Alka Jain

Three small integrin binding ligand N-linked glycoproteins (SIBLINGs) bind and activate specific matrix metalloproteinases

Matrix metalloproteinases (MMPs) are critical for development wound healing and for the progression of cancer. It is generally accepted that MMPs are secreted in a latent form (proMMP) and are activated only upon removal of their inhibitory propeptides. This report shows that three members of the SIBLING (Small Integrin‐Binding LIgand N‐linked Glycoprotein) family can specifically bind (Kd≈nM) and activate three different MMPs. Binding of SIBLING to their corresponding proMMPs is associated with structural changes as indicated by quenching of intrinsic tryptophan fluorescence increased susceptibility to plasmin cleavage and decreased inhibition by specific natural and synthetic inhibitors. Activation includes both making the proMMPs enzymatically active and the reactivation of the TIMP (tissue inhibitors of MMP) inhibited MMPs. Bone sialoprotein specifically binds proMMP‐2 and active MMP‐2 while osteopontin binds proMMP‐3 and active MMP‐3 and dentin matrix protein‐1 binds proMMP‐9 and active MMP‐9. Both pro and active MMP‐SIBLING complexes are disrupted by the abundant serum protein complement Factor H thereby probably limiting SIBLING‐ mediated activation to regions immediately adjacent to sites of secretion in vivo. These data suggest that the SIBLING family offers an alternative method of controlling the activity of at least three MMPs.

Small Integrin Binding Ligand N-Linked Glycoprotein Gene Family Expression in Different Cancers

Purpose: Members of the small integrin binding ligand N-linked glycoprotein (SIBLING) gene family have the capacity to bind and modulate the activity of matrix metalloproteinases (MMPs). The expression levels of five SIBLING gene family members [bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein 1 (DMP1), matrix extracellular phosphoglycoprotein (MEPE), and dentin sialophosphoprotein (DSPP)] and certain MMPs were determined using a commercial cancer array. Experimental Design: Cancer profiling arrays containing normalized cDNA from both tumor and corresponding normal tissues from 241 individual patients were used to screen for SIBLING and MMP expression in nine distinct cancer types. Results: Significantly elevated expression levels were observed for BSP in cancer of the breast, colon, stomach, rectum, thyroid, and kidney; OPN in cancer of the breast, uterus, colon, ovary, lung, rectum, and thyroid; DMP1 in cancer of the breast, uterus, colon, and lung; and dentin sialophosphoprotein in breast and lung cancer. The degree of correlation between a SIBLING and its partner MMP was found to be significant within a given cancer type (e.g., BSP and MMP-2 in colon cancer, OPN and MMP-3 in ovarian cancer; DMP1 and MMP-9 in lung cancer). The expression levels of SIBLINGs were distinct within subtypes of cancer (e.g., breast ductal tumors compared with lobular tumors). In general, SIBLING expression increased with cancer stage for breast, colon, lung, and rectal cancer. Conclusions: These results suggest SIBLINGs as potential markers of early disease progression in a number of different cancer types, some of which currently lack vigorous clinical markers.

Bone Sialoprotein Mediates the Tumor Cell–Targeted Prometastatic Activity of Transforming Growth Factor β in a Mouse Model of Breast Cancer

Transforming growth factor betas (TGF-beta) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as prometastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-beta antagonists can suppress metastasis without the predicted toxicities. To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer. Treatment of mice with a monoclonal anti-TGF-beta antibody (1D11) significantly suppressed metastasis of 4T1 cells to the lungs. When metastatic 4T1 cells were recovered from lungs of 1D11-treated and control mice, the most differentially expressed gene was found to be bone sialoprotein (Bsp). Immunostaining confirmed the loss of Bsp protein in 1D11-treated lung metastases, and TGF-beta was shown to regulate and correlate with Bsp expression in vitro. Functionally, knockdown of Bsp in 4T1 cells reduced the ability of TGF-beta to induce local collagen degradation and invasion in vitro, and treatment with recombinant Bsp protected 4T1 cells from complement-mediated lysis. Finally, suppression of Bsp in 4T1 cells reduced metastasis in vivo. We conclude that Bsp is a plausible mediator of at least some of the tumor cell-targeted prometastatic activity of TGF-beta in this model and that Bsp expression in metastases can be successfully suppressed by systemic treatment with anti-TGF-beta antibodies.

Fibroblast Growth Factor‐23 Is Regulated by 1α,25‐Dihydroxyvitamin D

Serum FGF‐23 regulation was studied in patients with hypoparathyroidism or pseudohypoparathyroidism treated with calcitriol. Serum FGF‐23 levels changed in parallel in response to changes in serum 1,25‐D, suggesting that FGF‐23 may be regulated by 1,25‐D. In addition, the phosphaturic effect of FGF‐23 may be diminished in the absence of PTH action on the kidney.

Serum Levels of the Immune Activation Marker Neopterin Change With Age and Gender and Are Modified by Race, BMI, and Percentage of Body Fat

BACKGROUND Neopterin, a GTP metabolite expressed by macrophages, is a marker of immune activation. We hypothesize that levels of this serum marker alter with donor age, reflecting increased chronic immune activation in normal aging. In addition to age, we assessed gender, race, body mass index (BMI), and percentage of body fat (%fat) as potential covariates. METHODS Serum was obtained from 426 healthy participants whose age ranged from 18 to 87 years. Anthropometric measures included %fat and BMI. Neopterin concentrations were measured by competitive ELISA. The paired associations between neopterin and age, BMI, or %fat were analyzed by Spearmans correlation or by linear regression of log-transformed neopterin, whereas overall associations were modeled by multiple regression of log-transformed neopterin as a function of age, gender, race, BMI, %fat, and interaction terms. RESULTS Across all participants, neopterin exhibited a positive association with age, BMI, and %fat. Multiple regression modeling of neopterin in women and men as a function of age, BMI, and race revealed that each covariate contributed significantly to neopterin values and that optimal modeling required an interaction term between race and BMI. The covariate %fat was highly correlated with BMI and could be substituted for BMI to yield similar regression coefficients. CONCLUSION The association of age and gender with neopterin levels and their modification by race, BMI, or %fat reflect the biology underlying chronic immune activation and perhaps gender differences in disease incidence, morbidity, and mortality.

Gene expression profiling identifies markers of ampullary adenocarcinoma

Ampullary adenocarcinoma is an aggressive cancer with a poor prognosis. Without surgical resection, ampullary adenocarcinomas can be difficult to distinguish from ampullary adenomas. The aim of this study was to identify differentially expressed genes in ampullary adenocarcinoma in order to identify candidate markers of the disease. The Affymetrix Human Genome U133 GeneChip set (HG-U133A and HG-U133B) was used to obtain gene expression profiles of 5 ampullary adenocarcinomas and 10 normal duodenal samples. Using fold change analysis we identified 235 fragments expressed at least fivefold higher in ampullary cancers than in normal duodenum. The expression profiles of eight candidate overexpressed genes (osteopontin, mesothelin, tissue inhibitor of metalloproteinases 1, mucin-1, mucin-5, fascin, heat shock protein 47, fibronectin 1) were confirmed by immunohistochemistry or in situ hybridization on tissue microarrays (TMA) containing 54 ampullary adenocarcinomas. One of these genes, osteopontin, was expressed 27-fold higher levels in ampullary adenocarcinomas compared to normal duodenum by genechip analysis. We therefore determined serum osteopontin levels in patients with ampullary neoplasms, patients with other periampullary diseases and in normal controls. Mean pre-operative serum osteopontin levels as measured by competitive ELISA were 906 ± 268 ng/ml in patients with ampullary cancer, 867 ± 160 ng/ml in patients with an ampullary adenoma, 327.1±195.6 ng/ml in patients with non-malignant periampullary diseases and 204 ± 65 ng/ml in age-matched healthy controls (P

WISP1/CCN4: A Potential Target for Inhibiting Prostate Cancer Growth and Spread to Bone

Prostate cancer (PC) is a leading cause of death in men however the factors that regulate its progression and eventual metastasis to bone remain unclear. Here we show that WISP1/CCN4 expression in prostate cancer tissues was up-regulated in early stages of the disease and, further, that it correlated with increased circulating levels of WISP1 in the sera of patients at early stages of the disease. WISP1 was also elevated in the mouse prostate cancer model TRAMP in the hypoplastic diseased tissue that develops prior to advanced carcinoma formation. When the ability of anti-WISP1 antibodies to reduce the spread of PC3-Luc cells to distant sites was tested it showed that twice weekly injections of anti-WISP1 antibodies reduced the number and overall size of distant tumors developed after intracardiac (IC) injection of PC3-Luc cells in mice. The ability of antibodies against WISP1 to inhibit growth of PC3-Luc cancer cells in mice was also evaluated and showed that twice weekly injections of anti-WISP1 antibodies reduced local tumor growth when examined in xenografts. To better understand the mechanism of action, the migration of PC3-Luc cells through membranes with or without a Matrigel™ barrier showed the cells were attracted to WISP1, and that this attraction was inhibited by treatment with anti-WISP1 antibodies. We also show the expression of WISP1 at the bone-tumor interface and in the stroma of early grade cancers suggested WISP1 expression is well placed to play roles in both fostering growth of the cancer and its spread to bone. In summary, the up-regulation of WISP1 in the early stages of cancer development coupled with its ability to inhibit spread and growth of prostate cancer cells makes it both a potential target and an accessible diagnostic marker for prostate cancer.

