Thyroid cancer is the most common human endocrine malignancy with increasing global incidence. Papillary thyroid carcinomas (PTC) and follicular thyroid carcinomas (FTC) are well-differentiated thyroid cancers (WDTC) accounting for 95% of all thyroid cancer cases, with survival rates of almost 100% when diagnosed early. Since PTC and FTC have different modes of metastasis, they require different treatment strategies. Standard diagnosis by fine needle aspiration with cytopathological examination can be inaccurate in approximately 10-30% of all cases and difficult to definitively classify as WDTC. Currently, there is no single or panel of biomarkers available for thyroid cancer diagnosis and classification. This study identified novel biomarkers for thyroid cancer diagnosis and classification using proteomics, which may be translated into a biomarker panel for clinical application. Two-dimensional SDS-PAGE and mass spectrometry were used to identify potential biomarkers in papillary and follicular thyroid carcinoma cell lines, and the biomarkers were validated in five PTC and five FTC tissues, with their adjacent normal tissues from Thai patients. Eight biomarkers could distinguish PTC from normal tissues, namely enolase 1, triose phosphate isomerase, cathepsin D, annexin A2, cofilin 1, proliferating cell nuclear antigen (PCNA), copine 1 and heat shock protein 27 kDa (HSP27). These biomarkers can also discriminate FTC from normal tissues, except for annexin A2. On the contrary, annexin A2, cofilin 1, PCNA and HSP27 can be used to classify the types of WDTC. These findings have potential for use as a novel multi-marker panel for more accurate diagnosis and classification to better guide physicians on thyroid cancer treatment. Moreover, our results suggest the involvement of proteins in cell growth and proliferation, and the p53 pathway in the carcinogenesis of WDTC, which may lead to targeted therapy for thyroid cancer.

Breast cancer is the most common type of cancer and leading cause of cancer-associated mortality in women worldwide. O-linked N-acetyl glucosaminylation (O-GlcNAcylation) is a dynamic post-translational modification of nuclear, cytoplasmic and mitochondrial proteins. Mounting evidence suggests that abnormal O-GlcNAcylation status is associated with cancer malignancy. In our previous study, it was reported that O-GlcNAc and O-GlcNAc transferase (OGT; an enzyme responsible for the addition of O-GlcNAc) were upregulated in breast cancer tissues and cells. Moreover, O-GlcNAcylation was required for resistance to anoikis and the anchorage-independent growth of breast cancer cells. However, the precise roles of this modification on the development of malignancy are yet to be elucidated. Therefore, in the present study, the effects of inhibiting O-GlcNAc on the malignant transformation of MCF-7 breast cancer cells under different culture conditions were determined, using monolayer (primary growth), anoikis resistance (spheroid growth) and reseeding (secondary growth) to mimic the metastatic process. Decreasing O-GlcNAc levels using small interfering (si)RNA targeting OGT resulted in a reduction in cell viability and invasiveness in anoikis resistant and reseeding conditions. Furthermore, gel-free quantitative proteomics was performed to identify the proteins affected by a reduction of O-GlcNAc. A total of 317 proteins were identified and compared, and the expression of 162 proteins was altered >1.5 fold in the siOGT treated cells compared with the siScamble (siSC) treated cells. Notably, 100 proteins involved in cellular metabolism, cellular localization, stress responses and gene expression were significantly altered in the reseeding condition. Among these differentially expressed proteins, the levels of small nuclear ribonucleoprotein Sm D1 exhibited the largest decrease in expression following knockdown of OGT, and this reduction in expression was associated with a significant decrease in the levels of mTOR expression, a protein which promotes tumor growth and progression. Taken together, the results of the present study demonstrate that decreasing O-GlcNAcylation altered protein expression, and ultimately influenced the metastatic processes, particulary regarding the invasion and reattached growth of MCF-7 breast cancer cells.

