Introduction: Lysyl oxidase (LOX) is an amine oxidase that is usually synthesized and secreted by fibrogenic cells. Four LOX-like (LOXL) genes have been identified so far in mammalian genomes, encoding four different LOX-like proteins: LOXL1, LOXL2, LOXL3 and LOXL4. All members of the LOX family show a highly conserved C-terminus region that contains the catalytic domain. The N-terminus of the LOX isoforms is less conserved among the different members and is thought to determine the individual role and tissue distribution of each isoenzyme. LOXL4, the least studied member of the LOX-like family enzymes, undergoes a process of alternative splicing in cancer, in a site- and stage-specific manner that we have previously shown. The purpose of the current study was to uncover the splicing mechanism that is responsible for this process.

Experimental procedures: I. ShRNAs for four splicing factors: SF2/ASF, SRp55, hnRNP-A1 and hnRNP-A2, were transfected in two cell lines: U-87 MG cell line (human glioblastoma) and NCI-H460 (human large-cell lung carcinoma). II. Over-expression of SF2 was performed in MST0-211H cell line (human malignant mesothelioma), HeLa cell line (human epithelial cervical cancer) and MCF10A cell line (human mammary epithelial line). III. Western blotting for SF2/ASF and tubulin. IV. RT-PCR for LOXL4 full length, splice-variant1 (splv1) and splice-variant2 (splv2) mRNA expression.

Results: We examined LOXL4 expression in U-87 MG cells. When untreated, these cells express the full length and splv2, almost equally. The silencing of two factors, SF2/ASF and hnRNP-A1, resulted in a significant change in the expression pattern of LOXL4. For both silenced factors, LOXL4 full-length mRNA expression was much stronger, while the shortest variant, splv2, completely vanished. The silencing of hnRNP-A2 led to a smaller decrease in splv2, while SRp55 silencing did not seem to change LOXL4 splicing. In NCI-H460 cells, which normally express small amounts of all variants, no significant changes were found following silencing. In an attempt to further establish the splicing factor responsible for LOXL4 splicing, we over-expressed SF2/ASF in MST0-211H cells, which normally express only the full length LOXL4. Expression of SF2/ASF resulted in the appearance of splv2, while dramatically reducing the expression of the full length. Similar results were seen in HeLa cells. Over-expression of SF2/ASF in MCF10A cells, which untreated, have the unique quality of expressing splv2 alone, caused only a slight increase in the expression of splv2.

Conclusions: These results demonstrate for the first time, that LOXL4 is a direct target of the splicing factor SF2/ASF. Furthermore, in concordance with our previous in-vivo findings, it can be concluded that LOXL4 splicing occurs similarly in other epithelial cancer types, such as breast cancer and mesothelioma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3147.