Faculty Bibliography

Maternal gene products drive early development when the newly formed embryo is transcriptionally inactive. During the maternal-zygotic transition, embryonic transcription is initiated and many maternal RNAs are degraded. Multiple mechanisms regulate the birth of zygotic RNAs and the death of maternal RNAs. Genome activation appears to rely in part on the sequestration of transcriptional repressors by the exponentially increasing amount of DNA during cleavage divisions. Maternal RNA degradation is induced by the binding of proteins and microRNAs to the 3' untranslated region of target RNAs.

The early embryo is formed by the fusion of two germ cells that must generate not only all of the nonreproductive somatic cell types of its body but also the germ cells for the next generation. Therefore, embryo cells face a crucial decision: whether to develop as germ or soma. How is this fundamental decision made and germ cell fate maintained during development? Studies in the nematode worm Caenorhabditis elegans and fruit fly Drosophila identify some of the decision-making strategies, including segregation of a specialized germ plasm and global transcriptional regulation

Nodal signals induce mesodermal and endodermal progenitors during vertebrate development. To determine the role of Nodal signaling at a genomic level, we isolated Nodal-regulated genes by expression profiling using macroarrays and gene expression databases. Putative Nodal-regulated genes were validated by in situ hybridization screening in wild type and Nodal signaling mutants. 46 genes were identified, raising the currently known number of Nodal-regulated genes to 72. Based on their expression patterns along the dorsoventral axis, most of these genes can be classified into two groups. One group is expressed in the dorsal margin, whereas the other group is expressed throughout the margin. In addition to transcription factors and signaling components, the screens identified several new functional classes of Nodal-regulated genes, including cytoskeletal components and molecules involved in protein secretion or endoplasmic reticulum stress. We found that x-box binding protein-1 (xbp1) is a direct target of Nodal signaling and required for the terminal differentiation of the hatching gland, a specialized secretory organ whose specification is also dependent on Nodal signaling. These results indicate that Nodal signaling regulates not only specification genes but also differentiation genes.

Mutations in human cartilage oligomeric matrix protein (COMP) have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia, however, the function of both wild-type and mutant COMP in the skeletogenesis remain unknown. In an effort to define the biological functions of COMP, a functional genetic screen based on the yeast two-hybrid system was performed. This led to the identification of Granulin-epithelin precursor (GEP), an autocrine growth factor, as a COMP-associated partner. COMP directly binds to GEP both in vitro and in vivo, as revealed by in vitro pull down and Co-immunoprecipitation assays. GEP selectively interacts with the EGF repeat domain of COMP but not with the COMP's other three functional domains. The Granulin A repeat unit of GEP is required and sufficient for association with COMP. COMP co-localizes with GEP predominantly in the pericellular matrix of transfected rat chondrosarcoma cell (RCS) and primary human chondrocytes. Staining of musculoskeletal tissues of day 19 mouse embryo with antibodies to GEP is restricted to chondrocytes and appears to be concentrated in areas where ossification will occur. Over expression of GEP stimulates the proliferation of chondrocytes and this stimulation is enhanced by COMP. In addition, COMP appears to be required for GEP-mediated chondrocyte proliferation, since chondrocyte proliferation induced by GEP is dramatically inhibited by an anti-COMP antiserum. These findings provide the first evidences linking the association of COMP and GEP, and identifying a previously unrecognized growth factor (i.e. GEP) in cartilage

Aiming at more precise detection of melanoma cells in sentinel lymph nodes and better understanding of the mechanisms underlying metastatic spread, expression of L1, CEACAM1, and binding of the lectins HPA, ML-I and PNA, was assessed in benign nevi (n=12), primary melanomas (PTs: n=67), their corresponding sentinel lymph nodes (SLNs: n=40), and distant metastases (DMs: n=35). Sensitivity and specificity of CEACAM1 (95-97%; 66%) and L1 (90-93%; 100%) exceeded that of the standard markers MelanA, S100, and HMB45 in single marker use. Lectin binding was found in PTs and DMs (HPA: 69% and 77%; ML-I: 82% and 77%, respectively), but rarely in SLNMs (HPA: 20%, ML-I: 20%, PNA: 5%, respectively). The highly specific and sensitive L1-11A against L1 and 4D1/C2 against CEACAM1 antibodies are a worthy completion to standard antibody panels for diagnosis of melanoma cells. Both CAMs seem to be functionally involved in lymphatic and haematogenous spread, and are thus promising target molecules for immunotoxins.

