Christiane Margue

Integrin alpha(v)beta3 expression confers on tumor cells a greater propensity to metastasize to bone.

The reasons why tumor cells metastasize to bone remain obscure. There is some evidence to support the theory that integrins (acting as cell surface adhesion receptors) play a role in mediating metastasis in certain organs. Here, we report that overexpression of a functionally active integrin αvβ3 in Chinese hamster ovary (CHO) tumor cells drastically increased the incidence, number, and area of bone metastases in nude mice compared with those observed in mock‐transfected CHO cells (CHO dhfr+) or in CHO cells expressing a functionally inactive integrin αvβ3 (CHO β3∆744). Moreover, a breast cancer cell line (B02) established from bone metastases caused by MDA‐MB‐231 cells constitutively overexpressed integrin αvβ3, whereas the cell surface expression level of other integrins remained unchanged. In vivo, the extent of bone metastases in B02‐bearing mice was significantly increased compared with that of MDAMB‐231‐bearing mice. In vitro, B02 cells and CHO cells expressing a functionally active integrin αvβ3 exhibited substantially increased invasion of and adhesion to mineralized bone, bone sialoprotein, and collagen compared with those found with MDA‐MB‐231, CHO dhfr+, and CHO β3∆744 cells, respectively. Overall, our findings suggest that integrin αvβ3 expression in tumor cells accelerates the development of osteolytic lesions, presumably through increased invasion of and adhesion to bone.

Signatures of MicroRNAs and Selected MicroRNA Target Genes in Human Melanoma

Small noncoding microRNAs (miRNA) regulate the expression of target mRNAs by repressing their translation or orchestrating their sequence-specific degradation. In this study, we investigated miRNA and miRNA target gene expression patterns in melanoma to identify candidate biomarkers for early and progressive disease. Because data presently available on miRNA expression in melanoma are inconsistent thus far, we applied several different miRNA detection and profiling techniques on a panel of 10 cell lines and 20 patient samples representing nevi and primary or metastatic melanoma. Expression of selected miRNAs was inconsistent when comparing cell line-derived and patient-derived data. Moreover, as expected, some discrepancies were also detected when miRNA microarray data were correlated with qPCR-measured expression levels. Nevertheless, we identified miRNA-200c to be consistently downregulated in melanocytes, melanoma cell lines, and patient samples, whereas miRNA-205 and miRNA-23b were markedly reduced only in patient samples. In contrast, miR-146a and miR-155 were upregulated in all analyzed patients but none of the cell lines. Whole-genome microarrays were performed for analysis of selected melanoma cell lines to identify potential transcriptionally regulated miRNA target genes. Using Ingenuity pathway analysis, we identified a deregulated gene network centered around microphthalmia-associated transcription factor, a transcription factor known to play a key role in melanoma development. Our findings define miRNAs and miRNA target genes that offer candidate biomarkers in human melanoma.

Complete loss of PTEN expression as a possible early prognostic marker for prostate cancer metastasis

The EGF/IGF growth factors are potent mitogens that regulate cell proliferation and cell survival and are involved in prostate cancer development. Using laser microdissection technology and real‐time PCR, together with immunohistochemistry, we have explored the growth factor and integrin dependent PI3‐kinase/PTEN/Akt signalling pathway in prostate cell lines and tumour samples by analysing EGF‐R, IGF1‐R, ILK, β3 integrin, PTEN and p‐Akt protein expression. We provide evidence that loss of PTEN expression rather than upregulated EGF/IGF1 receptor expression was responsible for increased p‐Akt in neoplastic prostate cells. We therefore compared PTEN expression in patient biopsies at first time diagnosis recruited prospectively (Study I, 112 patients) and patients with confirmed metastasis recruited retrospectively from the Luxembourg cancer registry (Study II, 42 patients). In Study I, loss of PTEN expression at first time diagnosis was found in 26 of 112 patients (23%). In Study II, 25 of the 42 patients (59%) with lymph node metastasis had complete loss of PTEN expression in both the neoplastic glands of the prostate and the invasive prostate cancer cells in the lymph node, and of these 13 (52%) exhibited already loss of PTEN expression at first diagnosis. These findings demonstrate that loss of PTEN expression is an important factor in progression towards metastatic disease and could potentially serve as an early prognostic marker for prostate cancer metastasis.

Transcriptional modulation of the anti-apoptotic protein BCL-XL by the paired box transcription factors PAX3 and PAX3/FKHR

The aberrant expression of the transcription factors PAX3 and PAX3/FKHR associated with rhabdomyosarcoma (RMS), solid tumors displaying muscle cell features, suggests that these proteins play an important role in the pathogenesis of RMS. We could previously demonstrate that one of the oncogenic functions of PAX3 and PAX3/FKHR in RMS is protection from apoptosis. BCL-XL is a prominent anti-apoptotic protein present in normal skeletal muscle and RMS cells. In the present study, we establish that BCL-XL is transcriptionally modulated by PAX3 and PAX3/FKHR, since enhanced expression of both PAX proteins stimulates transcription of endogenous BCL-XL mRNA in a cell type specific manner. Further, we present evidence that both PAX3 and PAX3/FKHR can transcriptionally activate the Bcl-x gene promoter in cotransfection assays. Using electrophoretic mobility shift assays, an ATTA binding site for PAX3 and PAX3/FKHR could be localized in the upstream promoter region (position −42 to −39). Finally, ectopic overexpression of either PAX3, PAX3/FKHR or BCL-XL can rescue tumor cells from apoptosis induced by antisense treatment. These results suggest that at least part of the anti-apoptotic effect of PAX3 and PAX3/FKHR is mediated through direct transcriptional modulation of the prominent anti-apoptotic protein BCL-XL.

