(Dido1) Is a BMP Target Gene and Promotes BMP-Induced Melanoma Progression

ORIGINAL ARTICLE Death inducer-obliterator 1 (Dido1) is a BMP target gene and promotes BMP-induced melanoma progression

S Braig and A-K Bosserhoff

Bone morphogenetic proteins (BMPs) are known to play an important role in melanoma development and progression. However, the downstream targets of BMPs have not been investigated thus far. Therefore, we treated melanoma cell lines with the Smad-specific BMP inhibitor Dorsomorphin and performed a cDNA microarray. We identified death inducer-obliterator 1 (Dido1) as a BMP-specific Smad-regulated target gene, which was confirmed by qRT--PCR, immunofluorescence staining and electrophoretic mobility shift assay experiments. An analysis of Dido1 expression revealed an upregulation of Dido1 levels in melanoma cell lines and tissues compared with normal melanocytes. Colony-formation assays showed that siDido1-transfected cells formed significantly smaller colonies when grown in soft agar compared with control cells. In addition, fluorescence- activated cell sorting and western blot experiments revealed that transfection of melanoma cells with Dido1 small interfering RNAs led to an upregulation of apoptosis. Furthermore, cell migratory and invasive potentials were strongly reduced in siDido1- transfected cells compared with control cells. Finally, we demonstrated that Dido1 induces the expression of Integrin aV, thereby promoting the attachment, migration, invasion and apoptosis resistance of melanoma cells.

Oncogene (2013) 32, 837--848; doi:10.1038/onc.2012.115; published online 2 April 2012 Keywords: malignant melanoma; bone morphogenic protein; Dido1; integrin aV; migration; invasion

INTRODUCTION as proliferation, differentiation, motility and cell death, BMPs are 6,7 Malignant melanomas represent B4% of all cutaneous cancers, also linked to tumor formation and progression. but account for 480% of skin cancer deaths.1 Moreover, the Our group was able to show that BMPs play an important role incidence of melanoma continues to rise, with a 460% increase during the formation and progression of malignant melanoma. having occurred over the past 30 years. The expression level of several BMPs, especially BMP4 and BMP7, Several studies have shown that the bone morphogenetic is upregulated in malignant melanoma cells compared with protein (BMP) signaling cascade is deregulated in malignant normal melanocytes. Functional analysis revealed that melanoma melanoma. BMPs are members of the transforming growth factor-b cell lines with diminished BMP activity, achieved either by family. BMPs bind to a heteromeric receptor complex consisting of overexpression of the BMP inhibitor Chordin or by the specific the membrane-bound type I and type II serine/threonine kinases downregulation of BMP4, exhibited reduced capacities for 8 (BMPRI and BMPRII). Depending on the receptor complex, migration and invasion. Consequently, subcutaneous injection different signaling pathways can be activated. After binding to of these melanoma cell lines in nude mice resulted in significantly 9 the preformed hetero-oligomeric complex, BMPs initiate the reduced tumor growth. phosphorylation and activation of Smad1/5/8, which then In this study, we aimed to identify and characterize new heterodimerize with the Co-Smad Smad4. The complex translo- Smad-dependent BMP target genes that promote melanoma cates to the nucleus and activates the transcription of progression by enhancing the migration and invasion capacities of BMP-specific target genes.2 melanoma cells. Apart from this classical Smad-dependent signaling cascade, BMPs are also able to activate mitogen-activated protein kinase pathways. Here, BMPs bind to the type I BMP receptor, which is RESULTS followed by recruitment of the type II receptor into the complex. Dido1 is a specific, Smad-regulated BMP target gene in malignant The type I BMP receptor associates with TAK1, TAB1 and XIAP, melanoma resulting in an activation of p38/mitogen-activated protein kinase, Previous studies have shown that members of the BMP family c-Jun N-terminal kinase or extracellular signal-regulated kinase promote melanoma progression by enhancing the migration and (ERK) that subsequently results in the stimulation of non-Smad- invasion of melanoma cells;8 however, the downstream targets of responsive genes.2,3 BMPs that mediate these oncogenic processes are largely The BMP signaling cascade is tightly regulated at several levels. unidentified. It is known that BMPs signal via ERK-dependent For instance, extracellular antagonists like Chordin or Noggin bind pathways, which are frequently used by numerous other to BMPs and block their ability to bind to the receptor. Intracellular oncogenes, as well as through Smad1/5/8-dependent cascades inhibitors like Smad6 and Smad7 compete with Smad1/5/8 for the that are only activated by BMPs. To identify new BMP target activated type I receptor binding site.4 Dorsomorphin acts as a genes, we treated the melanoma cell lines Mel Im and Mel Ju with Smad-specific BMP inhibitor by blocking the kinase activity of 2 mM Dorsomorphin, which is known to specifically inhibit Smad- BMPRI.5 Apart from their role in numerous cellular processes, such dependent BMP signaling. A study by Boergermann et al.10

University of Regensburg Medical School, Institute of Pathology, Regensburg, Germany. Correspondence: Professor A-K Bosserhoff, Institute of Pathology, University of Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg, Bavaria D-93053, Germany. E-mail: [email protected] Received 11 October 2011; revised 23 February 2012; accepted 26 February 2012; published online 2 April 2012 Dido1 promotes melanoma progression S Braig and A-K Bosserhoff 838 questioned whether Dorsomorphin is speciﬁc for Smad- unchanged (Figure 1a). These results suggest that the effect of dependent BMP signaling pathways; however, as shown by Dorsomorphin is strongly cell-type and concentration dependent. western blot analysis, treatment of melanoma cells with Microarray analysis revealed that Dorsomorphin-treated melano- Dorsomorphin speciﬁcally blocked the phosphorylation and ma cells exhibited an approximately fourfold reduction in Dido1 activation of Smad signaling, whereas ERK activation remained (Death inducer-obliterator 1) expression levels compared with

