Oral squamous cell carcinoma (OSCC) remains one of the most common cancers worldwide, and the mortality rate of this disease has increased in recent years. No molecular markers are available to assist with the early detection and therapeutic evaluation of OSCC; thus, identification of differentially expressed proteins may assist with the detection of potential disease markers and shed light on the molecular mechanisms of OSCC pathogenesis. We performed a multidimensional ¹⁶O/¹⁸O proteomics analysis using an integrated ESI-ion trap and MALDI-TOF/TOF MS system and a computational data analysis pipeline to identify proteins that are differentially expressed in microdissected OSCC tumor cells relative to adjacent non-tumor epithelia. We identified 1233 unique proteins in microdissected oral squamous epithelia obtained from three pairs of OSCC specimens with a false discovery rate of <3%. Among these, 977 proteins were quantified between tumor and non-tumor cells. Our data revealed 80 dysregulated proteins (53 up-regulated and 27 down-regulated) when a 2.5-fold change was used as the threshold. Immunohistochemical staining and Western blot analyses were performed to confirm the overexpression of 12 up-regulated proteins in OSCC tissues. When the biological roles of 80 differentially expressed proteins were assessed via MetaCore™ analysis, the interferon (IFN) signaling pathway emerged as one of the most significantly altered pathways in OSCC. As many as 20% (10 of 53) of the up-regulated proteins belonged to the IFN-stimulated gene (ISG) family, including ubiquitin cross-reactive protein (UCRP)/ISG15. Using head-and-neck cancer tissue microarrays, we determined that UCRP is overexpressed in the majority of cheek and tongue cancers and in several cases of larynx cancer. In addition, we found that IFN-β stimulates UCRP expression in oral cancer cells and enhances their motility in vitro. Our findings shed new light on OSCC pathogenesis and provide a basis for the future development of novel biomarkers.

BACKGROUND: A previous comparative tissue proteomics study by the authors of the current study led to the identification of caldesmon (CaD) as one of the proteins associated with cervical metastasis of oral cavity squamous cell carcinoma (OSCC). In the current investigation, the authors focused on the potential functions of CaD in patients with OSCC.METHODS: CaD expression was examined in tissue samples from 155 patients using immunohistochemical analysis. The expression of CaD variants was determined by Western blot analysis and reverse transcriptase‐polymerase chain reaction. In addition, the specific effects of CaD gene overexpression and silence were determined in OSCC cell lines.RESULTS: CaD expression was found to be significantly higher in tumor cells from metastatic lymph nodes compared with primary tumor cells, and was nearly absent in normal oral epithelia. Higher CaD expression was found to be correlated with positive N classification, poor differentiation, perineural invasion, and tumor depth (P = .001, P = .029, P = .001, and P = .031, respectively). In survival analyses, OSCC patients with higher CaD expression were found to have poorer prognosis with regard to disease‐specific survival and disease‐free survival (P = .003 and P = .014, respectively). Multivariate analyses further indicated that higher CaD expression was an independent predictor of disease‐specific survival (P = .043). Serum CaD levels were found to be significantly higher in patients with OSCC, but this finding was not associated with clinicopathological manifestations. Data obtained from in vitro suppression, rescue, and overexpression of CaD in OEC‐M1 cells indicated that CaD promotes migration and invasive processes in OSCC cells.CONCLUSIONS: The findings of the current study collectively suggest that the low‐molecular‐weight CaD expression in OSCC tumors is associated with tumor metastasis and patient survival. Cancer 2013;119:4003–4011. © 2013 American Cancer Society.

P62 is a receptor that facilitates selective autophagy by interacting simultaneously with cargoes and LC3 protein on the autophagosome to maintain cellular homeostasis. However, the regulatory mechanism(s) behind this process and its association with breast cancer remain to be elucidated. Here, we report that Flightless-I (FliI), a novel p62-interacting protein, promotes breast cancer progression by impeding selective autophagy. FliI was highly expressed in clinical breast cancer samples, and heterozygous deletion of FliI retarded the development of mammary tumors in PyVT mice. FliI induced p62-recruited cargoes into Triton X-100 insoluble fractions (TI) to form aggregates, thereby blocking p62 recognition of LC3 and hindering p62-dependent selective autophagy. This function of Flil was reinforced by Akt-mediated phosphorylation at Ser436 and inhibited by phosphorylation of Ulk1 at Ser64. Obstruction of autophagic clearance of p62-recruited cargoes by FliI was associated with the accumulation of oxidative damage on proteins and DNA, which could contribute to the development of cancer. Heterozygous knockout of FliI facilitated selectively autophagic clearance of aggregates, abatement of ROS levels, and protein oxidative damage, ultimately retarding mammary cancer progression. In clinical breast cancer samples, Akt-mediated phosphorylation of FliI at Ser436 negatively correlated with long-term prognosis, while Ulk1-induced FliI phosphorylation at Ser64 positively correlated with clinical outcome. Together, this work demonstrates that FliI functions as a checkpoint protein for selective autophagy in the crosstalk between FliI and p62-recruited cargoes, and its phosphorylation may serve as a prognostic marker for breast cancer. Significance: Flightless-I functions as a checkpoint protein for selective autophagy by interacting with p62 to block its recognition of LC3, leading to tumorigenesis in breast cancer.Cancer Res; 78(17); 4853–64. ©2018 AACR.

Absatract: Along with the constant improvement in high-throughput sequencing technology, an increasing number of transcriptome sequencing projects are carried out in organisms without decoded genome information and even on environmental biological samples. To study the biological functions of novel transcripts, the very first task is to identify their potential functions. We present a web-based annotation tool, FunctionAnnotator, which offers comprehensive annotations, including GO term assignment, enzyme annotation, domain/motif identification and predictions for subcellular localization. To accelerate the annotation process, we have optimized the computation processes and used parallel computing for all annotation steps. Moreover, FunctionAnnotator is designed to be versatile, and it generates a variety of useful outputs for facilitating other analyses. Here, we demonstrate how FunctionAnnotator can be helpful in annotating non-model organisms. We further illustrate that FunctionAnnotator can estimate the taxonomic composition of environmental samples and assist in the identification of novel proteins by combining RNA-Seq data with proteomics technology. In summary, FunctionAnnotator can efficiently annotate transcriptomes and greatly benefits studies focusing on non-model organisms or metatranscriptomes. FunctionAnnotator, a comprehensive annotation web-service tool, is freely available online at: http://fa.cgu.edu.tw/. This new web-based annotator will shed light on field studies involving organisms without a reference genome.