thymosin-beta(4) and Breast-Neoplasms

Intratumoral hypoxia has been correlated with distant metastatic potential. Two hypoxia inducible factors (HIFs), HIF-1α and HIF-2α, are induced by hypoxia, and high expression of these proteins has been correlated to angiogenesis and distant metastasis. Thymosin β4 (Tβ4) is frequently highly expressed in cancer, and this overexpression correlates with malignant progression. The objective of this study was to investigate the clinical correlation of HIF-α with Tβ4 and the intracellular functional roles of Tβ4 on HIF-α activation. We examined HIF-1α, HIF-2α and Tβ4 expressions in clinical human breast carcinoma (n=70) by immunohistochemistry. We show that high expression of HIF-1α and HIF-2α strongly correlates with Tβ4 expression (P≤0.0001) and overexpression of Tβ4 correlates significantly with patients with lymph node metastasis (P<0.05) of human breast cancer. Additionally, we demonstrate that hypoxia up-regulates intracellular Tβ4 protein, which then affects HIF-α activity, which is the key in regulating VEGF expression. We confirmed that hypoxia-induced intracellular Tβ4 and HIF-α activities were reduced by interference of Tβ4 expression using Tβ4 shRNA lentivirus. Vascular epidermal growth factor (VEGF)-A, a well-recognized lymphangiogenic cytokine, was also down-regulated, but VEGF-C and VEGF-D expressions were not affected. These findings suggest that the overexpression of Tβ4 is strongly associated with HIF-1α and HIF-2α expression and is also clinicopathologically involved with lymph node metastatic potential of breast cancer through the modulation of HIF-αactivation and induction of VEGF-A. Ultimately, these results highlight Tβ4 as a potentially therapeutic target in malignant cancers.

Topics: Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Cell Hypoxia; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Lymphatic Metastasis; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; Thymosin

Previous studies have shown that the G-actin sequestering polypeptide thymosin beta4 frequently is overexpressed in cancers and that such overexpression correlates to malignant progression. However, the localization of thymosin beta4 in human cancers has not been determined. We now demonstrate that there is a considerable heterogeneity in the cellular distribution of thymosin beta4 in breast cancer. In most tumors examined, cancer cells showed low or intermediate reactivity for thymosin beta4, whereas leukocytes and macrophages showed intense reactivity. In addition, endothelial cells showed variable reactivity to thymosin beta4, whereas myofibroblasts were negative. There was no correlation between the intensity of tumor cell staining and histological grade, whereas there was a tendency toward a correlation between endothelial cell staining and grade. These results demonstrate that multiple cell types within the tumor microenvironment produce thymosin beta4 and that such expression varies from tumor to tumor. Such heterogeneity of expression should be taken into account when the role of thymosin beta4 in tumor biology is assessed.

Topics: Breast Neoplasms; Endothelial Cells; Female; Fluorescent Antibody Technique; Humans; Leukocytes; Macrophages; Stromal Cells; Thymosin

Overexpression of thymosin beta-4 has been linked to malignant progression but the localization of this polypeptide within tumors is incompletely known. We therefore examined breast cancers for thymosin beta-4 using immunofluorescence. Reactive cells were identified with monoclonal cell marker antibodies. A very heterogeneous staining pattern for thymosin beta-4 was observed. Thus, while leukocytes and macrophages showed intense reactivity for this polypeptide, cancer cells, and endothelial cells showed a much more variable reactivity. A similar heterogeneous staining was observed also in colorectal carcinomas. The degree of staining of breast cancer cells for thymosin beta-4 correlated neither to histological grade nor to endothelial cell staining. However, there was a tendency toward correlation (P = 0.07) between staining of endothelial cells and histological grade. Treatment of cultured breast cancer cells (SK-BR-3) with 1-4 microg thymosin beta-4/mL significantly increased cell numbers, as determined by MTT-assays. These data reveal an unexpected cellular heterogeneity of thymosin beta-4 expression in breast and colonic carcinomas and suggest that local release of this polypeptide in the tumor microenvironment may modulate tumor behavior.

