1. Splicing Regulations of CD44v3 in Breast Cancer Metastasis Hayley Baines City of Hope Summer Student Program May 27 – August 1, 2003 Mentor: Tracy Li, M.D., M.S. PI: RJ Lin, PhD Molecular Biology

2. City of Hope Summer Student Program zDr. RJ Lin’s Molecular Biology Lab at the Beckman Research Institute at the City of Hope, Duarte, CA z10 weeks of working on my own CD44 research project with Tracy Li, my mentor, a third year PhD student. - Tracy is investigating several variants of CD44 and their splicing regulations, my project focused on CD44v3. zWeekly summer student meetings and seminars.

3. Splicing DNA Pre-mRNAmRNAProtein TranscriptionSplicingTranslation Alternative splicing Diversity of products Human Genome  Human Genome 30,000-40,000 protein-coding genes 59% genes have two or more alternative transcripts average of 2.6 distinct transcripts per gene (Nature 409; 860-, 2001) -Therefore, research investigating the alternatively spliced isoforms of a single gene is very important.

4. Alternative Splicing Mutually exclusive alternative splicing Alternative 5 ´ splice sites Alternative 3 splice sites Exon exclusion/inclusion Alternative splicing of pre-mRNA is an important mechanism for generating functionally distinct protein isoforms from a single gene.

5. CD44 Alternative Splicing Ex 6 Ex 15 ExtracellularTMCytoplasmic CD44 has 20 exons and the middle 10 exons are subjected to alternative splicing. CD44 is a cell surface adhesion molecule involved in cell-cell and cell- matrix interactions. CD44 is type I multifunctional receptor. Hyaluronic acid (HA) is one of the common ligands. CD44 also transmits signals mediating hematopoiesis and apoptosis. Stickeler E. et al. (1999) Oncogene 18:3574-3582

6. CD44 Splicing Variants CD44s (CD44H) is the smallest molecule which lacks the entire variable region, also the most commonly expressed type of CD44. CD44E (CD44v8-10) is preferentially expressed on epithelial cells. CD44v are widely found in cancer cell types as well as their metastases. v3, v5, v6 and v7 CD44 variants are related to cancer progression and metastasis. Nato D. et al. (1997) Adv Cancer Res 71:241 Ex 6 Ex 15 ExtracellularTMCytoplasmic

7. CD44 Signaling in Tumor Cells Turley et al. (2002) JBC 277: 4589-4592

8. Why Study CD44v3??? CD44 splice variants HMEC MDA231 MCF7 HMEC MDA231 MCF7 CD44v3 HMEC MDA231MCF7 HMEC MDA231 MCF7 CD44s V3 Exon5 Exon16 Exon5 Exon16 Exon4 V3 Exon 5 Exon 16  CD44v3 HMEC MCF7 MDA231 HMEC MCF7 MDA231 MCF-7 MCF-7:Non-metastatic MDA MDA:Metastatic HMEC HMEC: Human Mammary Epithelial Cells 30 cycles35 cycles

9. CD44 Gene Organization and Minigene Construction 4356220512462835475272514711861174811913402 V5V6V2V1V3V7V4V8V10V9 120129126 11411712913210290204 Exon 5Exon 16 Multiple cloning site PL53In

10. CD44 Minigene Construction cont. PL53In V3 BamHIXba I 126bp ~ 200bp PCR amplification Directional cloning

11. CD44 Minigene Construction and Transfection Splicing Inclusion: 372bp ~ CD44v3 insert + Insulin exons Splicing Skipping: 246bp ~ Insulin exons Linear Perspective of PL53In Exontrap Vector  Linear Perspective of PL53In Exontrap Vector RSV Promoter Insulin exon 2 Insulin exon 3 MCS SV40 Poly A CD44v3 Primer 1Primer 2 Intron Insert

12. Changing Strategy zAlthough the construction of the vector and v3 exon plasmid was successful, the E5-v3-E16 plasmid was not. zTherefore, a new strategy was devised using a new enzyme, pvu II. zInstead of inserting the entire E5-v3-E16 PCR fragment, used the v3 and vector plasmid and inserted the individual exons 5 and 16 in separate digestions. - Less time efficient, but more successful. - For the rest of the week, I will continue to test the transfection and RT-PCR of this new strategy.

13. Conclusions (?) zSuccessfully constructed a v3 minigene and an E5-v3- E16 minigene. zCurrently in the process of transfection of E5-v3-E16 minigene into MDA and MCF-7 cells. zPrevious transfection assays experienced some difficulty due to the low transfection efficiency of MDA cells. Future experiments will improve the transfection protocol in hopes of improving its efficiency with MDA. -If the repetitions of the transfection and RT-PCR experiments are not successful, it might be worthwhile to test other common breast cancer cell lines.

14. Expectations z What I hope to see is that there will be more CD44v3 included during splicing in the metastatic MDA cells than their non-metastatic MCF-7 counterparts. This would indicate that the CD44v3 minigene could serve as an indicator of metastatic breast cancer.

15. Future Experiments zContinue with transfection assay and RT-PCR. zDevelop the molecular mechanisms of the alternative splicing patterns of CD44v3. zUse a luciferase reporter for time efficiency. zInvestigate the cis-elements of CD44v3.  Find a preventative treatment for metastatic breast cancer (a cure would be nice too).

16. Acknowledgements Thanks to… zDr. RJ Lin’s Molecular Biology Lab at Beckman Research Institute at City of Hope: Dr. Lin (PI), for hiring me. Tracy Li (Mentor), for teaching me everything and being patient with me and all of my questions. Ken Dery, Mitsuo Kato, Ayaka Maeda, Ed Silverman, Sean Upchurch zSummer Students in Kaplan-Black zSummer Students in Kaplan-Black: Anna, Ashley, Nicole, Tim, Toby, Tracy, and Xin. zEveryone at COH Summer Student Program zOxy’s URC, Dr. Craney for sending me that letter back about COH in April. zThanks for your attention!