Possible role of stromal-cell-derived factor-1/CXCR4 signaling on lymph node metastasis of oral squamous cell carcinoma

We examined the role of chemokine signaling on the lymph node metastasis of oral squamous cell carcinoma (SCC) using lymph node metastatic (HNt and B88) and nonmetastatic oral SCC cells. Of 13 kinds of chemokine receptors examined, only CXCR4 expression was up-regulated in HNt and B88 cells. CXCR4 ligand, stromal-cell-derived factor-1alpha (SDF-1alpha; CXCL12), induced characteristic calcium fluxes and chemotaxis only in CXCR4-expressing cells. CXCR4 expression in metastatic cancer tissue was significantly higher than that in nonmetastatic cancer tissue or normal gingiva. Although SDF-1alpha was undetectable in either oral SCC or normal epithelial cells, submandibular lymph nodes expressed the SDF-1alpha protein, mainly in the stromal cells, but occasionally in metastatic cancer cells. The conditioned medium from lymphatic stromal cells promoted the chemotaxis of B88 cells, which was blocked by the CXCR4 neutralization. SDF-1alpha rapidly activated extracellular signal-regulated kinase (ERK)1/2 and Akt/protein kinase B (PKB), and their synthetic inhibitors attenuated the chemotaxis by SDF-1alpha. SDF-1alpha also activated Src family kinases (SFKs), and its inhibitor PP1 diminished the SDF-1alpha-induced chemotaxis and activation of both ERK1/2 and Akt/PKB. These results indicate that SDF-1/CXCR4 signaling may be involved in the establishment of lymph node metastasis in oral SCC via activation of both ERK1/2 and Akt/PKB induced by SFKs.     

Involvement of miR-518c-5p to Growth and Metastasis in Oral Cancer

We have previously demonstrated that a stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral cancer. Recently, small non coding RNAs, microRNAs (miRNAs) have been shown to be involved in the metastatic process of several types of cancers. However, the miRNAs that contribute to metastases induced by the SDF-1/CXCR4 system in oral cancer are largely unknown. In this study, we examined the metastasis-related miRNAs induced by the SDF-1/CXCR4 system using B88-SDF-1 oral cancer cells, which exhibit functional CXCR4 and distant metastatic potential in vivo. Through miRNA microarray analysis, we identified the upregulation of miR-518c-5p in B88-SDF-1 cells, and confirmed the induction by real-time PCR analysis. Although an LNA-based miR-518c-5p inhibitor did not affect cell growth of B88-SDF-1 cells, it did significantly inhibit the migration of the cells. Next, we transfected a miR-518c expression vector into parental B88 cells and CAL27 oral cancer cells and isolated stable transfectants, B88-518c and CAL27-518c cells, respectively. The anchorage-dependent and -independent growth of miR-518c transfectants was significantly enhanced compared with the growth of mock cells. Moreover, we detected the enhanced migration of these cells. The LNA-based miR-518c-5p inhibitor significantly impaired the enhanced cell growth and migration of miR-518c transfectants, indicating that these phenomena were mainly dependent on the expression of miR-518c-5p. Next, we examined the function of miR-518c-5p in vivo. miR-518c transfectants or mock transfectants were inoculated into the masseter muscle or the blood vessels of nude mice. Tumor volume, lymph nodes metastasis, and lung metastasis were significantly increased in the mice inoculated with the miR-518c transfectants. These results indicated that miR-518c-5p regulates the growth and metastasis of oral cancer as a downstream target of the SDF-1/CXCR4 system.     

