Investigating the effect of a small molecule, Inhibitor of Nuclear Import (INI-43) as a pan-cancer treatment

Rahman, Shabita

Investigating the effect of a small molecule, Inhibitor of Nuclear Import (INI-43) as a pan-cancer treatment

Thesis / Dissertation

2024

Abstract

Karyopherin beta 1 (KPNβ1) is a nuclear import protein that transports cargo molecules from the cytoplasm into the nucleus and plays an important role in other cellular processes such as mitosis and mitotic spindle assembly, DNA replication and cell cycle progression. Previous studies in our laboratory have reported that KPNβ1 is overexpressed in cervical and oesophageal cancer and is important for cancer cell survival and function, suggesting that inhibiting KPNβ1 may be a potential targeted anti-cancer strategy. An in silico screen identified a small molecule inhibitor, Inhibitor of Nuclear Import (INI-43) as an inhibitor of KPNβ1-mediated nuclear import and showed anti-cancer effects against cervical and oesophageal cancer cells using both cell culture and animal models. In this study, we expanded our investigations by determining KPNβ1 expression using bioinformatic analyses and Western blotting, and by investigating the effects of INI-43 in multiple cancer types grown in cell culture to ascertain the potential of KPNβ1 as a pan-cancer therapeutic target. In addition, we determined the ability of INI-43 to interact with KPNβ1 using the biophysical assay - Cellular Thermal Shift Assay (CETSA). Bioinformatic analyses using patient data revealed that KPNβ1 mRNA was significantly overexpressed in multiple cancer tissue compared to non-cancer tissue, including breast cancer, colon cancer, lung cancers and uterine cancers. Interestingly, we also found using bioinformatic approaches, that more than 50% of the cancers, for which data was available to us, show poorer patient survival when KPNβ1 is highly expressed. In vitro analysis using Western blotting on chosen cultured cell lines of different tissue origin revealed that KPNβ1 is overexpressed in multiple cancer types at the proteomic level, including liver cancer, gastric cancer, osteosarcoma and fibrosarcoma. These results suggest that KPNβ1 is a promising diagnostic/prognostic marker for a broad range of cancer types. Cancer cell lines of different tissue origin were found to be more sensitive to INI-43 treatment compared to the non-cancer epithelial cell line (ARPE-19) with EC50 values of ~ 5 - 15 µM for the cancer cell lines and ~ 25 µM for the non-cancer cell line, ARPE-19, which is at least two- RHMSHA007 fold higher than the EC50 values of majority of the cancer cell lines. INI-43 treatment also resulted in cancer cell death by apoptosis as observed by Caspase activity. These results support that INI-43 has potential as a pan-cancer therapeutic agent for multiple cancer types. To investigate KPNβ1:INI-43 interactions in living cells, we analysed thermal melt profiles of treated and untreated KPNβ1 containing protein lysates using the biophysical assay, CETSA. Our study found that treatment with INI-43 has a stabilising effect on KPNβ1, suggesting that INI-43 physically binds to KPNβ1. Furthermore, we obtained data to show that INI-43 does not appear to bind to other nuclear transport proteins that are associated with cancer, including CAS, CRM1, KPNα2, TNPO1 and GAPDH (control). Interestingly, we found that INI43 appears to bind to another nuclear import protein, IPO5. In conclusion, this study is a first to show that KPNβ1 is expressed at high levels in multiple cancer types of different tissue origin and that INI-43, a small molecule inhibitor with antiKPNβ1 activity, has potential as a pan-cancer treatment. The study shows that INI-43 interacts with KPNβ1 in living cells. These results provide evidence that KPNβ1 is a potential anti-cancer therapeutic target for multiple cancer types.

Keywords

Medicine

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