These authors contributed equally to this work.Keywords: autophagy, autophagosome, BECN1, LC3, PtdIns3KAbbreviations: ATG, autophagy related; BafA1, bafilomycin A 1 ; BCL2L1/Bcl -xL, BCL2-like 1; BECN1, Beclin 1, autophagy related; BECN1P1/BECN2, Beclin 1, autophagy related, pseudogene 1; EM, electron microscopy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; KO, knockout; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MAP1LC3-I/LC3-I, soluble, proteolytically processed microtubule-associated protein 1 light chain 3; MAP1LC3-II/LC3-II, proteolytically processed and lipid-modified microtubule-associated protein 1 light chain 3; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 b; PtdIns3K, phosphatidylinositol 3-kinase; PIK3C3/VPS34, phosphatidylinositol 3-kinase, catalytic subunit type 3; PIK3R4/VPS15, phosphoinositide-3-kinase, regulatory subunit 4; SQSTM1/p62, sequestosome 1; TUBB, tubulin, b class I; TALEN, transcription activator-like effector nuclease; UVRAG, UV radiation resistance associated; ZFYVE1/DFCP1, zinc finger, FYVE domain containing 1.BECN1/Beclin 1 is regarded as a critical component in the class III phosphatidylinositol 3-kinase (PtdIns3K) complex to trigger autophagy in mammalian cells. Despite its significant role in a number of cellular and physiological processes, the exact function of BECN1 in autophagy remains controversial. Here we created a BECN1 knockout human cell line using the TALEN technique. Surprisingly, the complete loss of BECN1 had little effect on LC3 (MAP1LC3B/LC3B) lipidation, and LC3B puncta resembling autophagosomes by fluorescence microscopy were still evident albeit significantly smaller than those in the wild-type cells. Electron microscopy (EM) analysis revealed that BECN1 deficiency led to malformed autophagosome-like structures containing multiple layers of membranes under amino acid starvation. We further confirmed that the PtdIns3K complex activity and autophagy flux were disrupted in BECN1 ¡/¡ cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.

No abstract

Despite the tremendous success of targeted and conventional therapies for lung cancer, therapeutic resistance is a common and major clinical challenge. RNF8 is a ubiquitin E3 ligase that plays essential roles in the DNA damage response; however, its role in the pathogenesis of lung cancer is unclear. Here, we report that RNF8 is overexpressed in lung cancer and positively correlates with the expression of p-Akt and poor survival of patients with non-small-cell lung cancer. In addition, we identify RNF8 as the E3 ligase for regulating the activation of Akt by K63-linked ubiquitination under physiological and genotoxic conditions, which leads to lung cancer cell proliferation and resistance to chemotherapy. Together, our study suggests that RNF8 could be a very promising target in precision medicine for lung cancer.

BackgroundCyclin-dependent kinase 7 (CDK7) is crucial for cell cycle progression and gene expression transcriptional regulation, which are often not assessed in cancer developing process. CDK7 inhibitors have emerged as promising drugs for treating diverse cancers, including breast cancer. However, the mechanism behind its anticancer effect has not been well investigated. Here, the possible mechanism of CDK7 inhibitors for treating human triple-negative breast cancer (TNBC) has been studied.MethodsThe effects of CDK7 inhibitors on breast cancer cells have been identified by measuring cell viability (Cell Counting Kit-8) and cell proliferation and calculating colony formation. The short hairpin RNA and short interfering RNA were used for the construction of knockdown cells. To assess the expression of associated proteins, western blot was used.ResultsThis study confirmed that, compared to hormone receptor-positive breast cancer cells, TNBC cells were more sensitive to THZ1, a novel CDK7 inhibitor. THZ1 treatment specifically downregulated mutated p53 in a dose- and time-dependent manner in TNBC cells with p53 mutation. Another CDK7 inhibitor, LDC4297, also potently interfered with the expression of mutated p53. Furthermore, endogenous CDK7 expression was positively correlated with the levels of mutated p53 in TNBC cells with p53 mutation. Downregulating mutated p53 expression significantly suppressed the proliferation of TNBC cells with p53 mutation.ConclusionOur findings demonstrated that targeting CDK7 was an effective approach for the treatment of TNBC with p53 mutation.