Hongmei Cui

Protein glycosylation is one of the most important post-translational modifications, implicated in the development of various diseases, including neurodegenerative diseases, diabetes, and cancers. However, the low content of glycoproteins in biological samples, the diversity and heterogeneity of glycan structures, and insensitive detection methods make glycosylation analysis challenging. As a result, efficient enrichment of glycopeptides from complex samples is a critical step. Efficient enrichment technology can increase the abundance of intact N-glycopeptides in complex biological samples, thereby improving the sensitivity and coverage of glycosylation analysis, which is of great significance for the accurate identification of biomarkers and the development of glycopeptide-based drugs. Among various separation methods for N-glycopeptides, hydrophilic interaction chromatography has received increasing attention, and a variety of enrichment materials have been developed. This article classifies and describes the relevant hydrophilic interaction chromatography materials and provides a comprehensive review of their applications in N-glycopeptide enrichment regarding selectivity, sensitivity, and enrichment performance. Future development trends of ideal glycopeptide enrichment materials are also discussed. Graphical Abstract

Paclitaxel (PTX) is one of the most efficient drugs for late-stage non-small cell lung cancer (NSCLC) patients. However, most patients gradually develop resistance to PTX with long-term treatments. The identification of new strategies to reverse PTX resistance in NSCLC is crucially important for the treatment. PTX is an agonist for the pregnane X receptor (PXR) which regulates PTX metabolism. Antagonizing PXR, therefore, may render the NSCLC more sensitive to the PTX treatment. In this study, we investigated the PXR antagonist SPA70 and its role in PTX treatment of NSCLC. In vitro, SPA70 and PTX synergistically inhibited cell growth, migration and invasion in both paclitaxel-sensitive and paclitaxel-resistant A549 and H460 lung cancer cells. Mechanistically, we found PTX and SPA70 cotreatment disassociated PXR from ABCB1 (MDR1, P-gp) promoter, thus inhibiting P-gp expression. Furthermore, the combination regimen synergistically enhanced the interaction between PXR and Tip60, which abrogated Tip60-mediated α-tubulin acetylation, leading to mitosis defect, S-phase arrest and necroptosis/apoptosis. Combination of PXT and SPA70 dramatically inhibited tumor growth in a paclitaxel-resistant A549/TR xenograft tumor model. Taken together, we showed that SPA70 reduced the paclitaxel resistance of NSCLC. The combination regimen of PTX and SPA70 could be potential novel candidates for the treatment of taxane-resistant lung cancer.

Background The mutation of BRAF V600E often occurred in melanoma and results in tumorigenesis. BRAF mutation drives hyperactivation of the RAF-MAPK-ERK pathway. The acquired drug resistance upon prolonged use of BRAF inhibitors (such as vemurafenib) still remains the main obstacle. Previously, we have found that E3 ligase Skp2 over-expresses vemurafenib-resistant melanoma cells, and knockdown of Skp2 enhances the anti-tumor effect of vemurafenib. Interestingly, the literature has reported that the selective USP14/UCHL5 inhibitor b-AP15 displays great potential in melanoma therapy; however, the molecular mechanism still remains unknown. Methods In vitro, the effect of the combination regimen of vemurafenib (Vem, PLX4032) and b-AP15 on vem-sensitive and vem-resistant melanoma has been investigated by wound healing, colony formation, transwell invasion assay, flow cytometry, lysosome staining, and ROS detection. In vivo, the combination effect on vem-resistant melanoma has been evaluated with a nude mice xenograft tumor model. GST-pulldown and co-immunoprecipitation (co-IP) assays have been applied to investigate the interactions between USP14, UCHL5, and Skp2. Cycloheximide (CHX) assay and ubiquitination assays have been used to explore the effect of USP14 on Skp2 protein half-life and ubiquitination status. Results In the present study, we have revealed that repression of USP14 sensitizes vemurafenib resistance in melanoma through a previously unappreciated mechanism that USP14 but not UCHL5 stabilizes Skp2, blocking its ubiquitination. K119 on Skp2 is required for USP14-mediated deubiquitination and stabilization of Skp2. Furthermore, the mutated catalytic activity amino acid cysteine (C) 114 on USP14 abrogates stabilization of Skp2. Stabilization of Skp2 is required for USP14 to negatively regulate autophagy. The combination regimen of Skp2 inhibitor vemurafenib and USP14/UCHL5 inhibitor b-AP15 dramatically inhibits cell viability, migration, invasion, and colony formation in vemurafenib-sensitive and vemurafenib-resistant melanoma. Vemurafenib and b-AP15 hold cells in the S phase thus leading to apoptosis as well as the formation of the autophagic vacuole in vemurafenib-resistant SKMEL28 cells. The enhanced proliferation effect of USP14 and Skp2 is mainly due to a more effective reduction of cell apoptosis and autophagy. Further evaluation of various protein alterations has revealed that the increased expression of cleaved-PARP, LC3, and decreased Ki67 are more obvious in the combination of vemurafenib and b-AP15 treatment than those in single-drug treatment. Moreover, the co-treatment of vemurafenib and b-AP15 dramatically inhibits the growth of vemurafenib-resistant melanoma xenograft in vivo. Collectively, our findings have demonstrated that the combination of Skp2 inhibitor and USP14 inhibitor provides a new solution for the treatment of BRAF inhibitor resistance melanoma.

