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Osteosarcoma is a highly invasive bone malignancy in which metastasis accounts for the vast majority of death and morbidity in patients. Understanding the mechanisms controlling metastasis is essential for improving patient survival in this disease. Recent studies have shown that Metadherin (MTDH) plays an essential role in mediating tumorigenesis and metastasis in a variety of human cancers. Our study assessed the role of MTDH in osteosarcoma metastasis and elucidated the mechanisms underlying its metastasis-promoting activity. We used western blot, qPCR, and flow cytometry to measure the expression of MTDH in a panel of osteosarcoma cell lines, and used immunohistochemistry to confirm expression in patient-derived material. In parallel experiments we used MTDH-specific shRNA to reduce endogenous MTDH expression, and blocked cell surface MTDH by anti-MTDH antibodies. The impact of MTDH inhibition was assessed in vitro using transwell migration assays and matrigel invasion assays. We also developed an orthotopic xenograft mouse model to study the relationship between MTDH expression and osteosarcoma pulmonary metastasis. To investigate the role of MTDH in cell-extracellular matrix (ECM) interaction, a series of adhesion assays were performed to assess the adhesion ability of osteosarcoma cells with or without MTDH inhibition to major components of ECM. Compared to normal human osteoblasts, all osteosarcoma cell lines tested displayed increased MTDH expression. MTDH knockdown significantly reduced migration and invasion in osteosarcoma cells. Blockade of cell surface MTDH with an anti-MTDH antibody targeting the extracellular domain of MTDH also profoundly impeded osteosarcoma cell motility and invasiveness. In the in vivo experiments, down-regulation of MTDH in osteosarcoma cells delayed primary tumor growth and prohibited pulmonary metastasis. Immunohistochemical analysis of tumors samples isolated from the mice showed that lung metastases had elevated MTDH expression level compared to primary tumors. Altogether, these data indicated an important role for Metadherin in osteosarcoma metastasis. Further, knockdown of MTDH by shRNA and inhibition of cell surface MTDH by anti-MTDH antibodies both profoundly decreased cell adhesion to matrigel and specific ECM proteins, which suggested that MTDH, particularly the proportion localized at the cell surface, may be involved in the interaction between tumor cells and the non-cellular matrix proteins that constitute tissue stroma. The interaction will be confirmed by future experiments. By identifying the ECM protein binding partners for MTDH, we may establish a new paradigm for the function of MTDH in mediating tumor cell migration and invasion in osteosarcoma. Citation Format: Limin Zhu, Adrianna Buford, Yanwen Yang, Pingyu Zhang, Wei-Lien Wang, Dennis P. Hughes. Metadherin mediates tumor invasion and interaction with extracellular matrix proteins in osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2013-2683

Osteosarcoma is the most common type of bone malignancy in children. It is a highly invasive mesenchymal tumor in which metastasis accounts for the vast majority of mortality and morbidity in patients. Understanding the mechanisms controlling metastasis in osteosarcoma and developing novel therapeutic options targeting metastasis are essential for improving patient survival in this disease. Recent studies have shown that Metadherin (MTDH) plays a key role in mediating metastasis in a variety of human cancers. This study examined the role of MTDH in osteosarcoma metastasis and elucidated the mechanisms underlying its oncogenic activity. We used western blot, qPCR, and flow cytometry to measure the expression of MTDH in a panel of osteosarcoma cell lines. Patient-derived osteosarcoma samples were also examined for MTDH expression using immunohistochemistry. In parallel experiments we used MTDH-specific shRNA to down-regulate endogenous MTDH expression, and blocked cell surface MTDH by anti-MTDH antibodies. The impact of MTDH inhibition was assessed in vitro using migration assays and matrigel invasion assays. We also developed an osteosarcoma orthotopic xenograft model of NOD/SCID/IL2Rγ-deficient mice through intratibial injection of osteosarcoma cells with or without MTDH knockdown to study the relationship between MTDH expression and pulmonary metastatic potential of osteosarcoma cells. Compared to normal human osteoblasts, all osteosarcoma cell lines tested displayed elevated MTDH expression as shown by western blot, qPCR and flow cytometry analyses. MTDH knockdown reduced migration and decreased invasion in matrigel. Inhibition of cell surface MTDH with an anti-MTDH antibody targeting the extracellular domain of MTDH also profoundly impeded osteosarcoma cell motility and invasiveness in vitro. In an orthotopic xenograft mouse model using CCHD cells, inhibition of MTDH delayed primary tumor growth and prohibited pulmonary metastasis. All mice in the control group developed lung metastases while fewer than 20% of the mice in the MTDH-knockdown group showed sign of metastasis at six weeks after inoculation. MTDH knockdown also reduced mean lung weight and mean number of pulmonary metastasis. Immunohistochemical analysis of tumor samples showed that lung metastases had elevated MTDH expression level compared to primary tumors. Altogether these studies suggested that MTDH plays a critical role in promoting pulmonary metastasis of osteosarcoma and may serve as an attractive target for therapeutic intervention. Citation Format: Limin Zhu, Adrianna S. Buford, Yanwen Yang, Pingyu Zhang, Wei-Lien Wang, Dennis P. Hughes. Metadherin mediates osteosarcoma invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B67.

