DNAprint Genomics (DNAG)

[SlopsterSlasher]

"Wistar institute" a name that lurks in the back ground But also is a Big Player with Dr. Sturm - http://www.wistar.upenn.edu/research_facilities/mherlyn/research.htm
Meenhard Herlyn, D.V.M.

Professor and Program Leader
Molecular and Cellular Oncogenesis Program
215-898-3950, Office
215-898-0980, Fax
[e-mail]
www.wistar.upenn.edu/herlyn

Introduction

Research in the laboratory of Meenhard Herlyn centers on the basic mechanisms that govern normal cell function, or homeostasis. Knowing how cells and tissues orchestrate their intertwined purposes helps researchers establish what happens when things go awry, such as in cancerous tumors.

Research Summary

Cells in normal tissues maintain a life-long homeostatic balance, in which growth, differentiation, and cell death are dynamically regulated. To establish and maintain normal organ structure and function, they must remain within their predestined locations within each organ. The delicate balance among cells within the scaffolding of matrix proteins is disturbed during tumor development. Transformed cells escape from homeostasis and destroy normal tissue architecture. Our laboratory is interested in defining normal tissue homeostasis and understanding pathological changes during cancer development and progression in order to develop new strategies for therapy.

Recent Scientific Advances

Intercellular crosstalk in normal skin and melanoma (Steve Kazianis, Ph.D., Mizuho Fukunaga M.D. Ph.D., Nikolas Haass, M.D., Ling Li, M.D., Akihiro Yoneta, M.D., Ronan McDaid, B.S., Bernard Herlyn): Using a three-dimensional model of normal human skin in vitro and in vivo, we are investigating how melanocytes and keratinocytes communicate with each other, how melanocytes transform when driven by growth factors and UVB, how melanoma cells have switched partners from keratinocytes to fibroblasts and endothelial cells and how we can establish new therapeutic strategies by forcing the malignant cells back under the control of normal keratinocytes.

Signaling in melanoma and therapeutic strategies (Keiran Smalley, Ph.D., Patricia Brafford, M.S.): We are defining the signal transduction pathways that are constitutively activate in melanoma cells through autocrine and paracrine growth factors (bFGF, ET-3, SCF, IGF-1, HGF, TGF-ß). Three-dimensional models are being developed to investigate tumor-to-fibroblast crosstalk in a tissue context. Through RNAi approaches we are selecting those genes in tumor cells and stromal fibroblasts that are potential targets for therapy. Artificial skin/melanoma and esophagus/carcinoma models are used for selecting small molecules as antagonists.

Stem cells, transformation, and microenvironment (Dong Fang, M.D., Ph.D., Rena Finko, M.S., Kim Leishear, B.S., Nga Nguyen, B.S., Angela Kulp): Human embryonic stem cells are differentiated into melanocytes to test the hypothesis that melanocyte progenitor cells are more prone to transformation than fully differentiated cells and that neighboring cells and matrix in the microenvironment play critical roles in differentiation and transformation. Bone marrow-derived stem cells give rise to hematopoietic, mesenchymal, endothelial cells, and, as in the case of multipotent adult progenitor cells (MAPCs), to a variety of ectodermal and neuroectodermal cells. We are investigating the biological significance for tumor progression of fibroblasts and endothelial cells derived from the bone marrow stem cell pool hypothesizing that rapidly expanding tumors attract stem cells to meet their needs for new vessels and matrix. We are also testing whether we can differentiate other skin cells from human bone marrow and embryonic stem cells.

The vascular phenotype in melanoma (Zhao-Jun Liu, M.D., Ph.D, Chelsea Pinnix, Klara Balint, M.D., Haiyan Chen, M.D., Cheeyong Pang): Active angiogenesis is one of the hallmarks of melanoma. We are interested in elucidating the molecular mechanisms of neovascularization in melanoma and are focusing on signaling through cell surface molecules including Notch, a v b3 integrin, and Mel-CAM (CD146). We are investigating their involvement in differentiation of bone marrow-derived stem cells into endothelial cells and their contribution to tumor angiogenesis. All three molecules are prototypes for expression on both melanoma and endothelial cells and we hypothesize that they play major roles in metastasis by regulating endothelial-melanoma cell interactions.

Cell-cell adhesion for normal tissue homeostasis:
Skin – Keratinocytes in the epidermis of the skin control proliferation of melanocytes and dictate which cell surface molecules are expressed for adhesion and migration. The lab’s working hypothesis is that melanocyte proliferation is possible if they decouple from keratinocytes by down-regulating E-cadherin and its co-receptor desmoglein-1, which will interrupt gap junctions formed through connexin 43 molecules. Dwon-regulation of expression of the cell-cell communication molecules is mediated through the production of HGF, ET-1, and PDGF. The melanocytes then retract their dendrites that had connected them to keratinocytes of the suprabasal layers by activating rac and rho genes. Cell division is likely initiated through activation by keratinocyte-derived growth factors such as bFGF, SCF, ET-1 or by fibroblast-derived factors such as HGF, IGF-1, or ET-3. After cell division, melanocytes separate and glide over the basement membrane using integrins such as alpha6ß1 or alpha7ß1 before repositioning singly among basal layer keratinocytes. Using the three-dimensional organotypic culture model of human skin consisting of dermis and epidermis we are retracing each step in the proliferation cascade of melanocytes to better understand dysregulation of growth and cell-cell communication in melanoma. The lab uses adenoviral and lentiviral vectors to transfer genes for overexpressing or inhibiting a function of interest. The unique model of human skin reconstruction in vitro and in vivo allows us to investigate signaling between melanocytes and keratinocytes for tissue homeostasis and its dysregulation during transformation to nevi and melanomas.Melanoma cells have escaped from keratinocytes by downregulating E-cadherin and upregulating N-cadherin. The cadherin switch allows a cell partner change because now melanoma cells can adhere through N-cadherin to fibroblasts and endothelial cells. Overexpression of E-cadherin in melanoma cells allows keratinocytes to adhere to them and regain control over proliferation and the expression of cell surface molecules, so the malignant cells revert to a non-malignant phenotype. The team expects in the next few years to identify and characterize transcriptional activators or repressors in normal melanocytes that are non-functional in melanoma cells but can be reactivated to control growth and invasion.

Esophagus – The squamous epithelium of the normal human esophagus follows a similar differentiation pattern compared to the normal epidermis except that different keratins are expressed. To better understand homeostasis in the normal esophagus the lab is overexpressing growth factors and disrupt/activate receptor function in either fibroblasts or esophageal keratinocytes. This is done by embedding the stromal cells in a three-dimensional collagen matrix and exposing overlayed keratinocytes to air to allow multi-layer formation and differentiation. The lab tests the hypothesis that continuous activation of keratinocytes through the local production of growth factors can induce a transformed phenotype. The lab hypothesizes that the EGF and TGF-ß receptor systems are dominant for maintaining the homeostatic balance in the normal esophageal mucosa and their dysfunction is critical for tumor development and progression and that fibroblasts and endothelial cells are recruited during tumor progression from the bone marrow stem cell pool.

Endothelium – Long-term objectives in this area include understanding the dynamic changes in gene expression during dysregulation of homeostasis in cancer and chronic wounds. The laboratory has developed an in vitro vessel reconstruction model that demonstrates the importance of fibroblasts for endothelial cell migration and differentiation. We can now systematically dissect the processes of blood vessel formation and tumor-stimulated angiogenesis by regulating gene expression in either fibroblasts or endothelial cells. The emphasis is on those growth factor and adhesion receptors that are shared between activated endothelial cells and melanoma cells including VEGFR2, VEGFR-1, a vß3, Mel-CAM (CD146), Notch-1, and EphA2.
Experimental transformation of melanocytes: The critical difference between uncontrolled and controlled melanocyte proliferation in skin is their permanent de-coupling from keratinocytes, the ability of cells to communicate with stromal cells and the constitutive production of growth factors. The lab hypothesizes that the growth factor bFGF needs to be activated if the melanocytes are to survive when they de-couple from the keratinocytes and migrate into the dermis. bFGF can transform melanocytes in a human skin graft model, when the skin is irradiated at the same time with UVB. If three growth factors -- bFGF, SCF and ET-3 -- are combined for overexpression in the dermis and the skin is irradiated with UVB, melanoma-like lesions are induced within three to four weeks. This unique carcinogenesis model allows the team to retrace the different steps of melanoma development from healthy epidermal melanocytes; to establish molecular markers for melanoma diagnosis and prognosis; and better investigate the most critical signaling pathways for transformation. It is becoming apparent that the MAP kinase signaling pathway is constitutively activated, either through autocrine and paracrine growth factors or through activating mutations of the BRAF and n-ras genes.

