Several lines of evidence suggest that chromosome 8 is likely to harbor tumor-suppressor genes involved in breast cancer. We showed previously that microcell-mediated transfer of human chromosome 8 into breast cancer cell line MDA-MB-231 resulted in reversion of these cells to tumorigenicity and was accompanied by changes in the expression of a breast cancer-relevant gene set. In the present study, we demonstrated that transfer of human chromosome 8 into another breast cancer cell line, CAL51, strongly reduced the tumorigenic potential of these cells. Loss of the transferred chromosome 8 resulted in reappearance of the CAL51 phenotype. Microarray analysis identified 78 probe sets differentially expressed in the hybrids compared with in the CAL51 and the rerevertant cells. This signature was also reflected in a panel of breast tumors, lymph nodes, and distant metastases and was correlated with several prognostic markers including tumor size, grading, metastatic behavior, and estrogen receptor status. The expression patterns of seven genes highly expressed in the hybrids but down-regulated in the tumors and metastases (MYH11, CRYAB, C11ORF8, PDGFRL, PLAGL1, SH3BP5, and KIAA1026) were confirmed by RT-PCR and tissue microarray analyses. Unlike with the corresponding nontumorigenic phenotypes demonstrated for the MDA-MB-231- and CAL51-derived microcell hybrids, the respective differentially expressed genes strongly differed. However, the majority of genes in both gene sets could be integrated into a similar spectrum of biological processes and pathways, suggesting that alterations in gene expression are manifested at the level of functions and pathways rather than in individual genes.
Purpose: Novel drugs including targeted approaches have changed treatment paradigms for multiple myeloma (MM) and may also have therapeutic potential in the poor-prognosis t(4;14) subset; t(4;14) results in overexpressed and activated fibroblast growth factor receptor 3 (FGFR3). Blocking this receptor tyrosine kinase (RTK) induces apoptosis in t(4;14)+ MM cells and decreases adhesion to bone marrow stromal cells (BMSC). Using combinations of novel drugs, we investigated potential enhancement of single-agent activities within the tumor cells, targeting of the marrow micromilieu, or circumvention of drug resistance in t(4;14)+ MM. Experimental Design: We tested effects on apoptosis and related signaling pathways in the t(4;14)+ MM subset, applying drug combinations including a FGFR3 tyrosine kinase inhibitor (RTKI), the proteasome inhibitor bortezomib, and dexamethasone. Results: RTKI, bortezomib, and dexamethasone were active as single agents in t(4;14)+ MM. RTK inhibition triggered complementary proapoptotic pathways (e.g., decrease of Mcl-1, downregulation of p44/42 mitogen-activated protein kinase, and activation of proapoptotic stressactivated protein/c-Jun NH 2 -terminal kinases). Synergistic or additive effects were found by combinations of RTKI with dexamethasone or bortezomib. In selected cases of t(4;14)+ MM, triple combinations were superior to dual combinations tested. Prevention from MM cell apoptosis by BMSC or exogenous interleukin-6 was circumvented by drug combinations. In t(4;14)+, N-ras^mutated NCI-H929 cells, resistance to RTKI was overcome by addition of dexamethasone. Notably, the combination of RTKI and dexamethasone showed additive proapoptotic effects in bortezomib-insensitive t(4;14)+ MM. Conclusions: Combining novel drugs in poor-prognosis t(4;14)+ MM should take into account at least bortezomib sensitivity and probably Ras mutational status.
NK cells have emerged as promising candidates for cancer immunotherapy, especially due to their ability to fight circulating tumor cells thereby preventing metastases formation. Hence several studies have been performed to generate and expand highly cytotoxic NK cells ex vivo , e.g., by using specific cytokines to upregulate both their proliferation and surface expression of distinct activating receptors. Apart from an enhanced activity, application of NK cells as immunotherapeutic agent further requires sufficient cell numbers and a high purity. All these parameters depend on a variety of different factors including the starting material, additives like cytokines as well as the culture system. Here we analyzed PBMC-derived NK cells of five anonymized healthy donors expanded under specific conditions in an innovative perfusion bioreactor system with respect to their phenotype, IFNγ production, and cytotoxicity in vitro . Important features of the meander type bioreactors used here are a directed laminar flow of medium and control of relevant process parameters. Cells are cultivated under “steady state” conditions in perfusion mode. Our data demonstrate that expansion of CD3 + T cell depleted PBMCs in our standardized system generates massive amounts of highly pure (>85%) and potent anti-cancer active NK cells. These cells express a variety of important receptors driving NK cell recruitment, adhesion as well as activation. More specifically, they express the chemokine receptors CXCR3, CXCR4, and CCR7, the adhesion molecules L-selectin, LFA-1, and VLA-4, the activating receptors NKp30, NKp44, NKp46, NKG2D, DNAM1, and CD16 as well as the death ligands TRAIL and Fas-L. Moreover, the generated NK cells show a strong IFNγ expression upon cultivation with K562 tumor cells and demonstrate a high cytotoxicity toward leukemic as well as solid tumor cell lines in vitro . Altogether, these characteristics promise a high clinical potency of thus produced NK cells awaiting further evaluation.
Adoptive transfer of autologous tumor-specific lymphocytes represents a viable treatment method for patients with advanced malignancies. Here, we report a patient’s case with metastatic hormone-refractory New York esophageal squamous cell carcinoma 1 (NY-ESO-1) expressing prostate cancer treated with in vitro expanded tumor-infiltrating lymphocytes (TILs) in conjunction with IL-2 and immune-checkpoint blockade. Complete and durable tumor remission was observed after three TIL infusions consisting of 1.4×109, 2.0×109, and 8.0×109T cells, respectively, lasting now for more than 3.5 years. Immunological correlates to the clinical development were the decrease of tumor-driven NY-ESO-1 serum antibody and the drop of prostate-specific antigen to <0.01 µg/L. TILs were reactive against cancer-testis antigen NY-ESO-1, individual tumor mutational proteins (eg, PRPF8, TRPS1), and the androgen receptor splice variant 12.
Previous studies have shown that transfer of chromosome 17 suppresses the tumorigenic phenotype of the breast cancer cell line CAL51, suggesting the presence of putative tumor suppressor genes on this chromosome. Suppression subtractive hybridization and oligonucleotide microarray analyses were performed to identify differentially expressed genes in nontumorigenic microcell hybrids, CAL/17-1 and CAL/17-3, when compared with CAL51 cells. In total, 263 differentially expressed transcripts were associated with these phenotypes. Of these, a high percentage is involved in various biological processes associated with tumorigenesis, including DNA-dependent regulation of transcription, regulation of cell cycle, signal transduction, and cell proliferation. Microarray analysis of selected chromosome 17 genes in a series of 25 human primary breast tumors showed associations with clinicopathologic parameters of the tumors. Of these genes, TOB1 (transducer of ERBB2) was selected for further expression analysis. Using RT-PCR and immunohistochemical staining of tissue microarrays, we could reveal a differential mRNA and protein expression of TOB1 in the majority of breast tumors and lymph node metastases compared with normal breast tissues, indicating a potential role of this protein in breast tumorigenesis.
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