MTDH (metadherin), an important oncogene that is widely overexpressed in various cancers, is a potential biomarker of tumor malignancy. Variants in MTDH have been associated with susceptibility to breast cancer. However, no studies assessing MTDH gene polymorphisms and their potential relationship to ovarian cancer susceptibility have been reported. Thus, we investigated the association of MTDH (-470G>A) polymorphism with ovarian cancer development in 145 ovarian cancer patients and 254 matched control subjects, using sequence analysis. We found that the MTDH (-470G>A) polymorphism was statistically correlated with ovarian cancer risk (under the additive genetic model, GG vs. GA vs AA, P = 0.042). Compared with genotypes containing the G allele (GG and GA), the AA genotype may decrease the risk of ovarian cancer (P = 0.0198, OR = 0.33, 95% CI [0.12∼0.78]). Compared with the G allele, the A allele is protective against ovarian cancer risk (P = 0.01756, OR = 0.66, 95% CI [0.46∼0.93]). Furthermore, a statistically significant association between the GG and GA+AA genotypes and the clinical stage was observed (P = 0.038). These data suggest that MTDH (-470G>A) could be a useful molecular marker for assessing ovarian cancer risk and for predicting ovarian cancer patient prognosis.

Epithelial-mesenchymal transition (EMT) is an initiating event in tumor cell invasion and metastasis. It has been shown to occur in resistance to a range of cancer therapies, including tamoxifen. MicroRNAs (miRNAs) have been associated with EMT as well as resistance to standard therapies. To investigate the role of miRNAs in the development of resistance to tamoxifen as well as accompanying EMT-like properties, we established a tamoxifen resistant (TamR) model by continually exposing MCF-7 breast cancer cells to tamoxifen. In addition to the molecular changes known to be involved in acquired tamoxifen resistance, TamR cells displayed mesenchymal features and had increased invasiveness. Genome-wide miRNA microarray analysis revealed that miRNA-375 was among the top down-regulated miRNAs in resistant cells. Re-expression of miR-375 was sufficient to sensitize TamR cells to tamoxifen and partly reversed EMT. A combination of mRNA profiling, bioinformatic analysis, and experimental validation identified metadherin/MTDH as a direct target of miR-375. Knockdown of MTDH partially phenocopied the effects of miR-375 on the sensitivity to tamoxifen and the reversal of EMT. We observed an inverse correlation between the expression of miR-375 and its target MTDH in primary breast cancer samples, implying the pathological relevance of targeting. Finally, tamoxifen-treated patients with higher expression of MTDH had a shorter disease-free survival and higher risk of relapse. As most cancer-related deaths occur due to resistance to standard therapies and metastasis, re-expression of miR-375 or targeting MTDH might serve as potential therapeutic approaches for the treatment of tamoxifen resistant breast cancer.

