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Most patients who die of cancer do so from its metastasis to other organs. The calcium-binding protein S100A4 can induce cell migration/invasion and metastasis in experimental animals and is overexpressed in most human metastatic cancers. Here, we report that a novel inhibitor of S100A4 can specifically block its increase in cell migration in rat (IC50, 46 µM) and human (56 µM) triple negative breast cancer (TNBC) cells without affecting Western-blotted levels of S100A4. The moderately-weak S100A4-inhibitory compound, US-10113 has been chemically attached to thalidomide to stimulate the proteasomal machinery of a cell. This proteolysis targeting chimera (PROTAC) RGC specifically eliminates S100A4 in the rat (IC50, 8 nM) and human TNBC (IC50, 3.2 nM) cell lines with a near 20,000-fold increase in efficiency over US-10113 at inhibiting cell migration (IC50, 1.6 nM and 3.5 nM, respectively). Knockdown of S100A4 in human TNBC cells abolishes this effect. When PROTAC RGC is injected with mouse TNBC cells into syngeneic Balb/c mice, the incidence of experimental lung metastases or local primary tumour invasion and spontaneous lung metastasis is reduced in the 10–100 nM concentration range (Fisher’s Exact test, p ≤ 0.024). In conclusion, we have established proof of principle that destructive targeting of S100A4 provides the first realistic chemotherapeutic approach to selectively inhibiting metastasis.

Fig S1 Whole Mounts, Fig S2 Westerns, Fig S3 Male/Female Bsk, Fig S4 Inhibitors, Fig S5 Breast Tumors, Fig S6 MMP2 + Other, Fig S7 MMP2/S100A4, Supp Methods, Figure 5 Legend.

supplemental materials and methods.

Table S1 MMPs/ S100A4, Table 2 Survival Times, Table S3 Cox Results, Table S4 2x2 Tables, Table S5 Logistic Association , Table S6 Same Area Staining.

Many human glandular cancers metastasize along nerve tracts, but the mechanisms involved are generally poorly understood. The calcium-binding protein S100A4 is expressed at elevated levels in human cancers, where it has been linked to increased invasion and metastasis. Here we report genetic studies in a Drosophila model to define S100A4 effector functions that mediate metastatic dissemination of mutant Ras-induced tumors in the developing nervous system. In flies overexpressing mutant RasVal12 and S100A4, there was a significant increase in activation of the stress kinase JNK and production of the matrix metalloproteinase MMP1. Genetic or chemical blockades of JNK and MMP1 suppressed metastatic dissemination associated with S100A4 elevation, defining required signaling pathway(s) for S100A4 in this setting. In clinical specimens of human breast cancer, elevated levels of the mammalian paralogs MMP2, MMP9, and MMP13 are associated with a 4- to 9-fold relative decrease in patient survival. In individual tumors, levels of MMP2 and MMP13 correlated more closely with levels of S100A4, whereas MMP9 levels correlated more closely with the S100 family member S100P. Overall, our results suggest the existence of evolutionarily conserved pathways used by S100A4 to promote metastatic dissemination, with potential prognostic and therapeutic implications for metastasis by cancers that preferentially exploit nerve tract migration routes. Cancer Res; 77(3); 780–9. ©2016 AACR.

S100P protein is a potent inducer of metastasis in a model system, and its presence in cancer cells of patients is strongly associated with their reduced survival times. A well-established Furth Wistar rat metastasis model system, methods for measuring cell migration, and specific inhibitors were used to study pathways of motility-driven metastasis. Cells expressing C-terminal mutant S100P proteins display markedly-reduced S100P-driven metastasis in vivo and cell migration in vitro. These cells fail to display the low focal adhesion numbers observed in cells expressing wild-type S100P, and the mutant S100P proteins exhibit reduced biochemical interaction with non-muscle myosin heavy chain isoform IIA in vitro. Extracellular inhibitors of the S100P-dependent plasminogen activation pathway reduce, but only in part, wild-type S100P-dependent cell migration, they are without effect on S100P-negative cells or cells expressing C-terminal mutant S100P proteins and have no effect on the numbers of focal adhesions. Recombinant wild-type S100P protein, added extracellularly to S100P-negative cells, stimulates cell migration, which is abolished by these inhibitors. The results identify at least two S100P-dependent pathways of migration, one cell surface and the other intracellularly-linked, and identify its C-terminal lysine as a target for inhibiting multiple migration-promoting activities of S100P protein and S100P-driven metastasis.

Background:Biomarkers for identification of endometrial cancers (ECs) with high risk of recurrence are required to reduce the rising EC-related mortality. AGR2 is a prognostic marker in several hormonally-regulated cancers. Aim:To assess the utility of AGR2 as a prognostic marker in EC. Methods:AGR2 immunoexpression was evaluated in 163 human endometrial samples. Change in AGR2 mRNA levels in response to oestrogen and dihydrotestosterone was studied in vitro. Results:Upregulation of AGR2 (protein and mRNA) was seen in low grade EC, compared to the postmenopausal endometrium (P = 0.013) and to the high-grade EC (P < 0.0001). Elevated AGR2 protein expression-scores were associated with a high expression of estrogen alpha (ERα), progesterone, androgen receptors and early clinical stages. Metastatic lesions maintained higher AGR2 expression relative to matched-primary tumors. High-AGR2 protein levels were associated with better overall survival (P = 0.02) in all ECs, but in highly-ERα-expressing ECs, AGR2 associated with unfavourable patient outcome. Androgen through its receptor, downregulated AGR2 mRNA in the Ishikawa cells. Conclusions:AGR2 is overexpressed in low grade ECs and positively associated with hormone receptors. The association between high AGR2 and progressive disease within the high-ERα-expressing ECs suggests that in this group of patients, AGR2 might be a potential biomarker of poor prognosis.

Background Breast cancer metastasis is a highly prevalent cause of death for European females. DNA microarray analysis has established that primary tumors, which remain localized, differ in gene expression from those that metastasize. Crossanalysis of these studies allow to revile the differences that may be used as predictive in the disease prognosis and therapy. Objective The aim of the project was to validate suggested prognostic and therapeutic markers using meta-analysis of data on gene expression in metastatic and primary breast cancer tumors. Method Data on relative gene expression values from 12 studies on primary breast cancer and breast cancer metastasis were retrieved from Genevestigator (Nebion) database. The results of the data meta-analysis were compared with results of literature mining for suggested metastatic breast cancer markers and vectors and consistency of their reported differential expression. Results Our analysis suggested that transcriptional expression of the COX2 gene is significantly downregulated in metastatic tissue compared to normal breast tissue, but is not downregulated in primary tumors compared with normal breast tissue and may be used as a differential marker in metastatic breast cancer diagnostics. RRM2 gene expression decreases in metastases when compared to primary breast cancer and could be suggested as a marker to trace breast cancer evolution. Our study also supports MMP1, VCAM1, FZD3, VEGFC, FOXM1 and MUC1 as breast cancer onset markers, as these genes demonstrate significant differential expression in breast neoplasms compared with normal breast tissue. Conclusion COX2 and RRM2 are suggested to be prominent markers for breast cancer metastasis. The crosstalk between upstream regulators of genes differentially expressed in primary breast tumors and metastasis also suggests pathways involving p53, ER1, ERB-B2, TNF and WNT, as the most promising regulators that may be considered for new complex drug therapeutic interventions in breast cancer metastatic progression.

Background. Osteoporosis is the most common metabolic bone disease in the world. Since osteoporosis is clinically symptomless until the first fracture occurs, early diagnosis is critical. Calcium, along with calcium-binding and calcium-associated proteins, plays an important role in homeostasis, maintaining healthy bone metabolism. This study is aimed at investigating the level of calcium-binding/associated proteins, annexin A1, S100A4, and TMEM64, in peripheral blood mononuclear cells associated with osteoporosis and its clinical significance. Methods. The levels of mRNAs of annexin A1, S100A4, and TMEM64 in human peripheral blood mononuclear cells were evaluated among 48 osteopenia and 23 osteoporosis patients compared to 17 nonosteoporotic controls. Total RNAs were isolated from clinical samples, and quantitation of mRNA levels was performed using real-time quantitative PCR. Results. The levels of mRNAs for calcium-binding proteins, annexin A1 and S100A4, and calcium-associated protein, TMEM64, in human peripheral blood mononuclear cells were significantly reduced in osteopenia and osteoporosis patients compared with nonosteoporotic controls (one-way ANOVA, P<0.0001, P=0.039, and P=0.0195, respectively). Annexin A1 and TMEM64 mRNAs were also significantly reduced in female osteoporosis patients over the age of 50 years compared to nonosteoporotic controls (one-way ANOVA, P=0.004 and P=0.0037, respectively). ROC analysis showed that the reduction in the level of mRNA for annexin A1, S100A4, or TMEM64 in the patients’ peripheral blood mononuclear cells has a good diagnostic value for osteoporosis. Conclusions. The results show for the first time that calcium-binding/associated proteins, annexin A1 and TMEM64, could be future diagnostic biomarkers for osteoporosis.

Osteoporosis, one of the most prevalent chronic ageing-related bone diseases, often goes undetected until the first fragility fracture occurs, causing patient suffering and cost to health/social care services. Osteoporosis arises from imbalanced activity of osteoclasts and osteoblasts. Since these cell lineages produce the protease, cathepsin Z, the aim of this study was to investigate whether altered cathepsin Z mRNA levels are associated with osteoporosis in clinical samples. Cathepsin Z mRNA in human peripheral blood mononuclear cells was significantly differentially-expressed among non-osteoporotic controls, osteopenia and osteoporosis patients (p P = 0.0016). Cathepsin Z mRNA level strongly correlated with low bone mineral density (BMD) (g/cm2), lumbar spine L2-L4 and femoral neck (T-scores) (P = 0.0149, 0.0002 and 0.0139, respectively). Importantly, cathepsin Z mRNA was significantly associated with fragility fracture in osteoporosis patients (P = 0.0018). The levels of cathepsin Z mRNA were not significantly higher in patients with chronic inflammatory disorders in these two groups compared to those without (P = 0.774 and 0.666, respectively). ROC analysis showed that cathepsin Z mRNA has strong diagnostic value for osteoporosis and osteoporotic fracture. The results show for the first time that cathepsin Z could be a future diagnostic biomarker for osteoporosis including female osteoporosis patients over the age of 50 years.

Osteoporosis is the most common age-related bone disease worldwide and is usually clinically asymptomatic until the first fracture happens. MicroRNAs are critical molecular regulators in bone remodelling processes and are stabilised in the blood. The aim of this project was to identify circulatory microRNAs associated with osteoporosis using advanced PCR arrays initially and the identified differentially-expressed microRNAs were validated in clinical samples using RT-qPCR. A total of 161 participants were recruited and 139 participants were included in this study with local ethical approvals prior to recruitment. RNAs were extracted, purified, quantified and analysed from all serum and plasma samples. Differentially-expressed miRNAs were identified using miRNA PCR arrays initially and validated in 139 serum and 134 plasma clinical samples using RT-qPCR. Following validation of identified miRNAs in individual clinical samples using RT-qPCR, circulating miRNAs, hsa-miR-122-5p and hsa-miR-4516 were statistically significantly differentially-expressed between non-osteoporotic controls, osteopaenia and osteoporosis patients. Further analysis showed that the levels of these microRNAs were associated with fragility fracture and correlated with the low bone mineral density in osteoporosis patients. The results show that circulating hsa-miR-122-5p and hsa-miR-4516 could be potential diagnostic biomarkers for osteoporosis in the future.