MiRNA-29: a microRNA family with tumor-suppressing and immune-modulating properties.

MicroRNAs (miRNAs) are ubiquitously expressed small, non-coding RNAs that negatively regulate gene expression at a post-transcriptional level. So far, over 1000 miRNAs have been identified in human cells and their diverse functions in normal cell homeostasis and many different diseases have been thoroughly investigated during the past decade. MiR-29, one of the most interesting miRNA families in humans to date, consists of three mature members miR-29a, miR-29b and miR-29c, which are encoded in two genetic clusters. Members of this family have been shown to be silenced or down-regulated in many different types of cancer and have subsequently been attributed predominantly tumor-suppressing properties, albeit exceptions have been described where miR-29s have tumor-promoting functions. MiR-29 targets expression of diverse proteins like collagens, transcription factors, methyltransferases and others, which may partake in abnormal migration, invasion or proliferation of cells and may favor development of cancer. Furthermore, members of the miR-29 family can be activated by interferon signaling, which suggests a role in the immune system and in host pathogen interactions, especially in response to viral infections. In this review, we summarize current knowledge on the genomic organization and regulation of the miR-29 family and we provide an overview of its implication in cancer suppression and promotion as well as in host immune responses. The numerous remarkable properties of these miRNAs and their often altered expression patterns might make the miR-29 family promising biomarkers and therapeutic targets for various diseases in future.

Interferon-γ-induced activation of Signal Transducer and Activator of Transcription 1 (STAT1) up-regulates the tumor suppressing microRNA-29 family in melanoma cells

BackgroundThe type-II-cytokine IFN-γ is a pivotal player in innate immune responses but also assumes functions in controlling tumor cell growth by orchestrating cellular responses against neoplastic cells. The role of IFN-γ in melanoma is not fully understood: it is a well-known growth inhibitor of melanoma cells in vitro. On the other hand, IFN-γ may also facilitate melanoma progression. While interferon-regulated genes encoding proteins have been intensively studied since decades, the contribution of miRNAs to effects mediated by interferons is an emerging area of research.We recently described a distinct and dynamic regulation of a whole panel of microRNAs (miRNAs) after IFN-γ-stimulation. The aim of this study was to analyze the transcriptional regulation of miR-29 family members in detail, identify potential interesting target genes and thus further elucidate a potential signaling pathway IFN-γ → Jak→ P-STAT1 → miR-29 → miR-29 target genes and its implication for melanoma growth.ResultsHere we show that IFN-γ induces STAT1-dependently a profound up-regulation of the miR-29 primary cluster pri-29a~b-1 in melanoma cell lines. Furthermore, expression levels of pri-29a~b-1 and mature miR-29a and miR-29b were elevated while the pri-29b-2~c cluster was almost undetectable. We observed an inverse correlation between miR-29a/b expression and the proliferation rate of various melanoma cell lines. This finding could be corroborated in cells transfected with either miR-29 mimics or inhibitors. The IFN-γ-induced G1-arrest of melanoma cells involves down-regulation of CDK6, which we proved to be a direct target of miR-29 in these cells. Compared to nevi and normal skin, and metastatic melanoma samples, miR-29a and miR-29b levels were found strikingly elevated in certain patient samples derived from primary melanoma.ConclusionsOur findings reveal that the miR-29a/b1 cluster is to be included in the group of IFN- and STAT-regulated genes. The up-regulated miR-29 family members may act as effectors of cytokine signalling in melanoma and other cancer cells as well as in the immune system.

Dual Role of the Jak1 FERM and Kinase Domains in Cytokine Receptor Binding and in Stimulation-Dependent Jak Activation

Jak1 is a tyrosine kinase that noncovalently forms tight complexes with a variety of cytokine receptors and is critically involved in signal transduction via cytokines. Jaks are predicted to have a 4.1, ezrin, radixin, moesin (FERM) domain at their N terminus. FERM domains are composed of three structurally unrelated subdomains (F1, F2, and F3) which are in close contact to one another and form the clover-shaped FERM domain. We generated a model structure of the Jak1 FERM domain, based on solved FERM structures and the alignments with other FERM domains. To destabilize different subdomains and to uncover their exact function, we mutated specific hydrophobic residues conserved in FERM domains and involved in hydrophobic core interactions. In this study, we show that the structural integrity of the F2 subdomain of the FERM domain of Jak1 is necessary to bind the IFN-γRα. By mutagenesis of hydrophobic residues in the hydrophobic core between the three FERM subdomains, we find that the structural context of the FERM domain is necessary for the inhibition of Jak1 phosphorylation. Thus, FERM domain mutations can have repercussions on Jak1 function. Interestingly, a mutation in the kinase domain (Jak1-K907E), known to abolish the catalytic activity, also leads to an impaired binding to the IFN-γRα when this mutant is expressed at endogenous levels in U4C cells. Our data show that the structural integrity of both the FERM domain and of the kinase domain is essential for both receptor binding and catalytic function/autoinhibition.

