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DEFINING THE ROLE OF MUTATED Hbraf IN MELANOGENESIS

DEFINING THE ROLE OF MUTATED Hbraf IN MELANOGENESIS. EUNICE ASARE Dr. ALAN HOUGHTON LABORATORY Memorial Sloan Kettering Cancer Center (MSKCC). INTRODUCTION. Melanoma is the deadliest skin cancer. It begins in melanocytes (cells responsible for skin pigmentation).

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DEFINING THE ROLE OF MUTATED Hbraf IN MELANOGENESIS

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  1. DEFINING THE ROLE OF MUTATED Hbraf IN MELANOGENESIS EUNICE ASARE Dr. ALAN HOUGHTON LABORATORY Memorial Sloan Kettering Cancer Center (MSKCC)

  2. INTRODUCTION • Melanoma is the deadliest skin cancer. It begins in melanocytes (cells responsible for skin pigmentation). • There is a frequency of Braf mutations in ras pathway found in most cases of melanoma, ~7O%. • Braf is a protein coding gene that enables a normal cell division.

  3. RISK FACTORS LEADING TO MELANOMA • CHANGING NEVUS(MOLE) • LARGE NUMBERS OF MOLES • FAMILY HISTORY OF MELANOMA • OVEREXPOSURE TO SUN ULTRAVOILET RADIATION • MUTATION OF GENE (Hbraf)

  4. CHARACTERISTICS OF MELANOMA

  5. Pathways that may lead to cell cycle dysregulation in melanocytes CELL CYCLE Ras Pten Pten CdKn2alocus Process by which a cell divides to Form a new cell. The gene product of braf interacts with other gene products in the cell for a normal cell division. A mutation in the pathway will cause the cell to divide abnormally, leading to cancer. There is a frequency of Braf mutations in ~70 of most cases of Melanoma.

  6. OBJECTIVE Generation of transgenic mice expressing the mutated Hbraf in melanocytes to mimic the situation in human melanoma patients with mutated Hbraf • Insertion of mutated Hbraf into expression vector • Expression of mutated Hbraf in melanocytes - Use of transgenic mice to express mutated Hbraf - Create transgenic mice susceptible in developing melanoma. • Vaccinate mice who develop melanoma with HGP75 optimized or HGP75 vaccine.

  7. STRATEGY TO INSERT Hbraf INTO EXPRESSION VECTOR

  8. MATERIALS and METHODS • PLASMID CONSTRUCTION : The oligo TRP2 1 Braf 1 and TRP2 Braf 2 is prepared from a sequence of DNA which contains the restriction sites for Xhol, NoTI, XbaI, HindIII, BAMHI to be ligated into the original plasmid. • INSERT OF TRP2: An obtained plasmid pPtrp-2/lacZ has the Hbraf gene promoter Trp-2. To attain only the Trp-2, we cleaved the plasmid of its other genes and promoters by digesting using NoTI.

  9. continued Plasmid vector pPtrp-2/lacZ containing the Hbraf gene promoter Trp-2.

  10. INSERT OF Hbraf.The final insert into our plasmid is Hbraf wild-type and Hbraf mutated. An acquired plasmid pcDNA 3.1(-) with a size of 5427bp contains both insert but also with other genes. we only want the wt and mut Hbraf genes. We digested the plasmid with PmeI, next agarose gel electrophoreses was run to check for the purity of the insert. Two separate ligations are done here; the first one is to insert the mut Hbraf into our constructed plasmid and the next ligation is to insert the wt Hbraf into our constructed plasmid without the mut Hbraf. Transformation is prepared with both newly constructed plasmids thus 62.1mod/oli/Trp2+mutH and 62.mod/oli/Trp2+wtHbraf. CONTINUED

  11. DESIGNING TRANSGENIC MICE

  12. FUTURE DIRECTIONS • In the course of the coming weeks our ongoing objectives will be headed for and completed. Since we have already constructed our expression vector with the desired gene mutHbraf, it will be expressed in melanocytes. • In the direction of inserting the mutHbraf in melanocytes, it will be sent to the Transgenic Mice Facility at MSKCC for this to be done. • The Embryonic Stem Procedure will be used to create a line of transgenic mice C57B/6 vulnerable in developing melanoma comparable to human melanoma patients with mutated Hbraf gene. • The C57B/6 mice will be tested for the presence of mutBraf gene. • The mice that test out positive for the gene will be followed to see if they develop melanoma. • The melanomas developed will be characterized and treated with the HGP75 optimized or HGP75 vaccine to suppress the melanoma.

