Pham Thuy Tien Vo

Supplementary Figure 1: cancer-associated HRAS mutations in HNSCC using data from The Cancer Genome Atlas (TCGA) and Represetive images of the clonogenic assays.

Supplementary Figure 2: metformin sensitivity in HRAS-mutant HNSCC cells

Supplementary Figure 3: Metabolic profiling of CAL27 Control and HRAS mutant cells reveals changes in ATP production and oxygen consumption rates.

Supplementary Figure 4: Synergistic effects of Metformin and Dichloroacetate (DCA) in HNSCC cell lines

Supplementary Figure 5: the orthotopic mouse model

Metformin administration has recently emerged as a candidate strategy for the prevention of head and neck squamous cell carcinoma (HNSCC). However, the intricate relationship between genetic alterations in HNSCC and metformin sensitivity is still poorly understood, which prevents the stratification of patients, harboring oral premalignant lesions that may benefit from the chemopreventive activity of metformin. In this study, we investigate the impact of prevalent mutations in HNSCC on response to metformin. Notably, we found that the expression of oncogenic HRAS mutants confers resistance to metformin in isogenic HNSCC cell systems, and that HNSCC cells harboring endogenous HRAS mutations display limited sensitivity to metformin. Remarkably, we found that metformin fails to reduce activation of the mTOR pathway in HRAS oncogene-expressing HNSCC cells in vitro and in vivo, correlating with reduced tumor suppressive activity. Mechanistically, we found that this process depends on the ability of HRAS to enhance glycolytic metabolism, thereby suppressing the requirement for oxidative phosphorylation to maintain the cellular energetic balance. Overall, our study revealed that HNSCC cells with oncogenic HRAS mutations exhibit diminished metformin sensitivity, thus shedding light on a potential mechanism of treatment resistance. This finding may also help explain the limited clinical responses to metformin in cancers with RAS mutations. Ultimately, our study underscores the importance of understanding the impact of the genetic landscape in tailoring precision cancer-preventive approaches in the context of HNSCC and other cancers that are characterized by the presence of a defined premalignant state, and therefore, are amenable to cancer interception strategies.Prevention Relevance:Our findings highlight the challenges of using metformin for cancer prevention in RAS-mutant cancers, where elevated glycolysis may reduce drug efficacy. This underscores the need to explore metformin’s potential in early, premalignant stages, before metabolic shifts render it less effective.

Supplementary Figure 6. Squamous cell carcinoma (SCC)-positive tongues from the metformin + naproxen treatment arm. (Related to Figure 4)

Supplementary Figure 1. Genome-wide CRISPR-Cas9 screen identifies PKA signaling as a metformin resistance pathway in HNSCC. (Related to Figure 1)

Supplementary Figure 2. Activation of PKA in HNSCC cells reduces sensitivity to metformin growth inhibition in vivo. (Related to Figure 1)

Supplementary Figure 3. Combination of metformin with NSAIDs, which inhibit COX2, enhances the inhibition of HNSCC cell growth in vitro. (Related to Figure 3)