Diverse chemokines and their receptors have been associated with tumor growth, tumor dissemination, and local immune escape. In different tumor entities, the level of chemokine receptor CXCR4 expression has been linked with tumor progression and decreased survival. The aim of this study was to evaluate the influence of CXCR4 expression on the progression of human renal cell carcinoma. CXCR4 expression of renal cell carcinoma was assessed by immunohistochemistry in 113 patients. Intensity of CXCR4 expression was correlated with both tumor and patient characteristics. Human renal cell carcinoma revealed variable intensities of CXCR4 expression. Strong CXCR4 expression of renal cell carcinoma was significantly associated with advanced T-status (P=.039), tumor dedifferentiation (P = .0005), and low hemoglobin (P = .039). In summary, strong CXCR4 expression was significantly associated with advanced dedifferentiated renal cell carcinoma.
1. Introduction
Renal cell carcinoma (RCC) is the sixth leading cause
of cancer-related deaths in the Western world and comprises 2-3% of all newly
diagnosed malignancies in adults. Among the different kidney neoplasms, it
represents with 85% the largest fraction [1]. The age-adjusted incidence of RCC
in Western nations is 5–12/100 000 in
women or men, respectively, with a peak incidence in the 6th decade [2]. In
practice, the only curable treatment is nephrectomy performed in early stages of
the disease. However, about 30–50% of patients
have already metastases at presentation, and approximately one third of the nephrectomized
patients relapse and progress with metastatic disease. The preferential sites
of metastasis are the regional lymph nodes, the lung, the liver, and the bones.
Survival strongly depends on the tumor stage at presentation. The 5-year
survival rate is approximately 50%, whereas the median survival in case of
metastasis is less than one year [3–5]. The current
standard treatment for metastasized RCC consists of the application of IFN-α
and IL-2 [6]. Recently, phase II clinical trials using receptor-tyrosine
kinase (RTK) inhibitors have shown more promising results and lead to
approval by the Food and Drug Administration (FDA) and European Medicines
Agency (EMEA) [2].
In vivo and in vitro results from different tumor
entities suggest that organ-specific metastasis is partially governed by
interactions of chemokine receptors on cancer cells and their corresponding
chemokines expressed in target organs and the tumor bed. This process is
considered to direct lymphatic and hematogenous spread and furthermore influences the
sites of metastatic growth [7]. Chemokines and their respective
G-protein-coupled receptors were initially described to mediate different pro-
and anti-inflammatory responses [8]. In particular, the high expression of
stromal cell derived factor 1α (SDF-1α), also known as CXCL12, by endothelial
cells, biliary epithelial cells, bone marrow stromal cells, and lymph nodes
results in a chemotactic gradient attracting CXCR4 expressing
lymphocytes into those organs [9–15]. Most recently, CXCR4 has shifted into focus as it
is the most common chemokine receptor expressed on cancer cells [16]. It was
suggested to play an important role in tumor spread of colorectal, breast, and oral
squamous cell carcinoma as all of them commonly metastasize to SDF-1α expressing organs [17–20]. Data
obtained from in vitro as well as from murine in vivo models, analyzing the
metastatic ability of CXCR4 in expressing cancer cells, underlined the
key role of CXCR4 for tumor cell malignancy, as activation of CXCR4 by SDF-1α induced migration, invasion, and angiogenesis of cancer
cells [21–23].
Therefore, we evaluated the
expression of CXCR4 in renal cancer cell lines and specimens and
correlated these results with the patients’ clinicopathological parameters and
survival.
2. Materials and Methods2.1. Tissue Samples
Renal cell
carcinoma samples were
intraoperatively obtained from 113 patients with renal clear cell carcinoma who
underwent surgery at the Department of Urology of the University of Mainz. The morphological classification of the carcinomas was conducted according to
World Health Organization (WHO) specifications. Patients were followed up on a
regular basis depending on the procedure performed.
2.2. Immunohistochemical Staining
The avidin-biotin-complex method
(LSAB+ System-HRP Kit, Dako Cytomation, Hamburg, Germany) was used to
detect the protein CXCR4 (anti-CXCR4, dilution 1 : 300; Capralogics
Inc., Mass, USA).
Formalin-fixed and paraffin-embedded tissues were deparaffinized and
subsequently microwaved (600 W, 15 minutes) in citrate buffer (ph 6.0). After
preincubation with hydrogen peroxide (LSAB+ System-HRP Kit, Dako Cytomation, Hamburg,
Germany) and human AB plasma (Department of Transfusion, University of Mainz,
Mainz, Germany), the primary antibodies were applied for one hour at room
temperature. After incubation with the secondary antibody (LSAB+ System-HRP
Kit, Dako Cytomation, Hamburg, Germany),
the avidin-biotin complex was added and the enzyme activity was visualized with
diaminobenzidine (LSAB+ System-HRP Kit, Dako Cytomation, Hamburg, Germany).
