Eva M Hernando-Monge, Ph.D.

miR-30b/30d Regulation of GalNAc Transferases Enhances Invasion and Immunosuppression during Metastasis
Gaziel-Sovran, Avital; Segura, Miguel F; Di Micco, Raffaella; Collins, Mary K; Hanniford, Douglas; Vega-Saenz de Miera, Eleazar; Rakus, John F; Dankert, John F; Shang, Shulian; Kerbel, Robert S; Bhardwaj, Nina; Shao, Yongzhao; Darvishian, Farbod; Zavadil, Jiri; Erlebacher, Adrian; Mahal, Lara K; Osman, Iman; Hernando, Eva
2011 Jul 12;20(1):104-118, Cancer cell
To metastasize, a tumor cell must acquire abilities such as the capacity to colonize new tissue and evade immune surveillance. Recent evidence suggests that microRNAs can promote the evolution of malignant behaviors by regulating multiple targets. We performed a microRNA analysis of human melanoma, a highly invasive cancer, and found that miR-30b/30d upregulation correlates with stage, metastatic potential, shorter time to recurrence, and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment. These data support a key role of miR-30b/30d and GalNAc transferases in metastasis, by simultaneously promoting cellular invasion and immunosuppression
— id: 135264, year: 2011, vol: 20, page: 104, stat: Journal Article,

Efficient in vivo microRNA targeting of liver metastasis
Huynh, C; Segura, M F; Gaziel-Sovran, A; Menendez, S; Darvishian, F; Chiriboga, L; Levin, B; Meruelo, D; Osman, I; Zavadil, J; Marcusson, E G; Hernando, E
2011 Mar 24;30(12):1481-1488, Oncogene
Targeting oncogenic microRNAs (miRNAs) is emerging as a promising strategy for cancer therapy. In this study, we provide proof of principle for the safety and efficacy of miRNA targeting against metastatic tumors. We tested the impact of targeting miR-182, a pro-metastatic miRNA frequently overexpressed in melanoma, the in vitro silencing of which represses invasion and induces apoptosis. Specifically, we assessed the effect of anti-miR-182 oligonucleotides synthesized with 2' sugar modifications and a phosphorothioate backbone in a mouse model of melanoma liver metastasis. Luciferase imaging showed that mice treated with anti-miR-182 had a lower burden of liver metastases compared with control. We confirmed that miR-182 levels were effectively downregulated in the tumors of anti-miR-treated mice compared with tumors of control-treated mice, both in the liver and in the spleen. This effect was accompanied by an upregulation of multiple miR-182 direct targets. Transcriptional profiling of tumors treated with anti-miR-182 or with control oligonucleotides revealed an enrichment of genes controlling survival, adhesion and migration modulated in response to anti-miR-182 treatment. These data indicate that in vivo administration of anti-miRs allows for efficient miRNA targeting and concomitant upregulation of miRNA-controlled genes. Our results demonstrate that the use of anti-miR-182 is a promising therapeutic strategy for metastatic melanoma and provide a solid basis for testing similar strategies in human metastatic tumors
— id: 138159, year: 2011, vol: 30, page: 1481, stat: Journal Article,

Correction: The Novel Gamma Secretase Inhibitor RO4929097 Reduces the Tumor Initiating Potential of Melanoma
Huynh, Chanh; Poliseno, Laura; Segura, Miguel F; Medicherla, Ratna; Haimovic, Adele; Menendez, Silvia; Shang, Shulian; Pavlick, Anna; Shao, Yongzhao; Darvishian, Farbod; Boylan, John F; Osman, Iman; Hernando, Eva
2011 ;6(11):?-?, PLoS ONE
[This corrects the article on p. e25264 in vol. 6.]
— id: 141637, year: 2011, vol: 6, page: ?, stat: Journal Article,

The Novel Gamma Secretase Inhibitor RO4929097 Reduces the Tumor Initiating Potential of Melanoma
Huynh, Chanh; Poliseno, Laura; Segura, Miguel F; Medicherla, Ratna; Haimovic, Adele; Menendez, Silvia; Shang, Shulian; Pavlick, Anna; Shao, Yongzhao; Darvishian, Farbod; Boylan, John F; Osman, Iman; Hernando, Eva
2011 ;6(9):e25264-e25264, PLoS ONE
Several reports have demonstrated a role for aberrant NOTCH signaling in melanoma genesis and progression, prompting us to explore if targeting this pathway is a valid therapeutic approach against melanoma. We targeted NOTCH signaling using RO4929097, a novel inhibitor of gamma secretase, which is a key component of the enzymatic complex that cleaves and activates NOTCH. The effects of RO4929097 on the oncogenic and stem cell properties of a panel of melanoma cell lines were tested both in vitro and in vivo, using xenograft models. In human primary melanoma cell lines, RO4929097 decreased the levels of NOTCH transcriptional target HES1. This was accompanied by reduced proliferation and impaired ability to form colonies in soft agar and to organize in tridimensional spheres. Moreover, RO4929097 affected the growth of human primary melanoma xenograft in NOD/SCID/IL2gammaR-/- mice and inhibited subsequent tumor formation in a serial xenotransplantation model, suggesting that inhibition of NOTCH signaling suppresses the tumor initiating potential of melanoma cells. In addition, RO4929097 decreased tumor volume and blocked the invasive growth pattern of metastatic melanoma cell lines in vivo. Finally, increased gene expression of NOTCH signaling components correlated with shorter post recurrence survival in metastatic melanoma cases. Our data support NOTCH inhibition as a promising therapeutic strategy against melanoma
— id: 138712, year: 2011, vol: 6, page: e25264, stat: Journal Article,

Integrative genomics identifies molecular alterations that challenge the linear model of melanoma progression
Rose AE; Poliseno L; Wang J; Clark M; Pearlman A; Wang G; Vega Y Saenz de Miera EC; Medicherla R; Christos PJ; Shapiro RL; Pavlick AC; Darvishian F; Zavadil J; Polsky D; Hernando E; Ostrer H; Osman I
2011 Apr 1;71(7):2561-2571, Cancer research
Superficial spreading melanoma (SSM) and nodular melanoma (NM) are believed to represent sequential phases of linear progression from radial to vertical growth. Several lines of clinical, pathological and epidemiologic evidence suggest, however, that SSM and NM might be the result of independent pathways of tumor development. We utilized an integrative genomic approach that combines single nucleotide polymorphism array (SNP 6.0, Affymetrix) with gene expression array (U133A 2.0, Affymetrix) to examine molecular differences between SSM and NM. Pathway analysis of the most differentially expressed genes between SSM and NM (N=114) revealed significant differences related to metabolic processes. We identified 8 genes (DIS3, FGFR1OP, G3BP2, GALNT7, MTAP, SEC23IP, USO1, ZNF668) in which NM/SSM-specific copy number alterations correlated with differential gene expression (P<0.05, Spearman's rank). SSM-specific genomic deletions in G3BP2, MTAP, and SEC23IP were independently verified in two external data sets. Forced overexpression of metabolism-related gene methylthioadenosine phosphorylase (MTAP) in SSM resulted in reduced cell growth. The differential expression of another metabolic related gene, aldehyde dehydrogenase 7A1 (ALDH7A1), was validated at the protein level using tissue microarrays of human melanoma. In addition, we show that the decreased ALDH7A1 expression in SSM may be the result of epigenetic modifications. Our data reveal recurrent genomic deletions in SSM not present in NM, which challenge the linear model of melanoma progression. Furthermore, our data suggest a role for altered regulation of metabolism-related genes as a possible cause of the different clinical behavior of SSM and NM
— id: 124135, year: 2011, vol: 71, page: 2561, stat: Journal Article,

Clinical relevance of SKP2 alterations in metastatic melanoma
Rose, Amy E; Wang, Guimin; Hanniford, Douglas; Monni, Stefano; Tu, Ting; Shapiro, Richard L; Berman, Russell S; Pavlick, Anna C; Pagano, Michele; Darvishian, Farbod; Mazumdar, Madhu; Hernando, Eva; Osman, Iman
2011 Feb;24(1):197-206, Pigment cell & melanoma research
In this study, we investigated the mechanism(s) of altered expression of protooncogene SKP2 in metastatic melanoma and its clinical relevance in patients with metastatic melanoma. The genomic status of SKP2 was assessed in cell lines by sequencing, single nucleotide polymorphism array, and genomic PCR. Copy number status was then evaluated for concordance with SKP2 mRNA and protein expression. SKP2 protein was further evaluated by immunohistochemistry in 93 human metastatic tissues. No mutations were identified in SKP2. Increased copy number at the SKP2 locus was observed in 6/14 (43%) metastatic cell lines and in 9/22 (41%) human metastatic tissues which was associated with overexpression of SKP2 protein. Overexpression of SKP2 protein in human tissues was associated with worse survival in a multivariate model controlling for the site of metastasis. Copy number gain is a major contributing mechanism of SKP2 overexpression in metastatic melanoma. Results may have implications for the development of therapeutics that target SKP2
— id: 138133, year: 2011, vol: 24, page: 197, stat: Journal Article,

Regulation of HMGA1 expression by microRNA296 affects prostate cancer growth and invasion
Wei JJ; Wu X; Peng Y; Shi G; Olca B; Yang X; Daniels G; Osman I; Ouyang J; Hernando E; Pellicer A; Rhim J; Melamed J; Lee P
2011 Mar 15;17(6):1297-1305, Clinical cancer research
PURPOSE: High mobility group AT-hook 1 (HMGA1) is a non-histone nuclear binding protein that is developmentally regulated. HMGA1 is significantly overexpressed in and associated with high grade and advance stage of prostate cancer (PC). The oncogenic role of HMGA1 is at least mediated through chromosomal instability and structural aberrations. However, regulation of HMGA1 expression is not well understood. Identification of microRNA mediated HMGA1 regulation will provide a promising therapeutic target in treating PC. Experimental Designs: In this study, we examined the functional relation between miR-296 and HMGA1 expression in several prostate cancer cell lines and a large prostate cancer cohort. We further examined the oncogenic property of HMGA1 regulated by miR-296. RESULTS: Here we report that miR-296, a microRNA predicted to target HMGA1, specifically represses HMGA1 expression by promoting degradation and inhibiting HMGA1translation . Repression of HMGA1 by miR-296 is direct and sequence-specific. Importantly, ectopic miR-296 expression significantly reduced prostate cancer cell proliferation and invasion, in part through the down-regulation of HMGA1. Examining prostate cancer patient samples, we found an inverse correlation between HMGA1 and miR-296 expression: high levels of HMGA1 were associated with low miR-296 expression and strongly linked to more advanced tumor grade and stage. CONCLUSIONS: Our results indicate miR-296 regulates HMGA1 expression and is associated with PC growth and invasion
— id: 115464, year: 2011, vol: 17, page: 1297, stat: Journal Article,

HMGA2 overexpression-induced ovarian surface epithelial transformation is mediated through regulation of EMT genes
Wu, Jingjing; Liu, Zhaojian; Shao, Changshun; Gong, Yaoqin; Hernando, Eva; Lee, Peng; Narita, Masashi; Muller, William; Liu, Jinsong; Wei, Jian-Jun
2011 Jan 15;71(2):349-359, Cancer research
The AT-hook transcription factor HMGA2 is an oncogene involved in the tumorigenesis of many malignant neoplasms. HMGA2 overexpression is common in both early and late-stage high-grade ovarian serous papillary carcinoma. To test whether HMGA2 participates in the initiation of ovarian cancer and promotion of aggressive tumor growth, we examined the oncogenic properties of HMGA2 in ovarian surface epithelial (OSE) cell lines. We found that introduction of HMGA2 overexpression was sufficient to induce OSE transformation in vitro. HMGA2-mediated OSE transformation resulted in tumor formation in the xenografts of nude mice. By silencing HMGA2 in HMGA2-overexpressing OSE and ovarian cancer cell lines, the aggressiveness of tumor cell growth behaviors was partially suppressed. Global gene profiling analyses revealed that HMGA2-mediated tumorigenesis was associated with expression changes of target genes and microRNAs that are involved in epithelial-to-mesenchymal transition (EMT). Lumican, a tumor suppressor that inhibits EMT, was found to be transcriptionally repressed by HMGA2 and was frequently lost in human high-grade serous papillary carcinoma. Our findings show that HMGA2 overexpression confers a powerful oncogenic signal in ovarian cancers through the modulation of EMT genes
— id: 133176, year: 2011, vol: 71, page: 349, stat: Journal Article,

Clinical variables and primary tumor characteristics predictive of the development of melanoma brain metastases and post-brain metastases survival
Zakrzewski, Jan; Geraghty, Laurel N; Rose, Amy E; Christos, Paul J; Mazumdar, Madhu; Polsky, David; Shapiro, Richard; Berman, Russell; Darvishian, Farbod; Hernando, Eva; Pavlick, Anna; Osman, Iman
2011 Apr 15;117(8):1711-1720, Cancer
BACKGROUND: Melanoma patients who develop brain metastases (B-Met) have limited survival and are excluded from most clinical trials. In the current study, the authors attempted to identify primary tumor characteristics and clinical features predictive of B-Met development and post-B-Met survival. METHODS: A prospectively accrued cohort of 900 melanoma patients was studied to identify clinicopathologic features of primary melanoma (eg, thickness, ulceration, mitotic index, and lymphovascular invasion) that are predictive of B-Met development and survival after a diagnosis of B-Met. Associations between clinical variables present at the time of B-Met diagnosis (eg, extracranial metastases, B-Met location, and the presence of neurological symptoms) and post-B-Met survival were also assessed. Univariate associations were analyzed using Kaplan-Meier survival analysis, and the effect of independent predictors was assessed using multivariate Cox proportional hazards regression analysis. RESULTS: Of the 900 melanoma patients studied, 89 (10%) developed B-Met. Ulceration and site of the primary tumor on the head and neck were found to be independent predictors of B-Met development on multivariate analysis (P = .001 and P = .003, respectively). Clinical variables found to be predictive of post-B-Met survival on multivariate analysis included the presence of neurological symptoms (P = .008) and extracranial metastases (P = .04). Ulceration was the only primary tumor characteristic that remained a significant predictor of post-B-Met survival on multivariate analysis (P = .04). CONCLUSIONS: Primary tumor ulceration was found to be the strongest predictor of B-Met development and remained an independent predictor of decreased post-B-Met survival in a multivariate analysis inclusive of primary tumor characteristics and clinical variables. The results of the current study suggest that patients with ulcerated primary tumors should be prospectively studied to determine whether heightened surveillance for B-Met can improve clinical outcome. Cancer 2011. (c) 2010 American Cancer Society
— id: 130314, year: 2011, vol: 117, page: 1711, stat: Journal Article,

A Differentiation-Based MicroRNA Signature Identifies Leiomyosarcoma as a Mesenchymal Stem Cell-Related Malignancy
Danielson, Laura S; Menendez, Silvia; Attolini, Camille Stephan-Otto; Guijarro, Maria V; Bisogna, Maria; Wei, Jianjun; Socci, Nicholas D; Levine, Douglas A; Michor, Franziska; Hernando, Eva
2010 Aug;177(2):908-917, American journal of pathology
Smooth muscle (SM) is a spontaneously contractile tissue that provides physical support and function to organs such as the uterus. Uterine smooth muscle-related neoplasia comprise common well-differentiated benign lesions called leiomyomas (ULM), and rare, highly aggressive and pleomorphic tumors named leiomyosarcomas (ULMS). MicroRNAs (miRNAs) are small non-coding RNAs that play essential roles in normal cellular development and tissue homeostasis that can be used to accurately subclassify different tumor types. Here, we demonstrate that miRNAs are required for full smooth muscle cell (SMC) differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). We also report a miRNA signature associated with this process. Moreover, we show that this signature, along with miRNA profiles for ULMS and ULM, are able to subclassify tumors of smooth muscle origin along SM differentiation. Using multiple computational analyses, we determined that ULMS are more similar to hMSCs as opposed to ULM, which are linked with more mature SMCs and myometrium. Furthermore, a comparison of the SM differentiation and ULMS miRNA signatures identified miRNAs strictly associated with SM maturation or transformation, as well as those modulated in both processes indicating a possible dual role. These results support separate origins and/or divergent transformation pathways for ULM and ULMS, resulting in drastically different states of differentiation. In summary, this work expands on our knowledge of the regulation of SM differentiation and sarcoma pathogenesis
— id: 111539, year: 2010, vol: 177, page: 908, stat: Journal Article,

The histone variant macroH2A suppresses melanoma progression through regulation of CDK8
Kapoor, Avnish; Goldberg, Matthew S; Cumberland, Lara K; Ratnakumar, Kajan; Segura, Miguel F; Emanuel, Patrick O; Menendez, Silvia; Vardabasso, Chiara; Leroy, Gary; Vidal, Claudia I; Polsky, David; Osman, Iman; Garcia, Benjamin A; Hernando, Eva; Bernstein, Emily
2010 Dec 23;468(7327):1105-1109, Nature
Cancer is a disease consisting of both genetic and epigenetic changes. Although increasing evidence demonstrates that tumour progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unclear. Histone variants replace conventional histones within the nucleosome and confer unique biological functions to chromatin. Here we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma. Loss of mH2A isoforms, histone variants generally associated with condensed chromatin and fine-tuning of developmental gene expression programs, is positively correlated with increasing malignant phenotype of melanoma cells in culture and human tissue samples. Knockdown of mH2A isoforms in melanoma cells of low malignancy results in significantly increased proliferation and migration in vitro and growth and metastasis in vivo. Restored expression of mH2A isoforms rescues these malignant phenotypes in vitro and in vivo. We demonstrate that the tumour-promoting function of mH2A loss is mediated, at least in part, through direct transcriptional upregulation of CDK8. Suppression of CDK8, a colorectal cancer oncogene, inhibits proliferation of melanoma cells, and knockdown of CDK8 in cells depleted of mH2A suppresses the proliferative advantage induced by mH2A loss. Moreover, a significant inverse correlation between mH2A and CDK8 expression levels exists in melanoma patient samples. Taken together, our results demonstrate that mH2A is a critical component of chromatin that suppresses the development of malignant melanoma, a highly intractable cutaneous neoplasm
— id: 134348, year: 2010, vol: 468, page: 1105, stat: Journal Article,

