Discovery and verification of head-and-neck cancer biomarkers by differential protein expression analysis using iTRAQ labeling, multidimensional liquid chromatography, and tandem mass spectrometry

Abstract

Multidimensional LC-MS/MS has been used for the analysis of biological samples labeled with isobaric mass tags for relative and absolute quantitation (iTRAQ) to identify proteins that are differentially expressed in human head-and-neck squamous cell carcinomas (HNSCCs) in relation to non-cancerous head-and-neck tissues (controls) for cancer biomarker discovery. Fifteen individual samples (cancer and non-cancerous tissues) were compared against a pooled non-cancerous control (prepared by pooling equal amounts of proteins from six non-cancerous tissues) in five sets by on-line and off-line separation. We identified 811 non-redundant proteins in HNSCCs, including structural proteins, signaling components, enzymes, receptors, transcription factors, and chaperones. A panel of proteins showing consistent differential expression in HNSCC relative to the non-cancerous controls was discovered. Some of the proteins include stratifin (14-3-3sigma); YWHAZ (14-3-3zeta); three calcium-binding proteins of the S100 family, S100-A2, S100-A7 (psoriasin), and S100-A11 (calgizarrin); prothymosin alpha (PTHA); L-lactate dehydrogenase A chain; glutathione S-transferase Pi; APC-binding protein EB1; and fascin. Peroxiredoxin2, carbonic anhydrase I, flavin reductase, histone H3, and polybromo-1D (BAF180) were underexpressed in HNSCCs. A panel of the three best performing biomarkers, YWHAZ, stratifin, and S100-A7, achieved a sensitivity of 0.92 and a specificity of 0.91 in discriminating cancerous from non-cancerous head-and-neck tissues. Verification of differential expression of YWHAZ, stratifin, and S100-A7 proteins in clinical samples of HNSCCs and paired and non-paired non-cancerous tissues by immunohistochemistry, immunoblotting, and RT-PCR confirmed their overexpression in head-and-neck cancer. Verification of YWHAZ, stratifin, and S100-A7 in an independent set of HNSCCs achieved a sensitivity of 0.92 and a specificity of 0.87 in discriminating cancerous from non-cancerous head-and-neck tissues, thereby confirming their overexpressions and utility as credible cancer biomarkers.

Figures

Fig. 1.

Flow diagram for on-line 2D LC-MS/MS analysis. In position 1, ports 1–2, 3–4, 5–6, 7–8, and 9–10 are connected; in position 2, ports 2–3, 4–5, 6–7, 8–9, and 10–1 are connected. In the diagram, the valves (A and B) are shown at the initial (time = 0 min) positions.

Fig. 2.

Receiver operating characteristic curves of a panel of three best performing biomarkers, YWHAZ, stratifin, and S100-A7: iTRAQ ratios (a) and IHC scores (b). See the text for details.

Fig. 3.

Immunohistochemical verification of iTRAQ-discovered potential cancer markers YWHAZ, stratifin, and S100-A7 in HNSCCs and non-cancerous head-and-neck tissues. Positive staining is brown and is intense in HNSCCs. The left panel shows the non-cancerous (histologically normal) tissues, and the right panel depicts the HNSCC tissue sections. A, the HNSCC sample shows intense cytoplasmic and nuclear staining for YWHAZ, whereas the normal mucosa shows no detectable immunostaining. B, the HNSCC tissue section shows cytoplasmic staining for stratifin in tumor cells, whereas the normal mucosa shows no detectable immunostaining. C, the HNSCC tissue section shows intense cytoplasmic staining for S100-A7 in tumor cells, whereas the normal mucosa shows no detectable immunoreactivity. All panels show ×200 magnifications.

Fig. 4.

Western blot analyses of YWHAZ, stratifin, and S100-A7 in HNSCCs and paired non-cancerous head-and-neck tissues. Equal amounts of protein lysates from HNSCCs and paired non-cancerous head-and-neck tissues were used. See the text for details. The panels show increased expression of YWHAZ (i), stratifin (ii), and S100-A7 (iii) in HNSCCs (C1–C3) as compared with paired non-cancerous head-and-neck tissues (N1–N3). α-Tubulin (iv) was used as the loading control.

Fig. 5.

RT-PCR analyses of YWHAZ, stratifin, and S100-A7 in HNSCCs and non-cancerous head-and-neck tissues. i shows increased levels of YWHAZ transcripts in HNSCCs (C1–C3) as compared with the non-cancerous head-and-neck tissues that show basal levels (N2 and N3) and no detectable level (N1) of YWHAZ transcripts. ii shows increased levels of stratifin transcripts in HNSCCs (C1–C3) as compared with the non-cancerous head-and-neck tissues that show a basal level (N3) and no detectable level (N1 and N2) of stratifin transcripts. iii shows increased levels of S100-A7 transcripts in HNSCCs (C1–C3) as compared with the non-cancerous head-and-neck tissues that show a basal level (N3) and no detectable level (N1 and N2) of S100-A7 transcripts. β-Actin (iv) was used as a control for normalizing the quantity of RNA used.

Fig. 6.

Specificity of the YWHAZ, stratifin, and S100-A7 panel against ESCC, invasive ductal carcinoma of the breast, and ovarian cancer: immunohistochemical analyses (top) and Western blot analyses (bottom). α-Tubulin (iv) was used as the loading control. The three-biomarker panel (i–iii) shows good specificity against breast and ovarian cancers although weaker discriminatory power against ESCC. See the text for details.