High Metastatic Variant Research Articles

EFFECT OF LACTATE DEHYDROGENASE INHIBITION BY OXAMATE ON LEWIS LUNG CARCINOMA CELLS WITH DIFFERENT METASTATIC POTENTIAL.

Today, the ability for metabolic reprogramming is considered one of the distinguishing features of metastatically active tumor cells, a classic example of which is aerobic glycolysis. Despite a large number of studies in this direction, the question of the relationship between the intensity of aerobic glycolysis and the metastatic potential of tumor cells remains almost completely open. The work aimed to investigate the effect of the lactate dehydrogenase (LDH) inhibitor on the viability and several characteristics of Lewis lung carcinoma cells with different metastatic potential. High-metastatic (LLC) and low-metastatic (LLC/R9) variants of Lewis lung carcinoma cells were used. After 24 h of tumor cells incubation with or without 40 mM sodium oxamate, cell viability, the concentration of glucose and lactate in the incubation medium, distribution of cells by the cell cycle phases, and intracellular ROS production were estimated. It was revealed that regardless of the metastatic potential, LLC cells are heterogeneous in terms of both the involvement of aerobic glycolysis in their growth and survival processes and the sensitivity to the cytotoxic/cytostatic action of an LDH inhibitor. 35% of cells of either LLC variant form an oxamate-resistant subpopulation while 65% are oxamate-sensitive. The rate of glucose consumption of LLC/R9 cells in the absence of oxamate is almost twice higher compared to LLC and, as a result, the sensitivity of these cells to the cytotoxic/cytostatic effect of oxamate also is significantly higher (the IC50 for LLC/R9 cells is by 35.8% lower than that for LLC cells, p < 0.05). Approximately one-third of the cells of both LLC and LLC/R9 variants can survive and proliferate when aerobic glycolysis is completely inhibited by oxamate. This indicates metabolic reprogramming (either pre-existing or dynamically arising in response to inhibition of glycolysis) of this subpopulation of cells, within which not only the survival of cells but also their proliferative activity is most likely based on glutamine metabolism. Such metabolic heterogeneity of metastatically active cells indicates that inhibition of glycolysis as monotherapy is insufficient for effective antimetastatic therapy. Presumably, more effective would be to involve various inhibitors of metabolic processes that ensure the metabolic plasticity of metastatic cells.

The disintegrin echistatin in combination with doxorubicin targets high-metastatic human osteosarcoma overexpressing ανβ3 integrin in chick embryo and nude mouse models.

Echistatin, a cyclic RGD peptide, which is an antagonist of αvβ3 integrin (disintegrin), inhibited human osteosarcoma in the chick chorioallontoic membrane (CAM) model and tumor growth and pulmonary metastases in a nude mouse orthotopic model. A high-metastatic variant of human osteosarcoma, 143B-LM4, overexpressing αvβ3 integrin was used. Tumor angiogenesis by high-metastatic variant 143B-LM4 cells in the CAM was significantly inhibited by echistatin (P<0.05) as was overall growth. A doxorubicin (DOX)-echistatin combination inhibited orthotopic tumor growth compared to untreated control (P<0.01) or DOX alone (P<0.05) in nude mice. Tumor-bearing mice treated with the DOX-echistatin combination survived longer than those treated with DOX alone or control PBS (P<0.01 and P<0.01, respectively). Echistatin also inhibited experimental lung metastasis of 143B-LM4 cells in nude mice. These results suggest that DOX in combination with a disintegrin has potential to treat osteosarcoma and that αvβ3 integrin may be a target for osteosarcoma.

Enhanced Metastatic Recurrence Via Lymphatic Trafficking of a High-Metastatic Variant of Human Triple-Negative Breast Cancer After Surgical Resection in Orthotopic Nude Mouse Models.

