BackgroundMetformin slows tumor growth and progression in vitro, and in combination with chemoradiotherapy, resulted in high overall survival in patients with head and neck cancer squamous cell carcinoma (HNSCC) in our phase 1 clinical trial (NCT02325401). Metformin is also postulated to activate an antitumor immune response. Here, we investigate immunologic effects of metformin on natural killer (NK) and natural killer T cells, including results from two phase I open-label studies in patients with HNSCC treated with metformin (NCT02325401,NCT02083692).MethodsPeripheral blood was collected before and after metformin treatment or from newly diagnosed patients with HNSCC. Peripheral immune cell phenotypes were evaluated using flow cytometry, cytokine expression by ELISA and/or IsoLight, and NK cell-mediated cytotoxicity was determined with a flow-based NK cell cytotoxicity assay (NKCA). Patient tumor immune infiltration before and after metformin treatment was analyzed with immunofluorescence. NK cells were treated with either vehicle or metformin and analyzed by RNA sequencing (RNA-seq). NK cells were then treated with inhibitors of significant pathways determined by RNA-seq and analyzed by NKCA, ELISA, and western blot analyses.ResultsIncreased peripheral NK cell activated populations were observed in patients treated with metformin. NK cell tumor infiltration was enhanced in patients with HNSCC treated with metformin preoperatively. Metformin increased antitumorigenic cytokines ex vivo, including significant increases in perforin. Metformin increased HNSCC NK cell cytotoxicity and inhibited the CXCL1 pathway while stimulating the STAT1 pathway within HNSCC NK cells. Exogenous CXCL1 prevented metformin-enhanced NK cell-mediated cytotoxicity. Metformin-mediated NK cell cytotoxicity was found to be AMP-activated protein kinase independent, but dependent on both mechanistic target of rapamycin and pSTAT1.ConclusionsOur data identifies a new role for metformin-mediated immune antitumorigenic function through NK cell-mediated cytotoxicity and downregulation of CXCL1 in HNSCC. These findings will inform future immunomodulating therapies in HNSCC.
Hepatocellular carcinoma (HCC) that is triggered by metabolic defects is one of the most malignant liver cancers. A much higher incidence of HCC among men than women suggests the protective roles of estrogen in HCC development and progression. To begin to understand the mechanisms involving estrogenic metabolic effects, we compared cell number, viability, cytotoxicity, and apoptosis among HCC-derived HepG2 cells that were treated with different concentrations of 2-deoxy-d-glucose (2-DG) that blocks glucose metabolism, oxamate that inhibits lactate dehydrogenase and glycolysis, or oligomycin that blocks ATP synthesis and mitochondrial oxidative phosphorylation. We confirmed that HepG2 cells primarily utilized glycolysis followed by lactate fermentation, instead of mitochondrial oxidative phosphorylation, for cell growth. We hypothesized that estrogen altered energy metabolism via its receptors to carry out its anticancer effects in HepG2 cells. We treated cells with 17β-estradiol (E2), 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) an estrogen receptor (ER) α (ERα) agonist, or 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), an ERβ agonist. We then used transcriptomic and metabolomic analyses and identified differentially expressed genes and unique metabolite fingerprints that are produced by each treatment. We further performed integrated multi-omics analysis, and identified key genes and metabolites in the gene–metabolite interaction contributed by E2 and ER agonists. This integrated transcriptomic and metabolomic study suggested that estrogen acts on estrogen receptors to suppress liver cancer cell growth via altering metabolism. This is the first exploratory study that comprehensively investigated estrogen and its receptors, and their roles in regulating gene expression, metabolites, metabolic pathways, and gene–metabolite interaction in HCC cells using bioinformatic tools. Overall, this study provides potential therapeutic targets for future HCC treatment.
Head and Neck Squamous Cell Carcinoma (HNSCC) is the 6th most common cancer worldwide. Even with intensive treatment, up to 50% of patients will relapse. Checkpoint inhibitors, including antibodies targeting programmed death 1 (PD-1), have dramatically improved survival outcomes but only elicit a response in 20% of patients. PD-1 is expressed on both T-cells and Natural Killer (NK) cells, and depletion of NK cells has been linked to poor prognosis in several solid tumors. NK cells are essential to the innate immune response, do not require MHC-1 expression for recognition, and can stimulate an anti-cancer response through release of cytotoxic granules and direct killing of cancer cells. Upregulation of several interleukins including IL-6, which induces the JAK/STAT3 pathway and subsequently inhibits cancer cell apoptosis, is correlated with a poor prognosis in HNSCC patients. Importantly, inhibition of phosphorylated STAT3 (pSTAT3) increases NK-specific cancer cytotoxicity, NK cell cytokine release, and anti-tumor cell chemokines. Therefore, we hypothesize that impaired NK cell cytotoxicity results in a poor response to immunotherapy. We have shown that a common anti-diabetes drug with anti-cancer properties, metformin, downregulates NK pSTAT3 with subsequent increase in cytotoxicity of peripheral NK cells in HNSCC patients. Metformin also decreases pSTAT3 pathway related cytokine release from HNSCC cell lines. However, it remains unclear whether metformin-mediated enhanced NK cytotoxicity was through a direct effect on NK cells or indirect through inhibition of pSTAT3 in tumor cells and subsequent decrease in inhibitory cytokine release. In addition, inhibiting pSTAT3 could lead to further activation of innate immunity through activation of NK cell cytotoxicity complementing current PD-1 inhibitor therapy and improved treatment response. Here we report the contribution of metformin-mediated direct and indirect effects on NK cell cytotoxic functions and the combined effect of metformin and pembrolizumab using patient samples from a clinical trial and ex vivo models. Understanding metformin-mediated regulation of pSTAT3 and downstream pathways will provide necessary insight to potentially overcome resistance to PD-1 inhibitors and identifying novel treatment combinations for this disease. Citation Format: McKenzie Crist, Maria Lehn, Trisha Wise-Draper. Metformin-mediated natural killer cell cytotoxicity in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1775.