Tumor-stroma interactions during tumor progression and metastasis: The unique RGP and VGP primary and metastatic melanoma cell lines that have been established in the last 20 years in the Herlyn laboratory are being used to investigate the roles of growth factors and adhesion receptors in tumor-stroma interactions,. The tumor stroma is composed of fibroblasts, blood vessels, inflammatory cells and matrix proteins such as collagens, fibronectin, laminins, and proteoglycans. Melanoma cells produce bFGF and HGF for autocrine (self) stimulation whereas PDGF, VEGF and TGF-ß are produced for stimulation of the tumor stroma. Stromal fibroblasts in turn provide growth factors such as IGF-1 or HGF for positive feed back (stimulation) of the malignant cells. The laboratory is developing for each stromal cell type – that is, fibroblasts, endothelial cells, monocytes, and neutrophils -- regulatory circuits for positive and negative cross talk to the malignant cells. Endothelial cells and fibroblasts in the tumor stroma are apparently derived from two separate pools, the resident pool recruited from tissues adjacent to the tumor, and the precursor pool that has migrated from bone marrow and peripheral blood. The team does not know how the precursor cells are recruited or the signals needed for differentiation, but expects that tumor cells provide appropriate signals and so are testing this hypothesis in melanoma, and esophageal carcinoma.

The plastic phenotype in metastasis: multiple roles for cell-cell and cell-matrix adhesion receptors for invasion and metastasis: Primary melanomas have numerous abnormalities within their genetic make-up, suggesting considerable genomic instability. No major additional genomic changes appear necessary for metastasis, because VGP melanomas are easily adapted to a metastatic phenotype by culture in growth factor-free medium and by induction of invasion through basement membrane-like matrix material. The lab is testing the hypothesis that micro-environmental changes in cell-matrix and cell-cell signaling are critical for the metastatic phenotype. One of the main players is the cell-cell adhesion molecule of the CAM family -- MUC18/Mel-CAM -- which is expressed by all melanoma cells and binds to an as-yet-unknown ligand also found on melanomas. Mel-CAM is one of at least six CAM -- ALCAM, VCAM-1, ICAM-1, CEA1-CAM, L1-CAM -- shared between melanoma and endothelial cells and we are delineating their biological functions for metastasis. Similarly, activated endothelial and melanoma cells share the integrin alphavß3 and cadherins N-cadherin and VE-cadherin, so understanding the contribution of these to the metastatic phenotype is also important. Based on preliminary studies it is also likely that the tumor-infiltrating endothelial cells are being recruited from the bone marrow stem cell pool. Thus another current experiment tests whether differentiating stem cells express unique molecules that can be targeted for therapy.

Selected Publications

Schaider, H.,* Oka, M., * Bogenrieder, T., Nesbit, M., Satyamoorthy, K., Berking, C., Matsushima, K., and Herlyn, M. Differential response of primary and metastatic melanomas to neutrophils attracted by IL-8. Int. J. Cancer 103: 335-343, 2003.* Equal contribution.

Gruss, C.J., Satyamoorthy, K., Berking, C., Lininger, J., Nesbit, M., Schaider, H., Liu, Z.-J., Oka, M., Hsu, M-Y., Shirakawa, T., Li, G., Bogenrieder, T., Carmeliet, P., El-Deiry, W.S., Eck, S.L., Rao, J.S., Baker, A.H., Bennett, J.T., Crombleholme, T.M., Velazquez, O., Karmacharya, J., Margolis, D.J., Wilson, J.M., Detmar, M., Skobe, M., Robbins, P.D., Buck, C., and Herlyn, M. Stroma formation and angiogenesis by induced expression of growth factors, cytokines, and proteolytic enzymes in human skin grafted to SCID mice. J. Invest. Derm. 120: 683-692, 2003.

