transforming-growth-factor-beta and Leiomyosarcoma

Hyaluronan and versican are extracellular matrix (ECM) components that are enriched in the provisional matrices that form during the early stages of development and disease. These two molecules interact to create pericellular "coats" and "open space" that facilitate cell sorting, proliferation, migration, and survival. Such complexes also impact the recruitment of leukocytes during development and in the early stages of disease. Once thought to be inert components of the ECM that help hold cells together, it is now quite clear that they play important roles in controlling cell phenotype, shaping tissue response to injury and maintaining tissue homeostasis. Conversion of hyaluronan-/versican-enriched provisional matrix to collagen-rich matrix is a "hallmark" of tissue fibrosis. Targeting the hyaluronan and versican content of provisional matrices in a variety of diseases including, cardiovascular disease and cancer, is becoming an attractive strategy for intervention.

Topics: Animals; Cell Proliferation; Cells, Cultured; Collagen; Disease Models, Animal; Extracellular Matrix; Female; Fibrosis; Gene Expression Regulation; Humans; Hyaluronic Acid; Leiomyosarcoma; Mice; Muscle, Smooth, Vascular; Platelet-Derived Growth Factor; RNA, Small Interfering; Transforming Growth Factor beta; Uterine Neoplasms; Versicans

Endoglin (CD105), an accessory receptor of transforming growth factor-β, is expressed in vascular endothelial cells. Recently, it was reported that endoglin expression was significantly associated with poorer survival in several cancers. In this study, we evaluated the role of endoglin in uterine leiomyosarcoma. We examined the expression of endoglin in 22 uterine leiomyosarcomas and the association between their expression and the outcome. Additionally, to evaluate the function of endoglin, we used SKN cells, a human uterine leiomyosarcoma cell line. We generated SKN cells stably transfected with plasmids encompassing shRNA targeting endoglin (shEng cells), and compared the ability of proliferation, migration, and invasion to control shRNA-transfected cells (shCon cells). We compared the level of VEGF and matrix metalloproteinases (MMP) in culture supernatants of shEndoglin and shControl cells. Nine patients were endoglin-positive and 13 patients were -negative. The endoglin-positive group had a significantly poorer overall survival and progression-free survival than the endoglin-negative group. In an in vitro study, there was no difference in cell proliferation between shEng and shCon cells. On the other hand, shEng cells showed a lower ability for migration and invasion than shControl cells. The activity of MMP-9 and VEGF level in the supernatant from shEng cells were lower than in shCon cells. In uterine leiomyosarcoma, endoglin expression was associated with a poor prognosis. It was suggested that endoglin up-regulated invasion and VEGF secretion. The investigation of endoglin may lead to a new strategy in uterine leiomyosarcoma therapy.

Topics: Adult; Aged; Antigens, CD; Cell Line, Tumor; Endoglin; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Immunohistochemistry; Leiomyosarcoma; Middle Aged; Neoplasm Invasiveness; Receptors, Cell Surface; Signal Transduction; Transforming Growth Factor beta; Uterine Neoplasms; Vascular Endothelial Growth Factor A

Aberrant expression of microRNAs (miRNAs), including miR-21, and alteration of their target genes stability have been associated with cellular transformation and tumorigenesis. We investigated the expression, regulation and function of miR-21 in leiomyomas which develop from myometrial cellular transformation. The results indicated that miR-21 is over-expressed in leiomyomas with specific elevation during the secretory phase of the menstrual cycle and in women who received Depo-Provera and oral contraceptives, but reduced due to GnRHa therapy (P < 0.05). Bioinformatic analysis of microarray gene expression profiles previously obtained from the above cohorts, and myometrial smooth muscle cells (MSMC) and leiomyoma smooth muscle cells (LSMC) treated with GnRHa, transforming growth factor (TGF)-beta and TGF-beta receptor type II (TGF-betaRII) antisense oligomer, indicated that a number of miR-21-predicted target genes were co-expressed and differentially regulated in these cohorts. Gain- and loss-of-function of miR-21 in MSMC, LSMC, transformed LSMC and leiomyosarcoma cell line (SKLM-S1) resulted in differential expression of many genes, including some of the miR-21-predicted/validated target genes, PTEN, PDCD4 and E2F1, and TGF-betaRII, in a cell-specific manner. Gain-of miR-21 function in MSMC and LSMC reduced TGF-beta-induced expression of fibromodulin and TGF-beta-induced factor (P < 0.05), and moderately altered the rate of cell growth and caspase-3/7 activity in these cells. We concluded that miR-21 is aberrantly expressed and hormonally regulated in leiomyomas where, through functional interaction with ovarian steroids and the TGF-beta signaling pathway, either directly or indirectly regulates a number of genes whose products are critical in leiomyoma growth and regression as well as their potential cellular transformation.

