Anja-Katrin Bosserhoff

The transcriptional coactivator FHL2 transmits Rho signals from the cell membrane into the nucleus

GTPases of the Rho family are transducers of extracellular signals and control cellular processes such as organization of the actin cytoskeleton, motility, adhesion and gene regulation. The Rho signalling pathway is activated, for example, by bioactive sphingolipids such as sphingosine‐1‐phosphate (SPP) or by overexpression of Rho family members in tumorigenesis and metastases. Here, we show that stimulation of the Rho signalling pathway induces translocation of the transcriptional LIM‐only coactivator FHL2 to the nucleus and subsequent activation of FHL2‐ and androgen receptor‐dependent genes. Interestingly, prostate tumours overexpress Rho GTPases and display altered cellular localization of FHL2 concomitant with tumour dedifferentiation. SPP‐induced FHL2 activation is mediated by Rho GTPases, but not by the GTPases Cdc42, Rac1 or Ras, and depends on Rho‐kinase. In addition, Rho signalling influences other transcriptional coactivators, thus pointing to a general regulatory role for Rho GTPases in cofactor function. In summary, our data propose a yet undescribed signalling pathway in which the coactivator FHL2 acts as a novel molecular transmitter of the Rho signalling pathway, thereby integrating extracellular cues into altered gene expression.

The Extracellular Human Melanoma Inhibitory Activity (Mia) Protein Adopts an SH3 Domain-Like Fold.

Melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma, as enhanced values diagnose metastatic melanoma stages III and IV. Here we show that the recombinant human MIA adopts an SH3 domain‐like fold in solution, with two perpendicular, antiparallel, three‐ and five‐stranded β‐sheets. In contrast to known structures with the SH3 domain fold, MIA is a single‐domain protein, and contains an additional antiparallel β‐sheet and two disulfide bonds. MIA is also the first extracellular protein found to have the SH3 domain‐like fold. Furthermore, we show that MIA interacts with fibronectin and that the peptide ligands identified for MIA exhibit a matching sequence to type III human fibronectin repeats, especially to FN14, which is close to an integrin α4β1 binding site. The present study, therefore, may explain the role of MIA in metastasis in vivo, and supports a model in which the binding of human MIA to type III repeats of fibronectin competes with integrin binding, thus detaching cells from the extracellular matrix.

Overexpression of melanoma inhibitory activity (MIA) enhances extravasation and metastasis of A-mel 3 melanoma cells in vivo

The secreted MIA protein is strongly expressed by advanced primary and metastatic melanomas but not in normal melanocytes. Previous studies have shown that MIA serum levels correlate with clinical tumour progression in melanoma patients. To provide direct evidence that MIA plays a role in metastasis of malignant melanomas, A-mel 3 hamster melanoma cells were transfected with sense- and antisense rhMIA cDNA and analysed subsequently for changes in their tumorigenic and metastatic potential. Enforced expression of MIA in A-mel 3 cells significantly increased their metastatic potential without affecting primary tumour growth, cell proliferation or apoptosis rate in hamsters, compared with control or antisense transfected cells. Additionally, MIA overexpressing transfectants showed a higher rate of both tumour cell invasion and extravasation. Cells transfected with MIA antisense generally exerted an opposite response. The above changes in function attributed to the expression of MIA may underlie the contribution of MIA to the malignant phenotype.

Comparison of Two Prognostic Markers for Malignant Melanoma: MIA and S100 β

It has recently been shown that the serum level of melanoma-inhibitory protein (MIA) provides useful information for the therapy and follow-up of patients with malignant melanoma. Previously, S100 β has been described as a useful tumor marker for malignant melanoma. In this study, we compare the significance of the two markers in follow-up, therapy outcome and prognosis by measuring MIA and S100 β serum levels in 50 melanoma patients. Serum levels were measured in patients with malignant melanomas of stages I–IV with at least 3 time points of measurement. Serial MIA and S100 β measurements were obtained from 32 patients with stage IV disease in parallel to chemotherapy and from 18 patients with a history of stage I and stage II disease during follow-up. The response to chemotherapy in stage IV disease and relapse of melanoma during follow-up correlated with changes in MIA and S100 β serum levels. In comparison, MIA revealed slightly higher specificity and sensitivity. In conclusion, both markers are useful for detection of progression from localized to metastatic disease during follow-up and for monitoring therapy of advanced melanomas.

Isolation of invasion-associated cDNAs in melanoma.

