Novel Human Tenascin-C Function-Blocking Camel Single Domain Nanobodies.

The extracellular matrix (ECM) molecule Tenascin-C (TNC) is well-known to promote tumor progression by multiple mechanisms. However, reliable TNC detection in tissues of tumor banks remains limited. Therefore, we generated dromedary single-domain nanobodies Nb3 and Nb4 highly specific for human TNC (hTNC) and characterized the interaction with TNC by several approaches including ELISA, western blot, isothermal fluorescence titration and negative electron microscopic imaging. Our results revealed binding of both nanobodies to distinct sequences within fibronectin type III repeats of hTNC. By immunofluroescence and immunohistochemical imaging we observed that both nanobodies detected TNC expression in PFA and paraffin embedded human tissue from ulcerative colitis, solid tumors and liver metastasis. As TNC impairs cell adhesion to fibronectin we determined whether the nanobodies abolished this TNC function. Indeed, Nb3 and Nb4 restored adhesion of tumor and mesangial cells on a fibronectin/TNC substratum. We recently showed that TNC orchestrates the immune-suppressive tumor microenvironment involving chemoretention, causing tethering of CD11c+ myeloid/dendritic cells in the stroma. Here, we document that immobilization of DC2.4 dendritic cells by a CCL21 adsorbed TNC substratum was blocked by both nanobodies. Altogether, our novel TNC specific nanobodies could offer valuable tools for detection of TNC in the clinical practice and may be useful to inhibit the immune-suppressive and other functions of TNC in cancer and other diseases.

Generation and characterization of dromedary Tenascin-C and Tenascin-W specific antibodies.

Tenascin-C (TNC) and tenascin-W (TNW), large hexameric glycoproteins overexpressed in the tumor microenvironment, are useful tumor biomarkers for theranostic applications. For now, polyclonal and monoclonal antibodies, as well as aptamers targeting TNC and TNW have been developed. However, the immunostaining sensitivity of antibodies is very heterogenous. The main aim of this study was to generate antibodies in dromedary that detect TNC and TNW, respectively. We show that immune sera from immunized dromedaries are able to specifically bind native TNC and TNW by ELISA and also to detect TNC and TNW in matrix tracks of mammary tumors by immunostaining. Furthermore, we demonstrate that purified IgG subtypes are able to interact specifically with TNC or TNW by ELISA and immunostaining. These camelid antibodies are a good basis to develop tools for the detection of TNC and TNW in the tumor microenvironment and could potentially have a broader application for early diagnosis of solid cancers.

Tenascin-W Is a Novel Stromal Marker in Biliary Tract Cancers.

Extrahepatic cancers of the biliary system are typically asymptomatic until after metastasis, which contributes to their poor prognosis. Here we examined intrahepatic cholangiocarcinomas (n = 8), carcinomas of perihilar bile ducts (n = 7), carcinomas of the gallbladder (n = 11) and hepatic metastasis from carcinomas of the gallbladder (n = 4) for the expression of the extracellular matrix glycoproteins tenascin-C and tenascin-W. Anti-tenascin-C and anti-tenascin-W immunoreactivity was found in all biliary tract tumors examined. Unlike tenascin-C, tenascin-W was not detected in normal hepatobiliary tissue. Tenascin-W was also expressed by the cholangiocarcinoma-derived cell line Huh-28. However, co-culture of Huh-28 cells with immortalized bone marrow-derived stromal cells was necessary for the formation and organization of tenascin-W fibrils in vitro. Our results indicate that tenascin-W may be a novel marker of hepatobiliary tumor stroma, and its absence from many normal tissues suggests that it may be a potential target for biotherapies.

Pivotal Role of Tenascin-W (-N) in Postnatal Incisor Growth and Periodontal Ligament Remodeling.

