Phosphoproteome Profiling of Isogenic Cancer Cell‐Derived Exosome Reveals HSP90 as a Potential Marker for Human Cholangiocarcinoma

The northeastern region of Thailand is well known to have a high incidence and mortality of cholangiocarcinoma (CCA). Protein phosphorylation status has been reported to reflect a key determinant of cellular physiology, but identification of phosphoproteins can be a problem due to the presence of phosphatase. Exosomes are stable toward circulating proteases and other enzymes in human blood and can be recognized before the onset of cancer progression. Here an in vitro metastatic model of isogenic CCA cells is used to provide insight into the phosphorylation levels of exosomal proteins derived from highly invasive cells. Gel‐based and gel‐free proteomics approaches are used to reveal the proteins differentially phosphorylated in relation to tumor cell phenotypes. Forty‐three phosphoproteins are identified with a significant change in phosphorylation level. Phos‐tag western blotting and immunohistochemistry staining are then employed to validate the candidate phosphoproteins. Heat shock protein 90 is successfully confirmed as being differentially phosphorylated in relation to tumor malignancy. Importantly, the aberrant phosphorylation of exosomal proteins might serve as a promising tool for the development of a biomarker for metastatic CCA.     

Global Gene Expression Profiling Reveals SPINK1 as a Potential Hepatocellular Carcinoma Marker

Background

Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets.

Methodology and Principal Findings

Global gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC.

Conclusion

HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated potential as a diagnostic HCC marker and should be evaluated in future studies.     

Coculture of Endothelial Cells with Human Pluripotent Stem Cell‐Derived Cardiac Progenitors Reveals a Differentiation Stage‐Specific Enhancement of Cardiomyocyte Maturation

Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are immature in their structure and function, limiting their potential in disease modeling, drug screening, and cardiac cellular therapies. Prior studies have demonstrated that coculture of hPSC‐derived CMs with other cardiac cell types, including endothelial cells (ECs), can accelerate CM maturation. To address whether the CM differentiation stage at which ECs are introduced affects CM maturation, the authors coculture hPSC‐derived ECs with hPSC‐derived cardiac progenitor cells (CPCs) and CMs and analyze the molecular and functional attributes of maturation. ECs have a more significant effect on acceleration of maturation when cocultured with CPCs than with CMs. EC coculture with CPCs increases CM size, expression of sarcomere, and ion channel genes and proteins, the presence of intracellular membranous extensions, and chronotropic response compared to monoculture. Maturation is accelerated with an increasing EC:CPC ratio. This study demonstrates that EC incorporation at the CPC stage of CM differentiation expedites CM maturation, leading to cells that may be better suited for in vitro and in vivo applications of hPSC‐derived CMs.     

Plasma Protein Profiling Reveal Osteoprotegerin as a Marker of Prognostic Impact for Colorectal Cancer

BACKGROUND: Due to difficulties in predicting recurrences in colorectal cancer stages II and III, reliable prognostic biomarkers could be a breakthrough for individualized treatment and follow-up. OBJECTIVE: To find potential prognostic protein biomarkers in colorectal cancer, using the proximity extension assays. METHODS: A panel of 92 oncology-related proteins was analyzed with proximity extension assays, in plasma from a cohort of 261 colorectal cancer patients with stage II-IV. The survival analyses were corrected for disease stage and age, and the recurrence analyses were corrected for disease stage. The significance threshold was adjusted for multiple comparisons. RESULTS: The plasma proteins expression levels had a greater prognostic relevance in disease stage III colorectal cancer than in disease stage II, and for overall survival than for time to recurrence. Osteoprotegerin was the only biomarker candidate in the protein panel that had a statistical significant association with overall survival (P = .00029). None of the proteins were statistically significantly associated with time to recurrence. CONCLUSIONS: Of the 92 analyzed plasma proteins, osteoprotegerin showed the strongest prognostic impact in patients with colorectal cancer, and therefore osteoprotegerin is a potential predictive marker, and it also could be a target for treatments.     

Proteomics Reveals Cell‐Surface Urokinase Plasminogen Activator Receptor Expression Impacts Most Hallmarks of Cancer

While metastasis is the primary cause of colorectal cancer (CRC) mortality, the molecular mechanisms underpinning it remains elusive. Metastasis is propagated through driver oncogene/suppressor gene mutations, accompanied by passenger mutations and underlying genomic instability. To understand cancer biology, a unifying framework called the “hallmarks of cancer” (HoCs) has been developed, which organizes cell biological alterations under ten key hallmarks. Underlying these HoCs, genome instability generates mutational diversity that is amplified by inflammation. Recognizing how critical cancer cell‐surface proteins influence, these HoCs have been proposed to accelerate precision medicine therapeutic development. A moderate decrease (43%↓) in HCT116 cell surface urokinase plasminogen activator receptor (uPAR) expression mitigates against many HoCs driven by these cell's KRAS and PIK3CA mutational signature. Comprehensive proteomics (whole cell lysis with two membrane protein enrichments) coupled with ingenuity pathway analysis (IPA) demonstrates that uPAR negates essential pathways across the HoC spectrum, particularly those associated with metastasis, resisting cell death, and sustaining proliferation, and parallels Cancer Hallmarks Analytics Tool analysis. Decreasing uPAR predominantly alters metastasis‐related and uPAR‐interactome protein expression (e.g., EGFR, caveolin, vitronectin, integrin β4). Collectively, it is demonstrated that uPAR is a lynchpin protein capable of regulating several HoC pathways in a classical CRC mutational background.     

Label‐Free Quantitative Phosphoproteomics Reveals Regulation of Vasodilator‐Stimulated Phosphoprotein upon Stathmin‐1 Silencing in a Pair of Isogenic Colorectal Cancer Cell Lines

In this communication, we present the phosphoproteome changes in an isogenic pair of colorectal cancer cell lines, viz., the poorly metastatic HCT‐116 and the highly metastatic derivative E1, upon stathmin‐1 (STMN1) knockdown. The aim was to better understand how the alterations of the phosphoproteins in these cells are involved in cancer metastasis. After the phosphopeptides were enriched using the TiO₂ HAMMOC approach, comparative proteomics analysis was carried out using sequential window acquisition of all theoretical mass spectra‐MS. Following bioinformatics analysis using MarkerView and OneOmics platforms, we identified a list of regulated phosphoproteins that may play a potential role in signaling, maintenance of cytoskeletal structure, and focal adhesion. Among these phosphoproteins, was the actin cytoskeleton regulator protein, vasodilator‐stimulated phosphoprotein (VASP), where its change in phosphorylation status was found to be concomitant with STMN1–associated roles in metastasis. We further showed that silencing of stathmin‐1 altered the expression, subcellular localization and phosphorylation status of VASP, which suggested that it might be associated with remodeling of the cell cytoskeleton in colorectal cancer metastasis.