD defining a pathway leading to the activation of PI3K, atypical PKC, and mTOR in AIPC cells. A. The activation of PLD activity in PC3 cells in response to the addition of 3 nmol/L rh-IL-8 was determined using colorimetric analysis of choline production. Columns, mean values determined in three individual experiments; bars, SE. B. Immunoblot showing that the rh-IL-8–promoted phosphorylation of the Thr308 residue of Akt in PC3 cells is attenuated by pretreatment of the cells for 5 min with 0.3% (v/v) butan-1-ol (1-ButOH), an inhibitor of PLD. Where indicated, the cells were stimulated with 3 nmol/L rh-IL-8 for 2 min. C. Left, immunoblot showing a time-dependent phosphorylation of the atypical PKC{zeta} isoform in PC3 cells in response to stimulation with 3 nmol/L rh-IL-8 (top); immunoblot confirming that the IL-8–induced phosphorylation of PKC{zeta} in PC3 cells is attenuated by addition of 0.3% (v/v) butan-1-ol, suggesting that the response is mediated in part by PA (middle top). Equal protein loading was shown by reprobing for the nonphosphorylated species of PKC{zeta} (middle bottom) or GAPDH (bottom). Right, the rh-IL-8 stimulated fold change in the relative phospho-PKC{zeta} (pPKC{zeta})/PKC{zeta} ratio in PC3 cells calculated by densitometry analysis of the respective immunoblots. D. Immunoblot showing a time-dependent phosphorylation of the mTOR substrate, the translation inhibitory protein 4E-BP1 in PC3 cells in response to stimulation with 3 nmol/L rh-IL-8. Each of the immunoblots shown in (B-D) is representative of three independent experiments.

 
Because PA is also a potent activator of PKC{zeta} (32), immunoblotting experiments were conducted to determine if PKC{zeta} was a downstream substrate of IL-8 signaling, examining the phosphorylation of PKC{zeta} on residue Thr410 in response to rh-IL-8 administration. Increased phosphorylation of PKC{zeta} was detected in PC3 cells between 5 to 45 min after stimulation with rh-IL-8 (Fig. 3C, left, top). Further analysis of these immunoblots by densitometry confirmed that IL-8 signaling increased the phosphorylation of this kinase by a factor of 3.4-fold relative to either PKC (Fig. 3C, right) or GAPDH expression, whereas densitometry confirmed that the expression of PKC did not change relative to GAPDH expression in these cells in response to rh-IL-8 administration (data not shown). The rh-IL-8–promoted potentiation of PKC{zeta} phosphorylation could not be shown when cells had been pretreated with 0.3% (v/v) butan-1-ol, suggesting that PA is an intermediate promoting the IL-8–stimulated phosphorylation of this kinase (Fig. 3C, left, middle top).

IL-8 Signaling Underpins the Phosphorylation of the mTOR Substrate Protein 4E-BP1
PA and Akt are proposed to be independent activators of mTOR (27, 28, 31), suggesting that mTOR may be a downstream target of IL-8 signaling cascades in PC3 cells. To test this, we determined the effect of IL-8 signaling on the phosphorylation status of the downstream mTOR substrate, 4E-BP1. Consistent with the rapid increase in both PLD activity and Akt phosphorylation, we observed increased phosphorylation of 4E-BP1 in PC3 cells (Fig. 3D) within 5 min of rh-IL-8 stimulation, with peak phosphorylation occurring between 20 to 30 min after stimulation. Immunoblotting and densitometry analysis confirmed that this increase could not be explained by changes in 4E-BP1 expression. Similarly, IL-8 signaling was shown to increase 4E-BP1 phosphorylation between 20 to 45 min in DU145 cells without altering 4E-BP1 expression (data not shown). Therefore, this suggests that IL-8 signaling activates mTOR in AIPC cells, inducing downstream phosphorylation of the mTOR substrate 4E-BP1.

IL-8 Signaling Underpins the Phosphorylation and Activation of Ribosomal S6 Kinase
Extracellular signal-regulated kinase (Erk), PKC{zeta}, PDK1, and mTOR all modulate the selective phosphorylation of ribosomal S6 kinase (rS6K) at distinct serine and threonine residues (34-36). Because prior experiments suggested that each of these kinases is IL-8 signaling intermediate, we examined the effect of IL-8 signaling on the phosphorylation status of rS6K using immunoblotting. We detected a time-dependent phosphorylation of the Thr421/Ser424 residues of both the cytoplasmic p70 and nuclear p85 S6K isoforms (upper and lower bands, respectively), in response to stimulation of PC3 cells with 3 nmol/L rh-IL-8 (Fig. 4A, top ). Phosphorylation of the Thr421/Ser424 residues was clearly detectable out to 30 min after stimulation with rh-IL-8. Stimulation with rh-IL-8 promoted a biphasic phosphorylation of the Thr389 residue of rS6K, with clearly distinguishable early and later peaks of phosphorylation (Fig. 4A, middle top). Although further experiments will be required to elucidate the mechanisms underpinning the early and late phosphorylation signals detected on this residue, it is possible that the initial response may be due to PA-initiated mTOR signaling, whereas the secondary response arises as a consequence of hierarchical phosphorylation promoted by Erk/PKC{zeta}–promoted phosphorylation of the Thr421/Ser424 residues. In addition, elevated phosphorylation on the catalytically active Thr229 residue of rS6K was detected 5 min after stimulation with rh-IL-8 and peaked between 10 and 20 min postchallenge. rS6K expression was unaffected by IL-8 signaling in PC3 cells.


