Prognostic Significance of Head-and-Neck Cancer Biomarkers

Prognostic Significance of Head-and-Neck Cancer Biomarkers Previously Discovered and Identified Using iTRAQ-Labeling and Multidimensional Liquid Chromatography-Tandem Mass Spectrometry Ajay Matta,† Leroi V. DeSouza,‡,§ Nootan Kumar Shukla,| Siddhartha D. Gupta,⊥ Ranju Ralhan,*,†,‡,§,# and K. W. Michael Siu‡,§ Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India, Department of Chemistry, Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3, Department of Surgical Oncology, Dr. BRA Institute Rotary Cancer Hospital, and Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India Received November 21, 2007

Diagnostic oncoproteomics is an emerging field; at present, studies on evaluation of prognostic utility of potential biomarkers identified using proteomic techniques are limited. Analysis with isobaric mass tags (iTRAQ) by multidimensional liquid chromatography-mass spectrometry (LC-MS/MS) to identify proteins that are differentially expressed in human head-and-neck/oral squamous cell carcinomas (HNOSCCs) versus noncancerous head-and-neck tissues has led to the discovery, identification, and verification of consistently increased expression of a panel of proteins, including stratifin (14-3-3σ) and YWHAZ (14-3-3ζ), that may serve as potential cancer biomarkers. Herein, we describe the prognostic utility of these two candidate biomarkers for head-and-neck/oral squamous cell carcinoma (HNOSCC). To determine the clinical significance of stratifin and YWHAZ in head-and-neck tumorigenesis, the expressions of these two proteins were analyzed in HNOSCCs (51 cases) and nonmalignant tissues (39 cases) using immunohistochemistry. Significant increase in stratifin expression was observed in the HNOSCCs as compared to the nonmalignant mucosa [p ) 0.003, Odd’s Ratio (OR) ) 3.8, 95% CI ) 1.6-9.2]. Kaplan-Meier survival analysis reveals correlation of stratifin overexpression with reduced disease-free survival of HNOSCC patients (p ) 0.06). The most intriguing finding is the significant decrease in median disease-free survival (13 months) in HNOSCC patients showing overexpression of both stratifin and YWHAZ proteins, as compared to patients that did not show overexpression of these proteins (median disease-free survival ) 38 months, p ) 0.019), underscoring their utility as adverse prognosticators for HNOSCCs. Co-immunoprecipitation assays show the formation of stratifin-YWHAZ heterodimers in HNOSCC cells and tissue samples, and interactions with NFκB, β-catenin, and Bcl-2 proteins. These results suggest the involvement of these proteins in the development of head-and-neck cancer and their association with adverse disease outcome. Keywords: Tissue proteomics • Stratifin (14-3-3σ) • YWHAZ (14-3-3ζ) • Head-and-neck/oral squamous cell carcinoma (HNOSCC) • Prognostic markers • Kaplan-Meier survival analysis

Introduction Functional proteomics combined with mass spectrometry (MS) offers great promise for unveiling the complex molecular events of tumorigenesis and transforming the management of * To whom correspondence should be addressed. Centre for Research in Mass Spectrometry, Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario, Canada M2J 1P3. Tel, (416)736-2100ext. 40048; fax, (416)736-5936; e-mail, [email protected]. † Department of Biochemistry, All India Institute of Medical Sciences. ‡ Department of Chemistry, York University. § Centre for Research in Mass Spectrometry, York University. | Department of Surgical Oncology, Dr. BRA Institute Rotary Cancer Hospital. ⊥ Department of Pathology, All India Institute of Medical Sciences. # R.R. is a recipient of the U.S. National Cancer Institute (NCI)-Novartis Translational Cancer Research Fellowship Award of the International Union Against Cancer (UICC) at York University; and Professor, Department of Biochemistry, All India Institute of Medical Sciences.