Functional expression of CXC chemokine recepter-4 mediates the secretion of matrix metalloproteinases from mouse hepatocarcinoma cell lines with different lymphatic metastasis ability

CXC chemokine recepter-4 (CXCR4) and its ligand, stromal cell-derived factor-1alpha (SDF-1alpha) have been implicated in the organ-specific metastasis of several malignancies. Hca-F and its syngeneic cell line Hca-P are mouse hepatocarcinoma cell lines with high and low potential of lymphatic metastasis, respectively. Previous studies showed that the secretion of matrix metalloproteinases (MMPs) associated with the metastatic ability of Hca-F and Hca-P cell line depending on the lymph node environment. However, the mechanism of this process has remained unclear. This study investigated the roles of CXCR4 on Hca-F cell and SDF-1alpha of lymph node in lymphatic metastasis. The RT-PCR and Flow cytometry analysis results show that Hca-F cells express higher level CXCR4 mRNA and cell-surface CXCR4 protein, as compared with Hca-P cells. Treatment of recombinant SDF-1alpha proteins induced greater amount of calcium-flux in Hca-F cells than that in Hca-P cells, demonstrating higher functional CXCR4 expression on Hca-F cells than that on Hca-P cells. Furthermore, both the cell-free extratcs of lymph node and recombinant SDF-1alpha proteins induced secretions of active MMP-9 and MMP-2 from Hca-F cells in vitro. But those secretions were significantly reduced by blockade of cell surface CXCR4 with rabbit anti-mouse CXCR4 polyclonal antibody (pAb) and neutralization of SDF-1alpha in lymph node extracts with rabbit anti-mouse SDF-1alpha pAb as well. These results suggest that the CXCR4/SDF-1alpha system mediates active MMP-9 and MMP-2 secretion from Hca-F and Hca-P cells, which facilitates lymphogenous metastasis of those cells consequently.     

Lower expression of CXCR4 in lymph node metastases than in primary breast cancers: potential regulation by ligand-dependent degradation and HIF-1alpha

Stromal-derived factor-1 (SDF-1) is a unique ligand of the CXC chemokine receptor 4 (CXCR4), which is critically involved in the metastasis of breast cancer. High levels of SDF-1 in the common destination organs of metastasis, such as the lymph nodes, lungs, liver, and bones, attract CXCR4-positive tumor cells. The interaction between SDF-1 and CXCR4 leads to the activation of specific signaling pathways, allowing for homing and metastatic progression. However, regulation of CXCR4 expression at the metastatic organ site is not well-documented. We detected the expression of CXCR4 and hypoxia inducible factor (HIF)-1alpha in breast tumor tissues by immunohistochemical staining and analyzed SDF-1 in primary tumors and lymph nodes using real-time RT-PCR. Compared to the corresponding metastasized tumors in the lymph nodes, primary invasive carcinomas showed more intense staining for CXCR4, particularly on the cellular membrane. Both primary tumors and lymph node metastases exhibited higher levels of CXCR4 expression compared to non-neoplastic breast tissues. Therefore, we hypothesized that the tumor environment in the lymph nodes may cause the reduction of CXCR4 levels in the metastatic tumor cells because of: (1) high SDF-1 levels and (2) lower levels of HIF-1alpha. Our in vitro data demonstrated that high levels of SDF-1 can induce the internalization and degradation of CXCR4 through the lysosome pathway. In addition, lower levels of HIF-1alpha in the lymph node metastases, probably induced by the less hypoxic environment, further lowered CXCR4 levels. These results indicate that ligand-dependent degradation and lower HIF-1alpha levels may be potential causes of lowered levels of CXCR4 in the lymph nodes compared to the primary tumors. Our study suggests that CXCR4 levels in tumor cells are regulated by its microenvironment. These findings may enhance our ability to understand the biological behavior of breast cancers.     