We previously developed and characterized a highly invasive and metastatic triple-negative breast cancer (TNBC) variant by serial orthotopic implantation of MDA-MB-231 human breast cancer cells in nude mice. Eventually, a highly invasive and metastatic variant of human TNBC was isolated after lymph node metastases was harvested and orthotopically re-implanted into the mammary gland of nude mice for two cycles. The variant thereby isolated is highly invasive in the mammary gland and metastasized to lymph nodes in 10 of 12 mice compared to 2 of 12 of the parental cell line. In the present report, we observed that high-metastatic MDA-MB-231H-RFP cells produced significantly larger subcutaneous tumors compared with parental MDA-MB-231 cells in nude mice. Extensive lymphatic trafficking by high-metastatic MDA-MB-231 cells was also observed. High-metastatic MDA-MB-231 developed larger recurrent tumors 2 weeks after tumor resection compared with tumors that were not resected in orthotopic models. Surgical resection of the MDA-MB-231 high-metastatic variant primary tumor in orthotopic models also resulted in rapid and enhanced lymphatic trafficking of residual cancer cells and extensive lymph node and lung metastasis that did not occur in the non-surgical mice. These results suggest that surgical resection of high metastatic TNBC can greatly increase the malignancy of residual cancer. J. Cell. Biochem. 118: 559-569, 2017. © 2016 Wiley Periodicals, Inc.

Disintegrin targeting of an αvβ3 integrin-over-expressing high-metastatic human osteosarcoma with echistatin inhibits cell proliferation, migration, invasion and adhesion in vitro.

The in vitro efficacy of the disintegrin echistatin was tested on a high-metastatic variant of 143B human osteosarcoma, 143B-LM4, which over-expresses αvβ3 integrin. Echistatin is an RGD cyclic peptide and an antagonist of αvβ3 integrin. In the present study, echistatin inhibited cell proliferation, migration, invasion, and adhesion of 143B-LM4 cells. 143B-LM4 cell proliferation decreased after treatment with echistatin in a time-dependent and dose-dependent manner (P <0.01). In vitro migration and invasion of 143B-LM4 cells were also inhibited by echistatin in a dose-dependent manner (P <0.01, respectively). Cell adhesion to vitronectin of 143B-LM4 cells was also inhibited by echistatin in a dose-dependent manner (P <0.01). These results suggest that αvβ3 integrin may be an effective target for osteosarcoma.

Real-Time In Vivo Confocal Fluorescence Imaging of Prostate Cancer Bone-Marrow Micrometastasis Development at the Cellular Level in Nude Mice.

In the present report, we demonstrate in vivo fluorescence imaging of bone-marrow micrometastasis of prostate cancer at the cellular level in nude mice. PC-3 human prostate cancer cells labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP) were injected into the left ventricle or intratibial bone marrow of nude mice. PC-3-GFP, as well as selected high metastatic variants of PC-3-GFP, PC-3-GFP-BM6 or PC-3-RFP were visualized by real-time fluorescence imaging, to traffic and grow in the bone marrow. Formation of bone marrow micrometastasis could be imaged at the single-cell level in live mice, using confocal microscopy. The ability to track bone marrow micrometastasis in real time at the cellular level provides a visual target for evaluating new therapeutics for this recalcitrant disease. J. Cell. Biochem. 117: 2533-2537, 2016. © 2016 Wiley Periodicals, Inc.

Prostate Cancer Heterogeneous High-Metastatic Multi-Organ-Colonizing Chemo-Resistant Variants Selected by Serial Metastatic Passage in Nude Mice Are Highly Enriched for Multinucleate Giant Cells.