2078 Journal of Proteome Research 2008, 7, 2078–2087 Published on Web 04/12/2008

cancer by identifying new markers for screening, diagnosis, prognosis, and monitoring response to therapy.1–5 These novel biomarkers have the potential to transform clinical practice by including cancer diagnosis and screening based on proteomic analysis as a complement to histopathology. Rapid advances in quantitative proteomic analysis based on isotope-dilution MS with mass-tagging reagents cICAT and iTRAQ plus multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) have revolutionized the field of biomarker discovery and identification.6–8 Biomarker discovery studies are typically conducted on a limited number of samples with verification of the candidate biomarkers taking place subsequently on a larger set of samples. To avoid possible analytical and methodological biases, a technology different from the one used for the discovery is typically employed. Immunohistochemistry is a robust, powerful technique that is frequently 10.1021/pr7007797 CCC: $40.75

 2008 American Chemical Society

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Prognostic Significance of Head-and-Neck Cancer Biomarkers selected for biomarker verification. This technique permits not only identification/verification of the biomarkers, but also quantification and localization of the biomarkers in the various possible cellular and structural compartments of the tissue.9 Studies on evaluation of prognostic utility of potential biomarkers identified using proteomic techniques are rare and are needed to substantiate the clinical utility of proteomics. Herein, we describe the potential for prognostic application of two candidate biomarkers for head-and-neck/oral squamous cell carcinoma (HNOSCC) identified using tissue proteomics. In a recent study, we employed multidimensional LC-MS/ MS following mass-tagging with iTRAQ to identify and quantify differentially expressed proteins in HNOSCCs with respect to noncancerous head-and-neck tissues.10 We identified a panel of three potential cancer biomarkers, two of which were stratifin (14-3-3σ) and YWHAZ (14-3-3ζ), that scored the highest in sensitivity and specificity among all differentially expressed proteins. This mass-tagging approach is powerful and optimal for biomarker discovery, but is nonideal for handling tens or hundreds of samples required for biomarker verification and eventual testing for diagnosis and prognosis of HNOSCC patients. The 14-3-3 proteins are a family of highly conserved eukaryotic proteins involved in a number of cellular processes, including redox regulation, regulation of metabolic pathways, transcription RNA processing, protein synthesis, folding and degradation, mitogenic and cell survival signaling, proliferation, differentiation, senescence cell cycle, apoptosis, epithelialmesenchymal transition, cell adhesion, invasion and metastasis, cytoskeletal organization, and cellular trafficking, by binding to phosphorylated sites in diverse target proteins.11–15 In mammalian cells, seven different isoforms have been identified (ζ, β, γ, , σ, η, and θ), each isoform having distinct tissue localization and function. The 14-3-3 proteins can form homoor heterodimers that allow them to function as an adapter, linker, scaffold, or coordinator in assembling signaling complexes.16–18 The 14-3-3 proteins associate with a number of different signaling proteins, including MEKK1 and PI-3 kinase, apoptosis regulatory proteins ASK-1, tumor suppressor p53, transcription regulatory proteins FKHRL1 and DAF-16, and histone deacetylase.19–26 The 14-3-3 proteins promote cell survival through their interactions with signaling proteins: EGFR, Raf-1, the pro-apoptotic protein BAD (Bcl-2/Bcl-XLantagonist causing cell death), and the cell cycle phosphatase cdc25.27,28 Furthermore, 14-3-3s may have multiple roles in connecting signaling pathways to the regulation of actin-based cellular changes in cytoskeleton and cell motility.29 Recent studies have suggested that 14-3-3 proteins are potential oncogenes.30 In this report, we describe the verification of a candidate biomarker, stratifin, in an independent set of clinical samples of HNOSCCs and noncancerous head-and-neck tissues by immunohistochemistry and investigate its potential as a prognostic marker for HNOSCC. To determine the clinical significance of stratifin in HNOSCCs, the expression of this protein was analyzed in HNOSCCs and nonmalignant (histologically normal) oral mucosa and correlated with clinico-pathological parameters and disease prognosis. YWHAZ (14-3-3ζ) is another candidate biomarker identified in our tissue proteomic analysis of HNOSCCs.10 Interestingly, YWHAZ was also identified earlier in our transcriptomic analysis of HNOSCCs by differential display and we recently showed its overexpression in oral cancer.31,32 The prognostic significance of the two potential

candidate biomarkers, stratifin and YWHAZ, in HNOSCC patients are pursued in this study.