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 4. IL-8 signaling promotes the phosphorylation and activation of ribosomal protein S6 kinase. A. Immunoblots showing time-dependent phosphorylation on three consensus phosphorylation sites of S6 kinase: Thr421/Ser424 (top), Thr389 (middle top), and Thr229 (middle) residues on ribosomal protein (p70) S6 kinase (lower band) and p85 S6 kinase (upper band) in PC3 cells in response to stimulation with 3 nmol/L rh-IL-8. All residues are numbered according to the structure of the p70 isoform of this kinase. B. Immunoblot showing time-dependent phosphorylation of ribosomal S6 protein in PC3 (left) and DU145 cells (right) in response to 3 nmol/L rh-IL-8. C. Immunoblot showing time-dependent phosphorylation of Ser422 of eIF4B in PC3 cells in response to stimulation with 3 nmol/L rh-IL-8. D. Effect of the PI3K inhibitor LY294002 (10 µmol/L) and the mTOR inhibitor rapamycin (Rap; 10 nmol/L) on IL-8–induced phosphorylation of ribosomal S6 protein in PC3 cells. Cells were equilibrated with the signal transduction inhibitors for 3 h before experimentation and then stimulated for 20 min with 3 nmol/L rh-IL-8. E. Effect of attenuating Akt and mTOR expression using siRNA on the IL-8–induced phosphorylation of ribosomal S6 protein in PC3 cells. Cells were transfected with kinase-targeted or scrambled oligonucleotides for 72 h and then stimulated for 20 min with 3 nmol/L rh-IL-8. All blots in (A-E) are representative of two or three independent experiments. Equivalent loading of protein samples was verified on all blots by detection of the nonphosphorylated protein species and/or GAPDH expression.

 
To determine whether IL-8 signaling increased S6K activity in AIPC cells, we examined the phosphorylation status of two substrate proteins, ribosomal S6 protein and eIF4B. Treatment of both PC3 cells (Fig. 4B, left) and DU145 cells (Fig. 4B, right) with 3 nmol/L rh-IL-8 induced a time-dependent phosphorylation of ribosomal S6 protein, a response that was sustainable for 45 min in both cell lines. Consistent with the peak phosphorylation of the catalytically active Thr229 residue of rS6K kinase following stimulation with rh-IL-8, the peak phosphorylation of ribosomal S6 protein was detected between 20 and 30 min after treatment with rh-IL-8. Although S6 protein expression was unchanged in the PC3 cell line, densitometry analysis of each replicate immunoblot confirmed that IL-8 signaling increased S6 protein expression in excess of 1.6- to 3.4-fold, 30 to 60 min after stimulation of DU145 cells (data not shown). Phosphorylation of eIF4B on the Ser422 residue was detectable between 2 and 5 min in PC3 cells following stimulation with 3 nmol/L rh-IL-8 (Fig. 4C), in parallel with the early phosphorylation of the Thr389 residue of rS6K.

In further experiments, administration of the PI3K inhibitor LY294002 and the mTOR inhibitor rapamycin attenuated IL-8–promoted phosphorylation of rS6 protein in PC3 cells (Fig. 4D). Suppression of Akt and mTOR expression using small interfering RNA (siRNA) also inhibited both the basal level and the 3 nmol/L rh-IL-8–promoted phosphorylation of rS6 protein in PC3 cells (Fig. 4E). In contrast, the inhibition of MAP/ERK kinase 1 (MEK1) expression using siRNA resulted in an increase in the phosphorylation of rS6 protein detected in both unstimulated and IL-8–stimulated cells.

Relationship of Constitutive IL-8, Akt, and mTOR to AIPC Cell Proliferation
Finally, experiments were conducted to show the relevance of endogenous IL-8 and each of the IL-8–promoted signaling pathways in regulating PC3 cell proliferation. An IL-8 siRNA strategy was adopted to deplete the endogenous levels of this CXC chemokine in these cells. Transfection of PC3 cells with 20 nmol/L of a commercial IL-8–targeted oligonucleotide pool reduced endogenous IL-8 secretion in PC3 cells from 40.7 ± 4.3 to 9.8 ± 1.4 ng/mL/106cells/72 h, as measured by ELISA. Conversely, transfection with a scrambled oligonucleotide at this concentration had no significant effect on IL-8 synthesis or secretion. When studied using cell count assays, the suppression of endogenous IL-8 expression reduced the rate of PC3 cell proliferation by 23.2 ± 6.8% relative to that observed in scrambled oligonucleotide-transfected cells over a 72-h period (P < 0.05; Fig. 5A ).


Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 5. Characterization of importance of endogenous IL-8 and multiple signaling pathways in underpinning AIPC cell proliferation. A. The suppression of endogenous IL-8 expression using a commercial siRNA strategy decreases the proliferation of PC3 cells. The effect of attenuating IL-8 expression using 20 nmol/L of an IL-8–targeted oligonucleotide pool is shown relative to the effect observed following transfection of PC3 cells with a scrambled oligonucleotide (Sc) at the same concentration or following mock transfection. Columns, mean value calculated from four independent experiments; bars, SE. B. The effect of various signal transduction pathway inhibitors, administered at indicated final concentrations, on the proliferation of PC3 cells was determined over the course of 72 h by cell count analysis. Columns, mean value calculated from four independent experiments; bars, SE. C. Immunoblots showing the siRNA-mediated depletion of MEK1, Akt, or mTOR expression in PC3 cells using 10, 50, or 100 nmol/L of the oligonucleotide pools. D. The effect on PC3 cell proliferation following siRNA-mediated depletion of MEK1, Akt, or mTOR expression in these cells. Data show the effects following transfection of PC3 cells with 10 nmol/L of the gene-targeted oligonucleotide pools or the scrambled oligonucleotide. Columns, mean value determined from four independent experiments; bars, SE. Statistical analysis of the data shown in (A), (B), and (D) was conducted using a Students t test with significance indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
PC3 cells were treated with established signal transduction inhibitors to establish the relationship of each of these IL-8–induced signaling pathways to PC3 cell proliferation. The effects of these inhibitors on cell proliferation were determined by cell count analysis 72 h after treatment. Treatment of the PC3 cells with the MEK1 inhibitor U0126 reduced the detected cell number to 65.1 ± 3.99% of control (P < 0.001). The PI3K inhibitor LY294002 decreased cell number to 47.9 ± 6.01% (P < 0.001) of control, whereas the mTOR inhibitor rapamycin reduced cell number to 61.6 ± 5.06% of control (P < 0.001; Fig. 5B). Treatment with the p38 MAPK inhibitor, SB203580, or the nonselective PKC inhibitor bisindolemaleimide had no demonstrable effect on PC3 cell proliferation. Parallel experiments confirmed that vehicle controls had no effect on cell proliferation at the concentrations used (data not shown).

