title: Unveiling the functions of Transcription Factor AP4 Isoform X3 (sAP4) in Cell Motility
Creators: name: Joyson, Sancy Mary
Publisher: name: University of Liverpool Repository
Year: 2028

description: Transcription factor AP4 (AP4) is a member of the basic helix-loop-helix leucine zipper subgroup of transcription factor. It is known to be involved in cell proliferation, migration, senescence, and apoptosis. Therefore, misregulation of AP4 leads to metastatic cancer and poor prognosis. Because of its role in tumour progression, it is essential to understand the functions of other AP4 isoforms which may have a similar role to AP4. According to The National Center for Biotechnology Information (NCBI), there are three AP4 isoforms: X1, X2, and X3, which have different protein sequences at N-termini compared to the full-length AP4. In this study, the characterisation of the AP4 X3 isoform (sAP4), which is an N-terminal truncated form (without the DNA binding domain) of AP4, was carried out. In contrast to AP4, which is found to localise in the nucleus, sAP4 was found to localise in the nucleus, cytoplasm, and plasma membrane. Accordingly, an investigation of the possible role of sAP4 in cell migration was carried out in this study.
Unlike other AP4 isoforms, sAP4 lacks a unique protein sequence for identification. Therefore, a stable cell line transfected with the FLAG-tagged sAP4 gene, which can be induced to overexpress in HT1080 cells was established. An immunofluorescence microscopy study revealed that the clustered form of plasma membrane associated sAP4-FLAG co-localises with the lipid microdomain marker caveolin-1 on the plasma membrane. Overexpressed sAP4 was also found to co-localise with the early endosome marker EEA-1, lysosome marker LAMP-1 and exosome marker CD63, but it was not observed to co-localise with the endoplasmic reticulum marker calnexin-1 and Golgi complex marker GM130. As sAP4 staining was not observed in the classical secretory pathway-related organelles, its involvement in the unconventional secretory pathway was further investigated.
Additionally, in the sAP4 transcriptional effect study, the overexpression of sAP4 did not show a dominant-negative effect on the AP4 target genes. However, the RNA sequencing analysis revealed that several intercellular and cell-substrate junction-related genes are downregulated, along with the upregulation of cellular metabolism-related genes, suggesting a distinct transcriptional function of sAP4. Overexpression of sAP4 led to reduced expression of integrin beta-8 and activated Akt, indicating that sAP4-ITGB8-Akt signalling pathway may give insights into the potential function of sAP4 in cell migration. During the functional analysis, a decrease in the cell migration rate was observed in sAP4 overexpressing cells and on the cells treated with sAP4-FLAG enriched exosomes. In addition, the invasive ability of sAP4 overexpressing cells was reduced by affecting the number of pseudopod branches and pseudopod length. However, sAP4 overexpression did not have a significant effect on cell proliferation and apoptosis.
In conclusion, sAP4 appears to play a role in cell motility and participates in cell-cell crosstalk through exosomes. Further proteomics investigation of sAP4-enriched exosomes needs to be
conducted, to gain insights into the inhibitory function of sAP4 on cell migration and its potential antagonistic role in cancer progression.