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Figure 1. Dido1 is a Smad-regulated BMP target gene. (a) Treatment of Mel Im melanoma cells with 2 mM Dorsomorphin for 6 h resulted in a reduced phosphorylation of Smad1/5/8, whereas the phosphorylation of ERK remained unchanged. (b) qRT--PCR and (c) immunofluorescence staining revealed a diminished expression of Dido1 in Mel Im and Mel Ju melanoma cells compared with dimethyl sulfoxide (DMSO)-treated cells, following incubation with 2 mM Dorsomorphin. DAPI (4,6-diamidino-2-phenylindole) was used to counterstain the nucleus. (d) Dido1 mRNA expression levels are significantly downregulated in Chordin (CHRD)- and asBMP4-overexpressing melanoma cell clones as determined by qRT--PCR. (e) Mel Im cells were transfected with siRNA against Smad4 (siSmad4) for 48 h, resulting in a reduced expression of Smad4 mRNA (left). Transfection of Mel Im cells with siSmad4 for 72 h strongly reduced Dido1 mRNA expression levels compared with control transfected cells (right). (f) A schematic representation of the BMP-specific Smad-binding sites within the Dido1 promoter sequence. Oligonucleotide sequences were used for electrophoretic mobility shift assay (EMSA) experiments. For simplicity, the complementary sequences are not shown. The core binding sites with homology to the consensus Smad-binding sequence are underlined and in uppercase. Dido1 101 mut and Dido1 205 mut represent the mutated oligonucleotides used for EMSA studies and competition experiments to show the specific binding of Smad to the wild-type sequences. The base-pair exchanges are indicated by lowercase letters. (g) EMSA confirmed the binding of Smad1/5/8 to the Dido1 promoter. Smad1/5/8 binding was shown using two oligonucleotides spanning BMP- specific Smad regions (Dido1 101 and Dido1 205). For competition experiments, the mutated oligonucleotides 101 mut and 205 mut were used. Generation of Dido1-Smad complexes was significantly inhibited by incubation of Mel Im nuclear extracts with Smad5, p-Smad1/5/8 and p-Smad1/5 antibodies, respectively. Additionally, nuclear extracts of Chordin-overexpressing Mel Im cell clones (CHRD1) were incubated with Dido1 oligonucleotides, resulting in strongly diminished complex formation compared with control cells. Bars show the mean±s.d. of three independent experiments. Measurements were performed in triplicate (*Po0.5; **Po0.01; ***Po0.001).