Small integrin-binding proteins as serum markers for prostate cancer detection.

Purpose: The small integrin-binding ligand N-linked glycoprotein (SIBLING) gene family includes bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE), and osteopontin (OPN). Previous studies have separately reported elevated expression of BSP, OPN, or DSPP in prostate tumor paraffin sections. We hypothesized that SIBLINGs may be informative serum markers for subjects with prostate cancer. Methods: Expression levels of SIBLINGs in biopsies of normal tissue and tumors from prostate were determined by cDNA array and by immunohistochemical staining with monoclonal antibodies. Competitive ELISAs for measuring total BSP, DSPP, MEPE, and OPN were applied to a test group of 102 subjects with prostate cancer and 110 normal subjects and a validation group of 90 subjects. Results: BSP, DMP1, DSPP, and OPN exhibited elevated mRNA expression and protein levels in biopsies. BSP, DSPP, and OPN were elevated in serum from prostate cancer subjects, with serum DSPP exhibiting the greatest difference, yielding an area under the receiver operator characteristic curve value of 0.98. Serum BSP and OPN levels were significantly elevated only in late stages, whereas DSPP was significantly elevated at all stages. Optimal serum value cutoff points derived for BSP, OPN, and DSPP were applied as a validation test to a new group of 90 subjects and DSPP yielded a sensitivity of 90% and a specificity of 100%. Conclusion: Of the SIBLING gene family members, DSPP appears to be a strong candidate for use in serum assays for prostate cancer detection. (Clin Cancer Res 2009;15(16):5199–207)

Comparison of conventional ELISA with electrochemiluminescence technology for detection of amyloid-β in plasma.

Plasma amyloid-β (Aβ) level could be useful as a non-invasive biomarker in Alzheimers disease research. We compared a multiplex electrochemiluminescence detection method with a well established ELISA method for plasma Aβ quantification. Compared to the ELISA method, the electrochemiluminescence detection method demonstrates a statistically significant, but modest correlation. The reasons for this may include the differences in the affinities of antibodies, and purity and source of Aβ peptides used as standards. However, the advantages of electrochemiluminescence detection technology include short processing time and small sample volume. This comparison demonstrates the need for a further study in optimizing this system.

Serum Chitotriosidase, a Putative Marker of Chronically Activated Macrophages, Increases With Normal Aging

BACKGROUND Chitotriosidase (ChT) is secreted by chronically activated macrophages in Gauchers disease. We hypothesize that circulating levels of ChT are altered with normal aging, reflecting age-related chronic macrophage activation. Potential sources that might contribute to altered levels were assessed by measuring systemic levels of ChT are α-naphthyl acetate esterase, a macrophage lysosomal enzyme; granulocyte-macrophage colony-stimulating factor (GM-CSF), which stimulates neutrophilic granule release of ChT; interleukin-6 (IL-6); and neopterin, a macrophage activation marker. METHODS Serum was obtained from 315 healthy participants whose age ranged from 18 to 92 years. Anthropometric measures included percent body fat and body mass index. ChT and α-naphthyl acetate esterase levels were measured by enzyme activity assays. GM-CSF, IL-6, and neopterin concentrations were measured by commercial enzyme-linked immunosorbent assays. Serum marker values were statistically analyzed using nonparametric tests. RESULTS Six percent of the participants had undetectable ChT levels. A positive association with age was observed for ChT and IL-6, whereas a negative correlation with age was seen for α-naphthyl acetate esterase and GM-CSF. ChT values were not associated with α-naphthyl acetate esterase or GM-CSF levels. ChT was independently associated with IL-6 and neopterin levels, but statistical significance was attenuated when controlled for age. CONCLUSIONS The data are consistent with increased serum ChT activity not arising from altered macrophage lysosomal enzyme trafficking or GM-CSF-stimulated release of neutrophil granule stores. The association of ChT with age remains significant after controlling for neopterin and IL-6 changes with age, suggesting that ChT levels reflect a macrophage state distinct from acute macrophage activation or inflammatory state.