Lina Yang

Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic

Significance Arsenic holds promise for treating a wide range of tumors. To understand arsenics antitumor mechanism further, we identified 360 arsenic-binding proteins using a human proteome microarray and found proteins of glycolysis to be highly enriched. In-depth in vitro and in vivo analysis revealed that glycolysis in general and the rate-limiting enzyme hexokinase-2 of the glycolytic pathway in particular play a key role in mediating the anticancer activity of arsenic. These findings shed light on the mode of action of arsenic, and the newly identified arsenic-binding proteins may serve as a rich resource for future studies. Arsenic is highly effective for treating acute promyelocytic leukemia (APL) and has shown significant promise against many other tumors. However, although its mechanistic effects in APL are established, its broader anticancer mode of action is not understood. In this study, using a human proteome microarray, we identified 360 proteins that specifically bind arsenic. Among the most highly enriched proteins in this set are those in the glycolysis pathway, including the rate-limiting enzyme in glycolysis, hexokinase-1. Detailed biochemical and metabolomics analyses of the highly homologous hexokinase-2 (HK2), which is overexpressed in many cancers, revealed significant inhibition by arsenic. Furthermore, overexpression of HK2 rescued cells from arsenic-induced apoptosis. Our results thus strongly implicate glycolysis, and HK2 in particular, as a key target of arsenic. Moreover, the arsenic-binding proteins identified in this work are expected to serve as a valuable resource for the development of synergistic antitumor therapeutic strategies.

Discovering cancer biomarkers from clinical samples by protein microarrays

Cancer biomarkers are of potential use in early cancer diagnosis, anticancer therapy development, and monitoring the responses to treatments. Protein‐based cancer biomarkers are major forms in use, as they are much easier to be monitored in body fluids or tissues. For cancer biomarker discovery, high‐throughput techniques such as protein microarrays hold great promises, because they are capable of global unbiased monitoring but with a miniaturized format. In doing so, novel and cancer type specific biomarkers can be systematically discovered at an affordable cost. In this review, we give a relatively complete picture on protein microarrays applied to clinical samples for cancer biomarker discovery, and conclude this review with the future perspectives.

Identification of serum biomarkers for gastric cancer diagnosis using a human proteome microarray

We aimed to globally discover serum biomarkers for diagnosis of gastric cancer (GC). GC serum autoantibodies were discovered and validated using serum samples from independent patient cohorts encompassing 1,401 participants divided into three groups, i.e. healthy, GC patients, and GC-related disease group. To discover biomarkers for GC, the human proteome microarray was first applied to screen specific autoantibodies in a total of 87 serum samples from GC patients and healthy controls. Potential biomarkers were identified via a statistical analysis protocol. Targeted protein microarrays with only the potential biomarkers were constructed and used to validate the candidate biomarkers using 914 samples. To provide further validation, the abundance of autoantibodies specific to the biomarker candidates was analyzed using enzyme-linked immunosorbent assays. Receiver operating characteristic curves were generated to evaluate the diagnostic accuracy of the serum biomarkers. Finally, the efficacy of prognosis efficacy of the final four biomarkers was evaluated by analyzing the clinical records. The final panel of biomarkers consisting of COPS2, CTSF, NT5E, and TERF1 provides high diagnostic power, with 95% sensitivity and 92% specificity to differentiate GC patients from healthy individuals. Prognosis analysis showed that the panel could also serve as independent predictors of the overall GC patient survival. The panel of four serum biomarkers (COPS2, CTSF, NT5E, and TERF1) could serve as a noninvasive diagnostic index for GC, and the combination of them could potentially be used as a predictor of the overall GC survival rate.

Construction of a metabolomics profile of arsenic trioxide effect in gastric carcinoma cell line SGC7901

Arsenic trioxide (ATO) is highly effective for treating acute promyelocytic leukemia. It also holds the promise for treating solid tumors, including gastric carcinoma. However, the molecular mechanism of the effectiveness of ATO to solid tumor is still poorly understood. In this study, we chosed gastric carcinoma as an example and tried to reveal the antitumor mechanism through metabolomics. Gastric carcinoma cell line SGC7901 was treated with ATO for 6, 12, and 24 h. The global metabolite profiles were monitored by metabolomics analysis using gas chromatography (GC)/mass spectrometry (MS) and liquid chromatography/MS/MS. A total of 281 certified metabolites were reliably detected. Bioinformatics analysis showed that glycerophospholipid synthesis, one-carbon synthesis, and glutathione synthesis were affected dramatically. Other cellular functions/pathways that had been affected included inflammatory response, nicotinamide adenine dinucleotide (NAD(+)), and polyamine biosynthesis pathway. The metabolomics data from this study, in combination with previous transcriptomics and proteomics data, could serve as valuable resources for the understanding of the specific antitumor mechanism of ATO treatment.

Skp1 in lung cancer: clinical significance and therapeutic efficacy of its small molecule inhibitors

Skp1 is an essential adaptor protein of the Skp1-Cul1-F-box protein complex and is able to stabilize the conformation of some ubiquitin E3 ligases. However, the role Skp1 plays during tumorigenesis remains unclear and Skp1-targeting agent is lacking. Here we showed that Skp1 was overexpressed in 36/64 (56.3%) of non-small cell lung cancers, and elevated Skp1 was associated with poor prognosis. By structure-based high-throughput virtual screening, we found some Skp1-targeting molecules including a natural compound 6-O-angeloylplenolin (6-OAP). 6-OAP bound Skp1 at sites critical to Skp1-Skp2 interaction, leading to dissociation and proteolysis of oncogenic E3 ligases NIPA, Skp2, and β-TRCP, and accumulation of their substrates Cyclin B1, P27 and E-Cadherin. 6-OAP induced prometaphase arrest and exerted potent anti-lung cancer activity in two murine models and showed low adverse effect. These results indicate that Skp1 is critical to lung cancer pathogenesis, and Skp1 inhibitor inactivates crucial oncogenic E3 ligases and exhibits significant therapeutic potentials.

Proteomic identification of the oncoprotein STAT3 as a target of a novel Skp1 inhibitor

The S phase kinase-associated protein 1 (Skp1), an adaptor protein of the Skp1-Cul1-F-box protein complex, binds the ubiquitin E3 ligase Skp2 and is critical to its biological functions. Targeting of Skp1 by a small compound 6-O-angeloylplenolin (6-OAP) results in dissociation and degradation of Skp2 and mitotic arrest of lung cancer cells. Here, by using a proteome microarray containing 16,368 proteins and a biotinylated 6-OAP, we identified 99 proteins that could bind 6-OAP, with Skp1 and STAT3 sitting at the central position of the 6-OAP interactome. 6-OAP formed hydrogen bonds with Ser611/Ser613/Arg609 at the SH2 domain of STAT3 and inhibited the constitutive and interleukin-6-induced phosphorylated STAT3 (pSTAT3), leading to inhibitory effects on lung cancer cells and suppression of Skp2 transcription. STAT3 was overexpressed in tumor samples compared to counterpart normal lung tissues and was inversely associated with prognosis of the patients. 6-OAP inhibited tumor growth in SCID mice intravenously injected with lung cancer cells, and downregulated both STAT3 and Skp2 in tumor samples. Given that 6-OAP is a Skp1 inhibitor, our data suggest that this compound may target Skp1 and STAT3 to suppress Skp2, augmenting its anti-lung cancer activity.