CXCR4/SDF1 mediate hypoxia induced chondrosarcoma cell invasion through ERK signaling and increased MMP1 expression
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Chondrosarcoma is a disease that does not respond to conventional cytotoxic chemotherapy and expression of MMP1 is a marker for a poor prognosis. The mechanism of increased MMP1 expression in chondrosarcoma is not completely known. Our goal is to identify molecular pathways that could serve as therapeutic targets. Chondrosarcoma become hypoxic as they grow, are capable of eliciting an angiogenic response, and typically metastasize to the lungs. The present study determined the effect of hypoxia and specifically HIF-1a on expression of CXCR4 and MMP1 and their role in chondrosarcoma cell invasion.
CXCR4 and its ligand, SDF1, are upregulated in primary chondrosarcoma tumors compared to normal articular cartilage, and CXCR4 was upregulated in chondrosarcoma cell line JJ compared to normal chondrocytes. Hypoxia and specifically HIF-1a increased CXCR4 and MMP1 expression in JJ cell line and chondrosarcoma invasion in vitro. The hypoxia mediated increase in MMP1 expression and chondrosarcoma invasion could be inhibited by siRNA directed at HIF-1a or CXCR4, the CXCR4 inhibitor AMD3100, as well as with ERK inhibitor U0126 and ERK siRNA.
Chondrosarcoma cell invasion is increased by hypoxia induced expression of CXCR4 and MMP1 and is mediated by HIF-1a and ERK. Both invasion and MMP1 can be inhibited with CXCR4 blockade, suggesting that CXCR4/SDF1 signaling may be a therapeutic target for chondrosarcoma.
KeywordsMMP1 Expression Chondrosarcoma CXCR4 Expression Chondrosarcoma Cell CXCR4 Signaling
Chondrosarcoma is the second most common primary malignant bone. It is a rare disease with a poor prognosis, usually occurs in adults, and the cure rate for this disease has not improved over the last several decades [1, 2]. For high grade tumors the cure rate has remained at 10-25%. The treatment for chondrosarcoma is surgical resection; chemotherapy and radiation therapy are not ordinarily used since chondrosarcoma are resistant to these adjuvant modalities. In contrast to chondrosarcoma, osteosarcoma, which usually occurs in adolescents, is sensitive to chemotherapy and the cure rate has increased from 20% to 75% with the advent of multiagent chemotherapy. However, in patients with either tumor, the majority of those who are not cured succumb to lung metastases. Our efforts are directed at elucidating the mechanisms of chondrosarcoma invasion and metastasis.
Invasion, angiogenesis, migration, and metastasis are intertwined processes regulated by overlapping molecular pathways. Chemokines and their receptors compose one such pathway and are involved with cell trafficking, migration, and proliferation. There are four groups of chemokine receptors: C, CC, CXC, and CX3C. Chemokine receptor four (CXCR4) is a seven-transmembrane G-protein-coupled receptor, whose activation leads to intracellular signaling cascades. CXCR4 is expressed in dendritic cells, naïve T cells, NK cells, and monocytes and is also the chemokine receptor most commonly expressed in tumors. Within normal cells chemokine receptors are important in immune cell function and migration of stem cells to sites of injury. Within tumor cells, chemokine receptor expression is related to development of metastases preferentially to sites with expression of the corresponding chemokine. The ligand for CXCR4 is the chemokine stromal cell derived factor one (SDF1) which is expressed in the lung and other sites of metastases. CXCR4/SDF1 also indirectly promotes tumor metastasis by mediating proliferation and migration of tumor cells and enhancing tumor-associated angiogenesis . The expression of chemokine receptors has been mostly investigated in carcinoma and increased levels of expression have been found in breast, gastric, colorectal, and lung cancer. CXCR4 expression has also been studied in melanoma, chondrosarcoma, and osteosarcoma. In the latter expression of CXCR4 correlates with overall survival, event-free survival, and metastasis free survival  For review see [7, 8].
Another factor that drives aggressive behavior in cancer is hypoxia. Hypoxia is a signal that develops as tumors outgrow their blood supply and results in a large number of adaptive changes aimed at surviving in the hypoxic environment as well as correcting the oxygen deficit. HIF-1 is a dimeric transcription factor composed of HIF-1 alpha and beta subunits. HIF-1 protein levels increase as a result of decreased degradation of the oxygen sensitive subunit HIF-1alpha. HIF-1 modulates changes in gene expression during hypoxia. One of the better characterized phenotypic changes induced by hypoxia is angiogenesis, largely mediated by HIF-1 and vascular endothelial growth factor (VEGF) which increases vessel ingrowth from surrounding tissue into the tumor. Our prior work has shown that grades II and III chondrosarcoma express higher levels of HIF-1 and VEGF than benign and grade I cartilage tumors Grades II and III chondrosarcoma are the tumors that metastasize and have poor survival. Hypoxia is also known to increase CXCR4 expression in other systems .
Tissue invasion by tumor cells and tumor induced blood vessels also requires matrix metalloproteinases. Specific tumors preferentially express different MMPs. In chondrosarcoma, MMP1 is the dominant metalloproteinase that is expressed and is a marker for poor prognosis . However, the mechanisms of increased MMP1 expression in chondrosarcoma are incompletely understood.
Therefore, we investigated the expression of CXCR4 in normal chondrocytes, normal cartilage, chondrosarcoma tissue, and chondrosarcoma cells (CS) and hypothesized that CXCR4 is overexpressed in chondrosarcoma, is upregulated by hypoxia and specifically by HIF-1, and increases the invasive phenotype by increasing expression of MMP1.
SDF1 and CXCR4 expression are increased in primary chondrosarcoma
Effect of hypoxia on endogenous CXCR4 expression in chondrosarcoma cell line
HIF-1a regulates CXCR4 expression
Effect of hypoxia, HIF-1a and CXCR4 knockdown, and CXCR4 blockade on invasion
Hypoxia and CXCR4 signaling increase MMP1 expression and activity
Downstream effects of hypoxia and CXCR4/SDF-1 are mediated through ERK signaling
A better understanding of the mechanisms underlying invasive behavior of a cancer is an important first step in developing improved treatment strategies. This study provides the first indication that CXCR4 is regulated by hypoxia and specifically HIF-1a in chondrosarcoma cells. We also show that increased CXCR4 signaling regulates expression of MMP1, a factor known to be involved with chondrosarcoma metastasis and a marker for poor prognosis. Overexpression of CXCR4 has been reported in a variety of tumors, primarily carcinoma. In carcinoma, CXCR4 expression mediates metastasis to bone, which has relatively high levels of SDF1. In chondrosarcoma, it is possible that local SDF1 stimulates local tumor growth in a paracrine manner, and for those cells which gain access to the circulation, may also partially account for the tendency of these tumors to develop lung metastases, since the lung also contains high levels of SDF1. Factors such as MMP1 mediate local migration out of the microenvironment, ie stroma for carcinoma and bone for chondrosarcoma, and into the circulation. Factors such as CXCR4 mediate homing and growth at distant sites. Within sarcoma, CXCR4 expression has been detected in osteosarcoma [6, 12]and recently in chondrosarcoma. Our results confirm the expression of CXCR4 in both chondrosarcoma tissue and cell lines and also show that CXCR4 expression was higher in high grade tumors, that hypoxia and HIF-1a enhance CXCR4/SDF1 mediated invasion through upregulation of CXCR4 expression, and that CXCR4/SDF1 signaling increases invasion through ERK mediated increase in MMP1 expression and activity.
Hypoxia, primarily acting through HIF-1a, elicits a wide spectrum of changes in gene expression that contribute to the metastatic phenotype of cancer cells. Hypoxia and Hif-1a have been shown to upregulate CXCR4 in carcinomas such as lung cancer , oral squamous cell carcinoma, breast carcinoma , and renal cell carcinoma . The mechanism of Hif-1a regulation of CXCR4 is through direct binding to the CXCR4 promoter . Our results show that HIF-1a also upregulates CXCR4 in chondrosarcoma. Interestingly, during chondrogenic differentiation CXCR4 is downregulated. Although chondrosarcoma share some markers of the cartilage phenotype, as cells become malignant, some repressed genes will be reexpressed. CXCR4 has been shown to be involved with cell migration and invasion in many systems. The data include in vitro invasion and migration assays as well as xenograft models of metastatic disease in which blockade of CXCR4 with drugs, peptides, or antibodies can inhibit development and growth of metastases. Independent of CXCR4, MMP1 has also been shown to be involved with tissue invasion and development of metastases. MMP1 is also known to be upregulated by hypoxia and HIF-1a in breast and lung cancer cells[20, 21], and also by CXCR4 in Nk cells  and prostate cancer cells . However, this project is the first to link the combined effects of HIF-1a on CXCR4 and MMP1 expression and the indirect effect of HIF-1a on MMP1 expression acting through CXCR4, which independently increases MMP1 in chondrosarcoma cells.