Oncogene (2013) 837 -- 848 & 2013 Macmillan Publishers Limited Dido1 promotes melanoma progression S Braig and A-K Bosserhoff 839 control cells. We were able to confirm these data by qRT--PCR and compared with those derived from control transfected cells immunofluorescence staining (Figures 1b and c). Additionally, (Figure 3c and Supplementary Figure S1). To investigate if the melanoma cell clones with diminished BMP activity, due to stable reduced colony-formation potential of siDido1-treated melanoma transfection of either antisense BMP4 constructs or a Chordin BMP cells was due to an induction of senescence, we performed inhibitor construct (constructs are described in Rothhammer senescence-associated b-galactosidase staining. Interestingly, we et al.8), showed diminished expression of Dido1 (Figure 1d). observed no differences in the cytoplasmic senescence-associated To confirm that Dido1 acts as a Smad-dependent BMP target b-galactosidase activities between siDido1-transfected Mel Ju and gene, we treated Mel Im melanoma cells with a small interfering Mel Im cells and control cells. Thus, Dido1 had no significant effect RNA (siRNA) against the common Smad, Smad4. This treatment on senescence induction in melanoma cells (Figure 3d and resulted in reduced expression of both Smad4 and Dido1 Supplementary Figure S2). (Figure 1e). Computer analysis revealed the presence of two putative BMP-specific Smad-binding sites within the promoter of Dido1 inhibits apoptosis in melanoma cells the Dido1 gene (Figure 1f). To demonstrate direct binding of To analyze the long-term effects of Dido1 knockdown, we Smads to the Smad-binding sites within the Dido1 promoter, we generated melanoma cell clones that were stably transfected performed electromobility shift assays with two labeled oligo- with an antisense Dido1 construct. Following clonal selection, Mel nucleotides that spanned the potential Smad-binding sites in the Im cells that had been stably transfected with antisense Dido1 Dido1 promoter. As shown in Figure 1g, incubation of Mel Im exhibited a decrease of up to 90% in Dido1 expression compared melanoma nuclear extracts with the oligonucleotides Dido1 101 or with pcDNA3 stably transfected control cells (Figure 4a). Surpris- Dido1 205 led to the generation of a complex, whereas incubation ingly, after cultivation for 2 weeks under selection conditions, a with oligonucleotides that harbored a mutated Smad-binding site re-expression of Dido1 in the asDido1 cell clones was observed. resulted in no complex formation. Additionally, incubation of Mel This level of re-expression was comparable to that observed in the Im nuclear extracts with Smad5 and p-Smad5 antibodies led to a pcDNA3 control cells (Figure 4b). Therefore, we speculated significant repression of complex formation by competition. whether knocking down Dido1 expression may induce apoptosis Furthermore, incubation of Dido1 101 or Dido1 205 oligonucleo- in melanoma cells. tides with CHRD1 nuclear extracts resulted in a strong reduction in It is known that Dido1 is involved in the regulation of complex generation compared with incubation with Mel Im apoptosis.11 Therefore, we investigated whether an inhibition of nuclear extracts (Figure 1g). These data demonstrate that the Dido1 expression in melanoma cells via specific siRNAs could Smad complex directly binds to the Smad-binding site within influence apoptotic behavior. By fluorescence-activated cell the Dido1 promoter, indicating that Dido1 is a Smad-dependent sorting (FACS) experiments, we were able to demonstrate that a BMP target gene. knockdown of Dido1 expression led to an induction of apoptosis (Figure 4c). Moreover, transfection of Mel Ju melanoma cells with Dido1 is overexpressed in melanoma cell lines and tissue samples a Dido1 expression construct strongly reduced the rate of compared with NHEMs apoptosis compared with control vector transfected cells Next, we analyzed the expression of Dido1 in different melanoma (Figure 4c). Furthermore, western blot analysis revealed an cell lines. Melanoma cell lines derived from metastases showed upregulation of cleaved PARP (poly (ADP-ribose) polymerase) particularly elevated levels of Dido1 mRNA expression compared protein levels in siDido1-transfected cells compared with control with normal human epidermal melanocytes (NHEMs; Figure 2a). cells (Figure 4d). Additionally, by immunofluorescence staining, we observed enhanced Dido1 protein expression in the nuclei of melanoma Dido1 induces the migration and invasion of melanoma cells cells derived from both primary and metastatic tumors in Because it is known that BMPs induce the migration and invasion comparison with NHEMs (Figure 2b). To verify these results of melanoma cells,8 we analyzed siDido1-treated Mel Im and Mel in vivo, we analyzed the expression levels of Dido1 in different Ju cells in Boyden chamber assays. These assays revealed a tissue samples. qRT--PCR revealed the presence of strongly significant decrease in the migratory potential of siDido1- increased Dido1 levels in melanoma tissue samples compared transfected melanoma cells compared with mock-transfected cells with NHEMs (Figure 2c). Immunohistochemical staining of (Figure 5a). This could be confirmed by xCELLigence experiments, melanoma tissue samples with a specific Dido1 antibody showed as shown in Supplementary Figure S3. In addition, the invasive strong expression of Dido1 in primary and metastatic melanoma behavior was clearly diminished in siDido1-transfected Mel Im and tissues, whereas immunofluorescence staining of melanocytes Mel Ju melanoma cells compared with control transfected cells, as located in the basal membrane of normal skin (identified by the determined by modified Boyden chamber assays (Figure 5b). melanocytic-specific marker tyrosine-related protein 2 (TRP2)) Furthermore, scratch assays performed with the xCELLigence displayed no nuclear expression of Dido1 protein (Figure 2d). instrument showed a strongly reduced migratory potential of the siDido1-transfected Mel Ju melanoma cells compared with The influence of Dido1 on the proliferation and senescence the mock-transfected cells (Supplementary Figure S4). Next, of melanoma cells we investigated the influence of Dido1 on melanoma cells grown To determine the functional relevance of Dido1 in malignant as three-dimensional spheroids embedded in collagen. In melanoma, we treated Mel Im and Mel Ju cells for 72 h with two accordance with our previous data, siDido1-transfected cells different siRNAs against Dido1, individually or in combination. showed a strongly reduced potential to invade into the Transfection led to a reduction in Dido1 expression of up to 50% in surrounding collagen matrix (Figure 5c). the case of Mel Im cells, and up to 80% in case of Mel Ju cells, To analyze the impact of Dido1 re-expression in cells with respectively, as determined by qRT--PCR (Figure 3a). In addition, diminished BMP activity, we used Mel Im melanoma cell clones immunofluorescence staining revealed diminished protein levels that were stably transfected with the general BMP-inhibitor of Dido1 in Mel Ju and Mel Im cells after transfection with specific Chordin (CHRD1 and CHRD9)8 and incubated with a Dido1 Dido1 siRNAs (Figure 3b). expression construct (kindly provided by Dr Martinez, Madrid, Next, the anchorage-independent growth of siDido1-trans- Spain). qRT--PCR experiments revealed strongly induced expres- fected cells was analyzed by a colony-formation assay. Following sion of Dido1 mRNA in Dido1-transfected cells compared with growth in soft agar, colonies derived from siDido1-transfected Mel related controls (Supplementary Figure S5). As determined by Ju and Mel Im melanoma cells were significantly smaller Boyden chamber assays, treatment of the CHRD1 and CHRD9 cell

& 2013 Macmillan Publishers Limited Oncogene (2013) 837 -- 848 Dido1 promotes melanoma progression S Braig and A-K Bosserhoff 840 Dido1 DAPI merge

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Figure 2. The expression of Dido1 in malignant melanoma. The expression of Dido1 mRNA was analyzed by quantitative RT--PCR (a) and immunofluorescence staining (b) in NHEMs and in different melanoma cell lines. (c) Dido1 mRNA expression was determined in tissue samples of primary melanomas (PT1--PT4) and melanoma metastases (Met1--Met4) by quantitative RT--PCR. (d) Dido1 protein expression was analyzed by immunohistochemistry in primary melanomas and metastases of malignant melanoma. Strong expression of Dido1 protein was detected in primary melanomas and metastases, as shown by red AEC staining (200-fold magnification). Melanocytes located in the basal membrane of normal skin were identified by immunofluorescence staining of the specific marker TRP2 (tyrosine-related protein 2). Nuclear Dido1 expression was absent in these cells. Bars show the mean±s.d. of three independent experiments. Measurements were performed in triplicate. NS, not significant (*Po0.5; **Po0.01; ***Po0.001).

