Prognostic biomarkers for immunotherapy with ipilimumab in metastatic melanoma.

New therapies, including the anti-cytotoxic T lymphocyte antigen (CTLA)-4 antibody, ipilimumab, is approved for metastatic melanoma. Prognostic biomarkers need to be identified, because the treatment has serious side effects. Serum samples were obtained before and during treatment from 56 patients with metastatic or unresectable malignant melanoma, receiving treatment with ipilimumab in a national Phase IV study (NCT0268196). Expression of a panel of 17 inflammatory-related markers reflecting different pathways including extracellular matrix remodeling and fibrosis, vascular inflammation and monocyte/macrophage activation were measured at baseline and the second and/or third course of treatment with ipilimumab. Six candidate proteins [endostatin, osteoprotegerin (OPG), C-reactive protein (CRP), pulmonary and activation-regulated chemokine (PARC), growth differentiation factor 15 (GDF15) and galectin-3 binding-protein (Gal3BP)] were persistently higher in non-survivors. In particular, high Gal3BP and endostatin levels were also independently associated with poor 2-year survival after adjusting for lactate dehydrogenase, M-stage and number of organs affected. A 1 standard deviation increase in endostatin gave 1·74 times [95% confidence interval (CI) = 1·10-2·78, P = 0·019] and for Gal3BP 1·52 times (95% CI = 1·01-2·29, P = 0·047) higher risk of death in the adjusted model. Endostatin and Gal3BP may represent prognostic biomarkers for patients on ipilimumab treatment in metastatic melanoma and should be further evaluated. Owing to the non-placebo design, we could only relate our findings to prognosis during ipilimumab treatment.

Enhanced targeting of triple-negative breast carcinoma and malignant melanoma by photochemical internalization of CSPG4-targeting immunotoxins.

Triple-negative breast cancer (TNBC) and malignant melanoma are highly aggressive cancers that widely express the cell surface chondroitin sulfate proteoglycan 4 (CSPG4/NG2). CSPG4 plays an important role in tumor cell growth and survival and promotes chemo- and radiotherapy resistance, suggesting that CSPG4 is an attractive target in cancer therapy. In the present work, we applied the drug delivery technology photochemical internalization (PCI) in combination with the novel CSPG4-targeting immunotoxin 225.28-saporin as an efficient and specific strategy to kill aggressive TNBC and amelanotic melanoma cells. Light-activation of the clinically relevant photosensitizer TPCS2a (fimaporfin) and 225.28-saporin was found to act in a synergistic manner, and was superior to both PCI of saporin and PCI-no-drug (TPCS2a + light only) in three TNBC cell lines (MDA-MB-231, MDA-MB-435 and SUM149) and two BRAFV600E mutated malignant melanoma cell lines (Melmet 1 and Melmet 5). The cytotoxic effect was highly dependent on the light dose and expression of CSPG4 since no enhanced cytotoxicity of PCI of 225.28-saporin compared to PCI of saporin was observed in the CSPG4-negative MCF-7 cells. The PCI of a smaller, and clinically relevant CSPG4-targeting toxin (scFvMEL-rGel) validated the CSPG4-targeting concept in vitro and induced a strong inhibition of tumor growth in the amelanotic melanoma xenograft A-375 model. In conclusion, the combination of the drug delivery technology PCI and CSPG4-targeting immunotoxins is an efficient, specific and light-controlled strategy for the elimination of aggressive cells of TNBC and malignant melanoma origin. This study lays the foundation for further preclinical evaluation of PCI in combination with CSPG4-targeting.

HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR.