The Role of Metabotropic Glutamate Receptor 5 on the Stromal Cell-Derived Factor-1/CXCR4 System in Oral Cancer

We have demonstrated that blocking CXCR4 may be a potent anti-metastatic therapy for CXCR4-related oral cancer. However, as CXCR4 antagonists are currently in clinical use to induce the mobilization of hematopoietic stem cells, continuous administration as an inhibitor for the metastasis may lead to persistent leukocytosis. In this study, we investigated the novel therapeutic downstream target(s) of the SDF-1/CXCR4 system, using B88-SDF-1 cells, which have an autocrine SDF-1/CXCR4 system and exhibit distant metastatic potential in vivo. Microarray analysis revealed that 418 genes were upregulated in B88-SDF-1 cells. We identified a gene that is highly upregulated in B88-SDF-1 cells, metabotropic glutamate receptor 5 (mGluR5), which was downregulated following treatment with 1,1’ -[1,4-Phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), a CXCR4 antagonist. The upregulation of mGluR5 mRNA in the SDF-1/CXCR4 system was predominately regulated by the Ras-extracellular signal-regulated kinase (ERK)1/2 pathway. Additionally, the growth of B88-SDF-1 cells was not affected by the mGluR5 agonist (S)-3,5-DHPG (DHPG) or the mGluR5 antagonists 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP). However, we observed that DHPG promoted B88-SDF-1 cell migration, whereas both MPEP and MTEP inhibited B88-SDF-1 cell migration. To assess drug toxicity, the antagonists were intraperitoneally injected into immunocompetent mice for 4 weeks. Mice injected with MPEP (5 mg/kg) and MTEP (5 mg/kg) did not exhibit any side effects, such as hematotoxicity, allergic reactions or weight loss. The administration of antagonists significantly inhibited the metastasis of B88-SDF-1 cells to the lungs of nude mice. These results suggest that blocking mGluR5 with antagonists such as MPEP and MTEP could prevent metastasis in CXCR4-related oral cancer without causing side effects.     

CXCR4–SDF-1 Signalling, Locomotion, Chemotaxis and Adhesion

Chemokines, small pro-inflammatory chemoattractant cytokines, that bind to specific G-protein-coupled seven-span transmembrane receptors present on plasma membranes of target cells are the major regulators of cell trafficking. In addition some chemokines have been reported to modulate cell survival and growth. Moreover, compelling evidence is accumulating that cancer cells may employ several mechanisms involving chemokine-chemokine receptor axes during their metastasis that also regulate the trafficking of normal cells. Of all the chemokines, stromal-derived factor-1 (SDF-1), an alpha-chemokine that binds to G-protein-coupled CXCR4, plays an important and unique role in the regulation of stem/progenitor cell trafficking. First, SDF-1 regulates the trafficking of CXCR4+ haemato/lymphopoietic cells, their homing/retention in major haemato/lymphopoietic organs and accumulation of CXCR4+ immune cells in tissues affected by inflammation. Second, CXCR4 plays an essential role in the trafficking of other tissue/organ specific stem/progenitor cells expressing CXCR4 on their surface, e.g., during embryo/organogenesis and tissue/organ regeneration. Third, since CXCR4 is expressed on several tumour cells, these CXCR4 positive tumour cells may metastasize to the organs that secrete/express SDF-1 (e.g., bones, lymph nodes, lung and liver). SDF-1 exerts pleiotropic effects regulating processes essential to tumour metastasis such as locomotion of malignant cells, their chemoattraction and adhesion, as well as plays an important role in tumour vascularization. This implies that new therapeutic strategies aimed at blocking the SDF-1-CXCR4 axis could have important applications in the clinic by modulating the trafficking of haemato/lymphopoietic cells and inhibiting the metastatic behaviour of tumour cells as well. In this review, we focus on a role of the SDF-1-CXCR4 axis in regulating the metastatic behaviour of tumour cells and discuss the molecular mechanisms that are essential to this process.     

Stromal Derived Factor-1 (SDF-1) and Its Receptors CXCR4 and CXCR7 in Endometrial Cancer Patients

Purpose

One of the most important function of stromal derived factor-1 (SDF-1) and its receptors, is regulating the process of metastasis formation. The aim of our study was to investigate the correlation between SDF-1, CXCR4 and CXCR7 protein levels measured by immunohistochemistry with the clinicopathological features and the survival of endometrial cancer patients.