Materials and Methods Tissues. Following institutional human ethics committee approval, 51 anonymized HNOSCCs and 39 nonmalignant head-and-neck tissues dating from 2002 and 2006 were retrieved from the Research Tissue Bank at All India Institute of Medical Sciences, New Delhi, India. The tissue specimens, surgically resected human HNOSCCs and nonmalignant tissues (taken from a distant site), had been collected from patients undergoing curative surgery (with prior written patient consents).32 After excision, tissues were immediately snap-frozen in liquid nitrogen and stored at -80 °C in the Research Tissue Bank. One piece from each patient was collected in 10% formalin and embedded in paraffin for histopathological analysis; the rest was banked. Clinical and pathological data were recorded in a predesigned performa; these included clinical TNM staging (tumor, node, and metastasis classification of malignant tumors of the International Union Against Cancer (UICC)),33 site of the lesion, histopathological differentiation, age, and gender. These data are provided as Supporting Information (Table S1). Follow-up Study. Fifty-one HNOSCC patients who underwent treatment of primary HNOSCC between 2002 and 2006 were investigated and evaluated in the head-and-neck cancer follow-up clinic. Survival status of the patients was verified and regularly updated from the records of the Tumor Registry, Institute Rotary Cancer Hospital, as of May 2007. Patients were monitored for a maximum period of 42 months. As per the hospital protocol, HNOSCC patients with T1 and T2 tumors were treated with radical radiotherapy or surgery alone, whereas a majority of patients with T3 and T4 diseases were treated using a combination of radical surgery followed by postoperative radical radiotherapy. The patients were revisited clinically on a regular basis and the time to recurrence was recorded. If a patient died, the survival time was censored at the time of death; the medical history, clinical examination, and radiological evaluation were used to determine whether the death had resulted from recurrent cancer (relapsing patients) or from any other cause. Disease-free survivors were defined as patients free from clinical and radiological evidence of local, regional, or distant relapse at the time of the last follow-up. Loco-regional relapse/death was observed in 17 of 51 (33%) patients monitored in this study. Thirty-four patients who did not show recurrence were alive until the end of the follow-up period. Only disease-free survival was evaluated in the present study, as the number of deaths due to disease progression did not allow a reliable statistical analysis. Diseasefree survival was expressed as the number of months from the date of surgery to the loco-regional relapse. Immunohistochemistry. Paraffin-embedded sections (5-µm thick) of human oral normal tissues (n ) 39) and HNOSCCs (n ) 51) were collected on gelatin-coated slides. For histopathological analysis, representative sections were stained with hematoxylin and eosin, whereas immunostaining was performed on serial sections as described previously.32 Briefly, the sections were deparaffinized in xylene, hydrated, and pretreated in a microwave oven in citrate buffer [0.01 M (pH ) 6.0)] for antigen retrieval. The sections were incubated with hydrogen peroxide (0.3% v/v) in methanol for 20 min to quench the endogenous peroxidase activity. Nonspecific binding was blocked with 1% bovine serum albumin (BSA) in phosphateJournal of Proteome Research • Vol. 7, No. 5, 2008 2079

