Recent studies have demonstrated that two SIBLING family members, bone sialoprotein (BSP) and osteopontin (OPN), are overexpressed in human prostate cancer. The expression of these proteins is associated with the acquisition of a metastatic phenotype by cancer cells and a poor prognosis for the patient. Dentin sialophosphoprotein (DSPP) shares several structural and genetic features with OPN and BSP. The presence of DSPP has been recently established in salivary glands, indicating that its expression is not restricted to mineralized tissues. However, its potential expression in human tumors has not been addressed yet. In this study, we sought to evaluate the expression of DSPP in human prostate cancer. Immunohistochemistry was performed on 69 prostate cancer specimens using LFMb-21 anti-DSPP monoclonal antibody. All of the prostate cancer lesions examined expressed detectable levels of DSPP, as compared with no or low level of expression in adjacent normal glands (p < 0.0001). High grade prostatic intraepithelial neoplasia (HGPIN) glands generally displayed DSPP expression levels that were similar to those found in neighboring cancer glands. DSPP expression was significantly associated with the pathological stage (p 5 0.0087) and the Gleason score (p 5 0.0176) of the tumors. Western Blot was performed on 5 representative prostate tumor extracts and 3 prostatic tumor cell lines (PC3, LNCaP and DU145). All tumor extracts and cell lines analyzed have been found to express DSPP. In addition, in situ hybridization was used to assess the presence of DSPP mRNA. DSPP was detected at the RNA level in both HGPIN and tumoral glands. This study shows for the first time that DSPP is ectopically expressed in human prostate cancer. The expression of this SIB-LING protein strongly correlates with conventional histopathological prognostic indicators of prostate cancer progression. ' 2005 Wiley-Liss, Inc.Key words: Osteomimetism; DSPP; prostate cancer; prognosis Accumulating data designate certain noncollagenous bone proteins as major players in the complex multistep process leading to cancer progression and metastasis. To date, 2 of these bone proteins, namely osteopontin (OPN) and bone sialoprotein (BSP), have been linked with different stages of tumor progression: cell growth, adhesion, migration and/or metastasis. On the basis of their common genetic and structural features, Fisher and collaborators have recently proposed to consider BSP, OPN and 3 other proteins named dentin sialophosphoprotein (DSPP), dentin matrix protein-1 (DMP1) and matrix extracellular phosphoglycoprotein (MEPE) as members of a unique family called SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein). This family is a conserved gene cluster with common exon-intron structures, the presence of the integrin-binding tripeptide Arg-Gly-Asp (RGD), as well as conserved phosphorylation and N-glycosylation sites. Immunohistochemical studies conducted by our group and others have demonstrated the expression of all SIBLINGs in cancer, with the exception...

Clinical practice reveals that osteoporotic women treated with BPs (bisphosphonates) show an increased bone mass density and a reduced risk of fractures. However, the mechanisms leading to these beneficial effects of BPs are still poorly understood. We hypothesized that ZOL (zoledronic acid), a potent third-generation BP, may induce the expression of proteins associated with the bone-forming potential of osteoblastic cells such as BSP (bone sialo-protein). Expression of BSP gene is up-regulated by hormones that promote bone formation and has been associated with de novo bone mineralization. Using real-time reverse transcriptase-PCR and Western-blot analysis, we demonstrated that ZOL increased BSP expression in Saos-2 osteoblast-like cells. Nuclear run-on and mRNA decay assays showed no effect at the transcriptional level but a stabilization of BSP transcripts in ZOL-treated cells. ZOL effect on BSP expression occurred through an interference with the mevalonate pathway since it was reversed by either mevalonate pathway intermediates or a Rho GTPase activator. We showed that ZOL impaired membrane localization of RhoA in Saos-2 cells indicating reduced prenylation of this protein. By the use of small interfering RNAs directed to RhoA and Rac1, we identified both Rho GTPases as negative regulators of BSP expression in Saos-2 cells. Our study demonstrates that ZOL induces BSP expression in osteoblast-like cells through inactivation of Rho GTPases and provides a potential mechanism to explain the favourable effects of ZOL treatment on bone mass and integrity.

The DNA of every cell is constantly exposed to insult mediated by endogenous and environmental factors that induced damage in its structure. To react to these attacks and maintain the integrity of the genome, eukaryotic cells are equipped with sophisticated mechanisms to detect, signal the presence of and repair DNA damage. The cellular response to DNA damage is a critical event for maintaining genomic stability and limiting neoplastic transformation. BRIT1, a newly identified protein, forms specific irradiation-induced nuclear foci. Our recent investigation demonstrates that BRIT1 functions as a proximal factor in the DNA damage checkpoints that control multiple damage sensors and early mediators. BRIT1 is also implicated in cell cycle checkpoints, controlling and regulating other important molecules and thus affecting the timing of mitosis. Depletion of BRIT1 abolishes the DNA damage response and results in centrosomal abnormalities and chromosomal aberrations. Moreover, aberrantly reduced expression of BRIT1 in human carcinomas implicates this protein in cancer initiation and progression. Together, the findings identify BRIT1 as a potential tumor suppressor. Fully elucidating the function of this intriguing protein may lead to new therapeutic approaches for the improved cancer treatment.