Materials and Methods

92 patients aged 37–84 (mean 65.1±9.5) were enrolled to our study between January 2000 and December 2007. After the diagnosis of endometrial cancer, all women underwent total abdominal hysterectomy, with bilateral salpingoophorectomy and pelvic lymph node dissection. In all patients clinical stage (according to FIGO classification), histologic grade, myometrial invasion, lymph node and distant metastases were determined.Furthermore, the survival time was assessed. Immunohistochemical analyses of SDF-1, CXCR4 and CXCR7 were performed on archive formalin fixed paraffin embedded tissue sections.

Results

Statistically significant correlations (p<0.01) were reported between SDF-1 and the clinical stage of disease, lymph node metastases, distant metastases, deep myometrial invasion (≥50%), cervical involvement, involvement of adnexa. Statistically significant correlation (p<0.01) was found between SDF-1 expression and the risk of the recurrence. Higher SDF-1 expression was associated with ahigher risk of recurrence (p = 0.0001). The results of CXCR4and CXCR7 expression didn''t reveal any significant differences(p>0.05) between the proteins expression in the primary tumor cells and the clinicopathological features. Moreover, the Kaplan-Meier analyses demonstrated a stepwise impairment of cancer overall survival (OS) with increasing SDF-1 expression.

Conclusion

The important role of SDF-1 as a predictor of negative clinicopathological characteristics of atumor suggests that the expression of this stromal factor should be included in the panel of accessory pathomorphological tests and could be helpful in establishing a more accurate prognosis in endometrial cancer patients.     

Transcutaneous Carbon Dioxide Induces Mitochondrial Apoptosis and Suppresses Metastasis of Oral Squamous Cell Carcinoma In Vivo

Squamous cell carcinoma (SCC) is the main histological type of oral cancer. Its growth rate and incidence of metastasis to regional lymph nodes is influenced by various factors, including hypoxic conditions. We have previously reported that transcutaneous CO₂ induces mitochondrial apoptosis and decreases lung metastasis by reoxygenating sarcoma cells. However, previous studies have not determined the sequential mechanism by which transcutaneous CO₂ suppresses growth of epithelial tumors, including SCCs. Moreover, there is no report that transcutaneous CO₂ suppresses lymphogenous metastasis using human cell lines xenografts. In this study, we examined the effects of transcutaneous CO₂ on cancer apoptosis and lymphogenous metastasis using human SCC xenografts. Our results showed that transcutaneous CO₂ affects expressions of PGC-1α and TFAM and protein levels of cleavage products of caspase-3, caspase-9 and PARP, which relatives mitochondrial apoptosis. They also showed that transcutaneous CO₂ significantly inhibits SCC tumor growth and affects expressions of HIF-1α, VEGF, MMP-2 and MMP-9, which play essential roles in tumor angiogenesis, invasion and metastasis. In conclusion, transcutaneous CO₂ suppressed tumor growth, increased mitochondrial apoptosis and decreased the number of lymph node metastasis in human SCC by decreasing intra-tumoral hypoxia and suppressing metastatic potential with no observable effect in vivo. Our findings indicate that transcutaneous CO₂ could be a novel therapeutic tool for treating human SCC.     

AMD3100 inhibits epithelial–mesenchymal transition,cell invasion,and metastasis in the liver and the lung through blocking the SDF-1α/CXCR4 signaling pathway in prostate cancer

Stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) have been found to be tightly correlated with the progression of prostate cancer (PC). In this study, we investigated the effects of an SDF-1α/CXCR4 inhibitor, AMD3100, on cell progression and metastasis potential of human PC cells. Human PC cell lines (LNCaP, PC3, and DU145) were cultured to detect SDF-1α/CXCR4, which showed higher SDF-1α and CXCR4 expression than the normal human prostate epithelial cell line, RWPE-1. AMD3100 was confirmed to be an inhibitor of SDF-1α, and to detect the effect of SDF-1α/CXCR4 inhibition on PC, PC cells were treated with AMD3100 or/and CXCR4 siRNA. The results suggested that inhibition of the SDF-1α/CXCR4 pathway could promote the E-cadherin level but inhibit the levels of invasion and migration of vimentin, N-cadherin and α5β1 integrin. Finally, tumor formation in nude mice was conducted, and the cell experiment results were verfied. These data show that AMD3100 suppresses epithelial–mesenchymal transition and migration of PC cells by inhibiting the SDF-1α/CXCR4 signaling pathway, which provides a clinical target in the treatment of PC.     