Melanoma is one of the most aggressive cancers, and its incidence is increasing. These tumors derive from the melanocyte lineage and remain incurable after metastasis. Here we report that SONIC HEDGEHOG (SHH)-GLI signaling is active in the matrix of human hair follicles, and that it is required for the normal proliferation of human melanocytes in culture. SHH-GLI signaling also regulates the proliferation and survival of human melanomas: the growth, recurrence, and metastasis of melanoma xenografts in mice are prevented by local or systemic interference of HH-GLI function. Moreover, we show that oncogenic RAS-induced melanomas in transgenic mice express Gli1 and require Hh-Gli signaling in vitro and in vivo. Finally, we provide evidence that endogenous RAS-MEK and AKT signaling regulate the nuclear localization and transcriptional activity of GLI1 in melanoma and other cancer cells. Our data uncover an unsuspected role of HH-GLI signaling in melanocytes and melanomas, demonstrate a role for this pathway in RAS-induced tumors, suggest a general integration of the RAS/AKT and HH-GLI pathways, and open a therapeutic approach for human melanomas.

The heat shock protein HSP90 chaperones proteins implicated in breast cancer progression, including Her2/neu. HSP90-targeting agents are in clinical trials for breast cancer. HSP90 expression is high in breast cancer cell lines, yet no large studies have been conducted on expression in human tumors and the association with clinical/pathologic variables. Tissue microarrays containing 10 cell lines and primary specimens from 655 patients with 10-year follow-up were assessed using our automated quantitative analysis (AQUA) method; we used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot and measured HSP90 expression within the mask using Cy5-conjugated antibodies. We similarly assessed estrogen receptor, progesterone receptor, and Her2/neu expression. HSP90 expression was more variable in human tumors than in cell lines (P < 0.0001). High HSP90 expression was associated with decreased survival (P = 0.0024). On multivariable analysis, high HSP90 expression remained an independent prognostic marker. High HSP90 expression was associated with high Her2/neu and estrogen receptor, large tumors, high nuclear grade, and lymph node involvement. Although HSP90 levels were high in all our cell lines, expression in tumors was more variable. High HSP90 expression in primary breast cancer defines a population of patients with decreased survival. Evaluation of HSP90 expression in early-stage breast cancer may identify a subset of patients requiring more aggressive or pathway-targeted treatment. Prospective studies are needed to confirm the prognostic role of HSP90, as well as the predictive role of HSP90 expression in patients treated with HSP90 inhibitors.

The unfolded protein response (UPR) is an intracellular signaling pathway that is activated in response to stress in the endoplasmic reticulum (ER). UPR can effectively cope with stress by reducing the amount of misfolded protein overload in this subcellular organelle. Significantly, ER-stress is associated with various neurodegenerative disorders, diabetes and cancer, where UPR affects the course of disease manifestation in many cases. While significant progress has been made in various experimental systems over the years, suitable models for in vivo analyses of UPR and disease remain scarce. In this regard, recent developments of Drosophila markers and genetic tools for UPR studies provide powerful means to investigate the connection between UPR and disease in vivo. Here, we review the molecular components of the Drosophila UPR as well as the disease models that may be affected by this signaling pathway

BACKGROUND: The regulation of the prostate size by androgens may be partly the result of androgen effects on the prostatic vasculature. We examined the effect of changes in androgen levels on the expression of a variety of angiogenic factors in the mouse prostate and determined if vascular endothelial growth factor (VEGF)-A and the angiopoietins are involved in the vascular response to androgens. METHODS: Expression of angiogenic factors in prostate was quantitated using real-time PCR at different times after castration and after administration of testosterone to castrated mice. Angiopoietins were localized in prostate by immunohistochemistry and in situ hybridization. The roles of VEGF and the angiopoietins in regeneration of the prostate were examined in mice inoculated with cells expressing soluble VEGF receptor-2 or soluble Tie-2. RESULTS: Castration resulted in a decrease in VEGF-A, VEGF-B, VEGF-C, placenta growth factor, FGF-2, and FGF-8 expression after 1 day. In contrast, VEGF-D mRNA levels increased. No changes in angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), hepatocyte growth factor, VEGF receptor-1, VEGF receptor-2, or tie-2 mRNA levels were observed. Administration of testosterone to castrated mice had the opposite effect on expression of these angiogenic factors. Ang-2 was expressed predominantly in prostate epithelial cells whereas Ang-1 was expressed in epithelium and smooth muscle. Inoculation of mice with cells expressing soluble VEGF receptor-2 or Tie-2 blocked the increase in vascular density normally observed after administration of testosterone to castrated mice. The soluble receptors also blocked the increase in prostate weight and proliferation of prostatic epithelial cells. CONCLUSION: VEGF-A and angiopoietins are required for the vascular response to androgens and for the ability of the prostate to regenerate in response to androgens