Inhibition of mTOR signaling and clinical activity of metformin in oral premalignant lesions

Abstract

BACKGROUNDThe aberrant activation of the PI3K/mTOR signaling circuitry is one of the most frequently dysregulated signaling events in head and neck squamous cell carcinoma (HNSCC). Here, we conducted a single-arm, open-label phase IIa clinical trial in individuals with oral premalignant lesions (OPLs) to explore the potential of metformin to target PI3K/mTOR signaling for HNSCC prevention.METHODSIndividuals with OPLs, but who were otherwise healthy and without diabetes, underwent pretreatment and posttreatment clinical exam and biopsy. Participants received metformin for 12 weeks (week 1, 500 mg; week 2, 1000 mg; weeks 3-12, 2000 mg daily). Pretreatment and posttreatment biopsies, saliva, and blood were obtained for biomarker analysis, including IHC assessment of mTOR signaling and exome sequencing.RESULTSTwenty-three participants were evaluable for response. The clinical response rate (defined as a ≥50% reduction in lesion size) was 17%. Although lower than the proposed threshold for favorable clinical response, the histological response rate (improvement in histological grade) was 60%, including 17% complete responses and 43% partial responses. Logistic regression analysis revealed that when compared with never smokers, current and former smokers had statistically significantly increased histological responses (P = 0.016). Remarkably, a significant correlation existed between decreased mTOR activity (pS6 IHC staining) in the basal epithelial layers of OPLs and the histological (P = 0.04) and clinical (P = 0.01) responses.CONCLUSIONTo our knowledge this is the first phase II trial of metformin in individuals with OPLs, providing evidence that metformin administration results in encouraging histological responses and mTOR pathway modulation, thus supporting its further investigation as a chemopreventive agent.TRIAL REGISTRATIONNCT02581137FUNDINGNIH contract HHSN261201200031I, grants R01DE026644 and R01DE026870.

Keywords: Clinical Trials; Head and neck cancer; Signal transduction.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. CONSORT diagram.

The study (M4OC Prevent) was a phase IIa single-arm, open-label trial of individuals with oral leukoplakia or erythroplakia to explore the potential of metformin for oral cancer prevention (NCT02581137). The primary endpoint was to determine whether 12–14 weeks of metformin intervention is associated with the clinical response of OPLs. The secondary endpoints included histologic response to metformin in the target lesion, pretreatment and posttreatment tissue-based biomarkers of molecular targets and dysregulated molecular mechanisms, modulation of circulating metabolic biomarkers, and serum and saliva metformin concentrations. A brief summary of the trial workflow, schedule of baseline evaluation, intervention, and postintervention evaluation, and agent used (metformin) is depicted.

Figure 2. Biomarker analysis.

Examples of lesions positive and negative for p53 are shown in the 2 boxes of the first column. In the positive lesions, the expression is limited to the nuclei of the dysplastic proliferating cells. Examples of high and low EGFR expression levels are depicted (second column). All lesions tested negative for p16 and expressed OCT3 throughout the epithelial layers (third column). Examples of positive and negative PTEN lesions are depicted (fourth column). Please see the corresponding data for each individual participant in Table 2.

Figure 3. Metformin concentration in blood and saliva and serum biomarkers.

Correlation analysis of metformin levels in serum and saliva in individuals treated with metformin.

Figure 4. Clinical and histological response to metformin.

(A) A waterfall plot of the clinical response to treatment is shown, depicting the percentage of change in lesion size. Histological responses are also indicated based on the column color. The individual participant responses as well as the smoking status are depicted following the same numbering as in Figure 1. (B) Summary of the clinical and histological responses of all participants evaluated. (C) Statistical analysis of the histological response in relationship to the smoking status (*P = 0.016, Fisher’s exact test). CR, complete response; PR, partial response; NC, no change; PD, progressive disease.

Figure 5. Impact of metformin on proliferation and mTOR pathway signaling in oral premalignant lesions.

(A and B) Quantification of the IHC of the proliferation marker KI67 (A) and pS6 (surrogate indicator of the mTOR pathway activity) (B) was evaluated and reported as the percentage of positive cells for Ki67 and H score for pS6 before and after metformin treatment. Statistical significance (nonparametric Wilcoxon’s matched-pairs signed-rank test) is indicated, and examples of staining before and after treatment are included in each case, with a higher magnification on the right. Notice the absence of pS6 staining in the basal layer of the oral premalignant lesion after metformin treatment. (C) Statistical analysis of the histological (left) and clinical (right) response in relationship to the changes in basal pS6 levels. Statistical significance is indicated (ordinal logistic regression). CR, complete response; PR, partial response; NC, no change; PD, progressive disease.