Splicing Regulations of CD44v3 in Breast Cancer Metastasis

1
Splicing Regulations of CD44v3in 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

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City of Hope Summer Student Program

• Dr. RJ Lins 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.

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Splicing

• ? 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.

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Alternative Splicing
Alternative 5 splice sites
Alternative 3? splice sites
Exon exclusion/inclusion
Mutually exclusive alternative splicing
Alternative splicing of pre-mRNA is an important
mechanism for generating functionally distinct
protein isoforms from a single gene.
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CD44Alternative Splicing
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 183574-3582
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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 71241
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CD44Signaling in Tumor Cells
Turley et al. (2002) JBC 277 4589-4592
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Why Study CD44v3???
CD44 splice variants
Exon5
Exon4
Exon16
MCF-7 Non-metastatic MDA Metastatic HMEC
Human Mammary Epithelial Cells
? CD44v3
CD44v3
CD44s
HMEC
HMEC
MCF7
MDA231
MCF7
MCF7
MDA231
MDA231
MDA231
HMEC
HMEC
MCF7
HMEC
HMEC
MDA231
MDA231
MCF7
MCF7
30 cycles
35 cycles
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CD44Gene Organization and Minigene Construction
PL53In
Multiple cloning site
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CD44Minigene Construction cont.
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CD44Minigene Construction and Transfection
? Linear Perspective of PL53In Exontrap Vector
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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.

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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.

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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.

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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).

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AcknowledgementsThanks to

• Dr. RJ Lins 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
• Summer Students in Kaplan-Black Anna, Ashley,
  Nicole, Tim, Toby, Tracy, and Xin.
• Everyone at COH Summer Student Program
• Oxys URC, Dr. Craney for sending me that letter
  back about COH in April.
• Thanks for your attention!