miR-7 and miR-130b are differentially regulated during Mesenchymal Stem Cell commitment
Palmer G.; Danielson L.S.; Attur M.; Abramson S.B.; Hernando E.
2010 ;62:1490-1490, Arthritis & rheumatism
Purpose: Stem cell-based therapies aimed at introducing progenitor cells into cartilage lesions hold great promise for the restoration of damaged articular surfaces following joint injury or osteoarthritis. Key to the generation of a functional repair tissue is the controlled differentiation into the desired phenotype. To this end microRNAs (miRNAs) may be important molecules that regulate this process. By acting as transcriptional repressors, their modulation during differentiation may enable commitment to a specific lineage by suppressing the expression of other lineage markers. In this study we profiled MSCs for miRNA expression following induction into the chondrocyte (C), osteoblast (O) and smooth muscle (SM) lineages. Results: Human bone marrow-derived Mesenchymal Stem Cells (MSCs) were obtained from NIH, or from the discarded hips of patients undergoing joint replacement surgery. SM differentiation was induced by treating monolayer cultures with 1 mM thromboxane-A2 [DP1] in the presence of 0.25% serum. C differentiation was induced by seeding MSCs in aggregate cultures in the presence of dexamethasone and TGF-b1 (10 ng/ml). O differentiation was induced by treatment of monolayer cultures with dexamethasone, ascorbate and beta-glycerolphosphate. Profiling of miRNAs by microarray (Agilent) or QPCR (SA Biosciences) revealed differential regulation of miR-7 and miR-130b, among 376 probes. Following SM and C differentiation, miR-7 expression was down-regulated up to 6.9-fold and 3-fold respectively. Conversely, during O differentiation, its expression was induced approximately 7-fold. Analysis of theoretical mRNA targets using TargetScan online software (www.targetscan.org) identified conserved sites in several genes associated with chondrocyte and myoblast lineages. Putative chondrogenic targets were found to include COL2A1, IGFR1, and GDF5, while potential smooth muscle modulators included EGFR1, PIK3CD, IRS1/IRS2, KLF4, CNN3 and IGF1R. Following a similar trend to miR-7, miR-130b was down-regulated up to 3.2-fold and 3.1-fold in C and SM differentiation respectively, while O differentiation induced its expression 2-fold. TargetScan analysis identified putative chondrogenic targets, TGF-BRII, Sox5, BMP-2 and IGF1; Potential smooth muscle regulators included ESR1, TGF-BRII, MBLN1, TGFBR1 and IGF2BP1. Together these observations suggest that miR-7 and miR-130b act to negatively regulate myogenic and chondrogenic cell fates via regulation of lineage specific genes. Conclusion: Our findings suggest that miR-7 and miR-130b, via the targeting of lineage specific molecules, regulate cell fate in adult human MSCs by inhibiting smooth muscle and chondrocyte differentiation, thereby promoting 'default' differentiation into the osteoblast lineage. [DP1]0.25% FBS 24 hours prior to addition of 1.0mM of the TxA2 chemical analog U46619
— id: 130943, year: 2010, vol: 62, page: 1490, stat: Journal Article,

MIR-7 AND MIR-130B ARE DIFFERENTIALLY REGULATED DURING MESENCHYMAL STEM CELL COMMITMENT
Palmer, G.; Danielson, L.; Attur, M.; Abramson, S. B.; Hernando, E.
2010 OCT ;18(12):S39-S39, Osteoarthritis & cartilage
— id: 120554, year: 2010, vol: 18, page: S39, stat: Journal Article,

Viral oncolysis that targets Raf-1 signaling control of nuclear transport
Riolobos, Laura; Valle, Noelia; Hernando, Eva; Maroto, Beatriz; Kann, Michael; Almendral, Jose M
2010 Feb;84(4):2090-2099, Journal of virology
The central role of Raf protein kinase isoforms in human cancer demands specific anti-Raf therapeutic inhibitors. Parvoviruses are currently used in experimental cancer therapy due to their natural oncotropism and lytic life cycle. In searching for mechanisms underlying parvovirus oncolysis, we found that trimers of the major structural protein (VP) of the parvovirus minute virus of mice (MVM), which have to be imported into the nucleus for capsid assembly, undergo phosphorylation by the Raf-1 kinase. Purified Raf-1 phosphorylated the capsid subunits in vitro to the two-dimensional pattern found in natural MVM infections. VP trimers isolated from mammalian cells translocated into the nucleus of digitonin-permeabilized human cells. In contrast, VP trimers isolated from insect cells, which are devoid of Raf-1, were neither phosphorylated nor imported into the mammalian nucleus. However, the coexpression of a constitutively active Raf-1 kinase in insect cells restored VP trimer phosphorylation and nuclear transport competence. In MVM-infected normal and transformed cells, Raf-1 inhibition resulted in cytoplasmic retention of capsid proteins, preventing their nuclear assembly and progeny virus maturation. The level of Raf-1 activity in cancer cells was consistent with the extent of VP specific phosphorylation and with the permissiveness to MVM infection. Thus, Raf-1 control of nuclear translocation of MVM capsid assembly intermediates provides a novel target for viral oncolysis. MVM may reinforce specific therapies against frequent human cancers with deregulated Raf signaling
— id: 133458, year: 2010, vol: 84, page: 2090, stat: Journal Article,

Melanoma MicroRNA Signature Predicts Post-Recurrence Survival
Segura, Miguel F; Belitskaya-Levy, Ilana; Rose, Amy E; Zakrzewski, Jan; Gaziel, Avital; Hanniford, Douglas; Darvishian, Farbod; Berman, Russell S; Shapiro, Richard L; Pavlick, Anna C; Osman, Iman; Hernando, Eva
2010 Mar 1;16(5):1577-1586, Clinical cancer research
PURPOSE: To identify a melanoma microRNA (miRNA) expression signature that is predictive of outcome and then evaluate its potential to improve risk stratification when added to the standard-of-care staging criteria. EXPERIMENTAL DESIGN: Total RNA was extracted from 59 formalin-fixed paraffin-embedded melanoma metastases and hybridized to miRNA arrays containing 911 probes. We then correlated miRNA expression with post-recurrence survival and other clinicopathologic criteria. RESULTS: We identified a signature of 18 miRNAs whose overexpression was significantly correlated with longer survival, defined as more than 18 months post-recurrence survival. Subsequent cross-validation showed that a small subset of these miRNAs can predict post-recurrence survival in metastatic melanoma with an estimated accuracy of 80.2% (95% confidence interval, 79.8-80.6%). In contrast to standard-of-care staging criteria, a six-miRNA signature significantly stratified stage III patients into 'better' and 'worse' prognostic categories, and a multivariate Cox regression analysis revealed the signature to be an independent predictor of survival. Furthermore, we showed that most miRNAs from the signature also showed differential expression between patients with better and worse prognoses in the corresponding paired primary melanoma. CONCLUSIONS: MiRNA signatures have potential as clinically relevant biomarkers of prognosis in metastatic melanoma. Our data suggest that molecularly based models of risk assessment can improve the standard staging criteria and support the incorporation of miRNAs into such models. Clin Cancer Res; 16(5); 1577-86
— id: 107357, year: 2010, vol: 16, page: 1577, stat: Journal Article,

Clinical variables and primary tumor characteristics predictive of the development of melanoma brain metastases and post-brain metastases survival
Zakrzewski J; Geraghty LN; Rose AE; Christos PJ; Mazumdar M; Polsky D; Shapiro R; Berman R; Darvishian F; Hernando E; Pavlick A; Osman I
2010 Nov 8;:?-?, Cancer
BACKGROUND:: Melanoma patients who develop brain metastases (B-Met) have limited survival and are excluded from most clinical trials. In the current study, the authors attempted to identify primary tumor characteristics and clinical features predictive of B-Met development and post-B-Met survival. METHODS:: A prospectively accrued cohort of 900 melanoma patients was studied to identify clinicopathologic features of primary melanoma (eg, thickness, ulceration, mitotic index, and lymphovascular invasion) that are predictive of B-Met development and survival after a diagnosis of B-Met. Associations between clinical variables present at the time of B-Met diagnosis (eg, extracranial metastases, B-Met location, and the presence of neurological symptoms) and post-B-Met survival were also assessed. Univariate associations were analyzed using Kaplan-Meier survival analysis, and the effect of independent predictors was assessed using multivariate Cox proportional hazards regression analysis. RESULTS:: Of the 900 melanoma patients studied, 89 (10%) developed B-Met. Ulceration and site of the primary tumor on the head and neck were found to be independent predictors of B-Met development on multivariate analysis (P = .001 and P = .003, respectively). Clinical variables found to be predictive of post-B-Met survival on multivariate analysis included the presence of neurological symptoms (P = .008) and extracranial metastases (P = .04). Ulceration was the only primary tumor characteristic that remained a significant predictor of post-B-Met survival on multivariate analysis (P = .04). CONCLUSIONS:: Primary tumor ulceration was found to be the strongest predictor of B-Met development and remained an independent predictor of decreased post-B-Met survival in a multivariate analysis inclusive of primary tumor characteristics and clinical variables. The results of the current study suggest that patients with ulcerated primary tumors should be prospectively studied to determine whether heightened surveillance for B-Met can improve clinical outcome. Cancer 2010. (c) 2010 American Cancer Society
— id: 141464, year: 2010, vol: , page: ?, stat: Journal Article,

Profiling and functional analyses of microRNAs and their target gene products in human uterine leiomyomas
Zavadil, Jiri; Ye, Huihui; Liu, Zhaojian; Wu, JingJing; Lee, Peng; Hernando, Eva; Soteropoulos, Patricia; Toruner, Gokce A; Wei, Jian-Jun
2010 ;5(8):e12362-e12362, PLoS ONE
BACKGROUND: Human uterine leiomyomas (ULM) are characterized by dysregulation of a large number of genes and non-coding regulatory microRNAs. In order to identify microRNA::mRNA associations relevant to ULM pathogenesis, we examined global correlation patterns between the altered microRNA expression and the predicted target genes in ULMs and matched myometria. METHODOLOGY/PRINCIPAL FINDINGS: Patterns of inverse association of microRNA with mRNA expression in ULMs revealed an involvement of multiple candidate pathways, including extensive transcriptional reprogramming, cell proliferation control, MAP kinase, TGF-beta, WNT, JAK/STAT signaling, remodeling of cell adhesion, and cell-cell and cell-matrix contacts. We further examined the correlation between the expression of the selected target gene protein products and microRNAs in thirty-six paired sets of leiomyomas and matched myometria. We found that a number of dysregulated microRNAs were inversely correlated with their targets at the protein level. The comparative genomic hybridization (CGH) in eight ULM patients revealed that partially shared deletions of two distinct chromosomal regions might be responsible for loss of cancer-associated microRNA expression and could thus contribute to the ULM pathogenesis via deregulation of target mRNAs. Last, we functionally tested the repressor effects of selected cancer-related microRNAs on their predicted target genes in vitro. CONCLUSIONS/SIGNIFICANCE: We found that some but not all of the predicted and inversely correlated target genes in ULMs can be directly regulated by microRNAs in vitro. Our findings provide a broad overview of molecular events underlying the tumorigenesis of uterine ULMs and identify select genetic and regulatory events that alter microRNA expression and may play important roles in ULM pathobiology by positively regulating tumor growth while maintaining the non-invasive character of ULMs
— id: 112544, year: 2010, vol: 5, page: e12362, stat: Journal Article,

