g(m2)-ganglioside and Kidney-Neoplasms

Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-alpha secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3-5 days of TNF-alpha pretreatment, a time frame paralleling that needed for TNF-alpha to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-alpha transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-alpha increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-alpha-treated tumor cells, and by the observation that small interfering RNA directed against TNF-alpha abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-alpha signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.

Topics: Adjuvants, Immunologic; Antibodies, Blocking; Apoptosis; Carcinoma, Renal Cell; Cell Line, Tumor; G(M2) Ganglioside; Gene Expression Regulation, Neoplastic; Glycosides; Humans; Kidney Neoplasms; Resting Phase, Cell Cycle; T-Lymphocyte Subsets; Transfection; Tumor Escape; Tumor Necrosis Factor-alpha; Up-Regulation

Multiple mechanisms have been proposed to account for immune escape by tumors. Although gangliosides have long been known to suppress T-cell immunity, few studies have examined the effect of human tumor-derived gangliosides on immune responses. Here, we show that gangliosides isolated from renal cell carcinoma (RCC) cell lines and clear cell tumor tissue can induce apoptosis in peripheral blood T cells. The RCC tissue-derived gangliosides also suppressed IFN-gamma and, in many cases, interleukin-4 production by CD4+ T cells at concentrations (1 ng/mL-100 pg/mL) well below those that induce any detectable T-cell death (4-20 microg/mL). Additional findings show that GM2 expressed by RCC plays a significant role in promoting T-cell dysfunction. This is supported by the demonstration that all RCC cell lines examined (n = 5) expressed GM2 as did the majority of tumors (15 of 18) derived from patients with clear cell RCC. Furthermore, an antibody specific for GM2 (DMF10.167.4) partially blocked (50-60%) T-cell apoptosis induced by coculturing lymphocytes with RCC cell lines or with RCC tissue-derived gangliosides. DMF10.167.4 also partially blocked the suppression of IFN-gamma production induced by RCC tissue-derived gangliosides, suggesting that GM2 plays a role in down-regulating cytokine production by CD4+ T cells.

Topics: Apoptosis; Carcinoma, Renal Cell; Cell Line, Tumor; Coculture Techniques; G(M2) Ganglioside; Humans; Kidney Neoplasms; T-Lymphocytes; Th1 Cells; Th2 Cells

Human renal cell carcinoma (RCC) has been characterized by remarkable changes in ganglioside composition. TOS1 cells, typical of metastatic RCC, are characterized by predominance of GM2 as monosialoganglioside, and beta 1,4GalNAc disialyl-Lc(4) (RM2 antigen) as disialoganglioside [J. Biol. Chem. 276 (2001) 16695]. In order to observe the functional role of gangliosides in RCC malignancy, TOS1 cells were transfected with short interfering RNA (siRNA) based on open reading frame sequence of beta 1,4GalNAc transferase (beta 1,4GalNAc-T), and its disordered sequence of siRNA (dsiRNA) as control. In siRNA transfectant, beta 1,4GalNAc-T mRNA level and GM2 expression were greatly reduced, whereby GM3 expression appeared. In contrast, RM2 antigen level was unchanged, even though it has the same beta 1,4GalNAc epitope at the terminus. dsiRNA transfectant showed no change of beta 1,4GalNAc-T mRNA and did not express GM3. Concomitant with reduction of GM2 and appearance of GM3, siRNA transfectant showed greatly reduced motility and invasiveness, although growth rate was unaltered. Both transfectants with siRNA and dsiRNA expressed the same level of tetraspanin CD9. Since CD9/GM3 complex is known to reduce integrin-dependent motility and invasiveness [Biochemistry 40 (2001) 6414], it is plausible that motility and invasiveness of siRNA transfectant of TOS1 cells may be reduced by enhanced formation of such complex.

Topics: Antigens; Antigens, CD; Carcinoma, Renal Cell; Cell Division; Cell Line, Tumor; Cell Movement; G(M2) Ganglioside; G(M3) Ganglioside; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Male; Membrane Glycoproteins; Middle Aged; N-Acetylgalactosaminyltransferases; Neoplasm Invasiveness; RNA; RNA, Messenger; RNA, Small Interfering; Tetraspanin 29; Transfection

Lung cancer is a major cause of cancer deaths, most of which can be attributed to distant multiorgan metastases. To examine the cellular and molecular mechanisms of lung cancer metastasis to distant organs, we have established novel models of human lung cancer (small cell and non-small cell lung cancer) metastasis in natural killer cell-depleted severe combined immunodeficient (SCID) mice. We investigated whether local production of the cytokines responsible for regulation of macrophage function at tumor growth sites affects the pattern of lung cancer metastasis in distant organs. Several lung cancer cell lines were genetically engineered to produce human macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein-1 (MCP-1), and their metastatic potentials were assessed. Interestingly, M-CSF gene transduction had an antimetastatic effect for the liver and lymph nodes, but not the kidneys. In contrast, MCP-1 gene-modified lung cancer cells and their parent cells had identical metastatic potentials. These findings indicate a possible role for cytokines and suggest that lung cancer has metastatic heterogeneity. Examining ways of controlling human lung cancer metastases, we investigated the antimetastatic effect of chimeric monoclonal antibodies (MAbs) against P-glycoprotein and ganglioside GM2 (MH162 and KM966, respectively). Both MAbs, when given on days 2 and 7, inhibited the development of distant metastases of lung cancer in a dose-dependent fashion. Combined use of anti-P-glycoprotein MAb with M-CSF or MCP-1 gene transduction caused complete inhibition of metastasis of H69/VP cells. The antimetastatic effect of these MAbs in vivo was mainly due to an antibody-dependent cell-mediated cytotoxicity reaction mediated by mouse macrophages. These findings suggest that the mouse-human chimeric MAb in combination with cytokine gene transduction may be useful for the eradication of lung cancer metastases in humans.

Topics: Animals; Antibodies, Monoclonal; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Chemokine CCL2; Cytokines; G(M2) Ganglioside; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Kidney Neoplasms; Killer Cells, Natural; Liver Neoplasms; Lung Neoplasms; Lymphatic Metastasis; Mice; Mice, SCID; Neoplasm Transplantation; Recombinant Fusion Proteins; Transduction, Genetic; Tumor Cells, Cultured