AbstractThe tumor suppressor LKB1 is mutated in 30% of non–small cell lung cancer (NSCLC) tumors and cell lines and is proposed to be a key regulator of epithelial cell polarity; however, how LKB1 regulates cancer cell polarity is not known. The experiments described herein show for the first time that LKB1 is a dynamic, actin-associated protein that rapidly polarizes to the leading edge of motile cancer cells. LKB1 proves to be essential for NSCLC polarity, because LKB1 depletion results in classic cell polarity defects, such as aberrant Golgi positioning, reduced lamellipodia formation, and aberrant morphology. To probe how LKB1 regulates these events, we show that LKB1 colocalizes at the cellular leading edge with two key components of the polarity pathway — the small rho GTPase cdc42 and its downstream binding partner p21-activated kinase (PAK). Importantly, LKB1 functionality is required for cdc42 polarization to the leading edge, maintaining active cdc42 levels, and downstream PAK phosphorylation. To do this, LKB1 interacts only with active form of cdc42 and PAK, but not with inactive cdc42. Taken together, these results show that LKB1 is a critical mediator of the NSCLC polarity program in lung cancer cells through a novel LKB1-cdc42-PAK pathway. [Cancer Res 2008;68(3):740–8]