Identification of a novel aspartic-like protease differentially expressed in human breast cancer cell lines

Four different human breast cancer cell lines were examined to search for genes associated with tumor growth and metastasis. Each of these cell lines, MDA-MB-453, MCF-7, MDA-MB-231 and MDA-MB-435, displays different phenotypic characteristics ranging from poorly to highly tumorigenic and metastatic. The differences in gene expression profiles of these cell lines generated by differential display technique should allow one to identify candidates as putative oncogenes or tumor/metastasis suppressor genes. A novel cDNA expressed in the highly tumorigenic and metastatic cell line, MDA-MB-435, was identified and isolated by this approach. The function for this gene, designated ALP56 (aspartic-like protease 56 kDa), in tumor progression is suggested by the homology of the encoded protein to aspartic proteases, such as cathepsin D. The amino acid residues in two catalytic domains of this family are highly conserved in those domains of ALP56. Northern hybridization indicated that the expression of ALP56 is associated with growth and metastasis of MDA-MB-435 tumors in immunodeficient mice. In situ hybridization of biopsies from breast cancer and colon cancer patients indicated that ALP56 is upregulated in human primary tumors and liver metastasis. These results suggest that this novel gene correlates with human tumor progression.     

Identification of hTLR10: a novel human Toll-like receptor preferentially expressed in immune cells

Herein we describe the isolation of a cDNA encoding a novel human Toll-like receptor (hTLR) that we term hTLR10. Human TLR10 contains 811 amino acid residues. Deduced amino acid sequence analysis reveals that like the other known hTLRs (hTLR1-9) it is characterized by a signal peptide followed by multiple leucine-rich repeats (LRRs), a cysteine-rich domain, a transmembrane sequence and a cytoplasmic domain homologous to that of the human interleukin-1 receptor. Phylogenetic analysis indicates that among all the hTLRs, hTLR10 is most closely related to hTLR1 and hTLR6; the overall amino acid identity is 50% and 49%, respectively. hTLR10 mRNA is most highly expressed in lymphoid tissues such as spleen, lymph node, thymus, and tonsil. Expression analysis of cell lines indicates a predominance in a variety of immune cell types. Thus, hTLR10 is preferentially expressed in tissues and cells involved in immune responses.     

A novel variant of ER-alpha, ER-alpha36 mediates testosterone-stimulated ERK and Akt activation in endometrial cancer Hec1A cells

Background  

Endometrial cancer is one of the most common gynecologic malignancies and its incidence has recently increased. Experimental and epidemiological data support that testosterone plays an important role in the pathogenesis of endometrial cancer, but the underlying mechanism has not been fully understood. Recently, we identified and cloned a variant of estrogen receptor (ER) alpha, ER-alpha36. The aim of the present study was to investigate the role of ER-alpha36 in testosterone carcinogenesis.     

Identification of a novel prostate cancer susceptibility variant in the KLK3 gene transcript

Genome-wide association studies (GWAS) have identified more than 30 prostate cancer (PrCa) susceptibility loci. One of these (rs2735839) is located close to a plausible candidate susceptibility gene, KLK3, which encodes prostate-specific antigen (PSA). PSA is widely used as a biomarker for PrCa detection and disease monitoring. To refine the association between PrCa and variants in this region, we used genotyping data from a two-stage GWAS using samples from the UK and Australia, and the Cancer Genetic Markers of Susceptibility (CGEMS) study. Genotypes were imputed for 197 and 312 single nucleotide polymorphisms (SNPs) from HapMap2 and the 1000 Genome Project, respectively. The most significant association with PrCa was with a previously unidentified SNP, rs17632542 (combined P?=?3.9?×?10(-22)). This association was confirmed by direct genotyping in three stages of the UK/Australian GWAS, involving 10,405 cases and 10,681 controls (combined P?=?1.9?×?10(-34)). rs17632542 is also shown to be associated with PSA levels and it is a non-synonymous coding SNP (Ile179Thr) in KLK3. Using molecular dynamic simulation, we showed evidence that this variant has the potential to introduce alterations in the protein or affect RNA splicing. We propose that rs17632542 may directly influence PrCa risk.     

Identification of a novel family of cell-surface proteins expressed in human vascular endothelium

Vascular endothelial cells (EC) play a key role in a variety of pathophysiologic processes, such as angiogenesis, inflammation, cancer metastasis, and vascular diseases. As part of a strategy to identify all genes expressed in human EC, a full-length cDNA encoding a potential secreted protein harboring 10 epidermal growth factor (EGF)-like domains and one CUB domain at the carboxyl terminus (termed, SCUBE1 for Signal peptide-CUB-EGF-like domain containing protein 1) was identified. SCUBE1 shares homology with several protein families, including members of the fibrillin and Notch families, and the anticoagulant proteins, thrombomodulin and protein C. SCUBE1 mRNA is found in several highly vascularized tissues such as liver, kidney, lung, spleen, and brain and is selectively expressed in EC by in situ hybridization. SCUBE1 is a secreted glycoprotein that can form oligomers and manifests a stable association with the cell surface. A second gene encoding a homologue (designated SCUBE2) was also identified and is expressed in EC as well as other cell types. SCUBE2 is also a cell-surface protein and can form a heteromeric complex with SCUBE1. Both SCUBE1 and SCUBE2 are rapidly down-regulated in EC after interleukin-1beta and tumor necrosis factor-alpha treatment in vitro and after lipopolysaccharide injection in vivo. Thus, SCUBE1 and SCUBE2 define an emerging family of human secreted proteins that are expressed in vascular endothelium and may play important roles in development, inflammation, and thrombosis.     

Identification of a novel high molecular weight protein preferentially expressed by sinusoidal endothelial cells in normal human tissues

Mouse mAb MS-1, raised against human spleen, detects an endothelial cell antigen abundantly expressed by the sinusoidal endothelia of spleen, lymph node, liver, and adrenal cortex, but absent from nonsinusoidal continuous endothelia in these organs. Immunoelectron microscopy of splenic tissue demonstrates that the MS-1 antigen is predominantly deposited at zones of intercellular contact between adjacent sinusoidal endothelial cells. mAb MS-1 also reacts with a variable proportion of high endothelial venules in tonsil, but not in other lymphoid tissues, and with an interstitial dendritic cell population most abundant in placenta. mAb MS-1 does not react with cultured resting or mediator- activated human umbilical vein endothelial cells, dermal fibroblasts, peripheral blood mononuclear cells, or the cell lines U937, HL-60, K562 or Mo7E; it does react with the primitive myeloid cell line KG-1. mAb MS-1 immunoprecipitates a major protein of 215 kD and minor proteins of 320 and 120 kD from splenic extracts as analyzed by SDS-PAGE with reduction. These proteins are soluble in aqueous buffers. Immunoprecipitation from KG-1 cell lysates detects four proteins of 280, 300, 205, and 120 kD; the 300-, 205-, and 120-kD species, presumably corresponding to the 320-, 215-, and 120-kD species in spleen, respectively, are secreted into the media. Under nonreducing conditions, immunoprecipitates from KG-1 cell lysates or conditioned media contain one predominant 300-kD species; upon isolation and reduction, this 300-kD species separates into the previously observed 300-, 205-, and 120-kD species. Pulse-chase experiments and limited proteolysis peptide mapping suggest that the 280-kD species is a precursor of the mature 300-kD species which may be subsequently cleaved to yield the 205- and 120-kD species. Because of its size, solubility and expression pattern, the antigen recognized by mAb MS-1 is likely to be an extracellular matrix protein utilized by endothelial cells of contorted, large caliber, or leaky microvessels that lack a well-formed basement membrane.     

Identification of differentially expressed genes associated with HER-2/neu overexpression in human breast cancer cells.

Amplification and resulting overexpression of the HER-2/ neu proto-oncogene is found in approximately 30% of human breast and 20% of human ovarian cancers. To better understand the molecular events associated with overexpression of this gene in human breast cancer cells, differential hybridization was used to identify genes whose expression levels are altered in cells overexpressing this receptor. Of 16 000 clones screened from an overexpression cell cDNA library, a total of 19 non-redundant clones were isolated including seven whose expression decreases (C clones) and 12 which increase (H clones) in association with HER-2/ neu overexpression. Of these, five C clones and 11 H clones have been confirmed to be differentially expressed by northern blot analysis. This group includes nine genes of known function, three previously sequenced genes of relatively uncharacterized function and four novel genes without a match in GenBank. Examination of the previously characterized genes indicates that they represent sequences known to be frequently associated with the malignant phenotype, suggesting that the subtraction cloning strategy used identified appropriate target genes. In addition, differential expression of 12 of 16 (75%) cDNAs identified in the breast cancer cell lines are also seen in HER-2/ neu -overexpressing ovarian cancer cells, indicating that they represent generic associations with HER-2/ neu overexpression. Finally, up-regulation of two of the identified cDNAs, one novel and one identified but as yet uncharacterized gene, was confirmed in human breast cancer specimens in association with HER-2/ neu overexpression. Further characterization of these genes may yield insight into the fundamental biology and pathogenetic effects of HER-2/ neu overexpression in human breast and ovarian cancer cells.     

Identification of a novel alternative splicing of human FGF receptor 4: soluble-form splice variant expressed in human gastrointestinal epithelial cells

Among four closely related members of the FGF receptor family, FGFR 1, 2, and 3 have alternative splicing forms encoded by different exons for the C-terminal half of the third Ig-like domain, but FGFR 4 has no such alternative exon. Furthermore, FGFR 1, 2, and 3 have another splice variant of nontransmembrane type; however, such a variant has not been reported for FGFR 4. While searching for a novel receptor-type tyrosine kinase by RT-PCR, we identified a non-transmembrane-type receptor of FGFR 4 in human intestinal epithelial cell lines (Intestine 407 and Caco-2). Sequence analysis of this receptor revealed that exon 9 coding the single transmembrane domain was displaced by intron 9. Consequently, this variant form was 120 bp shorter than the normal form and had no transmembrane portion. Moreover, the signal sequence in exon 2 was maintained, suggesting that this splice variant is a soluble receptor. This soluble receptor was detected in human gastrointestinal epithelial cells and pancreas, and also in gastric, colon, and pancreatic cancer cell lines. Single cell RT-PCR showed that this soluble receptor was expressed simultaneously with the transmembrane-type receptor in the same cell. Western blot analysis revealed that this receptor was secreted from the transfected COS7 cells. Thus, a soluble-form splice variant of FGFR 4 was identified in human gastrointestinal epithelial cells and cancer cells. This is the first report of alternative splicing of FGFR 4.