S100P protein in human breast cancer cells is associated with reduced patient survival and, in a model system of metastasis, it confers a metastatic phenotype upon benign mammary tumour cells. S100P protein possesses a C-terminal lysine residue. Using a multiwell in vitro assay, S100P is now shown for the first time to exhibit a strong, C-terminal lysine-dependent activation of tissue plasminogen activator (tPA), but not of urokinase-catalysed plasminogen activation. The presence of 10 μM calcium ions stimulates tPA activation of plasminogen 2-fold in an S100P-dependent manner. S100P physically interacts with both plasminogen and tPA in vitro, but not with urokinase. Cells constitutively expressing S100P exhibit detectable S100P protein on the cell surface, and S100P-containing cells show enhanced activation of plasminogen compared with S100P-negative control cells. S100P shows C-terminal lysine-dependent enhancement of cell invasion. An S100P antibody, when added to the culture medium, reduced the rate of invasion of wild-type S100P-expressing cells, but not of cells expressing mutant S100P proteins lacking the C-terminal lysine, suggesting that S100P functions outside the cell. The protease inhibitors, aprotinin or α-2-antiplasmin, reduced the invasion of S100P-expressing cells, but not of S100P-negative control cells, nor cells expressing S100P protein lacking the C-terminal lysine. It is proposed that activation of tPA via the C-terminal lysine of S100P contributes to the enhancement of cell invasion by S100P and thus potentially to its metastasis-promoting activity.

ObjectiveTranslational endometrial cancer (EC) research benefits from an in vitro experimental approach using EC cell lines. We demonstrated the steps that are required to examine estrogen-induced proliferative response, a simple yet important research question pertinent to EC, and devised a pragmatic methodological workflow for using EC cell lines in experimental models.MethodsComprehensive review of all commercially available EC cell lines was carried out, and Ishikawa cell line was selected to study the estrogen responsiveness with HEC1A, RL95-2, and MFE280 cell lines as comparators where appropriate, examining relevant differential molecular (steroid receptors) and functional (phenotype, anchorage-independent growth, hormone responsiveness, migration, invasion, and chemosensitivity) characteristics in 2-dimensional and 3-dimensional cultures in vitro using immunocytochemistry, immunofluorescence, quantitative polymerase chain reaction, and Western blotting. In vivo tumor, formation, and chemosensitivity were also assessed in a chick chorioallantoic membrane model.ResultsShort tandem repeat analysis authenticated the purchased cell lines, whereas gifted cells deviated significantly from the published profile. We demonstrate the importance of prior assessment of the suitability of each cell line for the chosen in vitro experimental technique. Prior establishment of baseline, nonenriched conditions was required to induce a proliferative response to estrogen. The chorioallantoic membrane model was a suitable in vivo multicellular animal model for EC for producing rapid and reproducible data.ConclusionsWe have developed a methodological guide for EC researchers when using endometrial cell lines to answer important translational research questions (exemplified by estrogen-responsive cell proliferation) to facilitate robust data, while saving time and resources.

Summary Filament assembly of nonmuscle myosin IIA (NMIIA) is selectively regulated by the small Ca2+-binding protein, S100A4, which causes enhanced cell migration and metastasis in certain cancers. Our NMR structure shows that an S100A4 dimer binds to a single myosin heavy chain in an asymmetrical configuration. NMIIA in the complex forms a continuous helix that stretches across the surface of S100A4 and engages the Ca2+-dependent binding sites of each subunit in the dimer. Synergy between these sites leads to a very tight association (KD ∼1 nM) that is unique in the S100 family. Single-residue mutations that remove this synergy weaken binding and ameliorate the effects of S100A4 on NMIIA filament assembly and cell spreading in A431 human epithelial carcinoma cells. We propose a model for NMIIA filament disassembly by S100A4 in which initial binding to the unstructured NMIIA tail initiates unzipping of the coiled coil and disruption of filament packing., Graphical Abstract Highlights ► S100A4 dimer binds single-myosin IIA molecule in an asymmetrical mode ► Synergy between Ca-dependent sites in S100A4 leads to high-affinity interaction ► Electron microscopy shows that two S100A4 dimers bind to the myosin coiled coil ► S100A4 has direct effect on formation of stress fibers and cell migration

BACKGROUND Endometriosis is a metastatic disease without obvious tumorigenesis. Expression of S100P, S100A4, osteopontin (OPN) or anterior gradient homologue 2 (AGR2) proteins can induce metastasis but fail to induce tumorigenesis per se. We now explore whether this group of metastasis-inducing proteins (MIPs) are associated with the pathogenesis of endometriosis. METHODS Eutopic endometrial biopsies were taken from 73 women (35 fertile women without endometriosis and 38 women with surgically diagnosed endometriosis). Ectopic endometriotic lesions were collected from eight of the women with endometriosis. The expression of MIPs at the cellular level was evaluated by immunohistochemistry and the presence of these proteins in the endometrial tissues was verified by western blotting and their gene expression was confirmed by RT–PCR. RESULTS All four MIPs were immunolocated in the endometrium of control women and S100P, AGR2 and OPN showed a cyclical variation. Proliferative phase eutopic endometrium of both groups showed a similar staining pattern for all MIPs, whereas secretory phase endometrium showed a differential expression between controls and cases. The secretory phase endometrial immunostaining of controls showed weak stromal and perivascular AGR2, and decreased stromal and glandular S100P. In contrast, immunostaining for all MIPs was increased in the late secretory endometrial samples of women with endometriosis and intense immunostaining was seen for S100A4 in the stroma (P< 0.05) and for S100P (P< 0.001) and AGR2 (P< 0.0001) in both glands and stroma (P< 0.001). All active peritoneal endometriotic lesions showed strong immunostaining for each of the MIPs studied. CONCLUSIONS We propose that these MIPs enhance endometrial cell invasiveness and contribute to the establishment of ectopic endometriotic deposits after retrograde menstruation.

Cell migration requires the initial formation of cell protrusions, lamellipodia and/or filopodia, the attachment of the leading lamella to extracellular cues and the formation and efficient recycling of focal contacts at the leading edge. The small calcium binding EF-hand protein S100A4 has been shown to promote cell motility but the direct molecular mechanisms responsible remain to be elucidated. In this work, we provide new evidences indicating that elevated levels of S100A4 affect the stability of filopodia and prevent the maturation of focal complexes. Increasing the levels of S100A4 in a rat mammary benign tumor derived cell line results in acquired cellular migration on the wound healing scratch assay. At the cellular levels, we found that high levels of S100A4 induce the formation of many nascent filopodia, but that only a very small and limited number of those can stably adhere and mature, as opposed to control cells, which generate fewer protrusions but are able to maintain these into more mature projections. This observation was paralleled by the fact that S100A4 overexpressing cells were unable to establish stable focal adhesions. Using different truncated forms of the S100A4 proteins that are unable to bind to myosin II A, our data suggests that this newly identified functions of S100A4 is myosin-dependent, providing new understanding on the regulatory functions of S100A4 on cellular migration.