We have previously shown that breast and prostate cancers express bone matrix proteins. DMP1 expression was evaluated in 59 human lung cancer samples at the protein and mRNA levels. It was detectable in 80% of the cases, suggesting a potential role for DMP1 in tumor progression and bone metastasis.Introduction: Previously, we and others have shown that bone extracellular matrix proteins such as bone sialoprotein (BSP) and osteopontin (OPN) are expressed in various types of cancer that are characterized by a high affinity for bone including breast, prostate, and lung adenocarcinoma. Based on biochemical and genetic features, BSP, OPN, dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP) have been recently classified in a unique family named SIBLING (small integrin-binding ligand, N-linked glycoprotein). Therefore, we investigated whether DMP1 could also be detected in osteotropic cancers. Materials and Methods: We first used a cancer array for evaluating the relative abundance of DMP1 transcript in a broad spectrum of human cancer tissues. This screening showed that DMP1 was strongly detectable in lung tumors compared with normal corresponding tissue. In a second step, we used an immunophosphatase technique and a specific polyclonal antibody directed against DMP1 to examine the expression of DMP1 in 59 human non-small cell lung cancer samples, including 29 squamous carcinoma, 20 adenocarcinoma, and 10 bronchioloalveolar carcinoma. Student's t-test was used to determine the statistical significance of immunostaining scores between the lung cancer histological groups studied and between cancer and normal lung tissues. Results: Our results show that DMP1 is detectable in 90% of the adenocarcinoma and squamous carcinoma analyzed while 8 of 10 bronchioloalveolar specimens were negative. DMP1 immunostaining intensity and extent scores were significantly higher in adenocarcinoma (p ϭ 0.0004) and squamous carcinoma (p Ͻ 0.0001) samples compared with adjacent normal lung tissue. In situ hybridization experiments confirmed that DMP1 mRNA is localized in lung cancer cells. Conclusion:In this study, we show that a third SIBLING protein is ectopically expressed in lung cancer. The role of DMP1 in lung cancer is largely unknown. Further studies are required to determine the implication of this protein, next to its sisters SIBLING proteins, in tumor progression and bone metastasis development.

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Small integrin-binding ligand N-linked glycoproteins (SIBLINGs) constitute a family of extracellular matrix proteins involved in bone homeostasis. Their pattern of expression has been primarily reported in bone and tooth and, more recently, in several cancer types. Dentin matrix protein 1 (DMP1), a SIBLING family member, expression was investigated by immunohistochemistry in a retrospective series of 148 primary human breast cancers. Correlations between DMP1 expression levels in the tumors and clinicopathologic features, bone metastases development and relapse of the disease were examined. DMP1 was expressed by 63.5% of the breast tumors analyzed. Significant inverse associations were found between DMP1 expression levels and the size and grade of the tumors (both, P < 0.0001). High DMP1 expression levels in the primary breast lesions were associated with a lower risk of subsequent development of skeletal metastases (P = 0.009). Patients with tumors expressing high levels of DMP1 had a significantly higher disease-free survival rate than those with low DMP1-expressing tumors (P = 0.0062). When DMP1 expression was examined in breast cancer cell lines, we found that non invasive MCF-7 and T47-D cells expressed higher levels than highly invasive MDA-MB-231 and Hs578T cells. Moreover, the specific inhibition of DMP1 expression in MCF-7 cells using siRNAs promoted significantly their migratory capability. Our data implicate for the first time DMP1 expression in breast cancer progression and bone metastases development.

Zoledronate exhibits antiangiogenic properties in vitro and in vivo. Integrins alphavbeta3 and alphavbeta5 are involved in angiogenesis. Because zoledronate inhibits endothelial cell adhesion, the authors explored the hypothesis that it could alter these integrins recruitment to focal adhesion sites. Human umbilical vein endothelial cells (HUVECs) were treated with zoledronate or with mevalonate pathway intermediates geranylgeraniol (GGOH) and farnesol (FOH). Zoledronate generated a significant decrease in alphavbeta3 and alphavbeta5 expression at HUVEC cell surface using flow cytometry and immunofluorescence. This inhibition was reversed by GGOH but not by FOH. Cells cotreated with zoledronate and GGOH were able to attach to vitronectin through alphavbeta3 and alphavbeta5, as confirmed by the use of specific function-blocking antibodies. The authors showed that zoledronate alters endothelial cell integrin-mediated adhesion. This effect is likely to contribute to the previously demonstrated antiangiogenic effect of zoledronate. Whether this mechanism of action also applies to metastatic tumor cells is under investigation.

Bone sialoprotein (BSP) is a secreted glycoprotein primarily found in the mineral compartment of developing bones. BSP is detected in a variety of human cancers, particularly those that metastasize to the skeleton. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. Since squamous cell carcinoma (SCCs) of the uterine cervix also frequently metastasizes to bone, we investigated whether BSP is expressed in human cervical cancer. We examined BSP expression in cervical tissue samples from 47 patients, including 19 normal tissues, 20 squamous intraepithelial lesions (SILs) (9 low and 11 high grade) and 8 invasive SCCs. BSP protein expression was evaluated by the immunophosphatase technique using a BSP polyclonal antibody in paraffin-embedded cervical biopsies. The abundance of BSP protein was significantly higher in invasive SCCs and high grade SILs than in normal cervix tissue samples and low grade SILs, which showed no or a low level of anti-BSP immunoreactivity. In situ hybridization experiments performed on representative cervix invasive SCCs frozen sections revealed that BSP transcripts were detectable in these lesions. Our study demonstrates that BSP expression is a common feature in high grade SILs and invasive SCCs of the uterine cervix. The prognostic value of BSP detection in these lesions and the potential role of BSP as an angiogenic factor in this type of cancer are currently under investigation.