To support the pharmacologic interventions, siRNA strategies were also used to examine the effect of attenuating Akt, Erk, and mTOR expression on the proliferation of PC3 cells. Specific protein knockdown was shown for each of the siRNA oligonucleotide pools by immunoblotting (Fig. 5C). Suppression of Akt expression reduced the detected cell number by 26.5 ± 6.7% of that of the scrambled oligonucleotide control (P < 0.01), whereas reducing mTOR expression decreased cell number by 44.2 ± 5.4% of the scrambled oligonucleotide control (P < 0.001; Fig. 5D). Surprisingly, the siRNA strategy to deplete MEK1 expression in PC3 cells failed to decrease the rate of cell proliferation, in marked contrast to the effect observed when cells were treated with U0126. Given the discrepancy between the molecular and pharmacologic inhibition of MEK1, the cell proliferation assay was repeated using PD98059, a further inhibitor of MEK1. Administration of 2 µmol/L PD98059 reduced cell number to 63.8 ± 7.7% of control (P < 0.001), comparable with the effect observed with U0126.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
We have previously reported elevated IL-8 and IL-8 receptor expression in human prostate cancer over that detected in normal prostate epithelium, the emergence of which was shown to correlate with markers of cell proliferation and microvessel density (25). Whereas angiogenesis has been proposed to contribute to IL-8–promoted tumor growth in vivo (21, 22), we have also shown a direct mitogenic effect of IL-8 on human prostate cancer cell lines in vitro (25). The aims of this current study were to provide evidence of a molecular link between IL-8 signaling and cyclin D1 expression and to further characterize the signaling pathways activated by IL-8 that underpin the mitogenic activity of this chemokine in prostate cancer cells.

Immunoblotting experiments were conducted on representative cell-based models of androgen-independent disease where immunohistochemistry and in situ hybridization studies suggest that autocrine IL-8 signaling may have its most pronounced effect (25, 37). Administration of exogenous rh-IL-8 was shown to potentiate the expression of cyclin D1 in both PC3 and DU145 cells. Furthermore, the suppression of endogenous IL-8 signaling in PC3 cells using a series of ligand and receptor-targeted antibodies was accompanied by a reduction in the basal expression of cyclin D1. The promotion of cyclin D1 expression by IL-8 signaling showed both a time dependence and a concentration dependence, adding further weight to the specificity of the response. The increase in cyclin D1 expression was observed to be rapid in both cells and was apparently inconsistent with a transcriptional regulation of the cyclin D1 gene because no detectable increase in cyclin D1 mRNA transcript levels could be detected using quantitative PCR analysis over the same time course. Furthermore, administration of the RNA synthesis inhibitor actinomycin D failed to attenuate this rapid rh-IL-8–stimulated increase in cyclin D1 expression, suggesting that the response is consistent with translational regulation of cyclin D1.

Our observation that IL-8 signaling promotes a translation-mediated increase in cyclin D1 expression in AIPC cells in vitro characterizes a molecular mechanism to explain our prior correlation of IL-8 expression with that of cyclin D1 in the tumor cells of human prostate biopsy tissue. Furthermore, the demonstration that IL-8 signaling increases cyclin D1 expression is consistent with the reported mitogenic function of this chemokine in prostate (25), ovarian (5), pancreatic (7), and colorectal cancer cell lines (15). Cyclin D1 expression in prostate cancer is related to several malignant cellular features, including tumor metastasis classification, histologic differentiation, perineural invasion, DNA ploidy, S-phase fraction, expression of Ki67, and mitotic index (38) and bone metastasis (39). Therefore, our in vitro observations, indicating that IL-8 signaling regulates the expression of the cyclin D1 oncogene, provides further insights about the importance of this CXC chemokine in modulating the tumorigenicity and metastasis of prostate cancer (20-22).

Our experiments also establish that IL-8 signaling regulates pathways central to the regulation of protein translation. Specifically, mTOR plays an important role in integrating the activation of the translational machinery through a combination of phosphorylation-mediated activation of stimulatory proteins and inhibition of negative regulators (27, 28). Immunoblotting experiments using phospho-specific antibodies show that IL-8 signaling promotes the phosphorylation of two characterized substrates of mTOR (i.e., 4E-BP1 and rS6K). In addition, we showed that IL-8 signaling induced PA accumulation and promoted the phosphorylation of Akt, in AIPC cells, each of which is upstream activator of mTOR. Treatment with rapamycin and LY294002, inhibitors of the PI3K/Akt and mTOR kinase activity, respectively, attenuated the IL-8–promoted phosphorylation of rS6K, showing the importance of these signaling pathways to the IL-8–promoted regulation of the translational machinery. Therefore, we propose that IL-8 signaling underpins mTOR activation in AIPC cells, a response that is likely mediated via both PLD-dependent and P13K/Akt–dependent signaling.