Ovarian cancer is often asymptomatic and is diagnosed at an advanced stage with poor survival rates, thus there is an urgent need to develop biomarkers for earlier detection of ovarian cancer. In the present study, we demonstrate for the first time that the previously reported metastasis-inducing protein AGR2 (anterior gradient protein 2) can be detected in the blood of ovarian cancer patients. Using a newly developed ELISA, we show significantly increased concentrations of AGR2 protein in plasma from cancer patients relative to normal controls. Plasma AGR2 concentrations were highest in stages II and III ovarian cancer patients and were similarly elevated in patients with both serous and non-serous tumours. The identification of elevated plasma concentrations of AGR2 may provide a useful biomarker to aid in the discrimination of normal and ovarian cancer patients particularly when used in combination with CA125.

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.

The secreted metastasis-inducing protein, human anterior gradient 2 (AGR2), has been independently reported to be associated with either a reduced or an increased survival of different groups of patients with breast cancer. We now aim to analyze the expression of AGR2 in a third completely independent group of patients using a specific AGR2 monoclonal antibody (mAb). Primary tumors from a group of 315 patients suffering from operable (stage I and II) breast cancer with 20-years follow-up were immunocytochemically stained with a specific mAb to AGR2 and associations with prognostic factors and patient survival were analyzed. The mAb specifically recognized AGR2 in Western blots, and positive staining for AGR2 was significantly associated with involved lymph nodes and staining for estrogen receptor α, progesterone receptor, and the metastasis-inducing proteins osteopontin, S100P, and S100A4. After 20 years of follow-up, only 26% of patients with AGR2-positive carcinomas survived compared with 96% of those with AGR2 negative carcinomas, with the highly significant difference in median survival times of 68 and >216 months, respectively (P < 0.0001). Cox’s multivariate regression analysis showed that staining for AGR2 was one of the most significant independent prognostic indicators, with a corrected relative risk of 9.4. The presence of AGR2 in the primary tumor is therefore a possible prognostic indicator of poor patient outcome in breast cancer.

Epidermal growth factor (EGF) receptor (EGFR) modulates mitosis and apoptosis through signaling by its high-affinity (HA) and low-affinity (LA) EGF-binding states. The prevailing model of EGFR activation-derived from x-ray crystallography-involves the transition from tethered ectodomain monomers to extended back-to-back dimers and cannot explain these EGFR affinities or their different functions. Here, we use single-molecule Förster resonant energy transfer analysis in combination with ensemble fluorescence lifetime imaging microscopy to investigate the three-dimensional architecture of HA and LA EGFR-EGF complexes in cells by measuring the inter-EGF distances within discrete EGF pairs and the vertical distance from EGF to the plasma membrane. Our results show that EGFR ectodomains form interfaces resulting in two inter-EGF distances ( approximately 8 nm and5.5 nm), different from the back-to-back EGFR ectodomain interface ( approximately 11 nm). Distance measurements from EGF to the plasma membrane show that HA EGFR ectodomains are oriented flat on the membrane, whereas LA ectodomains stand proud from it. Their flat orientation confers on HA EGFR ectodomains the exclusive ability to interact via asymmetric interfaces, head-to-head with respect to the EGF-binding site, whereas LA EGFRs must interact only side-by-side. Our results support a structural model in which asymmetric EGFR head-to-head interfaces may be relevant for HA EGFR oligomerization.

X-box binding protein 1 (XBP-1) is stimulated by endoplasmic reticulum stress as part of the unfolded protein response (UPR), which can promote apoptosis or cell survival. Non-conventional splicing, stimulated during the UPR, converts mRNA for "unspliced" XBP-1U to "spliced" XBP-1S mRNA. XBP-1 mRNA is oestrogen-responsive, but XBP-1S confers oestrogen independence and anti-oestrogen resistance to breast cancer cell lines. We therefore evaluated XBP-1 mRNA splicing as a factor in response of breast cancer patients to endocrine treatment. XBP-1 isoforms were measured by quantitative RT-PCR in 100 primary breast cancer patients treated with adjuvant tamoxifen (including 30 ER alpha-negative cases). In ER alpha-positive cases, levels of XBP-1U mRNA correlated with ER alpha mRNA levels and were lower in grade 3 tumors. Higher levels of XBP-1U mRNA were significantly associated with breast cancer survival (Log-rank p = 0.002; Cox hazard ratio (HR) 0.2, p = 0.005), independent of grade, size, nodal status and progesterone receptor status. However, in the full cohort, higher ratios of XBP-1S/XBP-1U mRNA (indicating enhanced splicing) were associated with poor survival (Log-rank p = 0.03; Cox HR 2.3, p = 0.03) and related factors: ER alpha-negative status, progesterone receptor negative status, grade 3 tumors and greater proliferation. Significant associations with poor outcome were also seen for XBP-1 splicing in ER alpha-positive cases. Our findings, that XBP-1 isoforms are differently associated with outcome of endocrine therapy for patients, can be explained by higher levels of dominant-negative XBP-1U favouring apoptosis of tumor cells and higher levels of XBP-1S increasing tumor survival.

Anterior gradient-2 (AGR2) expression was examined in a series of prostate cell lines and in an archival set of prostate tissues. The relative levels of AGR2 expression in the malignant cell lines PC-3 and PC-3M were, respectively, 5.3+/-0.1 and 3.8+/-0.2 times that detected in the benign cell line PNT-2. Immunohistochemical staining in 106 cases showed that amongst seven normal cases, one (14.3%) was unstained, five (71.4%) stained weakly positive and one (14.3%) stained moderately positive. Amongst 34 benign prostate hyperplastic (BPH) cases, 12 (35.3%) were unstained, 18 (52.9%) stained weakly positive and four (11.8%) stained moderately positive. Amongst 65 carcinomas, three (4.6%) were unstained, 14 (21.5%) stained weakly positive, 19 (29.2%) stained moderately positive and 29 (44.9%) stained strongly positive. AGR2 expression in carcinomas was significantly higher than that in BPH (chi(2)-test, P0.001). Kaplan-Meier survival analysis showed that increased AGR2 expression was significantly (log rank test, P=0.007) associated with reduced patient-survival time. Increased joint Gleason score (GS) was significantly (log rank test, P=0.001) associated with poor patient survival. However, neither prostate specific antigen (PSA) level, nor androgen receptor (AR) index, was significantly associated with patient-survival time. Increased AGR2 expression was significantly correlated with high GS (two-sided Fisher's exact test, P0.001) and PSA levels (Mann-Whitney U-test, P=0.047), but not significantly related to the level of AR (Mann-Whitney U-test, P=0.286). These results suggest that increased AGR2 expression is a valuable prognostic factor to predict the clinical outcome of the prostate cancer patients.

AGR2 is overexpressed in multiple cancers, particularly those arising from breast and prostate tissues, and higher levels of AGR2 are associated with earlier patient death. Although AGR2 is normally resident within the endoplasmic reticulum, the protein has been found in the extracellular space in several model systems. However, it has never been expressly demonstrated that this extracellular form of the protein is secreted and does not just accumulate in the extracellular space as a result of cell lysis. We show in this paper that AGR2 protein is secreted by both human and rat mammary epithelial cells in culture. Furthermore, this secreted form of AGR2 becomes O-glycosylated, with no detectable presence of N-glycosylation. Importantly, this post-translationally modified AGR2 is only detected in the conditioned medium from non-leaky cells, suggesting that membrane integrity must be maintained to allow AGR2 glycosylation. The results suggest a possible role for O-glycosylation in modulating the extracellular functions of AGR2.

The anterior gradient protein-2 (AGR2) is inducible by oestrogen and itself can induce metastasis in a rat model for breast cancer. Here, a rabbit antibody to recombinant human AGR2 was used to assess its prognostic significance in a retrospective cohort of 351 breast cancer patients treated by adjuvant hormonal therapy. The antibody stains 66% of breast carcinomas to varying degrees. The percentage of positive carcinoma cells in tumours directly correlates with the level of AGR2 mRNA (Spearman's rank correlation, P = 0.0007) and protein (linear regression analysis r2 = 0.95, P = 0.0002). There is a significant association of staining of carcinomas for AGR2 with oestrogen receptor alpha (ERalpha) staining and with low histological grade (both Fisher's Exact test P0.0001). In the ERalpha-positive cases, but not the ERalpha-negative cases, when subdivided into the separate staining classes for AGR2, there is a significantly progressive decrease in patient survival with increased staining (log rank test, P = 0.006). The significant association of staining for AGR2 with patient death over a 10-year period (log rank test P = 0.007, hazard ratio = 3) only becomes significant at 6 years of follow-up. This may be due to the cessation of adjuvant hormonal therapy at an earlier time, resulting in adverse re-expression of the metastasis-inducing protein AGR2.

PURPOSE: S100A4 and the estrogen-inducible osteopontin are alone capable of inducing angiogenesis and metastasis in rodent models for breast cancer. The present study assesses the relationship of S100A4 and osteopontin with vessel density and estrogen receptor alpha (ERalpha) in primary tumors and with survival of patients to ascertain their involvement in metastatic breast cancer. EXPERIMENTAL DESIGN: Primary tumors from 312 patients treated for minimally invasive human breast cancer were immunocytochemically stained and then assessed for the significance of their association with each other using Fisher's exact test or with patient survival over 18 years of follow-up using Kaplan-Meier plots and Wilcoxon-Gehan statistics. RESULTS: Antibodies to S100A4 significantly stained endothelial cells of vessels adjacent to S100A4-staining groups of carcinoma cells, and antibodies to osteopontin significantly stained groups of carcinoma cells staining for ERalpha (P < 0.0001). There was a significant association of tumors staining for S100A4 with those with high vessel density (P = 0.021) and of tumors staining for osteopontin with those staining for ERalpha (P = 0.034). The association of staining for S100A4, osteopontin, or vessel density with patient death was significant (P < 0.0001, P = 0.005, and P = 0.014, respectively). The difference in cumulative proportion surviving between S100A4-positive patients with higher or lower vessel density increased up to about 12 years, but thereafter decreased to virtually zero after 18 years of follow-up. Patients with both S100A4-positive and osteopontin-positive primary tumors showed a statistically significant reduction in survival time over those with either one alone (P < 0.019), although in multivariate regression analysis, only staining for S100A4 was significant (P < 0.001). CONCLUSIONS: It is suggested that in human breast cancer, S100A4 exerts some of its effects through angiogenesis, and that osteopontin is dependent on ERalpha for its expression.

S100P, an EF-hand calcium-binding protein, has been reported to be associated with the progression of many types of cancers. Transfection of an expression vector for S100P into a benign, nonmetastatic rat mammary cell line causes a 4- to 6-fold increase in its level in all four transformant cell clones. When the resultant transformant cell lines are introduced in turn into the mammary fat pads of syngeneic Furth-Wistar rats, there is a significant 3-fold increase in local muscle invasion and a significant induction of metastasis in 64% to 75% of tumor-bearing animals. In a group of 303 breast cancer patients followed for up to 20 years, antibodies to S100P immunocytochemically stain 161 primary tumors. Survival of patients with S100P-positive carcinomas is significantly worse by about 7-fold than for those with negatively stained carcinomas. There is also a significant association between the class level of immunocytochemical staining of the carcinoma cells and decreased patient survival. Positive staining for S100P is significantly associated with that for two other metastasis-inducing proteins, S100A4 and osteopontin. Patients with tumors that stained positively for both S100P and S100A4 have a significantly reduced survival of 1.1% over patients with either S100 protein alone. Multivariate regression analysis identifies S100P, S100A4, and osteopontin as the most significant independent indicators of death in this group of patients. These results suggest that stratification of patients into groups according to expression of multiple metastasis-inducing proteins may lead to a more accurate prediction of patient survival. (Cancer Res 2006; 66(2): 1199-207)

A metastatic phenotype can be induced in benign rat mammary cells (Rama 37 cells) by transfecting them with metastasis-inducing DNAs (Met-DNAs). Stable transfection of Met-DNAs increases the level of the metastasis-associated protein, osteopontin. Randomly picked clonal cell lines have been established from the pool of Rama 37 cells transfected with one metastasis-inducing DNA, C9-Met-DNA. In these cell lines, moderate correlation is observed between the copy number of C9-Met-DNA and their metastatic potential (linear regression coefficient, R(2)=0.48). A very close correlation is observed between the cell lines' metastatic potential in vivo and the osteopontin mRNA levels in vitro (R(2)=0.74), but not with another metastasis-associated protein in this system, S100A4 (R(2)=0.21). A close correlation is also observed between osteopontin mRNA levels and the adhesive potential (R(2)=0.91) of the cells, but not with their growth rate in vitro (R(2)=0.03). These observations support the previous suggestion that osteopontin is the direct effector of C9-Met-DNA and that the presence of C9-Met-DNA is necessary, if not sufficient, for the induction of metastasis in vivo in this system. Additionally, these results suggest that Rama 37 cells with increased osteopontin mRNA levels become metastatic not through an increased growth rate, but through an increase in cellular adhesiveness.

S100P is a small calcium-binding protein of the S100 EF-hand-containing family of proteins. Elevated levels of its mRNA are reported to be associated with the progression to hormone independence and metastasis of prostate cancer and to be associated with loss of senescence in human breast epithelial cells in vitro. The first structure of human recombinant S100P in calcium-bound form is now reported at 2.0A resolution by X-ray diffraction. A flexible linker connects the two EF-hand motifs. The protein exists as a homodimer formed by non-covalent interactions between large hydrophobic areas on monomeric S100P. Experiments with an optical biosensor to study binding parameters of the S100P monomer interaction showed that the association rate constant was faster in the presence of calcium than in their absence, whereas the dissociation rate constant was independent of calcium. The K(d) values were 64(+/-24)nM and 2.5(+/-0.8) microM in the presence and in the absence of calcium ions, respectively. Dimerization of S100P is demonstrated in vivo using the yeast two-hybrid system. The effect of mutation of specific amino acids suggests that dimerization in vivo can be affected by amino acids on the dimer interface and in the hydrophobic core.

Two suppression subtracted cDNA libraries have been constructed, one containing cDNAs to mRNAs present at a higher level in a benign human breast tumour-derived cell line relative to the malignant mammary cell line, MCF-7, and the other containing cDNAs present at a higher level in the MCF-7 cells relative to the benign cells. Randomly-picked cloned DNAs have been sequenced yielding 29 and 128 different cDNAs from the benign and malignant libraries, respectively. Using reverse Northern hybridisation, 76% and 83% of the cDNAs were differentially expressed by greater than two-fold, whilst 14% and 11% of cDNAs in the respective libraries were differentially expressed by more than 15-fold. Amongst these were oestrogen-responsive cDNAs and expressed sequence tags. One such oestrogen-responsive expressed sequence tag, M41, is transcribed from a gene located on chromosome 21q22.3, within an intron of a larger gene. The M41 gene contains oestrogen response elements, one of which is associated with alu repeats. M41 mRNA is expressed at a statistically significantly higher level in human breast cancer specimens than in normal human breast and benign lesions. In carcinomas, its up-regulation is associated with the development of the malignant cell. British Journal of Cancer (2002) 87, 423–431. doi:10.1038/sj.bjc.6600456 www.bjcancer.com © 2002 Cancer Research UK

The presence of the EF-hand-calcium-binding protein S100A4 in the carcinoma cells of the primary tumour is associated with a shorter survival time of a group of breast cancer patients. In colon cancer, primary tumours as well as metastases to the liver can be studied. Here we show, using quantitative PCR applied to RNA from 24 normal colon, four liver tissues, 24 colon carcinoma specimens, and 24 livers containing colonic carcinoma metastases, that the level of S100A4 mRNA was significantly higher in the carcinomas compared to normal specimens (Mann-Whitney U-test, P=0.05), and in liver metastases compared to carcinoma specimens (P=0.039). The latter comparison included seven liver metastases and their matched primary carcinomas (P

The abnormalities of lipid metabolism observed in cancer cachexia may be induced by a lipid-mobilizing factor produced by adenocarcinomas. The specific molecules and metabolic pathways that mediate the actions of lipid-mobilizing factor are not known. The mitochondrial uncoupling proteins-1, -2 and -3 are suggested to play essential roles in energy dissipation and disposal of excess lipid. Here, we studied the effects of lipid-mobilizing factor on the expression of uncoupling proteins-1, -2 and -3 in normal mice. Lipid-mobilizing factor isolated from the urine of cancer patients was injected intravenously into mice over a 52-h period, while vehicle was similarly given to controls. Lipid-mobilizing factor caused significant reductions in body weight (−10%, P=0.03) and fat mass (−20%, P

The calcium-binding protein S100A4 induces the metastatic phenotype in rodent models of breast cancer, and its expression strongly correlates with reduced survival in human breast and bladder cancer. We have established an orthotopic model of bladder cancer by injecting a cell line derived from a carcinogen-induced rat bladder tumor into the muscular wall of syngeneic rats. MYU-3L cells produce rapidly growing, invasive tumors in the bladder wall but they fail to metastasize. Transfection of MYU-3L cells with a plasmid vector directing overexpression of the S100A4 gene generates variants in which S100A4 expression is elevated by up to sevenfold in comparison with the untransfected cells. Variants overexpressing S100A4 produce primary tumors at similar frequencies and latencies to the parental cell line, a significant number of which metastasize to the para-aortic lymph nodes or lungs. Expression of S100A4 protein in the primary tumors was heterogeneous, but was stronger and more consistent in the metastases, suggesting that transfectants overexpressing S100A4 possess an enhanced ability to form metastatic lesions. We conclude that overexpression of S100A4 can induce the metastatic phenotype in this rodent model of bladder cancer. Taken together with the results from our parallel studies of human bladder cancer, these data suggest a significant role for S100A4 in bladder cancer metastasis and identify a potential new target for systemic therapy in patients with this disease.

