Myoferlin regulates cellular lipid metabolism and promotes metastases in triple-negative breast cancer.

Myoferlin is a multiple C2-domain-containing protein that regulates membrane repair, tyrosine kinase receptor function and endocytosis in myoblasts and endothelial cells. Recently it has been reported as overexpressed in several cancers and shown to contribute to proliferation, migration and invasion of cancer cells. We have previously demonstrated that myoferlin regulates epidermal growth factor receptor activity in breast cancer. In the current study, we report a consistent overexpression of myoferlin in triple-negative breast cancer cells (TNBC) over cells originating from other breast cancer subtypes. Using a combination of proteomics, metabolomics and electron microscopy, we demonstrate that myoferlin depletion results in marked alteration of endosomal system and metabolism. Mechanistically, myoferlin depletion caused impaired vesicle traffic that led to a misbalance of saturated/unsaturated fatty acids. This provoked mitochondrial dysfunction in TNBC cells. As a consequence of the major metabolic stress, TNBC cells rapidly triggered AMP activated protein kinase-mediated metabolic reprogramming to glycolysis. This reduced their ability to balance between oxidative phosphorylation and glycolysis, rendering TNBC cells metabolically inflexible, and more sensitive to metabolic drug targeting in vitro. In line with this, our in vivo findings demonstrated a significantly reduced capacity of myoferlin-deficient TNBC cells to metastasise to lungs. The significance of this observation was further supported by clinical data, showing that TNBC patients whose tumors overexpress myoferlin have worst distant metastasis-free and overall survivals. This novel insight into myoferlin function establishes an important link between vesicle traffic, cancer metabolism and progression, offering new diagnostic and therapeutic concepts to develop treatments for TNBC patients.

HDAC7 inhibition resets STAT3 tumorigenic activity in human glioblastoma independently of EGFR and PTEN: new opportunities for selected targeted therapies.

To date, the mutational status of EGFR and PTEN has been shown as relevant for favoring pro- or anti-tumor functions of STAT3 in human glioblastoma multiforme (GBM). We have screened genomic data from 154 patients and have identified a strong positive correlation between STAT3 and HDAC7 expression. In the current work we show the existence of a subpopulation of patients overexpressing HDAC7 and STAT3 that has particularly poor clinical outcome. Surprisingly, the somatic mutation rate of both STAT3 and HDAC7 was insignificant in GBM comparing with EGFR, PTEN or TP53. Depletion of HDAC7 in a range of GBM cells induced the expression of tyrosine kinase JAK1 and the tumor suppressor AKAP12. Both proteins synergistically sustained the activity of STAT3 by inducing its phosphorylation (JAK1) and protein expression (AKAP12). In absence of HDAC7, activated STAT3 was responsible for significant imbalance of secreted pro-/anti-angiogenic factors. This inhibited the migration and sprouting of endothelial cells in paracrine fashion in vitro as well as angiogenesis in vivo. In a murine model of GBM, induced HDAC7-silencing decreased the tumor burden by threefold. The current data show for the first time that silencing HDAC7 can reset the tumor suppressor activity of STAT3, independently of the EGFR/PTEN/TP53 background of the GBM. This effect could be exploited to overcome tumor heterogeneity and provide a new rationale behind the development of specific HDAC7 inhibitors for clinical use.

Consequences of chondrocyte hypertrophy on osteoarthritic cartilage: potential effect on angiogenesis.

OBJECTIVE: The aim of this study was to investigate the link between the hypertrophic phenotype of chondrocytes and angiogenesis in osteoarthritis (OA) and more particularly to demonstrate that OA hypertrophic chondrocytes potentially express a phenotype promoting angiogenesis through the expression of factors controlling endothelial cells migration, invasion and adhesion. METHOD: Human OA chondrocytes were cultivated in alginate beads in medium supplemented with 10% fetal bovine serum (FBS) to induce chondrocyte hypertrophy. The hypertrophic phenotype was characterized throughout 28 days of culture by measuring the expression of specific genes and by a microscopic observation of cellular morphology. The effect of media conditioned by OA hypertrophic chondrocyte on endothelial cells migration, invasion and adhesion was evaluated in functional assays. Moreover, hypertrophic OA chondrocytes were tested for the expression of angiogenic factors by real-time RT-PCR. RESULTS: Specific markers of hypertrophy and observation of cellular morphology attested of the hypertrophic phenotype of chondrocytes in our culture model. Functional angiogenesis assays showed that factors produced by hypertrophic chondrocytes stimulated migration, invasion and adhesion of endothelial cells. Among the evaluated angiogenic factors, bone sialoprotein (BSP) was the most highly upregulated in hypertrophic chondrocytes. The inhibition of endothelial cell adhesion by a GRGDS peptide confirmed the implication of RGD domain proteins, like BSP, in hypertrophic chondrocyte-induced adhesion of endothelial cells. CONCLUSION: Hypertrophic differentiation of chondrocyte may promote angiogenesis. Our findings established the relation of BSP with OA chondrocyte hypertrophy and suggested that this factor could constitute a potential target to control cartilage neovascularisation in OA.

HDAC4 represses p21(WAF1/Cip1) expression in human cancer cells through a Sp1-dependent, p53-independent mechanism.

Cancer cells have complex, unique characteristics that distinguish them from normal cells, such as increased growth rates and evasion of anti-proliferative signals. Global inhibition of class I and II histone deacetylases (HDACs) stops cancer cell proliferation in vitro and has proven effective against cancer in clinical trials, at least in part, through transcriptional reactivation of the p21(WAF1/Cip1)gene. The HDACs that regulate p21(WAF1/Cip1) are not fully identified. Using small interfering RNAs, we found that HDAC4 participates in the repression of p21(WAF1/Cip1) through Sp1/Sp3-, but not p53-binding sites. HDAC4 interacts with Sp1, binds and reduces histone H3 acetylation at the Sp1/Sp3 binding site-rich p21(WAF1/Cip1) proximal promoter, suggesting a key role for Sp1 in HDAC4-mediated repression of p21(WAF1/Cip1). Induction of p21(WAF1/Cip1) mediated by silencing of HDAC4 arrested cancer cell growth in vitro and inhibited tumor growth in an in vivo human glioblastoma model. Thus, HDAC4 could be a useful target for new anti-cancer therapies based on selective inhibition of specific HDACs.

CREB-1 and AP-1 transcription factors JunD and Fra-2 regulate bone sialoprotein gene expression in human breast cancer cells.

Bone sialoprotein (BSP) expression is detected in a variety of human osteotropic cancers. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. In this study, we examined the transcriptional regulation of BSP gene expression in MDA-MB-231 and MCF-7 human breast cancer cells compared with Saos-2 human osteoblast-like cells. BSP human promoter deletion analyses delineated a -56/-84 region, which comprises a cAMP response element (CRE) that was sufficient for maximal promoter activity in breast cancer cell lines. We found that the basic fibroblast growth factor response element (FRE) also located in the proximal promoter was a crucial regulator of human BSP promoter activity in Saos-2 but not in breast cancer cells. Promoter activity experiments in combination with DNA mobility shift assays demonstrated that BSP promoter activity is under the control of the CRE element, through CREB-1, JunD and Fra-2 binding, in MDA-MB-231, MCF-7 and in Saos-2 cells. Forskolin, a protein kinase A pathway activator, failed to enhance BSP transcriptional activity suggesting that CRE site behaves as a constitutive rather than an inducible element in these cell lines. Over-expression of JunD and Fra-2 increased BSP promoter activity and upregulated endogenous BSP protein expression in MCF-7 and Saos-2 cells while siRNA-mediated inhibition of both factors expression significantly reduced BSP protein level in MDA-MB-231. Collectively, these data provide with new transcriptional mechanisms, implicating CREB and AP-1 factors, that control BSP gene expression in breast cancer cells.

Zoledronate inhibits alphavbeta3 and alphavbeta5 integrin cell surface expression in endothelial cells.

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.

Transcriptome analysis reveals an osteoblast-like phenotype for human osteotropic breast cancer cells.

