Role of OCT-3 on metformin action in oral carcinogenesis The long-term goal of this proposal is to identify the target patient population who will benefit from the use of metformin, or related biguanides, in oral cancer prevention and treatment. In the United States, roughly one person dies every hour of each day because of complications associated with oral cancer, mainly oral squamous cell carcinoma (OSCC). Unfortunately, improvements in OSCC patient survival rates have remained unchanged for decades. Late diagnosis and field cancerization with multifocal potentially malignant dysplastic lesions or secondary primary OSCCs are major factors compromising standard treatments. In this regard, long- term chemopreventive targeting of at risk oral premalignant lesions may offer a great opportunity to control OSCC development and progression. We recently reported that metformin, a FDA-approved biguanide used as first-line treatment for type 2 diabetes, significantly prevented the conversion of carcinogen-induced oral premalignant lesions into OSCC tumors in immunocompetent mice. Despite significant progress, a critical issue that remains elusive is whether metformin acts directly on the tumor cells, or affects cancer development by controlling hormonal responses at extratumoral sites. This gap in knowledge holds significant clinical implications because, as a highly hydrophilic cationic drug, metformin intracellular uptake relies on tissue- specific mechanisms facilitated by a group of polyspecific cell membrane organic cation transporters (OCTs) belonging to the solute carrier 22A (SLC22A) gene family. Although, it is known that OCT-1, OCT-2 and OCT-3 mediate metformin uptake in different normal tissues, it is still unclear which role OCTs play on the antineoplastic effects of metformin, or other related biguanides. Our preliminary studies point to OCT-3 as the uptake transporter of metformin in oral carcinogenesis. We found variable OCT-3 expression in OSCC cell lines derived from human oral premalignant lesions and OSCC tumors. Interestingly, strong OCT-3 expression was commonly observed in oral dysplasias and well-differentiated OSCC, but progressively declined in more atypical, less differentiated OSCC tumors. These results suggest that OCT-3 expression is linked to the degree of tumor cell differentiation, and points to this previously unidentified association as a potential mechanism of resistance to metformin in oral carcinogenesis. In contrast, phenformin, a more hydrophobic biguanide, appears to be less dependent on OCT-3-mediated uptake and likely a better alternative to target established, OCT-3 negative OSCC tumors. Through in vitro and in vivo approaches, we will test the overall hypothesis that tumor growth inhibition by metformin or related biguanides such as phenformin is dependent on OCT-3 uptake activity at the primary site of oral carcinogenesis. Three specific aims are proposed. Aim 1 will define the contribution of OCT-3 on OSCC cell proliferation in response to metformin. Aim 2 will determine the impact of OCT-3 on metformin chemopreventive action in oral carcinogenesis. Aim 3 will identify dependence of OCT-3 for the antineoplastic activity of phenformin in oral carcinogenesis. By elucidating mechanisms of biguanide uptake and activity, we envision that the outcomes of the proposed studies may ultimately impact the selection of the most suitable patients who can benefit from these drugs in oral cancer chemoprevention and treatment.