Chemokine receptor CXCR4-beta1 integrin axis mediates tumorigenesis of osteosarcoma HOS cells.

It is known that beta1 integrins mediate the migratory response of cells to chemokine stimulation. Also, both beta1 integrins and chemokines have roles in tumor development. In the present study, the beta1 integrin-chemokine axis is assessed using human osteosarcoma (HOS) transfectant cells expressing the CXCR4 receptor for chemokine SDF-1 (CXCL12). We first identified in vitro the specific beta1 integrins that mediated the migratory response to SDF-1 stimulation. Results showed that on collagen type I and laminin, the chemotactic response to SDF-1 was predominantly mediated by alpha2beta1 integrin. On fibronectin, SDF-1-stimulated chemotaxis involved both alpha4beta1 and alpha5beta1 integrins. A comparison of the transfectant clones expressing CXCR4 at low, intermediate, and high levels and the control transfectant revealed that the transfectant clones migratory response in vitro and their ability to form tumors in vivo was related to their levels of CXCR4 expression. In addition, treatment by injection with mAbs to CXCR4, integrin alpha2beta1, or integrin alpha5beta1 effectively inhibited the growth of HOS-CXCR4 transfectant cells in vivo. Therefore, our results show that the beta1 integrins that mediated the migratory response were also functionally linked to the enhanced tumor growth of CXCR4-expressing HOS transfectant cells.     

CXCR4/SDF-1 pathway is crucial for TLR9 agonist enhanced metastasis of human lung cancer cell

Accumulating data suggested that CXCR4/SDF-1 pathway may play an important role in the metastasis of tumor. We previously demonstrated that CpG ODN could enhance the metastasis of human lung cancer cell via TLR9. Here we further evaluated the possible role of CXCR4/SDF-1 pathway in the enhanced metastasis of human lung cancer 95D cells induced by CpG ODN. Our data showed down-regulation of CXCR4 expression using siRNA against CXCR4 could significantly reduce the enhanced metastasis of 95D cells induced by CpG ODN both in vitro and in vivo. These results suggested that TLR9 agonist might promote the metastasis of human lung cancer cells via CXCR4/SDF-1 pathway.     

KITENIN increases invasion and migration of mouse squamous cancer cells and promotes pulmonary metastasis in a mouse squamous tumor model

KAI1 C-terminal interacting tetraspanin (KITENIN) is reported to promote metastasis in mouse colon cancer models. We investigated the role of KITENIN on the progression of squamous cell carcinoma (SCC). In a preliminary clinical study using resected tissues from head and neck SCC patients, KITENIN was highly expressed in tumors and metastatic lymph nodes, while KAI1 was more increased in adjacent mucosa than in tumor. KITENIN-transfected mouse squamous cancer (SCC VII/KITENIN) cells showed significantly higher invasion, migration, and proliferation than empty vector-transfected cells. In syngeneic mouse squamous tumor models, more increased tumor volume and enhanced lung metastasis were found in SCC VII/KITENIN cells-injected mice. Thus, KITENIN increases invasion and migration of squamous cancer cells and thereby promotes distant metastasis in mouse squamous tumor models.     

