Details

Autor(en) / Beteiligte

• Crist, McKenzie C

Titel

Understanding Metformin Mediated Natural Killer Cell Activation in Head and Neck Squamous Cell Carcinoma

Ort / Verlag

ProQuest Dissertation & Theses

Erscheinungsjahr

2023

Link zum Volltext

• ProQuest

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Head and neck cancer (HNC) is the 6th most common cancer worldwide, the majority of which are head and neck squamous cell carcinomas (HNSCC). Recurrent or metastatic HNSCC are often treated with immune checkpoint inhibitors (ICIs), which have dramatically increased survival, but unfortunately, only 20% respond to treatment necessitating improved therapies. The diabetes drug, metformin, was shown previously to have both anti-cancer and immune stimulating (primarily T cells) activity. In a phase I clinical trial (NCT02325401), metformin was administered to HNSCC patients prior to standard of care treatment. Metformin specifically increased NK cell numbers as well as activation more robustly than T cells in HNSCC patients. Therefore, we studied the direct effects of metformin on NK cells. Pre- and post-metformin- treated tumor tissue from a clinical trial (NCT02083692), was analyzed by immunofluorescence. Post-metformin tissue exhibited an increase in infiltrating NK cells. Ex vivo metformin treatment of HNSCC NK cells resulted in increased perforin production correlating with higher cytotoxicity of tumor cells. To determine pathways in which metformin may be regulating in order to promote cytotoxicity in NK cells, we utilized bulk RNA-sequencing (RNA-seq) on ex vivo pre- and post-metformin treated HNSCC patient NK cells. The chemokine, CXCL1, expression was significantly reduced with metformin treatment. Exogenous CXCL1 prevented metformin-mediated NK cell perforin release, but this could be reversed by an inhibitor of the CXCL1 receptor, CXCR2. We next investigated the mechanism in which metformin mediated CXCL1 inhibition may be preventing perforin release and subsequent cytotoxicity. Given metformin is known to inhibit mTOR and pSTAT3, we investigated the roles of these pathways in perforin release. Perforin was decreased by pSTAT1 inhibition and increased by mTOR inhibition suggesting that both pathways may be important. In order to understand if the CXCR2/CXCL1 pathway regulated the STAT pathways, NK cells were treated ex vivo with metformin, CXCL1, and a CXCR2 inhibitor and phosphorylation of STATs was determined by western blot analyses. Metformin resulted in increased pSTAT1 and decreased pSTAT3 as expected. However, CXCL1 reversed the effect of metformin mediated pSTAT3 inhibition which was then rescued by a CXCR2 inhibitor. We gathered supporting evidence that metformin directly increases NK cell infiltration, and perforin production by inhibiting CXCL1 likely through reduction of pSTAT3 and subsequent inhibition of CXCL1. Importantly, CXCR2 inhibition could reverse CXCL1 mediated inhibition of perforin release. In the future, CXCR2 inhibition in NK cells could be further explored to determine if knockdown of CXCR2 in NK cells could result in increased NK cell function and anti-tumor activity. Finally, we have identified a novel pathway by which metformin increases NK cell cytotoxicity, which could be utilized in the treatment of HNSCC in future studies.