Abstract Metastasis and invasion are key steps in the systemic spread of tumor cells. To identify the genes involved in this process we recently selected highly invasive and weakly invasive cell clones from a melanoma cell line. Both cell clones showed a stable phenotype over more than 40 passages and previous analyses revealed a fivefold difference in their invasive potential in vitro and in tumorigenesis in vivo. To compare gene expression of the two cell clones a cDNA array system (Clontech human cancer cDNA array) was used. Exact quantification of differentially expressed genes in each cell clone was performed by real-time RT-PCR. An evaluation of the array data revealed a total of 36 genes that were more than 1.5-fold differentially expressed, and 26 (72%) of these showed a differential expression pattern by quantitative RT-PCR. Previously known differences in expression patterns, including loss of p16 and HLA I, or equal expression of p73, and RAR α, β and γ were confirmed by the array data. In addition, reduced expression levels of several cytoskeletal proteins, such as vimentin, γ-actin, desmin and cytokeratins, in the highly invasive cell clone were reproducibly identified. Other genes strongly upregulated in the highly invasive cell clone included jagged 2, STAT1, tPA and c-myc, whereas MDA-6 (p21), caspase 2 and semaphorin were found to be downregulated. In conclusion, comparative hybridization of cDNA arrays identified a series of novel invasion-associated changes in gene expression and confirmed previously known expression patterns.

Melanoma inhibitory activity (MIA), a serological marker of malignant melanoma.

Melanoma inhibitory activity (MIA) was originally identified as an 11 kDa protein secreted from malignant melanoma cells. We have shown that MIA is strongly expressed in melanoma and melanoma cell lines but not in melanocytes and normal skin. We also observed that MIA mRNA expression correlates with progressive malignancy of melanocytic tumors. Measuring MIA in serum or plasma by a sensitive and quantitative ELISA and investigating the potential of MIA serum levels as a novel marker for malignant melanomas showed that the protein can be used to monitor therapy and follow-up. The present study measured the variations in blood concentrations of MIA in 84 patients with stage II-IV melanoma by ELISA. Patients treated with repeated injections of a polyvalent melanoma vaccine (PMV), interferon-alpha-2b (IFN-alpha 2b) or interleukin-2 (IL-2) were followed during treatment duration. Before treatment, patients treated with PMV or IFN-alpha 2b had comparable low MIA concentrations, whereas most IL-2-treated patients had higher MIA levels. At the end of treatment, MIA concentrations were higher in patients with progressive disease (PD) than in patients with no clinical evidence of melanoma (NPD) for PMV, IFN-alpha 2b or IL-2 therapy (3.7 +/- 0.2 vs 11.5 +/- 5.4 ng/ml, 3.8 +/- 0.2 vs 8.3 +/- 1.7 ng/ml, and 2.3 +/- 0.7 vs 20.2 +/- 7.4 ng/ml, respectively, p < 0.05). In contrast to the stable MIA concentrations measured in NPD patients, significant increase in MIA levels were observed in PD patients over time regardless of treatment. For PMV- and IFN-alpha 2b-treated patients, a rise in MIA levels occurred significantly earlier than clinical diagnosis of melanoma recurrence. In conclusion, our data suggest that quantitation of MIA serum levels may be used for detection of both clinically apparent and non-apparent metastatic melanoma disease and for monitoring therapy.

Upregulation of the cytoskeletal-associated protein Moesin in the neointima of coronary arteries after balloon angioplasty: a new marker of smooth muscle cell migration?

Migrating cells like coronary smooth muscle cells in restenosis change their cell shape and form cellular protrusions called filopodia. A prerequisite for filopodia formation is the rearrangement of the actin cytoskeleton. An essential role of the 78-kDa protein Moesin is described for Rho- and Rac-dependent assembly of actin filaments. In vivo Moesin is not observed in mature smooth muscle cells. The objective of this study was to demonstrate that Moesin is upregulated in migrating coronary smooth muscle cells during restenosis development. In vivo expression of Moesin was upregulated in neointimal coronary smooth muscle cells of dilated porcine coronary arteries compared to the undilated left circumflex coronary artery of the same swine. Concordant to these results Moesin expression was upregulated in migrating and invading human arterial smooth muscle cells in vitro analyzed by FACS, Western blotting and RT-PCR. In addition, the invasive potential of Moesin-positive Mel Im cells transfected with Moesin sense DNA increased by 28% as compared to mock-transfected control, whereas antisense transfected cells had a decreased invasive potential of 32%. Transfection of Moesin-negative HepG2 with Moesin sense cDNA increased the invasive potential by 43%. Finally, transfection of human arterial smooth muscle cells with Moesin sense cDNA caused an increased invasive potential of 30%. Transfection of haSMCs with antisense cDNA decreased the invasive potential by 37% in comparison to mock-transfected control. These results demonstrate for the first time an upregulation of Moesin expression in coronary smooth muscle cells of the neointima after arterial injury. The increased migrative and invasive potential of cells transfected with Moesin confirmed the functional role of Moesin in cell migration. This indicates an important role of Moesin during restenosis development.

