Proteome-wide analysis of head and neck squamous cell carcinomas using laser-capture microdissection and tandem mass spectrometry

ArticleinOral Oncology 41(2):183-99 · March 2005with7 Reads
DOI: 10.1016/j.oraloncology.2004.08.009 · Source: PubMed
Remarkable progress has been made to identify genes expressed in squamous cell carcinomas of the head and neck (HNSCC). However, limited information is available on their corresponding protein products, whose expression, post-translational modifications, and activity are ultimately responsible for the malignant behavior of this tumor type. We have combined laser-capture microdissection (LCM) with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify proteins expressed in histologically normal squamous epithelium and matching SCC. The protein fraction from approximately 10,000-15,000 normal and tumor cells was solubilized, digested with trypsin, and the resulting peptides were analyzed by LC-MS/MS. Database searching of the resulting sequence information identified 30-55 proteins per sample. Keratins were the most abundant proteins in both normal and tumor tissues. Among the proteins differentially expressed, keratin 13 was much lower in tumors, whereas heat-shock (Hsp) family members were highly expressed in neoplastic cells. Wnt-6 and Wnt-14 were identified in both normal and tumor tissues, respectively, and placental growth factor (PIGF) was detected only in tumors. Immunohistochemical analysis of HNSCC tissues revealed lack of keratin 13 in tumor tissues, and strong staining in normal epithelia, and high expression of Hsp90 in tumors. Our study, by combining LCM and proteomic technologies, underscores the advantages of this approach to investigate complex changes at the protein level in HNSCC, thus complementing existing and emerging genomic technologies. These efforts may likely result in the identification of new biomarkers for HNSCC that can be used to diagnose disease, predict susceptibility, and monitor progression in individual patients.
    • "Activation of the beta-catenin pathway is a frequent event in different human cancers including colon, kidney, prostate and thyroid. Although little is known about the role of this signaling pathway in HNSCC, several components of the wnt-pathway such as wnt receptors, frizzleds and their downstream targets, dishevelled and wnt14 are highly expressed in HNSCC tumors (Baker et al., 2005; Leethanakul et al., 2000). Accordingly, the wnt-signaling pathway may play a role in HNSCC, however the precise contribution of this change to HNSCC progression and pathogenesis remains to be defined. "
    [Show abstract] [Hide abstract] ABSTRACT: Despite significant improvements in therapeutic protocols, head and neck squamous cell carcinoma (HNSCC) remains a major health problem worldwide. The 5-year post-therapeutic survival rate is among the lowest of the major cancers with loco-regional relapse being the main cause of death. Moreover, in most instances, the quality of life of the afflicted patient is severely compromised. The poor prognosis for HNSCC is primarily due to disease detection at advanced stages. Accordingly, development of early detection and preventive strategies are essential. Recent advances in our understanding of the molecular biology and etiology of HNSCC should facilitate development of improved intervention and therapeutic approaches. The present review discusses the potential role of such factors for developing preventive and early diagnostic strategies for HNSCC management.
    Full-text · Article · Feb 2012
    • "The network with the highest significance also contained several cancer-related genes with high expression levels, including DSG2, PRKCA, PLA2G10, and Grp94. Considerable evidence has shown a significant association between the heat-shock protein 90 (Hsp90) and a wide range of human malignancies, including head and neck cancers (Ito et al, 1998; Baker et al, 2005; Yin et al, 2005). Grp94, also known as gp96, is the ER-resident member of the Hsp90 family constitutively expressed in virtually all cell types (Van et al, 1989) and the most abundant ER chaperon protein showing high homology (50%) to cytosolic counterpart Hsp90 (Sorger and Pelham, 1987). "
    [Show abstract] [Hide abstract] ABSTRACT: To characterise Ca(2+) -binding protein gene expression changes in oral squamous cell carcinomas (OSCCs), we compared the gene expression profiles in OSCC-derived cell lines with normal oral tissues. One hundred Ca(2+) -binding protein genes differentially expressed in OSCCs were identified, and genetic pathways associated with expression changes were generated. Among genes mapped to the network with the highest significance, glucose-regulated protein 94 kDa (Grp94) was evaluated further for mRNA and protein expression in the OSCC cell lines, primary OSCCs, and oral premalignant lesions (OPLs). A significant (P<0.001) overexpression of Grp94 protein was observed in all cell lines compared to normal oral epithelium. Immunohistochemical analysis showed highly expressed Grp94 in primary OSCCs and OPLs, whereas most of the corresponding normal tissues had no protein immunoreaction. Real-time quantitative reverse transcriptase-PCR data agreed with the protein expression status. Moreover, overexpression of Grp94 in primary tumours was significantly (P<0.001) correlated with poor disease-free survival. The results suggested that Grp94 may have potential clinical application as a novel diagnosis and prognostic biomarker for human OSCCs.
    Article · Sep 2007
    • "Several tissue microdissection technologies, including laser capture microdissection (Emmert-Buck et al. 1996; Bonner et al. 1997), laser catapulting (Schutze et al. 1998), laser microdissection (DeSouza et al. 2005), and laser-free microdissection (Furuta et al. 2004; Zhuang et al. 2004), have been developed to provide a rapid, straightforward method for procuring homogeneous subpopulations of cells or structures for biochemical and molecular biological analyses. However, the smaller quantities of samples available for microdissected populations have, to this point, restricted protein analyses to the use of only a single chromatography separation prior to tandem mass spectrometry (MS/ MS) analysis and have limited the ability to mine deeper into the tissue proteome in recent studies (Wu et al. 2003; Zang et al. 2004; Baker et al. 2005; Crockett et al. 2005; Hood et al. 2005; Palmer-Toy et al. 2005). Because the sizes of human tissue biopsies are becoming significantly smaller due to the advent of minimally invasive methods and early detection and treatment of lesions, a more effective discovery-based proteome technology is critically needed to enable sensitive studies of protein profiles that will have diagnostic and therapeutic relevance. "
    [Show abstract] [Hide abstract] ABSTRACT: Targeted proteomics research, based on the enrichment of disease-relevant proteins from isolated cell populations selected from high-quality tissue specimens, offers great potential for the identification of diagnostic, prognostic, and predictive biological markers for use in the clinical setting and during preclinical testing and clinical trials, as well as for the discovery and validation of new protein drug targets. Formalin-fixed and paraffin-embedded (FFPE) tissue collections, with attached clinical and outcome information, are invaluable resources for conducting retrospective protein biomarker investigations and performing translational studies of cancer and other diseases. Combined capillary isoelectric focusing/nano-reversed-phase liquid chromatography separations equipped with nano-electrospray ionization-tandem mass spectrometry are employed for the studies of proteins extracted from microdissected FFPE glioblastoma tissues using a heat-induced antigen retrieval (AR) technique. A total of 14,478 distinct peptides are identified, leading to the identification of 2733 non-redundant SwissProt protein entries. Eighty-three percent of identified FFPE tissue proteins overlap with those obtained from the pellet fraction of fresh-frozen tissue of the same patient. This large degree of protein overlapping is attributed to the application of detergent-based protein extraction in both the cell pellet preparation protocol and the AR technique.
    Article · Aug 2007
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