Metastasis-derived Cell Lines Research Articles

Down-regulated GAS1 expression correlates with recurrence in stage II and III colorectal cancer

Growth arrest-specific gene 1 had been associated with cell-cycle arrest, proliferation, and apoptosis. The aim of this study was to investigate the correlations between clinicopathologic factors and survival time and growth arrest-specific gene 1 expression in patients with stage II and III colorectal cancer (CRC). Quantitative real-time polymerase chain reaction was performed in 64 fresh CRC tissues to examine growth arrest-specific gene 1 mRNA expression. Six metastasis-derived and primary-derived cell lines were subjected to quantitative real-time polymerase chain reaction and Western blotting for further examination of both mRNA and protein concentrations. Growth arrest-specific gene 1 protein was immunostained in 118 paraffin-embedded specimens. Growth arrest-specific gene 1 expression was down-regulated both in tissues with recurrence and in metastasis-derived cell lines. Expression was unrelated to sex, age, tumor grade, or lymphovascular or perineural invasion. However, it was positively related to disease-free survival time (P < .05). Furthermore, lower growth arrest-specific gene 1 expression indicated a poorer survival rate (P < .05; log-rank test). Multivariate analysis also showed weak growth arrest-specific gene 1 protein expression to be an independent adverse prognosticator (P < .05). Taken together, our results support the idea that growth arrest-specific gene 1 contributes to predicting metastasis or recurrence in stage II and III CRC.

Analysis of RGS2 expression and prognostic significance in stage II and III colorectal cancer

The role of RGS2 (regulator of G-protein signalling 2) has been studied in several tumours. The purpose of the present study is to investigate the correlations between clinicopathological factors and patients' survival time and RGS2 expression in stage II and III CRC (colorectal cancer) patients. Real-time quantitative PCR was performed in 36 CRC tissues with recurrence and 28 without recurrence, and in three CRC-metastasis-derived cell lines (SW620, LoVo and Colo205) and 3 primary-CRC-derived ones (SW480, Caco-2 and HCT116) to examine RGS2 mRNA expression. In addition, to provide visualized evidence for RGS2 mRNA expression, random CRC samples were also performed with RT-PCR (reverse transcription-PCR). RGS2 protein was detected by immunostaining in 118 paraffin-embedded specimens, and the correlations between clinicopathological factors and survival time and RGS2 expression were analysed. We found that RGS2 mRNA was down-regulated both in CRC tissues with recurrence and metastasis-derived cell lines, and the expression level of RGS2 was unrelated to gender, age, tumour grade, or lymphovascular or perineural invasion. However, it was positively related to disease-free survival time (P<0.05). Furthermore, low RGS2 expression indicated a poorer survival rate (P<0.05, log-rank test). Multivariate analysis also showed that weak RGS2 protein expression was an independent adverse prognosticator in CRC (P<0.05). Taken together, we suggested that down-regulation of RGS2 might play an important role in CRC metastasis and predict poor prognosis in stage II and III CRC patients.

Abstract SY02-03: Studying tumor evolution in mouse models of cancer

Abstract Like other solid tumors, lung adenocarcinoma progresses through multiple stages including tumor initiation, expansion in the lung, and metastasis of the primary tumor to distant organs. Dissection of this process requires quantitative and tractable models that recapitulate the human disease at the genetic and histological level. We have employed autochthonous mouse models of lung adenocarcinoma that recapitulate tumor initiation, progression and metastasis in the human disease both genetically, and pathologically. We have established oncogenic Kras as the initiating event in lung adenoma and amplification of the oncogenic signaling is associated with progression to adenocarcinoma. The p53 tumor suppressor limits tumor progression in this model. Restoration of the Trp53 locus has pinpointed the adenoma-adenocarcinoma transition as the stage in which p53 function is critical for tumor suppression. Expression of oncogenic Kras in the absence of p53 leads to the development of distant metastases after long latency. Identification of the lentiviral integration site in each tumor allows the clonal relationship between metastases and the primary tumors to be unequivocally established. Gene expression and DNA copy number analyses of metastasis-derived cell lines and primary lung tumors have uncovered a metastasis associated gene expression signature and recurrent genomic alterations. Integration of these datasets, other genome-wide analyses and human tumor data are providing insights into the molecular mechanism and biology of lung cancer progression and metastasis. Citation Format: Tyler Jacks. Studying tumor evolution in mouse models of cancer [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY02-03