The calcium-binding protein S100A4 induces the metastatic phenotype in rodent models of breast cancer and its expression correlates strongly with reduced survival in human breast cancer. The expression of S100A4 in normal bladders and 101 bladder tumours has been studied using immunocytochemistry. Moderate or strong expression of S100A4 was found in 28% of the tumours, whilst the remaining tumours and normal urothelium either failed to stain or showed weak staining. S100A4 staining was more frequently observed in invasive bladder tumours than in non-invasive tumours (p

The major problem with cancer is the ability of cancer cells to infiltrate surrounding tissue (invasion) or to spread to distant organs (metastasis), decreasing patient survival. One of the Ca2+ binding proteins of the S100 family, S100A4, is expressed at elevated level in several forms of cancer and has the ability to induce metastasis and invasion in benign mammary cells. In order to generate a genetically tractable experimental model of S100A4-mediated metastasis, we have expressed the human S100A4 in the fruit fly Drosophila melanogaster to uncover players in tumour progression, invasion and metastasis. Here we demonstrate that the expression of full open reading frame of human S100A4 wild type protein in cooperation with RasVal12 into Drosophila, developed metastatic phenotypes. Fly larvae expressing the human S100A4 as determined by Western blot exhibit metastasis in the ventral nerve cord when crossed with Drosophila strains expressing oncogenic RasVal12 targeted to the optic lobes by the UAS-GAL4 system. However, the mutant of S100A4 (S100A4/∆2) missing the last two lysine reduced the tumour dissemination into the ventral nerve cord in Drosophila flies expressing RasVal12 oncogene. Downstream genes associated with the metastatic behaviour including JNK and MMPs (metalloproteinase) proteins are also investigated. We showed that the MMPs and the JNK are also activated in the flies with S100A4/ RasVal12 genotype. Loss this activation in flies with S100A4∆2/RasVal12 genotype indicated that MMPs and JNK proteins are essential in tumour progression and metastasis pathways. Therefore, MMPs and JNK are excellent targets for cancer therapy.

A suppression subtraction cDNA library representing mRNAs expressed at a higher level in a benign breast tumour-derived cell line relative to the malignant MCF-7A cell line contained cDNAs corresponding to mRNAs for plasminogen activator inhibitor I, annexin VIII and the EF-hand protein S100A2. S100A2 protein has previously been shown to be expressed in normal human breast epithelium, but not in human breast carcinoma cell lines. Using a PCR-based assay and in situ hybridization on histological sections of human breast specimens, the mRNA for S100A2 was shown to be present in all benign breast lesions examined as well as in normal epithelium. S100A2 mRNA was detectable in 37% of specimens of carcinoma in situ, but in less than 15% of carcinoma specimens. The results suggest that the loss of S100A2 is associated with the development of malignant cells and is not associated with early tumour development. © 2000 Cancer Research Campaign http://www.bjcancer.com

The tumour suppressor gene, p53, and genes coding for positive signal transduction factors can influence transit through cell-cycle checkpoints and modulate radiosensitivity. Here we examine the effects of RAF1 protein on the rate of exit from a G2/M block induced by γ-irradiation in relation to intrinsic cellular radiosensitivity in human cell lines expressing wild-type p53 (wtp53) protein as compared to mutant p53 (mutp53) protein. Cell lines which expressed mutp53 protein were all relatively radioresistant and exhibited no relationship between RAF1 protein and cellular radiosensitivity. Cell lines expressing wtp53 protein, however, showed a strong relationship between RAF1 protein levels and the radiosensitivity parameter SF2. In addition, when post-irradiation perturbation of G2/M transit was compared using the parameter T50 (time after the peak of G2/M delay at which 50% of the cells had exited from a block induced by 2 Gy of irradiation), RAF1 was related to T50 in wtp53, but not mutp53, cell lines. Cell lines which expressed wtp53 protein and high levels of RAF1 had shorter T50s and were also more radiosensitive. These results suggest a cooperative role for wtp53 and RAF1 protein in determining cellular radiosensitivity in human cells, which involves control of the G2/M checkpoint. © 2000 Cancer Research Campaign

Rodent S100A4 (p9Ka) induces a metastatic phenotype in benign rat mammary tumour cells and cooperates with the neu oncogene to produce metastatic tumours in a transgenic mouse model system. Human S100A4 possesses similar metastasis-inducing properties. S100A4 mRNA is now sought in human breast tumour-derived cell lines and tumour specimens. S100A4 mRNA is present in some cell lines derived from malignant breast cancers, but is not detectable in cells derived from benign breast tumours, In human tumour specimens, using in situ hybridisation, the mRNA for S100A4 is localised to the epithelial cells of carcinoma specimens, and in some normal breast specimens, to a stromal region surrounding the epithelial ducts, In carcinoma specimens, S100A4 mRNA is also found in the stromal region surrounding islands of cancer cells. For both the epithelial and stromal components, S100A4 mRNA is present at a higher level in carcinomas relative to benign breast tumour specimens. In general, there is a concordance between the S100A4 mRNA signal from the epithelial and stromal elements of the same carcinoma specimens. Using Northern blotting techniques, these results have been extended to a panel of 137 benign and malignant breast tumour specimens. The results show that S100A4 mRNA occurs in the more-malignant, rather than in the more-benign tumour specimens. Int. J, Cancer 86: 219-228, 2000. (C) 2000 Wiley-Liss, Inc.

