PHOSPHORYLATION STABILIZES ALTERNATIVELY SPLICED CD44 MESSENGER-RNA TRANSCRIPTS IN BREAST-CANCER CELLS - INHIBITION BY ANTISENSE COMPLEMENTARY TO CASEIN KINASE-II MESSENGER-RNA

CD44, the predominant vertebrate cell surface receptor for hyaluronan, exists in a variety of isoforms resulting from alternative splicing o f a single gene. Particular spliced variants of CD44 correlate with in creased cell motility, and with poor clinical prognosis in several kin ds of carcinomas. Combinations of 9 variant exons that confer this enh anced motility on tumor cells are inserted into a single site in the m iddle of the extracellular domain of CD44. Evidence suggests that phos phorylation of 2 serine residues in the intracellular domain of CD44 a re involved in controlling these events. However, evidence is lacking as to the nature of such kinases. Acidic amino acids in close proximit y to these 2 serine residues suggests casein kinase II (CKII) is invol ved. We now show an antisense phosphorothioate oligonucleotide designe d to hybridize to the AUG translation initiation codon of subunit CKII alpha' mRNA blocks in vivo phosphorylation of CD44 in MDA231 breast t umor cells, and at the protein level decreases ectopic expression of t otal CD44 as well as the metastatic v-7 CD44 isoform. Furthermore subp lateau RT-PCR analysis demonstrated antisense transfected MDA231 tumor cells had significant down-regulated or eliminated mRNA transcripts o f metastatic CD44 isoforms. CKII as a CD44-associated serine kinase th erefore may serve as an important molecule in a signaling cascade that produces a variety of cellular responses in MDA231 breast cancer cell s. Since the 3'-untranslated region of CD44 mRNA contain 4 dispersed A UUUA sequences which serve as signals targeting mRNA for rapid turnove r, a mechanism is proposed by which CD44 phosphorylation mediates labi le message stabilization, hence providing insights into the processes involved in cancer cell growth, invasion and metastasis.