The effects of mTOR activation in response to promotion of IL-8 signaling in AIPC cells resulted in concurrent phosphorylation of the translation inhibitory protein 4E-BP1 and Thr389 of p70 rS6K. The phosphorylation of 4E-BP1 decreases the avidity of this protein to bind and sequester its substrate, the eIF4E protein, permitting eIF4E to interact with additional substrates to form the eIF4F complex and promote cap-dependent translation. With regard to rS6K, immunoblotting experiments confirmed that IL-8 signaling promotes phosphorylation of each of the serine and threonine residues implicated in the hierarchical sequence of ordered kinase-dependent activation of this protein (35, 36, 40), including the phosphorylation of Thr229 residue, which is proposed to unleash the full catalytic activity to the kinase. Consistent with this, IL-8 signaling also promoted the phosphorylation of two downstream substrates of rS6K, the ribosomal S6 protein (rS6) and eIF4B (41). In each of our immunoblotting experiments examining the phosphorylation status of proteins associated with protein translation, the temporal relationship of these phosphorylation responses is consistent with (a) the identity of the upstream substrates (i.e., Erk, Akt, and mTOR) as known effectors of IL-8 signaling, (b) the kinetics with which IL-8 activates these upstream stimuli, and (c) the observation that the phosphorylation of the downstream substrates, rS6 and eIF4B, is detected in parallel with phosphorylation of the Thr229 residue of the S6 kinase.

Therefore, our data suggest that IL-8 signaling regulates the assembly of both the 48S ribosomal protein complex and the eIF4F complex, each of which binds mRNA transcripts for translation (Fig. 6 ). In particular, our demonstration that IL-8 signaling promotes the phosphorylation of 4E-BP1 is consistent with the promotion of cap-dependent translation of cyclin D, providing a molecular basis to support our initial indication that IL-8 signaling regulates the translation of this oncogene in AIPC cells.


Figure 6
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 6. Schematic summary of IL-8–promoted signal transduction pathways and the link to mRNA translation in AIPC cells. Proteins shown in this study to be phosphorylated or activated and/or whose expression was increased in response to IL-8 signaling in AIPC cells are shown.

 
The final objective of the article was to show the relevance of IL-8–induced signaling pathways to the promotion of AIPC cell proliferation. siRNA-mediated suppression of endogenous IL-8 expression retarded PC3 cell proliferation, by a level consistent with that previously observed in these cells following the administration of a neutralizing antibody to this chemokine (25). Subsequently, siRNA-mediated depletion of mTOR and Akt expression and/or pharmacologic inhibition of mTOR and PI3K/Akt signaling using rapamycin and LY294002 identified the importance of these signaling pathways to AIPC cell proliferation. As reported previously in studies conducted in tumor cells and neutrophils (5, 42, 43), IL-8 signaling was also shown to induce the activation of MAPK signaling pathway in PC3 and DU145 cells, exemplified by time-dependent Erk phosphorylation. Pharmacologic inhibition of this pathway using each of the MEK1 inhibitors U0126 and PD98059 attenuated AIPC cell proliferation, suggesting that activation of MAPK signaling may also underpin the IL-8–promoted proliferation of AIPC cells, in a manner similar to that reported previously in ovarian cancer cells (5). Interestingly, siRNA-mediated inhibition of MEK1 failed to reduce the cell proliferation rate of these cells and also resulted in an increased level of rS6 phosphorylation detected in these cells, suggesting that the oligonucleotide pool used to target this enzyme may have significant off-target and nonspecific effects that may invalidate its use in these cells.

As suggested by our prior study on human prostate biopsy tissue, the coexpression of IL-8 and IL-8 receptors on prostate cancer cells indicates that these cells are subject to a continuous autocrine/paracrine stimulus (25). Our current characterization of the signaling pathways activated by this chemokine in AIPC cells provides further insights into the relevance and importance of IL-8 in promoting the progression of prostate cancer. First, we have shown that IL-8 signaling increases both the expression and the activation of Akt in AIPC cells. Specifically, IL-8 signaling resulted in increased phosphorylation of both the Thr308 and Ser473 residues of Akt in PC3 and DU145 cells, although the response was more prolonged in the PC3 cells, most likely due to the absence of the endogenous phosphatase PTEN in these cells. The IL-8–induced phosphorylation of the Thr308 residue also seemed to lag that of the phosphorylation on Ser473 in either cell line, being most noticeable in the DU145 cells. Further detailed experiments will be required to map and characterize the pathways underpinning IL-8–induced activation of the PI3K/Akt pathway in each of these AIPC cells to explain the different kinetics of this response, focusing on the receptor selectivity, G proteins, and the various primary effectors responsible for inducing the phosphorylation of this kinase. In addition, the prolonged phosphorylation of Thr308 detected in the DU145 cells may result from cross-talk with other IL-8–induced signaling pathways. That said, the biological importance of PI3K/Akt signaling to human prostate cancer initiation and progression is increasingly acknowledged. Increased Akt phosphorylation has been detected using immunohistochemical and protein microarray studies on primary prostate tumors (44, 45). Other studies report that the progressive transition from histologically normal epithelium to prostate intraepithelial neoplasia is associated with increasing phosphorylation of Akt (46), whereas the induction of Akt activity in prostate cancer cells promotes androgen-independent growth and survival of prostate cancer cells (47). Furthermore, Akt1 deficiency dramatically inhibits prostate neoplasia development in PTEN+/– mice (48). Consistent with our current results, the activation of Akt has been shown to potentiate cyclin D1 expression in xenograft prostate cancer tissue (47), whereas in vitro studies have reported that pharmacologic inhibition of PI3K/Akt activity by LY294002 promotes G(1) cell cycle arrest and decreased expression of G(1)-associated proteins, including cyclin D1 and cyclin-dependent kinase 4 in prostate cancer cells (49). Second, we have shown that IL-8 signaling induces mTOR activity, a kinase identified as an important therapeutic target in prostate cancer. mTOR has been shown to mediate Akt-promoted G1-S cell cycle progression (49), whereas the inhibition of mTOR activity (a) attenuates the repopulation of surviving prostate cancer cells following chemotherapy in prostate cancer xenograft models (50), (b) sensitizes PTEN-deficient prostate cancer cells to chemotherapy (51) and radiotherapy (52), and (c) reverses the Akt-promoted prostate intraepithelial neoplasia phenotype in mice (53).