In order to further understand the role of tumor heterogeneity in metastasis and chemo-resistance, high metastatic PC-3 human prostate cancer variants were selected by injecting parental PC-3 cells, expressing green fluorescent protein (GFP) in the footpad of nude mice, which then metastasize to inguinal lymph nodes. The PC-3-GFP cells which metastasized to the inguinal lymph nodes were collected and were re-injected to the footpad. After 6 such cycles, the PC-3-GFP cells collected from inguinal lymph nodes (PC-3-GFP-LN) were again injected to the footpad. PC-3-GFP-LN showed 100% metastasis to major lymph nodes (popliteal, inguinal, axillary, and cervical), and 100% metastasis to bone and lung. The percent of giant cell variants was enriched in PC-3-GFP-LN-6 compared to parental cells and increased with each cycle of selection, which in turn had increased metastasis. PC-3-GFP-LN-6 cells were resistant to 5-fluorouracil, doxorubicin and cisplatinum, compared to parental PC-3. However, PC-3-GFP-LN-6 was sensitive to the traditional Chinese medicine (TCM) herbal mixture LQ, similar to the parental cells. These results suggest that PC-3 tumors are heterogenous and that subpopulations of highly metastatic, drug-resistant cells can be step-wise selected using a mouse model of tumor progression.

Abstract 4128: Extensive morphological heterogeneity of circulating and selected high metastatic variants and differential chemosensitivity of human prostate cancer in mouse models

Abstract Circulating PC-3-GFP human prostate cancer cells were collected from the blood from nude mice orthotopically implanted with PC-3-GFP. In parallel experiments, a highly metastatic PC-3-GFP cell line was developed by injecting parental PC-3-GFP to the footpad of nude mice which metastasized to inguinal lymph nodes from which the cancer cells were collected and were re-injected to the footpad. After 6 cycles, the PC-3-GFP cells collected from inguinal lymph nodes (PC-3-GFP-LN) and were injected to the footpad. PC-3-GFP-LN showed 100% metastasis to major lymph nodes (popliteal, inguinal, axillary, and cervical), and 100% metastasis to bone and lung. PC-3-GFP-CTC and PC-3-GFP-LN were cultured in vitro, and their morphology and chemo-sensitivity were comparing to the parental PC-3-GFP cells. Highly pleomorphic subpopulations of prostate cancer cells were identified in PC-3-GFP-CTC and PC-3-GFP-LN. The different cell subtypes included characteristic high malignancy features such as high nucleus to cytoplasmic (N/C) ratios and giant cell formation as a result of abnormal mitoses. Variations in growth rate, plating efficiency, size, shape, and number of the nuclei and nucleoli were observed. The results suggest extensive tumor heterogeneity in both sublines. The giant cells, contain multiple nuclei, cannot survive by themselves and need small cells around them in order to proliferate. However, giant cells also can produce smaller cells, which divide further. The ratio of giant cells/total cells were significantly higher in PC-3-GFP-LN compared to parental cells. However, the morphology of cells from lung metastasis and bone metastasis showed lung and bone epithelial features such as squamous on shape and tightly packed together. PC-3-GFP-CTC had similar sensitivity to both cisplatinum and docetaxel when compared to PC-3-parental cells. PC-3-GFP-LN was resistant. In contrast, PC-3-GFP-LN was sensitive to the traditional Chinese medicine (TCM) herbal mixture LQ similar to the parental cells. These results demonstrate that circulating tumor cells (CTCs) and selected high metastatic variants can be extensively heterogeneous and have different chemosensitivity profiles, suggesting new approaches to treatment such as with TCM. Citation Format: Lei Zhang, Chengyu Wu, Robert. M Hoffman. Extensive morphological heterogeneity of circulating and selected high metastatic variants and differential chemosensitivity of human prostate cancer in mouse models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4128. doi:10.1158/1538-7445.AM2015-4128

Acidic extracellular pH promotes epithelial mesenchymal transition in Lewis lung carcinoma model.