research articles buffered saline (PBS, 0.01M, pH ) 7.2) for 1 h. Thereafter, slides were incubated with the primary antibody, (1 µg/mL of anti14-3-3σ, goat polyclonal antibody, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 16 h at 4 °C and washed with PBS. The primary antibody was detected using the strepavidin-biotin complex (Dako LSAB plus kit, Dako, Copenhagen, Denmark) and diaminobenzidine as the chromogen. All incubations were performed at room temperature in a moist chamber. Slides were washed with 3× Tris-buffered saline (TBS, 0.1M, pH ) 7.4) after every step. Finally, the sections were counterstained with Mayer’s hematoxylin and mounted with DPX mountant. In negative controls, the primary antibody was replaced by nonimmune mouse IgG of the same isotype to ensure specificity. Serial sections of these HNOSCCs and normal oral tissues were used for immunohistochemical analysis of YWHAZ in our recent study.32 Positive Criteria for Immunohistochemical Staining. Immunopositive staining was evaluated in five areas of the tissue section. For stratifin expression, sections were scored as positive if epithelial cells showed immunopositivity in the cytoplasm, plasma membrane, and/or nucleus as evaluated by two independent scorers blinded to the clinical outcome (the slides were coded and the scorers did not have prior knowledge of the local tumor burden, lymphonodular spread, and grading of the tissue samples). These sections were rated based on the percentage of cells showing immunopositivity as follows: 0, < 10%; 1, 10-30%; 2, 30-50%; 3, 50-70%; and 4, >70%. Sections were also rated on the basis of stain intensity as follows: 0, none; 1, mild; 2, moderate; 3, intense, as described by Perathoner et al.34 Finally, a total score (ranging from 0 to 7) was obtained by adding the scores of percentage positivity and intensity. The sections were considered positive if the total score was >5.32 Cell Culture. Human oral squamous carcinoma cell line, HSC2, was used in this study.12 Cells were grown in monolayer cultures in Dulbecco’s modified eagle medium (DMEM-F12) supplemented with 10% fetal bovine serum (FBS, SigmaAldrich, MO), 100 µg/mL streptomycin and 100 U/mL penicillin in a humidified incubator (5% CO2, 95% air) at 37 °C as described.32 Co-Immunoprecipitation and Western Blotting. Co-immunoprecipitation (Co-IP) assays were carried out as described earlier.32 Briefly, oral cancer cells, HSC2 and surgically resected HNOSCC tissues were rinsed in ice-cold PBS and lyzed in lysis buffer. Lysates were incubated on ice for 30 min and cell debris was removed by centrifugation. Lysates were precleared by adding 20 µL of Protein A-Sepharose (GE Healthcare Biosciences, Sweden), followed by overnight incubation with polyclonal stratifin, YWHAZ, and NFκB antibodies (1 µg/mL each), or monoclonal β-catenin and Bcl-2 antibodies (2 µg/ mL each) (Santa Cruz Biotechnology, CA) on a rocker at 4 °C. Immunocomplexes were pulled down by incubating with Protein A-Sepharose for 2 h at 4 °C, followed by washing with 4× ice-cold lysis buffer to eliminate nonspecific interactions. In negative controls, the primary antibody was replaced by nonimmune mouse IgG of the same isotype to ensure specificity. Protein A-Sepharose-bound immunocomplexes were then resuspended in Laemelli sample buffer (10 mM Tris, 10% v/v glycerol, 2% w/v SDS, 5 mM EDTA, 0.02% bromophenol blue, and 6% β-mercaptoethanol, pH ) 7.4), boiled for 5 min, and analyzed by Western blotting using specific antibodies.32 The proteins were electro-transferred onto polyvinylidenedifluoride (PVDF) membrane. After blocking with 5% nonfat powdered 2080

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Matta et al. milk in TBS (0.1 M, pH ) 7.4), blots were incubated with specific antibodies for stratifin, YWHAZ, NFκB, β-catenin and Bcl-2 at 4 °C overnight. Membranes were incubated with secondary antibody, HRP-conjugated goat/rabbit/mouse antiIgG (Dako CYTOMATION, Denmark), diluted at an appropriate dilution in 1% BSA, for 2 h at room temperature. After each step, blots were washed with 3×Tween (0.2%)-TBS (TTBS). Protein bands were detected by the enhanced chemiluminescence method (Santa Cruz Biotechnology, CA) on XO-MAT film. Statistical Analysis. The immunohistochemical data were subjected to statistical analysis using SPSS 10.0 software. The relationship between the protein expression and clinicopathological parameters were tested by Chi-Square and Fischer’s exact test. Two sided p-values were calculated and p e 0.05 was considered to be significant. Box plots were prepared to determine the distribution of total score of stratifin expression in HNOSCCs and nonmalignant tissues. The correlation of stratifin and/or YWHAZ staining with patient survival was evaluated using life tables constructed from survival data with Kaplan-Meier plots.