In summary, we report that IL-8 regulates cyclin D1 expression at the level of protein translation in AIPC cells, substantiating our prior correlation of IL-8 with cyclin D1 expression in human prostate biopsy tissue. Second, we provide evidence that IL-8 signaling in AIPC cells induces the activation of key regulators of both cap-dependent mRNA translation and 5'-oligopyrimidine tract mRNA translation. Finally, our experiments confirm that IL-8 signaling is coupled to the activation of PI3K/Akt/mTOR and MAPK signaling pathways that regulate the proliferation of these AIPC cells.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Chemicals and Reagents
LY294002 and rapamycin were obtained from Cell Signaling Technology. rh-IL-8 was sourced from PeproTech. All other chemicals were sourced from the Sigma Chemical Co. unless otherwise stated.

Cells and Cell Culture
PC3 and DU145 human prostate adenocarcinoma cells were purchased from the American Type Culture Collection and cultured in RPMI 1640 and DMEM, respectively, supplemented with 10% fetal bovine serum and 2 mmol/L L-glutamine (Invitrogen Life Technologies) in a humidified atmosphere of 5% CO2 at 37°C.

Immunoprecipitation-Western Blotting
Cells were lysed in radioimmunoprecipitation assay buffer [10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1% (w/v) Triton X-100, 0.1% SDS, 1 protease inhibitor tablet/10 mL (Roche Diagnostics, Inc.)], and the lysates were cleared by centrifugation at 15,000 x g for 20 min at 4°C. The supernatant fraction containing 0.1 or 1.0 mg protein was added to protein A-Sepharose beads carrying 20 µL anti-CXCR1 rabbit polyclonal (Serotec) or anti-CXCR2 rabbit polyclonal antibody (Santa Cruz Biotechnology Ltd.), respectively. Immunoprecipitation was done for 18 h at 4°C, following which the immunoprecipitated fraction was washed thrice with radioimmunoprecipitation assay buffer. For immunoblotting, protein samples retained on protein A-Sepharose beads were boiled in Laemmli's SDS sample buffer, resolved by SDS-PAGE, and electroblotted onto Hybond membranes (Hybond-P, Amersham Pharmacia). Membranes were probed with the following primary antibodies: (a) 1:500 dilution of anti-CXCR1 mouse monoclonal (Biosource) or (b) 1:500 dilution of anti-CXCR2 mouse monoclonal (Biosource) and subsequently with a 1:2,000 dilution of horseradish peroxidase–linked antirabbit IgG as the secondary antibody (Amersham Pharmacia). Antibody staining was detected using a chemiluminescence detection system (SuperSignal, Pierce).

Cell Count Proliferation Assay
Cells were seeded in 24-well plates at a density of 3 x 104 cells per well and allowed to adhere overnight. Cells were replenished by the addition of 1 mL fresh serum-free RPMI 1640 to each well, following which the signal transduction inhibitors were added. The cells were incubated at 37°C in a humidified 5% CO2 atmosphere during experimentation. Cells were trypsinized and counted in triplicate using a Coulter Z Series particle count and size analyzer (Beckman Coulter). Cell numbers were normalized to control values and statistical analysis of the data was done using GraphPad Prism 3.0 software.

Immunoblotting
Cells were lysed in radioimmunoprecipitation assay buffer containing 100 nmol/L Na3VO4, and protein lysates were subjected to SDS-PAGE as described above. Hybond-P membranes were probed with the following primary antibodies sourced from Cell Signaling Technology at a dilution of 1:1,000 unless otherwise indicated: anti–phospho-Thr202/Tyr204 p44/p42 Erk kinase, anti–p44/p42 Erk kinase, anti–phospho-Thr308-Akt, anti–phospho-Ser473-Akt, anti-Akt, anti–phospho-Thr37/Thr46-4E-BP1, anti-4E-BP1, anti–phospho-Thr421/Ser424-S6 kinase, anti–phospho-Thr389-S6 kinase, anti–phospho-Thr229-S6 kinase, anti-S6 kinase, anti–phospho-Ser235/Ser236-ribosomal S6 protein, anti-S6 protein, anti–phospho-Ser422 eIF4B, anti–phospho-Thr403/Thr410-PKC{zeta}/{lambda}, anti-PKC{zeta}/{lambda}, and mouse monoclonal anti-cyclin D1 (1:500 dilution; Oncogene Research Products). Antibody staining was detected using the SuperSignal chemiluminescence detection system.