Low response rate to radiotherapy remains a problem for liver and colorectal cancer patients due to inappropriate DNA damage response in tumors. Here, we report that pregnane X receptor (PXR) contributes to irradiation (IR) resistance by promoting activating transcription factor 3 (ATF3)-mediated ataxia-telangiectasia-mutated protein (ATM) activation. PXR stabilized ATF3 protein by blocking its ubiquitination. PXR–ATF3 interaction is required for regulating ATF3, as one mutant of lysine (K) 42R of ATF3 lost binding with PXR and abolished PXR-reduced ubiquitination of ATF3. On the other hand, threonine (T) 432A of PXR lost binding with ATF3 and further compromised ATM activation. Moreover, the PXR–ATF3 interaction increases ATF3 stabilization through disrupting ATF3–murine double minute 2 (MDM2) interaction and negatively regulating MDM2 protein expression. PXR enhanced MDM2 auto-ubiquitination and shortened its half-life, therefore compromising the MDM2-mediated degradation of ATF3 protein. Structurally, both ATF3 and PXR bind to the RING domain of MDM2, and on the other hand, MDM2 binds with PXR on the DNA-binding domain (DBD), which contains zinc finger sequence. Zinc finger sequence is well known for nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) playing E3 ligase activity to degrade nuclear factor κB (NFκB)/p65. However, whether zinc-RING sequence grants E3 ligase activity to PXR remains elusive. Taken together, these results provide a novel mechanism that PXR contributes to IR resistance by promoting ATF3-mediated ATM activation through stabilization of ATF3. Our result suggests that targeting PXR may sensitize liver and colon cancer cells to IR therapy.

Pregnane x receptor (PXR) as a nuclear receptor is well-established in drug metabolism, however, it has pleiotropic functions in regulating inflammatory responses, glucose metabolism, and protects normal cells against carcinogenesis. Most studies focus on its transcriptional regulation, however, PXR can regulate gene expression at the translational level. Emerging evidences have shown that PXR has a broad protein-protein interaction network, by which is implicated in the cross signaling pathways. Furthermore, the interactions between PXR and some critical proteins (e.g., p53, Tip60, p300/CBP-associated factor) in DNA damage pathway highlight its potential roles in this field. A thorough understanding of how PXR maintains genome stability and prevents carcinogenesis will help clinical diagnosis and finally benefit patients. Meanwhile, due to the regulation of CYP450 enzymes CYP3A4 and multidrug resistance protein 1 (MDR1), PXR contributes to chemotherapeutic drug resistance. It is worthy of note that the co-factor of PXR such as RXRα, also has contributions to this process, which makes the PXR-mediated drug resistance more complicated. Although single nucleotide polymorphisms (SNPs) vary between individuals, the amino acid substitution on exon of PXR finally affects PXR transcriptional activity. In this review, we have summarized the updated mechanisms that PXR protects the human body against carcinogenesis, and major contributions of PXR with its co-factors have made on multidrug resistance. Furthermore, we have also reviewed the current promising antagonist and their clinic applications in reversing chemoresistance. We believe our review will bring insight into PXR-targeted cancer therapy, enlighten the future study direction, and provide substantial evidence for the clinic in future.

More than half of all cancer patients receive chemotherapy, however, some of them easily acquire drug resistance. Resistance to chemotherapy has become a massive obstacle to achieve high rates of pathological complete response during cancer therapy. S-phase kinase-associated protein 2 (Skp2), as an E3 ligase, was found to be highly correlated with drug resistance and poor prognosis. In this review, we summarize the mechanisms that Skp2 confers to drug resistance, including the Akt-Skp2 feedback loop, Skp2-p27 pathway, cell cycle and mitosis regulation, EMT (epithelial-mesenchymal transition) property, enhanced DNA damage response and repair, etc. We also addressed novel molecules that either inhibit Skp2 expression or target Skp2-centered interactions, which might have vast potential for application in clinics and benefit cancer patients in the future.

Paclitaxel (PTX) is a first-line drug for late-stage non-small cell lung cancer (NSCLC) patients who do not benefit from targeted therapy or immunotherapy. However, patients invariably develop resistance to PTX upon prolonged treatments. Although diverse mechanisms leading to PTX resistance have been well-documented in the literature, strategies to overcome PTX resistance in NSCLC based on these mechanisms are still challenging. In this article, we reviewed recent advancements elucidating major mechanisms of PTX resistance in NSCLC, including the overexpression of ABC transporters, alternations to tubulin structures, and the involvement of cytokines, miRNAs, kinase signaling pathways, and epithelial-mesenchymal transition. Potential markers of PTX resistance or PTX response that could help to direct treatment decisions and restore cellular sensitivity to PTX were also discussed. Finally, we summarized the corresponding strategies to overcome PTX resistance in NSCLC cells, which might provide new insights into clinical trials and benefit lung cancer patients in the future.