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Targeted protein degradation is a promising strategy for drug design and functional assessment. Several small molecule approaches have been developed that localize target proteins to ubiquitin ligases, inducing ubiquitination and subsequent degradation by the 26S proteasome. We discovered that the degradation of a target protein can also be induced by a recognition ligand linked to tert-butyl carbamate (Boc3)-protected arginine (B3A). Here we show that this process requires the proteasome, but does not involve ubiquitination of the target protein. B3A does not perturb the structure of the target protein; instead a B3A-ligand stabilizes its target protein. B3A ligands stimulate activity of purified 20S proteasome, emonstrating that the tag binds directly to the 20S proteasome. Moreover, purified 20S proteasome is sufficient to degrade target proteins in the presence of their respective B3A-linked recognition ligands. These observations suggest a simple model for B3A-mediated degradation wherein the B3A tag localizes target proteins directly to the 20S proteasome. Thus B3A ligands are the first example of a ubiquitin-free strategy for targeted protein degradation.

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Gastric cancer is one of the most common types of human cancer, and it is additionally one of the leading causes of cancer-associated mortality worldwide. Previous studies have suggested that interleukin (IL)-10 may contribute to the pathogenesis of gastric cancer. However, the underlying mechanisms remain unclear. In the present study, it was observed that the expression of IL-10 was significantly upregulated in gastric tumor tissues and serum samples of patients with gastric cancer. Furthermore, IL-10 was increased in the cell culture supernatant of cancer-associated macrophages (CAMs). Treatment with cell culture supernatant from CAMs induced a significant increase in proliferation and migration, while it suppressed apoptosis, in MGC-803 and BGC-823 gastric cancer cells. Notably, application of an inhibitory IL-10 antibody partially blocked the cell culture supernatant of CAM-induced oncogenic effects. RNA-sequencing analysis was then performed to identify the differentially expressed genes in MGC-803 cells treated with IL-10. Based on the sequencing results and in vitro analysis, it was demonstrated that IL-10-induced carcinogenic behaviors in MGC-803 cells were potentially mediated by activation of the c-Met/STAT3 signaling pathway. In conclusion, the present results demonstrated that IL-10 secreted by CAMs may be involved in the pathogenesis of gastric cancer, suggesting that IL-10 may serve as a potential therapeutic target for the treatment of gastric cancer.

Purpose Hepatocellular carcinoma (HCC) ranks as the fourth leading cause of cancer-related deaths worldwide. N6-methyladenosine (m6A) RNA methylation is the most common modification of messenger RNAs (mRNAs). The prognosis of HCC patients with metastasis remains poor. Our study aimed to elucidate the regulatory role of m6A on HCC metastasis. Patients and Methods All HCC patients were enrolled from The Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical University. The expression levels of gene were tested by quantitative polymerase chain reaction (qPCR), Western blot, or immunohistochemistry (IHC) analysis. Wound healing assay, Transwell invasion assay, and lung metastasis model were implemented to investigate the migration and invasion ability of HCC cells. Candidate targets were selected by a comprehensive analysis of RNA-sequencing and m6A-sequencing of HepG2 cells. Results In this study, we demonstrated that METTL14 was significantly downregulated in HCC and significantly associated with the prognosis of HCC patients. METTL14 knockdown promoted the migration, invasion, and epithelial–mesenchymal transition (EMT) of HCC cells in vitro and in vivo. In addition, overlapping RNA-sequencing and m6A-sequencing data, we identified EGFR as a direct target of METTL14 in HCC. Mechanistically, METTL14 was found to inhibit HCC cell migration, invasion, and EMT through modulating EGFR/PI3K/AKT signaling pathway in an m6A-dependent manner. Conclusion Targeting METTL14/EGFR/PI3K/AKT signaling pathway may facilitate the development of a new treatment strategy against the metastasis of HCC.

Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cellular metabolism, growth, and proliferation. mTORC1 has been implicated in many diseases such as cancer, diabetes, and neurodegeneration, and is a target to prolong lifespan. Here we report a small molecule inhibitor (Cbz-B3A) of mTORC1 signaling. Cbz-B3A inhibits the phosphorylation of eIF4E-binding protein 1 (4EBP1) and blocks 68% of translation. In contrast, rapamycin preferentially inhibits the phosphorylation of p70S6k and blocks 35% of translation. Cbz-B3A does not appear to bind directly to mTORC1, but instead binds to ubiquilins 1, 2, and 4. Knockdown of ubiquilin 2, but not ubiquilins 1 and 4, decreases the phosphorylation of 4EBP1, suggesting that ubiquilin 2 activates mTORC1. The knockdown of ubiquilins 2 and 4 decreases the effect of Cbz-B3A on 4EBP1 phosphorylation. Cbz-B3A slows cellular growth of some human leukemia cell lines, but is not cytotoxic. Thus Cbz-B3A exemplifies a novel strategy to inhibit mTORC1 signaling that might be exploited for treating many human diseases. We propose that Cbz-B3A reveals a previously unappreciated regulatory pathway coordinating cytosolic protein quality control and mTORC1 signaling.

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