The continuously growing mouse incisor provides a fascinating model for studying stem cell regulation and organ renewal. In the incisor, epithelial and mesenchymal stem cells assure lifelong tooth growth. The epithelial stem cells reside in a niche known as the cervical loop. Mesenchymal stem cells are located in the nearby apical neurovascular bundle and in the neural plexus. So far, little is known about extracellular cues that are controlling incisor stem cell renewal and guidance. The extracellular matrix protein tenascin-W, also known as tenascin-N (TNN), is expressed in the mesenchyme of the pulp and of the periodontal ligament of the incisor, and is closely associated with collagen 3 fibers. Here, we report for the first time the phenotype of tenascin-W/TNN deficient mice, which in a C57BL/6N background exhibit a reduced body weight and lifespan. We found major defects in the alveolar bone and periodontal ligament of the growing rodent incisors, whereas molars were not affected. The alveolar bone around the incisor was replaced by a dense scar-like connective tissue, enriched with newly formed nerve fibers likely leading to periodontal pain, less food intake and reduced body weight. Using soft food to reduce mechanical load on the incisor partially rescued the phenotype. In situ hybridization and Gli1 reporter mouse experiments revealed decreased hedgehog signaling in the incisor mesenchymal stem cell compartment, which coordinates the development of mesenchymal stem cell niche. These results indicate that TNN deficiency in mice affects periodontal remodeling and increases nerve fiber branching. Through periodontal pain the food intake is reduced and the incisor renewal and the neurovascular sonic hedgehog secretion rate are reduced. In conclusion, tenascin-W/TNN seems to have a primary function in rapid periodontal tissue remodeling and a secondary function in mechanosensation.

Preparation and Application of a Decellularized Extracellular Matrix for Identification of ADAMTS Substrates.

Here we describe the use of a decellularized ECM produced in vitro by BALB/c 3T3 fibroblasts for the identification of ADAMTS substrates. Seeding of ADAMTS protease-producing HEK cells on top of the cell-free ECM followed by analysis of the conditioned medium by liquid chromatography tandem mass spectrometry (LC-MS/MS), allows for screening of ADAMTS substrates without prior purification of full-length protease.

A Selective Extracellular Matrix Proteomics Approach Identifies Fibronectin Proteolysis by A Disintegrin-like and Metalloprotease Domain with Thrombospondin Type 1 Motifs (ADAMTS16) and Its Impact on Spheroid Morphogenesis.

Secreted and cell-surface proteases are major mediators of extracellular matrix (ECM) turnover, but their mechanisms and regulatory impact are poorly understood. We developed a mass spectrometry approach using a cell-free ECM produced in vitro to identify fibronectin (FN) as a novel substrate of the secreted metalloprotease ADAMTS16. ADAMTS16 cleaves FN between its (I)5 and (I)6 modules, releasing the N-terminal 30 kDa heparin-binding domain essential for FN self-assembly. ADAMTS16 impairs FN fibrillogenesis as well as fibrillin-1 and tenascin-C assembly, thus inhibiting formation of a mature ECM by cultured fibroblasts. Furthermore ADAMTS16 has a marked morphogenetic impact on spheroid formation by renal tubule-derived MDCKI cells. The N-terminal FN domain released by ADAMTS16 up-regulates MMP3, which cleaves the (I)5-(I)6 linker of FN similar to ADAMTS16, therefore creating a proteolytic feed-forward mechanism. Thus, FN proteolysis not only regulates FN turnover, but also FN assembly, with potential long-term consequences for ECM assembly and morphogenesis.

The teneurin C-terminal domain possesses nuclease activity and is apoptogenic.

Teneurins are type 2 transmembrane proteins expressed by developing neurons during periods of synaptogenesis and apoptosis. Neurons expressing teneurin-1 synapse with other teneurin-1-expressing neurons, and neurons expressing teneurin-2 synapse with other teneurin-2-expressing neurons. Knockdowns and mutations of teneurins lead to abnormal neuronal connections, but the mechanisms underlying teneurin action remain unknown. Teneurins appear to have evolved via horizontal gene transfer from prokaryotic proteins involved in bacterial self-recognition. The bacterial teneurin-like proteins contain a cytotoxic C-terminal domain that is encapsulated in a tyrosine-aspartic acid repeat barrel. Teneurins are likely to be organized in the same way, but it is unclear if the C-terminal domains of teneurins have cytotoxic properties. Here we show that expression of teneurin C-terminal domains or the addition of purified teneurin C-terminal domains leads to an increase in apoptosis in vitro The C-terminal domains of teneurins are most similar to bacterial nucleases, and purified C-terminal domains of teneurins linearize pcDNA3 and hydrolyze mitochondrial DNA. We hypothesize that yet to be identified stimuli lead to the release of the encapsulated teneurin C-terminal domain into the intersynaptic region, resulting in programmed cell death or the disruption of mitochondrial DNA and the subsequent pruning of inappropriate contacts.

Comprehensive list of SUMO targets in Caenorhabditis elegans and its implication for evolutionary conservation of SUMO signaling.