clones with the Dido1 expression construct enhanced their mice as described in Rothhammer et al.9 Immunohistological migratory and invasive potentials compared with control trans- analysis revealed strongly diminished protein expression of fected cells (Figure 5d). Subsequently, we treated Mel Im and Mel p-Smad1/5/8, Dido1 and Integrin aV in asBMP4 and Chordin- Ju melanoma cells with the Smad-dependent BMP inhibitor overexpressing cell clones compared with the control tumors Dorsomorphin that resulted in a strongly reduced migratory and (Figure 6d). invasive potential, as expected. Transfection of these cells with the xCELLigence experiments showed a reduction in the attachment Dido1 expression construct significantly enhances the migration capacity of siDido1-transfected Mel Ju cells compared with control and invasion compared with control transfected pEGFP melanoma cells (Figure 6e). To analyze if the observed impact of Dido1 on cells (Figure 5e), thereby further indicating that the Smad-specific attachment was mediated via Integrin aV, we performed attachment BMP target gene Dido1 is the main mediator of BMP-induced assays on vitronectin-coated plates. Mel Ju cells transfected with migration and invasion of melanoma cells. either siRNAs against Dido1 or control siRNA were treated with 20 mM of an inhibitory Integrin aVb3 antibody. Treatment with the Dido1 regulates the expression of Integrin aV inhibitory Integrin aVb3 antibody strongly reduced the attachment As sequence analyses of Rojas et al.12 revealed that Dido1 contains of control siRNA-transfected cells to vitronectin, whereas a similar several known transcription factor domains, we screened for treatment of siDido1-transfected cells resulted in only marginal potential Dido1 target genes in melanoma that may be involved in effects on the attachment potential of the cells (Figure 6f). the regulation of migration and invasion (Supplementary Figure S6). Finally, we analyzed the migratory potential of Chordin- Through this screen, we identified Integrin aV as being strongly overexpressing cell clones after incubation with the inhibitory reduced in siDido1-transfected Mel Ju cells compared with mock- integrin antibody. As shown in Figure 5d, the migratory potential transfected cells, as determined by qRT--PCR and western blot of CHRD cell clones transfected with a Dido1 expression construct assays (Figures 6a and b). Furthermore, melanoma cell clones with was strongly enhanced. Additional treatment with the inhibitory diminished BMP activity showed significantly decreased expres- Integrin aVb3 antibody reduced the migration of the CHRD1 cells sion of Integrin aV (Figure 6c). Additionally, control cells, asBMP4, to 41.53%, whereas the migration of the CHRD9 cell clones was and Chordin-overexpressing cell clones were injected into nude decreased to 57.69%.

Oncogene (2013) 837 -- 848 & 2013 Macmillan Publishers Limited Dido1 promotes melanoma progression S Braig and A-K Bosserhoff 841 Dido1DAPI merge

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0 Mel Ju ctrl siDido1 siDido1 siDido1 #1 #2 #12 Figure 3. Dido1 enhances the proliferative behavior of melanoma cells. (a, b) A reduction of Dido1 expression was achieved by transient transfection of Mel Im and Mel Ju cells with different siRNAs against Dido1 (siDido1 #1, siDido1 #2, siDido1 #1/2). Transfection with a scrambled siRNA served as a control. A strong reduction of Dido1 expression was observed after 72 h in the melanoma cell lines Mel Im and Mel Ju at mRNA (a) and protein (b) level, as shown by qRT--PCR analysis and immunofluorescence staining, respectively. (c) Anchorage- independent growth is strongly diminished in siDido1-transfected Mel Ju cells as determined by a colony-formation assay. Representative images of colonies are shown on the left. The diameters of at least 10 colonies were determined and quantified (right). (d) b-Galactosidase staining revealed no significant differences in the amount of senescent cells between those transfected with the control siRNA or with the siRNAs against Dido1. Representative images of Mel Ju cells are shown on the left, and the quantification of senescent cells is displayed on the right. Bars show the mean±s.d. of three independent experiments. Measurements were performed in triplicate. NS, not significant (*Po0.5; **Po0.01; ***Po0.001).

DISCUSSION demonstrated that the overexpression of BMP4 induces the BMPs play an important role in the formation and progression migration and invasion of melanoma cells. Furthermore, BMP4 of malignant melanoma.13 -- 15 Studies from our own group promotes neoangiogenesis and tumor growth;8,9 however, the

& 2013 Macmillan Publishers Limited Oncogene (2013) 837 -- 848 Dido1 promotes melanoma progression S Braig and A-K Bosserhoff 842

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0 β-actin /#2 control pEGFP +Dido1 siDido1 #1 siDido1 #2 siDido1 #1 Figure 4. The effect of Dido1 on apoptosis in melanoma cells. (a) Stable transfection of Mel Im cells with asDido1 expression constructs resulted in strongly reduced Dido1 mRNA expression compared with pcDNA3 control transfected cells (i). PCR analysis conﬁrmed expression of the asDido1 construct (ii). (b) qRT--PCR experiments showed that cultivation of stably transfected asDido1 cell clones for over 2 weeks led to re-expression of Dido1 (i). After 2 weeks, the asDido1cell clones showed no expression of the asDido1 construct, as determined by PCR (ii). (c) FACS analysis revealed an induction of apoptosis in siDido1-transfected melanoma cells, whereas transfection of melanoma cells with a Dido1 expression construct resulted in reduced apoptosis compared with pEGFP control transfected cells. Representative measurements (upper panel) are shown as a graph (lower panel). (d) Knocking down Dido1 expression resulted in an upregulation of cleaved PARP, as shown by western blot. Bars show the mean±s.d. of three independent experiments. Measurements were performed in triplicate (*Po0.5; **Po0.01; ***Po0.001).