The role of MMP1 in chondrosarcoma invasion and its role as a poor prognostic indicator have been known for some time [11, 24]. Inhibition of MMP1 with siRNA has been shown to decrease chondrosarcoma cell invasion [25, 26, 27]. We have shown that one mechanism of increased MMP1 in chondrosarcoma is mediated through CXCR4 signaling, which is amplified by hypoxia, and is mediated by ERK, but not other MAP kinases. siRNA directed against HIF-1a, CXCR4, ERK; CXCR4 blockade with AMD3100; or ERK inhibitor U0126 all efficiently inhibited the increase in invasion of chondrosarcoma cells during hypoxia. A previous study of CXCR4 in chondrosarcoma invasion during normoxia showed that CXCR signaling increased expression of alphavbeta3 integrin, also through ERK, and that alphavbeta3 integrin antibodies could also inhibit chondrosarcoma invasion in vitro. Therefore, CXCR4 affects chondrosarcoma invasion through upregulation of multiple genes including alphavbeta3 integrin and MMP1. In other tumors and chondrosarcoma (Additional file 2), CXCR4 signaling upregulates other MMPs such as MMP-2, 8 and -9 and -13[28, 29]. Since CXCR signaling upregulates multiple genes related to metastasis and since clinical MMP inhibition is not currently feasible, whereas CXCR4 blockade is possible with drugs such as AMD3100, CXCR4 may be a fruitful therapeutic target to inhibit some of the metastatic potential of chondrosarcoma cells.
We present data that shows hypoxia mediated increase in MMP1 expression and chondrosarcoma invasion is partially mediated by CXCR4 signaling. CXCR4 blockade can inhibit the effects of hypoxia on MMP1 expression and chondrosarcoma invasion in vitro, suggesting that CXCR4 blockade could be a therapeutic target to inhibit chondrosarcoma invasion and metastasis. The effectiveness of this strategy requires in vivo confirmation.
Articular cartilage, chondrosarcoma tissue, and cancellous bone were obtained from surgical specimens, and either preserved in RNAlater Solution (Applied Biosystems, Foster City, CA) or snap frozen in liquid nitrogen for later use. There were 8 articular cartilage specimens and 16 chondrosarcoma (3 grade I, 5 grade II, and 8 grade III). IRB approval was obtained.
Cell lines and cell culture
Human chondrocytes isolated from normal adult articular cartilage and chondrosarcoma (CS) cell line JJ (a gift from Dr. Joel Block, Rush Medical School, Chicago, IL) were cultured in complete medium (40% DMEM, 40% MEM, 20% F12) with 10% FBS. All cells were cultured in a humidified incubator (NuAire Inc, Plymouth MN) under 5% CO2 and either normoxia (ambient oxygen) or hypoxia (2% O2) . JJ was derived from a human grade II chondrosarcoma. The drugs and inhibitors used were: AMD3100 (Sigma Chemical Company St. Louis, MO), human recombinant SDF-1 (R&D Systems, Minneapolis, MN), MMP inhibitor O-phenanthroline (Sigma), MAP kinase inhibitors: MEK1/2 inhibitor U0126 (Cell Signaling, Beverly, MA), JNK inhibitor SP600125 (A.G. Scientific Inc., San Diego, CA), p38 inhibitor SB203580 (BioMol International, Plymouth Meeting, PA) or DMSO (Sigma), solvent for the inhibitors.
Cells were transiently transfected with an expression construct for human Hif-1a in pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA), (a gift from Hirouki Kato PhD, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan), or empty vector using Fugene HD (Roche, Indianapolis, IN) in 6 or 12 well plates 24 h after seeding. Cells were then incubated for 48 h and harvested for the following experiments.
RNA interference (siRNA)
Cells were transfected with Hif-1a siRNA (Qiagen, Valencia, CA), CXCR4 siRNA (Dharmacon, Lafayette, CO), ERK1/2 siRNA(Cell Signaling Technology, Inc., Beverly, MA) or control siRNA by HiPerFect transfection reagent (Qiagen). RNA and protein were obtained 72 h after transfection for qRT-PCR and Western Blot analysis.