Topics: Adult; Blotting, Western; Cell Proliferation; Cells, Cultured; Computational Biology; E2F1 Transcription Factor; Female; Gonadotropin-Releasing Hormone; Humans; Leiomyoma; Leiomyosarcoma; MicroRNAs; Middle Aged; Myocytes, Smooth Muscle; Oligonucleotide Array Sequence Analysis; PTEN Phosphohydrolase; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

Smooth muscle tumors of uterus have been reported to contain considerable number of mast cells, especially cellular leiomyoma. However, to our knowledge the mechanism by which mast cells increased in them is not known. The purpose of this study was to reveal the different mast cell subsets in smooth muscle tumors of uterus and to investigate the mechanism of local increase of mast cells.. Tissue sections from 85 uterine smooth muscle tumors were studied using immunohistochemical double labeling techniques, including 40 cases of ordinary leiomyomas, 30 cases of cellular leiomyomas and 15 cases of leiomyosarcomas. The sections were double immunostained for mast cell tryptase and chymase, mast cell tryptase and ki-67, mast cell tryptase and chemokines (i.e., CCL2, CCL5, CCL11, TGFbeta), as well as tryptase and CCR3.. MC(TC)-type of mast cells was the predominant type in ordinary leiomyoma and cellular leiomyoma, whereas MC(T)-type was seldom found in them. There was no MC(C) in smooth muscle tumors. The total intratumoral number of mast cells in cellular leiomyoma group was significantly higher than that in both leiomyosarcoma and ordinary leiomyoma (P<0.01). Mast cells proliferation was rarely detected in smooth muscle tumors, as revealed by constant negative labeling of the proliferation marker Ki-67 in mast cells. Almost all mast cells (tryptase positive) in smooth muscle tumors were also CCL2, CCL5, CCL11 and TGFbeta positive. Expressions of CCL5 and CCL11 in tumor cells in cellular leiomyoma were all significantly higher than that in both ordinary leiomyoma and leiomyosarcoma (P<0.01). While the expression of TGFbeta in tumor cells in cellular leiomyoma was not significantly different from that in ordinary leiomyoma, expression of CCL2 was not observed in smooth muscle tumor cells. There were positive correlations between CCL5 and the number of mast cells (r(s)=0.801, P<0.01) and between CCL11 and the number of mast cells (r(s)=0.744, P<0.01) in smooth muscle tumors as well. The vast majority of the mast cells in cellular leiomyoma were CCR3 positive.. Using the monoclonal anti-mast cell tryptase antibody could detect all mast cells in smooth muscle tumor. The increased intratumoral mast cell counts in cellular leiomyoma might be the result of mast cells recruitment from the peripheral blood rather than local mast cells proliferation. CCL5 and CCL11, which are expressed by smooth muscle tumor cells, are possibly responsible for the recruitment of mast cells in uterine cellular leiomyoma. Whether they combine to CCR3 expressed by mast cells need further study.

Topics: Chemokine CCL11; Chemokine CCL2; Chemokine CCL5; Chemokines, CC; Female; Humans; Ki-67 Antigen; Leiomyoma; Leiomyosarcoma; Mast Cells; Transforming Growth Factor beta; Uterine Neoplasms

The small leucine-rich proteoglycan biglycan is involved in several physiological and pathophysiological processes through the ability of its core protein to interact with other extracellular matrix molecules and transforming growth factor-beta (TGF-beta). To learn more about the regulation of biglycan core protein expression, we have cloned and sequenced 1218 base pairs from the 5'-flanking region of the human biglycan gene, demonstrated functional promoter activity, and investigated the molecular mechanisms through which various agents modulate its transcriptional activity. Sequencing revealed the presence of several cis-acting elements including multiple AP-2 sites and interleukin-6 response elements, a NF-kappaB site, a TGF-beta negative element, and an E-box. The TATA and CAAT box-lacking promoter possesses many features of a growth-related gene, e.g. a GC-rich immediate 5' region, many Sp1 sites, and the use of multiple transcriptional start sites. Transient transfections of the tumor cell lines MG-63, SK-UT-1, and T47D with various biglycan 5'-flanking region-luciferase reporter gene constructs showed that the proximal 78 base pairs are sufficient for full promoter activity. Several agents among them interleukin-6, and tumor necrosis factor-alpha. were capable of altering biglycan promoter activity. However, in MG-63 cells, TGF-beta1 failed to increase either activity of the biglycan promoter constructs or specific transcription from the endogenous biglycan gene. Since TGF-beta1 also did not alter the stability of cytoplasmic biglycan mRNA as determined from Northern analysis after inhibition of transcription with 5,6-dichloro-1beta-D-ribofuranosylbenzimidazole, an as yet unidentified nuclear post-transcriptional mechanism was considered responsible for the TGF-beta effect in this cell type. These results might help to elucidate the molecular pathways leading to pathological alterations of biglycan expression observed in atherosclerosis, glomerulonephritis, and fibrosis.

Topics: Base Sequence; Biglycan; Breast Neoplasms; Cells, Cultured; Extracellular Matrix Proteins; Gene Expression Regulation; Humans; Interleukin-6; Leiomyosarcoma; Male; Molecular Sequence Data; Promoter Regions, Genetic; Proteoglycans; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Testis; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The DDT1 MF2 smooth muscle cell line was derived from an estrogen/androgen-induced leiomyosarcoma arising in the hamster ductus deferens. Growth of this cell line is arrested in G0/G1 by treatment with glucocorticoids. To facilitate the study of the mechanism of glucocorticoid-induced cell growth arrest, a glucocorticoid-resistant variant cell line, DDT1 MF2 GR1 (GR1), was developed by genetic selection. Growth of this mutant cell line is completely resistant to the inhibitory action of glucocorticoids. However, we now demonstrate that both primary and secondary glucocorticoid-induced events still exist in the GR1 cell line. By analyzing the expression and genetic pattern of glucocorticoid receptor, no detectable rearrangement of the glucocorticoid receptor gene was found although the expression of both mRNA and protein levels of the receptor were lower in the variant compared to wild-type cells. In addition, we found that the expression of two growth-associated genes, Ha-ras and transforming growth factor beta 1 (TGF-beta 1) are down-regulated by glucocorticoids in wild-type DDT1 MF2 cells but not in GR1 cells. These results indicated that the function or activity of glucocorticoid receptor in the GR1 cells is not qualitatively altered. Our data suggest that a lower glucocorticoid receptor level is not the real cause or at least not the single cause for the GR1 cell's loss of sensitivity to the inhibitory action of glucocorticoid. Instead, we postulate the existence of a defect downstream of the primary site of action of glucocorticoid receptor complexes in GR1 cells.

Topics: Animals; Cell Division; Cricetinae; Drug Resistance; Gene Expression; Genes; Genes, ras; Glucocorticoids; In Vitro Techniques; Leiomyosarcoma; Muscle, Smooth; Receptors, Glucocorticoid; Restriction Mapping; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured