Expression and localization of osteopontin, homing cell adhesion molecule/CD44, and integrin αvß3 in mucoepidermoid carcinoma and acinic cell adenocarcinoma of salivary gland origin.

OBJECTIVE: Osteopontin (OPN) plays a role in tumor progression. This study aimed to determine the expression of OPN, CD44, and integrin αvß3 in pleomorphic adenoma (PA), acinic cell adenocarcinoma (ACA), and mucoepidermoid carcinoma (MEC). STUDY DESIGN: Immunohistochemistry was used to semiquantify the levels of expression of OPN and its receptors in normal salivary glands (NSG) (n = 20), PA (n = 20), ACA (n = 11), and MEC (n = 29). RESULTS: OPN expression was increased in ACA and MEC compared with PA and NSG (median scores, 6, 6, 4, and 4, respectively). CD44 expression was increased in ACA and reduced in MEC and PA compared with NSG (median scores, 8, 4, 3, and 5, respectively). Integrin αvß3 median scores were 5 in ACA, 1 in MEC, and 0 in PA and NSG. CONCLUSIONS: OPN is expressed in salivary gland tumors and is at higher levels in ACA and MEC.

Expression and localization of osteopontin, homing cell adhesion molecule/CD44, and integrin αvß3 in pleomorphic adenoma, polymorphous low-grade adenocarcinoma, and adenoid cystic carcinoma.

OBJECTIVES: Osteopontin (OPN) plays a role in tumor progression. This study aimed to determine the expression of OPN, CD44, and integrin αvß3 in pleomorphic adenoma (PA), polymorphous low-grade adenocarcinoma (PLGA), and adenoid cystic carcinoma (ACC). STUDY DESIGN: Immunohistochemistry was used to semiquantify the level of expression of OPN and its receptors in normal salivary glands (NSG; n = 20), PA (n = 20), PLGA (n = 16), and ACC (n = 22). RESULTS: OPN expression was increased in PLGA and intermediate-/high-grade ACC compared with PA and NSG (median scores, 6, 5, 4, and 4, respectively). CD44 expression was reduced in PA, PLGA, and ACC. OPN expression levels were moderately correlated with CD44 in PLGA. Integrin αvß3 was not expressed in PA and ACC and was seen in only 1 case of PLGA. CONCLUSIONS: OPN is expressed in salivary gland tumors but does not correlate well with CD44 and αvß3.

Plasma osteopontin as a biomarker of prostate cancer aggression: relationship to risk category and treatment response.

BACKGROUND: High plasma osteopontin (OPN) has been linked to tumour hypoxia, metastasis, and poor prognosis. This study aims to assess whether plasma osteopontin was a biomarker of increasing progression within prostate cancer (PCa) prognostic groups and whether it reflected treatment response to local and systemic therapies. METHODS: Baseline OPN was determined in men with localised (n=199), locally recurrent (n=9) and castrate-resistant, metastatic PCa (CRPC-MET; n=37). Receiver-operating curves (ROC) were generated to describe the accuracy of OPN for distinguishing between localised risk groups or localised vs metastatic disease. We also measured OPN pre- and posttreatment, following radical prostatectomy, external beam radiotherapy (EBRT), androgen deprivation (AD) or taxane-based chemotherapy. RESULTS: The CRPC-MET patients had increased baseline values (mean 219; 56-513 ng ml(-1); P<0.0001) compared with the localised, non-metastatic group (mean 72; 12-438 ng ml(-1)). The area under the ROC to differentiate localised vs metastatic disease was improved when OPN was added to prostate-specific antigen (PSA) (0.943-0.969). Osteopontin neither distinguished high-risk PCa from other localised PCa nor correlated with serum PSA at baseline. Osteopontin levels reduced in low-risk patients after radical prostatectomy (P=0.005) and in CRPC-MET patients after chemotherapy (P=0.027), but not after EBRT or AD. CONCLUSION: Plasma OPN is as good as PSA at predicting treatment response in CRPC-MET patients after chemotherapy. Our data do not support the use of plasma OPN as a biomarker of increasing tumour burden within localised PCa.

