Investigating the role of nuclear transport proteins secreted by cancer cells

Whitehouse, Alexandra

Investigating the role of nuclear transport proteins secreted by cancer cells

Thesis / Dissertation

2025

Publisher

University of Cape Town

Department

Department of Integrative Biomedical Sciences (IBMS)

Faculty

Faculty of Health Sciences

Abstract

Previous studies in our laboratory have shown that the nuclear transport protein Importin 1 (Kpnb1) is upregulated in several types of cancer and is crucial to cancer cell survival. Recent findings from our laboratory showed that Kpnb1, amongst other proteins is secreted into the extracellular space by cancerous and non-cancerous cells. Whilst the role of endogenous Kpnb1 has been relatively well described in the literature; little is known of the role of secreted Kpnb1 in the extracellular space. Proteins secreted into the extracellular space have been reported to play a role in intercellular signaling that may have an impact on the biology of the cell type of origin or on cells of different origin, referred to as autocrine or paracrine respectively. This study is aimed at investigating the effect of Kpnb1 bound to enhanced Green Fluorescent Protein (Kpnb1-eGFP) containing secreted protein isolates obtained from cervical cancer cell lines on the biology of cell lines of cervical cancer and non-cancer origin. Conditioned media collected from control, eGFP-expressing or Kpnb1-eGFP expressing HeLa and CaSki cervical cancer cells was used to treat recipient cells, both cancerous and non-cancerous. This was followed by monitoring the uptake of Kpnb1-eGFP and effects on biological phenotypes including proliferation and migration. Our study observed that the internalisation of Kpnb1-eGFP from conditioned media treatments varied across the cell lines used. The treatment of cervical cancer cell lines with conditioned media (control and Kpnb1-eGFP containing) in an autocrine manner resulted in an increase in cell proliferation and migration. These effects however, appeared independent of Kpnb1 as the Kpnb1-eGFP-containing conditioned media did not confer a growth or migratory advantage to the cancer cell lines when compared to the eGFP-containing conditioned media. The effect on non-cancerous epithelial or fibroblast cells had cell line specific effects that appeared dependent on the origin of the secreted Kpnb1-eGFP, with the non-cancerous epithelial cell line showing increased cell migration whilst the non-cancerous fibroblasts showed an increase in proliferation in response to treatment with Kpnb1-eGFP-containing conditioned media obtained from CaSki cells. As Kpnb1 is a nuclear transport protein that binds to and transports a variety of cargo proteins, we postulated that secreted Kpnb1 could also be bound to cargo proteins in the extracellular space. These cargo proteins, if any, may also affect the biology of cells targeted by conditioned media treatments. In this study, we thus investigated Kpnb1 binding partners in the secretome of cancer cell lines. Previous studies in the literature identified endogenous Kpnb1 binding partners and found that many of these are involved in protein synthesis amongst other cellular processes and are localised in the nucleus, cytoplasm or ribosome. An analysis of these studies identifying endogenous Kpnb1-binding partners found that the use of immunoprecipitation mass spectrometry (IP-MS) identified a high number of proteins compared to other methods. As such our study made use of immunoprecipitation of secreted Kpnb1 to identify Kpnb1-binding partners in the extracellular space. Two approaches were used; firstly, the use of Western blot analysis to determine if previously described endogenous Kpnb1-binding partners could be identified as bound to Kpnb1 the extracellular space and secondly using IP-MS to identify novel Kpnb1-binding partners in the extracellular space. Western blot analysis for the immunoprecipitated Kpnb1 revealed that Kpnb1 is bound to Exportin 1 (CRM1) in the secretome of cancerous and non-cancerous cells. It also showed that Kpnb1 secreted from CaSki, WHCO5, Kyse30 and hTERT RPE-1 is bound to a Kpnb1 adaptor protein, Karyopherin subunit alpha 2 (Kpna2). IP-MS of Kpnb1's binding partners secreted from cancerous and non-cancerous cells, although having a relatively low yield, identified Histones, an Actin protein (Actin cytoplasmic 1, N-terminally processed (ACTB)) and Laminin b. These binding partners have known functions in the extracellular space that may play a role in intercellular signaling. In conclusion, this study shows that the secretome of cervical and oesophageal cancer cells contain Kpnb1 and Kpnb1-binding partners. An analysis of secreted Kpnb1's binding partners suggest possible mechanistic pathways that may have a role in intercellular communication. Our results suggest that secreted cellular fractions containing overexpressed Kpnb1 has cell line specific effects, with some biological features of non-cancerous cells possibly being affected.

Keywords

Cancer cells

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