Overexpression of metadherin (MTDH/AEG-1) has been documented in many solid tumors and is implicated in metastasis and chemoresistance. MTDH has been detected at the plasma membrane as well as in the cytoplasm and nucleus, and the function of MTDH in these locales remains under investigation. In the nucleus, MTDH acts as a transcription co-factor to induce expression of chemoresistance-associated genes. However, MTDH is predominantly cytoplasmic in solid tumors, and this localization has been shown to correlate with poor prognosis. In this study, we used endometrial cancer cells as a model system to define a new role for MTDH in the cytoplasm. First, MTDH was primarily localized to the cytoplasm in endometrial cancer cells, and the N-terminal region of MTDH was required to maintain cytoplasmic localization. Depletion of cytoplasmic MTDH in endometrial cancer cells restored chemosensitivity to various DNA damaging agents including mitomycin C (MMC) and doxorubicin. Next, we identified novel binding partners for cytoplasmic MTDH, including ribosome protein RPL4, ribosome biogenesis-related protein NPM1, and a component of the RNA induced silencing complex SND1. Association of MTDH with these cytoplasmic proteins was disrupted by nuclease treatment, demonstrating that nucleic acid is required for the interaction of cytoplasmic MTDH with these RNA metabolism and processing proteins. Furthermore, RNA binding protein-chromatin IP (RIP-chIP) analysis demonstrated that MTDH interacted with multiple mRNAs, and treatment with the PI3K and mTOR dual inhibitor BEZ235 altered the pattern of mRNAs that associate with MTDH. Knockdown of MTDH promoted an increase in protein levels of some MTDH-associated mRNAs. Finally, increased stress granule formation was observed in MTDH knockdown cells in response to heat shock, suggesting that one mechanism by which MTDH promotes chemoresistance is prevention of stress granule formation. Taken together, our data identify MTDH as an active RNA binding protein in the cytoplasm of cancer cells. Our findings suggest that the RNA binding properties of cytoplasmic MTDH serve to regulate genes involved in the drug resistance and metastasis of tumors and highlight MTDH as an ideal target for future drug development. Acknowledgements: This work was supported by NIH Grant R01CA99908 to K.K.L., and in part by the Department of Obstetrics and Gynecology Research Development Fund, the Institutional Research Grant Number IRG-77-004-31 from the American Cancer Society to XM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3053. doi:1538-7445.AM2012-3053

Understanding the molecular mechanisms of chemoresistance is vital to design therapies to restore chemosensitivity. In particular, metadherin (MTDH) has been demonstrated to have a critical role in chemoresistance. Over-expression of MTDH has recently been correlated with poor clinical outcomes in breast cancer, neuroblastoma, hepatocellular carcinoma and prostate cancer. In endometrial cancer patients, lymph node metastasis and increase of mortality were significantly associated with chromosome 8q22-q23copy number gains, which is where MTDH is localized. However, it is unclear how MTDH overexpression mechanistically contributes to endometrial cancer development, progression, or response to therapy. In this present study, we detected overexpression of MTDH in endometrial cancer tissues compared to normal endometrium. Studies in endometrial cancer cell lines indicate that MTDH depletion enhanced sensitivity to chemotherapy agents as well as cell death induced by a combinatorial treatment of histone deacetylase (HDAC) inhibitor and tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). TRAIL promotes death of cancerous cells of the human reproductive tract, and HDAC inhibitors, such as LBH 589 used in this study, have been shown to increase sensitivity of cancer cells to TRAIL-induced apoptosis. Inhibition of PI3K/AKT survival pathway such as PDK1 and AKT phosphorylation, increased degradation of anti-apoptosis protein XIAP, and increased expression of pro-apoptosis protein Bim and cleaved active isoforms of caspase 8 and caspase 3 were all observed during cell death induced by TRAIL and LBH 589 combination treatment in MTDH depleted endometrial cancer cells. However, in cells with normal MTDH expression, TRAIL/LBH 589 treatment had no effect on these pro-survival factors. Interestingly, MTDH was also found to be involved in G2/M checkpoint regulation. Treatment of MTDH-depleted cells with TRAIL and LBH589 resulted in an increased number of cells arrested in G2/M compared to cells with expressing MTDH. Using microarray technology, 57 genes with greater than two-fold gene expression alteration were identified after MTDH depletion, including Calbindin 1 and Galectin 1, which may contribute to MTDH-mediated drug resistance. Therefore, MTDH mediates drug resistance by regulating expression of genes required for the control of apoptosis and cell cycle. These findings indicate that sensitivity to chemotherapy agents and combination treatment with HDAC inhibitor and TRAIL can be restored or enhanced by manipulating MTDH, and hence depletion of MTDH is a potentially novel avenue for effective cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 71. doi:10.1158/1538-7445.AM2011-71

Plausible genome-wide associations for episodic neurological diseases (such as migraine, epilepsy and ataxias) have been slow to emerge. The first such association was reported in a recent genome-wide association study of migraine, with quantitative expression analysis linking the variant to a nearby regulatory gene, MTDH/AEG-1. This putative mechanism, regulating the expression of the primary glutamate transporter in the brain, EAAT2/GLT-1, has interesting implications bridging the gap between Mendelian and common forms in this key group of disorders.

Rationale Mannitol dehydrogenase (MtDH) has previously been shown to be the major allergen of Cladosporium herbarum. Since cross-reactivity has been demonstrated for several fungal allergens we have cloned the homolog protein of Alternaria alternata and tested it in respect to its IgE-reactivity. Methods A. alternata MtDH was cloned by screening of an in vivo excised A. alternata cDNA library (pBluescript SK) using the C. herbarum homolog as a hybridization probe. In order to obtain a recombinant non fusion and a 6xHIS fusion protein, the coding sequence was subcloned into the pMW172 and the pHIS-parallel2 vector, respectively. Expression of the recombinant proteins was performed in the E. coli strain Bl21(DE3). The recombinant non fusion protein was purified by ammonium sulphate precipitation, hydrophobic interaction chromatography, anion exchange chromatography and Cibacron Blue F3G-A affinity chromatography. IgE immunoblots were performed utilizing the 6xHIS fusion protein purified by Ni2+ complexed Chelating Sepharose affinity chromatography. Results A. alternata MtDH has been cloned. The coding sequence of the full-length cDNA clone comprises 798 bp and 266 amino acids, respectively. Protein sequence analysis revealed an identity of 74% and a homology of 80,5% between the MtDHs of A. alternata and C. herbarum. The purified recombinant protein was enzymatically active as shown by spectrophotometrical analysis. In IgE-immunoblots 9 out of 28 (34%) A. alternata patients reacted with the 6xHIS-MtDH. Conclusion Mannitol dehydrogenase was shown to be a new important allergen of A. alternata.

Affinity-purified, electrophoretically homogeneous NADP + -mannitol dehydrogenase (MtDH; EC 1.1.1.138), isolated from fruit bodies of Agaricus bisporus (Lange) Sing., was used to produce polyclonal antibodies. Antiserum against MtDH and the affinity-purified antibody inhibited enzyme activity in a dose-dependent manner, with about 15 mol of purified antibody required for 50% inhibition of 1 mol MtDH. Immunological specificity of the antisera was demonstrated by double immunodiffusion, the dotimmunobinding assay, and immunoblotting. Controls (preimmune sera and preadsorbed antisera) were negative. An enzyme-linked immunosorbent assay (ELISA) was established to quantitate immunobinding in various cell fractions of the fungus. The bound protein was found predominantly in the soluble fraction (150,000 g ), where it contributed 5% to the total protein of the fruit body and 1% to that of the mycelium. Though only to a minor extent, the anti-MtDH antiserum also bound to the supernatants obtained following treatment of a “mixed membrane fraction” and the cell wall fraction (54,000 g and 4,000 g sediments, respectively; both rigorously washed with buffer) with digitonin or sodium dodecyl sulfate/heat. In sporocarp extracts, the antibody bound specifically to a protein of M r 30K (corresponding to the subunit molecular weight of MtDH) as shown by immunoblotting, whereas mycelial extracts contained three (30K, 26K, 24K) protein bands, which cross-reacted with the anti-MtDH antiserum.