downstream targets of BMPs that mediate these oncogenic because Dorsomorphin has no impact on the alternative ERK functions are poorly characterized. Until now, only a few BMP signaling cascades. Dorsomorphin-treated melanoma cell lines target genes have been discovered. Melanoma cells with were analyzed using a gene microarray, which led to the diminished BMP activity show reduced mRNA expression levels identification of the potential BMP target gene Dido1. Incubation of EphA2, VE-Cadherin, Id-1 and TEK/Tie-2,9 resulting in a decreased of melanoma cells with Dorsomorphin resulted in a down- development of the vascular network and angiogenesis.16 -- 18 regulation of Dido1 expression, which was verified by qRT--PCR Detailed analyses revealed that VE-Cadherin, EphA2 and TEK/Tie-2 and immunofluorescence staining. Additionally, diminished ex- are indirect target genes of the BMP signaling pathway, whereas pression of Dido1 was observed in melanoma cell clones stably Id-1 acts as a direct BMP target gene.9 In addition, we were able to transfected with either antisense BMP4 or Chordin constructs. To show that BMPs induce the expression of several matrix further validate the Smad-dependent regulation of Dido1, cells metalloproteinases, thereby contributing to the dissemination of were treated with an siRNA against Smad4. Dido1 expression was melanoma cells from the primary tumor.19 significantly reduced in siSmad4-treated cells compared with To identify further BMP target genes, melanoma cell lines were mock-transfected melanoma cells. Additionally, electrophoretic treated with Dorsomorphin, which is known as a specific Smad- mobility shift assay experiments confirmed the direct binding of dependent inhibitor of the BMP signaling cascade. Usage of this Smad1/5/8 to the Dido1 promoter. These experiments showed BMP inhibitor enabled us to identify BMP-specific target genes, that Dido1 is a Smad-dependent BMP target gene in malignant

Oncogene (2013) 837 -- 848 & 2013 Macmillan Publishers Limited Dido1 promotes melanoma progression S Braig and A-K Bosserhoff 843 120 120 100 100 80 80 60 * ** 60 ***

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25 25 % relative to related control % relative to related control 0 0 Mel lm Mel lm Mel Ju Mel Ju mel lm Mel lm Mel Ju Mel Ju +pEGFP +Dido1 +pEGFP +Dido1 +pEGFP +Dido1 +pEGFP +Dido1 Figure 5. Dido1 increases the migratory and invasive potentials of melanoma cells. (a) Migration and (b) invasion assays using the Boyden chamber model revealed a significant reduction in the migratory and invasive potentials of melanoma cells after the siRNA-mediated downregulation of Dido1 expression. (c) siDido1-transfected Mel Ju cells that were grown as spheroids and embedded in collagen showed a diminished migration capacity compared with control siRNA-transfected cells. (d) Stable Chordin-overexpressing melanoma cell clones (CHRD1 and CHRD9) were transfected with a Dido1 expression construct. Boyden chamber assays showed strongly increased migratory and invasive potentials of Dido1-overexpressing CHRD Mel Im cells compared with mock-transfected cell clones. (e) Mel Im and Mel Ju cells were treated with 2 mM Dorsomorphin leading to reduced migratory and (f) invasive behavior as determined by Boyden chamber assays. Additional transfection of these cells with a Dido1 expression construct enhanced (e) migration and (f) invasion of Dorsomorphin treated cells compared with pEGFP transfected cells. Bars show the mean±s.d. of three independent experiments. Measurements were performed in triplicate (*Po0.5; **Po0.01; ***Po0.001). melanoma. As far as we know, this is the first study demonstrating like RNA interference or protein stabilization are also possible. that a signaling cascade regulates the expression of Dido1. Immunofluorescence staining revealed the expression of Because, until now, no data were available concerning the Dido1 in the nucleus of melanoma cells, which is in contrast to expression levels of Dido1 in different tumor types, we determined studies by Garcia-Domingo et al.,11 who observed the localization the Dido1 mRNA levels in numerous melanoma cell lines and of Dido1 in the nucleus of mouse embryonic fibroblasts only were able to show enhanced Dido1 mRNA expression mainly in under apoptotic conditions. A possible explanation for this melanoma cell lines derived from metastases. However, at the discrepancy may be that the localization and function of Dido1 protein level, Dido1 expression was strongly increased in depends on cell type or culture conditions. Expression levels of melanoma cells that had been derived from both primary tumors Dido1 mRNA and protein were enhanced in melanoma tissue and metastases compared with normal melanocytes, indicating samples compared with normal melanocytes, thus further that, apart from the observed transcriptional regulation of indicating an impact of Dido1 on the progression of malignant Dido1 expression, posttranscriptional regulation mechanisms melanoma.

& 2013 Macmillan Publishers Limited Oncogene (2013) 837 -- 848 Dido1 promotes melanoma progression S Braig and A-K Bosserhoff 844

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20 % relative to related control 0

ITG

+inhib. ITGsiDido1 #1 siDido1 #2 +inhib. ITG Mel Ju sictrl + inhib. + inhib. ITG siDido1 #1/#2 Figure 6. Dido1 upregulates the expression of Integrin aV, thereby influencing the attachment, apoptosis and migration of melanoma cells. (a) Knocking down Dido1 expression in Mel Ju cells resulted in a strong decrease in the expression of Integrin aV mRNA and (b) protein, as determined by qRT--PCR and western blot, respectively. (c) Chordin- and asBMP4-overexpressing cell clones showed reduced Integrin aV mRNA levels compared with mock-transfected cells. (d) asBMP4- and Chordin-overexpressing cell clones were injected into nude mice. Immunohistological analysis showed weakened staining of p-Smad1/5/8, Dido1 and Integrin aV in asBMP4- and Chordin-overexpressing cell clones compared with pcDNA control tumors. (e) xCELLigence experiments revealed a significantly diminished attachment potential of siDido1-transfected Mel Ju cells compared with control cells (left). The quantitative analysis of the migratory behavior during the marked timeframe is shown on the right. (f) Treatment of siDido1-transfected Mel Ju cells with an inhibitory Integrin aV antibody (inhib. ITG) had only minor effects on cell attachment to vitronectin-coated plates, whereas incubation of control cells with the inhibitory Integrin aV antibody resulted in significantly decreased cell attachment. Bars show the mean±s.d. of three independent experiments. Measurements were performed in triplicate. NS, not significant (*Po0.5; **Po0.01; ***Po0.001).