Results The amino acid sequences of stratifin and YWHAZ with peptides identified by MS and MS/MS are given in Figure 1, panels a and b, respectively. Immunohistochemical Analysis of Stratifin in HNOSCCs and Nonmalignant Tissues. Results of the immunohistochemical analysis of stratifin expression in HNOSCCs and nonmalignant mucosa, and the relationship with clinicopathological parameters, are summarized in Table 1. The detailed data are provided as Supporting Information (Table S1). Chi-Square analysis shows significant increase in stratifin expression in HNOSCCs as compared to nonmalignant mucosa (p ) 0.003, Odd’s Ratio (OR) ) 3.8, 95% CI ) 1.6-9.2). In histologically normal oral tissues, 31% of the cases show weak immunostaining of stratifin (Figure 2a). Increased stratifin expression was observed in 63% of HNOSCCs. Intense nuclear/membranous staining, in addition to cytoplasmic staining, was observed in the epithelial cells of HNOSCCs (Figure 2b). No immunostaining was observed in tissue sections used as negative controls where the primary antibody was replaced by isotype specific IgG (Figure 2c). No significant correlation was observed between stratifin overexpression and clinicopathological parameters including age, gender, histological differentiation, tumor stage, and nodal status of HNOSCCs (Table 1). However, it should be noted that the numbers of poorly differentiated tumors and early stage tumors (T1) analyzed in this study are too low for any definitive conclusions. Increased expressions of stratifin were observed in HNOSCCs with a median score of 6 (range 4-7), as compared to the nonmalignant (histologically normal) oral tissues median score of 5 (range 3-6) shown in the box-plot analysis in Figure 3. Co-Immunoprecipitation. To determine the functional significance of stratifin in head-and-neck carcinogenesis, we identified its binding partners in oral cancer cells, HSC2, and in clinical specimens of HNOSCCs, using co-IP assays followed by Western blotting. IP of stratifin reveals its binding to YWHAZ, NFκB, β-catenin, and Bcl-2 proteins in oral cancer cells HSC2 as well as in HNOSCC tissue samples as shown in Figure 4a. Reverse IP assay using specific antibodies for these proteins followed by Western blotting with YWHAZ (Figure 4b), NFκB (Figure 4c), β-catenin (Figure 4d) and Bcl-2 (Figure 4e) confirmed their binding to stratifin. No band was observed in the

Prognostic Significance of Head-and-Neck Cancer Biomarkers

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Figure 1. Identification of stratifin and YWHAZ in HNOSCCs by mass spectrometry. The peptides whose MS/MS spectra are shown are colored red and in a larger font. Those that are common between stratifin and YWHAZ are shown in purple. Other peptides observed are in blue. The matched b ions are shown in green, and the matched y ions in red.

immunoblot analysis of the negative controls (Figure 4a). It is noteworthy that all these proteinssstratifin, YWHAZ, NFκB, β-catenin and Bcl-2shave 14-3-3-binding motif, Mode 1, as reported earlier by us.32 Association of Stratifin and YWHAZ Expression with Disease Outcome. Kaplan-Meier Survival analysis reveals reduced disease-free survival for HNOSCC patients overexpressing stratifin (p ) 0.06, Figure 5a). The median diseasefree survival was 19 months in HNOSCC patients showing

stratifin overexpression as compared to 38 months in HNOSCC patients who did not. Patients with YWHAZ-positive tumors had a shorter disease-free survival (median ) 23 months) than those with YWHAZ-negative tumors (median ) 35 months; p ) 0.08, Figure 5b). Most remarkably, HNOSCC patients showing overexpressions of both stratifin and YWHAZ have a significantly decreased median disease-free survival of 13 months in HNOSCCs (p ) 0.019, Figure 5c), as compared to patients showing no overexpression of these two proteins (median ) Journal of Proteome Research • Vol. 7, No. 5, 2008 2081

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Table 1. Analysis of Stratifin and YWHAZ in HNOSCCs: Correlation with Clinicopathological Parameters stratifin+

total cases

stratifin+-YWHAZ+

stratifin+/YWHAZ+

clinicopathological features

N

n

(%)

n

(%)

n

(%)

Nonmalignant tissue HNOSCCa Differentiationb WDSCC MDSCC PDSCC Tumor Stage T1 T2 T3 T4 Nodal Status NN+

39 51

12 32

(31) (63)

8 28

(20) (55)

25 43

(64) (84)

