Synuclein γ protects Akt and mTOR and renders tumor resistance to Hsp90 disruption.

Heat shock protein (Hsp)90 regulates many key pathways in oncogenesis, including Akt and mammalian target of rapamycin (mTOR). The strengths of disruption of Hsp90 in cancer therapy include their versatility in inhibiting a wide range of oncogenic pathways. The present study demonstrated that synuclein γ (SNCG) protects the functions of Akt and mTOR in the condition when the function of Hsp90 is blocked. Disruption of Hsp90 abolished Akt activity and mTOR signaling. However, expression of SNCG restored Akt activity and mTOR signaling. SNCG bound to Akt and mTOR in the presence and absence of Hsp90. Specifically, the C-terminal (Gln106-Asp127) of SNCG bound to the loop connecting αC helix and ß4 sheet of the kinase domain of Akt. SNCG renders resistance to 17-AAG-induced apoptosis both in vitro and in tumor xenograft. A clinical follow-up study indicates that patients with an SNCG-positive breast cancer have a significantly shorter disease-free survival and overall survival than patients with SNCG-negative tumors. The present study indicates that SNCG protects Hsp90 client proteins of Akt and mTOR, and renders drug resistance to Hsp90 disruption.

Chaperoning of estrogen receptor and induction of mammary gland proliferation by neuronal protein synuclein gamma.

Synucleins are emerging as central players in the formation of pathologically insoluble deposits characteristic of neurodegenerative diseases. However, synuclein gamma (SNCG), previously identified as a breast cancer specific gene (BCSG1), is also highly associated with breast cancer progression. Using transgenic mouse model, we demonstrated a role of SNCG in induction of highly proliferative pregnancy-like phenotype of mammary epithelial cells and branching morphology. SNCG participated in the heat shock protein-based multiprotein chaperone complex for steroid receptor signaling. Expression of SNCG in mammary epithelium resulted in a significant stimulation of ERalpha transcriptional activity. SNCG-induced mammary gland proliferation can be effectively blocked by antiestrogen and ovariectomy, indicating that the induced proliferation is mediated by ERalpha signaling and requires estrogen stimulation. These data indicate the chaperone activity of SNCG on stimulation of steroid receptor signaling in mammary gland and, thus induces extensive mammary gland proliferation and contributes to the hormonal impact on mammary tumorigenesis.

An epidemiological investigation of congenital Schistosoma japonicum transmission in Hubei Province, PR China.

A cohort study was conducted in Hubei Province, China, following serious flooding of the Yangtze River in the autumn of 1998 to investigate the possibility of congenital transmission of Schistosoma japonicum in humans. The cohort investigated was comprised of 205 women and their 208 infants born between 1 September and 30 December 1998. Blood and fecal samples from all the women and their infants were collected and examined for S. japonicum infection. Positive specific antibody titers were found in 14 (6.8%) of the mothers, but no fecal egg excretion was observed. All infants had negative specific antibody titers and no S. japonicum eggs were found in their feces. Hence, the present study coud not confirm congenital S. japonicum transmission in humans. Further studies are highly wanted to study the impact of prenatal exposure of S. japonicum on the offspring.

Inhibition of Wilms' tumor growth by intramuscular administration of tissue inhibitor of metalloproteinases-4 plasmid DNA.

Extracellular matrix (ECM) degrading matrix metalloproteinases (MMPs) lead to ECM turnover, a key event in cancer growth and progression. The tissue inhibitors of matrix metalloproteinases (TIMPs) limit the activity of MMPs, which suggests their use for cancer gene therapy. Here we report that systemic administration of naked TIMP-4 DNA significantly inhibited Wilms' tumor growth in nude mice. TIMP-4, whose expression was lost in Wilms' tumor, inhibited the growth of G401 Wilms' tumor cells at a concentration lower than those required for MMP inhibition. This inhibition was associated with internalization of exogenous recombinant TIMP-4. Electroporation-mediated intramuscular injection of TIMP-4 expression plasmid resulted in sustained plasma TIMP-4 levels and significant tumor suppression. Our data demonstrate a tumor suppressive effect of TIMP-4 against Wilms' tumor and the potential utility of intramuscular delivery of TIMP gene for treatment of kidney derived cancers.

Stimulation of mammary tumorigenesis by systemic tissue inhibitor of matrix metalloproteinase 4 gene delivery.

Tissue inhibitors of matrix metalloproteinase (TIMPs) are multifunctional proteins with both matrix metalloproteinase (MMP) inhibitory effects and growth-regulatory activity. TIMPs inhibit MMP activity, suggesting a use for cancer gene therapy. However, here we report that systemic administration of human TIMP-4 by electroporation-mediated i.m. injection of naked TIMP-4 DNA stimulates tumorigenesis of human breast cancer cells in nude mice. Consistent with tumor stimulation, TIMP-4 up-regulates Bcl-2 and Bcl-X(L) protein. TIMP-4 also inhibits apoptosis in human breast cancer cells in vitro and mammary tumors in vivo. A synthetic MMP inhibitor BB-94 did not have such antiapoptotic effect. Analysis of TIMP-4 expression in human mammary specimens indicates that TIMP-4 protein is increased in mammary carcinoma cells compared with normal mammary epithelial cells. These data indicate an antiapoptotic activity in breast cancer cells and a tumor-stimulating effect of TIMP-4 when administrated systemically.

Development and characterization of a new polyclonal antibody specifically against tissue inhibitor of metalloproteinases 4 in human breast cancer.

Tissue inhibitors of metalloproteinases (TIMPs) may regulate extracellular matrix turnover and cellular functions by modulating matrix metalloproteinase (MMP) activity and cell proliferation and apoptosis. To investigate the locations and functions of TIMP-4 in human breast cancer, a highly specific polyclonal anti-TIMP-4 peptide antibody (pAb-T4-S61) was developed. The potency and specificity of the purified IgG were characterized by an enzyme-linked immunosorbent assay, immunoblot, and immunohistochemistry. The optimal IgG concentration range was 0.1-10 microg/ml. pAb-T4-S61 did not cross-react with TIMP-1 and TIMP-2 and should not react with TIMP-3 according to the sequence analysis. Parental MDA-MB-435 breast cancer cells were TIMP-4 negative and a TIMP-4 transfected clone, TIMP-4-435-12, produced TIMP-4. Membrane type-1 MMP was detected although TIMP-2 was not found in these cells. Interestingly, the TIMP-4 protein was detected by immunohistochemical staining in infiltrating breast carcinoma cells in tumor tissues. Thus, pAb-T4-S61 is a useful tool to investigate expression patterns and functions of TIMP-4 in cancers.

[Construction of multivalent DNA vaccine against Schistosoma japonicum].

OBJECTIVE: To construct a multivalent DNA vaccine. METHODS: The multivalent DNA vaccine candidates pBK-Sj26-Sj23, pBK-Sj32-Sj23 were constructed based on the plasmids pBluescript-Sj26, pBluescript-Sj32 and pBluescript-Sj23 with three pairs of specific primers using DNA recombinant technique. In the primers, a synthetic linker sequence encoding a peptide was designed, and the antigen genes Sj26 and Sj23, Sj32 and Sj23 were then ligated. After identification, the quadriceps muscle of mice were immunized with the multivalent antigen genes. Four weeks after immunization, the multivalent antigen genes were present in the muscular tissue of mice by PCR. RESULTS: The eukaryotic plasmids including multivalent antigens of S. japonicum were constructed successfully, and the plasmids including multivalent antigen gene could be stably existing in the muscle tissue of mice and the multivalent antigens could be expressed in the muscle tissue cells of mice. CONCLUSION: A multivalent S. japonicum DNA vaccine has been established.

Induction of mammary differentiation by mammary-derived growth inhibitor-related gene that interacts with an omega-3 fatty acid on growth inhibition of breast cancer cells.