Counterstaining was performed with haematoxylin (Roth, Karlsruhe, Germany).
For negative controls only the secondary antibody was used. A negative control
was performed for each sample (N=113). For positive controls formalin-fixed and
paraffin-embedded tissue samples of the human spleen were applied.
2.3. Evaluation of Immunostaining
Immunostaining was evaluated by three authors
independently (T.C. Wehler, C. Graf, S. Biesterfeld), blinded to patient
outcome and all clinicopathologic findings. The immunohistochemical staining
was analyzed according to a scoring method as previously validated and
described [17]. The tumors were classified into four groups based on
the homogeneous staining intensity: 0, absent; 1, weak; 2, intermediate; 3,
strong staining. In the case of heterogeneous staining within the same sample, the respective higher score was chosen, if more than 50% of cells revealed a higher staining intensity. If expression intensity was exactly in between two scores, the authors agreed on 0.5 point-steps. If evaluations
did not agree, specimens were re-evaluated and reclassified according to the
assessment given most frequently by the observers.
2.4. Statistics
The correlation of CXCR4 staining
intensity with clinicopathological patterns was assessed with the χ2 test and with the unpaired Student t-test (one/two sided), when appropriate. Survival rates were visualized
applying Kaplan-Meier curves, and P-values were determined by log-rank
test. P<.05 was considered significant and P<.001
highly significant in all statistical analyses.
3. Results3.1. Tumor Characteristics and Patient Profiles
The selected group of patients represents the
typical characteristics of renal cell carcinoma in industrialized countries.
3.2. Immunohistochemical Staining of CXCR4 in Renal Cell Carcinoma
The staining of normal human kidney tissue for CXCR4 revealed a cytoplasmatic expression and in only few specimens an additional weak membranous location of CXCR4 (see Figure 1). A nuclear staining of CXCR4 was not observed. In renal
cell carcinoma, the respective expression rate for CXCR4 was 100%
(113/113) and varied from weak (34%), intermediate (42%), to strong (24%).
Negative controls of human renal cancer remained negative for all tissue
samples (N=113, not shown). Glomeruli did not reveal any CXCR4 expression and thus served as internal negative control. As internal positive control, splenic lymphocytes
(strong CXCR4 expression) and tubuli cells (intermediate CXCR4 expression) were used.
Similarly, inflammatory infiltrates in kidney tissue (data not shown) depicted
a strong CXCR4 expression.
The
figure depicts CXCR4 expression in healthy kidney and cancer samples. While
glomeruli did not depict any CXCR4 expression, tubuli did reveal a
medium-strong predominantly cytoplasmic CXCR4 expression. All cancer
samples did reveal a cytoplasmatic expression of CXCR4 ranging from weak
(34%) to medium (42%) and strong (24%).
3.3. Relevance of CXCR4 Expression in Renal Cell Carcinoma
Strong CXCR4 expression significantly correlated with
dedifferentiated (P=.0005) and progressed renal cell carcinoma, indicated by T-status (P=.039;
see Table 1). Furthermore, strong CXCR4 expression revealed a
significant association with low hemoglobin values (P=.039) and a nonsignificant trend towards
increased thrombocytes (P=.089/P=.18, resp.). No
correlation was seen for age, size, survival, or creatinine values.
Patient and tumor characteristics dependent on intensity of CXCR4 expression.
CXCR4 expression
Statistics
Weak
Medium
Strong
Total number
39 (34%)
47 (42%)
27 (24%)
Average age (years)
63.8
66.3
n.s.
Gender
Female
36 (42%)
8 (30%)
n.s.
Male
50 (58%)
19 (70%)
Grading
1/2
65 (78%)
11 (41%)
P=.0005
3/4
18 (22%)
15 (59%)
T-status
1/2
64 (76%)
15 (56%)
P=.039
3/4
20 (24%)
12 (44%)
Average size (cm)
5.7
6.0
n.s.
Survival (months)
29.7
36.8
n.s.
Average creatinin (mg/dl)
1.11
1.07
n.s.
Average hemoglobin (g/dl)
14.32
13.25
P=.019(1-sided)
P=.039(2-sided)
Average thrombocytes (/nl)
271
313
P=.089(1-sided)
P=.18(2-sided)
4. Discussion
The
expression of the chemokine receptor CXCR4 has been reported in various epithelial, mesenchymal, and hematopoietic tumors.
