Proteomics of Cancer Secretome Yields Biomarkers in Head and Neck Cancer

With an annual incidence of over 640,000 new cases worldwide, head and neck squamous cell cancer (HNSCC) is the sixth most common cancer in terms of sheer numbers. Individuals who smoke or chew tobacco, consume alcohol, carry human papilloma virus (HPV) infections, or are genetically predisposed may be at an elevated risk for HNSCC. It includes cancers of the nasal passages, sinuses, lips, mouth, salivary glands, larynx and pharynx.¹

Proteins secreted by cancer cells or tissues have been sources of potential biomarkers for esophageal, breast and other cancers.^2,3Identification of secreted biomarkers from serum or plasma is difficult, however, due to the complex nature of the molecules. Although cell lines have been helpful, the limited success using label-free quantitative proteomic approaches, combined with gene expression analysis, has been attributed to poor reliability and the need for replicate analysis.⁴

Researchers from the Institute of Bioinformatics in Bangalore (India) have, therefore, used isobaric tags for relative and absolute quantitation (iTRAQ) labelling methodology, combined with high-resolution mass spectrometry (MS), to identify and quantitate secreted proteins from head and neck cancer cell lines.⁵After the labelling of samples with iTRAQ reagents (iTRAQ Reagents Multiplex kit; Applied Biosystems/MDS Sciex, Foster City, CA), the labelled peptides were fractionated using strong cation exchange (SCX) chromatography. A total of 96 fractions were detected at a wavelength of 214 nm using a Variant Wavelength Detector module of the high-performance liquid chromatography (LC) system. The fractions were pooled into 20 fractions based on chromatographic peaks. After additional steps, researchers analyzed the pooled fractions using an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, Bremen, Germany) interfaced with Proxeon Easy nLC system (Thermo Scientific, Bremen, Germany).

Following LC–MS/MS analysis of the HNSCC secretome, the researchers used the Proteome Discoverer (version 1.3.0.339) software (Thermo Scientific, Bremen, Germany) and searched, using the Sequest search algorithm, against human protein database NCBI RefSeq (version 52 containing 33,985 protein sequences and known contaminants). They identified 2,472 proteins from 23 fractions. Among these fractions, 225 were differentially secreted, with 148 proteins occurring in four-fold higher quantities and 77 other proteins in lower amounts in the cancer cell derived secretome.

After a search against the Human Protein Reference Database (www.hprd.org),⁶ the researchers identified 567 proteins containing an N-terminal signal peptide and known to pass through the classical secretory pathway releasing the protein to the extracellular space. They found that 755 other proteins were secreted by non-classical mechanisms involving endosomal recycling, plasma membrane transporter proteins, membrane blebbing and other membrane-related events.

Olfactomedin (OLFM4), also known as GW112, was one of the upregulated proteins occurring at levels exceeding 12-fold in the current study. Previous studies reported the overexpression of OLFM4 in cancers of the digestive tract, gastric, prostate, breast and lung, as well as pancreas. OLFM4 is an extracellular matrix glycoprotein that mediates cell adhesion and that also is a regulator of cell death, playing a key role in tumour cell survival and growth.

The protein insulin-like growth factor-binding protein (IGFBP3), which plays a role in angiogenesis, was present more than seven-fold in the cancer cell secretome. IGFBP2 and IGFBP4, seen in thyroid cancer, were present at 2.7-fold and 2.4-fold higher levels, respectively. The XRCC6 and XRCC5 levels were 4.6-fold and 6.4-fold higher in the current study. The investigators also observed increased levels of caveolin-1 (CAV1), kallikrein-related peptidase 6 (KLK6), and hepatoma-derived growth factor (HDGF) in HNSCC. CAV1 plays a role in the cell cycle progression through Ras-ERK pathway; KLK6 is a trypsin-like serine protease involved in receptor activation and regulation of apoptosis; HDGF is involved in cell proliferation and differentiation. The expression of hepatocyte growth factor activator (HGFAC), which plays a significant role in the invasive growth of tumour cells, was increased up to 4.8-fold in the cancer cell secretome. Other proteins, such as inter-alpha-trypsin inhibitor heavy chain 2 (ITIH2), which was elevated 6.6-fold, may be involved in tumour initiation or progression.

“Quantitation of these proteins in the sera of HNSCC patients and characterization of their expression may serve as the next step toward evaluating the suitability of these proteins as potential HNSCC biomarkers,” the researchers write. The cancer secretome analysis might offer an alternate source of novel biomarkers; advanced MS analyses should validate the in-vitro findings. Additional studies to determine the sensitivity and specificity of biomarkers in a clinical setting may help elucidate their roles in the early detection of cancer.

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