Molecular mechanisms regulating the tumor-targeting potential of splice-activated gene expression

Abstract

Previous studies have suggested that differences in the ability of normal and malignant cells to process certain alternatively spliced pre-mRNA transcripts can be exploited as a potentially powerful means of targeting the expression of therapeutic genes to tumor cells in vivo and in vitro. Specifically, it was shown that efficient processing of minigene constructs containing the alternatively spliced CD44 exons v9 and v10 only occurs in tumor cells that express CD44 isoforms that incorporate these exons (e.g. CD44R1). In the present study, efforts were made to define the molecular mechanisms that underlie the apparent specificity of this process. RT-PCR analysis and DNA sequencing were used to characterize the various splicing events that occur between CD44 exons v8, v9 and v10 following transfection of minigene constructs containing these various exons into CD44R1-positive (PC3) and CD44R1-negative (T24) cell lines. The results obtained confirm that although the v8-v9 intron is efficiently removed in both CD44R1-positive and CD44R1-negative cells, the corresponding v9-v10 intron is accurately spliced and the exons appropriately joined only in lines that express v10-containing CD44 isoforms (e.g. PC3). In CD44R1-negative cell lines (e.g. T24) alternative 5 0 and 3 0 splice sites located within the v9-v10 intron are preferentially used, resulting in various portions of the intron being retained within the final processed mRNA product. It is proposed that identification of these functionally important intronic sequence elements will facilitate the development of second generation ''splice activated gene expression'' vectors that may prove useful in various cancer gene therapy applications.

Unbalanced alternative splicing and its significance in cancer

“…CD44 has ten alternative exons encoding its cell surface region and several recent studies have further elucidated the mechanisms leading to their observed combinations in cancer. (94,95) As the variable exons are all in the extracellular region of CD44, they are potential therapeutic targets (96,97) (Fig. 3).…”

Splooce

“…24 Further CASE examples include the gene CD44, which is a known marker of malignancy and invasiveness and has about ten ASEs that can occur in different combinations in its region coding for the extra-cellular portion of the protein. 20,21,23 In spite of the efforts of other groups, 25-27 a simple and efficient nomenclature to take into account all the variability generated by alternative splicing, especially for CASEs, is still missing. Here, we present a web portal, SPLOOCE, which uses a method based on regular expressions with an associated syntax.…”

“…In humans, some ASEs and CASEs occurring in oncogenes and tumor suppressor genes have already been associated to cancer. 6,[20][21][22][23] For example, the gene NTRK1 (nerve growth factor) has a sequence variant, TrkAIII, which is common in certain tumors and lacks three exons that affect a regulatory immunoglobulinlike domain. 24 Further CASE examples include the gene CD44, which is a known marker of malignancy and invasiveness and has about ten ASEs that can occur in different combinations in its region coding for the extra-cellular portion of the protein.…”

Identification of a novel human tissue factor splice variant that is upregulated in tumor cells

“…19 As a consequence, a number of interventions that either alter or exploit alternative splicing are currently under active investigation for cancer therapy. 27,28 These strategies include targeting of the fibronectin extra domain B 29 and antibody therapy against CD44v6. 30 In summary, we report here that human TF is expressed in the form of 6 distinct transcripts including wild-type TF, TF-A, TF-B, asTF, asTF-A and asTF-B (Fig.…”