To study the functional relevance of Dido1 in melanoma, the remained unchanged (data not shown). Further studies showed expression of Dido1 was inhibited by siRNA transfection. First, that this effect is caused by an inhibition of proliferation instead of we investigated whether Dido1 modulates the colony-forming an induction of senescence. potential of melanoma cells. Here, we were able to demonstrate For subsequent experiments, stable antisense Dido1 melanoma that siDido1-transfected cells form signiﬁcantly smaller colonies cell clones were generated. Because cultivation over 2 weeks compared with control cells, whereas the number of colonies resulted in the re-expression of Dido1 in these cells, we assumed

Oncogene (2013) 837 -- 848 & 2013 Macmillan Publishers Limited Dido1 promotes melanoma progression S Braig and A-K Bosserhoff 845 that Dido1 influences apoptosis. FACS experiments and western aV signaling diminished the invasion of melanoma cells,30,31 the blot analysis verified our hypothesis. Inhibition of Dido1 expres- treatment of Chordin-overexpressing melanoma cell clones with a sion by siRNA transfection resulted in an induction of apoptosis, Dido1 expression construct alongside with an inhibitory Integrin whereas overexpression of Dido1 using a Dido1 expression aV resulted in significantly reduced migratory behavior compared construct led to decreased apoptosis, thereby demonstrating that with control transfected cell clones. Dido1 mediates melanoma cell resistance to apoptosis. These Moreover, we demonstrated that Dido1 contributes to the observations are in contrast to the study performed by the ability of melanoma cells to resist apoptosis. With regard to the Martinez group,11 who showed that the overexpression of Dido1 study of Koistinen et al.,32 which showed that the depletion of in mouse embryonic fibroblasts resulted in the translocation of Integrin aV resulted in the detachment of melanoma cells from Dido1 from the cytoplasm into the nucleus, which subsequently the extracellular matrix and a subsequent induction of apoptosis, led to the activation of apoptosis. Moreover, this group demon- the influence of Dido1 on apoptosis resistance is most likely strated enhanced Dido1 expression during the early steps of mediated via an induction of Integrin aV expression. apoptosis.20 As discussed in detail below, Dido1 regulates the In summary, we identified Dido1 as a new Smad-dependent expression of Integrin aV in melanoma cells. Several studies have BMP target gene that contributes to the progression of melanoma shown an association between Integrin signaling and apoptosis/ by inducing the expression of Integrin aV, thereby enhancing the anoikis.21,22 For example, Zhang et al.23 described that treatment attachment, migration, invasion and anchorage-independent of melanoma cells with an inhibitor of Integrin aVb3 promotes growth of melanoma cells and promoting tumor resistance to anoikis and activates caspase cascades. Hence, it is feasible that apoptosis. Dido1-dependent upregulation of Integrin aV in malignant melanoma results in an enhanced resistance to apoptosis. Because it is known that melanoma cells exhibit a high MATERIALS AND METHODS resistance to apoptosis, which often results in the failure of Cell culture 24,25 therapy, an inhibition of Dido1 expression might be a The melanoma cell lines Mel Ei, Mel Wei, Mel Ho, Mel Juso, Mel Im, Mel Ju, promising therapeutic approach. Such inhibition may result in SkMel 3, SkMel 28 and 501Mel were described previously.33 The cell lines an increase in apoptosis in response to conventional chemother- Mel Ei, Mel Wei, Mel Ho and Mel Juso were derived from a primary apy, allowing one to overcome the common problem of cutaneous melanoma, whereas Mel Im, Mel Ju, SkMel3, SkMel28 and chemoresistance. Apart from malignant melanoma, the inhibition 501Mel cell lines were derived from metastases of malignant melanomas. of Dido1 may also be therapeutic in other cancer types. All cell lines were cultured as described previously.33 NHEMs were derived Previous studies from our group showed that BMPs play an from neonatal skin. The cultivation of NHEMs was described previously.34 important role in the migration and invasion capacities of Melanocytes of at least three different donors were used, and the mean 19 melanoma cells. Because Dido1 is a BMP target gene, we values are displayed in the graphs. Mel Im melanoma cells lines stably analyzed the migration and invasion of siDido1-treated melanoma transfected with antisense BMP4 or Chordin expression constructs were cells. Subsequently, we were able to demonstrate that siDido1- described by Rothhammer et al.8 transfected cells exhibited strongly diminished migration and invasion potentials in both two- and three-dimensional cultures. Microarray The opposite was also shown: the treatment of melanoma cell clones possessing diminished BMP-activity with a Dido1 expres- To identify Smad-regulated BMP target genes in malignant melanoma, the sion construct increased their migration and invasion behavior cell lines Mel Im and Mel Ju were treated with 2 mM of the Smad-specific compared with control cells. Thus, therapeutic treatment with BMP inhibitor Dorsomorphin (Sigma-Aldrich, Hamburg, Germany) or with Dido1 inhibitors may also serve to counteract the metastasis and dimethyl sulfoxide as a control. After incubation for 6 h, RNA was isolated, progression of malignant melanoma. processed and hybridized to a Human Gene 1.0 ST Array (Affymetrix, Santa Sequence analysis indicated that Dido1 may act as a transcrip- Clara, CA, USA). Detailed analysis of the array data was performed using the tion factor.12 Therefore, we screened for potential Dido1 target programs ChipInspector and Bibliosphere (Genomatix, Munich, Germany). genes that influence the migration and invasion of melanoma cells by analyzing the expression levels of matrix metalloprotei- Transfection experiments nases, cadherins, Mia, Ski, Sno and integrins in melanoma cells For siRNA transfections, 200 000 Mel Im or Mel Ju cells were seeded into transfected with siRNAs against Dido1. We were able to identify each well of a six-well plate. Using 18 ml HiPerFect transfection reagent Integrin aV as being strongly affected in melanoma cells with (Qiagen, Hilden, Germany) per well, cells were transfected with 1500 ng of diminished Dido1 expression, thereby showing for the first time Smad4 siRNA, 375 ng of Dido1 siRNA (siDido1 1 and siDido1 2, Qiagen), a that BMPs regulate the expression of integrins in malignant combination of siDido1 1 and 2 or a negative control siRNA. The cells were melanoma. Interestingly, overexpression of Dido1 in primary lysed 72 h after transfection, and the mRNA was isolated and transcribed melanocytes (NHEMs) resulted in only slightly increased into complementary DNA before qRT--PCR analysis. Mel Im, Mel Ju and ITGaV mRNA expression levels (data not shown). It seems that in stable Chordin-overexpressing Mel Im melanoma cell clones were healthy melanocytes additional molecules beside of Dido1 transfected with a Dido1 expression construct (kindly provided by regulate the expression of Integrin aV. Dr Martinez, and described in Trachana et al.35) using the Lipofectamine BMP-dependent regulation of integrins might be an essential Plus Reaction Kit (Invitrogen, Groningen, The Netherlands) according to the step in the progression of other types of cancer that show manufacturer’s instructions. All transfections were repeated at least three deregulated expression of BMPs and integrins, although this times. remains to be studied. It is known that Integrin aV binds to extracellular matrix components such as fibronectin and vitro- Analysis of gene expression by quantitative PCR nectin, thereby regulating tumor cell adhesion, migration and Quantitative real time--PCR was performed on a Lightcycler (Roche, survival.26 -- 29 The attachment of siDido1-transfected melanoma Mannheim, Germany) as described previously.19 Annealing and melting cells is significantly reduced compared with control transfected temperatures were optimized for each primer set (Table 1). cells, as determined by xCELLigence experiments. Attachment assays performed with specific vitronectin-coated plates confirmed that the diminished adhesion capacity of the siDido1- Generation of stable asDido1 cell clones transfected cells is mediated by Integrin aV. Consistent By stable transfection of melanoma cells, a panel of Mel Im cell clones with previous studies showing that an inhibition of Integrin expressing antisense Dido1 was established. Here, Mel Im cells were

