Splicing Regulations of CD44v3

in Breast Cancer Metastasis

Hayley BainesCity of Hope Summer Student

ProgramMay 27 – August 1, 2003

Mentor: Tracy Li, M.D., M.S.PI: RJ Lin, PhD Molecular Biology

City of Hope Summer Student Program

Dr. RJ Lin’s Molecular Biology Lab at the Beckman Research Institute at the City of Hope, Duarte, CA

10 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.

Weekly summer student meetings and seminars.

Splicing

DNA Pre-mRNA mRNA ProteinTranscription Splicing Translation

Alternative splicingDiversity of products

Human GenomeHuman Genome30,000-40,000 protein-coding genes59% 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.

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.

CD44Alternative Splicing

Ex 6 Ex 15

Extracellular TM Cytoplasmic

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

CD44Splicing 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

Extracellular TM Cytoplasmic

CD44Signaling in Tumor Cells

Turley et al. (2002) JBC 277: 4589-4592

Why Study CD44v3???

V3Exon 5 Exon 16

CD44v3

0

2

4

6

8

10

12

14

30 35

HMEC

MCF7

MDA

CD44v3

HMEC

MCF7MDA231

HMEC

MCF7MDA231

MCF-7MCF-7: Non-metastatic

MDAMDA: Metastatic

HMECHMEC: Human Mammary Epithelial Cells

30 cycles 35 cycles

CD44Gene Organization and Minigene Construction

4356

2205

1246

2835

475

2725

1471

1861

1748

1191

3402

V5 V6V2V1 V3 V7V4 V8 V10V9

120 129 126 114 117 129 132 102 90 204

Exon 5 Exon 16

Multiple cloning site

PL53In

CD44Minigene Construction cont.

PL53In

V3

BamHI Xba I126bp

~200bp ~200bpPCR amplification

Directional cloning

CD44Minigene Construction and Transfection

Splicing Inclusion: 372bp ~ CD44v3 insert + Insulin exons

Splicing Skipping: 246bp ~ Insulin exons

Linear Perspective of PL53In Exontrap VectorLinear Perspective of PL53In Exontrap Vector

RSV PromoterInsulin exon 2

Insulin exon 3

MCS MCS

SV40 Poly A

CD44v3

Primer 1 Primer 2

Intron Intron

Insert

Changing Strategy

Although the construction of the vector and v3 exon plasmid was successful, the E5-v3-E16 plasmid was not.

Therefore, a new strategy was devised using a new enzyme, pvu II.

Instead 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.

Conclusions (?)

Successfully constructed a v3 minigene and an E5-v3-E16 minigene.

Currently in the process of transfection of E5-v3-E16 minigene into MDA and MCF-7 cells.

Previous 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.

Expectations

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.

Future Experiments

Continue with transfection assay and RT-PCR.Develop the molecular mechanisms of the

alternative splicing patterns of CD44v3.Use a luciferase reporter for time efficiency. Investigate the cis-elements of CD44v3.Find a preventative treatment for metastatic

breast cancer (a cure would be nice too).

AcknowledgementsThanks to…

Dr. RJ Lin’s Molecular Biology Lab at Beckman Dr. RJ Lin’s Molecular Biology Lab at Beckman Research Institute at City of Hope: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

Summer Students in Kaplan-BlackSummer Students in Kaplan-Black: Anna, Ashley, Nicole, Tim, Toby, Tracy, and Xin.

Everyone at COH Summer Student ProgramEveryone at COH Summer Student Program Oxy’s URC, Dr. Craney for sending me that letter Oxy’s URC, Dr. Craney for sending me that letter

back about COH in April.back about COH in April.

Thanks for your attention!Thanks for your attention!