29 19 3

18 12 2

(62) (63) (67)

16 10 2

(55) (53) (67)

26 15 2

(90) (79) (67)

6 15 13 17

5 6 9 12

(83) (40) (69) (71)

5 4 8 11

(83) (27) (61) (65)

6 11 12 14

(100) (73) (92) (82)

28 23

17 15

(61) (65)

15 13

(54) (56)

23 20

(82) (87)

a For HNOSCCs vs nonmalignant tissues, (i) stratifin+ (p ) 0.003, OR ) 3.8, 95% CI ) 1.6-9.2) ; (ii) YWHAZ+ (p ) 0.024, OR ) 2.8, 95% CI ) 1.2-6.8); (ii) stratifin+_-YWHAZ+ (p ) 0.001, OR ) 4.7, 95% CI ) 1.8-12.2); (iv) SFN+/YWHAZ+ (p ) 0.027, OR ) 3.1, 95% CI ) 1.1-8.2). b WDSCC, well differentiated squamous cell carcinoma; MDSCC, moderately differentiated squamous cell carcinoma; PDSCC, poorly differentiated squamous cell carcinoma.

Figure 2. Immunohistochemical analysis of stratifin in head-and-neck cancer tissues. Paraffin-embedded HNOSCC tissue sections and nonmalignant mucosa were stained using anti-stratifin antibody as described in Materials and Methods. (a)Normal oral mucosa with no detectable stratifin immunostaining; (b) a representative HNOSCC section showing strong cytoplasmic and nuclear stratifin immunostaining in the tumor cells; (c) a HNOSCC section stained with isotype specific IgG serving as a negative control that shows no detectable stratifin immunostaining in the tumor cells (a-c, original magnification ×100).

38 months), underscoring the utility of these proteins as adverse prognosticators for HNOSCCs.

Discussion To our knowledge, this investigation is one of the very few studies that demonstrate the prognostic utility of candidate biomarkers identified using MS-based proteomics. Recently, a comparison of protein profiles in tumor-distant head-and-neck tissues with clinical outcomes was reported to reveal a significant association between aberrant profiles and tumor-relapse events, suggesting that proteomic profiling in conjunction with protein identification may have significant predictive power for clinical outcome.35 The concept of using a panel of biomarkers for the purpose of improved diagnostics has taken hold in recent years. For example, DeSouza et al.7 and Dube´ et al.8 demonstrated that the use of a panel of three biomarkerss chaperonin 10, pyruate kinase M2, and R-1-antitrypsins markedly increases the sensitivity and specificity for differentiating between endometrial carcinoma and nonmalignant endometrial tissues. For head-and-neck cancers, we have recently determined that a panel of biomarkerssstratifin, YWHAZ, and S100 A7sperforms better than any of the individual biomarkers for the detection of HNOSCC.10 In this 2082


current study, we find that a panel of two biomarkerssstratifin and YWHAZsshows promise as prognostic markers for HNOSCCs. The enhanced performance of the combination of stratifin and YWHAZ versus either protein individually, in prognosticating the clinical outcome of HNOSCCs, can be understood on the basis of their biological functions detailed below. Results of our studies using oral cancer cells and tissue samples of HNOSCCs demonstrate the formation of stratifin-YWHAZ heterodimers and binding of stratifin to NFκB, β-catenin, and Bcl-2, thereby implicating stratifin’s involvement in many cellular processes associated with tumorigenesis. Similar to our findings, Bhawal et al.36 have very recently reported increased expression of the stratifin transcript and protein in OSCCs. These results are in accordance with the findings of Chen et al.37 and Lo et al.38,39 who reported overexpression of stratifin in OSCCs. In addition, Lo et al.39 reported HPV18 positivity in 11 of 82 (13%) OSCCs and demonstrated 10-fold increase in stratifin expression in HPV18positive OSCCs, in comparison with HPV18-negative OSCCs.39 In comparison, several studies from India report HPV16/18 positivity in 14-74% OSCCs.40–47 This raises the possibility that there may be an association between HPV and overexpression of stratifin, It is of note that, although all these studies are on


Figure 3. Box-Plot analysis: Box plots showing distribution of total immunostaining scores of stratifin determined by immunohistochemistry in paraffin-embedded sections of HNOSCCs and nonmalignant head-and-neck tissues. The vertical axis gives the total immunostaining score, obtained as described in Materials and Methods. Box plots showed increased expressions of stratifin in HNOSCCs with a median score (bold horizontal line) of 6 (range 4-7, as shown by vertical bars), as compared to nonmalignant (histologically normal) oral tissues with a median score of 5 (range 3-6).