We previously identified and characterized a novel tumor growth inhibitor and a fatty acid-binding protein in human mammary gland and named it the mammary-derived growth inhibitor-related gene (MRG). Here, the effects of MRG on mammary gland differentiation and its interaction with omega-3 polyunsaturated fatty acids (omega-3 PUFAs) on growth inhibition were investigated. MRG protein expression was associated with human mammary gland differentiation, with the highest expression observed in the differentiated alveolar mammary epithelial cells from the lactating gland. Overexpression of MRG in human breast cancer cells induced differentiation with changes in cellular morphology and a significant increase in the production of lipid droplets. Treatment of mouse mammary gland in organ culture with MRG protein resulted in a differentiated morphology and stimulation of beta-casein expression. Treatment of human breast cancer cells with the omega-3 PUFA docosahexaenoic acid resulted in a differential growth inhibition proportional to their MRG expression. MRG-transfected cells or MRG protein treated cells were much more sensitive to docosahexaenoic acid-induced growth inhibition than MRG-negative or untreated control cells. Our results suggest that MRG is a candidate mediator of the differentiating effect of pregnancy on breast epithelial cells and may play a major role in omega-3 PUFA-mediated tumor suppression.

Transcriptional suppression of synuclein gamma (SNCG) expression in human breast cancer cells by the growth inhibitory cytokine oncostatin M.

Previously, we have shown that synuclein gamma (SNCG), a member of the brain protein synuclein family, is highly expressed in human infiltrating breast carcinomas but not expressed in normal or benign breast tissues. The SNCG mRNA was also detected in several human breast cancer cell lines with the highest expression found in H3922, a cell line derived from an infiltrating ductal carcinoma. In this study, we show that expression of SNCG mRNA in H3922 cells is significantly decreased by treating cells with the cytokine oncostatin M (OM) who has a growth-inhibitory effect on these cells. A decrease in SNCG mRNA level can be detected as early as 30 min after OM addition. By 4 h OM treatment, the level of SNCG mRNA was decreased to 70% of control, and by 24 h the mRNA was below detectable level. Western blot analysis further demonstrated the suppression of SNCG protein expression by OM. The level of SNCG protein in H3922 cells was reduced more than 90% by OM after 2 days. Since OM-induced growth inhibition occurs after 3-4 days, the down-regulation of SNCG expression appears to proceed the effect of OM on cell growth. Additional experiments to measure the transcriptional rates of SNCG indicate that the observed OM-induced down-regulation of SNCG mRNA occurs mainly at the transcriptional level. In an attempt to examine the role of SNCG gene in the proliferation of breast cancer cells, SNCG cDNA was stably transfected into MCF-7 cells that do not express endogenous SNCG gene. Examination of cell growth under anchorage-dependent and anchorage-independent conditions demonstrates that over expression of SNCG gene significantly stimulated the growth of MCF-7 cells both in monolayer culture and in soft agar. These data together suggest that SNCG may play a role in cell proliferation.

Pathology and course of natural Schistosoma japonicum infection in pigs: results of a field study in Hubei province, China.

In order to obtain information on the natural course of porcine infection with Schistosoma japonicum, pigs were exposed to the cercariae of this parasite in a highly endemic region of China. Five, 5-month-old pigs previously infected with S. japonicum (group A) and 10, schistosome-naïve piglets (group B) were allowed on a pasture infested with Oncomelania snails for one transmission period (approximately 5.5 months). All the piglets rapidly acquired infection, and both groups remained infected throughout the study period. Group B showed fever, diarrhoea and anorexia in the early egg-excretion phase, and marked growth reduction. In both groups, post-mortem examination revealed live schistosomes and lesions associated with dead worms in the intestinal and mesenteric vasculature, and egg-related pathology in the large intestine and liver. Major findings were exudative lesions connected with egg excretion in the intestine, and granulomatous obstruction of portal veins in the liver. Signs of granuloma modulation were found in the liver, but not in the intestine. In conclusion, the study showed that field exposure of pigs to S. japonicum for one transmission period resulted in clinical disease and growth retardation in the youngest pigs, and significant pathology in both groups. Self cure, prominent in experimental porcine infections produced with single, high-dose inocula, was not induced in either group.

TIMP-4 is regulated by vascular injury in rats.

The role of basement membrane-degrading matrix metalloproteinases (MMPs) in enabling vascular smooth muscle cell migration after vascular injury has been established in several animal models. In contrast, the role of their native inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), has remained unproven despite frequent coregulation of MMPs and TIMPs in other disease states. We have investigated the time course of expression and localization of TIMP-4 in rat carotid arteries 6 hours, 24 hours, 3 days, 7 days, and 14 days after balloon injury by in situ hybridization, immunohistochemistry, and Western blot analysis. TIMP-4 protein was present in the adventitia of injured carotid arteries from 24 hours after injury. At 7 and 14 days after injury, widespread immunostaining for TIMP-4 was observed throughout the neointima, media, and adventitia of injured arteries. Western blot analysis confirmed the quantitative increase in TIMP-4 protein at 7 and 14 days. In situ hybridization detected increased expression of TIMP-4 as early as 24 hours after injury and a marked induction in neointimal cells 7 days after injury. We then studied the effect of TIMP-4 protein on the migration of smooth muscle cells through a matrix-coated membrane in vitro and demonstrated a 53% reduction in invasion of rat vascular smooth muscle cells. These data and the temporal relationship between the upregulation of TIMP-4, its accumulation, and the onset of collagen deposition suggest an important role for TIMP-4 in the proteolytic balance of the vasculature controlling both smooth muscle migration and collagen accumulation in the injured arterial wall.

Suppression of breast cancer growth and metastasis by a serpin myoepithelium-derived serine proteinase inhibitor expressed in the mammary myoepithelial cells.

A serpin was identified in normal mammary gland by differential cDNA sequencing. In situ hybridization has detected this serpin exclusively in the myoepithelial cells on the normal and noninvasive mammary epithelial side of the basement membrane and thus was named myoepithelium-derived serine proteinase inhibitor (MEPI). No MEPI expression was detected in the malignant breast carcinomas. MEPI encodes a 405-aa precursor, including an 18-residue secretion signal with a calculated molecular mass of 46 kDa. The predicted sequence of the new protein shares 33% sequence identity and 58% sequence similarity to plasminogen activator inhibitor (PAI)-1 and PAI-2. To determine whether MEPI can modulate the in vivo growth and progression of human breast cancers, we transfected a full-length MEPI cDNA into human breast cancer cells and studied the orthotopic growth of MEPI-transfected vs. control clones in the mammary fat pad of athymic nude mice. Overexpression of MEPI inhibited the invasion of the cells in the in vitro invasion assay. When injected orthotopically into nude mice, the primary tumor volumes, axillary lymph node metastasis, and lung metastasis were significantly inhibited in MEPI-transfected clones as compared with controls. The expression of MEPI in myoepithelial cells may prevent breast cancer malignant progression leading to metastasis.

Stimulation of breast cancer invasion and metastasis by synuclein gamma.

We recently identified and cloned novel breast cancer-specific gene BCSG1 by direct differential cDNA sequencing. BCSG1 has a great sequence homology with the Alzheimer's disease related neural protein synuclein (SNC); thus, it was also named SNC-gamma. Overexpression of SNC-gamma in breast cancer cells leads to a significant increase in motility and invasiveness in vitro and a profound augmentation of metastasis in vivo. Our data suggest that this member of the neural protein SNCs might have important functions outside the central nervous system and may play a role in breast cancer progression.

RIZ1, but not the alternative RIZ2 product of the same gene, is underexpressed in breast cancer, and forced RIZ1 expression causes G2-M cell cycle arrest and/or apoptosis.