Real-time RT-PCR (qPCR)
RNA was isolated from cells with RNAqueous Kit (Ambion, Austin, TX) or tissues with Trizol Reagent (Invitrogen). After treatment with TURBO DNase (Ambion), one microgram of RNA was reverse transcribed with random hexamers to obtain first-strand cDNA using iScript cDNA kit (Bio-Rad, Philadelphia, PA). The quantification of mRNA for Hif-1a, CXCR4, SDF-1, and MMP1 was performed by two-step real-time quantitative RT-PCR (Qiagen). Primers (5'-3') for Hif-1a were: forward, ctc aaa gtc gga cag cct ca; reverse, ccc tgc agt agg ttt ctg ct; for CXCR4, forward: gtc cac gcc acc aac ag, reverse: ctg ttg gtg gcg tgg ac; for SDF-1, forward: cgt gct ggt cct cgt gct gac; reverse: gct ttc tcc agg tac tcc tg; for MMP1, forward: gag caa aca cat ctg acc tac agg a; and reverse, ttg tcc cga tga tct ccc ctg aca; 18S was used as an internal control since it has been shown to be the optimal reference gene. Amplification conditions were as follows: 2 min preincubation at 50°C, 10 minutes at 95°C for enzyme activation, and 40 cycles at 95°C denaturation for 10 s, 55°C annealing for 30 s (59°C for MMP1) and 72°C extension for 30 s. The comparative threshold cycle (Ct) method, i.e., 2-ΔΔCt method was used for the calculation of fold amplification. Each experiment was evaluated with three PCR reactions and each experiment was repeated three times. Data are presented as mean value ± SD.
Western Blot Analysis
Protein from cell lysates of tissues or cells were separated via SDS-PAGE and probed with antibodies for CXCR4, MMP1, actin (Santa Cruz Biotechnology, Santa Cruz, CA); and antibodies for Hif-1a, p-ERK, ERK, p-JNK, JNK, p-p38, and p38 (Cell Signaling Technology, Inc.). Western Blot analyses were performed as previously described . Protein concentrations were determined using the Bio-Rad Quick Start Bradford protein assay (Bio-Rad, Hercules, CA) and the equivalent of forty μg of protein were subjected to SDS-PAGE (Lonza Inc., Allendale, NJ).
After treatment, cells were cultured O/N in FBS free medium and the conditioned media (CM) from CS cells was concentrated using Centricon-30 centrifugal filter device (Millipore, Billerica, MA). The amount of active MMP1 was detected using Human Active MMP1 Fluorescent Assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Active MMP1 in the CM was measured in duplicate for each sample and normalized to the cell number at the end of the culture period. Each experiment was repeated three times.
Tumor cell invasion assay
Invasive activity of CS cells was analyzed with matrigel coated BD Falcon Cell Culture Inserts (BD Biosciences, Bedford, MA). Briefly, 180 μl of BD Matrigel Matrix Growth Factor Reduced (BD Biosciences, Bedford, MA) diluted 1:3 with serum-free medium was used to coat 8 μm pore size 12-well inserts and incubated at 37°C for 2 h. After various treatments, during which cells are cultured O/N without FBS, the cells were harvested by trypsinization, counted, and resuspended in complete medium containing 1% FBS at a concentration of 106/ml. 800 μl containing 8 × 105 cells were added to each of the upper wells. 1.5 ml of 5% FBS complete medium containing recombinant SDF1 (50 ng/ml, R&D Systems) was added to the lower wells. After incubating for 72 h in hypoxia, cells that had invaded across the membrane were stained with Cell Stain Solution (Millipore), washed, photographed, then lysed and cell number quantitated by absorbance at 560 nm on a standard microplate reader. The invasion index was calculated by normalizing to the number of cells invading when the lower well has no SDF1 or FBS.
All the experiments were repeated at least 3 times. Statistical analysis was performed with GraphPad Prism, v 3.0 (GraphPad Software, San Diego, CA). ELISA results and CXCR4 expression in different grades of chondrosarcoma were analyzed with one-way ANOVA. Post test comparisons were made with Bonferroni correction. Experiments with two groups were analyzed with the Student's t-test. The null hypothesis of no difference was rejected at a significance level of 5%.
The project described was supported by Grant Number 1P20RR024484-01, from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH) and its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. This work was also supported by the RIH Orthopaedic Foundation. RMT thanks Jack Wands, MD for advice and mentorship This paper was presented in abstract form at the 54th Annual Meeting of the Orthopaedic Research Society, San Francisco, 2008.
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