Martin Degen

Tenascin-C is a novel RBPJkappa-induced target gene for Notch signaling in gliomas.

Tenascin-C (TNC) expression is known to correlate with malignancy in glioblastoma (GBM), a highly invasive and aggressive brain tumor that shows limited response to conventional therapies. In these malignant gliomas as well as in GBM cell lines, we found Notch2 protein to be strongly expressed. In a GBM tumor tissue microarray, RBPJk protein, a Notch2 cofactor for transcription, was found to be significantly coexpressed with TNC. We show that the TNC gene is transactivated by Notch2 in an RBPJk-dependent manner mediated by an RBPJk binding element in the TNC promoter. The transactivation is abrogated by a Notch2 mutation, which we detected in the glioma cell line Hs683 that does not express TNC. This L1711M mutation resides in the RAM domain, the site of interaction between Notch2 and RBPJk. In addition, transfection of constructs encoding activated Notch2 or Notch1 increased endogenous TNC expression identifying TNC as a novel Notch target gene. Overexpression of a dominant negative form of the transcriptional coactivator MAML1 or knocking down RBPJk in LN319 cells led to a dramatic decrease in TNC protein levels accompanied by a significant reduction of cell migration. Because addition of purified TNC stimulated glioma cell migration, this represents a mechanism for the invasive properties of glioma cells controlled by Notch signaling and defines a novel oncogenic pathway in gliomagenesis that may be targeted for therapeutic intervention in GBM patients.

Tenascin-W is found in malignant mammary tumors, promotes alpha8 integrin-dependent motility and requires p38MAPK activity for BMP-2 and TNF-alpha induced expression in vitro.

Tenascins represent a family of extracellular matrix glycoproteins with distinctive expression patterns. Here we have analyzed the most recently described member, tenascin-W, in breast cancer. Mammary tumors isolated from transgenic mice expressing hormone-induced oncogenes reveal tenascin-W in the stroma around lesions with a high likelihood of metastasis. The presence of tenascin-W was correlated with the expression of its putative receptor, α8 integrin. HC11 cells derived from normal mammary epithelium do not express α8 integrin and fail to cross tenascin-W-coated filters. However, 4T1 mammary carcinoma cells do express α8 integrin and their migration is stimulated by tenascin-W. The expression of tenascin-W is induced by BMP-2 but not by TGF-β1, though the latter is a potent inducer of tenascin-C. The expression of tenascin-W is dependent on p38MAPK and JNK signaling pathways. Since preinflammatory cytokines also act through p38MAPK and JNK signaling pathways, the possible role of TNF-α in tenascin-W expression was also examined. TNF-α induced the expression of both tenascin-W and tenascin-C, and this induction was p38MAPK- and cyclooxygenase-dependent. Our results show that tenascin-W may be a useful diagnostic marker for breast malignancies, and that the induction of tenascin-W in the tumor stroma may contribute to the invasive behavior of tumor cells.

Tenascin-W Is a Novel Marker for Activated Tumor Stroma in Low-grade Human Breast Cancer and Influences Cell Behavior

This is the first report about human tenascin-W, the fourth and final member of the extracellular matrix protein family of tenascins. Sixty-three human breast tumor extracts were analyzed by Western blotting for the presence of tenascin-W and compared with tenascin-C, an established marker of tumor stroma. Interestingly, we found tenascin-W expression in the majority of the tumor tissues, but no detectable expression in the normal mammary parenchyma. Eighty-one percent of the breast tumor samples were tenascin-W positive and 86% showed expression of tenascin-C. However, tenascin-W and tenascin-C amounts varied greatly between tumors and some contained either tenascin-W or tenascin-C exclusively, indicating independent mechanisms regulating their expression. Although there was no difference between high- or low-grade tumors with respect to the presence of tenascin-C, tenascin-W was more prominent in low-grade tumors. For 42 of the breast cancer tissues, a frozen tumor microarray was available to confirm the Western blot data by immunohistochemistry. Similar to tenascin-C, tenascin-W was detected in the tumor stroma. Fibroblasts adhered to tenascin-W in a beta(1) integrin-dependent manner and spread with a distinctive morphology under conditions where they remained round on tenascin-C. CHOB2 cells expressing alpha(v)beta(1) or alpha4beta(1) integrins were able to spread on tenascin-W. Furthermore, addition of tenascin-W to the culture medium increased migration of breast cancer cells toward a fibronectin substratum in vitro. These data imply that tenascin-W expression in the activated tumor stroma facilitates tumorigenesis by supporting the migratory behavior of breast cancer cells.

Tenascin-W, a new marker of cancer stroma, is elevated in sera of colon and breast cancer patients.