The retinoblastoma protein-interacting zinc finger gene RIZ maps to the distal short arm of human chromosome 1 (1p36), a region thought to harbor tumor suppressor genes for a variety of human cancers including breast cancer. The RIZ gene normally produces two protein products of different length, RIZ1 and RIZ2. RIZ2 is generated by an internal promoter and lacks an NH2-terminal motif of RIZ1, the PR domain conserved in a subfamily of zinc finger genes that function as negative regulators of tumorigenesis. We have here studied whether the RIZ gene may play a role in human neoplasia. We found that expression of RIZ1 is commonly decreased or at undetectable levels in breast cancer tissues and cell lines. Decreased RIZ1 expression was also found in other tumor types including neuroblastoma and lung cancer. Remarkably, RIZ2 is normally expressed in all cases examined, suggesting that the abnormality observed for RIZ1 is specific. Forced RIZ1 expression in breast cancer cells caused cell cycle arrest in G2-M and/or programmed cell death. These observations suggest an exclusive negative selection for RIZ1 but not RIZ2 in breast cancer and a role for RIZ1 in tumor suppression.

Antitumor activity of the novel human breast cancer growth inhibitor, mammary-derived growth inhibitor-related gene, MRG.

A novel human tumor growth inhibitor was identified by differential cDNA sequencing. The predicted amino acid sequence of this tumor-suppressing factor has a significant sequence homology to mouse mammary-derived growth inhibitor and thus was named mammary-derived growth inhibitor-related gene (MRG). MRG was found to be expressed in normal and benign human breast tissues but not in breast carcinomas. In situ hybridization analysis demonstrated a stage-specific MRG expression as follows. MRG was barely detectable in breast carcinomas, showed partial and weak expression in benign hyperplasia, but was expressed at a high level in normal breast epithelial cells. To determine if MRG can modulate in vivo growth of human breast cancers, we transfected a full-length MRG cDNA into MDA-MB-231 human breast cancer cells and studied the orthotopic growth of MRG transfectants versus control transfectants in the mammary fat pad of athymic nude mice. Overexpression of MRG in human breast cancer cells significantly suppressed cell proliferation in vitro and tumor growth in an orthotopic nude mouse model. These results suggest that MRG has tumor-suppressing activity, and the loss of MRG expression may be involved in the development and progression of breast cancer.

Preparation and characterization of recombinant tissue inhibitor of metalloproteinase 4 (TIMP-4).

TIMP-4, a novel human tissue inhibitor of metalloproteinase, was identified and cloned (Greene, J., Wang, M., Raymond, L. A., Liu, Y. E., Rosen, C., and Shi, Y. E. (1996) J. Biol. Chem. 271, 30375-30380). In this report, the production and characterization of recombinant TIMP-4 (rTIMP4p) are described. rTIMP4p, expressed in baculovirus-infected insect cells, was purified to homogeneity by a combination of cation exchange, hydrophobic, and size-exclusion chromatographies. The purified protein migrated as a single 23-kDa band in SDS-polyacrylamide gel electrophoresis and in Western blot using a specific anti-TIMP-4 antibody. Inhibition of matrix metalloproteinase (MMP) activities by rTIMP4p was demonstrated in five MMPs. Enzymatic kinetic studies revealed IC50 values (concentration at 50% inhibition) of 19, 3, 45, 8, and 83 nM for MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, respectively. Purified rTIMP4p demonstrated a strong inhibitory effect on the invasion of human breast cancer cells across reconstituted basement membranes. Thus, TIMP-4 is a new enzymatic inhibitor in MMP-mediated extracellular matrix degradation and may have therapeutic potential in treating cancer malignant progression.

Inhibition of tumor growth and metastasis of human breast cancer cells transfected with tissue inhibitor of metalloproteinase 4.