Liu, Z.-J., Shirakawa, T., Li, Y., Souma, A., Oka , M., Dotto, G.P., Fairman, R., Velazquez, O.C., and Herlyn, M. Regulation of notch1 and dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis. Mol. Cell Biol. 23: 14-25, 2003.

Satyamoorthy, K.,* Li, G.,* Gerrero, M.R., Brose, M.R., Volpe, P., Weber, B.L., Van Belle, P.A., Elder, D.E., and Herlyn, M. Constitutive mitogen-activated protein kinase activation in melanoma by both BRAF mutations and autocrine growth factor stimulation. Cancer Res. 63: 756-759, 2003. *Equal contribution

Kalabis, J., Patterson, M.J., Gimotty, P., Enders, G.H., Marian, B., Iozzo, R.V., Rogler, G., and Herlyn, M. Stimulation of human colonic epithelial cells by leukemia inhibitory factor is dependent on collagen-embedded fibroblasts in organotypic culture. FASEB J. 17: 1115-1117, 2003.

Li, G., Kalabis, J., Xu, X., Meier, F., Oka, M., Bogenrieder, T., Herlyn, M. Reciprocal regulation of MelCAM and AKT in melanoma. Oncogene 22: 6891-6899, 2003.

Berking, C., Takemoto, R., Satyamoorthy, K., Eskandarpour, M., Shirakawa, T, Hanson, J., vanBelle, P.A., Elder, D.E., Herlyn, M: Induction of melanoma phenotypes in human skin by growth factors and ultraviolet B. Cancer Res., 64: 807-811, 2004.

Reviews

Velazquez, O., and Herlyn, M. The vascular phenotype of melanoma metastasis. Clin. Exp. Met. 20 : 229-235, 2003. (R)

Li, G., Meier, F., Berking, C., Satyamoorthy, K., Bogenrieder, T., and Herlyn, M. Function and regulation of melanoma-stromal fibroblast interactions: when seeds meet soil. Oncogene 22: 3162-3171, 2003. (R)

Tuveson, D. A., Weber, B.L., and Herlyn, M. BRAF as a potential therapeutic target in melanoma and other malignancies. Cancer Cell 4 :95-8, 2003 (R).

Liu, Z.-J., and Herlyn, M. Slit-Robo: Neuronal guides signal tumor angiogenesis. Cancer Cell 4: 1-2, 2003 (R).

Bogenrieder, T., Elder, D.E., and Herlyn, M. Molecular and cellular biology. In: Cutaneous Melanoma, 4 th edition, C. M. Balch, A.N. Houghton, A.J. Sober, S-j Soong, eds., Quality Medical Publ., St Louis, MO, pp. 713-751, 2003 (R).

Bogenrieder, T., and Herlyn, M. Axis of evil: Molecular mechanisms of cancer metastasis. Oncogene 42 : 6524-6536, 2003 (R).

Satyamoorthy, K., and Herlyn, M. p16 INK4A and familial melanoma. Methods Mol. Biol. 222: 185-195, 2003. (R).

Hsu, M-Y., Ling, L., and Herlyn, M. Cultivation of normal human epidermal melanocytes in the absence of phorbol esters. In: Methods in Molecular Medicine: Human Cell Culture Protocols (G.E. Jones ed.). Humana Press, Inc., Totowa, NJ, in press.

Perlis, C., and Herlyn, M: Recent advances in melanoma biology. Oncologist, in press, 2004 (R).

Smalley, K.S., and Herlyn, M. The great escape: Another way for melanoma to leave physiological control? J. Invest. Dermatol. 121: ix, 2003 (R).

Liu, X.-J., and Herlyn, M. Molecular biology of cutaneous melanoma. In: 7 th edition of Cancer: Principles and Practice of Oncology, (V.T. DeVita, Jr., S. Hellman, S.A. Rosenberg, eds.). Lippincott Williams &Wilkins, Philadelphia, PA, in press. (R)

Herlyn, M., and Guerry, D. IV. Meeting Report: First International Melanoma Research Congress. Cancer Biol. Therapy 2: 721-724 , 2003. (R)

Haass, N.K., Smalley, K. S. M, and Herlyn, M: The role of altered cell-cell communication in melanoma progression. J. Mol. Histo., in press. (R)