Cutaneous squamous cell carcinoma metastatic to chronic lymphocytic leukaemia: diagnostic potential of fine needle aspiration cytology

OBJECTIVE: The phenomenon of cancer-to-cancer metastasis of cutaneous squamous cell carcinoma (SCC) and chronic lymphocytic leukaemia (CLL)/small lymphocytic lymphoma (SLL) is a rare event and only occasionally documented in the medical literature. METHODS: Two patients with SCC of the skin that were previously treated for CLL are presented. Both had palpable lymph nodes in the neck and fine needle aspiration cytology (FNAC) was performed to evaluate the pathological process. In addition, the literature on cutaneous SCC metastatic to CLL/SLL with special emphasis on the role of FNAC in this specific clinical situation was reviewed. RESULTS: On examination of the FNAC smear, cancer-to-cancer metastasis of cutaneous SCC to lymph node replaced by CLL was found. In one of the patients, light microscopy examination of the smear was complemented by immunostaining of atypical cells with cytokeratin antibodies and immunophenotyping of lymphoid cells by flow cytometry. In addition to our two patients, nine cases of cutaneous SCC metastatic to CLL/SLL have been reported in the literature, and in only one was the diagnosis made by FNAC. CONCLUSION: FNAC supported by ancillary immunocytological techniques could also be used in diagnosis of specific clinical situations such as cancer-to-cancer metastasis of the tandem of SCC-CLL/SLL.     

人肝癌原位移植转移模型特性实验研究

目的　建立人肝癌细胞系HCC-9724(简称H)淋巴结转移模型,研究肿瘤转移机理。方法　采用裸鼠肝脏原位移植法,接种肿瘤细胞,取其淋巴结转移灶反复肝内接种,连续传三代后,观察其转移特性,采用SABC法测定淋巴结中nm23和Ⅳ型胶原酶表达。结果　裸鼠原位接种50d,肝内长出约1.7cm×6.0cm大小的肿瘤,呈分叶状,质地较软,周围血供丰富,瘤组织与邻近脏器粘连,有明显的浸润和转移,经裸鼠三次筛选后肿瘤潜伏期短(15d),瘤体大,形成广泛的肠系膜淋巴结转移,淋巴结中Ⅳ型胶原酶表达呈强阳性;而nm23呈弱阳性。结论　采用裸鼠肝原位移植法,反复筛选,获得了人肝癌淋巴结高转移模型。     

SDF-1alpha promotes invasion of head and neck squamous cell carcinoma by activating NF-kappaB

CXCL12/stromal cell-derived factor-1alpha (SDF-1alpha), a chemokine ligand for the G protein-coupled receptor CXCR4, plays an important role in the directed movement of cells. Many studies have documented the importance of CXCR4 in tumor progression and organ-specific metastasis. Recently, several studies have implicated a role for SDF-1alpha in head and neck squamous cell carcinoma (HNSCC) metastasis, but currently there is little information about how SDF-1alpha promotes HNSCC metastasis. In this report we show that the NF-kappaB signaling pathway is activated in response to SDF-1alpha in HNSCC while primary and immortalized keratinocytes show no SDF-1alpha-mediated NF-kappaB activity. We found that SDF-1alpha-mediated NF-kappaB signaling is independent of phosphoinositide 3-kinase/Akt and ERK/MAPK pathways. We observed that SDF-1alpha induces IkappaBalpha phosphorylation and degradation and the nuclear translocation of NF-kappaB in HNSCC cell lines, suggesting that SDF-1alpha activates the classical NF-kappaB signaling pathway. Contrary to previous reports, SDF-1alpha-induced NF-kappaB activation is not mediated by tumor necrosis factor alpha. Furthermore, blocking the NF-kappaB signaling pathway with an IKKbeta inhibitor significantly reduces SDF-1alpha-mediated HNSCC invasion. Taken together, our data suggest SDF-1alpha/CXCR4 may promote HNSCC invasion and metastasis by activating NF-kappaB and that targeting NF-kappaB may provide therapeutic opportunities in preventing HNSCC metastasis mediated by SDF-1alpha.     