BackgroundEpithelial mesenchymal transition (EMT) is thought to be an essential feature of malignant tumor cells when they spread into the stroma. Despite the extracellular acidity of tumor tissues, the effect of acidic extracellular pH (pHe) on EMT in carcinoma models, including the Lewis lung carcinoma (LLC) model, remains unclear.MethodsHigh and low metastatic LLC variants were generated by repeated tail vein injection of metastatic cells. DMEM/F12 medium, which has been supplemented with 15 mM HEPES, 4 mM phosphoric acid, and 1 g/L NaHCO3 and adjusted to the desire pH with HCl or NaOH, was used for cell culture. EMT marker gene expression was determined by quantitative reverse transcription-polymerase chain reaction. Migration and invasion activities were analyzed by wound healing assay and the Boyden chamber assay through Matrigel®, respectively.ResultsLow metastatic variant LLCm1 cells showed a cobble-stone like morphology at pHe 7.4. At pHe 6.8, however, their morphology became fibroblastic, similar in shape to high metastatic variant LLCm4 cells. Steady state levels of matrix metalloproteinase-9 (Mmp9) mRNA were induced by acidic pHe, maximizing at pH 6.8, with the levels of Mmp9 mRNA higher in LLCm4 than in LLCm1 cells. Both variants showed decreased levels of E-cadherin and increased levels of vimentin at pHe 6.8. Acidic pHe also induced expression of mRNAs encoding the E-cadherin repressors, Zeb2, Twist1 and Twist2, as well as enhancing cell motility and in vitro invasion through Matrigel®.ConclusionsAcidic pHe can induce EMT in some types of carcinoma.

Tumor-targeting Salmonella typhimurium A1-R prevents experimental human breast cancer bone metastasis in nude mice.

Bone metastasis is a lethal and morbid late stage of breast cancer that is currently treatment resistant. More effective mouse models and treatment are necessary. High bone-metastatic variants of human breast cancer cells were selected in nude mice by cardiac injection. After cardiac injection of a high bone-metastatic variant of breast cancer, all untreated mice had bone metastases compared to only 20% with parental cells. Treatment with tumor-targeting Salmonella typhimurium A1-R completely prevented the appearance of bone metastasis of the high metastatic variant in nude mice (P < 0.001). After injection of the highly bone-metastatic breast cancer variant to the tibia of nude mice, S. typhimurium A1-R treatment significantly reduced tumor growth in the bone (P < 0.001). These data indicated that S. typhimurium A1-R is useful to prevent and inhibit breast cancer bone metastasis and should be of future clinical use for breast cancer in the adjuvant setting.

The effect of antineoplastic drug NSC631570 on immunogenicity of B16 melanoma

Objectives: NSC631570 is a cytotoxic drug with the ability to be selectively accumulated in tumor tissue and activate apoptosis only in malignant cells and not in normal cells. Therapy with NSC631570 is accompanied by the stimulation of anticancer immune responses. It is known that cytotoxic anticancer drugs have additional effects on the immune system of tumor-bearing organism by increasing the immunogenic properties of tumor cells. This study aimed to investigate the immunogenicity of melanoma B16 after treatment with NSC631570. Methods: Two melanoma B16 sublines with different metastatic potentials were used. For in vivo growth cells were inoculated intravenously into C57BL/6 mice. The anticancer effect was calculated according to the growth inhibition index. Cell viability was determined by the MTT test. Cell apoptosis and necrosis were assessed by flow cytometry. HMGB1 expression, the serum level of cytokines and cytokine profile in tumor tissue were determined by ELISA. TAP1 and TAP2 expression was evaluated in RT-PCR and by Western blot. Results: Treatment of melanoma B16 cells with NSC631570 at apoptogenic concentrations induced tumor cell death accompanied by dose-dependent HMGB1 release in vitro. At the non-apoptogenic concentration the preparation caused an increase in TAP expression. The therapeutic efficacy of NSC631570 was also associated with strong release of HMGB1 in the serum of tumor-bearing mice and was more expressed in the case of the high-metastatic tumor variant. The therapeutic effect was accompanied by an increase of levels of Th1 cytokines in the serum and in tumor tissue of treated animals. Conclusion: In addition to the direct induction of tumor cell apoptosis, the preparation can increase the tumor immunogenicity.