Cancer patients infected with the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have a higher mortality rate compared to non-cancer patients. Recent anticancer treatment, including immunotherapy, is associated with severe infection including development of acute respiratory distress syndrome (ARDS) and high levels of cytokine release resulting in cytokine storm. Immune checkpoint inhibitors (ICIs) are approved for use in multiple cancer types and function by blocking the interaction between PD-1 and its ligand PD-L1, activating antitumor cytotoxic immune cells. However, ICIs can also increase inflammatory cytokine secretion, which may predispose to the development of cytokine storm. In fact, we have shown via single-cell cytokine secretion analysis that pembrolizumab (anti-PD-1 antibody) increases cytokine secretion by polyfunctional strength index, a measure of the percentage of cells secreting multiple functional cytokines. Therefore, we hypothesize that ICIs may worsen inflammatory cytokine secretion and potentiate cytokine storm and downstream complications in COVID-19 patients. Peripheral blood mononuclear cells (PBMCs) were isolated via Ficoll density gradient centrifugation from healthy donors, head and neck cancer (HNC) patients, and COVID-19-infected cancer patients. Flow cytometry was performed on patient PBMCs, after staining for viability and immune cell markers including CD3, CD8, CD19, and CD45. PBMCs were also activated overnight with low-dose IL-2, cocultured with Cal27 or HN5 cell lines, and subjected to various treatment conditions. For non-COVID-19 patients, PBMCs were exposed to 25 nM SARS-CoV-2 recombinant spike (S) protein, a virulent protein associated with cytokine storm, or control prior to drug treatments. Preliminary flow cytometry analysis showed that a COVID-19-positive patient with thyroid cancer had an increased proportion of CD8+ cells compared with a COVID-negative ovarian cancer patient and healthy donor. Recombinant SARS-CoV-2 S protein caused increased secretion of IL-6, IL-2, perforin, and MIP-1b from PBMCs isolated from both healthy donors and HNC patients, which was measured by IsoLight Codeplex bulk cytokine analysis or ELISA. We have previously shown that metformin, a commonly prescribed antidiabetes drug, decreases the proportion of cells that secrete inflammatory cytokines such as IL-6, which is thought to be an important cytokine for cytokine storm. Interestingly, we observed that metformin treatment resulted in decreased IL-6 secretion from PBMCs isolated from a COVID-19-positive patient. Results from this project suggest that ICIs may potentiate cytokine storm, and ongoing investigation will be informative to oncologists as to whether ICI treatment should be postponed in severe COVID-19 infections. In addition, metformin may be a novel potential treatment for COVID-19 patients to prevent and treat cytokine storm. Citation Format: Layne Weatherford, Maria Lehn, McKenzie Crist, Chelsea Wendling, Kristin Hudock, Vinita Takiar, Trisha Wise-Draper. SARS-CoV-2 induces inflammatory cytokine release, which may be exacerbated by immune checkpoint blockade [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr S02-03.
Purpose: The efficacy of cetuximab is poor in metastatic head and neck squamous cell carcinoma (HNSCC). Cetuximab initiates natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity, with resultant recruitment of immune cells and suppression of anti-tumor immunity. We hypothesized that adding an immune checkpoint inhibitor (ICI) could overcome this and lead to an enhanced anti-tumor response. Patients and Methods: A phase II study of cetuximab and durvalumab in metastatic HNSCC was conducted. Eligible patients had measurable disease. Patients who had received both cetuximab and an ICI were excluded. The primary endpoint was objective response rate (ORR) by RECIST 1.1 at six months. Results: As of April 2022, 35 patients enrolled, of whom 33 received at least 1 dose of durvalumab and were included in the response analysis. Eleven patients (33%) had received prior platinum-based chemotherapy, 10 an ICI (30%), and 1 patient (3%) cetuximab. ORR was 39% (13/33) with a median duration of response of 8.6 months (95% CI: 6.5, 16.8). Median progression-free and overall survivals were 5.8 months (95% CI: 3.7 to 14.1) and 9.6 months (95% CI: 4.8 to 16.3), respectively. There were sixteen grade 3 treatment-related adverse events (TRAEs) and one grade 4 TRAE, with no treatment-related deaths. Overall and progression-free survival did not correlate with PD-L1 status. NK cell cytotoxic activity was increased by cetuximab and further increased with the addition of durvalumab in responders. Conclusions: The combination of cetuximab and durvalumab demonstrated durable activity with a tolerable safety profile in metastatic HNSCC and warrants further investigation.
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