Post-translational modification by small ubiquitin-related modifier (SUMO) is a key regulator of cell physiology, modulating protein-protein and protein-DNA interactions. Recently, SUMO modifications were postulated to be involved in response to various stress stimuli. We aimed to identify the near complete set of proteins modified by SUMO and the dynamics of the modification in stress conditions in the higher eukaryote, Caenorhabditis elegans. We identified 874 proteins modified by SUMO in the worm. We have analyzed the SUMO modification in stress conditions including heat shock, DNA damage, arsenite induced cellular stress, ER and osmotic stress. In all these conditions the global levels of SUMOylation was significantly increased. These results show the evolutionary conservation of SUMO modifications in reaction to stress. Our analysis showed that SUMO targets are highly conserved throughout species. By comparing the SUMO targets among species, we approximated the total number of proteins modified in a given proteome to be at least 15-20%. We developed a web server designed for convenient prediction of potential SUMO modification based on experimental evidences in other species.

KLF4α stimulates breast cancer cell proliferation by acting as a KLF4 antagonist.

Krüppel-like factor 4 (KLF4), a transcription factor involved in both tumor suppression and oncogenesis in various human tumors, is subject to alternative splicing that produces KLF4α. KLF4α is primarily expressed in the cytoplasm because it lacks exon 3 of KLF4, which contains the nuclear localization signal. The role of KLF4 in breast cancer remains unclear and nothing is known yet about the expression and function of the isoform KLF4α. Here, we show that KLF4α is expressed in normal and tumoral tissue of the breast and provide evidence that the KLF4α/KLF4(full-length) (FL) ratio is increased in tumors compared to corresponding normal tissue. Forced increase of the KLF4α/KLF4(FL) ratio in the metastatic breast cancer cell line MDA-MB-231 decreases the levels of E-Cadherin, p21Cip1, and p27Kip1, three known KLF4(FL) target genes, and stimulates cell proliferation. We suggest that cytoplasmic KLF4α binds to KLF4(FL) and retains it in the cytoplasm thereby antagonizing the gene regulatory activities of KLF4(FL) in the nucleus. Our results establish KLF4α as a KLF4 isoform that opposes the function of KLF4(FL) and as an important factor in the complex and unresolved role of KLF4(FL) in breast carcinogenesis.

The expression of tenascin-C and tenascin-W in human ossicles.

The ossicles of the middle ear (the malleus, incus and stapes) transmit forces resulting from vibrations of the tympanic membrane to the cochlea where they are coded as sound. Hearing loss can result from diseases such as rheumatoid arthritis (RA) that affect the joints between the ossicles or degenerative processes like otosclerosis that lead to ankylosis of the footplate of the stapes in the oval window of the cochlea. In this study, immunohistochemistry was used to determine if the extracellular matrix glycoproteins tenascin-C or tenascin-W are expressed in the incudomalleolar and incudostapedial joints of ossicles dissected from human cadavers. Tenascin-C, which is expressed during inflammatory conditions including RA, was seen in the articular cartilage of the incudomalleolar joints and the head of the stapes. Tenascin-W, in contrast, was enriched in the annular ligament that anchors the footplate of the stapes into the oval window of the cochlea.

Missense mutations in TENM4, a regulator of axon guidance and central myelination, cause essential tremor.

Essential tremor (ET) is a common movement disorder with an estimated prevalence of 5% of the population aged over 65 years. In spite of intensive efforts, the genetic architecture of ET remains unknown. We used a combination of whole-exome sequencing and targeted resequencing in three ET families. In vitro and in vivo experiments in oligodendrocyte precursor cells and zebrafish were performed to test our findings. Whole-exome sequencing revealed a missense mutation in TENM4 segregating in an autosomal-dominant fashion in an ET family. Subsequent targeted resequencing of TENM4 led to the discovery of two novel missense mutations. Not only did these two mutations segregate with ET in two additional families, but we also observed significant over transmission of pathogenic TENM4 alleles across the three families. Consistent with a dominant mode of inheritance, in vitro analysis in oligodendrocyte precursor cells showed that mutant proteins mislocalize. Finally, expression of human mRNA harboring any of three patient mutations in zebrafish embryos induced defects in axon guidance, confirming a dominant-negative mode of action for these mutations. Our genetic and functional data, which is corroborated by the existence of a Tenm4 knockout mouse displaying an ET phenotype, implicates TENM4 in ET. Together with previous studies of TENM4 in model organisms, our studies intimate that processes regulating myelination in the central nervous system and axon guidance might be significant contributors to the genetic burden of this disorder.

Tenascin-C: Its functions as an integrin ligand.

This review summarizes the experimental evidence of tenascin-C/integrin interactions, emphasizing the identification of integrin binding sites and the effects of specific interactions on cell behavior. At least four integrins appear to bind to the third fibronectin-type 3 domain of tenascin-C: α9ß1, αVß3, α8ß1 and αVß6. The α9ß1 integrin recognizes a highly conserved IDG motif in this domain, while the others recognize an RGD motif. There is also significant evidence that the collagen receptor α2ß1 can bind to tenascin-C, but the interacting site is unknown. Tenascin-C interactions with α9ß1 and αVß3 can promote cell proliferation and interactions with αVß3 can also inhibit apoptosis. Interactions with α7ß1 integrin, which may bind to the alternatively spliced domain of tenascin-C, and α9ß1 integrin are able to influence the differentiation of mesenchymal stem cells into the neuronal lineage. This illustrates the potential for using our knowledge of tenascins and their integrin receptors in stem cell-based therapies.

Mechanism of irradiation-induced mammary cancer metastasis: A role for SAP-dependent Mkl1 signaling.

Radiotherapy is a standard treatment after conservative breast cancer surgery. However, cancers relapsing within a previously irradiated area have an increased probability to metastasize. The mechanisms responsible for this aggressiveness remain unclear. Here, we used the clinically relevant 4T1 breast cancer model mimicking aggressive local relapse after radiotherapy to identify differences between tumors grown in untreated versus preirradiated mammary glands. Tumors grown within preirradiated beds were highly enriched in transcripts encoding collagens and other proteins building or modifying the extracellular matrix, such as laminin-332, tenascins, lysyl oxidases and matrix metalloproteinases. Type I collagen, known to directly contribute to tissue stiffening, and the pro-metastatic megakaryoblastic leukemia-1 (Mkl1) target gene tenascin-C were further investigated. Mammary tissue preirradiation induced Mkl1 nuclear translocation in the tumor cells in vivo, indicating activation of Mkl1 signaling. Transcript profiling of cultured 4T1 cells revealed that the majority of the Mkl1 target genes, including tenascin-C, required serum response factor (SRF) for their expression. However, application of dynamic strain or matrix stiffness to 4T1 cells converted the predominant SRF/Mkl1 action into SAP domain-dependent Mkl1 signaling independent of SRF, accompanied by a switch to SAP-dependent tumor cell migration. 4T1 tumors overexpressing intact Mkl1 became more metastatic within preirradiated beds, while tumors expressing Mkl1 lacking the SAP domain exhibited impaired growth and metastatic spread, and decreased Mkl1 target gene expression. Thus, we identified SAP-dependent Mkl1 signaling as a previously unrecognized mediator of aggressive progression of mammary tumors locally relapsing after radiotherapy, and provide a novel signaling pathway for therapeutic intervention.

Transcriptional regulation of tenascin-W by TGF-beta signaling in the bone metastatic niche of breast cancer cells.

Tenascin-W is a matricellular protein with a dynamically changing expression pattern in development and disease. In adults, tenascin-W is mostly restricted to stem cell niches, and is also expressed in the stroma of solid cancers. Here, we analyzed its expression in the bone microenvironment of breast cancer metastasis. Osteoblasts were isolated from tumor-free or tumor-bearing bones of mice injected with MDA-MB231-1833 breast cancer cells. We found a fourfold upregulation of tenascin-W in the osteoblast population of tumor-bearing mice compared to healthy mice, indicating that tenascin-W is supplied by the bone metastatic niche. Transwell and co-culture studies showed that human bone marrow stromal cells (BMSCs) express tenascin-W protein after exposure to factors secreted by MDA-MB231-1833 breast cancer cells. To study tenascin-W gene regulation, we identified and analyzed the tenascin-W promoter as well as three evolutionary conserved regions in the first intron. 5'RACE analysis of mRNA from human breast cancer, glioblastoma and bone tissue showed a single tenascin-W transcript with a transcription start site at a noncoding first exon followed by exon 2 containing the ATG translation start. Site-directed mutagenesis of a SMAD4-binding element in proximity of the TATA box strongly impaired promoter activity. TGFß1 induced tenascin-W expression in human BMSCs through activation of the TGFß1 receptor ALK5, while glucocorticoids were inhibitory. Our experiments show that tenascin-W acts as a niche component for breast cancer metastasis to bone by supporting cell migration and cell proliferation of the cancer cells.