Abstract IA-4: Studying tumor evolution in mouse models of cancer

Abstract Like other solid tumors, lung adenocarcinoma progresses through multiple stages including tumor initiation, expansion in the lung, and metastasis of the primary tumor to distant organs. Dissection of this process requires quantitative and tractable models that recapitulate the human disease at the genetic and histological level. We have employed autochthonous mouse models of lung adenocarcinoma that recapitulate tumor initiation, progression and metastasis in the human disease both genetically, and pathologically. We have established oncogenic Kras as the initiating event in lung adenoma and amplification of the oncogenic signaling is associated with progression to adenocarcinoma. The p53 tumor suppressor limits tumor progression in this model. Restoration of the Trp53 locus has pinpointed the adenoma-adenocarcinoma transition as the stage in which p53 function is critical for tumor suppression. Expression of oncogenic Kras in the absence of p53 leads to the development of distant metastases after long latency. This suggests that unknown cooperating genetic events are required for metastatic spread and we are utilizing multiple tools to identify these changes. Use of lentiviral vectors that drive stable GFP expression in tumors allows for identification and purification of tumor cells. Additionally, identification of the lentiviral integration site in each tumor allows the clonal relationship between metastases and the primary tumors to be unequivocally established. Gene expression and DNA copy number analyses of metastasis-derived cell lines and primary lung tumors have uncovered a metastasis associated gene expression signature and recurrent genomic alterations. Integration of these datasets, other genome-wide analyses and human tumor data are providing insights into the molecular mechanism and biology of lung cancer progression and metastasis. Citation Information: Cancer Res 2009;69(23 Suppl):IA-4.

Differential baseline and response profile to IFN-γ gene transduction of IL-6/IL-6 receptor-α secretion discriminate primary tumors versus bone marrow metastases of nasopharyngeal carcinomas in culture

BackgroundUnderstanding of immunobiology of bone marrow metastases (designated BM-NPC) versus primary tumors (P-NPC) of the nasopharynx is far from complete. The aim of this study was to determine if there would be differences between cultured P-NPCs and BM-NPCs with respect to (i) constitutive IL-6 and the IL-6 receptor gp80 subunit (IL-6Rα) levels in the spent media of nontransduced cells, and (ii) IL-6 and IL-6Rα levels in the spent media of cells transduced with a retroviral vector containing the IFN-γ gene.MethodsA panel of NPC cell lines were transduced with the IFN-γ gene through a retroviral vector. Four clonal sublines were isolated via limiting dilution methods. Cytofluorometric analysis was performed for the detection of cell surface antigens of HLA class I, HLA class II and ICAM-1. ELISA was used to assay for IFN-γ, IL-6 and IL-6Rα in the spent media of cultured cell lines.ResultsOur results showed that in day 3 culture supernatants, low levels of soluble IL-6 were detected in 5/5 cultured tumors derived from P-NPCs, while much higher constitutive levels of IL-6 were detected in 3/3 metastasis-derived NPC cell lines including one originated from ascites; the difference was significant (p = 0.025). An inverse relationship was found between IL-6Rα and IL-6 in their release levels in cultured P-NPCs and metastasis-derived NPCs. In IFN-γ-transduced-P-NPCs, IL-6 production increased and yet IL-6Rα decreased substantially, as compared to nontransduced counterparts. At variance with P-NPC cells, the respective ongoing IL-6 and IL-6Rα release patterns of BM-NPC cells were not impeded as much following IFN-γ transduction. These observations were confirmed by extended kinetic studies with representative NPC cell lines and clonal sublines. The latter observation with the clonal sublines also indicates that selection for high IL-6 or low IL-6Rα producing subpopulations did not occur as a result of IFN-γ-transduction process. P-NPCs, which secreted constitutively only marginal levels of IFN-γ (8.4 ~ 10.5 pg/ml), could be enhanced to produce higher levels of IFN-γ (6.8- to 10.3-fold increase) after IFN-γ transduction. Unlike P-NPCs, BM-NPCs spontaneously released IFN-γ at moderate levels (83.8 ~ 100.7 pg/ml), which were enhanced by 1.3- to 2.2-fold in the spent media of their IFN-γ-transduced counterparts.ConclusionOur results showed that cultured P-NPCs and BM-NPCs could be distinguished from one another on the basis of their differential baseline secretion pattern of IFN-γ, IL-6 and IL-6Rα, and their differential response profiles to IFN-γ gene transfer of the production of these three soluble molecules. These results suggest that the IL-6 and IFN-γ pathways in a background of genetic instability be involved in the acquisition of metastatic behaviour in BM-NPCs.

A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene

Metastasis suppressor genes inhibit one or more steps required for metastasis without affecting primary tumor formation. Due to the complexity of the metastatic process, the development of experimental approaches for identifying genes involved in metastasis prevention has been challenging. Here we describe a genome-wide RNAi screening strategy to identify candidate metastasis suppressor genes. Following expression in weakly metastatic B16-F0 mouse melanoma cells, shRNAs were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increased metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1 (Growth arrest-specific 1), because we found that it was substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributed to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples. Thus, we developed a genome-wide shRNA screening strategy that enables the discovery of new metastasis suppressor genes.

Increased expression of p73Δex2 transcript in uveal melanoma with loss of chromosome 1p

The loss of chromosome 1p and chromosome 3 is associated with metastatic disease and decreased survival of uveal melanoma (UM) patients. The p53 homologues, p73 and p63, are located on chromosomes 1p and 3q, respectively. Both are able to activate p53 target genes, resulting in growth arrest, apoptosis and differentiation. N-terminally truncated isoforms of these genes may act as dominant negative inhibitors of wild-type p53 and transactivating activity. Although, p53 is frequently involved in several malignancies it does not play a major role in UM. Altered expression has been reported for both p63 and p73 in various malignancies. In this study, fluorescent in-situ hybridization was performed to identify gains or losses of p63 and p73 loci in UM. The expression of the different p63 and p73 isoforms was evaluated by reverse transcriptase PCR followed by Southern blot analysis. Furthermore, the expression pattern of the various DeltaTAp73 transcripts was analysed in seven primary UMs and 11 UM-derived cell lines using isoform-specific real-time PCR. Our results indicated that the isoform p73Deltaex2/3 was abundantly expressed and a relative loss of the p73 locus was associated with the upregulation of p73Deltaex2 and TAp73 transcripts. N-terminal transactivation forms of both p73 and p63 were observed in primary and metastasis-derived cell lines, as well as in primary melanomas, but in only one of the cell lines a DeltaNp63 mRNA transcript was observed. Our data suggest a potential function of p73 deletion transcripts in UM progression.

Chemokine Receptor Expression in Oral Squamous Cell Carcinoma: Correlation with Growth Factor- and Cytokine-mediated Cell Migration in vitro

Metastasis is the chief cause of mortality in cancer patients. Recently, chemokines and chemokine receptors were shown to play an important role in the metastasis of various cancers. We examined the role of chemokine receptor-mediated signaling in the invasion potential of human oral squamous cell carcinoma (OSCC) cell lines that were derived from 5 primary tumors and 6 cervical lymph node metastases. Comprehensive analysis of the mRNAs for human chemokine receptors showed that the OSCC cell lines had uniform expression patterns of chemokine receptors. Overall, there were no consistent differences in the expression of chemokine receptors between primary site- and lymph node metastasis-derived cell lines. However, a highly invasive OSCC cell line (SAS-H1) expressed up-regulation of CCR5, CCR6, CCR7, CXCR1, CXCR6 and CX3CR1 compared to a poorly invasive OSCC cell line (SAS-L1). Then we examined whether factors in the tumor microenvironment regulated chemokine receptor expression in SAS-H1 cells. Specifically, transforming growth factor (TGF) -β1 enhanced the expression of CCR5, CCR6, CCR7 and CX3CR1. Pretreatment of SAS-H1 cells with transforming growth factor (TGF) -β1 increased the expression of CCR7 and CX3CR1, and then enhanced CCL21- and CX3CL1-induced directional migration (1.5-fold enhancement as compared with untreated control). In addition, CX3CL1 increased the adhesion of SAS-H1 cells on uncoated tissue culture plates. Neither chemokine stimulated cell proliferation. Treatment of SAS-H1 cells with CX3CL1 activated the phosphotidylinositol-3-kinase (PI3K) and MEK signal transduction pathways. Our results suggest that chemokine receptor-mediated signaling is involved in the local invasion and metastasis of human OSCC.