PLD Assay
Cells were initially seeded at a density of 5 x 105 cells per well in RPMI 1640, supplemented with 10% FCS and 20 mmol/L L-glutamine. The medium was replaced with serum-free RPMI 1640 and the cells rested for a further 24 h at 37°C in a 5% CO2 humidified atmosphere. rh-IL-8 (3 nmol/L) was added to cells for predetermined times (1, 2, 5, 10, or 30 min) and the reactions terminated by aspiration of the medium and two cell washes in ice-cold PBS. Cells were harvested by scraping in radioimmunoprecipitation assay buffer (250 µL) and were immediately snap frozen in liquid nitrogen. On thawing, cell debris was pelleted by centrifugation at 13,000 rpm for 5 min at 4°C and the supernatant was decanted for experimentation. PLD activity was measured using the Amplex Red PLD assay kit (Molecular Probes, Inc.) according to the manufacturer's protocol. The resulting fluorescence was detected using a fluorescence microplate reader at an excitation wavelength of 563 nm and an emission wavelength of 587 nm (Applied Biosystems Cytofluor 4000).

Real-time PCR Analysis
RNA was harvested from cultured cells using RNAStat60 and cDNA was synthesized from 2 µg total RNA as per manufacturer's instructions (Invitrogen Life Technologies). Quantitative real-time PCR analysis was done on the DNA Engine Opticon 2 continuous fluorescence detector (MJ Research) with product amplification determined by SYBR Green 1 fluorescence detection (Finnzymes). The forward and reverse primers are as shown: cyclin D1, 5'-TTGCATGTTCGTGGCCTCTA-3' (forward) and 5'-AAATCGTGCGGGGTCTAA-3' (reverse), and 18S, 5'-CATTCGTATTGCGCCGCTA-3' (forward) and 5'-CGACGGTATCTGATCGTC-3' (reverse). Standard cycling procedures were used with an annealing temperature of 55°C for all primer pairs tested. Specific amplicon formation with each primer pair was confirmed by melt curve analysis. Gene expression was quantified relative to an 18S housekeeping gene.

siRNA Transfection
Cells were seeded into either 24-well plates for cell count analysis or p90 tissue culture dishes for Western blot analysis at densities of 3 x 104 and 2 x 105 cells per well, respectively, and allowed to attach overnight. Akt1, MAPK kinase, FRAP-1 SMARTpool siRNA, and siCONTROL nontargeting siRNA 1 were obtained from Dharmacon. PC3 cells were transfected with 10, 50, or 100 nmol/L of the oligonucleotides using the Dharmafect 2 transfection reagent (Dharmacon) in accordance with the manufacturer's instructions. For investigation of IL-8 inhibition, IL-8 SMARTpool siRNA (Dharmacon) and siCONTROL nontargeting siRNA 1 were used at a concentration of 20 nmol/L. For quantification of protein knockdown, protein lysates were prepared as described previously and Western blot analysis was done 72 h after transfection to validate efficiency of the Akt1, MAPK kinase, and mTOR siRNA. Inhibition of IL-8 gene function by IL-8–targeted siRNA was assessed by specific ELISA, as described previously (25).

Data Analysis
Differences observed in functional assays between control and treated groups were analyzed for statistical significance using a two-tailed Student's t test comparison (GraphPad Prism).


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Grant support: Ulster Cancer Foundation (P.G. Johnston and D.J.J. Waugh), the Royal Society grant 22865 (D.J.J. Waugh), the Northern Ireland HPSS R&D Office grant RRG9.14 (D.J.J. Waugh), and The Wellcome Trust grant 067104/Z/02/Z (D.J.J. Waugh).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: C.F. MacManus and J. Pettigrew contributed equally to this work.