Metastatic breast cancer cells exhibit the selective ability to seed and grow in the skeleton. We and others have previously reported that human breast tumors which metastasize to the skeleton overexpress bone matrix extracellular proteins. In an attempt to reveal the osteoblast-like phenotype of osteotropic breast cancer cells, we performed a microarray study on a model of breast cancer bone metastasis consisting of the MDA-MB-231 human cell line and its variant B02 selected for its high capacity to form bone metastases in vivo. Analysis of B02 cells transcriptional profile revealed that 11 and 9 out of the 50 most up- and down-regulated mRNAs, respectively, corresponded to genes which expression has been previously associated with osteoblastic differentiation process. Thus, osteoblast specific cadherin 11 which mediates the differentiation of mesenchymal cells into osteoblastic cells is up-regulated in B02. While S100A4, recently described as a key negative regulator of osteoblast differentiation, is the most down-regulated gene in B02 cells. RT-PCR and western blotting experiments allowed the validation of the modulation of several genes of interest. Using immunohistochemistry, performed on human breast primary tumors and their matched liver and bone metastases, we were able to confirm that the osteoblast-like pattern of gene expression observed in our model holds true in vivo. This is the first report demonstrating a gene-expression pattern corresponding to the acquisition of an osteomimetic phenotype by bone metastatic breast cancer cells.

Low dentin matrix protein 1 expression correlates with skeletal metastases development in breast cancer patients and enhances cell migratory capacity in vitro.

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.

Anti-proliferative properties of prenylated flavonoids from hops (Humulus lupulus L.) in human prostate cancer cell lines.

Chalcones xanthohumol (X) and desmethylxanthohumol (DMX), present in hops (Humulus lupulus L.), and the corresponding flavanones isoxanthohumol (IX, from X), 8-prenylnaringenin (8-PN, from DMX), and 6-prenylnaringenin (6-PN, from DMX), have been examined in vitro for their anti-proliferative activity on human prostate cancer cells PC-3 and DU145. X proved to be the most active compound in inhibiting the growth of the cell lines with IC50 values of 12.3+/-1.1 microM for DU145 and 13.2+/-1.1 microM for PC-3. 6-PN was the second most active growth inhibitor, particularly in PC-3 cells (IC50 of 18.4+/-1.2 microM). 8-PN, a highly potent phytoestrogen, exhibited pronounced anti-proliferative effects on PC-3 and DU145 (IC50 of 33.5+/-1.0 and 43.1+/-1.2 microM, respectively), and IX gave comparable activities (IC50 of 45.2+/-1.1 microM for PC-3 and 47.4+/-1.1 microM for DU145). DMX was the least active compound. It was evidenced for the first time that this family of prenylated flavonoids from hops effectively inhibits proliferation of prostate cancer cells in vitro.

Detection of bone sialoprotein in human (pre)neoplastic lesions of the uterine cervix.

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.

Dentin matrix protein 1 is expressed in human lung cancer.

UNLABELLED: 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 = 0.0004) and squamous carcinoma (p < 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.

Bone sialoprotein, bone morphogenetic protein 6 and thymidine phosphorylase expression in localized human prostatic adenocarcinoma as predictors of clinical outcome: a clinicopathological and immunohistochemical study of 43 cases.

PURPOSE: Skeletal metastases are the hallmark of advanced prostate cancer and recurrence after local surgery is common. Currently to our knowledge no biological markers predict the risk of disease progression in individuals with localized prostate cancer. In a search for predictive markers we evaluated the expression of bone sialoprotein and bone morphogenetic protein 6, 2 bone related proteins, and the angiogenic factor thymidine phosphorylase. MATERIALS AND METHODS: The study population included 43 men who presented with localized prostate cancer treated with radical prostatectomy. Bone sialoprotein, bone morphogenetic protein 6 and thymidine phosphorylase expression was assessed by immunohistochemical testing. Results were analyzed in relation to pathological disease stage, Gleason score and clinical outcome. Clinical followup was 4.3 to 11.4 years after surgery (median 7.9). RESULTS: Disease did not progress in 17 of the 43 cases, while recurrence and/or metastasis developed in the other 26 at a median of 6.5 and 6.9 years, respectively. Bone sialoprotein and bone morphogenetic protein 6 expression detected in 28 (65%) and 29 (67%) of the 43 samples, respectively, was significantly associated (p = 0.0001). Thymidine phosphorylase detected in 26 samples (60%) was not related to bone sialoprotein and/or bone morphogenetic protein 6 positivity. Bone sialoprotein and/or bone morphogenetic protein 6 expression correlated with bone metastasis, while thymidine phosphorylase expression was related to local recurrence (p = 0.002 and/or 0.007, and 0.00007, respectively). On multivariate analysis only the correlation of thymidine phosphorylase expression with recurrence remained statistically significant (p = 0.002). Co-expression of the 3 markers was observed in the samples of 10 of the 11 patients (90%) with bone metastases and only in 5 of the 17 (29%) who were disease-free. CONCLUSIONS: This study indicates that the expression of bone sialoprotein, bone morphogenetic protein 6 and thymidine phosphorylase determined at a clinically early stage of disease by a simple immunohistochemical technique would enable subgroups of patients to be identified that are at different risks of bone metastasis or recurrence. Detection of such markers would provide additional prognostic information that would be useful for patients with intermediate or low Gleason score or stage disease. These patients would benefit from a more adapted clinical follow-up.

Increased expression of bone sialoprotein in bone metastases compared with visceral metastases in human breast and prostate cancers.

The recent demonstration that bone sialoprotein (BSP) is expressed in osteotropic cancers suggests that this bone matrix protein might be implicated in the preferential seed and growth of metastatic cells in bone. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. The exact mechanisms by which BSP may favor bone metastases formation are not clearly established yet. Although BSP expression has been detected in breast, prostate, lung, thyroid, and neuroblastoma primary tumors, no information regarding its expression in metastases is available to date. In this study, we have examined BSP expression in 15 bone and 39 visceral metastatic lesions harvested from 8 breast cancer patients and 7 prostate cancer patients who died of disseminated disease. We were able to retrieve the primary lesions from 5 of the 8 breast cancer patients as well as from all 7 prostate cancer patients. All the primary breast tumor patients and 5 of the 7 primary prostate cancer patients expressed a detectable level of BSP. Bone metastases from all 8 breast cancer patients and from 5 out of 7 prostate cancer patients exhibited detectable levels of the protein. Metastatic cells in close contact with bone trabeculae usually were highly positive for BSP. BSP also was detected in secondary lesions developed at visceral sites including liver, thyroid, lung, and adrenal glands. However, BSP expression was significantly lower in visceral metastases than in skeletal ones (Mann-Whitney test, p < 0.05). Our data represent the first demonstration of an increased expression of BSP in bone metastases compared with nonskeletal metastases in human breast and prostate cancers and add weight to the body of evidence attributing a significant role to this protein in the genesis of bone metastases.

Bone sialoprotein mediates human endothelial cell attachment and migration and promotes angiogenesis.

Bone sialoprotein (BSP) is a secreted glycoprotein primarily found in sites of biomineralization. Recently, we demonstrated that BSP is strongly upregulated in osteotropic cancers and particularly those that exhibit microcalcifications. BSP contains an Arg-Gly-Asp (RGD) motif found in other adhesive molecules that interact with cellular integrins. In bone, BSP has been shown to mediate the attachment of osteoblasts and osteoclasts via alpha(v)beta(3) integrin receptors. Ligands for alpha(v)beta(3) integrin are considered to play a central role during angiogenesis. Therefore, we used human umbilical vein endothelial cells (HUVECs) to study the potential role of BSP in angiogenesis. We found that purified eukaryotic recombinant human BSP (rhBSP) is able to promote both adhesion and chemotactic migration of HUVECs in a dose-dependent manner. These interactions involve HUVEC alpha(v)beta(3) integrin receptors and the RGD domain of BSP. Indeed, HUVECs attach to a recombinant BSP fragment containing the RGD domain, whereas this response is not observed with the same fragment in which RGD has been mutated to Lys-Ala-Glu (KAE). A cyclic RGD BSP peptide inhibits both adhesion and migration of HUVECs to rhBSP. Moreover, anti-alpha(v)beta(3) but not anti-alpha(v)beta(5) monoclonal antibodies also prevent BSP-mediated adhesion and migration of HUVECs. We observed that both rhBSP and the RGD BSP recombinant fragment stimulated ongoing angiogenesis on the chorioallantoic chick membrane assay. BSP angiogenic activity was inhibited by anti-alpha(v)beta(3) antibody, and the KAE BSP fragment was inactive. Our findings represent the first report implicating BSP in angiogenesis. BSP could play a critical role in angiogenesis associated with bone formation and with tumor growth and metastatic dissemination.

Bone sialoprotein mRNA and protein expression in human multiple myeloma cell lines and patients.

Bone sialoprotein (BSP) is a glycoprotein essentially found in mineralizing connective tissues. We have recently demonstrated that BSP is ectopically expressed by carcinomas that metastasize to bone with high frequency. Multiple myeloma (MM) is characterized by the localization of tumour plasma cells in the bone marrow. In this study, BSP expression was evaluated in human myeloma cell lines and in bone marrow aspirates and one ascites fluid from MM patients. BSP was detectable in conditioned media of MM cell lines. Using FACS analysis and in situ hybridization, we demonstrated that tumour cells from all MM patients and cell lines analysed express BSP at both the protein and the mRNA level.

Ectopic expression of bone sialoprotein in human thyroid cancer.

Bone sialoprotein (BSP) is a small, highly posttranslationally modified integrin binding protein found in the mineral compartment of developing bone. The recent discovery that BSP can be detected in a variety of human cancers, particularly those that metastasize preferentially to the skeleton, shed light on potential new biological functions for this protein. The demonstration of a positive association between BSP expression in primary breast tumors and the development of bone metastases suggests that this glycoprotein could play a role in the selective implantation of breast cancer cells in bone. BSP is also expressed in most lung and prostate cancers as well as in multiple myeloma, three other osteotropic malignancies. Because thyroid carcinoma also metastasizes preferentially to the skeleton, we decided to look at the expression of BSP in a collection of 145 thyroid malignant lesions including 24 follicular thyroid carcinomas (FTCs), 55 papillary thyroid carcinomas (PTCs), 19 medullary thyroid carcinomas (MTCs), 23 anaplastic carcinomas (ACs), and 24 poorly differentiated carcinomas (PDCs). BSP expression was evaluated by immunoperoxidase technique using two specific polyclonal antibodies. Most of the thyroid carcinomas (72%) examined expressed high levels of BSP. Expression of BSP was significantly lower in FTCs and MTCs compared with PDCs, which are more aggressive (p = 0.0009 and 0.0003, respectively). Our study demonstrates for the first time that ectopic BSP expression is a common feature of thyroid cancer. The prognostic value of BSP detection in thyroid adenocarcinoma and the potential role of BSP in the propension of this type of cancer to metastasize to bone are currently under investigation.

Prognostic value of bone sialoprotein expression in clinically localized human prostate cancer.

BACKGROUND: Bone sialoprotein (BSP), a bone matrix protein, was recently found to be expressed ectopically in breast cancer and to have a statistically significant association with poor prognosis and the development of bone metastases in that disease. These data prompted us to investigate whether BSP might also be expressed in human prostate cancer, which often metastasizes to bone, and be predictive for progression risk. METHODS: Tissue sections from 180 patients who had undergone a radical prostatectomy for localized prostate cancer were analyzed immunohistochemically for BSP expression. Biochemical progression was defined as an increasing serum prostate-specific antigen level of 0.5 ng/mL or more. Statistical analysis was used to assess associations between pathologic findings and level of BSP expression, and a Cox proportional hazards model was used to determine which clinical and histologic parameters, including stage, Gleason score, and BSP expression (immunostaining intensity and extent), were independently associated with biochemical progression. All P values were two-sided. RESULTS: Most of the prostate cancer lesions examined (78.9%) expressed detectable levels of BSP, compared with no or low expression in the adjacent normal glandular tissue. A statistically significant association was found between BSP expression and biochemical progression in both univariate and multivariate analyses. After a follow-up interval of 3 years, the biochemical relapse rate was 36.7% (95% confidence interval [CI] = 23.4%-47.7%) in patients whose tumors expressed high levels of BSP compared with 12.1% (95% CI = 2.3%-20.8%) in patients whose tumors expressed no or a low detectable level of the protein (logrank test, P = .0014). BSP expression status could identify those patients at higher risk of biochemical progression (logrank test, P<.05) among patients with moderately differentiated tumors or with pathologically confined tumors. CONCLUSIONS: To our knowledge, this study is the first to demonstrate BSP expression in human prostate cancer and to highlight the protein's statistically significant prognostic value in patients with clinically confined prostate adenocarcinomas.

Evidence that breast cancer associated microcalcifications are mineralized malignant cells.