Removal of a titanium ring using a dental saw.

Titanium is becoming increasingly popular in the manufacture of jewellery. Titanium rings are notoriously difficult to remove in an emergency situation and there is speculation amongst the general public that amputation is sometimes necessary due to the indestructibility of this material. We present the case of removal of a titanium ring using a dental saw allowing the patient's finger to be preserved.

Volume measurement variability in three-dimensional high-frequency ultrasound images of murine liver metastases.

The identification and quantification of tumour volume measurement variability is imperative for proper study design of longitudinal non-invasive imaging of pre-clinical mouse models of cancer. Measurement variability will dictate the minimum detectable volume change, which in turn influences the scheduling of imaging sessions and the interpretation of observed changes in tumour volume. In this paper, variability is quantified for tumour volume measurements from 3D high-frequency ultrasound images of murine liver metastases. Experimental B16F1 liver metastases were analysed in different size ranges including less than 1 mm3, 1-4 mm3, 4-8 mm3 and 8-70 mm3. The intra- and inter-observer repeatability was high over a large range of tumour volumes, but the coefficients of variation (COV) varied over the volume ranges. The minimum and maximum intra-observer COV were 4% and 14% for the 1-4 mm3 and <1 mm3 tumours, respectively. For tumour volumes measured by segmenting parallel planes, the maximum inter-slice distance that maintained acceptable measurement variability increased from 100 to 600 microm as tumour volume increased. Comparison of free breathing versus ventilated animals demonstrated that respiratory motion did not significantly change the measured volume. These results enable design of more efficient imaging studies by using the measured variability to estimate the time required to observe a significant change in tumour volume.

Osteopontin expression in salivary gland tumours.

Osteopontin (OPN) is expressed in numerous carcinomas and plays a role in tumour development, invasion and metastasis. This study examines by immunohistochemistry the expression of OPN in normal salivary gland tissue and three types of salivary gland tumour: pleomorphic adenoma (PA), adenoid cystic carcinoma (ACC) and polymorphous low grade adenocarcinoma (PLGA). PAs and PLGAs demonstrated higher levels of OPN than normal salivary gland tissue, while ACC, although showing a trend towards increased OPN, was not significantly different. The results of this study indicate that OPN expression is present in normal salivary gland tissue, and is increased in certain salivary gland tumours, but further investigation is necessary to clarify its role.

Genomic amplification of MET with boundaries within fragile site FRA7G and upregulation of MET pathways in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EA) is characterized by a poor prognosis making the identification of clinically targetable proteins essential for improving patient outcome. We report the involvement of multiple alterations of the MET pathway in EA development and progression. Microarray analysis of Barrett's metaplasia, dysplasia, and EA revealed overexpression of the MET oncogene in EAs but only those with MET gene amplification. STS-amplification mapping revealed that the boundary of the MET amplicon in these EAs is defined by fragile site FRA7G. We also identified an amplicon at 11p13 that resulted in amplification and overexpression of CD44, a gene involved in MET autophosphorylation upon HGF stimulation. Tissue microarrays with phospho-MET-specific antibodies demonstrated a uniformly high abundance of MET activation in primary EA and cells metastatic to lymph nodes but to a lesser extent in a subset of metaplastic and dysplastic Barrett's samples. Increased expression of multiple genes in the MET pathway associated with invasive growth, for example, many MMPs and osteopontin, also was found in EAs. Treatment of EA-derived cell lines with geldanamycin, an inhibitor for tyrosine kinases including MET receptor kinase, reduced cell migration and induced EA cell apoptosis. The data indicate that upregulation of the MET pathway may contribute to the poor outcome of EA patients and that therapeutic agents targeting this pathway may help improve patient survival.

Role of osteopontin in tumour progression.

Since its first identification as a transformation-associated protein, osteopontin (OPN) has been recognised as important in the processes of tumorigenicity and metastasis. Here, we review the evidence that OPN might be considered as a candidate prognostic marker in human cancer. In animal systems, evidence from cell injection experiments and genetically manipulated mice suggest an important but complex role for the protein in tumour progression. Moreover, studies in a variety of human cancers associate high levels of OPN expression in tumours or in blood with more advanced cancers. The mechanism of action of OPN in promoting cancer is still unclear, and we consider aspects of OPN biology that can complicate interpretation of human studies. Nevertheless, growing evidence supports a role for OPN as a potential prognostic factor for various human cancers.

Osteopontin and its receptor alphavbeta(3) integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially.

Osteopontin is an arginine-glycine-aspartic acid-containing acidic glycoprotein component of the extracellular matrix that is postulated to bind to integrin receptors at the cell surface to mediate cellular adhesion and migration during embryo implantation. The primary aim of this study was to examine the uterine expression of osteopontin throughout the menstrual cycle in normal fertile controls sampled prospectively based on urinary LH surge detection. Expression of osteopontin was documented using Northern blot analysis, in situ hybridization, and immunohistochemistry. Furthermore, the temporal pattern of osteopontin expression was compared with that of its receptor, the alphavbeta3 integrin. Using Ishikawa cells, a well differentiated endometrial adenocarcinoma cell line, the in vitro regulation of osteopontin and its receptor alphavbeta3 integrin was studied. By Northern blot analysis, osteopontin mRNA appears during the early secretory phase, with maximal expression occurring in mid to late secretory-phase endometrium. The in situ hybridization analyses showed that osteopontin mRNA specifically localized in epithelial cells within the endometrium. Immunostaining of osteopontin was detected in the glandular secretions and on the apical portions of surface (luminal) epithelium. The patterns of expression of osteopontin by Northern blotting, in situ hybridization, and immunohistochemistry are remarkably similar to the pattern for the alphavbeta3 integrin. Despite these similarities in distribution, in vitro studies demonstrate that osteopontin and beta3 integrin subunit expression are differentially regulated. The expression of osteopontin was primarily induced in response to progesterone, whereas the beta3 integrin subunit was up-regulated by epidermal growth factor or heparin-binding epidermal growth factor. The differential regulation of these two endometrial proteins suggests the existence of two separate pathways regulating epithelial gene expression in human endometrium during the window of implantation. In adhesion assays using Ishikawa cells, alphavbeta3 but not alphavbeta5 or beta1 integrins appear to be the primary receptors for osteopontin. These findings may better define the factors that favor the development of a receptive endometrium.

Solitary cancer cells as a possible source of tumour dormancy?

Metastasis can occur many years after primary tumour treatment. However, the status of the tumour during this period of dormancy is poorly understood. As part of our ongoing experimental studies on mechanisms of metastasis, we have discovered that large numbers of disseminated single cells may persist in secondary sites for extended time periods. Identification of these cells was facilitated by in vivo techniques developed to quantify the fate of individual cells during the metastatic process. Here we review these in vivo techniques and findings. We also discuss the potential clinical implications if dormant solitary cells exist in appreciable numbers in cancer patients.

Critical steps in hematogenous metastasis: an overview.

Metastasis is responsible for most cancer deaths. A better understanding of the process provides opportunities to develop new treatments to prevent metastasis. This article summarizes findings from experimental in vivo videomicroscopy and quantitative studies on metastatic inefficiency, which indicate that early steps in hematogenous metastasis may be quite efficient, but that regulation of cancer cell growth in secondary sites determines metastatic outcome. The authors have identified three key stages of this growth regulation: survival of a subset of single cells, proliferation of a subset of these cells to form preangiogenic micrometastases, and persistence of growth of a subset of these to form vascularized metastases. Formation of clinically relevant metastases is determined by the proportion of cells that proceeds successfully through each stage, and surviving single cells and preangiogenic micrometastases both represent possible sources of tumor dormancy.

The role of osteopontin in breast cancer: clinical and experimental studies.