In vivo serial selection of human pancreatic cancer cells in orthotopic mouse models produces high metastatic variants irrespective of Kras status

IntroductionKras mutations have been thought to play an important role in pancreatic cancer progression. In this study, we evaluated how serially passaging primary pancreatic tumors with and without Kras mutations, in nude mice, can generate more aggressive variants of human pancreatic cancer. Materials & methodsOrthotopic mouse models of human pancreatic cancer were established by injecting 1 × 106 cells of the Kras wildtype BxPC-3 cell line, expressing red fluorescent protein or the Kras mutant Panc-1 cell line expressing green fluorescent protein, into the pancreas. Pancreatic tumors were harvested from premorbid mice to establish cell lines. One million passaged cells were then orthotopically injected into another set of mice. Serial passaging continued until decreasing lifespan of the implanted mice stabilized, which occurred by six passages. Mice harboring serially-passaged cell lines were followed with weekly imaging. ResultsSerially passaging generated more aggressive variants of both human pancreatic cancer cell lines, one of which was Kras wild-type (BXPC-3) and the other Kras mutant, Panc-1, which displayed faster tumor growth and shortened survival time. Overall survival decreased from 18 wk in mice with the parental cell line (passage 0) tumor to ∼6 wk in mice by passage 6. Average time to metastasis was shortened from 14 wk to ∼3 wk or less. At termination, mice with the passaged tumor demonstrated a greater extent of distant metastasis. ConclusionsSerial passaging of tumor creates more aggressive variants of human pancreatic cancer cell lines regardless of Kras mutation. The aggressive variants can be used to study the molecular basis of highly malignant pancreatic cancer and to screen for effective agents against this disease.

Abstract C7: Role of β1,6 branched N linked oligosaccharides and associated terminal substitutions in organ-specific metastasis.

Abstract Metastasis involves complex interplay of multitude of events which aid dissemination of tumour cells from primary site to a distant, non-contiguous organ site. The cell surface molecules along with their associated posttranslational modifications are known to play an important role in these events. One such widely studied modification is altered expression of β1,6 branched N-glycans on cell surface glycoproteins. These oligosaccharides have been strongly associated with invasive as well as metastastic potential of human and murine tumours and cancer cell lines. One of the interesting features of the cell lines which express these oligosaccharides is that they either metastasize to the lungs or to the liver. The cells expressing β1,6 branched N-oligosaccharides substituted with Lewis antigens have been shown to metastasize preferentially to liver, the mechanism by which they facilitate lung metastasis is not clear. Low (B16F1) and high (B16F10) metastatic variants of B16 Melanoma has been used as a model to investigate the role of these oligosaccharides in lung metastasis. The model provides cell lines with vastly different metastatic potential which metastasize in organ specific manner to lungs and also express β1,6 branched N-glycans. Previously, poly N-acetyl lactosamine (polylacNAc) substituted β1,6 branched N-glycans were shown to facilitate lung homing via galectin-3. Galectin-3 was shown to be expressed in highest amounts on the lungs on all the major compartments and constitutively on the surface of lung vascular endothelium. Galectin-3 not only provides initial adhesion on the lung vascular endothelium, but also stabilizes adhesion by promoting spreading of cells. Further, it facilitates degradation of vascular basement membrane and mobility into organ parenchyma. To confirm the role of polylacNAc on β1,6 branched N-glycans, we generated inducible lentiviral (pTRIPZ) mediated shRNA clones which target galactosyl transferase genes (GalTI and GalTV) involved in synthesis of polylacNAc. Real time PCR results showed reduction in expression of GalTI and GalTV genes at transcript levels. The cell surface levels of polylacNAc were analysed by flow cytometry using biotinylated Lycopersicum Esculantum (LEA) lectin as well as by biotinylated galectin-3.The downregulation of polylacNAc on B16F10 cells also results in inhibition of processes critical for metastasis including cell adhesion, cell spreading and motility of cells on galectin-3 and galectin-3 induced secretion of MMP-9. In summary, polylacNAc on β1,6 branched N-glycans is an important determinant of lung specific metastasis of murine melanoma cells. Citation Format: Manohar Dange, Nithya Srinivasan, Rajiv D. Kalraiya. Role of β1,6 branched N linked oligosaccharides and associated terminal substitutions in organ-specific metastasis.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C7.