Tenascins are extracellular matrix proteins present during the development of organisms as well as in pathological conditions. Tenascin‐W, the fourth and last member of the tenascin family remains the least well‐characterized one. Our study aimed to evaluate the potential significance of tenascin‐W as cancer biomarker by monitoring its presence in the serum of colorectal and breast cancer patients and its expression in colorectal tumor tissues. To measure serum tenascin‐W levels, a sensitive sandwich‐ELISA was established. Mean tenascin‐W concentration in sera of patients with nonmetastatic colorectal cancer at time of diagnosis was highly increased compared to that of healthy volunteers. A similar tendency was observed for tenascin‐C in the same patient cohort. However, the increase was much more striking for tenascin‐W. We also detected elevated tenascin‐W levels in sera of breast cancer patients. Furthermore, we could show a prominent expression of tenascin‐W in extracts from colorectal tumor tissues by immunoblot analysis, whereas tenascin‐W was not detectable in the corresponding normal colon mucosa. To confirm the western blot results, we performed immunohistochemistry of frozen sections of the same patients as well as of an additional, independently chosen collection of colorectal cancer tissues. In all cases, similarly to tenascin‐C, tenascin‐W was detected in the tumor stroma. Our results reveal a clear association between elevated levels of tenascin‐W and the presence of cancer. These results warrant further studies to evaluate the potential value of serum and tissue tenascin‐W levels as diagnostic, prognostic or monitoring biomarker in colorectal, breast and possibly other solid cancers.

Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro

The microenvironment hosting a tumor actively participates in regulating tumor cell proliferation, migration, and invasion. Among the extracellular matrix proteins enriched in the stroma of carcinomas are the tenascin family members tenascin‐C and tenascin‐W. Whereas tenascin‐C overexpression in gliomas is known to correlate with poor prognosis, the status of tenascin‐W in brain tumors has not been investigated so far. In the present study, we analyzed protein levels of tenascin‐W in 38 human gliomas and found expression of tenascin‐W in 80% of the tumor samples, whereas no tenascin‐W could be detected in control, nontumoral brain tissues. Double immunohistochemical staining of tenascin‐W and von Willebrand factor revealed that tenascin‐W is localized around blood vessels, exclusively in tumor samples. In vitro, the presence of tenascin‐Wincreased the proportion of elongated human umbilical vein endothelial cells (HUVECs) and augmented the mean speed of cell migration. Furthermore, tenascin‐W triggered sprouting of HUVEC spheroids to a similar extent as the proangiogenic factor tenascin‐C. In conclusion, our study identifies tenascin‐W as a candidate biomarker for brain tumor angiogenesis that could be used as a molecular target for therapy irrespective of the glioma subtype.—Martina, E., Degen, M., Ruegg, C., Merlo, A., Lino, M. M., Chiquet‐Ehrismann, R., Brellier, F. Tenascin‐W is a specific marker of glioma‐associated blood vessels and stimulates angiogenesis in vitro. FASEB J. 24, 778–787 (2010). www.fasebj.org

Clinical Classification of Cancer Cachexia: Phenotypic Correlates in Human Skeletal Muscle

Background Cachexia affects the majority of patients with advanced cancer and is associated with a reduction in treatment tolerance, response to therapy, and duration of survival. One impediment towards the effective treatment of cachexia is a validated classification system. Methods 41 patients with resectable upper gastrointestinal (GI) or pancreatic cancer underwent characterisation for cachexia based on weight-loss (WL) and/or low muscularity (LM). Four diagnostic criteria were used >5%WL, >10%WL, LM, and LM+>2%WL. All patients underwent biopsy of the rectus muscle. Analysis included immunohistochemistry for fibre size and type, protein and nucleic acid concentration, Western blots for markers of autophagy, SMAD signalling, and inflammation. Findings Compared with non-cachectic cancer patients, patients with LM or LM+>2%WL, mean muscle fibre diameter was reduced by about 25% (p = 0.02 and p = 0.001 respectively). No significant difference in fibre diameter was observed if patients had WL alone. Regardless of classification, there was no difference in fibre number or proportion of fibre type across all myosin heavy chain isoforms. Mean muscle protein content was reduced and the ratio of RNA/DNA decreased in patients with either >5%WL or LM+>2%WL. Compared with non-cachectic patients, SMAD3 protein levels were increased in patients with >5%WL (p = 0.022) and with >10%WL, beclin (p = 0.05) and ATG5 (p = 0.01) protein levels were increased. There were no differences in phospho-NFkB or phospho-STAT3 levels across any of the groups. Conclusion Muscle fibre size, biochemical composition and pathway phenotype can vary according to whether the diagnostic criteria for cachexia are based on weight loss alone, a measure of low muscularity alone or a combination of the two. For intervention trials where the primary end-point is a change in muscle mass or function, use of combined diagnostic criteria may allow identification of a more homogeneous patient cohort, reduce the sample size required and enhance the time scale within which trials can be conducted.

Avian tenascin-W: expression in smooth muscle and bone, and effects on calvarial cell spreading and adhesion in vitro.