Osteopontin (OPN) is a secreted, integrin-binding protein which has been implicated in cancer, as well as other pathologies and some aspects of normal development. Here we focus on the role of OPN in breast cancer. We describe studies that have shown that OPN plays a role in normal mammary gland development as well as in progression of breast cancer. We also summarize studies that have shown that OPN can play a functional role in malignancy of breast cancer. At least some of these effects are mediated by specific cell surface integrins (alpha(v)beta3 vs. alpha(v)beta1 and alpha(v)beta5) and lead to increased cell migration, activation of growth factor/receptor pathways (e.g. HGF and EGF), and increased proteolytic enzyme activity (e.g. uPA). We also summarize clinical studies that show that OPN levels in tumors and blood are elevated in women with metastatic breast cancer and may offer promise as prognostic markers in breast cancer.

Osteopontin(OPN)-induced increase in human mammary epithelial cell invasiveness is urokinase (uPA)-dependent.

We have recently shown that either exogenous or endogenous, transfected OPN induces both uPA expression and increased invasiveness of 21 PT (non-tumorigenic) and 21 NT (tumorigenic) human mammary epithelial cells. Here we asked whether uPA contributes functionally to the increased invasiveness of these cells. The most invasive OPN-transfected cells were assessed for migration through Matrigel in transwell assays, in the presence or absence of various blocking antibodies and uPA inhibitors. Antibodies to both uPA and uPA receptor (uPAR) were shown to significantly inhibit cell invasion, as did the uPA inhibitors (plasminogen activator inhibitor-1 [PAI-1], p-aminobenzamidine [PABN], aprotinin, and amiloride). Both anti-uPA and anti-uPAR antibodies inhibited invasion to a level comparable to that of the control vector transfected cells. In contrast, non-specific IgG showed no antiinvasive effect. Cell migration experiments performed with the parental cell lines in the presence or absence of anti-uPA or anti-uPAR antibodies showed that uPA is also required for migratory responsiveness to exogenous OPN. These data thus provide direct evidence that OPN-induced invasion and migration of these cells requires uPA.

The functional and clinical roles of osteopontin in cancer and metastasis.

Osteopontin (OPN) is a secreted and integrin-binding protein that has been implicated in a number of pathologies. In this review we will focus on the functional and clinical roles of OPN in cancer and metastasis, with a particular emphasis on breast cancer. While much evidence has suggested that OPN is associated with cancer, its functional contribution to cancer remains poorly understood. Here we will review evidence for mechanisms by which OPN may act to enhance malignancy, including evidence that signaling pathways directly induced by OPN, as well as interactions with growth factor receptor pathways, can combine to activate expression of genes and functions that contribute to metastasis. OPN has been shown to be over-expressed in a variety of human tumors and is present in elevated levels in the blood of some patients with metastatic cancers. We also will discuss recent clinical evidence that suggests that OPN is not only associated with several tumor types, but that levels of OPN in cancer patients' blood or tumors may provide prognostic information.

Functional analysis of bone sialoprotein: identification of the hydroxyapatite-nucleating and cell-binding domains by recombinant peptide expression and site-directed mutagenesis.

Mammalian bone sialoprotein (BSP) is a mineralized tissue-specific protein containing an RGD (arginine-glycine-aspartic acid) cell-attachment sequence and two distinct glutamic acid (glu)-rich regions, with each containing one contiguous glu sequence. These regions have been proposed to contribute to the attachment of bone cells to the extracellular matrix and to the nucleation of hydroxyapatite (HA), respectively. To further delineate the domains responsible for these activities, porcine BSP cDNA was used to construct expression vectors coding for two partial-length recombinant BSP peptides: P2S (residues 42-87), containing the first glutamic acid-rich domain; and P1L (residues 69-300), containing the second glutamic acid-rich region and the RGD sequence. These peptides were expressed in Escherichia coli as his-tag fusion proteins and purified by nickel affinity columns and FPLC chromatography. Digestion with trypsin released the his-tag fusion peptide, which generated P2S-TY (residues 42-87) and P1L-TY (residues 132-239). Using a steady-state agarose gel system, P2S-TY promoted HA nucleation, whereas P2S, P1L, and P1L-TY did not. This implies that the minimum requirement for nucleation of HA resides within the amino acid sequence of the first glutamic acid-rich domain, whereas the second glutamic acid-rich domain may require posttranslational modifications for activity. P1L, but not P2S, promoted RGD-mediated attachment of human gingival fibroblasts in a manner similar to that of native BSP. Deletion of the RGD domain or conversion of it to RGE (arginine-glycine-glutamic acid) abolished the cell-attachment activity of P1L. This suggests that, at least for human gingival fibroblasts, the major cell-attachment activity in the recombinant BSP peptides studied (residues 42-87 and 69-300) requires the RGD sequence located at the C-terminal domain.