Interaction of ligand-receptor system between stromal-cell-derived factor-1 and CXC chemokine receptor 4 in human prostate cancer: a possible predictor of metastasis

Interaction of ligand-receptor systems between stromal-cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) is closely involved in the organ specificity of cancer metastasis. We hypothesized that SDF-1-CXCR4 ligand-receptor system plays an important role in prostate cancer metastasis. To test this hypothesis, expression level of SDF-1 and CXCR4 was analyzed in prostate cancer (PC) cell lines (LNCaP, PC3, and DU145) and normal prostate epithelial cell line (PrEC). We also performed migration assay and MTT assay to investigate the chemotactic effect and growth-promoting effect of SDF-1 on DU145 and PC3 cells, respectively. Furthermore, we performed immunohistochemical analysis of CXCR4 expression in tissues from 35 cases of human prostate cancer. CXCR4 expression was detected in all three prostate cancer cell lines, but not in PrECs. SDF-1 significantly enhanced the migration of PC3 and DU145 cells in a dose-dependent manner, and anti-CXCR4 antibody inhibited this chemotactic effect. However, SDF-1 itself did not significantly stimulate the cell growth rate of prostate cancer cell lines. Positive CXCR4 protein was found in 20 out of 35 clinical PC samples (57.1%). Three patients with lung metastasis showed definitely positive CXCR4 immunostaining. Logistic regression analysis revealed that positive expression of CXCR4 protein was an independent and superior predictor for bone metastasis to Gleason sum (P < 0.05). Furthermore, among PC patients with PSA greater than 20 ng/mL, the positive rate of CXCR4 protein was significantly higher in patients with bone metastasis than in those with no bone metastasis (P = 0.017). These findings suggest that the interaction between SDF-1 and CXCR4 ligand-receptor system is involved in the process of PC metastasis by the activation of cancer cell migration. This is the first report to investigate the role of interaction of ligand-receptor systems between SDF-1 and CXCR4 in prostate cancer metastasis.     

Development of an experimental model for spontaneous lymph node metastasis of human esophageal carcinoma in nude mice--histopathological analysis]

Three human squamous cell carcinoma cell lines (HPL-EsC-1-K, -S, and -M) originated from a male patient with esophageal carcinoma were established and were studied on their tumorigenic and metastatic properties in nude mice. All cell lines grew in the hind foot pads following subcutaneous inoculation and produced popliteal lymph node metastasis dose (2-8 x 10(6)/mouse)-dependently. Based on the histopathological findings on serial sections of the lymph nodes, the stages of lymph node invasion by cancer cells were classified into 4 stages (St. 0-III). The time course of lymph node metastasis of EsC-K cells were examined. Advanced stage of metastasis increased according to the time elapsed after tumor cell inoculation. Incidence of metastasis of EsC-K cells were not affected by host factors such as sex differences, anti-asialo GM1 antibody treatment on the hosts. Today, there are few experimental models for studies on spontaneous lymph node metastasis of human carcinomas. This experimental model provides a useful research tool for studies on the biology and therapy for lymph node metastasis of esophageal cancer.     

Expression of stromal derived factor-1 (SDF-1) and chemokine receptor (CXCR4) in bone metastasis of renal carcinoma

Renal cancer is a relatively common malignant carcinoma that metastasizes to bone. The chemokine stromal derived factor-1 (SDF-1) and its corresponding receptor CXCR4 have been shown to regulate organ-specific metastasis in other cancer types. Based on this observation, we predicted that the expressions of SDF-1 and CXCR4 play a role in renal carcinoma metastasis to bone. To investigate the expressions of SDF-1 and CXCR4, and to assess the correlation between SDF-1 and CXCR4 immunoreactivity in bone metastasis of renal carcinoma, we collected 10 in situ renal carcinoma samples and 30 bone metastasis samples. We analyzed SDF-1 and CXCR4 expression with immunohistochemical analysis on paraffin-embedded sections. Compared with primary renal carcinomas, the SDF-1 expression in bone metastases was significantly higher [80% (24/30) vs. 30% (3/10), P = 0.006]; the expression of CXCR4 was also higher [83.3% (25/30) vs. 40% (4/10), P = 0.014]. Pearson correlation analysis supports a positive correlation between SDF-1 and CXCR4 in bone metastasis of renal carcinoma. In addition, RT-PCR demonstrated that, as compared with in situ renal carcinoma tissues, SDF-1 expression was predominant in the bone metastasis samples (P = 0.001), while CXCR4 was overexpressed in the bone metastasis tissues (P = 0.028). Western blot analysis confirmed these trends. Our data suggest that the expression of SDF-1/CXCR4 is high in bone metastases and over-expression of SDF-1/CXCR4 may play important roles in the bone metastasis of renal carcinoma.     