Loss of p14ARF expression in melanoma.

Abstract. Lack of p14ARF expression or its functional inactivation has been observed in human and murine carcinomas. Although very few mutations of p14ARF have been detected in some cancer types, changes in expression seem to play an important role in the development of other human cancers such as mesotheliomas. To examine the p14ARF gene and expression of p14ARF protein in melanomas, we screened eight human melanoma cell lines and primary human melanocytes by RT-PCR, sequencing and immunoblotting. All melanoma cell lines analyzed expressed wild-type p14ARF mRNA as well as protein. P14ARF expression was investigated by immunohistochemical staining of 32 tissue samples of benign melanocytic nevi (n=14), melanomas (n=12) and melanoma metastases (n=6). In contrast to the results obtained from cell lines in vitro the immunohistochemical stainings revealed a correlation between the progression of melanoma and the lack of the p14ARF protein expression. Positive p14ARF protein staining was observed in 11 of 14 benign nevi, in 3 of 12 melanomas and in 0 of 6 melanoma metastases. In summary, we demonstrated a significant inverse correlation between p14ARF protein expression and progression of melanocytic tumors since the amount of p14ARF protein staining decreased from benign melanocytic nevi to metastatic melanoma in situ. These results suggest that p14ARF inactivation is important in the development of melanomas.

[Melanoma inhibitory activity (MIA). Evaluation of a new tumor-associated antigen as a serum marker for uveal melanomas].

ZusammenfassungHintergrund. Für das Tumor- und Metastasenmonitoring des uvealen Melanoms steht bisher kein spezifischer, serologischer Tumormarker zur Verfügung. Das neue tumorassoziierte Antigen Melanoma inhibitory activity (MIA) wird im uvealen Melanom und seinen Metastasen exprimiert und ist immunhistochemisch nachweisbar. Patienten und Methode. Wir berichten über unsere serologischen Untersuchungen von MIA bei 38 Patienten mit uvealem Melanom. 4 Patienten wiesen bereits eine systemische Metastasierung auf. Zur Bestimmung der MIA-Serumspiegel wurde ein ELISA verwendet. Resultat. Bei den 34 Patienten ohne systemische Metastasierung betrug der Mittelwert (±1 Standardabweichung [SD]) der MIA-Serumkonzentration 3,6±1,0 ng/ml. Bei den 4 Patienten mit systemischer Metastasierung betrug der Mittelwert (±1 SD) 27,7±3,0 ng/ml. Die Differenz war statistisch hochsignifikant (/ldquo;student t test”: p=0,0001). Ergebnisse. MIA wird von uvealen Melanomen und deren Metastasen exprimiert. Patienten mit systemischer Metastasierung weisen signifikant erhöhte MIA-Serumspiegel auf, sodass MIA eine viel versprechende Rolle als Tumormarker bei der Überwachung und Nachsorge von Patienten mit uvealem Melanom spielen könnte.SummaryBackground. Currently no serological marker for the monitoring of uveal melanoma and its metastases is available. The novel tumorassociated antigen Melanoma inhibitory activity (MIA) is expressed in the uveal melanoma and its metastatic lesions. Method. We report about the serum samples of 38 patients with uveal melanomas. 4 of these patients had overt metastatic disease. A nonradioactive one step ELISA was used to quantify the MIA serum levels. Results. In the 34 patients without overt metastatic disease the serum concentration of MIA was mean (±1 SD) 3.6±1.0 ng/ml. In the 4 patients with overt metastatic disease the serum concentration of MIA was mean (±1 SD) 27.7±3.0 ng/ml. The difference was statistically highly significant (student t test: p=0.0001). Conclusion. MIA is expressed in primary and metastatic lesions of uveal melanomas. The elevation of MIA serum levels in patients with metastatic disease from melanomas suggests a promising role as a serum marker for monitoring patients with uveal melanoma.