CCN4/WISP1 (WNT1 inducible signaling pathway protein 1): a focus on its role in cancer.

The matricellular protein WISP1 is a member of the CCN protein family. It is induced by WNT1 and is a downstream target of ß-catenin. WISP1 is expressed during embryonic development, wound healing and tissue repair. Aberrant WISP1 expression is associated with various pathologies including osteoarthritis, fibrosis and cancer. Its role in tumor progression and clinical outcome makes WISP1 an emerging candidate for the detection and treatment of tumors.

Transcriptional regulation of tenascin genes.

Extracellular matrix proteins of the tenascin family resemble each other in their domain structure, and also share functions in modulating cell adhesion and cellular responses to growth factors. Despite these common features, the 4 vertebrate tenascins exhibit vastly different expression patterns. Tenascin-R is specific to the central nervous system. Tenascin-C is an "oncofetal" protein controlled by many stimuli (growth factors, cytokines, mechanical stress), but with restricted occurrence in space and time. In contrast, tenascin-X is a constituitive component of connective tissues, and its level is barely affected by external factors. Finally, the expression of tenascin-W is similar to that of tenascin-C but even more limited. In accordance with their highly regulated expression, the promoters of the tenascin-C and -W genes contain TATA boxes, whereas those of the other 2 tenascins do not. This article summarizes what is currently known about the complex transcriptional regulation of the 4 tenascin genes in development and disease.

The intracellular domain of teneurin-1 induces the activity of microphthalmia-associated transcription factor (MITF) by binding to transcriptional repressor HINT1.

Teneurins are large type II transmembrane proteins that are necessary for the normal development of the CNS. Although many studies highlight the significance of teneurins, especially during development, there is only limited information known about the molecular mechanisms of function. Previous studies have shown that the N-terminal intracellular domain (ICD) of teneurins can be cleaved at the membrane and subsequently translocates to the nucleus, where it can influence gene transcription. Because teneurin ICDs do not contain any intrinsic DNA binding sequences, interaction partners are required to affect transcription. Here, we identified histidine triad nucleotide binding protein 1 (HINT1) as a human teneurin-1 ICD interaction partner in a yeast two-hybrid screen. This interaction was confirmed in human cells, where HINT1 is known to inhibit the transcription of target genes by directly binding to transcription factors at the promoter. In a whole transcriptome analysis of BS149 glioblastoma cells overexpressing the teneurin-1 ICD, several microphthalmia-associated transcription factor (MITF) target genes were found to be up-regulated. Directly comparing the transcriptomes of MITF versus TEN1-ICD-overexpressing BS149 cells revealed 42 co-regulated genes, including glycoprotein non-metastatic b (GPNMB). Using real-time quantitative PCR to detect endogenous GPNMB expression upon overexpression of MITF and HINT1 as well as promoter reporter assays using GPNMB promoter constructs, we could demonstrate that the teneurin-1 ICD binds HINT1, thus switching on MITF-dependent transcription of GPNMB.

The evolution of tenascins and fibronectin.

Tenascins are extracellular matrix glycoproteins that act both as integrin ligands and as modifiers of fibronectin-integrin interactions to regulate cell adhesion, migration, proliferation and differentiation. In tetrapods, both tenascins and fibronectin bind to integrins via RGD and LDV-type tripeptide motifs found in exposed loops in their fibronectin-type III domains. We previously showed that tenascins appeared early in the chordate lineage and are represented by single genes in extant cephalochordates and tunicates. Here we have examined the genomes of the coelacanth Latimeria chalumnae, the elephant shark Callorhinchus milii as well as the lampreys Petromyzon marinus and Lethenteron japonicum to learn more about the evolution of the tenascin gene family as well as the timing of the appearance of fibronectin during chordate evolution. The coelacanth has 4 tenascins that are more similar to tetrapod tenascins than are tenascins from ray-finned fishes. In contrast, only 2 tenascins were identified in the elephant shark and the Japanese lamprey L. japonicum. An RGD motif exposed to integrin binding is observed in tenascins from many, but not all, classes of chordates. Tetrapods that lack this RGD motif in tenascin-C have a similar motif in the paralog tenascin-W, suggesting the potential for some overlapping function. A predicted fibronectin with the same domain organization as the fibronectin from tetrapods is found in the sea lamprey P. marinus but not in tunicates, leading us to infer that fibronectin first appeared in vertebrates. The motifs that recognize LDV-type integrin receptors are conserved in fibronectins from a broad spectrum of vertebrates, but the RGD integrin-binding motif may have evolved in gnathostomes.