Abstract C25: A five-gene signature is associated with tumor progression and poor prognosis in colorectal cancer

Abstract In order to develop an experimentally derived molecular signature closely associated with survival of colorectal cancer (CRC) patients from mouse tumor models, we isolated single cell-derived progenies (SCPs) from SW480 CRC cell line and compared their metastatic potential in an orthotopic implantation model of murine CRC. We compared the differences in the gene expression profiles of a high metastatic variant SCP51, a low metastatic variant SCP58 and their parent SW480/EGFP. We found that 143 genes were commonly differentially expressed between SCP51 and SCP58 cells, SCP58 and SW480/EGFP. Using Gene-annotation enrichment analysis of DAVID, 80 genes were found in top ten clusters of the analysis. Thirty-two genes of above the 80-gene set were found to be involved in metastasis by Genclip, a text mining program. We selected five genes (LYN, SDCBP, MAP4K4, DKK1 and MID1) for immunohistochemical analysis in a cohort of 181 CRC patients. Results showed those LYN, SDCBP, MAP4K4, MID1 and five-gene signatures were significantly correlated with overall survival in patients with CRC. We also found that LYN, MAP4K4 and MID1 but not SDCBP and DKK1 expression was closely correlated with lymph node metastasis and Dukes classification in patients with CRC. Our five-gene signature was closerly associated with survival of CRC patients and could be used as a potential prognostic factor for CRC. Citation Information: Cancer Res 2009;69(23 Suppl):C25.

Upregulation of thrombospondin-1 and angiogenesis in an aggressive human pancreatic cancer cell line selected for high metastasis

Pancreatic cancer remains a leading cause of death despite its relatively low incidence. As in many other solid tumors, angiogenesis is critical to the growth and metastasis of this cancer. Through serial in vivo passages in mice, we have developed a highly aggressive variant of human pancreatic cancer cell line XPA-1 which shows more rapid primary tumor growth, faster time to metastasis, and more rapid lethality than the parental cell line. The high-metastatic variant developed a much denser tumor vasculature early during growth within the pancreas. Interestingly, examination of the in vitro growth of this aggressive variant yielded no significant difference from the parental cell line. Real-time PCR evaluation of genes involved in angiogenesis revealed a 24-fold increase in Thrombospondin-1 expression in cells derived from the high-metastatic variant when compared with the parental cell line. These findings provide direct evidence that elevated capability for angiogenesis, mediated by specific changes in gene expression, can lead to a large increase in cancer aggressiveness and resulting metastasis. These findings have important implications for the treatment of metastatic disease.

Differences in the Expression of Cathepsin B in B16 Melanoma Metastatic Variants Depend on Transcription Factor Sp1

Cathepsin B contributes to the invasiveness of B16 melanoma cells in mice, with the highly metastatic B16a melanoma producing six- to eightfold more cathepsin B mRNA and protein than the less metastatic B16F1 variant. The proximal promoter region of the cathepsin B (Ctsb) gene (-149 to +94) was previously found to be capable of reproducing this pattern of differential gene activation in B16 melanoma variants. The binding of B16 melanoma nuclear proteins to this promoter region has now been mapped to three GC-boxes (Sp1 transcription factor binding sites) and a potential X-box [tax response element (TRE)/c-AMP responsive element (CRE) site]. Mutation of the GC-boxes at -55 and -37 independently decreased the expression of a luciferase reporter gene in B16a cells to the level observed in B16F1 cells. Promoter activity was also attenuated by mutations within the GC-rich segment between +6 and +16, but not by mutation of the putative X-box. Both Sp1 and Sp3 bound the GC-boxes in the Ctsb promoter, and western blotting showed the level of Sp1 to be greater in B16a compared to B16F1 cells. B16F1 cells that were made to express Sp1 at levels observed in B16a cells produced corresponding increased amounts of endogenous cathepsin B mRNA and enzyme activity. Thus, the difference in cathepsin B expression between high and low metastatic B16 melanoma variants is largely due to different levels of Sp1.