& 2013 Macmillan Publishers Limited Oncogene (2013) 837 -- 848 Dido1 promotes melanoma progression S Braig and A-K Bosserhoff 846 Table 1. Oligonucleotide sequences (Zymed, San Francisco, CA, USA) was used to stain the membranes. For quantification, western blot membranes were scanned, and band density Gene Primer sequences (fwd/rev) was evaluated by pixel count in relation to the loading control. Dido1 50-TTCCATCCAAACTCTTGCCCTTT-30 50-CCAAGAATTATATTCGGACGTGGTG-30 Immunohistochemistry 0 0 Smad4 5 -TAGCTCCAGCTATCAGTCTGTCAGC-3 Paraffin-embedded preparations of mouse tumor sections, as well as 0 0 5 -TCTGCAATCGGCATGGTATGAAGTAC-3 human primary melanomas and metastases were screened for Dido1 and Integrin aV 50-GATGTTGGGCCAGTTGTTCAGCACATCTATG-30 Integrin aV protein expression using the Envision system (DAKO, Hamburg, 50-CAGACGACTTCAGAGAATAGGAATGATTCTG-30 Germany). The tissues were deparaffinized, rehydrated and incubated with Tris-EDTA for 5 min at 120 1C. After treatment with H2O2, the tissues were b-Actin 50-CTACGTCGCCCTGGACTTCGAGC-30 incubated with a primary polyclonal Dido1 antibody (1:100, sc-25264, 50-GATGGAGCCGCCGATCCACACGG-30 Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-Integrin aV antibody (1:500, AB 1930, Millipore), respectively, for 30 min at room temperature. Tissues were then incubated with the secondary antibody transfected with antisense Dido1 expression plasmids (consisting of 349 bp (labeled polymer, horseradish peroxidase, supplied with the kit) for 30 min of the human Dido1 gene cloned into the pcDNA3 vector) using the at room temperature. Staining of the paraffin-embedded mouse tumor Lipofectamine Plus Reaction Kit (Invitrogen) as previously described.19 sections was performed using Histofine Simple Stain MAX PO (Nichirei Bioscience, Tokyo, Japan) and anti-phospho Smad1/5/8 (1:100, no. 9511, Cell Signaling). For each group, at least three tumors were sectioned into RNA isolation and reverse transcription 4-mm slices, and four sections were evaluated. Antibody binding was Total cellular RNA was isolated from cultured cells using the E.Z.N.A. Total visualized using the DAB þ substrate-chromogen solution (DAKO). Finally, RNA Kit I (Omega Bio-Tek, VWR, Darmstadt, Germany) according to the the tissues were counterstained by hemalaun. manufacturer’s instructions. Complementary DNAs were generated by a reverse transcriptase reaction (500 ng of total RNA) using the SuperScript II Reverse Transcriptase Kit (Invitrogen). Immunofluorescence Immunofluorescence assays were performed as described previously.37 Electrophoretic mobility shift assay Briefly, cells were seeded in chamber slides, washed with PBS, fixed with ice-cold acetone for 10 min at À20 1C, permeabilized using 0.1% Triton Nuclear extracts (6 mg) were incubated with radiolabeled double-stranded X-100 for 5 min, washed again and blocked for 1 h with 1% bovine serum oligonucleotides comprising the BMP-specific Smad-binding sites in the albumin/PBS. Subsequently, cells were incubated with anti-Dido1 antibody Dido1 promoter. Wild-type or mutated binding sites (bold type) for Smad (1:50, sc-25264, Santa Cruz Biotechnology) overnight at 4 1C. After washing, were used. The following oligonucleotides were employed: Dido1 205 fwd: cells were incubated with the secondary antibody (1:50, fluorescein 50-GCCGCTTGGGCCGCCCTCGGG; Dido1 205 rev: 50-TGGGCGGACGGCCACC isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin) for 2 h, GAGAT-30; Dido1 101 fwd: 5-ATCTCGGTGGCCGTCCGCCCA-30; Dido1 101 followed by rinsing with PBS and mounting with Vectashield Hard Set rev: 5-TGGGCGGACGGCCACCGAGAT-30; Dido1 101 mut fwd: 50-ATCTCGG Mounting Medium with DAPI H-1500 (Vector Laboratories, Burlingame, CA, TGGAAGTCCGCCCA-30; Dido1 101 mut rev: 50-TGGGCGGACTTCCACCGA USA). Images were collected by immunofluorescence microscopy using an GAT-30; Dido1 205 mut fwd: 50-GCCGCTTGGGAAGCCCTCGGG-30; Dido1 205 Axio Imager Zeiss Z1 fluorescence microscope (Axiovision Rel. 4.6.3, Carl mut rev 205: 50-CCCGAGGGCTTCCCAAGCGGC-30. The oligonucleotides Zeiss AG, Oberkochen, Germany). Cryosections of normal skin were fixed, were end-labeled with T4-polynucleotide kinase (Roche, Penzberg, permeabilized, blocked and co-incubated with anti-Dido1 (1:50, Santa Cruz Germany) and [g-32P]-ATP (PerkinElmer, Waltham, MA, USA). Band shifts Biotechnology) and anti-TRP2 (1:500, ab74073, Abcam, Cambridge, UK) were performed as described previously.36 The specificity of the probe was overnight at 4 1C. Following incubation with the secondary antibodies, evaluated by incubating the nuclear extracts with a 40 Â excess of Alexa Fluor 594-labeled goat anti-rabbit (1:150, Invitrogen) and FITC- unlabeled oligonucleotide. Antibodies against Smad5, p-Smad1/5 and conjugated anti-mouse antibody (1:40, DAKO), sections were mounted p-Smad1/5/8 (Cell Signaling, Danvers, MA, USA) were used for antibody with Vectashield Hard Set Mounting Medium with DAPI H-1500. interference experiments.