Cervical cancer is one of the most common causes of cancer-associated mortality in females worldwide. Serum biomarkers are important tools for diagnosis, disease staging, monitoring treatment and detecting recurrence in different types of cancer. However, only a small number of established biomarkers have been used for clinical diagnosis of cervical cancer. Therefore, the identification of minimally invasive, sensitive and highly specific biomarkers for detection of cervical cancer may improve outcomes. In the present pilot study, changes in disease-relevant proteins in 31 patients with cervical cancer were compared with 16 healthy controls. The Human 14 Multiple Affinity Removal system was used to deplete the 14 most abundant serum proteins to decrease sample complexity and to enrich proteins that exhibited decreased levels of abundance in the serum samples. Immunoaffinity-depleted serum samples were analyzed by in-gel digestion, followed by liquid chromatography mass spectrometry analysis and data processing. Automated quantitative western blot assays and receiver operating characteristic (ROC) curves were used to evaluate the differential protein expression levels between the two groups. Capillary electrophoresis-based western blot analysis was performed to quantitatively determine serum levels of the candidate biomarkers. Significantly increased levels of α-1-antitrypsin (A1AT) and pyrroline-5-carboxylate reductase 2 (PYCR2) were detected, whereas the levels of transthyretin (TTR), apolipoprotein A-I (ApoA-I), vitamin D binding protein (VDBP) and multimerin-1 (MMRN1) were significantly decreased in patients with cervical cancer compared with the healthy controls. ROC curve analysis indicated that the sensitivity and specificity was improved through the combination of the 6 candidate biomarkers. In summary, the results demonstrated that 6 candidate biomarkers (A1AT, PYCR2, TTR, ApoA-I, VDBP and MMRN1) exhibited significantly different expression between serum samples from healthy controls and patients with cervical cancer. These proteins may represent potential biomarkers for distinguishing patients with cervical cancer from healthy controls and for differentiation of patient subgroups.

Due to the invasive procedure associated with Pap smears for diagnosing cervical cancer and the conservative culture of developing countries, identifying less invasive biomarkers is of great interest. Quantitative label-free mass spectrometry was performed to identify potential biomarkers in the urine samples of patients with cervical cancer. This technique was used to study the differential expression of urinary proteomes between normal individuals and cancer patients. The alterations in the levels of urinary proteomes in normal and cancer patients were analyzed by Progenesis label-free software and the results revealed that 60 proteins were upregulated while 73 proteins were downregulated in patients with cervical cancer. This method could enrich high molecular weight proteins from 100 kDa. The protein-protein interactions were obtained by Search Tool for the Retrieval of Interacting Genes/Proteins analysis and predicted the biological pathways involving various functions including cell-cell adhesion, blood coagulation, metabolic processes, stress response and the regulation of morphogenesis. Two notable upregulated urinary proteins were leucine-rich α-2-glycoprotein (LRG1) and isoform-1 of multimerin-1 (MMRN1), while the 3 notable downregulated proteins were S100 calcium-binding protein A8 (S100A8), serpin B3 (SERPINB3) and cluster of differentiation-44 antigen (CD44). The validation of these 5 proteins was performed by western blot analysis and the biomarker sensitivity of these proteins was analyzed individually and in combination with receiver operator characteristic curve (ROC) analysis. Quantitative mass spectrometry analysis may allow for the identification of urinary proteins of high molecular weight. The proteins MMRN1 and LRG1 were presented, for the first time, to be highly expressed urinary proteins in cervical cancer. ROC analysis revealed that LRG1 and SERPINB3 could be individually used, and these 5 proteins could also be combined, to detect the occurrence of cervical cancer.

Background/Aim: Resistance to anoikis is a prerequisite step in metastasis, a major cause of death in patients with cancer, including thyroid cancer. Impairing anoikis resistance is a possible strategy for therapy of metastatic cancer. We, therefore, we aimed to investigate the key players of anoikis resistance. Materials and Methods: Papillary-type (BCPAP), follicular-type (FTC133), and anaplastic-type (ARO) thyroid carcinoma cells, cultured in poly(2-hydroxyethyl methacrylate)-coated plates to mimic circulating cells, were used as model systems in this study. Flow cytometry and softagar assays were used to determine cells exhibiting anoikis resistance. Proteomics was used to identify candidate proteins and validated using western blot and siRNA knockdown. Results: Only ARO cells showed both anoikis resistance potential and anchorage-independent growth ability. Tumor susceptibility gene 101 protein (TSG101) was identified to be potentially important in anoikis resistance, which was confirmed by an increase in anoikis and expression of a proapoptotic protein (BCL-2 like protein 4) and an apoptotic marker (cleaved poly-ADP ribose polymerase) in floating siTSG101-knockdown cells. Conclusion: To our knowledge, this is the first study that implicates the importance of TSG101 in anoikis resistance of thyroid cancer.