Microcalcifications are often associated with both benign and malignant human breast lesions. Around 40% of mammary carcinoma present such ectopic mineralization and frequently, they are the only mammographic feature that indicate the presence of a tumoral lesion. Microcalcifications associated with breast cancer are usually composed of hydroxyapatite, the bone specific mineral. The mechanisms responsible for the formation of such crystals within breast malignant tissue have not been elucidated. A possible clue could be provided by the recent demonstration that breast cancer cells express several bone matrix proteins including osteonectin, osteopontin and bone sialoprotein (BSP). This latter phospho-protein is involved in the initiation of hydroxyapatite crystallisation and its expression in breast cancer has been associated to the presence of hydroxyapatite microcalcifications. We examined 10 human breast cancer lesions which were characterized by the presence of microcalcifications and high expression of BSP. Histological examination of the lesions suggested, in most of the cases, that the microcalcifications were breast cancer cells which became mineralized. Hydroxyapatite stained in blue by hematoxylin appears concentrated around single of associated cancer cells. Staining of these tissue sections with 4',6 diamidino-2-phenylindole which specifically labels DNA led us to demonstrate that the mineralizated structures contain cells. These data are the first direct demonstration that breast microcalcifications are fossils of cancer cells. The mechanisms for such a phenomenon remain to be demonstrated. We speculate that the high expression of BSP could create an appropriate microenvironment for the crystallisation of calcium and phosphate into hydroxyapatite.

Expression of bone sialoprotein in human lung cancer.

Lung cancer belongs to the group of malignant lesions that specifically select bone as secondary implantation site. The molecular bases for this property, defined as osteotropism, is still largely unknown. The recent demonstration that human breast cancer cells express and attach to bone sialoprotein (BSP), a sulfated phosphoprotein rich in bone and other mineralized tissues, could provide a clue to elucidating bone metastases formation. BSP contains the integrin binding peptide Arg-Gly-Asp (RGD), as well as non-RGD cell attachment domain. Using an immunoperoxidase technique and a specific polyclonal antibody directed against a BSP synthetic peptide, we examined the expression of BSP in 48 lung lesions including 25 squamous carcinoma, 21 adenocarcinoma, and 2 bronchioloalveolar cancers, as well as 38 human ovarian carcinoma that constitute a group of generally nonosteotropic cancers. BSP was not specifically detected in normal lung tissue with the exception of cartilage associated with bronchi. Most of the adenocarcinoma (74%) and all squamous carcinoma of the lung examined exhibited detectable levels of BSP. Staining was mainly cytoplasmic and membrane associated. The two bronchioloalveolar lung cancers examined did not show detectable amounts of BSP. When microcalcifications were observed in pulmonary malignant lesions, they were usually associated with cancer cells expressing BSP. Only 21% of the ovarian cancers examined contained malignant cells with 2+ or 3+ positivity for BSP. We further demonstrated that in 8 of 10 additional lung cancers, BSP was detected at the mRNA level. Our observation is the first demonstration that BSP is expressed in non-small cell lung carcinoma. Lung cancer cells are now the second type of osteotropic malignant cells described to express BSP. Added to the observation that BSP expression is not frequent in ovarian carcinoma, a low osteotropic cancer, our study supports our hypothesis that BSP could play a role in determining the affinity of cancer cells to bone.

Expression of bone matrix proteins in human breast cancer: potential roles in microcalcification formation and in the genesis of bone metastases.

The skeleton is the privileged target of metastatic human breast cancer cells. Bone metastases are indeed found in virtually all advanced breast cancer patients and generate major morbidity. The high osteotropism of breast cancer cells suggests that they exhibit a selective affinity for mineralized tissues. The observation that mammary malignant cells are able to induce hydroxyapatite crystals deposition within the primary tumour suggests that they can generate a microenvironment that favors the crystallization of calcium and phosphate ions into the bone specific hydroxyapatite. Osteonectin (OSN), osteopontin (OPN) and bone sialoprotein (BSP), 3 bone matrix proteins involved in bone matrix mineralization, are expressed in human breast cancers. BSP, an RGD (Arg-Gly-Asp) containing phosphoprotein, initiates hydroxyapatite deposition and mediates attachment of osteoclast to the same crystals prior to their resorption. Detection of BSP at both the protein and the mRNA levels in human breast cancer and in human breast cancer cell lines (MCF-7, T47-D and MDA-MB 231) indicates that mammary malignant cells synthesize directly BSP rather than uptaking it from the serum. Interestingly, the level of BSP expression correlates with the development of bone metastases and with poor survival. These data suggest that the ectopic expression of bone matrix proteins could be involved in conferring osteotropic properties to circulating metastatic breast cancer cells. These observations open new alleys of investigation for the identification of the molecular mechanisms responsible for the genesis of bone metastases.