Tenascins are glycoproteins found primarily in the embryonic extracellular matrix. Here we have characterized the fourth and final member of the tenascin family in birds: tenascin‐W. Avian tenascin‐W has 3.5 epidermal growth factor‐like repeats, 6 fibronectin type III domains, and a C‐terminal fibrinogen‐related domain. Immunohistochemistry reveals that avian tenascin‐W is expressed transiently in developing smooth muscle, tendons, and ligaments, but the primary site of tenascin‐W expression during development is in the extracellular matrix of bone and the cellular periosteum. In bony matrix, tenascin‐W‐coated fibrils partly overlap with fibrils that contain tenascin‐C. The anti‐tenascin‐W also labels fibrils in cultures of osteogenic embryonic chicken calvarial cells. Primary calvarial cells cultured on purified tenascin‐W become rounded, and fewer of these cells spread on fibronectin when tenascin‐W is added to the medium when compared with calvarial cells cultured on fibronectin alone. Moreover, tenascin‐W reduces the adhesion of calvarial cells to collagen type I in a shear force assay. We conclude that tenascin‐W is likely to play a phylogenetically conserved role in developing bone and that it shares some of the basic anti‐adhesive and matrix modulatory properties as tenascin‐C. Developmental Dynamics 235:1532–1542, 2006.

Tenascin-W is a better cancer biomarker than tenascin-C for most human solid tumors

BackgroundTenascins are large glycoproteins found in the extracellular matrix of many embryonic and adult tissues. Tenascin-C is a well-studied biomarker known for its high overexpression in the stroma of most solid cancers. Tenascin-W, the least studied member of the family, is highly expressed in the stroma of colon and breast tumors and in gliomas, but not in the corresponding normal tissues. Other solid tumors have not been analyzed. The present study was undertaken to determine whether tenascin-W could serve as a cancer-specific extracellular matrix protein in a broad range of solid tumors.MethodsWe analyzed the expression of tenascin-W and tenascin-C by immunoblotting and by immunohistochemistry on multiple frozen tissue microarrays of carcinomas of the pancreas, kidney and lung as well as melanomas and compared them to healthy tissues.ResultsFrom all healthy adult organs tested, only liver and spleen showed detectable levels of tenascin-W, suggesting that tenascin-W is absent from most human adult organs under normal, non-pathological conditions. In contrast, tenascin-W was detectable in the majority of melanomas and their metastases, as well as in pancreas, kidney, and lung carcinomas. Comparing lung tumor samples and matching control tissues for each patient revealed a clear overexpression of tenascin-W in tumor tissues. Although the number of samples examined is too small to draw statistically significant conclusions, there seems to be a tendency for increased tenascin-W expression in higher grade tumors. Interestingly, in most tumor types, tenascin-W is also expressed in close proximity to blood vessels, as shown by CD31 co-staining of the samples.ConclusionsThe present study extends the tumor biomarker potential of tenascin-W to a broad range of solid tumors and shows its accessibility from the blood stream for potential therapeutic strategies.

Effects of tenascin-W on osteoblasts in vitro

Tenascin-W is a glycoprotein secreted into the extracellular matrix of developing bones. Here, we have examined possible roles for tenascin-W in osteogenesis. Purified recombinant tenascin-W, like tenascin-C, increases the number of mineralized foci in primary cultures of avian osteoblasts and increases alkaline phosphatase activity in vitro. In addition, tenascin-W in solution promotes the migration of primary osteoblasts across fibronectin-coated filters. The sixth fibronectin type III domain of chicken tenascin-W contains a phylogenetically conserved KGD motif that is predicted to be available to integrin binding. To determine whether this motif is potentially functional, we have cultured osteoblasts on KGD-containing peptides and control peptides. Osteoblasts cultured on peptides with the KGD motif acquire a multipolar phenotype with pseudopods tipped with actin-rich ruffles, which is similar to the morphology of osteoblasts cultured on recombinant tenascin-W. Moreover, the KGD peptides, but not the control peptides, promote proliferation in cultured osteoblasts but not alkaline phosphatase activity or migration. Finally, explanted embryonic frontal bones are significantly thicker when cultured in the presence of tenascin-W, suggesting that tenascin-W can accelerate the formation of new bone in a complex multicellular environment.

Loss of oxidative defense and potential blockade of satellite cell maturation in the skeletal muscle of patients with cancer but not in the healthy elderly

Muscle wasting in old age or cancer may result from failed myofiber regeneration and/or accelerated atrophy. This study aimed to determine from transcriptomic analysis of human muscle the integrity of the cellular stress response system in relation to satellite cell differentiation or apoptosis in patients with cancer (weight-stable (CWS) or weight-losing (CWL)) or healthy elderly (HE) when compared with healthy middle-aged controls (HMA). 28 patients with cancer (CWS: 18 and CWL: 10), HE: 21 and HMA: 20 underwent biopsy of quadriceps muscle. The expression of transcription factors for muscle regeneration (Pax3, Pax7 and MyoD) was increased in CWS and HE compared with HMA (p<0.001). In contrast, the expression of the late myogenic differentiation marker MyoG was reduced in CWS and CWL but increased in HE (p<0.0001). Bax was significantly increased in CWS, CWL and HE (P<0.0001). Expression of the oxidative defense genes SOD2, GCLM, and Nrf2 was decreased in CWS and CWL but increased in HE (p<0.0001). There is evidence for blockade of satellite cell maturation, upregulation of apoptosis and reduced oxidative defense in the muscle of cancer patients. In the healthy elderly the potential for differentiation and oxidative defense is maintained.