Prostaglandin F(2alpha) (PGF(2alpha)), a mitogen for Swiss 3T3 cells, triggers cyclin D1 mRNA/protein expression prior to cellular entry into the S phase, but fails to raise cdk4 or cyclin D3 levels, while 1-oleoyl-2-diacylglycerol (OAG), a protein kinase C (PKC) and tyrosine kinase (TK) activator, induces only cyclin D1 expression with no mitogenic response. In contrast, in PKC-depleted or -inhibited cells, PGF(2alpha), but not OAG, increases cyclin D1 expression with no mitogenic response. Finally, OAG, in the presence of orthovanadate (Na(3)VO(4)) or TGF(beta1), induces DNA synthesis. Thus, it appears that PGF(2alpha) triggers cyclin D1 expression via two independent signaling events that complement with TGF(beta1)-triggered events to induce DNA synthesis.

Our aim was to compare the occurrence and prognostic significance over 14-20 years of immunocytochemically detected S100A4 and other tumour variables in primary tumours from 349 patients with operable breast cancer. For a cut-off of 1% staining of the malignant cells, the antibody to S100A4 stains positively 56% of the carcinomas. There was a significant association of staining for S100A4 with tumours fixed to the chest wall, staining for c-erbB-2, c-erbB-3, pS2, cathepsin D and, inversely, at borderline levels with staining for estrogen receptor. Using Wilcoxon statistics in univariate analyses, staining for S100A4, nodal status, tumour class, histological grade and staining for c-erbB-2, p53 were associated negatively and staining for estrogen receptor, progesterone receptor were associated positively with patient survival times. The survival times of patients with S100A4-negative carcinomas with or without one of the other tumour variables showed no significant differences, whilst those of patients with S100A4-positive carcinomas showed significant differences in a negative or a positive way. Multivariate regression analysis for 137 patients showed that staining for S100A4 is most highly correlated with patient deaths, but involved lymph nodes, fixed tumours, high histological grade and staining for progesterone receptor were also significant independent prognostic variables. Our results suggest that in this set of patients, the tumour variable most tightly correlated with patient death is S100A4.

A panel of human breast cancer specimens was examined for single base change mutations by DNA sequencing and for larger deletions using a PCR-based assay. In the cancer specimens examined, no sequencing variants were detected other than a previously characterized polymorphism. Although most of the specimens contained estrogen receptor (ER) variants at a low level, 2 of 118 specimens exhibited variants which, after amplification, constituted most of the amplified ER cDNA. One specimen contained a single variant form, and there was little evidence of the wild-type ER mRNA by PCR, Northern blotting or immunocytochemistry. The second specimen, despite the presence of a normal-sized mRNA by Northern blotting and normal immunocytochemical staining for ER, contained at least 5 different variant forms as well as the wild-type ER. All but 1 of the variant forms were processing variants, and 3 of these processing variants have not been described before. One variant, although lacking exons 2–4, has break points in exons 1 and 5 that do not correspond to intron–exon boundaries. This variant might reflect more widespread damage to the genome in this breast cancer specimen. The low level of occurrence of variants suggests that ER variant forms, at least in the coding region, do not contribute generally to the progression of breast cancer. Int. J. Cancer 88:209–216, 2000. © 2000 Wiley-Liss, Inc.

The S100 proteins contain two EF-hand motifs and are of generally unknown function. One of these proteins, S100A4, is an intracellular calcium-binding protein that is present in normal rodent and human cells. In cultured rodent mammary cells, S100A4 is expressed at a higher level in some metastatic epithelial cells than in non-metastatic counterparts. Similarly, in human breast cell lines, S100A4 is present at a higher level in cultured cells from the more malignant, than in those from the more benign tumours. Gene transfer experiments have shown that rodent or human S100A4 is able to induce metastatic capability in otherwise non-metastatic breast tumour cells. Furthermore, expression of rodent S100A4 transgenes can induce metastasis of benign tumours arising in transgenic model systems. Possible mechanisms for the metastasis-inducing effect of S100A4 and the relevance of these observations to human cancer are discussed.

Background S100P is an EF-hand calcium-binding protein. Like S100A4 and S100A6, S100P is expressed at a high level in cancers, particularly in those of the prostate, pancreas, and breast. We have demonstrated that S100P is a metastasis-inducing protein and that its upregulation enhances cell migration. When studying the molecular mechanisms, we found that S100P interacts separately with two major components of cytoskeleton, non-muscle myosin IIA (NMIIA) and tubulin. The aim of this study was to understand how S100P enhances cell migration through these protein interactions. Methods S100P-inducible cell lines and specific siRNAs were used to control the expression of S100P in cells. We used specific chemicals to disrupt actin stress fibres and microtubules and peptides to block the protein interactions. Results S100P differentially regulates NMIIA and NMIIB filaments, which reduced cell adhesion and enhanced cell migration. This process involves Rho, RAC1, and focal adhesion kinase. S100P upregulation simultaneously altered the dynamics of tubulin polymerisation to facilitate cell migration. Interpretation S100P directly regulates the dynamics of both actin-myosin filaments and microtubules, and the co-regulation of these cytoskeletons may synergistically enhance cell migration and promote cancer cell metastasis.

The rodent and human nonpregnant mammary glands contain epithelial, intermediate and myoepithelial cells which have all been isolated as cell lines in vitro. Transforming growth factor-alpha (TGF alpha) and basic fibroblast growth factor (bFGF) are produced by myoepithelial cells and can stimulate the growth of intermediate stem cells in vitro. Epithelial and intermediate cells behave like stem cells in vitro, since they can differentiate into alveolar-like an myoepithelial cells. The myoepithelial differentiation pathway is associated with the early expression of a calcium-binding regulatory protein called p9Ka and the protease, Cathepsin D. Myoepithelial cells are also present in benign lesions but not in malignant mammary carcinomas of rats or humans, whose resultant cell lines fail to differentiate completely along the myoepithelial cell pathway. Loss of the myoepithelial cell in some invasive carcinomas may be compensated, at least in part, by changes in malignant cells. Over-expression of TGF alpha and/or erbB receptors may reduce the requirement for TGF alpha, whilst ectopic production of bFGF and its receptors and p9Ka/Cathespin D may assist in tumorigenesis and in metastasis, respectively. Thus compensation for, or retention of, molecules potentially involved in the differentiation of mammary cells may be a mechanism by which malignancy progresses in some human invasive carcinomas.