Oncogenic PIK3CA (p110␣), the catalytic subunit of class IA PI3K, plays a major role in PI3K-related cancer progression. The mechanisms underlying the dynamic regulation of PIK3CA protein levels remain unknown. Here we demonstrated that PIK3CA is regulated by polyubiquitination. We identified NEDD4L as the E3 ligase that catalyzes PIK3CA polyubiquitination, leading to its proteasome-dependent degradation. NEDD4L ubiquitinates both the free and regulatory subunitbound PIK3CA but does not ubiquitinate the regulatory subunit of PI3K. Overexpression of NEDD4L accelerates the turnover rate of PIK3CA, whereas suppression of NEDD4L results in not only the accumulation of PIK3CA but also a paradoxical decrease of AKT activation. Thus, we propose that NEDD4L negatively regulates PIK3CA protein levels via ubiquitination and is required for the maintenance of PI3K-AKT signaling pathway.Class IA phosphoinositide 3-kinases (PI3Ks) are lipid kinases that integrate signals from growth factors and hormones to regulate multiple cellular process including growth, proliferation, migration, and survival (1). In response to activation of various growth factor receptors, PI3K is recruited to the cell membrane to catalyze the production of phosphoinositide-3,4,5-trisphosphate, a second messenger required for the activation of a series of downstream kinases (1, 2). Class IA PI3K is composed of a catalytic subunit (p110) and a regulatory subunit (p85), which are demonstrated to form an obligatory heterodimer (3). PIK3CA (p110␣), a major isoform of the catalytic subunits, is widely expressed in various tissues (4). Somatic mutations in PIK3CA occur frequently in cancers of the breast, colon, endometrium, and prostate as well as glioblastomas (5-7). Gain of PIK3CA copy number leads to increased PIK3CA protein levels and is also associated with human cancers (4,8,9). These observations indicate that dysregulation of PI3K plays an important role in cancer emergence and development, underscoring the significance of understanding the regulation mechanisms of PI3K signaling pathway.PI3K function can be modulated via its gene expression or its association with Ras, receptor-tyrosine kinases and other adaptor proteins such as insulin receptor substrate1/2 (IRS1/2). It can also be regulated through post-translational modifications. For instance, previous reports demonstrate that the p85 regulatory subunit can be polyubiquitinated and negatively regulated by the E3 ubiquitin ligase Cbl-b in T cells without affecting its protein level (10, 11). In addition, recent work shows that dephosphorylation of the p110-free p85␤ regulatory subunit leads to its degradation through F-box protein FBXL2 (12). Importantly, the catalytic subunit PIK3CA may also be subject to a similar regulation: that is, a dynamic cycle of proteasomedependent degradation and resynthesis of PIK3CA was observed in response to the stimulation of epidermal growth factor (13).To further understand the regulation of PIK3CA, we developed an in vitro ubiquitination assay for PIK3CA. Usin...

Endometrial cancer (EC) is a common leading cause of cancer-related death in women, which is associated with the increased level of estrogen in the body. Artesunate (ART), an active compound derived from Artemisia annua L., exerted antitumor properties in several cancer types. However, the role of artesunate and the molecular basis on EC remains unclear. Here, we aimed to explore the effects and mechanisms of artesunate. Our results identified that estrogen receptor-α (ER-α) was a key factor for the type I EC (ER-α-positive), which might suppress the downstream LKB1/AMPK/mTOR pathway. Besides, we found ART significantly inhibited tumor proliferation in a dose-dependent manner. Mechanistic studies identified that ART led to tumor cell apoptosis and cell cycle arrest by downregulating the ER-α expression and activating the LKB1/AMPK/mTOR pathway. In addition, we found ART could increase the expression of heart and neural crest derivatives expressed 2 (HAND2) in the ER-α-positive EC cells, which could interact with ER-α. Through the gain-and loss-function experiments, we showed that over expression of HAND2 repressed the proliferation and migration of ER-α-positive EC cells via inhibition of ER-α expression. HAND2 knockdown increased ER-α expression and alleviated the antitumor effect of ART in vitro and in vivo. Overall, our study first showed that ART could be an effective antitumor agent through modulating ER-α-mediated LKB1/AMPK/mTOR pathway in the HAND2 dependent manner. Our findings provide an effective therapeutic agent for ER-α-positive EC treatment.