A critical role for choline kinase-alpha in the aggressiveness of bladder carcinomas
Hernando, E; Sarmentero-Estrada, J; Koppie, T; Belda-Iniesta, C; Ramirez de Molina, V; Cejas, P; Ozu, C; Le, C; Sanchez, J J; Gonzalez-Baron, M; Koutcher, J; Cordon-Cardo, C; Bochner, B H; Lacal, J C; Ramirez de Molina, A
2009 Jul 2;28(26):2425-2435, Oncogene
Bladder cancer is one of the most common causes of death in industrialized countries. New tumor markers and therapeutic approaches are still needed to improve the management of bladder cancer patients. Choline kinase-alpha (ChoKalpha) is a metabolic enzyme that has a role in cell proliferation and transformation. Inhibitors of ChoKalpha show antitumoral activity and are expected to be introduced soon in clinical trials. This study aims to assess whether ChoKalpha plays a role in the aggressiveness of bladder tumors and constitutes a new approach for bladder cancer treatment. We show here that ChoKalpha is constitutively altered in human bladder tumor cells. Furthermore, in vivo murine models, including an orthotopic model to mimic as much as possible the physiological conditions, revealed that increased levels of ChoKalpha potentiate both tumor formation (P< or =0.0001) and aggressiveness of the disease on different end points (P=0.011). Accordingly, increased levels of ChoKalpha significantly reduce survival of mice with bladder cancer (P=0.05). Finally, treatment with a ChoKalpha-specific inhibitor resulted in a significant inhibition of tumor growth (P=0.02) and in a relevant increase in survival (P=0.03)
— id: 107405, year: 2009, vol: 28, page: 2425, stat: Journal Article,

Expression of insulin-like growth factors (IGFs) and IGF signaling: molecular complexity in uterine leiomyomas
Peng, Lan; Wen, Yong; Han, Yulong; Wei, Anran; Shi, Guizhi; Mizuguchi, Masashi; Lee, Peng; Hernando, Eva; Mittal, Khush; Wei, Jian-Jun
2009 Jun;91(6):2664-2675, Fertility & sterility
OBJECTIVE: To study whether dysregulation of insulin-like growth factors (IGFs) and IGF signaling are common molecular changes in symptomatic leiomyomas (fibroids) and whether IGFs are associated with large fibroids. DESIGN: Examination of IGFs and IGF pathway genes in a large cohort of fibroids at transcriptional and translational levels. Mechanisms leading to alterations of IGFs and related genes were also analyzed. SETTING: University clinical research laboratory. PATIENT(S): Hysterectomies for symptomatic fibroids were collected: 180 cases from paraffin-embedded tissues and 50 cases from fresh-frozen tissues. INTERVENTION(S): Tissue microarray and immunohistochemistry, DNA methylation analysis, reverse-transcriptase polymerase chain reaction, and Western blot. MAIN OUTCOME MEASUREMENT(S): Transcription and translation analyses of IGF-1/2, p-AKT, p-S6K, and TSC1/2 in fibroids and matched myometrium. RESULT(S): Insulin-like growth factors and downstream effectors were dysregulated in approximately one third of fibroids. All except for IGF-2 seemed to be abnormally regulated at translation levels. Up-regulation of IGF-2 messenger RNAs was contributed by all four alternating slicing promoters. There was a positive correlation of IGF-1 and p-AKT over-expression with fibroid size. Insulin-like growth factor 1 but not IGF-2 levels directly correlated with activation of p-AKT and p-S6K. CONCLUSION(S): Altered expressions of IGFs and their related downstream proteins were found in one third of fibroids. Large fibroids show high levels of IGF-1 and p-AKT activity compared with small ones
— id: 78574, year: 2009, vol: 91, page: 2664, stat: Journal Article,

Gamma-secretase inhibitors reverse glucocorticoid resistance in T cell acute lymphoblastic leukemia
Real, Pedro J; Tosello, Valeria; Palomero, Teresa; Castillo, Mireia; Hernando, Eva; de Stanchina, Elisa; Sulis, Maria Luisa; Barnes, Kelly; Sawai, Catherine; Homminga, Irene; Meijerink, Jules; Aifantis, Iannis; Basso, Giuseppe; Cordon-Cardo, Carlos; Ai, Walden; Ferrando, Adolfo
2009 Jan;15(1):50-58, Nature medicine
Gamma-secretase inhibitors (GSIs) block the activation of the oncogenic protein Notch homolog-1 (NOTCH1) in T cell acute lymphoblastic leukemia (T-ALL). However, limited antileukemic cytotoxicity and severe gastrointestinal toxicity have restricted the clinical application of these targeted drugs. Here we show that combination therapy with GSIs plus glucocorticoids can improve the antileukemic effects of GSIs and reduce their gut toxicity in vivo. Inhibition of NOTCH1 signaling in glucocorticoid-resistant T-ALL restored glucocorticoid receptor autoupregulation and induced apoptotic cell death through induction of the gene encoding BCL-2-like apoptosis initiator-11 (BCL2L11). GSI treatment resulted in cell cycle arrest and accumulation of goblet cells in the gut mediated by upregulation of the gene encoding the transcription factor Kruppel-like factor-4 (Klf4), a negative regulator of the cell cycle required for goblet cell differentiation. In contrast, glucocorticoid treatment induced transcriptional upregulation of cyclin D2 (Ccnd2) and protected mice from developing the intestinal goblet cell metaplasia typically induced by inhibition of NOTCH signaling with GSIs. These results support a role for glucocorticoids plus GSIs in the treatment of glucocorticoid-resistant T-ALL
— id: 105355, year: 2009, vol: 15, page: 50, stat: Journal Article,

The unique molecular signatures of nodular and superficial spreading melanoma
Rose A.E.; Wang J.; Pearlman A.; Doudican N.; Hernando E.; J. Orlow S.; Polsky D.; Ostrer H.; Osman I.
2009 ;27(15 Suppl 1):9047-9047, Journal of clinical oncology
Background: Primary nodular melanoma (NM) patients have a relatively poor prognosis compared to superficial spreading melanoma (SSM) patients. The disparity is generally attributed solely to NM's advanced thickness at presentation. In this study we attempted to define molecular signatures of NM and SSM that may explain their clinical differences. Methods: We performed an in silico gene expression analysis of 2 public data sets consisting of 36NM and 54 SSM primary melanoma tissues (CCR 2007;13 and JNCI 2006;98). We then utilized DNA microarray to generate gene expression profiles of a panel of 22 melanoma cell lines (2SSM, 4 NM, 12 met, 4 melanocytes). Differentially expressed genes and over-represented pathways in NM and SSM were identified based on a pooled analysis of the 3 data sets. We then used SNP array to define genomic alterations unique to NM and SSM but not altered in normal melanocytes. Finally, we correlated SNP array with gene expression. Results: Genes significantly overexpressed (P<0.05) in NM showed over-representation of pathways related to MAPK signaling (p=0.05) and cytoskeleton organization (p=0.02), while SSM showed over-representation of cell communication (p=0.05) and primary metabolic processes (p=0.002). Notable correlations between gene expression and copy number alteration in NM include increased copy number/overexpression of SOX5 (transcription factor related to embryonic development and cell fate) and the downregulation/deletion of ST14 (suppression of tumorigenicity 14). SSM demonstrated concordance of increased copy number/overexpression of EZR (cell adhesion protein implicated in human cancer) as well as PALLD (a protein related to motility, adhesion, and extracellular matrix interactions). Notable SSM genes showing correlation between downregulation/deletion include BNIP3 (a pro-apoptotic protein) and MTAP (often co- deleted with tumor suppressor p16). Conclusions: Simultaneous integration of gene expression with SNP array revealed molecular signatures characteristic of NM and SSM. These results suggest that NM and SSM are distinct biologic entities and that molecularly targeted adjuvant therapy may be more effective if tailored to the molecular signatures of melanoma subtypes. Validation is necessary to draw further conclusions
— id: 111806, year: 2009, vol: 27, page: 9047, stat: Journal Article,

Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor
Segura, Miguel F; Hanniford, Douglas; Menendez, Silvia; Reavie, Linsey; Zou, Xuanyi; Alvarez-Diaz, Silvia; Zakrzewski, Jan; Blochin, Elen; Rose, Amy; Bogunovic, Dusan; Polsky, David; Wei, Jianjun; Lee, Peng; Belitskaya-Levy, Ilana; Bhardwaj, Nina; Osman, Iman; Hernando, Eva
2009 Feb 10;106(6):1814-1819, Proceedings of the National Academy of Sciences of the United States of America
The highly aggressive character of melanoma makes it an excellent model for probing the mechanisms underlying metastasis, which remains one of the most difficult challenges in treating cancer. We find that miR-182, member of a miRNA cluster in a chromosomal locus (7q31-34) frequently amplified in melanoma, is commonly up-regulated in human melanoma cell lines and tissue samples; this up-regulation correlates with gene copy number in a subset of melanoma cell lines. Moreover, miR-182 ectopic expression stimulates migration of melanoma cells in vitro and their metastatic potential in vivo, whereas miR-182 down-regulation impedes invasion and triggers apoptosis. We further show that miR-182 over-expression promotes migration and survival by directly repressing microphthalmia-associated transcription factor-M and FOXO3, whereas enhanced expression of either microphthalmia-associated transcription factor-M or FOXO3 blocks miR-182's proinvasive effects. In human tissues, expression of miR-182 increases with progression from primary to metastatic melanoma and inversely correlates with FOXO3 and microphthalmia-associated transcription factor levels. Our data provide a mechanism for invasion and survival in melanoma that could prove applicable to metastasis of other cancers and suggest that miRNA silencing may be a worthwhile therapeutic strategy
— id: 92154, year: 2009, vol: 106, page: 1814, stat: Journal Article,

