transforming-growth-factor-beta and Carcinoma--Medullary

MicroRNAs (miRNAs) represent a class of small, non-coding RNAs that control gene expression by targeting mRNA and triggering either translational repression or RNA degradation. The objective of our study was to evaluate the involvement of miRNAs in human medullary thyroid carcinoma (MTC) and to identify the markers of metastatic cells and aggressive tumour behaviour. Using matched primary and metastatic tumour samples, we identified a subset of miRNAs aberrantly regulated in metastatic MTC. Deregulated miRNAs were confirmed by quantitative real-time PCR and validated by in situ hybridisation on a large independent set of primary and metastatic MTC samples. Our results uncovered ten miRNAs that were significantly expressed and deregulated in metastatic tumours: miR-10a, miR-200b/-200c, miR-7 and miR-29c were down-regulated and miR-130a, miR-138, miR-193a-3p, miR-373 and miR-498 were up-regulated. Bioinformatic approaches revealed potential miRNA targets and signals involved in metastatic MTC pathways. Migration, proliferation and invasion assays were performed in cell lines treated with miR-200 antagomirs to ascertain a direct role for this miRNA in MTC tumourigenesis. We show that the members of miR-200 family regulate the expression of E-cadherin by directly targeting ZEB1 and ZEB2 mRNA and through the enhanced expression of tumour growth factor β (TGFβ)-2 and TGFβ-1. Overall, the treated cells shifted to a mesenchymal phenotype, thereby acquiring an aggressive phenotype with increased motility and invasion. Our data identify a robust miRNA signature associated with metastatic MTC and distinct biological processes, e.g., TGFβ signalling pathway, providing new potential insights into the mechanisms of MTC metastasis.

Topics: Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoma, Medullary; Cell Adhesion; Cell Proliferation; Gene Expression Profiling; Gene Ontology; Homeodomain Proteins; Humans; Immunoenzyme Techniques; In Situ Hybridization; Lymphatic Metastasis; MicroRNAs; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thyroid Neoplasms; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Zinc Finger E-box Binding Homeobox 2; Zinc Finger E-box-Binding Homeobox 1

Permanent human tumor cell lines are an important tool for the study of breast cancer. Two new breast cancer cell lines (BrCa-MZ-01 and BrCa-MZ-02) were isolated from a solid tumor and a pleural effusion, respectively. One cell line was established from a medullary carcinoma, the other from a ductal carcinoma. These cells exhibit ultrastructural and immunohistochemical features of epithelial cells of mammary origin. Intermediate filament and cytokeratin typing showed a clear predominance of the simple-epithelial cytokeratins CK 8, CK 18 and CK 19, although the expression was reduced in comparison to the hormone receptor-positive reference cell lines MCF-7 and ZR-75-1. Both cell lines produced slow-growing tumors after subcutaneous (s.c.) transplantation of 1 x 10(7) viable tumor cells into nude mice. The cell line BrCa-MZ-01 expresses the estrogen and progesterone receptor, whereas the cell line BrCa-MZ-02 remains negative. Both cell lines are positive for secretion of platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), whereas interleukin-6 (IL-6) is only secreted by the cell line BrCa-MZ-02.

Topics: Aged; Aged, 80 and over; Animals; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Medullary; Cell Division; Cell Line; Epithelial Cells; Female; Humans; Immunohistochemistry; Interleukin-6; Intermediate Filament Proteins; Keratins; Mice; Mice, Nude; Platelet-Derived Growth Factor; Receptors, Estrogen; Receptors, Progesterone; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Cells, Cultured

Medullary thyroid cancer (MTC) is an endocrine tumor of the thyroid C-cells which provides an important experimental model for studies of tumor differentiation and progression. We investigated the effects of transforming growth factor-beta 1 (TGF beta 1) on the growth and functional characteristics of a human medullary thyroid carcinoma cell line (TT). Because the c-myc protooncogene may play an important role in the growth inhibition induced by TGF beta 1, we also assessed steady state c-myc messenger RNA (mRNA) levels in these cells. A 6-day exposure of TT cells to TGF beta 1 resulted in a dose-dependent inhibition of cell proliferation. In addition, TGF beta 1 exposure led to a 3-fold increase in nonadherent floating TT cells in the culture supernatants. The floating cells exhibited ultrastructural features of dying or apoptotic cells, including chromatin condensation, cytoplasmic and nuclear vesicularization, and DNA degradation with evidence of internucleosomal DNA "laddering." Despite inhibition of cell proliferation, steady state c-myc mRNA levels were 3.6 +/- 0.6-fold higher in cells exposed to TGF beta 1 compared to those in control cells (P < 0.001). Exposure of cells to a 15-base antisense c-myc oligonucleotide (10 microM) resulted in an attenuation of the TGF beta 1-induced growth inhibition and induction of cell death. TGF beta 1 also resulted in an approximately 3-fold decrease in steady state calcitonin and calcitonin gene-related peptide mRNA levels. Finally, using a sensitive bioassay for TGF beta, TT cells were shown to produce and activate significant amounts of TGF beta, particularly under conditions of serum deprivation. Our data thus indicate that TGF beta 1 has multiple effects on TT cell growth and function. It induces growth inhibition in the presence of an increase in steady state mRNA levels of the c-myc protooncogene, which is usually associated with cell proliferation. In addition, TGF beta 1 accelerates apoptosis in TT cells.

Topics: Base Sequence; Blotting, Northern; Carcinoma, Medullary; Cell Division; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Humans; Microscopy, Electron; Molecular Sequence Data; Proto-Oncogene Proteins c-myc; RNA, Messenger; Thyroid Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured