Phosphorylation stabilizes alternatively spliced CD44 mRNA transcripts in breast cancer cells: inhibition by antisense complementary to casein kinase II mRNA.
CD44, the predominant vertebrate cell surface receptor for hyaluronan, exists in a variety of isoforms resulting from alternative splicing of a single gene. Particular spliced variants of CD44 correlate with increased cell motility, and with poor clinical prognosis in several kinds of carcinomas. Combinations of 9 variant exons that confer this enhanced motility on tumor cells are inserted into a single site in the middle of the extracellular domain of CD44. Evidence suggests that phosphorylation of 2 serine residues in the intracellular domain of CD44 are involved in controlling these events. However, evidence is lacking as to the nature of such kinases. Acidic amino acids in close proximity to these 2 serine residues suggests casein kinase II (CKII) is involved. We now show an antisense phosphorothioate oligonucleotide designed to hybridize to the AUG translation initiation codon of subunit CKII alpha' mRNA blocks in vivo phosphorylation of CD44 in MDA231 breast tumor cells, and at the protein level decreases ectopic expression of total CD44 as well as the metastatic v-7 CD44 isoform. Furthermore subplateau RT-PCR analysis demonstrated antisense transfected MDA231 tumor cells had significant down-regulated or eliminated mRNA transcripts of metastatic CD44 isoforms. CKII as a CD44-associated serine kinase therefore may serve as an important molecule in a signaling cascade that produces a variety of cellular responses in MDA231 breast cancer cells. Since the 3'-untranslated region of CD44 mRNA contain 4 dispersed AUUUA sequences which serve as signals targeting mRNA for rapid turnover, a mechanism is proposed by which CD44 phosphorylation mediates labile message stabilization, hence providing insights into the processes involved in cancer cell growth, invasion and metastasis.
Formby
B
B
Sansum Medical Research Institute, Molecular Oncology Laboratory, Santa Barbara, CA 93105, USA.
Stern
R
R
D017398:Alternative Splicing / Q000187:drug effects; D001940:Breast / Q000166:cytology; D001943:Breast Neoplasms; D047390:Casein Kinase II; D018387:Codon, Initiator / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D014176:Protein Biosynthesis / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured
D018387:Codon, Initiator; D018960:Hyaluronan Receptors; D016376:Oligonucleotides, Antisense; D020033:Protein Isoforms; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases