CPPA: A Web Tool for Exploring Proteomic and Phosphoproteomic Data in Cancer.

A tremendous amount of proteomic and phosphoproteomic data has been produced over the years with the development of mass spectrometry techniques, providing us with new opportunities to explore and understand the proteome and phosphoproteome as well as the function of proteins and protein phosphorylation sites. However, a lack of powerful tools that we can utilize to explore these valuable data limits our understanding of the proteome and phosphoproteome, particularly in diseases such as cancer. To address these unmet needs, we established CPPA (Cancer Proteome and Phosphoproteome Atlas), a web tool to mine abnormalities of the proteome and phosphoproteome in cancer based on published data sets. All analysis results are presented in CPPA with a flexible web interface to provide key customization utilities, including general analysis, differential expression profiling, statistical analysis of protein phosphorylation sites, correlation analysis, similarity analysis, survival analysis, pathological stage analysis, etc. CPPA greatly facilitates the process of data mining and therapeutic target discovery by providing a comprehensive analysis of proteomic and phosphoproteomic data in normal and tumor tissues with a simple click, which helps to unlock the precious value of mass spectrometry data by bridging the gap between raw data and experimental biologists. CPPA is currently available at https://cppa.site/cppa.

USP29 enhances chemotherapy-induced stemness in non-small cell lung cancer via stabilizing Snail1 in response to oxidative stress.

Chemotherapy remains an essential part of diverse treatment regimens against human malignancies. However, recent progressions have revealed a paradoxical role of chemotherapies to induce the cancer stem cell-like features that facilitate chemoresistance and tumor dissemination, with the underlying mechanisms underinvestigated. The zinc-finger transcription factor Snail1 is a central regulator during the epithelial-mesenchymal transition process and is closely implicated in cancer progression. Snail1 expression is strictly regulated at multiple layers, with its stability governed by post-translational ubiquitylation that is counterbalanced by the activities of diverse E3 ligases and deubiquitylases. Here we identify the deubiquitylase USP29 as a novel stabilizer of Snail1, which potently restricts its ubiquitylation in a catalytic activity-dependent manner. Bioinformatic analysis reveals a reverse correlation between USP29 expression and prognosis in lung adenocarcinoma patients. USP29 is unique among Snail1 deubiquitylases through exhibiting chemotherapy-induced upregulation. Mechanistically, oxidative stresses incurred by chemotherapy stimulate transcriptional activation of USP29. USP29 upregulation enhances the cancer stem cell-like characteristics in lung adenocarcinoma cells to promote tumorigenesis in athymic nude mice. Our findings uncover a novel mechanism by which chemotherapy induces cancer stemness and suggest USP29 as a potential therapeutic target to impede the development of chemoresistance and metastasis in lung adenocarcinoma.

Dynasore potentiates c-Met inhibitors against hepatocellular carcinoma through destabilizing c-Met.

c-Met receptor is frequently overexpressed in hepatocellular carcinoma and thus considered as an attractive target for pharmacological intervention with small molecule tyrosine kinase inhibitors. Albeit with the development of multiple c-Met inhibitors, none reached clinical application in the treatment of hepatoma so far. To improve the efficacy of c-Met inhibitors towards hepatocellular carcinoma, we investigated the combined effects of the dynamin inhibitor dynasore with several c-Met inhibitors, including tivantinib, PHA-665752, and JNJ-38877605. We provide several lines of evidence that dynasore enhanced the inhibitory effects of these inhibitors on hepatoma cell proliferation and migration, accompanied with increased cell cycle arrest and apoptosis. Mechanically, the combinatorial treatments decreased c-Met levels and hence markedly disrupted downstream signaling, as revealed by the dramatically declined phosphorylation of AKT and MEK. Taken together, our findings demonstrate that the candidate agent dynasore potentiated the inhibitory effects of c-Met inhibitors against hepatoma cells and will shed light on the development of novel therapeutic strategies to target c-Met in the clinical management of hepatocellular carcinoma patients.

Endocytic degradation of ErbB2 mediates the effectiveness of neratinib in the suppression of ErbB2-positive ovarian cancer.

The tyrosine kinase receptor ErbB2 is frequently found to be overexpressed in multiple cancer types. Targeted therapeutic approaches against ErbB2 have shown promising results and received FDA approvals in the treatment of breast cancer. However, this approach has not been granted in ovarian cancers till now. In order to assess the validity of ErbB2-targeted therapy in ovarian cancer, we investigated the effectiveness of two FDA-approved tyrosine kinase inhibitors of ErbB2, lapatinib and neratinib, on the growth of ovarian cancers. We observed that both lapatinib and neratinib displayed inhibitory effects towards the proliferation and migration of ErbB2-positive ovarian cancer cells in vitro, with neratinib showing stronger suppression in general. Neratinib treatment led to the reduction of ErbB2 protein levels, with concomitant attenuation of the phosphorylation of AKT, MEK, and ERK1/2. Immunofluorescence assays revealed that neratinib induced the internalization and lysosomal degradation of ErbB2, which was accompanied by its hyperubiquitylation. Lapatinib and neratinib also repressed the in vivo growth of SKOV3 cells, and neratinib downregulated ErbB2 levels in xenograft tumors to cause potent inhibition. Therefore, the ubiquitylation-mediated endocytic degradation of ErbB2 incurred by neratinib treatment conferred potent inhibition of ovarian cancer growth. Clinical investigations of neratinib in ErbB2-positive ovarian cancer are warranted.

Amplification of USP13 drives non-small cell lung cancer progression mediated by AKT/MAPK signaling.

USP13 is emerging as a potential target in cancer therapy. However, the effect of USP13 on tumor progression is controversial. Here we focused on non-small cell lung cancer (NSCLC), a common cancer with high mortality, and studied the role of USP13 in tumor growth. By analysis of multi-level genetic database, we found USP13 is high expressed in heart among healthy primary tissues and is most amplified in lung cancer. Clinical samples of NSCLC showed tumor exhibited high USP13 level compared with adjacent normal tissues. We further utilized lung adenocarcinoma A549 and squamous carcinoma H226 cells as cell model and investigated USP13 effect by USP13 knockdown. As a results, downregulation of USP13 dramatically inhibited A549 and H226 cell proliferation by AKT/MAPK signaling and suppressed tumor growth in nude mice. Collectively, we identified USP13 as a tumor promoter in NSCLC and provide a promising target in cancer therapy.

Cholesterol content in cell membrane maintains surface levels of ErbB2 and confers a therapeutic vulnerability in ErbB2-positive breast cancer.

BACKGROUND: ErbB2 overexpression identifies a subset of breast cancer as ErbB2-positive and is frequently associated with poor clinical outcomes. As a membrane-embedded receptor tyrosine kinase, cell surface levels of ErbB2 are regulated dynamically by membrane physical properties. The present study aims to investigate the influence of membrane cholesterol contents on ErbB2 status and cellular responses to its tyrosine kinase inhibitors. METHODS: The cholesterol abundance was examined in ErbB2-positive breast cancer cells using filipin staining. Cellular ErbB2 localizations were investigated by immunofluorescence with altered membrane cholesterol contents. The inhibitory effects of the cholesterol-lowering drug lovastatin were assessed using cell proliferation, apoptosis, immunoblotting and immunofluorescence assays. The synergistic effects of lovastatin with the ErbB2 inhibitor lapatinib were evaluated using an ErbB2-positive breast cancer xenograft mouse model. RESULTS: Membrane cholesterol contents positively correlated with cell surface distribution of ErbB2 through increasing the rigidity and decreasing the fluidity of cell membranes. Reduction in cholesterol abundance assisted the internalization and degradation of ErbB2. The cholesterol-lowering drug lovastatin significantly potentiated the inhibitory effects of ErbB2 kinase inhibitors, accompanied with enhanced ErbB2 endocytosis. Lovastatin also synergized with lapatinib to strongly suppress the in vivo growth of ErbB2-positive breast cancer xenografts. CONCLUSION: The cell surface distribution of ErbB2 was closely regulated by membrane physical properties governed by cholesterol contents. The cholesterol-lowering medications can hence be exploited for potential combinatorial therapies with ErbB2 kinase inhibitors in the clinical treatment of ErbB2-positive breast cancer.

Dynasore-induced potent ubiquitylation of the exon 19 deletion mutant of epidermal growth factor receptor suppresses cell growth and migration in non-small cell lung cancer.

Lung cancer is a leading cause of death worldwide, with mutations in EGFR frequently detected that render this receptor tyrosine kinase constantly active. Targeted therapy against EGFR has proved effective in lung cancer treatment, but secondary mutations in EGFR frequently cause drug resistance. In the efforts made to investigate alternative ways to inhibit mutant EGFR, we observed that the dynamin inhibitor dynasore effectively suppressed the exon 19-deleted mutant of EGFR. This agent inhibited cell proliferation, colony formation, cell migration, and cell cycle progression of HCC827 and H1650 cells driven by the exon 19-deleted EGFR mutant. From a mechanistic point of view, dynasore suppressed the activation of AKT and MEK in HCC827 and H1650 cells. However, dynasore failed to alter the subcellular distribution of EGFR, and another dynamin inhibitor, dyngo-4a, did not phenocopy the effects of dynasore, suggesting a dynamin activity-independent effect of dynasore. Finally, we show that dynasore induced the potent ubiquitylation of the exon 19-deleted mutant of EGFR. Our observations will shed light on the development of alternative therapeutic strategies that target mutant EGFR in lung cancer.

Apigenin suppresses PD-L1 expression in melanoma and host dendritic cells to elicit synergistic therapeutic effects.

BACKGROUND: The PD-L1/PD-1 pathway blockade-mediated immune therapy has shown promising efficacy in the treatment of multiple cancers including melanoma. The present study investigated the effects of the flavonoid apigenin on the PD-L1 expression and the tumorigenesis of melanoma. METHODS: The influence of flavonoids on melanoma cell growth and apoptosis was investigated using cell proliferation and flow cytometric analyses. The differential IFN-γ-induced PD-L1 expression and STAT1 activation were examined in curcumin and apigenin-treated melanoma cells using immunoblotting or immunofluorescence assays. The effects of flavonoid treatment on melanoma sensitivity towards T cells were investigated using Jurkat cell killing, cytotoxicity, cell viability, and IL-2 secretion assays. Melanoma xenograft mouse model was used to assess the impact of flavonoids on tumorigenesis in vivo. Human peripheral blood mononuclear cells were used to examine the influence of flavonoids on PD-L1 expression in dendritic cells and cytotoxicity of cocultured cytokine-induced killer cells by cell killing assays. RESULTS: Curcumin and apigenin showed growth-suppressive and pro-apoptotic effects on melanoma cells. The IFN-γ-induced PD-L1 upregulation was significantly inhibited by flavonoids, especially apigenin, with correlated reductions in STAT1 phosphorylation. Apigenin-treated A375 cells exhibited increased sensitivity towards T cell-mediated killing. Apigenin also strongly inhibited A375 melanoma xenograft growth in vivo, with enhanced T cell infiltration into tumor tissues. PD-L1 expression in dendritic cells was reduced by apigenin, which potentiated the cytotoxicity of cocultured cytokine-induced killer cells against melanoma cells. CONCLUSIONS: Apigenin restricted melanoma growth through multiple mechanisms, among which its suppression of PD-L1 expression exerted a dual effect via regulating both tumor and antigen presenting cells. Our findings provide novel insights into the anticancer effects of apigenin and might have potential clinical implications.

The exon 19-deleted EGFR undergoes ubiquitylation-mediated endocytic degradation via dynamin activity-dependent and -independent mechanisms.

BACKGROUND: The epidermal growth factor receptor (EGFR) is closely implicated in cancer, and sequencing analyses have revealed a high mutation rate of EGFR in lung cancer. Recent advances have provided novel insights into the endocytic regulation of wild-type EGFR, but that of mutated EGFR remains elusive. In the present study, we aim to investigate the endocytic degradation of a frequently occurred exon 19-deleted mutant in lung cancer. METHODS: The EGF-induced endocytic degradation of EGFR was examined in a panel of lung cancer cells using immunoblotting. The subcellular distribution of internalized EGFR was investigated using immunofluorescence and confocal microscopy. The effects of dynamin were assessed using its small molecule inhibitors, while the influence of RTN3 was tested using shRNA-mediated knockdown. Finally the ubiquitylation status of EGFR mutant was studied using immunoprecipitation under steady state and tyrosine kinase inhibitor-treated conditions. RESULTS: EGF induced various rates of EGFR endocytic degradation in lung cancer cells. Interestingly, the exon 19 deletion mutant is constantly internalized and sorted to lysosome for degradation, and this process is independent of dynamin activity. EGF stimulation and HSP90 inhibition further enhance the endocytic degradation of the exon 19 deletion mutant, in a dynamin activity-dependent and -independent manner, respectively. Albeit with different modes of internalization, the uptake of the exon 19-deleted EGFR is mediated through receptor ubiquitylation. CONCLUSIONS: The internalized EGFR mutant is constantly routed through endosome to lysosome for degradation. The endocytosis of EGFR mutant occurs through both dynamin activity-dependent and -independent mechanisms. Our findings gain novel insights into the endocytic regulation of mutated EGFR and may have potential clinical implications.