Topics: Breast Neoplasms; Cell Nucleus; Colorectal Neoplasms; Cytoplasm; Female; Fluorescent Antibody Technique; Humans; Keratins; Leukocytes; Thymosin

A method has been developed for high sequence coverage analysis of proteins isolated from breast cancer cell lines. Intact proteins are isolated using multidimensional liquid-phase separations that permit the collection of individual protein fractions. Protein digests are then analyzed by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting and by capillary electrophoresis-electrospray ionization (CE-ESI)-TOF MS peptide mapping. These methods can be readily interfaced to the relatively clean proteins resulting from liquid-phase fractionation of cell lysates with little sample preparation. Using combined sequence information provided by both mapping methods, 100% sequence coverage is often obtained for smaller proteins, while for larger proteins up to 75 kDa, over 90% coverage can be obtained. Furthermore, an accurate intact protein MW value (within 150 ppm) can be obtained from ESI-TOF MS. The intact MW together with high coverage sequence information provides accurate identification. More notably the high sequence coverage of CE-ESI-TOF MS together with the MS/MS information provided by the ion trap/reTOF MS elucidates posttranslational modifications, sequence changes, truncations, and isoforms that may otherwise go undetected when standard MALDI-MS peptide fingerprinting is used. This capability is critical in the analysis of human cancer cells where large numbers of expressed proteins are modified, and these modifications may play an important role in the cancer process.

Topics: Amino Acid Sequence; Breast Neoplasms; Cell Line, Tumor; Chromatography, High Pressure Liquid; Electrophoresis, Capillary; Electrophoresis, Gel, Two-Dimensional; Humans; Isoelectric Focusing; Lamins; Molecular Sequence Data; Molecular Weight; Protein Isoforms; Protein Processing, Post-Translational; Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thymosin; Time Factors; Trypsin

To identify genes that are involved in breast cancer, suppression subtractive hybridization (SSH) was utilized to construct a breast cancer subtracted library. Differential screening of the library isolated 28 genes which by Northern analysis were highly expressed in the breast cancer cell line MDA-MB-231 compared to the normal breast cell line MCF12A. Sequence analysis revealed that 15 clones coded for previously described genes such as SNAP43, Cyr61, Thymosin beta4, tra1, elongation factor 1alpha, BSF-2/IL6, BiP, and GDP/GTP exchange protein. The remaining 13 clones did not match sequences in GenBank/EMBL database, indicating that they may be novel genes. SNAP43, a subunit of the TBP-TAF complex, was expressed 20-fold higher in MDA-MB-231 compared to MCF12A and several breast cancer cell lines, implying that SNAP43 may be involved in tumorigenesis of a specific subset of breast cancers. Amplification of SNAP43 was not found by Southern analysis. However, genetic alterations of MDA-MB-231 included a deletion of chromosome 14 with a reciprocal translocation t(6;14) and two additional translocations [t(12;14) and t(14;15)] as determined by fluorescent in situ hybridization (FISH) with YAC 823G8 located at chromosome 14q23 which contained SNAP43. Because of the numerous alterations observed by FISH in MDA-MB-231, we further explored the genetic abnormalities in this breast cancer cell line using multiplex FISH (M-FISH) and comparative genomic hybridization (CGH). These cells were replete with numerous complex structural rearrangements and had DNA copy-number imbalances involving multiple chromosomes including gains on chromosomes 2p, 2q31-q32, 3p14-pter, 5q, 6p, 7q36-qter, 11, 14q21-q24, 17p11.2-pter, 17q21-qter, 19, 20, Xp11-q13 and losses on chromosomes 4pter-q32, 8p, 9p21-p24, 10q26-qter, 16p13-pter, 18q12-qter, 22, Xp11.3-p22.1, Xq13-qter. In summary, SSH revealed a number of genes that were either novel or previously not associated with breast cancer. In addition, we found that breast cancer cells abounded with abnormalities as observed by M-FISH and CGH. Together, these results may facilitate defining the genetic alterations associated with breast cancer progression.

Topics: Breast Neoplasms; Chromosome Aberrations; Chromosomes, Human, Pair 4; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization, Fluorescence; Nucleic Acid Hybridization; Thymosin; Transcription Factors; Translocation, Genetic; Tumor Cells, Cultured

Elevated expression of the beta-thymosin isotypes T beta(4), T beta(10), and T(15) appears to be involved in the manifestation of a malignant phenotype of human tumor cells, including those of mammary carcinomas. This has evoked an interest in these peptides as diagnostic/prognostic tumor markers. If increased levels of beta-thymosins correspond to tumor malignancy, the question arises whether tumor growth inhibition induced by chemotherapeutic drugs would reduce their expression.. Two human breast cancer cell lines, the estrogen receptor(ER)-positive MCF-7 and the ER-negative MDA-MB231, were thus analyzed for the amount of beta-thymosin mRNAs by RNase protection assay and for the respective peptide levels by HPLC following different hormonal and drug treatments.. Both cell lines, growing in medium with 10% FCS, contain T beta(4) (400-500 fg/cell) and Tbeta(10) (about 100 fg/cell), but no T beta(15). Incubating MCF-7 cells with tamoxifen (1 microM) for 5 days resulted in about 80% growth inhibition and in reduction of intracellular T beta(4) and T beta(10) concentrations by about 40%. Levels of T beta(4) and T beta(10)-mRNA were reduced by about 60%. In contrast, cisplatin (2 microM) changed neither the peptide concentrations nor the mRNA levels of beta-thymosins, in spite of marked growth inhibition. In addition, no changes in beta-thymosin expression were observed in MDA-MB231 cells treated with either drug. MCF-7 cells maintained in estrogen-poor medium (10% horse serum) or stimulated to grow with estradiol (1 nM) had Tbeta(4) and T beta(10) concentrations reduced by about 30%, but changes in T beta(4)- and T beta(10)-mRNA levels did not correspond to those of the peptide.. Expression of T beta(4) and T beta(10) mRNAs and their peptides is differentially regulated and does not correlate with growth. Instead, reduced beta-thymosin expression may be linked to more intensive TRITC-phalloidin staining of F-actin lining the membrane at sites of intimate cell-cell contacts, while increased beta-thymosin levels appear in cells with more extensive substrate adhesion. This suggests that beta-thymosins play a role in cell surface dynamics.

Topics: Actins; Antineoplastic Agents; Breast Neoplasms; Cytoskeleton; Female; Humans; Receptors, Estrogen; RNA, Messenger; Thymosin; Tumor Cells, Cultured

Thymosins beta 4 and beta 10 are 2 structurally related polypeptides originally defined in the rat immune system. To date, no truly unambiguous functions have been formally ascribed to these small (less than 4.9 kDa) acidic proteins. Previous research has demonstrated relationships between expression of these genes and cell growth/differentiation. These observations prompted the present study which has used cDNA and synthetic oligonucleotide probes in combination with high-performance liquid chromatography (HPLC) to examine the differential expression of these 2 genes in normal and neoplastic human tissues and in the developing human kidney. Low levels of beta 4 and beta 10 mRNA species prevailed in normal tissues; in contrast, these gene transcripts were notably more abundant in malignant renal tumors and in the normal human embryonic kidney. These findings show that the thymosin beta 4 and beta 10 genes are constitutively expressed at higher levels in embryonic/neoplastic as compared to normal/benign tissues and that thymosin in beta 10 in particular may be a new molecular marker for renal-cell carcinoma as well as other malignancies.

Topics: Aging; Base Sequence; Blotting, Northern; Breast Neoplasms; Chromatography, High Pressure Liquid; Cloning, Molecular; Female; Gene Expression; Humans; Infant; Kidney; Kidney Neoplasms; Middle Aged; Molecular Sequence Data; Neoplasm Proteins; Neoplasms; Nucleic Acid Hybridization; Oligonucleotide Probes; Ovarian Neoplasms; Reference Values; RNA, Messenger; Thymosin