Let-7 repression leads to HMGA2 overexpression in uterine leiomyosarcoma
Shi, Guizhi; Perle, Mary Ann; Mittal, Khush; Chen, Hua; Zou, Xuanyi; Narita, Masashi; Hernando, Eva; Lee, Peng; Wei, Jian-Jun
2009 Sep;13(9B):3898-3905, Journal of cellular & molecular medicine
Overexpression of HMGA2 is common in uterine leiomyomas (ULM). The expression of HMGA2 in its malignant counterpart - uterine leiomyosarcomas (ULMS) remains undetermined. Recently it has been shown that repression of HMGA2 by microRNA let-7s is a critical molecular regulatory mechanism associated with tumour growth in many tumours and cell types, including leiomyomas. To test whether HMGA2 and let-7s play a role in ULMS, we examined the levels of endogenous HMGA2 and let-7 expression and found a significant correlation between these two molecules in a case-matched cohort of human ULMS. We found that overexpression of HMGA2 and let-7-mediated HMGA2 repression is a relevant molecular alteration in ULMS. Disrupting the control of HMGA2 and let-7 pairs promotes ULMS cell growth in vitro
— id: 114731, year: 2009, vol: 13, page: 3898, stat: Journal Article,

Developing a multidisciplinary prospective melanoma biospecimen repository to advance translational research
Wich, Lindsay G; Hamilton, Heather K; Shapiro, Richard L; Pavlick, Anna; Berman, Russell S; Polsky, David; Goldberg, Judith D; Hernando, Eva; Manga, Prashiela; Krogsgaard, Michelle; Kamino, Hideko; Darvishian, Farbod; Lee, Peng; Orlow, Seth J; Ostrer, Harry; Bhardwaj, Nina; Osman, Iman
2009 ;1(1):35-43, American Journal of Translational Research
Several challenges face the development and operation of a biospecimen bank linked to clinical information, a critical component of any effective translational research program. Melanoma adds particular complexity and difficulty to such an endeavor considering the unique characteristics of this malignancy. We describe here a review of biospecimen bank and our experience in establishing a multi-disciplinary, prospective, integrated clinicopathological-biospecimen database in melanoma. The Interdisciplinary Melanoma Cooperative Group (IMCG), a prospective clinicopathological and biospecimen database, was established at the New York University (NYU) Langone Medical Center. With patients' informed consent, biospecimens from within and outside NYU, clinicopathological data, and follow-up information are collected using developed protocols. Information pertaining to biospecimens is recorded in 35 fields, and clinicopathological information is recorded in 371 fields within 5 modules in a virtual network system. Investigators conducting research utilizing the IMCG biospecimen resource are blind to clinicopathological information, and molecular data generated using biospecimens are linked independently with clinicopathological data by biostatistics investigators. This translational research enterprise acts as a valuable resource to efficiently translate laboratory discoveries to the clinic
— id: 105566, year: 2009, vol: 1, page: 35, stat: Journal Article,

Chemokine signaling via the CXCR2 receptor reinforces senescence
Acosta, Juan C; O'Loghlen, Ana; Banito, Ana; Guijarro, Maria V; Augert, Arnaud; Raguz, Selina; Fumagalli, Marzia; Da Costa, Marco; Brown, Celia; Popov, Nikolay; Takatsu, Yoshihiro; Melamed, Jonathan; d'Adda di Fagagna, Fabrizio; Bernard, David; Hernando, Eva; Gil, Jesus
2008 Jun 13;133(6):1006-1018, Cell
Cells enter senescence, a state of stable proliferative arrest, in response to a variety of cellular stresses, including telomere erosion, DNA damage, and oncogenic signaling, which acts as a barrier against malignant transformation in vivo. To identify genes controlling senescence, we conducted an unbiased screen for small hairpin RNAs that extend the life span of primary human fibroblasts. Here, we report that knocking down the chemokine receptor CXCR2 (IL8RB) alleviates both replicative and oncogene-induced senescence (OIS) and diminishes the DNA-damage response. Conversely, ectopic expression of CXCR2 results in premature senescence via a p53-dependent mechanism. Cells undergoing OIS secrete multiple CXCR2-binding chemokines in a program that is regulated by the NF-kappaB and C/EBPbeta transcription factors and coordinately induce CXCR2 expression. CXCR2 upregulation is also observed in preneoplastic lesions in vivo. These results suggest that senescent cells activate a self-amplifying secretory network in which CXCR2-binding chemokines reinforce growth arrest
— id: 90815, year: 2008, vol: 133, page: 1006, stat: Journal Article,

Control of chromosome stability by the beta-TrCP-REST-Mad2 axis
Guardavaccaro, Daniele; Frescas, David; Dorrello, N Valerio; Peschiaroli, Angelo; Multani, Asha S; Cardozo, Timothy; Lasorella, Anna; Iavarone, Antonio; Chang, Sandy; Hernando, Eva; Pagano, Michele
2008 Mar 20;452(7185):365-369, Nature
REST/NRSF (repressor-element-1-silencing transcription factor/neuron-restrictive silencing factor) negatively regulates the transcription of genes containing RE1 sites. REST is expressed in non-neuronal cells and stem/progenitor neuronal cells, in which it inhibits the expression of neuron-specific genes. Overexpression of REST is frequently found in human medulloblastomas and neuroblastomas, in which it is thought to maintain the stem character of tumour cells. Neural stem cells forced to express REST and c-Myc fail to differentiate and give rise to tumours in the mouse cerebellum. Expression of a splice variant of REST that lacks the carboxy terminus has been associated with neuronal tumours and small-cell lung carcinomas, and a frameshift mutant (REST-FS), which is also truncated at the C terminus, has oncogenic properties. Here we show, by using an unbiased screen, that REST is an interactor of the F-box protein beta-TrCP. REST is degraded by means of the ubiquitin ligase SCF(beta-TrCP) during the G2 phase of the cell cycle to allow transcriptional derepression of Mad2, an essential component of the spindle assembly checkpoint. The expression in cultured cells of a stable REST mutant, which is unable to bind beta-TrCP, inhibited Mad2 expression and resulted in a phenotype analogous to that observed in Mad2(+/-) cells. In particular, we observed defects that were consistent with faulty activation of the spindle checkpoint, such as shortened mitosis, premature sister-chromatid separation, chromosome bridges and mis-segregation in anaphase, tetraploidy, and faster mitotic slippage in the presence of a spindle inhibitor. An indistinguishable phenotype was observed by expressing the oncogenic REST-FS mutant, which does not bind beta-TrCP. Thus, SCF(beta-TrCP)-dependent degradation of REST during G2 permits the optimal activation of the spindle checkpoint, and consequently it is required for the fidelity of mitosis. The high levels of REST or its truncated variants found in certain human tumours may contribute to cellular transformation by promoting genomic instability
— id: 78365, year: 2008, vol: 452, page: 365, stat: Journal Article,

Cancer. Aneuploidy advantages?
Hernando, Eva
2008 Oct 31;322(5902):692-693, Science
— id: 90814, year: 2008, vol: 322, page: 692, stat: Journal Article,

Implications of cellular senescence in tissue damage response, tumor suppression, and stem cell biology
Krizhanovsky, V; Xue, W; Zender, L; Yon, M; Hernando, E; Lowe, S W
2008 ;73:513-522, Cold Spring Harbor symposia on quantitative biology
Cellular senescence is characterized by an irreversible cell cycle arrest that, when bypassed by mutation, contributes to cellular immortalization. Activated oncogenes induce a hyperproliferative response, which might be one of the senescence cues. We have found that expression of such an oncogene, Akt, causes senescence in primary mouse hepatoblasts in vitro. Additionally, AKT-driven tumors undergo senescence in vivo following p53 reactivation and show signs of differentiation. In another in vivo system, i.e., liver fibrosis, hyperproliferative signaling through AKT might be a driving force of the senescence in activated hepatic stellate cells. Senescent cells up-regulate and secrete molecules that, on the one hand, can reinforce the arrest and, on the other hand, can signal to an innate immune system to clear the senescent cells. The mechanisms governing senescence and immortalization are overlapping with those regulating self-renewal and differentiation. These respective control mechanisms, or their disregulation, are involved in multiple pathological conditions including fibrosis, wound healing, and cancer. Understanding extracellular cues that regulate these processes may enable new therapies for these conditions
— id: 107358, year: 2008, vol: 73, page: 513, stat: Journal Article,