In several entities, its expression was linked to tumor dissemination and poor
prognosis [20, 24, 25]. CXCR4 expression can be increased as a result of intracellular second messengers such
as calcium [26] and cyclic AMP [27, 28] by the inactivation of the tumor
suppressor gene p53 and
overexpression of NFκB [29–31], by cytokines like IL-2, IL-10, or TGF-1β [26, 32] and by growth factors such as VEGF and EGF [33, 34]. In addition, Staller and colleagues could
demonstrate that CXCR4 is a hypoxia
inducible gene with a HIF-1α binding domain, and that its overexpression
in clear-cell renal cell carcinoma is due to a loss-of-function of the von
Hippel-Lindau (VHL) tumor suppressor
protein, which under normoxic conditions directs HIF-1α to ubiquitin-mediated degradation [35]. Loss of VHL stabilizes HIF-1α leading to
increased expression of hypoxia-response genes including VEGFA, CXCR4, its ligand SDF1α, and HIF-1α itself [36, 37]. They also
reported a positive correlation between strong CXCR4 expression and poor
tumor-specific survival independent of tumor stage and differentiation grade.
The latter is in contrast to the results obtained in our study.
We analyzed
the expression profile of CXCR4 in a
series of human renal cell carcinoma cell lines and 113 patients’ samples for
which exact tumor staging and followup data were available and correlated the
expression profile with clinicopathological data. The human renal cell
carcinoma tumor samples that are analyzed revealed varying intensities of CXCR4 expression ranging from weak to
strong, as previously described for pancreatic and colorectal cancer [38].
Interestingly, CXCR4 expression was downregulated in 34% and upregulated
in 24% of renal cell carcinoma as compared to original tubuli cells. 42% of
cancers revealed the identical expression intensity of CXCR4 as tubuli
cells. A cytoplasmatic staining of CXCR4 was observed in all cancers, whereas fewer cases depicted an additional
membranous localization of CXCR4.
These observations are in line with a recently published study by
Zagzag and coworkers [44]. Furthermore, it was reported that CXCR4 surface expression
was higher in permanent cell lines than in primary tumor samples [39]. Noteworthy, an inducible translocation of CXCR4 from the cytoplasm to the membrane
has been reported previously in [29]. In addition, at least in breast cancer
cells, inhibited CXCR4 ubiquitination
was described as another mechanism contributing to increased CXCR4 surface levels [40].
In our
renal cell carcinoma patients, a strong CXCR4 expression was
significantly associated as well with progressed cancer as indicated by the
T-status as with dedifferentiation. Our results are furthermore in line with
recent reports from our group and others, describing a similar effect of CXCR4 on disease progression in other
tumor entities [17, 41]. Hence, our data suggest a relevant influence of CXCR4 on proliferation and
differentiation of renal cell carcinoma with regard to the in vivo situation.
This hypothesis is strengthened by observations in a murine model, where the
metastatic capability of CXCR4-expressing
RCC cells strongly correlated with CXCR4 protein level on cancer cells and the SDF-1α expression in the target organs [23]. Therefore, CXCR4-expressing cancer cells are
certainly attracted to the typical “homing organs” such as lungs, bone marrow,
liver, and lymph-nodes showing a high SDF-1α expression [13, 42]. A pathophysiological
relevant fact worthwhile to be mentioned is that endothelial cells coexpress SDF-1α and VCAM-1,
thus mediating tumor-cell/endothelial cell attachment. CXCR4 activation by SDF-1α induces β-integrin expression, binding VCAM-1 on endothelial cell
[43, 44]. Similar pathophysiological processes must be proposed for renal cell
carcinoma dissemination.
Therefore, CXCR4 might be an interesting
therapeutic target in a multimodal therapy of renal clear cell carcinoma.
AbbreviationsCXCR4:
Chemokine receptor 4
EMEA:
European Medicines Agency
FDA:
Food and Drug Administration
HIF:
Hypoxia induced factor
IL:
Interleukin
RCC:
Renal cell carcinoma
RTK:
Receptor-tyrosine kinases
SDF-1α:
Stromal cell derived factor 1α
VHL:
Von Hippel Lindau
WHO:
World health organization.
Acknowledgment
The authors
thank the Sparkasse Pforzheim-Calw, Pforzheim, Germany, for
supporting their work.