The members of the epidermal growth factor receptor (EGFR) kinase family are important players in breast morphogenesis and cancer. EGFR2/HER2 and EGFR expression have a prognostic value in certain subtypes of breast cancer such as HER2-amplified, basal-like and luminal type B. Many clinically approved small molecular inhibitors and monoclonal antibodies have been designed to target HER2, EGFR or both. There is, however, still limited knowledge on how the two receptors are expressed in normal breast epithelium, what effects they have on cellular differentiation and how they participate in neoplastic transformation. D492 is a breast epithelial cell line with stem cell properties that can undergo epithelial to mesenchyme transition (EMT), generate luminal- and myoepithelial cells and form complex branching structures in three-dimensional (3D) culture. Here, we show that overexpression of HER2 in D492 (D492(HER2)) resulted in EMT, loss of contact growth inhibition and increased oncogenic potential in vivo. HER2 overexpression, furthermore, inhibited endogenous EGFR expression. Re-introducing EGFR in D492(HER2) (D492(HER2/EGFR)) partially reversed the mesenchymal state of the cells, as an epithelial phenotype reappeared both in 3D cultures and in vivo. The D492(HER2/EGFR) xenografts grow slower than the D492(HER2) tumors, while overexpression of EGFR alone (D492(EGFR)) was not oncogenic in vivo. Consistent with the EGFR-mediated epithelial phenotype, overexpression of EGFR drove the cells toward a myoepithelial phenotype in 3D culture. The effect of two clinically approved anti-HER2 and EGFR therapies, trastuzumab and cetuximab, was tested alone and in combination on D492(HER2) xenografts. While trastuzumab had a growth inhibitory effect compared with untreated control, the effect of cetuximab was limited. When administered in combination, the growth inhibitory effect of trastuzumab was less pronounced. Collectively, our data indicate that in HER2-overexpressing D492 cells, EGFR can behave as a tumor suppressor, by pushing the cells towards epithelial differentiation.

Clinical significance of disseminated tumour cells in non-small cell lung cancer.

BACKGROUND: Early-stage non-small cell lung cancer (NSCLC) patients have a high risk of disease relapse despite curatively intended surgical resection, and the detection of tumour cells in the bone marrow could be one method of determining the presence of the disseminated disease in its early stages. METHODS: Bone marrow aspirates were collected from 296 patients at the time of surgery, and the presence of disseminated tumour cells was determined with the help of immunomagnetic selection (IMS) using the MOC31-antibody recognising EpCAM and with the help of standard immunocytochemistry (ICC) using the anti-cytokeratin (CK) antibodies AE1/AE3. RESULTS: Disseminated tumour cells were found in 152 of 252 (59%) bone marrow samples using IMS and in 25 of 234 (11%) samples using ICC. No association between the two detection methods was observed. The presence of EpCAM⁺ cells was not associated with any clinicopathological parameters, whereas a higher frequency of CK⁺ cells was found in patients with an advanced pT status. Disseminated tumour cells, as detected using IMS, had no prognostic impact. Patients with CK⁺ cells in the bone marrow had a reduced relapse-free survival, but the difference was not statistically significant. CONCLUSION: Our findings do not support the further development of DTC detection for clinical use in early-stage NSCLC. Future studies should include the molecular characterisation of DTCs, along with an attempt to identify subpopulations of cells with biological and clinical significance.

EMMPRIN is associated with S100A4 and predicts patient outcome in colorectal cancer.

BACKGROUND: Proteolytic enzymes and their regulators have important biological roles in colorectal cancer by stimulating invasion and metastasis, which makes these factors attractive as potential prognostic biomarkers. METHODS: The expression of extracellular matrix metalloproteinase inducer (EMMPRIN) was characterised using immunohistochemistry in primary tumours from a cohort of 277 prospectively recruited colorectal cancer patients, and associations with expression of S100A4, clinicopathological parameters and patient outcome were investigated. RESULTS: One hundred and ninety-eight samples (72%) displayed positive membrane staining of the tumour cells, whereas 10 cases (4%) were borderline positive. EMMPRIN expression was associated with shorter metastasis-free, disease-specific and overall survival in both univariate and multivariate analyses. The prognostic impact was largely confined to TNM stage III, and EMMPRIN-negative stage III patients had an excellent prognosis. Furthermore, EMMPRIN was significantly associated with expression of S100A4, and the combined expression of these biomarkers conferred an even poorer prognosis. However, there was no evidence of direct regulation between the two proteins in the colorectal cancer cell lines HCT116 and SW620 in siRNA knockdown experiments. CONCLUSION: EMMPRIN is a promising prognostic biomarker in colorectal cancer, and our findings suggest that it could be used in the selection of stage III patients for adjuvant therapy.

Microarray analysis of phosphatase gene expression in human melanoma.