Identification and cloning of genes displaying elevated expression as a consequence of metastatic progression in human melanoma cells by rapid subtraction hybridization

Although extensively investigated, the complete repertoire of genes associated with and causative of metastasis remain largely unknown. We developed an efficient approach for identifying differentially expressed genes that involves rapid subtraction hybridization (RaSH) of cDNA clones prepared from two cell populations, a driver and a tester. This RaSH approach has previously documented high sensitivity and effectiveness in identifying genes that are differentially expressed as a function of induction of terminal differentiation in human melanoma cells, resistance or sensitivity to human immunodeficiency virus-1 (HIV-1) infection of human T cells and perturbation in gene expression in normal human fetal astrocytes infected with HIV-1 or treated with HIV-1 gp120 viral envelope glycoprotein or tumor necrosis factor-α (TNF-α). In the present study, RaSH has been applied to a metastatic melanoma model, which mimics the early events of metastasis in humans, comprising weakly metastatic vs. immunosuppressed newborn rat-selected highly metastatic variants. This has now resulted in the identification of eight genes displaying elevated expression in the high metastatic variants vs. normal immortal melanocytes or weakly metastatic parental clones. These include six known genes, 67-kDa laminin receptor (67LR), endothelin receptor B (ENDRB), Na +/K +-ATPase, Ku antigen, interleukin-receptor-associated kinase-1 (IRAK-1) and ribosomal protein RPLA, which may contribute to the complex process of melanoma metastasis. Additionally, two unknown genes (not reported in current databases) that may also impact on the metastatic phenotype have also been identified. These studies provide additional support of the use of the RaSH approach, in this application in the context of closely related variant cell lines with different metastatic potential, for effective differential gene identification and elucidate eight previously unrecognized genes whose role in melanoma progression to metastatic competence can now be scrutinized.

Differential gene expression in metastasizing cells shed from kidney tumors.

We developed a novel orthotopic mouse tumor model of renal cell carcinoma to collect and characterize cells spontaneously shed from SN12C (renal cell carcinoma) and SN12L1 (high metastatic variant of SN12C) tumors grown in kidneys of severe combined immunodeficient mice. Viability of the shed cell population was greater for SN12L1 tumors (25%) compared with SN12C tumors (11%, P < 0.05). Gene array analysis of 23 genes involved in metastasis showed that CD44, alpha3 integrin, and caveolin were down-regulated in the shed tumor cells compared with their primary counterparts, and blocking alpha3 integrin or CD44 function inhibited attachment and migration of both cell lines. These results suggest that cohesion of the cells within the primary tumor mediated by CD44 and alpha3 integrins hinders metastasis and that shedding is a passive process not necessarily mediated by cell migration in these tumors. Furthermore, resistance to apoptosis may enhance metastasis in the higher metastatic tumor.

Synthesis of platelet-activating factor (PAF) in transformed cell lines of a different origin

Interest in the possible involvement of the platelet-activating factor (PAF) in tumor growth and invasiveness has been stimulated by the recognition that PAF influences various biological responses relevant to metastatic diffusion, such as angiogenesis, adhesiveness to endothelia and cellular motility. In the present study, we investigated the extent to which PAF is synthesized by a series of human and murine transformed cell lines of a different histotype. Synthesis of PAF was studied by combining the 14 C -acetate incorporation into PAF with the quantitative analysis of PAF performed by a procedure based on gas chromatography–mass spectrometry with a negative ion chemical ionization. In the presence of the Ca 2+ ionophore A23187, cultures of human melanoma (Hs294T), fibrosarcoma (HT1080) and colon carcinoma (LS180) cell lines synthesized conspicuous amounts of PAF, comparable to those produced by resident peritoneal macrophages. Substantial quantities of PAF were also synthesized by the murine melanoma (F10-M3 cells). PAF synthesis was rather limited in RSV-transformed Balb/c3T3 (B77-3T3) cells and in one of their high metastatic variants (B77-AA6 cells), although it was more abundant in the latter. We also investigated whether certain cytokines, such as TNFα and IFNγ might induce PAF synthesis in our systems of cell lines which we found to express mRNAs encoding receptors for these cytokines. We observed that PAF synthesis was enhanced in human melanoma and colon carcinoma cell lines and in the murine B77-AA6 cells to levels comparable to those obtained with the Ca 2+ ionophore. Synthesis of PAF was not inducible by TNFα in murine F10-M3 melanoma cells. IFNγ also stimulated PAF synthesis in human and murine melanoma lines, and in human LS180 colon carcinoma line, but not in the B77-AA6 cells. PAF synthesis was also inducible by exogenous PAF in the human and murine melanoma lines, and in the human LS180 colon carcinoma line, all of which expressed cell surface PAF receptors. PAF synthesis was not inducible by exogenous PAF in the B77-AA6 cells, which do not express PAF receptors.

