Abstract 1776 Nuclear APLF in the regulation of breast cancer metastasis

Journal of Biological Chemistry   | Volume 300, Issue 3, Supplement,2024 | doi.org/10.1016/j.jbc.2024.106688.

Debparna Nandy; Pallavi Varghese; Mayur Shirude; Anjali Devarajan, and Debasree Dutta

ABSTRACT

Abstract Objective- To study the role of APLF localization in regulating breast cancer metastasis. Methodology- Immunohistochemistry was carried out in tissue biopsy cores. Cell culture- HEK293T, MCF10A, MCF7, BT474, SKBR3 and MDAMB231 cell lines were used for experiments. Protein isolation- Total cell protein and nuclear-cytoplasmic protein fractions were isolated from the above-mentioned cell lines. Western blotting- was done to check protein expression. Immunofluorescence- Fluorophore tagged secondary antibody was used to check the protein expression and localization of proteins in different cell lines. Cloning- APLF was engineered into Flag and mCherry-NLS vector for over-expression and nuclear localization, while APLF sh was used for the down-regulation of APLF. Transfection- Transfection was done using Lipofectamine 2000 reagent for over- expression and nuclear localization system. Lentiviral mediated transfection procedure was used for the Aplf knockdown system. FACS and Single-cell clonal expansion- Fluorescently tagged cells are sorted by FACs and serially diluted to obtain a population from single cell clone. Scratch assay is carried out to check the migratory property. Transwell membrane assay- is carried out to check the invasive property. Immunoprecipitation- Protein lysates were pulled down with sepharose beads to study interacting partners and identify possible pathways responsible for such mechanisms. Pathway analysis is carried out using the DAVID pathway and Reactome database. Result- APLF localization is both cytoplasmic and nuclear in the cell line MDAMB231, while APLF is cytoplasmically located in MCF10A, MCF7, BT474 and SKBR3 cell lines. Immunoprecipitation result indicated the possible association of PARP1 in the nuclear shuttling of APLF in MDAMB 231, which needs further experimentation. Upon tagging NLS, APLF enters inside the nucleus in the MCF 7 cell line. Immunofluorescence data confirmed again the co-localization of NLS-tagged APLF with PARP1 further corroborating the role of PARP1 in APLF nuclear shuttling. Functional assays performed showed nuclear APLF is associated with increased invasive potential and significantly increased migratory potential in-vitro. Protein expression for the scratched assay samples indicated the higher expression of metastatic genes in NLS-tagged APLF compared to its vector. Nuclear APLF might regulate EMT via PARP1. Conclusion- Nuclear localization of APLF can serve as an indicator of breast cancer metastasis. APLF is transported inside the nucleus, possibly by PARP1.

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