Received 1/19/07; revised 3/15/07; accepted 5/ 1/07.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Huang S, Mills L, Mian B, et al. Fully humanized neutralizing antibodies to interleukin-8 (ABX-IL8) inhibit angiogenesis, tumor growth, and metastasis of human melanoma. Am J Pathol 2002;161:125–34.[Abstract/Free Full Text]
  2. Zhang XY, Chan WY, Whitney BM, et al. Changes of interleukin expression correlate with Helicobacter pylori infection and lymph node metastases in gastric carcinoma. Diagn Mol Pathol 2002;11:135–9.[Medline]
  3. Slaton JW, Inoue K, Perrotte P, et al. Expression levels of genes that regulate metastasis and angiogenesis correlate with advanced pathological stage of renal cell carcinoma. Am J Pathol 2001;158:735–43.[Abstract/Free Full Text]
  4. Xu L, Fidler IJ. Interleukin 8: an autocrine growth factor for human ovarian cancer. Oncol Res 2000;12:97–106.[Medline]
  5. Venkatakrishnan G, Salgia R, Groopman JE. Chemokine receptors CXCR-1/2 activate mitogen-activated protein kinase via the epidermal growth factor receptor in ovarian cancer cells. J Biol Chem 2000;275:6868–75.[Abstract/Free Full Text]
  6. Herrera CA, Xu L, Bucana CD, et al. Expression of metastasis-related genes in human epithelial ovarian tumors. Int J Oncol 2002;20:5–13.[Medline]
  7. Takamori H, Oades ZG, Hoch OC, Burger M, Schraufstatter IU. Autocrine growth effect of IL-8 and GRO{alpha} on a human pancreatic cancer cell line, Capan-1. Pancreas 2000;21:52–6.[CrossRef][Medline]
  8. Nomura H, Nishimori H, Yasoshima T, et al. A new liver metastatic and peritoneal dissemination model established from the same human pancreatic cancer cell line: analysis using cDNA macroarray. Clin Exp Metastasis 2002;19:391–9.[Medline]
  9. Shi Q, Abbruzzese JL, Huang S, Fidler IJ, Xiong Q, Xie K. Constitutive and inducible interleukin 8 expression by hypoxia and acidosis renders human pancreatic cancer cells more tumorigenic and metastatic. Clin Cancer Res 1999;5:3711–21.[Abstract/Free Full Text]
  10. De Larco JE, Wuertz BR, Rosner KA, et al. A potential role for interleukin-8 in the metastatic phenotype of breast carcinoma cells. Am J Pathol 2001;158:639–46.[Abstract/Free Full Text]
  11. Salcedo R, Martins-Green M, Gertz B, Oppenheim JJ, Murphy WJ. Combined administration of antibodies to human interleukin 8 and epidermal growth factor receptor results in increased antimetastatic effects on human breast carcinoma xenografts. Clin Cancer Res 2002;8:2655–65.[Abstract/Free Full Text]
  12. Miller LJ, Kurtzman SH, Wang Y, Anderson KH, Lindquist RR, Kreutzer DL. Expression of interleukin-8 receptors on tumor cells and vascular endothelial cells in human breast cancer tissue. Anticancer Res 1998;18:77–81.[Medline]
  13. Bendre MS, Gaddy-Kurten D, Mon-Foote T, et al. Expression of interleukin 8 and not parathyroid hormone-related protein by human breast cancer cells correlates with bone metastasis in vivo. Cancer Res 2002;62:5571–9.[Abstract/Free Full Text]
  14. Li A, Varney ML, Singh RK. Expression of interleukin 8 and its receptors in human colon carcinoma cells with different metastatic potentials. Clin Cancer Res 2001;7:3298–304.[Abstract/Free Full Text]
  15. Brew R, Erikson JS, West DC, Kinsella AR, Slavin J, Christmas SE. Interleukin-8 as an autocrine growth factor for human colon carcinoma cells in vitro. Cytokine 2000;12:78–85.[CrossRef][Medline]
  16. Inoue K, Slaton JW, Kim SJ, et al. Interleukin 8 expression regulates tumorigenicity and metastasis in human bladder cancer. Cancer Res 2000;60:2290–9.[Abstract/Free Full Text]
  17. Sheryka E, Wheeler MA, Hausladen DA, Weiss RM. Urinary interleukin-8 levels are elevated in subjects with transitional cell carcinoma. Urology 2003;62:162–6.[Medline]
  18. Karashima T, Sweeney P, Kamat A, et al. Nuclear factor-{kappa}B mediates angiogenesis and metastasis of human bladder cancer through the regulation of interleukin-8. Clin Cancer Res 2003;9:2786–97.[Abstract/Free Full Text]
  19. Mian BM, Dinney CP, Bermejo CE, et al. Fully human anti-interleukin 8 antibody inhibits tumor growth in orthotopic bladder cancer xenografts via down-regulation of matrix metalloproteases and nuclear factor-{kappa}B. Clin Cancer Res 2003;9:3167–75.[Abstract/Free Full Text]
  20. Inoue K, Slaton JW, Eve BY, et al. Interleukin 8 expression regulates tumorigenicity and metastases in androgen-independent prostate cancer. Clin Cancer Res 2000;6:2104–19.[Abstract/Free Full Text]
  21. Kim SJ, Uehara H, Karashima T, McCarty M, Shih N, Fidler IJ. Expression of interleukin-8 correlates with angiogenesis, tumorigenicity, and metastasis of human prostate cancer cells implanted orthotopically in nude mice. Neoplasia 2001;3:33–42.[CrossRef][Medline]
  22. Moore BB, Arenberg DA, Stoy K, et al. Distinct CXC chemokines mediate tumorigenicity of prostate cancer cells. Am J Pathol 1999;154:1503–12.[Abstract/Free Full Text]
  23. Veltri RW, Miller MC, Zhao G, et al. Interleukin-8 serum levels in patients with benign prostatic hyperplasia and prostate cancer. Urology 1999;53:139–47.[CrossRef][Medline]
  24. McCarron SL, Edwards S, Evans PR, et al. Influence of cytokine gene polymorphisms on the development of prostate cancer. Cancer Res 2002;62:3369–72.[Abstract/Free Full Text]
  25. Murphy C, McGurk M, Pettigrew J, et al. Non-apical and cytoplasmic expression of interleukin-8, CXCR1, and CXCR2 correlates with cell proliferation and microvessel density in prostate cancer. Clin Cancer Res 2005;11:4117–27.[Abstract/Free Full Text]
  26. Rosenwald IB. The role of translation in neoplastic transformation from a pathologist's point of view. Oncogene 2004;23:3230–47.[CrossRef][Medline]
  27. Fingar DC, Blenis J. Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 2004;23:3151–71.[CrossRef][Medline]