Expression of proangiogenic chemokine Gro 1 in low and high metastatic variants of Pam murine squamous cell carcinoma is differentially regulated by IL-1alpha, EGF and TGF-beta1 through NF-kappaB dependent and independent mechanisms.

We previously reported that chemokine Growth Regulated Oncogene 1 (Gro 1) is over-expressed in murine squamous cell carcinoma (SCC) with metastatic tumor progression. The enhanced expression of Gro-1 gene by SCC is regulated by activation of nuclear factor-kappaB (NF-kappaB), leading to accelerated tumor growth, angiogenesis and metastasis in vivo. In our study, we investigated the effect of the regulatory cytokines, IL-1alpha, EGF and TGF-beta1 on activation of NF-kappaB and Gro1 in primary and metastatic sublines of the murine SCC Pam 212. We found that Gro 1 expression could be induced by IL-1alpha or EGF in the low cytokine producing Pam 212 cells, but no significant induction was observed in high cytokine producing and metastatic LY-2 cells. Conditioned medium from LY-2 containing functional IL-1alpha induced Gro 1 expression in Pam 212 cells, which can be blocked by IL-1 receptor antagonist (IL-1RA). IL-1RA, however, had a minimal effect on constitutive Gro 1 production by LY-2 cells. TGF-beta1 suppressed constitutive as well as IL-1alpha and EGF-inducible Gro 1 production in both Pam 212 and LY-2 cells. IL-1alpha and EGF, but not TGF-beta1, were found to activate NF-kappaB in Pam 212, whereas none of the stimulants showed a significant effect on constitutive activation of NF-kappaB in LY-2 cells. Overexpression of a super repressor IkappaBalphaM in Pam 212 inhibited NF-kappaB binding activity, which led to impaired Gro 1 induction by IL-1alpha and EGF. These results demonstrate that IL-1alpha, EGF, and TGF-beta1 are important modulators of Gro 1 expression in SCC. Different responses to these modulators observed along with SCC metastatic progression may suggest a transition mechanism(s) for Gro 1 expression from host factor dependent to an independent stage involving NF-kappaB activation. Published 2001 Wiley-Liss, Inc.

Isolation of a Novel Actin-Related Gene Expressed in Low-Metastatic PC-14 Human Lung Adenocarcinoma

In this article, we report the identification of differentially expressed genes associated with the metastatic potential of PC-14 human lung adenocarcinoma. The mRNA differential display method was applied to compare mRNAs from six cloned PC-14 cell lines representing different metastatic potentials. A novel gene was identified as being expressed in low-metastatic cells but not in high-metastatic cells. Sequence analysis revealed that this novel gene has an open reading frame of 210 amino acid residues showing 89% amino acid identity with human Arp3, which is one of the actin-related proteins. This gene was designated the Arp11 gene (the 11th Actin-related gene). The Arp11 gene was mapped to human chromosome 7q32–36. Southern blot and PCR analyses show that all of the high-metastatic variant cells have not rearrangements and deletions of Arp11 allele. Expression of the Arp11 gene may regulate the metastatic potential of PC-14 human lung adenocarcinoma.