Production d'hyaluronidase par les cellules cancéreuses humaines en culture

The presence of hyaluronidase was detected at pH 3.8 in eight out of twelve human cancer cell line culture media. Eight cell lines derived from primary tumours and four from metastases. In three culture media the enzymatic activity was lower than 0.035 pU/cell/h. In five others (in a hepatoma cell line and in four metastasis-derived cell lines) the activity was higher than 0.057 pU/cell/h. A tumour-derived fibroblast culture was negative. The optimal activity was observed at a pH comprised between 3.6 and 4. Salt inhibition of hyaluronidase was reversible. The enzyme was denaturated by a 10-min heating at 70 °C. The enzyme was not strictly specific for hyaluronan hydrolysis but also digested chondroitin sulfates. PH20, a spermatozoid protein that has homologies with the bee venom hyaluronidase, was not expressed by cell lines tested.

Spontaneous but not experimental metastatic activities differentiate primary tumor-derived vs metastasis-derived mouse prostate cancer cell lines.

We previously developed an in vivo mouse prostate reconstitution (MPR) model of metastatic prostate cancer using p53 'knockout' mouse urogenital sinus tissue for retroviral transduction of ras and myc oncogenes (Thompson et al., Oncogene, 10, 869, 1995). We further demonstrated contrasting responses to transforming growth factor beta-1 (TGF-beta1) in three matched pairs of early passage cell lines derived from primary prostate tumors and lung metastases generated by this model system (Sehgal et al., Cancer Res, 56, 3359, 1996). In this study we tested these cell lines for growth potential in subcutaneous and orthotopic (dorso-lateral prostate) locations and metastatic activities in both spontaneous and experimental assays. Subcutaneous and orthotopic tumors produced by cell lines derived from metastatic lesions tended to grow less rapidly but demonstrated greater spontaneous metastatic potential than the cell lines derived from primary tumors. In contrast all cell lines produced lung colonies in an experimental metastasis assay (tail vein inoculation) with the primary tumor-derived cell lines yielding higher activities in two of three matched pair analyses. The ability of all cell lines to produce lung metastases in the experimental assay, while only the metastasis-derived cell lines retain the ability to initiate and complete the entire metastatic pathway in the spontaneous assay, suggests that intravasation may be the rate-limiting step in metastasis in this model system.

Oncogene expression in related cancer lines differing in metastatic capacity.

Various murine tumor cell lines with different metastatic capacities were tested in vitro for oncogene expression, especially of the p21-Ha-ras protein. Small differences were seen in the expression of several distinct oncogenes in the case of a high metastatic lymphoma variant (ESb) and its low metastatic parental line (Eb). In one instance we observed a 30-fold Ha-ras gene amplification in a metastasis-derived cell line from a spontaneous mouse mammary carcinoma. In spite of this amplification we did not find an increased p21 expression in these cells.

Development and Biologic Properties of Malignant Cell Sublines and Clones of a Spontaneously Metastasizing Rat Mammary Adenocarcinoma23

With the use of the rat 13762 mammary adenocarcinoma tumor, an animal model for spontaneous mammary tumor metastasis was developed. The parental tumor implanted sc into the mammary fat pads of female Fischer 344 rats metastasized at low frequency to lymph nodes and lung and did not metastasize to other organs. Oophorectomy did not modify the metastatic properties of the parental 13762 tumor. Cell lines were adapted to tissue culture from lymph node or lung metastases, and these were compared to parental tumor transplants in spontaneous metastasis assays. Only the cell lines established from metastases were spontaneously metastatic within 23 days after sc implantation, indicating that tumor cell populations from metastases are more metastatic than are cells from the parental tumor. Several individual parental tumor-derived and lung metastasis-derived clones were compared for spontaneous metastatic potentials, cell culture morphologies, histologic structures at primary implant and secondary metastatic sites, and growth characteristics in vivo and in vitro. There was no correlation between any of these tumor and cellular properties and metastatic potential. The parental mammary tumor-derived, lymph node metastasis-derived, and lung metastasis-derived cell lines and clones with differing spontaneous metastatic properties should prove useful in studies on the roles of tumor cell and host properties in lymphatic and blood-borne metastasis.