Investigations into novel antagonists of the CXCR4 chemokine receptor to prevent the migration of cancerous cells

Hamshaw, Isabel (2020) Investigations into novel antagonists of the CXCR4 chemokine receptor to prevent the migration of cancerous cells. Doctoral thesis, University of East Anglia.

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Abstract

Chemokines are signalling molecules that enable cell migration. The chemokine CXCL12 and its receptor CXCR4 have major roles in neutrophil homeostasis. However, CXCR4 is overexpressed on cancer cells, leading to aberrant downstream signalling. This study identifies the importance of two downstream proteins, PKC and PKD, in CXCL12-stimulated PC3 prostate cancer migration. Additionally, novel CXCR4 antagonists for in situ click chemistry were developed and 3D printed materials for migration assays were designed. Finally, cellular expression of ACKR3, which also binds CXCL12, and its role in migration was investigated.

Experiments used MCF-7 breast cancer cells, PC3 cells, Jurkat leukemic T-lymphocytes and THP-1 AML cells which naturally express CXCR4 and ACKR3 and SKMEL28 melanoma cells which naturally express CXCR4. PKC/PKD inhibitors, CXCR4 antagonists and CXCR4/ACKR3 antibodies were used in migration assays and calcium release assays to measure cellular responses. Immunofluorescence, flow cytometry and copper-catalysed alkyne-azide cycloaddition (CuAAC) determined CXCR4 expression and/or internalisation. Actin expression was analysed after incubation with PKC/PKD inhibitors or CXCR4 antagonists.

PKC/PKD inhibition prevented CXCL12-stimulated cell migration. The novel CXCR4 antagonists AZ6-2, IS4 and IS7 are more potent than AMD3100. IS4 is more stable than AZ6-2 and can be used in CuAAC. IS7 fluorescently labels CXCR4. ACKR3 has no effect upon cellular migration using chemotaxis assays. ACKR3 has faster turnover rates in PC3 and THP-1 cells than MCF-7 and Jurkat cells. ACKR3 internalisation in MCF-7 and Jurkat cells occurs via caveolin-dependant endocytosis. In the presence of CXCL12, ACKR3 internalisation occurs via clathrin/caveolin independent endocytosis in Jurkat cells. 3D printing can be used to print stoppers for the ORISTM Cell Migration assay.

CXCL12-stimulated migration can be modulated by PKC/PKD inhibitors and CXCR4 antagonists. IS4/IS7 are potential future therapeutics or laboratory tools. ACKR3 turnover and internalisation vary in different cell types. 3D printing could be a useful for the development of migration assays.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Chris White
Date Deposited: 14 Apr 2021 09:38
Last Modified: 14 Apr 2021 09:38
URI: https://ueaeprints.uea.ac.uk/id/eprint/79737
DOI:

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