BACKGROUND: Tyrosine phosphate is abnormally elevated in malignant melanoma, and this has been interpreted to reflect the activity of oncogenic protein tyrosine kinases. However, elevation may also arise due to decreased protein tyrosine phosphatase (PTP) expression. OBJECTIVES: To survey phosphatase gene expression in melanoma cell lines, a benign naevus and normal melanocytes: we searched for downregulation of phosphatase gene expression in malignant cells that may indicate a role as melanoma suppressor genes. METHODS: Microarray analysis was used to compare gene expression for 133 phosphatase genes, comprising 39 PTPs, 16 dual-specificity phosphatases (DSPs), 47 serine/threonine phosphatases and 31 acid/alkaline and lipid-based phosphatases. Northern blotting analysis was used to study gene expression in human melanoma biopsies. RESULTS: There was decreased expression of four DSP genes (including PTEN); eight receptor PTP genes were downregulated in melanoma, among which were PTP-KAPPA and PTP-PI (consistent with our previous data). In addition, PTP-RF/LAR was downregulated in 13 of 22 metastatic melanomas. CONCLUSIONS: The expression of multiple PTP receptors is decreased in melanoma; this may be a mechanism which stimulates autonomous growth in advanced melanoma.

Transgene expression is increased by photochemically mediated transduction of polycation-complexed adenoviruses.

Poor efficiency of adenoviral gene transfer to target cells is a major limitation to adenoviral gene therapy. Inefficient gene transfer occurs in the absence of coxsackie- and adenovirus receptor (CAR) on the cell surface, and can be overcome by enhancing viral entry with cationic molecules. Recombinant adenovirus (Ad) noncovalently complexed with polycations imply a lack of transduction specificity. Therefore, we have investigated the potential of a novel light-specific treatment, named photochemical internalization (PCI), to enhance gene delivery of adenovirus serotype 5 (Ad5) complexed with the cationic agents poly-L-lysine (PLL) and SuperFect trade mark. Cell lines differing in their receptiveness to Ad5 were infected with amounts of virus transducing about 2% of the cells by conventional Ad infection. The combination of polycations and photochemical treatment enabled a substantial increase in reporter gene expression, resulting in up to 75% positive cells. The effect was most prominent in cell lines expressing moderate to low levels of CAR. Furthermore, we show that PCI enables proper gene delivery of fiberless Ad5 at viral concentrations and infection times where transduction of photochemically untreated cells was negligible, both in the absence and presence of PLL. Thus, we conclude that the photochemically induced transduction by adenoviral vectors complexed with polycations present an opportunity to obtain high cell-infectivity levels with low viral doses, also without the fiber-CAR interaction.

Interferon-gamma suppresses S100A4 transcription independently of apoptosis or cell cycle arrest.

The S100A4 protein has been associated with increased metastatic capacity of cancer cells, and recent studies have suggested a correlation between the expression level of S100A4 and the prognostic outcome for patients with various types of cancer. The knowledge about the mechanisms underlying the metastasis-promoting effects is still limited, and the aim of the present study was to elucidate signal transduction pathways involved in the regulation of S100A4. After treatment of human carcinoma cells with interferon-gamma (IFN-gamma), we observed downregulation of S100A4 both at mRNA and protein levels. The effect was not dependent on IFN-gamma-induced apoptosis or IFN-gamma-mediated cell cycle arrest. Moreover, IFN-gamma-mediated decrease in mRNA stability could not account for the observed decrease in S100A4 transcript level. Finally, microarray analysis suggests ISGF3G, ETV5, ZNF133 and CEBPG as possible candidate genes involved in IFN-gamma-mediated repression of S100A4.

Light directed gene transfer by photochemical internalisation.

