Modification of Alternative Splicing of Bcl-x Pre-mRNA in Prostate and Breast Cancer Cells

Abstract

There is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5-splice site of bcl-x L , an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-x L to Bcl-x S , a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-x L , Bcl-x S , and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells.

MCL1 provides a window on the role of the BCL2 family in cell proliferation, differentiation and tumorigenesis

“…In fact, BCL2 was first identified at the t(14;18) chromosome translocation breakpoint characteristic of follicular lymphoma, and BCL2 is highly expressed in other cancers such as chronic B cell lymphocytic leukemia (CLL) and diffuse large cell lymphoma. [106][107][108][109] Recent findings suggest that, beyond the alteration in BCL2, follicular lymphoma involves an imbalance between anti-vs pro-apoptotic BCL2 family members. 28 Specifically, the lymphoma cells differ from normal germinal center cells both in the expression of multiple anti-apoptotic family members (BCL2, BCLX, and MCL1) and in the lack of expression of pro-apoptotic BAX and BAD.…”

“…Approaches could then be developed to turn off or block the effects of anti-apoptotic gene products that are inappropriately up-regulated in the cancer cells (eg through the use of antisense oligonucleotides or small molecules 115,116 ). Additional approaches could be aimed at promoting their alternative splicing to pro-apoptotic forms, 91,109 altering their activity through post-translational modification, 94 or at identifying stimuli that turn on pro-apoptotic gene products.…”

“…128,129 Additional research is examining means of delivering antisense reagents, such as by targeting oligonucleotides to the cancer cells (eg through conjugation to peptides that bind to the surface of the tumor, or the use of liposomes or antisense ribozymes in adenovirus vectors 116,130,131 ). Since some family members, including BCLX and MCL1, can be alternately spliced to yield pro-instead of anti-apoptotic gene products, yet another approach is to use antisense oligonucleotides that favor this alternate splicing (eg antisense oligonucleotides that match a splice junction necessary for splicing of the anti-apoptotic gene product 109 ). Since anti-apoptotic BCL2 family members are also subject to post-translational modification through multiple pathways, such as phosphorylation, it may be possible to develop means of converting them to death-inducing forms.…”

“…[120][121][122][123][124][125][126] This approach can also be used in combination with cytotoxic chemotherapeutic agents, resulting in enhanced tumor cell death. 109,120,123,127 A recent creative approach involves the use of bispecific oligonucleotides that target a sequence in common in BCL2 and BCLX. 128,129 Additional research is examining means of delivering antisense reagents, such as by targeting oligonucleotides to the cancer cells (eg through conjugation to peptides that bind to the surface of the tumor, or the use of liposomes or antisense ribozymes in adenovirus vectors 116,130,131 ).…”

Identification of Two RNA cis-Elements That Function to Regulate the 5′ Splice Site Selection of Bcl-x Pre-mRNA in Response to Ceramide

“…Many reports in the literature demonstrate that the physiological fate of the cell can be determined by the ratio of antiapoptotic Bcl-x(L) to pro-apoptotic Bcl-x(s), and induction of pro-apoptotic Bcl-x(s) sensitizes cells to chemotherapeutic agents (35)(36)(37). This has been demonstrated by specifically redirecting the alternative 5Ј splice site selection of Bcl-x to increase the expression of Bcl-x(s) at the expense of the levels of Bcl-x(L) (36,37).…”