A developmental model of sarcomagenesis defines a differentiation-based classification for liposarcomas
Matushansky, Igor; Hernando, Eva; Socci, Nicholas D; Matos, Tulio; Mills, Joslyn; Edgar, Mark A; Schwartz, Gary K; Singer, Samuel; Cordon-Cardo, Carlos; Maki, Robert G
2008 Apr;172(4):1069-1080, American journal of pathology
The importance of adult stem cells in the development of neoplastic diseases is becoming increasingly well appreciated. We hypothesized that sarcomas of soft tissue could be categorized by their developmental/differentiation status from stem cell to mature tissue, similar to the hematological malignancies. We conducted gene expression analyses during in vitro differentiation of human mesenchymal stem cells into adipose tissue, as a representative mature connective tissue, and identified genes whose expression changed significantly during adipogenesis. Gene clustering and distance correlation analysis allowed the assignment of a unique time point during adipogenesis that strongly correlates to each of the four major liposarcoma subtypes. Using a novel gene expression strategy, in which liposarcomas are compared to their corresponding adipocytic maturing cells, we identified a group of genes overexpressed in liposarcomas that indicate the stage of differentiation arrest, ie, sharing a similar expression profile to adipocytic cells at a corresponding stage of differentiation, and a distinct set of genes overexpressed in liposarcomas that are not found in the corresponding stage of differentiation. We propose that the latter set is enriched for candidate transformation-associated genes. Our results indicate that a degree of developmental maturity can be quantitatively assigned to solid tumors, supporting the notion that transformation of a solid tumor stem cell can occur at distinct stages of maturation
— id: 90816, year: 2008, vol: 172, page: 1069, stat: Journal Article,

Tissue-specific and reversible RNA interference in transgenic mice
Dickins, Ross A; McJunkin, Katherine; Hernando, Eva; Premsrirut, Prem K; Krizhanovsky, Valery; Burgess, Darren J; Kim, Sang Yong; Cordon-Cardo, Carlos; Zender, Lars; Hannon, Gregory J; Lowe, Scott W
2007 Jul;39(7):914-921, Nature genetics
Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice. Transgenic mice harboring a tet-responsive RNA polymerase II promoter driving a microRNA-based short hairpin RNA targeting the tumor suppressor Trp53 reversibly express short hairpin RNA when crossed with existing mouse strains expressing general or tissue-specific 'tet-on' or 'tet-off' transactivators. Reversible Trp53 knockdown can be achieved in several tissues, and restoring Trp53 expression in lymphomas whose development is promoted by Trp53 knockdown leads to tumor regression. By leaving the target gene unaltered, this approach permits tissue-specific, reversible regulation of endogenous gene expression in vivo, with potential broad application in basic biology and drug target validation.
— id: 72890, year: 2007, vol: 39, page: 914, stat: Journal Article,

Declining p53 function in the aging process: A possible mechanism for the increased tumor incidence in older populations
Feng, Zhaohui; Hu, Wenwei; Teresky, Angelika K; Hernando, Eva; Cordon-Cardo, Carlos; Levine, Arnold J
2007 Oct 16;104(42):16633-16638, Proceedings of the National Academy of Sciences of the United States of America
Cancer is a disease of aging. The accumulation of mutations in individual cells over a lifetime is thought to be the reason. In this work, we explored an additional hypothesis: could p53 function decline with age, which would contribute to an enhanced mutation frequency and tumorigenesis in the aging process? The efficiency of the p53 response to gamma-irradiation was found to decline significantly in various tissues of aging mice from several inbred strains, including lower p53 transcriptional activity and p53-dependent apoptosis. This decline resulted from a decreased stabilization of the p53 protein after stress. The function of the Ataxia-telangiectasia mutated (ATM) kinase declined significantly with age, which may then be responsible for the decline of the p53 response to radiation. Declining p53 responses to other stresses were also observed in the cultured splenocytes from aging mice. Interestingly, the time of onset of this decreased p53 response correlated with the life span of mice; mice that live longer delay their onset of decreased p53 activity with time. These results suggest an enhanced fixation of mutations in older individuals because of the declining fidelity of p53-mediated apoptosis or senescence in response to stress, and they suggest a plausible explanation for the correlation between tumorigenesis and the aging process
— id: 74438, year: 2007, vol: 104, page: 16633, stat: Journal Article,

microRNAs and cancer: role in tumorigenesis, patient classification and therapy
Hernando, E
2007 Mar;9(3):155-160, Clinical & translational oncology
MicroRNAs (miRNAs) are small non-coding RNAs that downregulate gene expression during various crucial cell processes such as apoptosis, differentiation and development. Recent work supports a role for miRNAs in the initiation and progression of human malignancies. Moreover, large high-throughput studies in patients revealed that miRNA profiling has the potential to classify tumours and predict patient outcome with high accuracy. Functional studies, some of which involve animal models, indicate that miRNAs act as tumour suppressors and oncogenes. This review examines the role of miRNAs in the pathogenesis of cancer as well as miRNA-profiling studies performed in human malignancies. Implications of these findings for the diagnosis and treatment of cancer patients are also discussed.
— id: 72854, year: 2007, vol: 9, page: 155, stat: Journal Article,

The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas
Hernando, Eva; Charytonowicz, Elizabeth; Dudas, Maria E; Menendez, Silvia; Matushansky, Igor; Mills, Joslyn; Socci, Nicholas D; Behrendt, Nille; Ma, Li; Maki, Robert G; Pandolfi, Pier Paolo; Cordon-Cardo, Carlos
2007 Jun;13(6):748-753, Nature medicine
We analyzed the PI3K-AKT signaling cascade in a cohort of sarcomas and found a marked induction of insulin receptor substrate-2 (IRS2) and phosphorylated AKT and a concomitant upregulation of downstream effectors in most leiomyosarcomas. To determine the role of aberrant PI3K-AKT signaling in leiomyosarcoma pathogenesis, we genetically inactivated Pten in the smooth muscle cell lineage by cross-breeding Pten(loxP/loxP) mice with Tagln-cre mice. Mice carrying homozygous deletion of Pten alleles developed widespread smooth muscle cell hyperplasia and abdominal leiomyosarcomas, with a very rapid onset and elevated incidence ( approximately 80%) compared to other animal models. Constitutive mTOR activation was restricted to the leiomyosarcomas, revealing the requirement for additional molecular events besides Pten loss. The rapamycin derivative everolimus substantially decelerated tumor growth on Tagln-cre/Pten(loxP/loxP) mice and prolonged their lifespan. Our data show a new and critical role for the AKT-mTOR pathway in smooth muscle transformation and leiomyosarcoma genesis, and support treatment of selected sarcomas by the targeting of this pathway with new compounds or combinations of these with conventional chemotherapy agents.Note: In the version of this article initially published online, the name of the fifth author was misspelled. The correct name is Matushansky. The error has been corrected for all versions of the article.
— id: 72853, year: 2007, vol: 13, page: 748, stat: Journal Article,

Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway
Matushansky, Igor; Hernando, Eva; Socci, Nicholas D; Mills, Joslyn E; Matos, Tulio A; Edgar, Mark A; Singer, Samuel; Maki, Robert G; Cordon-Cardo, Carlos
2007 Nov;117(11):3248-3257, Journal of clinical investigation
Malignant fibrous histiocytoma (MFH), now termed high-grade undifferentiated pleomorphic sarcoma, is a commonly diagnosed mesenchymal tumor, yet both the underlying molecular mechanisms of tumorigenesis and cell of origin remain unidentified. We present evidence demonstrating that human mesenchymal stem cells (hMSCs) are the progenitors of MFH. DKK1, a Wnt inhibitor and mediator of hMSC proliferation, is overexpressed in MFH. Using recombinant proteins, antibody depletion, and siRNA knockdown strategies of specific Wnt elements, we show that DKK1 inhibits hMSC commitment to differentiation via Wnt2/beta-catenin canonical signaling and that Wnt5a/JNK noncanonical signaling regulates a viability checkpoint independent of Dkk1. Finally, we illustrate that hMSCs can be transformed via inhibition of Wnt signaling to form MFH-like tumors in nude mice, and conversely, MFH cells in which Wnt signaling is appropriately reestablished can differentiate along mature connective tissue lineages. Our results provide mechanistic insights regarding the cell of origin of MFH, establish what we believe is a novel tumor suppressor role for Wnt signaling, and identify a potential therapeutic differentiation strategy for sarcomas
— id: 90817, year: 2007, vol: 117, page: 3248, stat: Journal Article,

Role of the chromobox protein CBX7 in lymphomagenesis
Scott, Clare L; Gil, Jesus; Hernando, Eva; Teruya-Feldstein, Julie; Narita, Masako; Martinez, Dolores; Visakorpi, Tapio; Mu, David; Cordon-Cardo, Carlos; Peters, Gordon; Beach, David; Lowe, Scott W
2007 Mar 27;104(13):5389-5394, Proceedings of the National Academy of Sciences of the United States of America
Chromobox 7 (CBX7) is a chromobox family protein and a component of the Polycomb repressive complex 1 (PRC1) that extends the lifespan of cultured epithelial cells and can act independently of BMI-1 to repress the INK4a/ARF tumor suppressor locus. To determine whether CBX7 might be oncogenic, we examined its expression pattern in a range of normal human tissues and tumor samples. CBX7 was expressed at high levels in germinal center lymphocytes and germinal center-derived follicular lymphomas, where elevated expression correlated with high c-Myc expression and a more advanced tumor grade. By targeting Cbx7 expression to the lymphoid compartment in mice, we showed that Cbx7 can initiate T cell lymphomagenesis and cooperate with c-Myc to produce highly aggressive B cell lymphomas. Furthermore, Cbx7 repressed transcription from the Ink4a/Arf locus and acted epistatically to the Arf-p53 pathway during tumorigenesis. These data identify CBX7 as a chromobox protein causally linked to cancer development and may help explain the low frequency of INK4a/ARF mutations observed in human follicular lymphoma.
— id: 72891, year: 2007, vol: 104, page: 5389, stat: Journal Article,

Mad2 overexpression promotes aneuploidy and tumorigenesis in mice
Sotillo, Rocio; Hernando, Eva; Diaz-Rodriguez, Elena; Teruya-Feldstein, Julie; Cordon-Cardo, Carlos; Lowe, Scott W; Benezra, Robert
2007 Jan;11(1):9-23, Cancer cell
Mad2 is an essential component of the spindle checkpoint that blocks activation of Separase and dissolution of sister chromatids until microtubule attachment to kinetochores is complete. We show here that overexpression of Mad2 in transgenic mice leads to a wide variety of neoplasias, appearance of broken chromosomes, anaphase bridges, and whole-chromosome gains and losses, as well as acceleration of myc-induced lymphomagenesis. Moreover, continued overexpression of Mad2 is not required for tumor maintenance, unlike the majority of oncogenes studied to date. These results demonstrate that transient Mad2 overexpression and chromosome instability can be an important stimulus in the initiation and progression of different cancer subtypes.
— id: 72892, year: 2007, vol: 11, page: 9, stat: Journal Article,

Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas
Xue, Wen; Zender, Lars; Miething, Cornelius; Dickins, Ross A; Hernando, Eva; Krizhanovsky, Valery; Cordon-Cardo, Carlos; Lowe, Scott W
2007 Feb 8;445(7128):656-660, Nature
Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma. We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth
— id: 70176, year: 2007, vol: 445, page: 656, stat: Journal Article,

Cyclin D3 is down-regulated by rapamycin in HER-2-overexpressing breast cancer cells
Garcia-Morales, Pilar; Hernando, Eva; Carrasco-Garcia, Estefania; Menendez-Gutierrez, Maria Piedad; Saceda, Miguel; Martinez-Lacaci, Isabel
2006 Sep;5(9):2172-2181, Molecular cancer therapeutics
Rapamycin and its analogues are being tested as new antitumor agents. Rapamycin binds to FKBP-12 and this complex inhibits the activity of FRAP/mammalian target of rapamycin, which leads to dephosphorylation of 4EBP1 and p70 S6 kinase, resulting in blockade of translation initiation. We have found that RAP inhibits the growth of HER-2-overexpressing breast cancer cells. The phosphorylation of mammalian target of rapamycin, p70 S6 kinase, and 4EBP1 is inhibited by rapamycin and cells are arrested in the G1 phase, as determined by growth assays, fluorescence-activated cell sorting analysis, and bromodeoxyuridine incorporation studies. Rapamycin causes down-regulation of cyclin D3 protein, retinoblastoma hypophosphorylation, loss of cyclin-dependent kinase (cdk) 4, cdk6, and cdk2 activity. The half-life of cyclin D3 protein decreases after rapamycin treatment, but not its synthesis, whereas the synthesis or half-life of cyclin D1 protein is not affected by the drug. Additionally, rapamycin caused accumulation of ubiquitinated forms of cyclin D3 protein, proteasome inhibitors blocked the effect of rapamycin on cyclin D3, and rapamycin stimulated the activity of the proteasome, showing that the effect of rapamycin on cyclin D3 is proteasome proteolysis dependent. This effect depends on the activity of HER-2 because Herceptin, a neutralizing antibody against HER-2, is able to block both the induction of proteasome activity and the cyclin D3 down-regulation due to rapamycin. Furthermore, inhibition of HER-2 gene expression by using small interfering RNA blocked the rapamycin effects on cyclin D3. These data indicate that rapamycin causes a G1 arrest in HER-2-overexpressing breast cancer cells that is associated with a differential destabilization and subsequent down-regulation of cyclin D3 protein
— id: 69225, year: 2006, vol: 5, page: 2172, stat: Journal Article,

p73beta-Mediated apoptosis requires p57kip2 induction and IEX-1 inhibition
Gonzalez, Susana; Perez-Perez, Manuel M; Hernando, Eva; Serrano, Manuel; Cordon-Cardo, Carlos
2005 Mar 15;65(6):2186-2192, Cancer research
Similarly to p53, p73alpha and p73beta induce growth arrest and/or apoptosis in response to DNA damage or when exogenously expressed. However, how they trigger apoptosis remains unresolved. After stable transduction of either p73alpha or p73beta, a greater apoptotic response was observed for p73beta in both primary and tumor cells. Consistently, blocking ectopic and endogenous p73beta expression by specific shRNA significantly decreased apoptotic levels after DNA damage. We found that p73beta targets the apoptotic program at multiple levels: (i) facilitating caspase activation through p53-dependent signals and (ii) inducing p57KIP2, while down-regulating c-IPA1 and IEX1 through a p53-independent mechanism. p73beta-mediated apoptosis was considerably reduced after inhibition of p57(KIP2) by small interfering RNA, IEX-1 overexpression, and in mouse embryo fibroblasts derived from p57-/- mice. Data from this study offer evidence for the apoptotic activity exclusive of p73beta. In the clinical context, these results might have potential therapeutic implications, because p73beta could induce alternative apoptotic responses in tumors harboring p53 mutations
— id: 69227, year: 2005, vol: 65, page: 2186, stat: Journal Article,

A microRNA polycistron as a potential human oncogene
He, Lin; Thomson, J Michael; Hemann, Michael T; Hernando-Monge, Eva; Mu, David; Goodson, Summer; Powers, Scott; Cordon-Cardo, Carlos; Lowe, Scott W; Hannon, Gregory J; Hammond, Scott M
2005 Jun 9;435(7043):828-833, Nature
To date, more than 200 microRNAs have been described in humans; however, the precise functions of these regulatory, non-coding RNAs remains largely obscure. One cluster of microRNAs, the mir-17-92 polycistron, is located in a region of DNA that is amplified in human B-cell lymphomas. Here we compared B-cell lymphoma samples and cell lines to normal tissues, and found that the levels of the primary or mature microRNAs derived from the mir-17-92 locus are often substantially increased in these cancers. Enforced expression of the mir-17-92 cluster acted with c-myc expression to accelerate tumour development in a mouse B-cell lymphoma model. Tumours derived from haematopoietic stem cells expressing a subset of the mir-17-92 cluster and c-myc could be distinguished by an absence of apoptosis that was otherwise prevalent in c-myc-induced lymphomas. Together, these studies indicate that non-coding RNAs, specifically microRNAs, can modulate tumour formation, and implicate the mir-17-92 cluster as a potential human oncogene
— id: 68897, year: 2005, vol: 435, page: 828, stat: Journal Article,

Structural determinants of tissue tropism and in vivo pathogenicity for the parvovirus minute virus of mice
Kontou, Maria; Govindasamy, Lakshmanan; Nam, Hyun-Joo; Bryant, Nathan; Llamas-Saiz, Antonio L; Foces-Foces, Concepcion; Hernando, Eva; Rubio, Mari-Paz; McKenna, Robert; Almendral, Jose M; Agbandje-McKenna, Mavis
2005 Sep;79(17):10931-10943, Journal of virology
Two strains of the parvovirus minute virus of mice (MVM), the immunosuppressive (MVMi) and the prototype (MVMp) strains, display disparate in vitro tropism and in vivo pathogenicity. We report the crystal structures of MVMp virus-like particles (MVMp(b)) and native wild-type (wt) empty capsids (MVMp(e)), determined and refined to 3.25 and 3.75 A resolution, respectively, and their comparison to the structure of MVMi, also refined to 3.5 A resolution in this study. A comparison of the MVMp(b) and MVMp(e) capsids showed their structures to be the same, providing structural verification that some heterologously expressed parvovirus capsids are indistinguishable from wt capsids produced in host cells. The structures of MVMi and MVMp capsids were almost identical, but local surface conformational differences clustered from symmetry-related capsid proteins at three specific domains: (i) the icosahedral fivefold axis, (ii) the 'shoulder' of the protrusion at the icosahedral threefold axis, and (iii) the area surrounding the depression at the icosahedral twofold axis. The latter two domains contain important determinants of MVM in vitro tropism (residues 317 and 321) and forward mutation residues (residues 399, 460, 553, and 558) conferring fibrotropism on MVMi. Furthermore, these structural differences between the MVM strains colocalize with tropism and pathogenicity determinants mapped for other autonomous parvovirus capsids, highlighting the importance of common parvovirus capsid regions in the control of virus-host interactions
— id: 69226, year: 2005, vol: 79, page: 10931, stat: Journal Article,

Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control
Hernando, Eva; Nahle, Zaher; Juan, Gloria; Diaz-Rodriguez, Elena; Alaminos, Miguel; Hemann, Michael; Michel, Loren; Mittal, Vivek; Gerald, William; Benezra, Robert; Lowe, Scott W; Cordon-Cardo, Carlos
2004 Aug 12;430(7001):797-802, Nature
Advanced human cancers are invariably aneuploid, in that they harbour cells with abnormal chromosome numbers. However, the molecular defects underlying this trait, and whether they are a cause or a consequence of the malignant phenotype, are not clear. Mutations that disable the retinoblastoma (Rb) pathway are also common in human cancers. These mutations promote tumour development by deregulating the E2F family of transcription factors leading to uncontrolled cell cycle progression. We show that the mitotic checkpoint protein Mad2 is a direct E2F target and, as a consequence, is aberrantly expressed in cells with Rb pathway defects. Concordantly, Mad2 is overexpressed in several tumour types, where it correlates with high E2F activity and poor patient prognosis. Generation of Rb pathway lesions in normal and transformed cells produces aberrant Mad2 expression and mitotic defects leading to aneuploidy, such that elevated Mad2 contributes directly to these defects. These results demonstrate how chromosome instability can arise as a by-product of defects in cell cycle control that compromise the accuracy of mitosis, and suggest a new model to explain the frequent appearance of aneuploidy in human cancer
— id: 69228, year: 2004, vol: 430, page: 797, stat: Journal Article,