  28. De Benedetti A, Graff JR. eIF-4E expression and its role in malignancies and metastases. Oncogene 2004;23:3189–99.[CrossRef][Medline]
  29. Sozzani S, Agwu DE, Ellenburg MD, et al. Activation of phospholipase D1 by interleukin-8 in human neutrophils. Blood 1994;84:3895–901.[Abstract/Free Full Text]
  30. Jones SA, Wolf M, Qin S, Mackay CR, Baggiolini M. Different functions for the interleukin-8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D1 are activated through IL-8R1 but not IL-8R2. Proc Natl Acad Sci U S A 1996;93:6682–6.[Abstract/Free Full Text]
  31. Fang Y, Vilella-Bach M, Bachmann R, Flanigan A, Chen J. Phosphatidic acid-mediated mitogenic activation of mTOR signaling. Science 2001;294:1942–5.[Abstract/Free Full Text]
  32. Limatola C, Schapp P, Moolenaar WH, van Blitterswijk WJ. Phosphatidic acid activation of protein kinase C-{zeta} overexpressed in COS cells: comparison with other protein kinase C isotypes and other acidic lipids. Biochem J 1994;304:1001–8.[Medline]
  33. Lim H-K, Choi Y-A, Park W, et al. Phosphatidic acid regulates systemic inflammatory responses by modulating the Akt-mammalian target of rapamycin-p70 S6 kinase 1 pathway. J Biol Chem 2003;278:45117–27.[Abstract/Free Full Text]
  34. Weng QP, Andrabi K, Kozlowski MT, Grove JR, Avruch J. Multiple independent inputs are required for activation of the p70 S6 kinase. Mol Cell Biol 1995;15:2333–40.[Abstract]
  35. Weng QP, Kozlowski M, Belham C, et al. Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies. J Biol Chem 1998;273:16621–9.[Abstract/Free Full Text]
  36. Pullen N, Dennis PB, Andjelkovic M, et al. Phosphorylation and activation of p70 S6 kinase by PDK1. Science 1998;279:707–10.[Abstract/Free Full Text]
  37. Uehara H, Troncoso P, Johnston D, et al. Expression of interleukin-8 gene in radical prostatectomy specimens is associated with advanced pathologic stage. Prostate 2005;64:40–9.[Medline]
  38. Aaltomaa S, Eskelinen M, Lipponen P. Expression of cyclin A and D1 proteins in prostate cancer and their relation to clinopathological variables and patient survival. Prostate 1999;38:175–82.[CrossRef][Medline]
  39. Drobnjak M, Osman I, Scher HI, Fazzari M, Cordon-Cardo C. Overexpression of cyclin D1 is associated with metastatic prostate cancer to bone. Clin Cancer Res 2001;6:1891–5.
  40. Sonenberg N, Gingras A-C. The mRNA 5' cap-binding protein eIF4E and control of cell growth. Curr Opin Cell Biol 2000;10:268–75.
  41. Raught B, Peiretti F, Gingras A-C, et al. Phosphorylation of eukaryotic translation initiation factor 4B Ser422 is modulated by S6 kinases. EMBO J 2004;23:1761–9.[CrossRef][Medline]
  42. Knall C, Young S, Nick JA, Buhl AM, Worthen GS, Johnson GL. Interleukin-8 regulation of the Ras/Raf/mitogen-activated protein kinase pathway in human neutrophils. J Biol Chem 1996;271:2832–8.[Abstract/Free Full Text]
  43. Knall C, Worthen GS, Johnson GL. Interleukin 8-stimulated phosphatidylinositol-3-kinase activity regulates the migration of human neutrophils independent of extracellular signal-regulated kinase and p38 mitogen-activated protein kinases. Proc Natl Acad Sci U S A 1997;94:3052–7.[Abstract/Free Full Text]
  44. Malik SN, Brattain M, Ghosh PM, et al. Immunohistochemical demonstration of phospho-Akt in high Gleason grade prostate cancer. Clin Cancer Res 2002;8:1168–71.[Abstract/Free Full Text]
  45. Paweletz CP, Charboneau L, Bichsel VE, et al. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene 2001;20:1981–9.[CrossRef][Medline]
  46. Majumder PK, Yeh JJ, George DJ, et al. Prostate intra-epithelial neoplasia induced by prostate restricted Akt activation: the MPAKT model. Proc Natl Acad Sci U S A 2003;100:7841–6.[Abstract/Free Full Text]
  47. Li B, Sun A, Youn H, et al. Conditional Akt activation promotes androgen-independent progression of prostate cancer. Carcinogenesis 2007;28:572–83. Epub 2006 Oct 10.[Abstract/Free Full Text]
  48. Chen ML, Xu PZ, Peng XD, et al. The deficiency of Akt1 is sufficient to suppress tumor development in Pten+/– mice. Genes Dev 2006;20:1569–74.[Abstract/Free Full Text]
  49. Gao N, Zhang Z, Jiang BH, Shi X. Role of PI3K/AKT/mTOR signaling in the cell cycle progression of human prostate cancer. Biochem Biophys Res Commun 2003;310:1124–32.[CrossRef][Medline]
  50. Wu L, Birle DC, Tannock IF. Effects of the mammalian target of rapamycin inhibitor CCI-779 used alone or with chemotherapy on human prostate cancer cells and xenografts. Cancer Res 2005;65:2825–31.[Abstract/Free Full Text]
  51. Grunwald V, DeGraffenried L, Russel D, Friedrichs WE, Ray RB, Hidalgo M. Pharmacodynamic evaluation of CCI-779, an inhibitor of mTOR, in cancer patients. Cancer Res 2002;62:6141–5.[Abstract/Free Full Text]
  52. Cao C, Subhawong T, Albert JM, et al. Inhibition of mammalian target of rapamycin or apoptotic pathway induces authophagy and radiosensitizes PTEN null prostate cancer cells. Cancer Res 2006;66:10040–7.[Abstract/Free Full Text]
  53. Majumder PK, Febbo PG, Bikoff R, et al. mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways. Nat Med 2004;10:594–6.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 CarcinogenesisHome page
A. Seaton, P. Scullin, P. J. Maxwell, C. Wilson, J. Pettigrew, R. Gallagher, J. M. O'Sullivan, P. G. Johnston, and D. J. J. Waugh
Interleukin-8 signaling promotes androgen-independent proliferation of prostate cancer cells via induction of androgen receptor expression and activation
Carcinogenesis, June 1, 2008; 29(6): 1148 - 1156.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
T. T. Murooka, R. Rahbar, L. C. Platanias, and E. N. Fish
CCL5-mediated T-cell chemotaxis involves the initiation of mRNA translation through mTOR/4E-BP1
Blood, May 15, 2008; 111(10): 4892 - 4901.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
1541-7786.MCR-07-0032v1
5/7/737    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow