Mint3-mediated L1CAM expression in fibroblasts promotes cancer cell proliferation via integrin α5ß1 and tumour growth.

Fibroblasts are some of the major cells in tumour tissues that influence tumour progression and drug resistance. However, our understanding on fibroblast-mediated tumour malignancy remains incomplete. Munc18-1-interacting protein 3 (Mint3) is known as an activator of hypoxia-inducible factor-1 (HIF-1) even during normoxia in cancer cells, macrophages and fibroblasts. Although Mint3 promotes ATP production via glycolysis by activating HIF-1 in cancer cells and macrophages, the biological role of Mint3-mediated HIF-1 activation in fibroblasts remains unclear. To address this, we examined whether Mint3 in fibroblasts contributes to tumour growth. Mint3 depletion in mouse embryonic fibroblasts (MEFs) decreased tumour growth of co-injected human breast cancer cells, MDA-MB-231 and epidermoid carcinoma A431 cells in mice. In MEFs, Mint3 also promoted cancer cell proliferation in vitro in a cell-cell contact-dependent manner. Mint3-mediated cancer cell proliferation depended on HIF-1, and further gene expression analysis revealed that the cell adhesion molecule, L1 cell adhesion molecule (L1CAM), was induced by Mint3 and HIF-1 in fibroblasts. Mint3-mediated L1CAM expression in fibroblasts stimulated the ERK signalling pathway via integrin α5ß1 in cancer cells, and promoted cancer cell proliferation in vitro and tumour growth. In cancer-associated fibroblasts (CAFs), knockdown of MT1-MMP, which promotes Mint3-mediated HIF-1 activation, or Mint3 decreased L1CAM expression. As MEFs, CAFs also promoted cancer cell proliferation in vitro, and tumour growth via Mint3 and L1CAM. In human breast cancer specimens, the number of fibroblasts expressing L1CAM, Mint3 and MT1-MMP was higher in cancer regions than in adjacent benign regions. In addition, more phospho-ERK1/2-positive cancer cells existed in the peripheral region surrounded by the stroma than in the central region of solid breast cancer nest. Thus, Mint3 in fibroblasts might be a good target for cancer therapy by regulating cancer cell-stromal cell communication.

Cartilage oligomeric matrix protein contributes to the development and metastasis of breast cancer.

Cartilage oligomeric matrix protein (COMP) is a soluble pentameric protein expressed in cartilage and involved in collagen organization. Tissue microarrays derived from two cohorts of patients with breast cancer (n=122 and n=498) were immunostained, revealing varying expression of COMP, both in the tumor cells and surrounding stroma. High levels of COMP in tumor cells correlated, independently of other variables, with poor survival and decreased recurrence-free survival. Breast cancer cells, MDA-MB-231, stably expressing COMP were injected into the mammary fat pad of SCID (CB-17/Icr-Prkdcscid/Rj) mice. Tumors expressing COMP were significantly larger and were more prone to metastasize as compared with control, mock-transfected, tumors. In vitro experiments confirmed that COMP-expressing cells had a more invasive phenotype, which could in part be attributed to an upregulation of matrix metalloprotease-9. Furthermore, microarray analyses of gene expression in tumors formed in vivo showed that COMP expression induced higher expression of genes protecting against endoplasmic reticulum stress. This observation was confirmed in vitro as COMP-expressing cells showed better survival as well as a higher rate of protein synthesis when treated with brefeldin A, compared with control cells. Further, COMP-expressing cells appeared to undergo a metabolic switch, that is, a Warburg effect. Thus, in vitro measurement of cell respiration indicated decreased mitochondrial metabolism. In conclusion, COMP is a novel biomarker in breast cancer, which contributes to the severity of the disease by metabolic switching and increasing invasiveness and tumor cell viability, leading to reduced survival in animal models and human patients.

Cancer-associated myofibroblasts possess various factors to promote endometrial tumor progression.

Myofibroblastic invasion associated with malignant epithelial cells of endometrial cancer as well as other cancers is often found in the interstitium. To assess the myofibroblastic-epithelial interaction, frozen sections from a total of 10 endometrial cancers with or without invasive myofibroblasts were immunohistochemically examined. Interestingly, the invasive myofibroblasts adjacent to malignant epithelial cells showed frequently intensive positive staining of several growth factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor I, and epidermal growth factor, the cognate receptors such as Fetal liver kinase-1/Kinase Insert Domain-containing receptor/VEGF receptor-2, fms-like tyrosine kinase-1/VEGF receptor-1, and epidermal growth factor receptor, several cell cycle regulators such as cyclins and cyclin dependent kinases, and estrogen receptor alpha. Moreover, we indicated that the majority of the myofibroblasts as well as cancer epithelial cells are proliferating because of their positive staining of proliferating cell nuclear antigen and Ki-67. Furthermore, the myofibroblasts were also positive of hypoxia-inducible factor 1 alpha, which is a marker protein of hypoxia, probably followed by activation of VEGF-Flk-1 and VEGF-fms-like tyrosine kinase-1 signals, which could initiate angiogenesis. These findings suggest directly that the myofibroblasts might participate in the progression of tumor cells in terms of cancer cell growth stimulation and also activated initiation of angiogenesis.

Molecular cloning of ring finger protein 21 (RNF21)/interferon-responsive finger protein (ifp1), which possesses two RING-B box-coiled coil domains in tandem.

We have cloned the full length of a novel cDNA, named ring finger protein 21 (RNF21), composed of the RING finger-B box-coiled coil (RBCC) domain and the B30.2 domain, which are characteristic of the RBCC-B30.2 family. As a structural feature, the RNF21 cDNA possessed at least three kinds of isoforms, due to alternative splicing, consisting of the long form with the RBCC-RBCC-B30.2 domain, the medium form with the RBCC-B30.2 domain, and the short form with only the RBCC domain. Moreover, respective transcripts corresponding to the three isoforms were detected in various human organs by reverse transcription-PCR and Northern blot analyses. Interestingly, the medium form of the RNF21 mRNA expressed most predominantly was dramatically up-regulated within 8-16 h by interferon stimulation of HeLa cells. These findings suggest that RNF21 is a downstream gene that may mediate interferon's biological action.

Molecular cloning of testis-abundant finger Protein/Ring finger protein 23 (RNF23), a novel RING-B box-coiled coil-B30.2 protein on the class I region of the human MHC.

We have identified a genomic DNA fragment, using the PCR method with degenerate oligonucleotide primers which contain the conserved sequence of the RING finger domain. Using the DNA fragment as a probe, a novel cDNA was cloned from human and mouse testis. The cDNA had a domain structure of the typical RING-B box-coiled coil (RBCC)-B30.2 domain and therefore was named testis-abundant finger protein (tfp). Indeed, the transcript was highly expressed in the testis, although it was also found ubiquitously in various organs by Northern blot analysis. The tfp gene was mapped at the class I region of the human MHC (major histocompatibility complex), within which some known RBCC-B30.2 proteins such as RFP, RFB30/HERF1, AFP, and HZF had been localized. These findings demonstrate that several RBCC-B30.2 proteins including tfp, which are non-HLA proteins, are clustered within the class I region of the human MHC.

Chromosome mapping of RNF16 and rnf16, human, mouse and rat genes coding for testis RING finger protein (terf), a member of the RING finger family.

RNF16 (ring finger protein 16; alias terf), a member of the RING finger family, has been shown to be exclusively expressed in the testis. Human RNF16 is located at 1q42 based on PCR-assisted analysis of both a human/rodent mono-chromosomal hybrid cell panel and a radiation hybrid-mapping panel. On the other hand, chromosomal mapping of the RNF16 gene by fluorescence in situ hybridization reveals that mouse Rnf16 is located at 11B1.2-B1.3 and rat Rnf16 at 10q22. These results provide additional evidence that the mouse 11B region displays conserved linkage homology with the rat 10q22 region, whereas in the case of RNF16, this homology is only conserved among rodents, distinct from the 1q42 region of the human genome.

Impaired estrogen sensitivity in bone by inhibiting both estrogen receptor alpha and beta pathways.

Although it is well established that estrogen deficiency causes osteoporosis among the postmenopausal women, the involvement of estrogen receptor (ER) in its pathogenesis still remains uncertain. In the present study, we have generated rats harboring a dominant negative ERalpha, which inhibits the actions of not only ERalpha but also recently identified ERbeta. Contrary to our expectation, the bone mineral density (BMD) of the resulting transgenic female rats was maintained at the same level with that of the wild-type littermates when sham-operated. In addition, ovariectomy-induced bone loss was observed almost equally in both groups. Strikingly, however, the BMD of the transgenic female rats, after ovariectomized, remained decreased even if 17beta-estradiol (E(2)) was administrated, whereas, in contrast, the decrease of littermate BMD was completely prevented by E(2). Moreover, bone histomorphometrical analysis of ovariectomized transgenic rats revealed that the higher rates of bone turnover still remained after treatment with E(2). These results demonstrate that the prevention from the ovariectomy-induced bone loss by estrogen is mediated by ER pathways and that the maintenance of BMD before ovariectomy might be compensated by other mechanisms distinct from ERalpha and ERbeta pathways.

Efp as a primary estrogen-responsive gene in human breast cancer.

We have previously isolated the efp (estrogen-responsive finger protein) that is required for the normal estrogen-induced cell proliferation. Here, we show the genomic organization of the human efp gene which consists of nine exons. The efp mRNA was expressed in human breast tumors and the estrogen-induced expression of the efp was found in MCF-7 human breast cancer cells. Moreover, efp promoter activity was enhanced through the estrogen-responsive element dependent on estrogen and estrogen receptor. These results suggest that the efp can mediate estrogen actions such as cell growth in human breast cancer as a primary responsive gene.

Estrogen activates telomerase.

Telomerase activity is present in most malignant tumors and provides a mechanism for the unlimited potential for division of neoplastic cells. Although telomerase is known to be a regulated enzyme, the factors and mechanisms involved in telomerase regulation are not well understood. In the present study, we examined the effects of estrogen on telomerase activity. Telomerase activity in estrogen receptor (ER)-positive MCF-7 cells was up-regulated by the treatment with 17beta-estradiol. This activation accompanied up-regulation of the telomerase catalytic subunit, hTERT mRNA. Gel shift assays revealed that the imperfect palindromic estrogen-responsive element in the hTERT promoter specifically binds to ER. Transient expression assays using luciferase reporter plasmids containing various fragments of hTERT promoter showed that this imperfect palindromic estrogen-responsive element is responsible for transcriptional activation by ligand-activated ER. We also found that estrogen activates c-Myc expression in MCF-7 cells and that E-boxes in the hTERT promoter that bind c-Myc/Max play additional roles in estrogen-induced transactivation of hTERT. Estrogen thus activates telomerase via direct and indirect effects on the hTERT promoter. These findings may help elucidate the mechanisms of hormonal control of telomerase activity and aid understanding of the roles of sex steroids in cellular senescence and aging as well as estrogen-induced carcinogenesis.

Underdeveloped uterus and reduced estrogen responsiveness in mice with disruption of the estrogen-responsive finger protein gene, which is a direct target of estrogen receptor alpha.

The biological roles of estrogen-responsive finger protein (efp) in vivo were evaluated in mice carrying a loss-of-function mutation in efp by gene-targeted mutagenesis. Although efp homozygous mice were viable and fertile in both sexes, the uterus that expressed abundant estrogen receptor alpha exhibited significant underdevelopment. When the ovariectomized homozygotes were subjected to 17beta-estradiol treatment, they showed remarkably attenuated responses to estrogen, as exemplified by decreased interstitial water imbibition and retarded endometrial cell increase, at least, attributable to the lower ratio of G1 to S-phase progression in epithelial cells. These results suggest that efp is essential for the normal estrogen-induced cell proliferation and uterine swelling as one of the direct targets of estrogen receptor alpha.

Molecular cloning of rat efp: expression and regulation in primary osteoblasts.

We have previously identified an estrogen-responsive gene, efp (estrogen-responsive finger protein), by genomic binding-site cloning method. Here, we isolated a rat homologue of efp cDNA that encodes an open reading frame of 644 amino acids sharing high homology with human efp (69% identity at the protein level) and mouse efp (80% identity at the protein level). The efp protein has a RING finger, a variant type of zinc finger motif, B1 box and B2 box, each having a pair of zinc fingers, and coiled-coil domain, belonging to the RING finger-B box-Coiled Coil (RBCC) family. Several members of RBCC family including efp have characteristic C-terminal domain, forming a subfamily. Next, we detected efp mRNA in primary osteoblasts, one of estrogen target cells, derived from the calvariae of rat fetus. An anti-efp antibody revealed the efp protein is expressed and regulated by estrogen in the primary osteoblasts. Interestingly, the efp protein in primary osteoblasts is down-regulated by 1alpha,25-dihydroxyvitamin D(3) treatment that promotes the differentiation of the cells, whereas it is up-regulated by TGF-beta1 treatment that inhibits the differentiation of the cells. These findings suggest the possible involvement of the efp in the differentiation of osteoblastic cells.

NMDA receptor type 2D gene as target for estrogen receptor in the brain.

Although it is well known that estrogen exerts its effect in the brain, the direct target genes transcriptionally regulated by estrogen or rather estrogen receptor (ER) are almost unknown. During the search for estrogen receptor-binding sites from human CpG island library, we found one genomic DNA fragment corresponding to the putative 3'-untranslated region of human NMDA receptor subunit 2D (NR2D) gene. It contained at least four half palindromic estrogen responsive elements (hEREs) within two hundred nucleotides, which was conserved also in the rat. Interestingly, the NR2D mRNA is co-localized with ERalpha and/or ERbeta mRNA in a number of regions of rat brain. We have also demonstrated that NR2D mRNA is up-regulated in rat hypothalamus by estrogen possibly via hEREs identified here. Thus, we suggest that NR2D is one of the direct targets of estrogen receptors which are involved in reproductive as well as non-reproductive actions in the brain.

Differential immunolocalization of estrogen receptor alpha and beta in rat ovary and uterus.

In order to investigate the localization of estrogen receptor (ER) alpha and ERbeta in the reproductive organs in the rat, polyclonal antibodies were raised to each specific amino acid sequence. The Western blot with anti-ERalpha antibody showed a 66 kDa band in rat ovary and uterus, while that with anti-ERbeta antibody detected a 55 kDa band in rat ovary, uterus and prostate. The ligand-independent nuclear localization of the two receptors was verified by immunocytochemistry. By immunohistochemistry, the nuclei of glandular and luminal epithelial cells in the uterus were stained with anti-ERalpha antibody, whereas only the nuclei of glandular epithelium cells were stained with anti-ERbeta antibody. In rat ovary, positive signals were shown with anti-ERbeta antibody in the nuclei of granulosacells. No specific immunostaining was observed with anti-ERalpha antibody. Although ERbeta was immunostained at the proestrous, metestrous and diestrous stages, the immunoreactivity of ERbeta was hardly detected at the estrous stage in rat ovary. Thus, we show differential expression of ERalpha and ERbeta in rat uterus and ovary at the protein level, which may provide a clue for understanding the roles of the two receptors in reproductive organs.

Molecular cloning, localization, and developmental expression of mouse brain finger protein (Bfp)/ZNF179: distribution of bfp mRNA partially coincides with the affected areas of Smith-Magenis syndrome.

Bfp (brain finger protein) is a member of the RING finger protein family, which is highly expressed in the brain. We have previously shown that one copy of the human bfp gene, mapped at 17p11.2, was actually deleted in six of six Smith-Magenis syndrome (SMS) patients. Now we have isolated the mouse bfp cDNA. Using in situ hybridization and immunohistochemistry, the distribution of mouse bfp mRNA and protein was identified especially in neural cells of the cerebral cortex, hippocampus, lateral amygdaloid nucleus, and ventromedial hypothalamus. In primary culture of the whole brain in a neonatal mouse, the Bfp protein was detected in both neuron and glial cells, and its subcellular localization was predominantly in the nucleus, but some amounts were also found in the cytoplasm. The bfp mRNA was also expressed strongly in the marginal zone of brain vesicles, optic stalk, and cartilage primordium, which are part of the critical tissues frequently involved in SMS patients, and in such tissues as nasal epithelium and primordium of follicles in a 13. 5-dpc embryo. Subsequently, its amount in the developing brain further increased during embryogenesis, reaching the highest level in the adult brain. These findings suggest a possibility that Bfp might be involved in the pathogenesis of Smith-Magenis syndrome as a regulator protein related to neural differentiation and function.

Molecular cloning of a novel RING finger-B box-coiled coil (RBCC) protein, terf, expressed in the testis.

RING finger is a variant zinc finger motif present in a new family of proteins including transcription regulators. Here, utilizing the polymerase chain reaction with degenerate primers, we isolated a genomic DNA fragment containing the RING finger motif. Using this fragment as a probe, we have identified a novel cDNA from rat testis library. Then, the human homologue of the terf cDNA was also isolated from a testis library. This gene was designated testis RING finger protein (terf) because the corresponding transcripts were detected almost exclusively in the testis by Northern blot analysis. Both cDNAs encode an open reading frame of 477 amino acids sharing high homology (74% identity at the protein level) between two species. The terf contains an N-terminal RING finger domain, one B-box domain, middle coiled-coil domain, and a C-terminal domain, belonging to the RING finger-B box-coiled coil (RBCC) family. Several RBCC proteins, such as PML, TIF1alpha and RFP, have transformation capabilities when found in chromosomal translocations. Among the members of the RBCC family, the terf shares highest homology (40% identity at the protein level) with RFP that is expressed only in the testis in normal tissues. Structural similarity raises the possibilities that the terf gene might be also involved in carcinogenesis or cell transformation.

Promoter analysis of mouse estrogen-responsive finger protein (efp) gene: mouse efp promoter contains an E-box that is also conserved in human.

The estrogen-responsive finger protein (efp) containing a RING finger motif has been identified as an estrogen-responsive gene in human and mouse. Here, we have characterized the basal promoter region of the mouse efp gene. The promoter lacks the TATA motif, and transcription initiation sites are found at positions -38T, -64A and -73C from the translation initiation site. Deletion analysis of the 5'-flanking region using Jyg-Mc(B) mouse breast cancer cells indicates that the sequence encompassing from -139 to -1 has a basal transcription activity. This region is GC-rich in both mouse and human promoters, and the E-box is precisely matched on the sequence alignment. A mutation experiment with E-box shows that the E-box is functionally active. An electrophoretic mobility shift assay using Jyg-Mc(B) nuclear extracts shows that a transcription factor, USF-1 binds to the E-box in the mouse efp promoter. It has been shown that the E-box in the human efp promoter is indispensable for basal transcriptional activity and binds USF-1. These findings suggest that the mouse efp promoter is regulated by a similar mechanism to that of the human. In mouse, however, we have not found a negative regulatory region that is present in human promoter.

Molecular cloning and characterization of human estrogen receptor betacx: a potential inhibitor ofestrogen action in human.

We have identified and characterized a novel human estrogen receptor (ER) beta isoform, ERbetacx, which is truncated at the C-terminal region but has an extra 26 amino acids due to alternative splicing. The ERbetacx transcript is expressed in testis, ovary, thymus and prostate as well as in human cultured cell lines such as HEC-1, HOS-TE85 and Saos-2 cells. ERbetacx protein is also immunodetectable in these human cells. Biochemical analysis reveals that the average dissociation constants ( K d) of ERalpha and ERbeta for 17beta-estradiol (E2) are 0.2 and 0.6 nM respectively, but ERbetacx has no ligand binding ability. ERalpha and ERbeta proteins bind to the estrogen response element, whereas ERbetacx does not form any shifted complex in gel shift assays. In a transient expression assay, ERbetacx shows no ligand-dependent transactivation ability of a basal promoter and also cannot interact with a cofactor, TIF1alpha, in the presence or absence of E2. ERbetacx preferentially forms a heterodimer with ERalpha rather than that with ERbeta, inhibiting DNA binding by ERalpha. Interestingly, however, it shows a significant dominant negative activity only against ERalpha transactivation. Thus, this study indicates that ERbetacx potentially inhibits ERalpha-mediated estrogen action and that alternative splicing of the C-terminal region and its inhibitory properties are characteristic of several members of nuclear receptor isoforms.

UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells.

We have obtained a novel transcriptional cofactor, termed undifferentiated embryonic cell transcription factor 1 (UTF1), from F9 embryonic carcinoma (EC) cells. This protein is expressed in EC and embryonic stem cells, as well as in germ line tissues, but could not be detected in any of the other adult mouse tissues tested. Furthermore, when EC cells are induced to differentiate, UTF1 expression is rapidly extinguished. In normal mouse embryos, UTF1 mRNA is present in the inner cell mass, the primitive ectoderm and the extra-embryonic tissues. During the primitive streak stage, the induction of mesodermal cells is accompanied by the down-regulation of UTF1 in the primitive ectoderm. However, its expression is maintained for up to 13.5 days post-coitum in the extra-embryonic tissue. Functionally, UTF1 boosts the level of transcription of the adenovirus E2A promoter. However, unlike the pluripotent cell-specific E1A-like activity, which requires the E2F sites of the E2A promoter for increased transcriptional activation, UTF1-mediated activation is dependent on the upstream ATF site of this promoter. This result indicates that UTF1 is not a major component of the E1A-like activity present in pluripotent embryonic cells. Further analyses revealed that UTF1 interacts not only with the activation domain of ATF-2, but also with the TFIID complex in vivo. Thus, UTF1 displays many of the hallmark characteristics expected for a tissue-specific transcriptional coactivator that works in early embryogenesis.