We recently identified, cloned, and characterized a novel human tissue inhibitor of metalloproteinases-4, TIMP-4 (Greene et al., 1996). To determine if TIMP-4 can modulate the in vivo growth of human breast cancers, we transfected a full-length TIMP-4 cDNA into MDA-MB-435 human breast cancer cells and studied the orthotopic growth of TIMP-4-transfected (TIMP4-435) versus control (neo-435) clones in the mammary fat pad of athymic nude mice. TIMP4-435 clones expressed TIMP-4 mRNA and produced anti-metalloproteinase (MMP) activity, while neo-435 clones did not express TIMP-4 mRNA or produce detectable anti-MMP activity. Overexpression of TIMP-4 inhibited the invasion potential of the cells in the in vitro invasion assay. When injected orthotopically into nude mice, TIMP-4 transfectants were significantly inhibited in tumor growth by 4-10-fold in primary tumor volumes; and in an axillary lymph node and lung metastasis as compared with controls. These results suggest the therapeutic potential of TIMP-4 in treating cancer malignant progression.

Specific, high affinity binding of tissue inhibitor of metalloproteinases-4 (TIMP-4) to the COOH-terminal hemopexin-like domain of human gelatinase A. TIMP-4 binds progelatinase A and the COOH-terminal domain in a similar manner to TIMP-2.

The binding properties of the newly described tissue inhibitor of metalloproteinases-4 (TIMP-4) to progelatinase A and to the COOH-terminal hemopexin-like domain (C domain) of the enzyme were examined. We present evidence for the first time of a specific, high affinity interaction between TIMP-4 and the C domain of human gelatinase A and show that TIMP-4 binds both progelatinase A and the C domain in a similar manner to that of TIMP-2. Saturable binding of recombinant C domain to TIMP-4 and to TIMP-2 but not to TIMP-1 was demonstrated using a microwell protein binding assay. The recombinant collagen binding domain of gelatinase A, comprised of the three fibronectin type II-like repeats, did not bind to TIMP-4, indicating that binding is mediated selectively by the C domain. Binding to TIMP-4 was of high affinity with an apparent Kd of 1.7 x 10(-7) M but slightly weaker than that to TIMP-2 (apparent Kd of 0.66 x 10(-7) M). Affinity chromatography confirmed the TIMP-4-C domain interaction and also showed that the complex could not be disrupted by 1 M NaCl or 10% dimethyl sulfoxide, thereby further demonstrating the tight binding. To verify the biological significance of this interaction, binding of full-length progelatinase A to TIMP-4 was investigated. TIMP-4 and TIMP-2 but not TIMP-1 bound specifically to purified TIMP-2-free human recombinant full-length progelatinase A and to full-length rat proenzyme from the conditioned culture medium of ROS 17/2.8 cells. Preincubation of the C domain with TIMP-2 was found to reduce subsequent binding to TIMP-4 in a concentration-dependent manner. Competition between TIMP-2 and TIMP-4 for a common or overlapping binding sites on the gelatinase A C domain may occur; alternatively TIMP-2 may prevent the binding of TIMP-4 by steric hindrance or induction of a conformational change in the C domain. We propose that the binding of progelatinase A to TIMP-4 represents a third TIMP-progelatinase interaction in addition to that of progelatinase A with TIMP-2 and progelatinase B with TIMP-1 described previously. This new phenomenon may be of important physiological significance in modulating the cell surface activation of progelatinase A.

Computational sequence analysis of the tissue inhibitor of metalloproteinase family.

The tissue inhibitor of metalloproteinase (TIMP) family regulates extracellular matrix turnover and tissue remodeling by forming tight-binding inhibitory complexes with matrix metalloproteinases (MMPs). MMPs and TIMPs have been implicated in many normal and pathological processes, such as morphogenesis, development, angiogenesis, and cancer metastasis. This minireview provides information that would aid in classification of the TIMP family and in understanding the similarities and differences among TIMP members according to the physical data, primary structure, and homology values. Calculations of molecular weight, isoelectric point values, and molar extinction coefficients are reported. This study also compares sequence similarities and differences among the TIMP members through calculations of homology within their individual loop regions and the mature region of the molecule. Lastly, this report examines structure-function relationships of TIMPs. Thorough knowledge of TIMP primary and tertiary structure would facilitate the uncovering of the molecular mechanisms underlying metalloproteinase, inhibitory activities and biological functions of TIMPs.