Abstract
Alterations in RNA splicing are associated with different malignancies, including leukemia, lymphoma, and solid tumors. The RNA splicing modulators such as FD-895 and pladienolide B have been investigated in different malignancies to target/modulate spliceosome for therapeutic purpose. Different cell lines were screened using an RNA splicing modulator to test in vitro cytotoxicity and the ability to modulate RNA splicing capability via induction of intron retention (using RT-PCR and qPCR). The Cignal Finder Reporter Array evaluated [pathways affected by the splice modulators in HeLa cells. Further, the candidates associated with the pathways were validated at protein level using western blot assay, and gene-gene interaction studies were carried out using GeneMANIA. We show that FD-895 and pladienolide B induces higher apoptosis levels than conventional chemotherapy in different solid tumors. In addition, both agents modulate Wnt signaling pathways and mRNA splicing. Specifically, FD-895 and pladienolide B significantly downregulates Wnt signaling pathway-associated transcripts (GSK3β and LRP5) and both transcript and proteins including LEF1, CCND1, LRP6, and pLRP6 at the transcript, total protein, and protein phosphorylation’s levels. These results indicate FD-895 and pladienolide B inhibit Wnt signaling by decreasing LRP6 phosphorylation and modulating mRNA splicing through induction of intron retention in solid tumors.
Highlights
RNA splicing and the protein machinery that guides this process, the spliceosome, constitute a very relevant biological target for cancer therapy [1, 2]
We previously reported the apoptotic activity of FD-895 and pladienolide B in chronic lymphocytic leukemia (CLL)-B, mantle cell lymphoma (MCL), and other B and T lymphoma cell lines
Using an expanded panel of cell lines, we found that FD-895 and pladienolide B IC50 ranged from 30.7 ± 2.2 to 415.0 ± 5.3 nM (Table 2) across the breast, colon, and cervix tumor cell lines (Figure 1A–1D)
Summary
RNA splicing and the protein machinery that guides this process, the spliceosome, constitute a very relevant biological target for cancer therapy [1, 2]. Within www.aging-us.com the major component, the SF3B core unit is comprised of spliceosome associated proteins including SF3B1 (Splicing Factor 3b Subunit 1), U2AF1 (U2 Small Nuclear RNA Auxiliary Factor 1), and SRSF2 (Serine And Arginine Rich Splicing Factor 2) that have been implicated in large a number of malignancies [6] including chronic lymphocytic leukemia (CLL) [7], uveal melanoma [8], and myelodysplastic syndrome [9]. Recent structural studies have shown that pladienolide B-related FD-895 polyketides and their analogs bind to a specific pocket within this SF3B core comprised of SF3B1, SF3B3 (Splicing factor 3B subunit 3), and PHF5A (PHD Finger Protein 5A) [10, 11]. SF3B1 and mutations in it have been exploited extensively as a therapeutic target in FLT3/ITD positive acute myeloid leukemia (AML), endometrial cancer, and hepatocellular carcinoma [12–14]
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