Targeting TRPC1: A Novel Approach to Treat Oral Cancer - Project Summary Oral squamous cell carcinoma (OSCC) is a deadly disease that makes up the majority of all head and neck cancers resulting in 11,000 deaths in the US each year. There are currently 199,670 people living with OSCC in the United States. Patient outcomes for OSCC have not changed in 50 years, due to a lack of targeted therapies. In fact, oral cancer is one of the most understudied cancer types there is resulting in a low 5-year survival rate that has changed very little in decades. Hence, there is a tremendous need to identify biological targets and develop new treatments against OSCC. Our laboratory developed a new class of anti-cancer drugs called 1,3- thioureas, to treat OSCC. We discovered that the lead compound, CIDD99, mediates Transient Receptor Potential Canonical 1 (TRPC1)-mediated calcium entry OSCC cells. Recent studies demonstrate that TRPC1 is overexpressed in OSCC and that siRNA knock-down (KD) of TRPC1 in OSCC cell lines reduced cell proliferation. STIM1 channels are localized in the endoplasmic reticulum and mediate calcium homeostasis via different interactions: 1) STIM1 and Ora1 interactions result in calcium release-activated calcium (Icrac) currents; and 2) TRPC1, STIM1, and Orai1 interactions result in calcium selective, store-operated (Isoc) currents. Isoc currents are the predominant current in OSCC cells, making TRPC1 an attractive therapeutic target. The goal of this study is to tease out the role of TRPC1 in oral cancer tumorigenesis and the feasibility of targeting TRPC1 in vivo. As immunotherapies gain momentum, we appreciate the importance of immune cell function in treating cancer. Icrac currents are critical to immune cell function and are undetectable in OSCC cells, which display Isoc currents. Since STIM1 and Orai1 are involved in both Icrac and Isoc calcium entry, TRPC1 is important to selectively kill oral cancer cells while protecting immune cells from anti-cancer therapeutics. Our overarching hypothesis is that TRPC1 inhibition will selectively kill oral cancer cells while leaving immune cell populations unharmed. Aim 1 will test the hypothesis that targeting TRPC1 will inhibit oral cancer cell proliferation/migration and calcium influx in vitro. Using small molecule inhibitors and CRISPR-Cas9 knock- out (KO) of TRPC1, we will evaluate the effects on OSCC cell proliferation/migration. The effects on STIM1 and Orai1 expression, subcellular localization, and activity will be assessed using QPCR, immunofluorescence, western blot analysis. We will also assess TRPC1 signaling (e.g. PI3K/AKT and MAPK). Calcium imaging and patch clamp electrophysiology will be used to evaluate the effect of TRPC1 inhibition on calcium influx. Aim 2 will test the hypothesis that targeting TRPC1 will reduce OSCC tumorigenesis in vivo. Using TRPC1 KO cell lines and TRPC1 small molecule inhibitors, we will assess the efficacy of targeting TRPC1 in mouse OSCC syngeneic models (immunocompetent). Using immunohistochemistry we will assess changes in STIM1 and Orai1 expression and key signaling pathways in OSCC tumors (+/-) TRPC1. Changes in immune cell populations will be evaluated using Flow Cytometry. Together, these aims will delineate the feasibility of targeting TRPC1 in OSCC.