SummaryThe Metadherin gene (MTDH) is prevalently amplified in breast cancer and associated with poor prognosis; however, its functional contribution to tumorigenesis is poorly understood. Using mouse models representing different subtypes of breast cancer, we demonstrated that MTDH plays a critical role in mammary tumorigenesis by regulating oncogene-induced expansion and activities of tumor-initiating cells (TICs), whereas it is largely dispensable for normal development. Mechanistically, MTDH supports the survival of mammary epithelial cells under oncogenic/stress conditions by interacting with and stabilizing Staphylococcal nuclease domain-containing 1 (SND1). Silencing MTDH or SND1 individually or disrupting their interaction compromises tumorigenenic potential of TICs in vivo. This functional significance of MTDH-SND1 interaction is further supported by clinical analysis of human breast cancer samples.

© Springer Science+Business Media Dordrecht 2014, Colorectal cancer (CRC) is the third most common malignant disease in men and the second in women worldwide. CRC relapse occurs mostly in liver and lungs, decreasing the 5-year survival to 6 %. Metadherin (MTDH) is overexpressed in several types of cancer, has been implicated in proliferation, invasion, metastasis, angiogenesis, and chemoresistance, and is a factor of poor prognosis in CRC. In this work we addressed the prognostic significance of MTDH expression in CRC progression to the lungs. We found that MTDH gene was more frequently amplified (copy number >1.8) in patients with CRC and relapse to the lung, when compared to patients without lung metastases (17.4 vs 100 %; p < 0.001). We observed a correlation between MTDH gene copy number and MTDH expression by IHC (p = 0.0001). Next we also analyzed MTDH expression by IHC in samples from 85 patients diagnosed with CRC, stage II or III, M0, with at least 3 years of follow-up. Kaplan-Meier survival analysis showed that lung relapse-free survival (HR 5.29, 95 % CI 1.90-14.77, p = 0.0004), liver relapse-free survival (HR 8.59, 95 % CI 0.99-74.18, p = 0.003), relapse-free survival (HR 4.85, 95 % CI 1.88-12.45, p = 0.0003) and overall survival (HR 3.75, 95 % CI 1.15-12.18, p = 0.018) were significantly lower in the group with high MTDH expression. Multivariate analysis showed that high MTDH expression was an independent factor for all outcomes. This study demonstrates that high MTDH expression is a biomarker of relapse in CRC, including lung-specific relapse. Determination of MTDH expression in primary CRC may be useful in the earlier detection of lung metastases in patients with high expression and increased risk., This project was supported by a Grant from Fundação para a Ciência e Tecnologia (PIC/IC/82821/2007).S. Casimiro was supported by a Post-Doctoral Grant (SFRH/BPD/34801/2007) and J. Tato-Costa by a PhD Grant (SFRH/BD/45219/2008) from Fundação para a Ciência e Tecnologia.

SummaryTargeted therapy for metastatic diseases relies on the identification of functionally important metastasis genes from a large number of random genetic alterations. Here we use a computational algorithm to map minimal recurrent genomic alterations associated with poor-prognosis breast cancer. 8q22 genomic gain was identified by this approach and validated in an extensive collection of breast tumor samples. Regional gain of 8q22 elevates expression of the metastasis gene metadherin (MTDH), which is overexpressed in more than 40% of breast cancers and is associated with poor clinical outcomes. Functional characterization of MTDH revealed its dual role in promoting metastatic seeding and enhancing chemoresistance. These findings establish MTDH as an important therapeutic target for simultaneously enhancing chemotherapy efficacy and reducing metastasis risk.