Differential expression of drug resistance genes and chemosensitivity in glial cell lineages correlate with differential response of oligodendrogliomas and astrocytomas to chemotherapy.

The two principal subtypes of glial neoplasms, astrocytomas and oligodendrogliomas, exhibit striking differences in response to chemotherapy. This differential chemosensitivity might be explained by the specific genetic alterations causing gliomas but could also be attributable to specific properties intrinsic to the cells from which gliomas arise. To examine the possibility that chemosensitivity might be associated with lineage-specific properties of potential ancestors of these tumors, we explored: (a) the expression of drug resistance genes in rat glial cells; (b) the sensitivity of rat glial subtypes to the bifunctional alkylating agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU); and (c) the effect of O6-methylguanine-DNA methyltransferase (MGMT) and glutathione modulation on resistance to BCNU. Astrocytes, O-2A progenitors, and oligodendrocytes each displayed a unique pattern of expression of six drug resistance genes: MGMT, GST mu, GST pi,p53, MDR, and MT. Oligodendrocytes were more sensitive to BCNU than either astrocytes or O-2A progenitors. The increased resistance of astrocytes in comparison to oligodendrocytes was modulated, at least in part, by both O6-benzylguanine (BG) and DL-buthionine-(S,R)-sulfoximine, suggesting a role for both MGMT and glutathione in the resistance of astrocytes to BCNU. The sensitivity of O-2A progenitors to BCNU following BG pretreatment is virtually indistinguishable from that of oligodendrocytes depleted of MGMT, suggesting that the down-regulation of MGMT is sufficient to account for the increased sensitivity of oligodendrocyte lineage cells to BCNU as they differentiate. These experiments provide support for the hypothesis that properties of glial cells retained in gliomas may contribute to the differential chemosensitivity of glial neoplasms.

The role of osteopontin in the development of granulomatous lesions in lung.

Osteopontin (OPN) has been shown to be expressed by cells in granulomas of various origins, but whether it plays a functional role in granuloma formation is not known. Here we used a cardiomyopathic hamster (TO2) model, to test the hypothesis that OPN contributes functionally to granuloma development. We immunized cardiomyopathic and normal hamsters by subcutaneous injection of bovine serum albumin in complete Freund's adjuvant, and assessed various tissues for both OPN RNA expression and granuloma formation. Cardiomyopathic hamsters expressed OPN, and formed granulomatous lesions, in heart tissue in both immunized and untreated animals. In addition, immunization induced expression of OPN in lung and lymph nodes of cardiomyopathic (but not normal) hamsters, and also induced granuloma formation in these organs. To test whether OPN expression could play a functional role in inducing granulomas, we produced an adenoviral vector containing the murine OPN gene, and introduced this vector intratracheally into the lungs of normal hamsters. The OPN-containing vector, but not the control vector, induced pulmonary granuloma formation. These studies provided direct in vivo evidence that OPN can contribute functionally to the formation of granulomatous lesions, and suggest that OPN expression may be a common factor involved in formation of granulomas of various origin.

Osteopontin-induced, integrin-dependent migration of human mammary epithelial cells involves activation of the hepatocyte growth factor receptor (Met).