Numerous gene therapy vectors, both viral and non-viral, are taken into the cell by endocytosis, and for efficient gene delivery the therapeutic genes carried by such vectors have to escape from endocytic vesicles so that the genes can further be translocated to the nucleus. Since endosomal escape is often an inefficient process, release of the transgene from endosomes represents one of the most important barriers for gene transfer by many such vectors. To improve endosomal escape we have developed a new technology, named photochemical internalisation (PCI). In this technology photochemical reactions are initiated by photosensitising compounds localised in endocytic vesicles, inducing rupture of these vesicles upon light exposure. The technology constitutes an efficient light-inducible gene transfer method in vitro, where light-induced increases in transfection or viral transduction of more than 100 and 30 times can be observed, respectively. The method can potentially be developed into a site-specific method for gene delivery in vivo. This article will review the background for the PCI technology, and several aspects of PCI induced gene delivery with synthetic and viral vectors will be discussed. Among these are: (i) The efficiency of the technology with different gene therapy vectors; (ii) use of PCI with targeted vectors; (iii) the timing of DNA delivery relative to the photochemical treatment. The prospects of using the technology for site-specific gene delivery in vivo will be thoroughly discussed, with special emphasis on the possibilities for clinical use. In this context our in vivo experience with the PCI technology as well as the clinical experience with photodynamic therapy will be treated, as this is highly relevant for the clinical use of PCI-mediated gene delivery. The use of photochemical treatments as a tool for understanding the more general mechanisms of transfection will also be discussed.

Expression of S100A4, E-cadherin, alpha- and beta-catenin in breast cancer biopsies.

In 66 breast cancer biopsies, the expression of the Ca(2+)-binding protein S100A4, E-cadherin, alpha- and beta-catenin was examined by immunohistochemistry, and the results were related to clinical and pathological parameters. High levels of S100A4 were found to significantly correlate with histological grade (P=0.030) and loss of oestrogen receptor (P=0.046), but not to the time interval between surgery and development of distant metastasis (P=0.51) or to patient survival (P=0.89). Loss of E-cadherin expression, associated with altered cell-cell adhesion, showed a highly significant association to overall survival (P=0.020) and metastasis-free period (P=0.0052). In multivariate analysis, only lymph node involvement was a more significant predictor of patient demise. No association was found between expression of S100A4 and any single member of the cadherin-catenin complex, but a trend (P=0.053) towards reduced expression of one or several of these proteins and S100A4 immunoreactivity was observed. In conclusion, although our results suggest an association between S100A4 expression and an aggressive tumour phenotype, no relationship to overall survival was found. Deregulation of E-cadherin expression, however, was of high prognostic significance.

Cyclin A expression in superficial spreading malignant melanomas correlates with clinical outcome.

The present study analysed by immunohistochemistry the protein level of cyclin A and Ki-67 in a panel of paraffin-embedded tissue obtained from 172 primary (110 superficial and 62 nodular) and 73 metastatic melanomas, and ten benign naevi. Since cyclin A exists in the same quaternary complex in the S-phase of the cell cycle as the cdk inhibitor p21WAF1/CIP1, the levels of the two proteins were compared. Cyclin A and Ki-67 were heterogeneously expressed in the malignant tumours, whereas in benign naevi, only rare positive cells were detected. In superficial spreading melanomas, the cyclin A level was related to tumour thickness, with less expression in thinner lesions (p<0.00001), and to Ki-67 (p<0.00001) and p21WAF1/CIP1 (p=0.01) scores. Multivariate analysis showed that in addition to the depth of the primary tumour, the protein level of cyclin A was an independent indicator of relapse-free period (thickness, p<0.00001; cyclin A, p=0.0003). In contrast, in nodular melanoma, the cyclin A level was associated with Ki-67 expression, but neither cyclin A nor Ki-67 was related to tumour thickness (cyclin A, p=0.06; Ki-67, p=0.61) and neither had any impact on relapse-free (cyclin A, p=0.64; Ki-67, p=0.32) or overall (cyclinA, p=0.94; Ki-67, p=0.45) survival. In conclusion, the results indicate that cyclin A is a strong prognostic factor for patients with superficial spreading melanomas. In nodular melanomas, the proliferation rate seems to have little impact on disease progression.

Protein tyrosine phosphatase genes downregulated in melanoma.

