Alternatively spliced isoforms of IL-32 differentially influence cell death pathways in cancer cell lines

Abstract

Alternative splicing is a biological mechanism that enables the synthesis of several isoforms with different or even opposite functions. This process must be tightly regulated to prevent unwanted isoform expression favoring pathological processes. Some isoforms of interleukin 32 (IL-32) are reported to be more potent in inducing inflammation, however the role in cell death remains to be investigated. This study demonstrates that IL-32γ and IL-32β can induce caspase-8-dependent cell death whereas this was not observed for IL-32α. Overexpression of IL-32β or IL-32γ but not IL-32α, resulted in enhanced expression of the survival cytokine IL-8. Furthermore, restoring the IL-8 signaling pathway by overexpressing CXCR1 in HEK293 cells, rescued IL-32β but not IL-32γ-induced cell death. Interestingly, IL-32γ was able to downregulate CXCR1 and thereby induce cell death. Subsequent studies into the role of IL-32 in thyroid cancer (TC) revealed that several IL-32 isoforms, IL-8, and CXCR1 are expressed in TC cell lines and specimens. Remarkably, TC cell lines were found to produce high concentrations of IL-8, indicating an important role for IL-8 in the survival-signaling pathway in these cells. Intriguingly, a significant correlation between the IL-8 receptor CXCR1 and IL-32γ was observed in TC specimens, while this was not observed for the other IL-32 splice variants. Blocking IL-32 alternative splicing by Isoginkgetin resulted in predominant expression of IL-32γ splice variants and cell death in TC cell lines. All together, modulation of IL-32 alternative splicing could represent a novel strategy for the treatment of malignancies, in particular thyroid cancer.

Interleukin-32 in Infection, Inflammation and Cancer Biology

“…One of the most noteworthy observations is that IL-32 is still not found in rodents, such as mice and rats. Recent discoveries in this area confirmed earlier hypothesis -alternative splicing can be a mighty regulator of isoform types which are produced in various conditions and tissue types (18).…”

“…IL-32θ inhibited epithelial-mesenchymal transition (EMT), resulting in the suppression of migratory and invasive capabilities of HT29 colon cancer cells (74). Higher levels of IL-32 were reported in many other cancers: melanoma, thyroid, renal cell (18,71,75). Recent studies have revealed higher expression of IL-32 in human pancreas, liver, and esophagus cancer tissues, compared with normal tissue or serum (76)(77)(78).…”

“…IL-32α is the most abundant while IL-32β is the most common transcript (12,18,19). The different isoforms of IL-32 originate by splicing of pre-mRNA of the isoform IL-32γ.…”

“…These signals regulate important biological cell functions, such as migration, proliferation, and survival (34,35,33). So far, we can conclude that there are at least two membrane binding proteins for extracellular IL-32 -PR3 and integrins, and another yet unknown receptor (18). Intracellulary, for the activity of this cytokine are responsible FAK-paxillin proteins, which after binding with IL-32 regulate various cell functions, such as cell growth, metabolism, cytokine production, cell adhesion, migration, proliferation, differentiation, angiogenesis and apoptosis (9,30,36,37).…”

“…Also, examination of effects in neonatal HUVEC (human umbilical vein endothelial cells) and adult pulmonary microvascular EC, using an in vivo and an in vitro angiogenesis assay, showed that induction of IL-32 in ECs was related with activation and proliferation of these cells, as well as angiogenesis (32). Angiogenic effect in certain concentrations on HUVEC was even greater than with VEGF, but was not dependent on VEGF (18,32). Authors conclude that IL-32 exerts its angiogenic properties in EC via integrins (at least in some part), requires second stimuli (with LPS, IFN-γ or unknown cofactor) for endothelial cell responsiveness.…”