Protein analysis in vitro (western blotting) Apoptosis assay A total of 3 million cells were lysed in 200 ml of RIPA buffer (Roche) and Apoptotic cells were analyzed by flow cytometry using the ApoDETECT incubated for 15 min at 4 1C. Insoluble fragments were removed by ANNEXIN V-FITC KIT (Zymed, Carlsbad, CA, USA) according to the centrifugation at 13 000 r.p.m. for 10 min, and the supernatant (lysate) was manufacturer’s instructions. The flow cytometry analysis was performed immediately shock frozen and stored at À80 1C. Cell lysates were loaded in a FACSCanto II cytometer (Becton Dickinson, Heidelberg, Germany). and separated on sodium dodecyl sulfate--polyacrylamide gel electro- FACS data were analyzed using FACSDiva software (Becton Dickinson). phoresis gels, and the proteins were subsequently blotted onto a polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA, USA). The membranes were then blocked in 5% nonfat dry milk/Tris-buffered saline/ Senescence-associated b-galactosidase staining 0.1% Tween-20 for 1 h and incubated with anti-Smad5 (no. 9517), To determine the fraction of senescent melanoma cells after transfection p-Smad1/5/8 (no. 9511), p44/42 ERK (no. 9102), p-p44/42 ERK (no. 4370) with siRNA against Dido1, cells were stained as described in Dimri et al.38 or PARP antibody (no. 9542; 1:1000; Cell Signaling) in 5% bovine serum Briefly, cells were fixed in 3.7% formaldehyde for 5 min and stained with albumin/Tris-buffered saline/0.1% Tween-20 or 5% nonfat dry milk/Tris- X-Gal at 37 1C overnight. After washing with PBS, senescent cells were buffered saline/0.1% Tween-20 overnight at 4 1C. A 1:2000 dilution of anti- counted. rabbit-horseradish peroxidase (Cell Signaling) was used as the secondary antibody. Staining was performed using the ECL Plus Western Blotting Detection kit (GE Healthcare, Buckinghamshire, UK). To analyze Integrin aV Migration and invasion assay using Boyden chambers protein expression, membranes were blocked in 3% nonfat dry milk/ Assays were performed as described previously using Boyden chambers phosphate-buffered saline (PBS) and incubated with anti-Integrin aV containing polycarbonate filters with an 8-mm pore size (Costar, antibody (1:5000, AB1930, Millipore, MA, Billerica, USA) followed by an anti- Bodenheim, Germany).33 Experiments were carried out in triplicate and rabbit-AP-conjugated secondary antibody (1:4000). The BCIP/NBT kit were repeated three times.

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Oncogene (2013) 837 -- 848 & 2013 Macmillan Publishers Limited