Phospho-tyrosine levels are increased in melanoma, apparently consistent with reports of elevated protein tyrosine kinase activity. Some protein tyrosine kinases are encoded by oncogenes and have been implicated in melanoma genesis. Decreased protein tyrosine phosphatase activity may also increase phospho-tyrosine. Protein tyrosine phosphatase genes are candidate tumor suppressors and loss of expression may contribute to melanoma genesis. Here we survey protein tyrosine phosphatase expression in pigment cells. Protein tyrosine phosphatase genes were cloned by reverse transcriptase polymerase chain reaction using degenerate primers based upon conserved sequences within the phosphatase catalytic domain. Reaction products were cloned and sequenced: 118 and 113 partial protein tyrosine phosphatase products were isolated from normal melanocytes and melanoma cells, respectively. Northern blotting analysis was used to study expression of 15 protein tyrosine phosphatase genes. Expression of PTP-kappa and PTP-pi was absent or downregulated in more than 20% of melanoma cell lines and in some unmanipulated melanoma biopsies. These closely related enzymes are members of the 2B receptor protein tyrosine phosphatase family previously implicated in contact inhibition. Loss of protein tyrosine phosphatase expression may contribute to the abnormal tyrosine phosphorylation seen in melanoma; these genes are candidate tumor suppressors.

Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas-a possible mechanism for altering the nm23-H1 activity.

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

Expression of matrix metalloproteinases and the metastasis-associated gene S100A4 in human neuroblastoma and primitive neuroectodermal tumor cells.

BACKGROUND: Matrix metalloproteinases (MMPs) and their endogenous inhibitors (tissue inhibitors of MMPs; TIMPs) have been shown to correlate with in vitro invasiveness and clinical outcome in several adult malignancies. The importance of MMP and TIMP expression in neuroblastoma (NB) and primitive neuroectodermal tumors (PNET) is incompletely understood. The aim of the current study was to relate in vitro invasion of NB and PNET cell lines with MMP and TIMP expression and evaluate the effect of a synthetic MMP inhibitor. Furthermore, S100A4 levels were determined because recent reports have suggested a possible association between MMPs, TIMPs, and the metastasis-associated gene S100A4. METHODS: Expression of MMPs, TIMPs, and S100A4 was evaluated at both mRNA and protein levels in 2 human NB and 2 PNET cell lines. In vitro invasion and effects of the synthetic MMP inhibitor Marimastat were assessed in the Transwell chamber assay. RESULTS: The most invasive cells expressed the highest levels of MMPs and S100A4. Marimastat reduced invasion by 30%. CONCLUSIONS: In vitro invasion correlated with MMP and S100A4 expression. The fact that Marimastat reduced in vitro invasion is encouraging for further studies on a possible therapeutic application for proteinase inhibitors.

Expression of a novel factor, com1, is regulated by 1,25-dihydroxyvitamin D3 in breast cancer cells.

Tumor cells and their surrounding microenvironment produce a variety of factors that promote tumor growth and metastasis. We recently identified a nuclear factor, termed com1, that is up-regulated in human breast carcinoma cells on formation of experimental metastatic tumors and is assumed to act as a growth-promoting factor in breast cancer. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is a potent inhibitor of growth in breast cancer both in vitro and in vivo. We compared the growth-regulatory mechanisms of nontumorigenic and estrogen-dependent MCF-7 cells with those of the tumorigenic and tamoxifen-resistant subline MCF7/ LCC2 in the presence of 1,25(OH)2D3. Proliferation of MCF7/LCC2 cells, which revealed constitutive com1 expression, was inhibited by 1,25(OH)2D3 (10(-7) M). This was strongly associated with cell cycle arrest in G1 phase, consistent with accumulation of the hypophosphorylated form of the retinoblastoma protein as well as the induction of the cyclin-dependent kinase inhibitor p21. These cell cycle events were preceded by a transient up-regulation (5-8-fold) of com1 mRNA. Furthermore, clonal growth of the MCF7/LCC2 cells was also inhibited by 1,25(OH)2D3 (10(-7) M), and when the com1-negative MCF-7 cells were stably transfected with com1, the resulting MCF7/com1 cells showed a significant decrease in colony formation. These results seem to indicate that rather than promoting growth, com1 may participate in the regulatory pathway involved in cellular growth inhibition when recruited by inhibitory signals.

Levels of cyclin D1 and D3 in malignant melanoma: deregulated cyclin D3 expression is associated with poor clinical outcome in superficial melanoma.

We examined 172 primary (110 superficial and 62 nodular) and 73 metastatic melanomas, as well as 10 benign nevi, for protein expression of cyclin D1 and cyclin D3 and evaluated the relationship between deregulated protein levels and clinical outcome. For both proteins, a heterogeneous nuclear staining pattern was observed. Cyclin D3 was expressed by 96% of primary and 97% of metastatic melanomas. The corresponding percentages for cyclin D1 were 62% and 29%, respectively. In benign nevi, only rare cyclin D3-positive cells and no cyclin D1-positive cells were observed. High levels of cyclin D3 (>5% of the cells stained) were detected in 26 of 62 (42%) nodular melanomas and in 22 of 110 (20%) superficial tumors, whereas no such difference was observed with respect to cyclin D1. In superficial melanomas, a significant concordant staining pattern was observed between cyclin D1 and cyclin D3 (P = 0.0009), cyclin D1 and Ki-67 (P = 0.0001), cyclin D1 and cyclin A (P = 0.02), cyclin D3 and Ki-67 (P < 0.00001), and cyclin D3 and cyclin A (P = 0.002). Kaplan-Meier analysis revealed that high levels of cyclin D3 were an indicator of early relapse and decreased overall survival for patients with superficial (P = 0.001 and P = 0.009, respectively) but not nodular (P = 0.64 and P = 0.23) melanoma. Cyclin D1 did not have any impact on disease-free and overall survival for either of the subtypes. In conclusion, our results suggest that deregulation of cyclin D3 expression leading to increased proliferation may be a prognostic factor for superficial melanoma, whereas deregulated cell cycle machinery seems to have little impact on disease progression of nodular melanoma.

Identification of nucleolar protein No55 as a tumour-associated autoantigen in patients with prostate cancer.

Four different genes were identified by immunoscreening of a cDNA expression library from the human prostate cancer cell line DU145 with allogeneic sera from four prostate cancer patients. A cDNA encoding the nucleolar protein No55 was further analysed and shown to be expressed at the mRNA level in several normal tissues, including ovaries, pancreas and prostate and in human prostate cancer cell lines PC-3, PC-3m and LNCaP. By reverse transcriptase/polymerase chain reaction, expression of No55 was several-fold higher in two out of nine prostate cancer primary tumours and two out of two metastatic lesions, compared to normal prostate tissue. Antibodies to No55 were detected in sera from seven out of 47 prostate cancer patients but not in sera from 20 healthy male controls. Sequence analysis of the No55 open reading frame from normal and tumour tissues revealed no tumour-specific mutations. The No55 gene was located to chromosome 17q21, a region reported to be partially deleted in prostate cancer. Considering the immunogenicity of the No55 protein in the tumour host, the expression profile and chromosomal localization of the corresponding gene, studies evaluating No55 as a potential antigen for immunological studies in prostate cancer may be warranted.

S100A4 involvement in metastasis: deregulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in osteosarcoma cells transfected with an anti-S100A4 ribozyme.

The biological function of the metastasis-associated gene S100A4 is not fully understood, although there is evidence indicating interactions between the gene product and the cytoskeleton. We have examined whether an association could exist between S100A4 and the regulation of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). For these studies, three clones of a highly metastatic human osteosarcoma cell line (OHS) transfected with a hammerhead ribozyme directed against the S100A4 gene transcript were used. The clones demonstrated different expression levels of S100A4 and also different metastatic capacity. In the clone with the most prominent down-regulation of S100A4, the mRNA levels of MMP2, membrane type (MT) 1-MMP, and TIMP-1 were significantly reduced in exponentially growing cultures. Western blots, gelatin zymography, and ELISA showed similar expression patterns of MMPs and TIMPs at the protein level. In the clones with an intermediate expression of S100A4, reduced expression of MT1-MMP and TIMP-1 was detected, whereas the expression of MMP-2 was at the same level as in the control cells. In contrast to the other factors, TIMP-2 was up-regulated in all of the clones independent of the extent of ribozyme-induced down-regulation of S100A4. The transwell chamber assay demonstrated that the capacity of the ribozyme-transfected cells to cross uncoated filters was reduced, relative to control cells, according to the reduction in the S100A4 expression level. The clone with the lowest reduction in S100A4 did not demonstrate different motility compared with control cells, whereas transfectants with only 5% S100A4 mRNA showed a 50% reduction in motility. Interestingly, this trend was even more striking when the capacity to cross Matrigel-coated filters was analyzed, as all the clones demonstrated between 40 and 75% reduced invasion. It is concluded that S100A4 may exert its effect on metastasis formation not only by stimulating the motility of tumor cells but also by affecting their invasive properties through influencing the expression of MMPs and their endogenous inhibitors.