  13. GENOTYPING TRANSGENIC MICE Presently in the lab our ongoing aim is to: • Use Polymerase Chain Reaction (PCR) method to detect the presence of MutHbraf in our obtained transgenic mice. • We will cross breed MutHbraf transgenic mice with mice whose Ink4a gene ( tumor suppressor gene) has been “Knocked Out”. • New mice breed from the cross will be monitored to see if they develop melanoma. • The melanomas developed will be characterized and treated with the HGP75 optimized or HGP75 vaccine to suppress the melanoma.

  14. Ras Pten Pten CdKn2alocus Pathways that may lead to cell cycle dysregulation in melanocytes

  15. GENOTYPING TRANSGENIC MICE Presently in the lab our ongoing aim is to: • Use Polymerase Chain Reaction (PCR) method to detect the presence of MutHbraf in our obtained transgenic mice. • We will cross breed MutHbraf transgenic mice with mice whose Ink4a gene ( tumor suppressor gene) has been “Knocked Out”. • New mice breed from the cross will be monitored to see if they develop melanoma. • The melanomas developed will be characterized and treated with the HGP75 optimized or HGP75 vaccine to suppress the melanoma.

  16. AKNOWLEDGEMENT • ELVA BARREDA and Dr TAHA MERGHOUB • MINYI TAN • SHELDON MOORE • BARTEK JABLONSKI • ALAN HOUGHTON LABORATORY OF IMMUNOLOGY • MSKCC • Dr. SAT BHATTACHARY HARLEM CHILDREN SOCIETY

  17. REFERENCE • (1) (1)MouseGENETICSANDTRANSGENICS • THE Practical Approach SERIES; B.D Hames • http:www.oup.co.uk/PAS • (2) Houghton AN, Polsky D. Focus on melanoma. Cancer Cell. 2002 Oct;2(4):275-8. Review • (3) Tuveson DA, Weber BL, Herlyn M.BRAF as a potential therapeutic target in melanoma and other malignancies. Cancer Cell. 2003 Aug;4(2):95-8. Review. • (4) JK. Related Links Is there more than one road to melanoma?Lancet. 2004 Feb 28;363(9410):728-30. Review. PMID: 15005091 [Pub Med - indexed for MEDLINE • (5) Immunity to cancer through immune recognition of altered self: Studies with melanoma. Jose A. Guevara-Patino, Mary Jo Turk, Jedd D. Wolchock, and Alan N. Houghton • MSKCC and the Weill Graduate school of Medical sciences and Medical school of Cornell University, 1275 York Avenue, New York, N.Y 10021 • (6)Mutations of the Braf gene in human cancer • HELEN DAVIES et al • Cancer Genome project, The welcome Trust Sanger Institute Welcome Trust Genome Campus, Hinxton CB10ISA,UK et. al • (7)American Cancer Society Web site; what are the key statistics for melanoma skin cancer? • http://www.cancer.org • (8) Cancer Reference Information • skin cancer –Melanoma • what are the risk factors for melanoma? • http:www.cancer.org/docroot/cri/content/cri_2_4_2x what are the risk • Melanoma • Escape dangers by learning to protect yourself • http://www.ftmeade.army.mil/soundoff/archives/SO2002/Jul18/html/Cover-story.htm • Incidence of Braf oncogene mutation and clinical relevance for Primary Cutaneous Melanomas • Masaru Shinozaki et. al • Department of Molecular oncology, John Wayne cancer institute, Saint John’s Health Center, Santa Monica, California

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