AllinenM.BeroukhimR.CaiL.Molecular characterization of the tumor microenvironment in breast cancer200461173210.1016/j.ccr.2004.06.010AmatoR. J.Chemotherapy for renal cell carcinoma2000272177186AryaM.manit_arya@hotmail.comPatelH. R. H.Expanding role of chemokines and their receptors in cancer200336749752BaggioliniM.Chemokines and leukocyte traffic1998392667656556810.1038/33340BalkwillF.f.balkwill@icrf.icnet.ukMantovaniA.mantovani@marionegri.itInflammation and cancer: back to Virchow?2001357925553954510.1016/S0140-6736(00)04046-0BleulC. C.SchultzeJ. L.SpringerT. A.springer@sprsgi.med.harvard.eduB lymphocyte chemotaxis regulated in association with microanatomic localization, differentiation state, and B cell receptor engagement1998187575376210.1084/jem.187.5.753BrigatiC.NoonanD. M.AlbiniA.BenelliR.Tumors and inflammatory infiltrates: friends or foes?200219324725810.1023/A:1015587423262BurgerM.GlodekA.HartmannT.Functional expression of CXCR4 (CD184) on small-cell lung cancer cells mediates migration, integrin activation, and adhesion to stromal cells200322508093810110.1038/sj.onc.1207097CardonesA. R.MurakamiT.HwangS. T.hwangs@mail.nih.govCXCR4 enhances adhesion of B16 tumor cells to endothelial cells in vitro and in vivo via β1 integrin2003632067516757ChinniS. R.schinni@med.wayne.eduSivaloganS.DongZ.CXCL12/CXCR4 signaling activates Akt-1 and MMP-9 expression in prostate cancer cells: the role of bone microenvironment-associated CXCL122006661324810.1002/pros.20318CristilloA. D.HighbargerH. C.DewarR. L.DimitrovD. S.GoldingH.BiererB. E.biererb@nih.govUp-regulation of HIV coreceptor CXCR4 expression in human T lymphocytes is mediated in part by a cAMP-responsive element200216335436410.1096/fj.01-0744comHaoL.ZhangC.QiuY.Recombination of CXCR4, VEGF, and MMP-9 predicting lymph node metastasis in human breast cancer20072531344210.1016/j.canlet.2007.01.005HelbigG.ChristophersonK. W.IIBhat-NakshatriP.NF-κB promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4200327824216312163810.1074/jbc.M300609200JöhrerK.Karin.Joehrer@uibk.ac.atZelle-RieserC.PerathonerA.Up-regulation of functional chemokine receptor CCR3 in human renal cell carcinoma20051172459246510.1158/1078-0432.CCR-04-0405KatoM.KitayamaJ.KazamaS.NagawaH.Expression pattern of CXC chemokine receptor-4 is correlated with lymph node metastasis in human invasive ductal carcinoma200355R144R150LandisS. H.MurrayT.BoldenS.WingoP. A.Cancer statistics, 1999199949183110.3322/canjclin.49.1.8LaverdiereC.HoangB. H.YangR.Messenger RNA expression levels of CXCR4 correlate with metastatic behavior and outcome in patients with osteosarcoma20051172561256710.1158/1078-0432.CCR-04-1089LiY. M.PanY.WeiY.Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis20046545946910.1016/j.ccr.2004.09.027MaynardM. A.OhhM.michael.ohh@utoronto.cavon Hippel-Lindau tumor suppressor protein and hypoxia-inducible factor in kidney cancer200424111310.1159/000075346MehtaS. A.ChristophersonK. W.BroxmeyerH. E.KopelovichL.GouletR. J.Jr.NakshatriH.Understanding the metastatic switch in breast cancer: role of tumor suppressor p53 on expression of CXCR4, a chemokine receptor involved in site-specific metastasisProceedings of the American Association for Cancer Research, vol. 45, Abstract #3331, 2004MoriT.DoiR.doir@kuhp.kyoto-u.ac.jpKoizumiM.CXCR4 antagonist inhibits stromall cell-derived factor 1-induced migration and invasion of human pancreatic cancer2004312937MoriuchiM.MoriuchiH.TurnerW.FauciA. S.Cloning and analysis of the promoter region of CXCR4, a coreceptor for HIV-1 entry1997159943224329MotzerR. J.BanderN. H.NanusD. M.Renal-cell carcinoma19963351286587510.1056/NEJM199609193351207MotzerR. J.BacikJ.MazumdarM.Prognostic factors for survival of patients with stage IV renal cell carcinoma: memorial Sloan-Kettering Cancer Center experience200410186302s6303s10.1158/1078-0432.CCR-040031MurdochC.CXCR4: chemokine receptor extraordinaire2000177117518410.1034/j.1600-065X.2000.17715.xPanJ.jpan@mednet.ucla.eduMestasJ.jm8es@virginia.eduBurdickM. D.mdb5b@virginia.eduStromal derived factor-1 (SDF-1/CXCL12) and CXCR4 in renal cell carcinoma metastasis20065, article 5611410.1186/1476-4598-5-56PatelP. H.ChagantiR. S. K.MotzerR. J.motzerr@mskcc.orgTargeted therapy for metastatic renal cell carcinoma200694561461910.1038/sj.bjc.6602978PhillipsR. J.BurdickM. D.LutzM.BelperioJ. A.KeaneM. P.StrieterR. M.rstrieter@mednet.ucla.eduThe stromal derived factor-1/CXCL12-CXC chemokine receptor 4 biological axis in non-small cell lung cancer metastases2003167121676168610.1164/rccm.200301-071OCPhillipsR. J.MestasJ.Gharaee-KermaniM.Epidermal growth factor and hypoxia-induced expression of CXC chemokine receptor 4 on non-small cell lung cancer cells is regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signaling pathway and activation of hypoxia inducible factor-1α200528023224732248110.1074/jbc.M500963200PremackB. A.SchallT. J.Chemokine receptors: gateways to inflammation and infection19962111174117810.1038/nm1196-1174RichardC. L.TanE. Y.BlayJ.Adenosin increases cell-surface CXCR4 expression on HT-29 human colorectal carcinoma cellsProceedings of the American Association for Cancer Research, vol. 45, Abstract #3330, 2004RosenbergS. A.LotzeM. T.MuulL. M.A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone198731615889897SalcedoR.WassermanK.YoungH. A.Vascular endothelial growth factor and basic fibroblast growth factor induce expression of CXCR4 on human endothelial cells: in vivo neovascularization induced by stromal-derived factor-1α1999154411251135SchimanskiC. C.dr_schimanski@yahoo.deSchwaldS.SimiantonakiN.Effect of chemokine receptors CXCR4 and CCR7 on the metastatic behavior of human colorectal cancer20051151743175010.1158/1078-0432.CCR-04-1195StallerP.SulitkovaJ.LisztwanJ.MochH.OakeleyE. J.KrekW.wilhelm.krek@cell.biol.ethz.chChemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL2003425695530731110.1038/nature01874Stetler-StevensonW. G.The role of matrix metalloproteinases in tumor invasion, metastasis, and angiogenesis2001102383392TeradaR.YamamotoK.kazuhide@md.okayama-u.ac.jpHakodaT.Stromal cell-derived factor-1 from biliary epithelial cells recruits CXCR4-positive cells: implications for inflammatory liver diseases2003835665672TwitchellD. D.LondonN. R.Jr.TomerD. P.TomerS.MurrayB. K.O'NeillK. L.Tannic acid prevents angiogenesis in vivo by inhibiting CXCR4/SDF-1 alpha binding in breast cancer cellsProceedings of the American Association for Cancer Research, vol. 45, Abstract #51, 2004UchidaD.daisuke@dent.tokushima-u.ac.jpBegumN.-M.AlmoftiA.Possible role of stromal-cell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma2003290228930210.1016/S0014-4827(03)00344-6WaldO.PappoO.SafadiR.Involvement of the CXCL12/CXCR4 pathway in the advanced liver disease that is associated with hepatitis C virus or hepatitis B virus20043441164117410.1002/eji.200324441WangJ.wangj@cber.fda.govGuanE.RoderiquezG.CalvertV.AlvarezR.NorcrossM. A.norcross@cber.fda.govRole of tyrosine phosphorylation in ligand-independent sequestration of CXCR4 in human primary monocytes-macrophages200127652492364924310.1074/jbc.M108523200WehlerT.WolfertF.SchimanskiC. C.Strong expression of chemokine receptor CXCR4 by pancreatic cancer correlates with advanced disease200616611591164YangL.JacksonE.WoernerB. M.PerryA.Piwnica-WormsD.RubinJ. B.Rubin_J@kids.wustl.eduBlocking CXCR4-mediated cyclic AMP suppression inhibits brain tumor growth in vivo200767265165810.1158/0008-5472.CAN-06-2762ZagzagD.KrishnamacharyB.YeeH.Stromal cell-derived factor-1α and CXCR4 expression in hemangioblastoma and clear cell-renal cell carcinoma: von Hippel-Lindau loss-of-function induces expression of a ligand and its receptor200565146178618810.1158/0008-5472.CAN-04-4406