Complete loss of the tumor suppressor MAD2 causes premature cyclin B degradation and mitotic failure in human somatic cells
Michel, Loren; Diaz-Rodriguez, Elena; Narayan, Gopeshwar; Hernando, Eva; Murty, Vundavalli V V S; Benezra, Robert
2004 Mar 30;101(13):4459-4464, Proceedings of the National Academy of Sciences of the United States of America
MAD2 inhibits the anaphase-promoting complex when chromosomes are unattached to the mitotic spindle. It acts as a tumor suppressor gene because MAD2+/-cells enter anaphase early and display chromosome instability, leading to the formation of lung tumors in mice. Complete MAD2 inactivation has not been identified in human tumors, although partial defects are prevalent. By employing RNA interference in human somatic cells, we found that severe reduction of MAD2 protein levels results in mitotic failure and extensive cell death arising from defective spindle formation, incomplete chromosome condensation, and premature mitotic exit leading to multinucleation. Cyclin B is degraded prematurely in the MAD2 short interfering RNA-treated cells but not in MAD2+/- cells, suggesting an explanation for the spindle failure and mitotic catastrophe in the MAD2 knockdown cells. Thus, anaphase-promoting complex substrates exhibit distinct sensitivities in the presence of different MAD2 doses, which in turn determine MAD2's role as either a tumor suppressor or an essential gene
— id: 69229, year: 2004, vol: 101, page: 4459, stat: Journal Article,

Tumor promotion by Mdm2 splice variants unable to bind p53
Fridman, Jordan S; Hernando, Eva; Hemann, Michael T; de Stanchina, Elisa; Cordon-Cardo, Carlos; Lowe, Scott W
2003 Sep 15;63(18):5703-5706, Cancer research
The Mdm2 oncoprotein physically associates with p53 and antagonizes its tumor suppressor functions. Previous studies indicate that some tumors express alternatively or aberrantly spliced Mdm2 variants that are unable to bind p53, but whether these actively contribute to carcinogenesis or are a byproduct of cancer progression has been unclear. In this study, we examined the ability of full-length Mdm2 and several tumor-derived splice variants to modulate tumor development in E micro -myc transgenic mice. We report that several tumor-derived Mdm2 splice variants promote tumorigenesis in a manner that is comparable with full-length Mdm2. Our results imply that the current paradigm for understanding Mdm2 action during oncogenesis is incomplete, and its splice variants contribute to human cancer
— id: 69230, year: 2003, vol: 63, page: 5703, stat: Journal Article,

An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo
Hemann, Michael T; Fridman, Jordan S; Zilfou, Jack T; Hernando, Eva; Paddison, Patrick J; Cordon-Cardo, Carlos; Hannon, Gregory J; Lowe, Scott W
2003 Mar;33(3):396-400, Nature genetics
The application of RNA interference (RNAi) to mammalian systems has the potential to revolutionize genetics and produce novel therapies. Here we investigate whether RNAi applied to a well-characterized gene can stably suppress gene expression in hematopoietic stem cells and produce detectable phenotypes in mice. Deletion of the Trp53 tumor suppressor gene greatly accelerates Myc-induced lymphomagenesis, resulting in highly disseminated disease. To determine whether RNAi suppression of Trp53 could produce a similar phenotype, we introduced several Trp53 short hairpin RNAs (shRNAs) into hematopoietic stem cells derived from E(mu)-Myc transgenic mice, and monitored tumor onset and overall pathology in lethally irradiated recipients. Different Trp53 shRNAs produced distinct phenotypes in vivo, ranging from benign lymphoid hyperplasias to highly disseminated lymphomas that paralleled Trp53-/- lymphomagenesis in the E(mu)-Myc mouse. In all cases, the severity and type of disease correlated with the extent to which specific shRNAs inhibited p53 activity. Therefore, RNAi can stably suppress gene expression in stem cells and reconstituted organs derived from those cells. In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an 'epi-allelic series' for dissecting gene function in vivo
— id: 69231, year: 2003, vol: 33, page: 396, stat: Journal Article,

Protein signals regulating the nuclear-cytoplasmic traffic of the parovovirus MVM capsid
Valle N; Ramirez JC; Lombardo E; Maroto B; Garcia J; Hernando E; Riolobos L; Almendral JM
2003 ;5:271-285, Recent research developments in virology
— id: 69235, year: 2003, vol: 5, page: 271, stat: Journal Article,

Separation of live cells in different phases of the cell cycle for gene expression analysis
Juan, Gloria; Hernando, Eva; Cordon-Cardo, Carlos
2002 Dec 1;49(4):170-175, Cytometry
BACKGROUND: Homogeneity of cell populations is a basic requirement for gene expression analyses of the cell cycle, such as those based on microarrays. The most common approach to obtain specific populations is the use of synchronization methods that increase the number of cells representing a certain cell cycle stage. On the one hand, conventional synchronization usually causes undesirable effects. On the other hand, cell separation methods may imply loss of RNA quality, another limiting factor for expression profiling. We describe a new strategy to specifically separate live cells in different phases of the cell cycle (G(1) and G(2)/M) to obtain good quality RNA for gene expression analyses. METHODS: The experimental design included sorting G(1) and G(2)/M cells with the vital fluorochrome Hoechst 33342, followed by RNA isolation from the sorted cells. RESULTS: Sorted living G(1) and G(2)/M cells, analyzed by immunocytochemistry and laser scanning cytometry, showed strong enrichment. The quality and specificity of the isolated RNA were demonstrated by northern blot. CONCLUSIONS: This new approach has many potential applications, such as expression profiling of specific cell populations after eliminating the irrelevant data produced by cells in other stages of the cycle
— id: 69232, year: 2002, vol: 49, page: 170, stat: Journal Article,

Molecular analyses of the mitotic checkpoint components hsMAD2, hBUB1 and hBUB3 in human cancer
Hernando, E; Orlow, I; Liberal, V; Nohales, G; Benezra, R; Cordon-Cardo, C
2001 Jul 20;95(4):223-227, International journal of cancer
During the metaphase-anaphase transition, the spindle checkpoint prevents segregation of chromosomes if the spindle assembly is perturbed. Critical components of this checkpoint are the MAD and BUB families of proteins, which prevent the proteolysis of Pds1 and B cyclins, producing mitotic arrest. In the present study, we first intended to resolve the role of the hsMAD2 gene in human cancer by determining the potential presence of hsMAD2 mutations in 44 primary bladder tumors, 42 soft-tissue sarcomas and 10 hepatocellular carcinomas. The entire coding region of the hsMAD2 gene was analyzed using PCR-SSCP and sequencing. One of the bladder tumor samples showed a point mutation consisting of a transition, ATC-->GTC (Ile-->Val) in codon 190 of hsMAD2. However, no differences were found in the mitotic arrest between cells transfected with mutant and wild-type MAD2 cDNA. We also identified mobility shifts in hsMAD2 in both normal and tumor DNA in 3 bladder tumors, 3 soft-tissue sarcomas and 1 hepatocellular carcinoma, consistent with a polymorphism at codon 143, CCA-->CCG (Pro-->Pro). Another polymorphism was identified in a hepatocellular carcinoma case at codon 22, GAG-->GAA (Glu-->Glu). In addition, a subgroup of 67 primary tumors was analyzed by Southern blot hybridization. No deletion or visible re-arrangements were detected by comparing tumor and normal DNA band signals. Two other important components of the spindle mitotic checkpoint, hBUB1 and hBUB3, were also screened for mutations: hBUB1 in 43 bladder tumors and 9 bladder cell lines and hBUB3 only in the cell lines. Two polymorphisms were found in hBUB1 at positions 144, CAG-->CAA (Gln-->Gln) in 1 primary tumor and 1 bladder cell line, and 913 (ATC-->ATT, Ile-->Ile) in 1 primary tumor. We did not find sequence alterations in hBUB3. These results suggest that mutations of the hsMAD2, hBUB1 and hBUB3 genes are very rare in bladder tumors and that hsMAD2 alterations are also infrequent in soft-tissue sarcomas and hepatocellular carcinomas
— id: 69233, year: 2001, vol: 95, page: 223, stat: Journal Article,

Biochemical and physical characterization of parvovirus minute virus of mice virus-like particles
Hernando, E; Llamas-Saiz, A L; Foces-Foces, C; McKenna, R; Portman, I; Agbandje-McKenna, M; Almendral, J M
2000 Feb 15;267(2):299-309, Virology
The VP-2 major capsid protein of the prototype strain of the parvovirus minute virus of mice (MVMp) was expressed, using a baculovirus vector, in Sf9 insect cells. Immunogold electron microscopy of infected Sf9 cells showed VP-2 localized in the nucleus and cytoplasm as is observed in mammalian cells during natural infections. The VP-2 subunits self-assembled into empty parvovirus-like particles (VLPs), which appeared morphologically similar to and immunogenically indistinguishable from native empty MVMp particles, which also contain the minor capsid protein, VP1. Incubations under different pH and temperature conditions showed that the MVMp VLPs and native empty MVMp capsids share comparable stability. Once heated the particles can be similarly and specifically cleaved by trypsin at the VP-2 N-terminal domain. This process mimics the further maturation of the 'rat-like' parvovirus virions, following viral DNA encapsidation, indicating that biologically relevant features of the MVMp capsid are maintained in the VLPs. Crystals have been obtained for the MVMp VLPs which were isomorphous to those used for the high-resolution structure determination of virions and native empty particles of the immunosuppressive strain of MVM (MVMi). The VLP crystals diffracted X rays to beyond 3-A resolution and are in space group C2 (a = 448.7, b = 416.6, c = 306.1 A, and beta = 95.9 degrees ). This is the first report of crystals from parvoviral particles produced in a heterologous system diffracting X rays to high resolution, indicating that VP-2 of some parvovirus capsids can self-assemble into ordered T = 1 icosahedral capsids in the absence of other viral and host cell functions
— id: 69234, year: 2000, vol: 267, page: 299, stat: Journal Article,