Osteopontin (OPN) is a secreted glycophosphoprotein which induces migration of mammary carcinoma cells, and has been implicated in the malignancy of breast carcinoma. Hepatocyte growth factor (HGF) induces cell migration of several mammary epithelial cell (MEC) lines, via activation of its cognate receptor (Met). This study examines the mechanism of OPN-induced MEC migration, in terms of the cell surface integrins involved and induction of the HGF/Met pathway. Three different MEC cell lines were used, representing different stages of tumor progression: 21PT, non-tumorigenic; 21NT, tumorigenic; non-metastatic; and MDA-MB-435, tumorigenic, highly metastatic. Human recombinant OPN was found to induce the migration of all three lines. OPN-induced migration of 21PT and 21NT cells was alphavbeta5 and beta1-integrin dependent, and alphavbeta3-independent, while that of MDA-MB-435 cells was alphavbeta3-dependent. HGF also induced migration of all three cell lines, and a synergistic response was seen to HGF and OPN together. The increased migration response to OPN was found to be associated with an initial increase in Met kinase activity (within 30 min), followed by an increase in Met mRNA and protein expression. OPN-induced cell migration is thus mediated by different cell surface integrins in MEC lines representing different stages of progression, and involves activation of the HGF receptor, Met.

Temporal progression of metastasis in lung: cell survival, dormancy, and location dependence of metastatic inefficiency.

Cancer metastasis is an inefficient process. The steps in metastasis responsible for this inefficiency and how metastatic inefficiency can vary in different locations within an organ remain poorly understood. B16F10 cells were injected to target mouse lung, and at sequential times thereafter we quantified in lung the time course of: (a) overall cell survival and metastatic development; and (b) local cell survival and growth with respect to the lung surface and specific interior structures. We found high rates of initial survival of cells trapped in the lung circulation, extravasation into lung tissue, and subsequent survival of extravasated solitary cells (74% at day 3) before metastasis formation. However, at the time of initial replication of metastatic cells a major loss of cells occurred. Although only a small proportion of injected cells started to form metastases, most of these developed into macroscopic tumors. Solitary cells found at later times were dormant. Thus, overall metastatic inefficiency was largely due to postextravasation events affecting solitary cells. Regionally within the lung, cells and metastases were randomly distributed to day 4, but by day 10 preferential tumor growth was found along the lung surface and around arterial and venous vessels. Thus, trapping and early growth of injected cells was unaffected by location within the lung, whereas subsequent metastatic growth was enhanced in specific microenvironments. This study: (a) quantifies early temporal and spatial progression of metastasis in lung; (b) documents persistence of solitary dormant cells; and (c) shows that metastatic inefficiency depends on the initiation of growth in a subset of extravasated cells, whereas continued growth of metastases occurs preferentially in specific tissue environments.

Clinical targets for anti-metastasis therapy.

Metastasis is responsible for most cancer deaths. Therapeutic strategies to prevent development of metastases thus have potential to impact on cancer mortality. Development of these therapies requires a better understanding of the biology and molecular events of the metastatic process. Metastasis is usually defined, clinically and experimentally, by evidence of the endpoint of the process, that is, the presence of metastatic tumors. Endpoint assays are suitable for determining if a therapeutic approach is effective, but can provide little information on how a treatment works in vivo and what steps in metastasis are affected. We describe here two methodological advances in the study of metastasis as a process: in vivo videomicroscopy, which permits direct observation of steps in metastasis, and a "cell accounting" technique that permits quantification of the fate of cells over time. These procedures have provided new and unexpected insights into the biology of the metastatic process. Based on these insights, we consider which steps in the metastatic process are biologically and clinically most appropriate as therapeutic targets for development of anti-metastasis therapies. We conclude that the most promising stage of the metastasis process for therapeutic targeting is the growth phase, after cancer cells have arrested in the microcirculation in secondary sites and have completed extravasation. Earlier phases in the process are either biologically inappropriate or clinically inaccessible, except in specific cases (e.g., chemoprevention strategies). The role of "seed" and "soil" in determining organ-specific metastasis is also discussed. The metastatic growth phase fortunately is a clinically broad target, and any treatment that limits growth of metastases prior to their causing irreversible harm to the patient has the potential to be clinically useful. A variety of therapeutic approaches to target this phase are under active development, including inhibition of angiogenesis or signal transduction pathways needed to support the growth of metastatic cells.