In Escherichia coli the great majority of one-carbon units enter the folate pathway as N5,N10-methylene-THF. This compound is utilized directly in the biosynthesis of thymidylate, converted to N5-methyl-THF for use in methionine biosynthesis, and to N10-formyl-THF, which provides carbons 2 and 8 of purines and the formyl group of f-met-tRNA. Since the proportions utilized for different end-products can vary widely with changing conditions of growth, the distribution of N5,N10-methylene-THF must be regulated in some way. We have studied the regulation of the conversion of N5,N10-methylene-THF to N10-formyl-THF which is catalyzed by N5,N10-methylene-THF dehydrogenase (MTDH) which converts N5,N10-methylene-THF to N5,N10-methenyl-THF, and N5,N10-methenyl-THF cyclohydrolase (MTC), which converts N5,N10-methenyl-THF to N10-formyl-THF. We concluded that the regulation could not be by control of enzyme synthesis since the specific activity of both enzymes increased with decreasing specific growth rate. N10-formyl-THF was found to be a noncompetitive, allosteric inhibitor of MTDH, and a competitive inhibitor of MTC. The value of Ki for both inhibitions was about 5 × 10−5m. The intracellular concentration of total folates is in the range of 2 to 5 × 10−4m in E. coli; thus the concentration of N10-formyl-THF will be such that significant inhibition will occur. The location of N10-formyl-THF in the folate pathway is such that its concentration can increase as its rate of utilization in purine biosynthesis decreases, as would be expected if the inhibition were to have regulatory significance; however, we do not yet have experimental proof that its concentration does change in the expected manner. Solutions have been calculated for the operation of a model folate pathway as it supplies the pathways of purine, deoxypyrimidine and methionine biosynthesis in a growing cell. The results of these calculations showed that inhibition of MTDH by N10-formyl-THF is advantageous to the cell, in that it permits a higher specific growth rate than if the inhibition did not occur; and that it can be essential, insofar as circumstances exist in which a steady state is impossible without the inhibition. However, it was also shown that inhibition of MTDH by N10-formyl-THF alone was insufficient to regulate the distribution of N5,N10-methylene-THF when de novo purine biosynthesis is reduced by the addition of exogenous purines. We concluded that N10-formyl-THF inhibition of MTDH is a significant element in the regulation of MTDH, but that other mechanisms of regulation exist which we have yet to identify.

Feline mammary carcinomas (FMCs) with anaplastic and malignant spindle cells histologically resemble the human metaplastic breast carcinoma (hMBC), spindle-cell subtype. hMBCs display epithelial-to-mesenchymal transition (EMT) characteristics. Herein we report the establishment and characterization of a cell line (TiHoCMglAdcar0906; TiHo-0906) exhibiting EMT-like properties derived from an FMC with anaplastic and malignant spindle cells. Copy-number variations (CNVs) by next-generation sequencing and immunohistochemical characteristics of the cell line and the tumour were compared. The absolute qPCR expression of EMT-related markers HMGA2 and CD44 was determined. The growth, migration, and sensitivity to doxorubicin were assessed. TiHo-0906 CNVs affect several genomic regions harbouring known EMT-, breast cancer-, and hMBCs-associated genes as AKT1, GATA3, CCND2, CDK4, ZEB1, KRAS, HMGA2, ESRP1, MTDH, YWHAZ, and MYC. Most of them were located in amplified regions of feline chromosomes (FCAs) B4 and F2. TiHo-0906 cells displayed an epithelial/mesenchymal phenotype, and high HMGA2 and CD44 expression. Growth and migration remained comparable during subculturing. Low-passaged cells were two-fold more resistant to doxorubicin than high-passaged cells (IC50: 99.97 nM, and 41.22 nM, respectively). The TiHo-0906 cell line was derived from a poorly differentiated cellular subpopulation of the tumour consistently displaying EMT traits. The cell line presents excellent opportunities for studying EMT on FMCs.

The enormity and complexity of cancer genome data present significant challenges in downstream validation of novel oncogenes and tumor suppressors. In this issue of Cancer Cell , Hu et al. evaluate candidate oncogenes in a recurrent amplification in poor-prognosis breast cancers. They identify and validate the prometastatic gene metadherin ( MTDH ) as a key modulator of endothelial adhesion and chemoresistance.