New Target Genes of MITF-Induced microRNA-211 Contribute to Melanoma Cell Invasion

The non-coding microRNAs (miRNA) have tissue- and disease-specific expression patterns. They down-regulate target mRNAs, which likely impacts on most fundamental cellular processes. Differential expression patterns of miRNAs are currently being exploited for identification of biomarkers for early disease diagnosis, prediction of progression for melanoma and other cancers and as promising drug targets, since they can easily be inhibited or replaced in a given cellular context. Before successfully manipulating miRNAs in clinical settings, their precise expression levels, endogenous functions and thus their target genes have to be determined. MiR-211, a melanocyte lineage-specific small non-coding miRNA, is located in an intron of TRPM1, a target gene of the microphtalmia-associated transcription factor (MITF). By transcriptionally up-regulating TRPM1, MITF, which is critical for both melanocyte differentiation and survival and for melanoma progression, indirectly drives the expression of miR-211. Expression of this miRNA is often reduced in melanoma samples. Here, we investigated functional roles of miR-211 by identifying and studying new target genes. We show that MITF-correlated miR-211 expression levels are mostly but not always reduced in a panel of 11 melanoma cell lines and in primary and metastatic melanoma compared to normal melanocytes and nevi, respectively. MiR-211 itself only marginally impacted on cell invasion and migration, while perturbation of some new miR-211 target genes, such as AP1S2, SOX11, IGFBP5, and SERINC3 significantly increased invasion. These results and the variable expression levels of miR-211 raise serious doubts on the value of miR-211 as a melanoma tumor-suppressing miRNA and/or as a biomarker for melanoma.

Recombinant interleukin-24 lacks apoptosis-inducing properties in melanoma cells.

IL-24, also known as melanoma differentiation antigen 7 (mda-7), is a member of the IL-10 family of cytokines and is mainly produced by Th2 cells as well as by activated monocytes. Binding of IL-24 to either of its two possible heterodimeric receptors IL-20R1/IL-20R2 and IL-22R/IL-20R2 activates STAT3 and/or STAT1 in target tissues such as lung, testis, ovary, keratinocytes and skin. To date, the physiological properties of IL-24 are still not well understood but available data suggest that IL-24 affects epidermal functions by increasing proliferation of dermal cells. In stark contrast to its “normal” and physiological behaviour, IL-24 has been reported to selectively and efficiently kill a vast variety of cancer cells, especially melanoma cells, independent of receptor expression and Jak-STAT signalling. These intriguing properties have led to the development of adenovirally-expressed IL-24, which is currently being evaluated in clinical trials. Using three different methods, we have analysed a large panel of melanoma cell lines with respect to IL-24 and IL-24 receptor expression and found that none of the investigated cell lines expressed sufficient amounts of functional receptor pairs and therefore did not react to IL-24 stimulation with Jak/STAT activation. Results for three cell lines contrasted with previous studies, which reported presence of IL-24 receptors and activation of STAT3 following IL-24 stimulation. Furthermore, evaluating four different sources and modes of IL-24 administration (commercial recombinant IL-24, bacterially expressed GST-IL-24 fusion protein, IL-24 produced from transfected Hek cells, transiently over-expressed IL-24) no induction or increase in cell death was detected when compared to appropriate control treatments. Thus, we conclude that the cytokine IL-24 itself has no cancer-specific apoptosis-inducing properties in melanoma cells.

IL-24: a classic cytokine and/or a potential cure for cancer?

IL‐24, a member of the IL‐10 family of cytokines, is produced by monocytes and Th2 cells. Interestingly, immune cells do not appear to express specific IL‐24 receptor chains (IL‐20R1/IL‐20R2 and IL‐22R/IL‐20R2), it is therefore unlikely that IL‐24 has classical immune‐modulating properties. Skin, on the other hand, seems to represent a major target tissue for IL‐24 and related cytokines such as IL‐19, ‐20, and ‐22. However, the initial interest in IL‐24 did not arise from its physiological signalling properties through its cognate receptors but rather because of its tentative ability to selectively kill different cancer cells. In an attempt to further investigate the signalling events underlying the IL‐24‐induced cancer cell death, we found that melanoma cell lines did not react in the expected and previously described way. Using several different forms and delivery modes of IL‐24, we were unable to detect any apoptosis‐inducing properties of this cytokine in melanoma cells. In the present ‘Point of view’ we will briefly summarizse these findings and put them in context of published reports stating that IL‐24 might be a long sought after treatment for several types of cancer.