The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine/threonine protein kinase, integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth, proliferation, survival, and autophagy. The mTOR pathway is frequently activated in many human cancers, mainly resulting from alterations in the upstream regulators, such as phosphoinositide 3-kinase (PI3K)/AKT activation, PTEN loss or dysregulation of mTOR-negative regulators (e.g., TSC1/2), leading to uncontrolled proliferation. Thus, inhibiting the PI3K/AKT/mTOR pathways is widely considered as an effective approach for targeted cancer therapy. Recently, we and others found that DEPTOR, a naturally occurring inhibitor of both mTORC1 and mTORC2, was degraded by SCF (Skp1-Cullin-F box proteins) E3 ubiquitin ligase, the founding member of cullin-RING-ligases (CRLs), resulting in mTOR activation and cell proliferation. In addition to DEPTOR, previous studies have demonstrated that several other negative regulators of mTOR pathway are also substrates of CRL/SCF E3s. Thus, targeting CRL/SCF E3s is expected to cause the accumulation of these mTOR signal inhibitors to effectively block the mTOR pathway. In this review, we will discuss mTOR signaling pathway, how DEPTOR regulates mTOR/AKT axis, thus acting as a tumor suppressor or oncogene in some cases, how DEPTOR is ubiquitinated and degraded by SCF(β-TrCP) E3, and how MLN4924, a small-molecule indirect inhibitor of CRL/SCF E3 ligases through blocking cullin neddylation, might be useful as a novel approach of mTOR pathway targeting for cancer therapy.