Tenascin-C is required for normal Wnt/ß-catenin signaling in the whisker follicle stem cell niche.

Whisker follicles have multiple stem cell niches, including epidermal stem cells in the bulge as well as neural crest-derived stem cells and mast cell progenitors in the trabecular region. The neural crest-derived stem cells are a pool of melanocyte precursors. Previously, we found that the extracellular matrix glycoproteins tenascin-C and tenascin-W are expressed near CD34-positive cells in the trabecular stem cell niche of mouse whisker follicles. Here, we analyzed whiskers from tenascin-C knockout mice and found intrafollicular adipocytes and supernumerary mast cells. As Wnt/ß-catenin signaling promotes melanogenesis and suppresses the differentiation of adipocytes and mast cells, we analyzed ß-catenin subcellular localization in the trabecular niche. We found cytoplasmic and nuclear ß-catenin in wild-type mice reflecting active Wnt/ß-catenin signaling, whereas ß-catenin in tenascin-C knockout mice was mostly cell membrane-associated and thus transcriptionally inactive. Furthermore, cells expressing the Wnt/ß-catenin target gene cyclin D1 were enriched in the CD34-positive niches of wild-type compared to tenascin-C knockout mice. We then tested the effects of tenascins on this signaling pathway. We found that tenascin-C and tenascin-W can be co-precipitated with Wnt3a. In vitro, substrate bound tenascins promoted ß-catenin-mediated transcription in the presence of Wnt3a, presumably due to the sequestration and concentration of Wnt3a near the cell surface. We conclude that the presence of tenascin-C in whiskers assures active Wnt/ß-catenin signaling in the niche thereby maintaining the stem cell pool and suppressing aberrant differentiation, while in the knockout mice with reduced Wnt/ß-catenin signaling, stem cells from the trabecular niche can differentiate into ectopic adipocytes and mast cells.

SAP domain-dependent Mkl1 signaling stimulates proliferation and cell migration by induction of a distinct gene set indicative of poor prognosis in breast cancer patients.

BACKGROUND: The main cause of death of breast cancer patients is not the primary tumor itself but the metastatic disease. Identifying breast cancer-specific signatures for metastasis and learning more about the nature of the genes involved in the metastatic process would 1) improve our understanding of the mechanisms of cancer progression and 2) reveal new therapeutic targets. Previous studies showed that the transcriptional regulator megakaryoblastic leukemia-1 (Mkl1) induces tenascin-C expression in normal and transformed mammary epithelial cells. Tenascin-C is known to be expressed in metastatic niches, is highly induced in cancer stroma and promotes breast cancer metastasis to the lung. METHODS: Using HC11 mammary epithelial cells overexpressing different Mkl1 constructs, we devised a subtractive transcript profiling screen to identify the mechanism by which Mkl1 induces a gene set co-regulated with tenascin-C. We performed computational analysis of the Mkl1 target genes and used cell biological experiments to confirm the effect of these gene products on cell behavior. To analyze whether this gene set is prognostic of accelerated cancer progression in human patients, we used the bioinformatics tool GOBO that allowed us to investigate a large breast tumor data set linked to patient data. RESULTS: We discovered a breast cancer-specific set of genes including tenascin-C, which is regulated by Mkl1 in a SAP domain-dependent, serum response factor-independent manner and is strongly implicated in cell proliferation, cell motility and cancer. Downregulation of this set of transcripts by overexpression of Mkl1 lacking the SAP domain inhibited cell growth and cell migration. Many of these genes are direct Mkl1 targets since their promoter-reporter constructs were induced by Mkl1 in a SAP domain-dependent manner. Transcripts, most strongly reduced in the absence of the SAP domain were mechanoresponsive. Finally, expression of this gene set is associated with high-proliferative poor-outcome classes in human breast cancer and a strongly reduced survival rate for patients independent of tumor grade. CONCLUSIONS: This study highlights a crucial role for the transcriptional regulator Mkl1 and its SAP domain during breast cancer progression. We identified a novel gene set that correlates with bad prognosis and thus may help in deciding the rigor of therapy.