Interleukin-1 alpha and basic fibroblast growth factor induction of matrix metalloproteinases and their inhibitors in osteosarcoma cells is modulated by the metastasis associated protein CAPL.

BACKGROUND: Several recent investigations have shown that the expression of the CAPL protein seems to be of importance in the metastatic potential in some types of cancer. However, the mechanisms behind this and other biological functions of CAPL are still largely unknown. The aim of the present work was to investigate whether CAPL could affect the expression of candidate proteolytic facilitators of the metastatic process, i.e. matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). MATERIALS AND METHODS: A highly metastatic osteosarcoma cell-line with a high expression of CAPL was transfected with either a vector containing a ribozyme against this transcript, or with the vector alone as a control. The expression of MMPs and TIMPs was investigated with ELISA and gelatin zymography. RESULTS: The cell-line with a low CAPL expression (III-14) responded to bFGF treatment by an increased synthesis of MMP-1 and MMP-9 and to Il-1 alpha treatment by an increased synthesis of MMP-9. In contrast, the cell-line with a high CAPL expression (pH beta-1) did not respond with an altered expression of these MMPs. Neither of these two cell-lines responded with an altered expression of MMP-2. bFGF treatment resulted in an increased expression of TIMP-1 in both cell-lines, while Il-1 alpha treatment resulted in a decreased production of TIMP-1 in pH beta-1 cells, and III-14 cells were unaffected. CONCLUSIONS: The CAPL protein expressed in cell-cultures appear to block the MMP induction by bFGF and Il-1 alpha. However, the induction of TIMP-1 by bFGF must proceed through a pathway different from the MMP induced pathway, i.e. a pathway unaffected by CAPL. In addition, CAPL appeared to act in synergy with Il-1 alpha to reduce the synthesis of TIMP-1.

Hybridization of a 99Tcm-labelled oligodeoxynucleotide to CAPL RNA.

Oligodeoxynucleotides (ODNs) labelled with an appropriate radionuclide could provide a means to identify serious diseases early on and thereby help initiate treatment at a very early phase. Regardless of important issues like in-vivo stability and membrane passage, the key issue for the oligonucleotide approach is the ability of the radiolabelled ODN to hybridize to the target mRNA. The secondary structure of mRNA does not permit all complementary ODNs to hybridize and a careful selection of the probe with consecutive testing is therefore necessary. This study was initiated to demonstrate hybridization of a 99Tcm-labelled 20-mer ODN to RNA of CAPL (S100A4), a gene reported to be overexpressed in metastatic cancers like breast carcinoma and osteosarcoma. The phosphodiester ODN GX-1 (antisense) and two control sequences (scrambled and random) were conjugated to the bifunctional chelating agent S-benzoyl-mercaptoacetyltriglycine (S-benzoyl-MAG3) and labelled with 99Tcm. The radiolabelled ODNs were purified on a C18 mini-column and characterized on a reverse-phase HPLC system. The radio-chemical purity was > 90% and the product was stable for > 6 h in aqueous medium. The hydrization properties of unlabelled, 32P-labelled and 99Tcm-labelled ODNs to transcribed RNA were studied using polyacrylamide gel electrophoresis (PAGE). Direct hybridization of GX-1 to transcribed RNA was demonstrated. A 50-fold excess of unlabelled ODN over transcribed RNA caused a near to complete consumption of RNA by RNase H activation. In 1:1 proportions of radiolabelled (32P and 99Tcm) ODNs to RNA, only radiolabelled GX-1 was found to hybridize to RNA in a PAGE system. The radiolabelled control ODNs did not show signs of hybridization. This study demonstrates that 3'-99Tcm-labelling of ODNs does not interfere with the hybridization properties of the ODNs in solution, making 99Tcm-labelling an attractive procedure for the future development of antisense technology in imaging.