Diverse signaling pathways through the SDF-1/CXCR4 chemokine axis in prostate cancer cell lines leads to altered patterns of cytokine secretion and angiogenesis

The establishment of metastatic bone lesions in prostate cancer (CaP) is a process partially dependent on angiogenesis. Previously we demonstrated that the stromal-derived factor-1 (SDF-1 or CXCL12)/CXCR4 chemokine axis is critical for CaP cell metastasis. In this investigation, cell lines were established in which CXCR4 expression was knocked down using siRNA technology. When CaP cells were co-transplanted with human vascular endothelial cells into SCID mice, significantly fewer human blood vessels were observed paralleling the reductions in CXCR4 levels. Likewise, the invasive behaviors of the CaP cells were inhibited in vitro. From these functional observations we explored angiogenic and signaling mechanisms generated following SDF-1 binding to CXCR4. Differential activation of the MEK/ERK and PI3K/AKT pathways that result in differential secretion IL-6, IL-8, TIMP-2 and VEGF were seen contingent on the cell type examined; VEGF and TIMP-2 expression in PC3 cells are dependent on AKT activation and ERK activation in LNCaP and LNCaP C4-2B cells leads to IL-6 or IL-8 secretion. At the same time, expression of angiostatin levels were inversely related to CXCR4 levels, and inhibited by SDF-1 stimulation. These data link the SDF-1/CXCR4 pathway to changes in angiogenic cytokines by different signaling mechanisms and, suggest that the delicate equilibrium between proangiogenic and antiangiogenic factors may be achieved by different signal transduction pathways to regulate the angiogenic phenotype of prostate cancers. Taken together, our results provide new information regarding expression of functional CXCR4 receptor-an essential role and potential mechanism of angiogenesis upon SDF-1 stimulation.     

Systematic analysis of genes involved in oral cancer metastasis to lymph nodes

Oral cancer remains a deadly disease worldwide. Lymph node metastasis and invasion is one of the causes of death from oral cancer. Elucidating the mechanism of oral cancer lymph node metastasis and identifying critical regulatory genes are important for the treatment of this disease. This study aimed to identify differentially expressed genes (gene signature) and pathways that contribute to oral cancer metastasis to lymph nodes. The GSE70604-associated study compared gene profiles in lymph nodes with metastasis of oral cancer to those of normal lymph nodes. The GSE2280-associated study compared gene profiles in primary tumor of oral cancer with lymph node metastasis to those in tumors without lymph node metastasis. There are 28 common differentially expressed genes (DEGs) showing consistent changes in both datasets in overlapping analysis. GO biological process and KEGG pathway analysis of these 28 DEGs identified the gene signature CCND1, JUN and SPP1, which are categorized as key regulatory genes involved in the focal adhesion pathway. Silencing expression of CCND1, JUN and SPP1 in the human oral cancer cell line OECM-1 confirmed that those genes play essential roles in oral cancer cell invasion. Analysis of clinical samples of oral cancer found a strong correlation of these genes with short survival, especially JUN expression associated with metastasis. Our study identified a unique gene signature – CCND1, JUN and SPP1 – which may be involved in oral cancer lymph node metastasis.