Metformin effects on head and neck squamous carcinoma microenvironment: Window of opportunity trial

Abstract

Objective: The tumor microenvironment frequently displays abnormal cellular metabolism, which contributes to aggressive behavior. Metformin inhibits mitochondrial oxidative phosphorylation, altering metabolism. Though the mechanism is unclear, epidemiologic studies show an association between metformin use and improved outcomes in head and neck squamous cell carcinoma (HNSCC). We sought to determine if metformin alters metabolism and apoptosis in HNSCC tumors.

Study design: Window of opportunity trial of metformin between diagnostic biopsy and resection. Participants were patients with newly diagnosed HNSCC. Fifty patients were enrolled, and 39 completed a full-treatment course. Metformin was titrated to standard diabetic dose (2,000 mg/day) for a course of 9 or more days prior to surgery.

Methods: Immunohistochemistry (IHC) for the metabolic markers caveolin-1 (CAV1), B-galactosidase (GALB), and monocarboxylate transporter 4 (MCT4), as well as the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay and Ki-67 IHC, were performed in pre- and postmetformin specimens. Exploratory mass spectroscopy imaging (MSI) to assess lactate levels also was performed in three subjects.

Results: Metformin was well tolerated. The average treatment course was 13.6 days. Posttreatment specimens showed a significant increase in stromal CAV1 (P < 0.001) and GALB (P < 0.005), as well as tumor cell apoptosis by TUNEL assay (P < 0.001). There was no significant change in stromal MCT4 expression or proliferation measured by Ki67. Lactate levels in carcinoma cells were increased 2.4-fold postmetformin (P < 0.05), as measured by MSI.

Conclusion: Metformin increases markers of reduced catabolism and increases senescence in stromal cells as well as carcinoma cell apoptosis. This study demonstrates that metformin modulates metabolism in the HNSCC microenvironment.

Level of evidence: 4. Laryngoscope, 127:1808-1815, 2017.

Trial registration: ClinicalTrials.gov NCT02083692.

Keywords: Head and neck cancer; metformin; squamous cell carcinoma; tumor metabolism.

Figure 1

(A–B). Effect of metformin on stromal CAV1 and GALB expression in HNSCC. CAV1 and GALB immunostaining were performed on paired premetformin (A and B, respectively) and postmetformin HNSCC samples, and a representative example is shown. Note that there is a postmetformin increase in CAV1 and GALB stromal staining. Original magnification: 40×. Waterfall plot of the change in CAV1 and GALB intensity between pre‐ and postmetformin samples also is shown. CAV1 = caveolin‐1; GALB = beta galactosidase; HNSCC = head and neck squamous cell carcinoma.

Figure 2

Effect of metformin on TUNEL staining in HNSCC. TUNEL was performed on paired premetformin and postmetformin HNSCC samples, and a representative example is shown. Note that there is a postmetformin increase in TUNEL staining in carcinoma cells. Original magnification: 40×. Waterfall plot of the change in GALB intensity between pre‐ and postmetformin samples also is shown. HNSCC = head and neck squamous cell carcinoma; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 3

(A–C) Mass spectroscopy imaging (MSI) comparing carcinoma regions premetformin and postmetformin. Hematoxylin and eosin staining was performed on HNSCC samples, and carcinoma regions were marked in pre‐ and postmetformin samples. Original magnification: 40× (A). Carcinoma cells have a 2.4‐fold reduction in lactate levels postmetformin (post‐rx) compared to premetformin (pre‐rx) in all samples (P < 0.05). Lactate levels were measured by MSI in the carcinoma regions pre‐ and postmetformin, and results were compared for each patient (B). Note that the black lines are the tracings for the premetformin samples and the red lines for the postmetformin samples. Effects of metformin on the tumor microenvironment of HNSCC (C). The current clinical trial demonstrates in HNSCC that metformin has anticancer activity. Metformin in carcinoma cells increases apoptosis, as measured by terminal deoxynucleotidyl transferase dUTP nick end labeling, and increases lactate levels. Metformin also alters metabolic markers in stromal cells. The tumor stroma after metformin exposure has increased CAV1 and GALB expression, which are markers of reduced catabolism and increased senescence. CAV1 = caveolin‐1; GALB = beta galactosidase; HNSCC = head and neck squamous cell carcinoma; MSI = mass spectroscopy imaging; rx = treatment.