Asian populations, recent reports show similar HPV prevalence in the range 19-74% in Mexican, Canadian, and North American populations as well.48–52 While the occurrence of viral infection, in particular HPV16 and 18, in HNOSCCs has not been determined in our study, a recent report from the Delhi population (with similar risk factors) showed the presence of HPV16 in 18/66 (27%) OSCCs.40 Overexpression of stratifin was observed in 32/51 (63%) HNOSCCs, and concomitant expression of stratifin and YWHAZ was observed in 28/51 (55%) tumors in our study. Furthermore, a recent prospective clinical trial reported improved survival of patients with HPV-positive HNOSCCs relative to HPV-negative patients,52 while our study showed reduced disease-free survival of patients that showed overexpression of stratifin and YWHAZ. Thus, there appears to be little, if any, evidence to associate HPV infection with stratifin and YWHAZ expressions in HNOSCCs. Our results and others who examined Asian populations reported overexpression of stratifin in HNSCCs.36–39 By contrast, one study on European population reported decreased expression of stratifin in HNSCCs.35 Thus, there appears to be different mechanisms at work in HNOSCC tumorigenesis that may or may not be associated with overexpression of stratifin. Whether these different mechanisms can be attributed to the presence or absence of concomitant diseases and/or differences in risk factors, such as smoking and drinking in the European and Western populations, and chewing of betel quid and/or tobacco in the Asian population remains to be determined. Overexpression of stratifin has also been observed in other human cancers. Perathoner et al.34 suggested that stratifin overexpression promotes tumor proliferation and/or prevents apoptotic signal transduction in colorectal carcinoma. Samuel et al.53 demonstrated the role of stratifin in prevention of apoptosis by influencing the subcellular distribution of the proapoptotic protein, Bax, in colorectal cancer cells. Deletion of

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stratifin has been correlated with increased sensitivity of colorectal cells to doxorubicin. Similarly, Guweidhi et al.54 proposed an antiapototic role for stratifin in pancreatic cancer cells by inhibiting bad-mediated apoptosis. Liu et al.55 showed that elevated stratifin expression contributes considerably to the observed drug resistance in MCF7/AdVp3000 cells. Stratifin has been shown to be a pivotal MDM2 regulator, involved in blocking a variety of MDM2 activities, including MDM2mediated cytoplasmic localization of p53. Stratifin overexpression leads to destabilization of MDM2 by enhancing its selfubiquitination and, thereby stabilizing cellular p53.56 In our previous studies, we reported that p53 mutations are infrequent in OSCCs in the Indian population; a currently unknown mechanism must be involved in stabilizing p53 in these OSCC patients.57 Our group has also reported overexpressions of MDM2 and cyclinD1 in OSCCs.58,59 Investigation of the relationship between stratifin overexpression, p53 stabilization, and MDM2 and cyclinD1 expressions in HNOSCCs is currently underway. In a recent study aimed at delineation of early changes in expression of proteins, we demonstrated increased expressions of NFκB and COX-2 in early premalignant stages of development of oral cancer and sustained elevation along the tumorigenic pathway.60 Furthermore, we showed increased expression of YWHAZ in different stages of the development of OSCC and YWHAZ’s involvement in cell-signaling pathways involved in inflammation, cell proliferation, and abrogation of apoptosis during oral carcinogenesis.32 Herein, we extend these findings by demonstrating the binding of stratifin with YWHAZ, thus, suggesting the formation of stratifin–YWHAZ heterodimers and binding to NFκB in oral cancer. Our findings are supported by the study of Aguilera et al.61 which showed the requirement of 14-3-3 proteins for efficient export of the p65 subunit of NFκB. Taken together with our earlier findings of YWHAZ, we hypothesize that 14-3-3 proteins may be an important link between chronic inflammation and cancer that warrants further investigation. The Co-IP results show stratifin binding also with β-catenin and Bcl-2 proteins. Earlier, we showed that these proteins interact with YWHAZ, thereby supporting our hypothesis that these complexes may be responsible for altered functions of stratifin. Fang et al.62 recently showed that AKT, which is activated downstream from EGFR signaling, phosphorylates β-catenin at Ser-552 in vitro and in vivo, causing its dissociation from cell-cell contacts and accumulation in both the cytosol and nucleus, and enhancing its interaction with YWHAZ via a binding motif containing Ser-552. This phosphorylation of β-catenin by AKT increases β-catenin’s transcriptional activity and promotes tumor cell invasion, indicating that AKT-dependent regulation of β-catenin plays a critical role in tumor invasion and development. The oncogenic role of YWHAZ has been proposed in a recent study using siRNA for knocking down its expression in cancer cells.63 Downregulation of YWHAZ sensitizes cells to stress-induced apoptosis and JNK/ p38 signaling; in addition, it enforces cell-cell contacts and expression of adhesion proteins. YWHAZ’s oncogenic properties is also supported by a Web-based meta-analysis (Onco-mine) that reveals its overexpression in various types of carcinomas.15,64 To unravel the functional significance of 14-3-3 proteins in tumor development, it will be imperative to delineate the precise contribution of each 14-3-3 isoform in this process. We are currently investigating the functional significance of the interactions between stratifin and YWHAZ, and their respective Journal of Proteome Research • Vol. 7, No. 5, 2008 2083

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Figure 4. Co-immunoprecipitation assay and Western blot analysis. Immunoprecipitation assays of stratifin, YWHAZ, NFκB, Bcl-2 and β-catenin proteins were carried out using specific antibodies in head-and-neck cancer cells, HSC2 and two HNOSCCs tissues (HNOSCC1 and HNOSCC2): (a) immunoblot analysis for stratifin, demonstrating the binding of stratifin with YWHAZ, NFκB, Bcl-2, and β-catenin, and the lack of binding in the negative control. Similarly, reverse immunoprecipitation assays were carried out using specific antibodies for YWHAZ, NFκB, Bcl-2, and β-catenin: The panels show the Co-IPs with different proteins obtained with specific antibodies mentioned at the top of each panel immunoblotted and probed with antibodies for (b) YWHAZ, (c) NFκB, (d) β-catenin, and (e) Bcl-2 confirming the binding of these proteins with stratifin in oral cancer cells, HSC2, and HNOSCC tissues (HNOSCC1 and HNOSCC2).

roles in the development and progression of HNOSCC. All indications are that targeting specific 14-3-3 isoforms may serve as a plausible strategy for cancer therapy. In conclusion, stratifin is overexpressed in HNOSCCs relative to normal tissues. Increased concomitant expression 2084


of stratifin and YWHAZ serves as adverse prognosticator in HNOSCCs and underscores the importance of these proteins in head-and-neck tumorigenesis. Increased expression of stratifin forming stratifin-YWHAZ heterodimers and binding to NFκB, β-catenin, and Bcl-2 proteins suggest the implica-


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Figure 5. Kaplan-Meier estimation of cumulative proportion of disease-free survival: (a) stratifin protein expression; the median time for disease-free survival (no recurrence/metastasis) in patients with stratifin-positive tumors was 19 months, whereas in those with stratifin-negative tumors, it was 38 months (p ) 0.06). (b) YWHAZ protein expression; the median time for disease-free survival (no recurrence/metastasis) in patients with YWHAZ-positive tumors was 23 months, whereas in those with YWHAZ-negative tumors, it was 35 months (p ) 0.08). (c) concomitant stratifin and YWHAZ expressions; the median time for disease-free survival of patients with HNOSCCs showing concomitant expressions of stratifin and YWHAZ (Stratifin+/YWHAZ+) was 13 months, as compared to patients with tumors that did not show increased expression of either of these proteins with the median time for disease-free survival being 38 months (p ) 0.019).

tion of these complexes in diverse cellular processes in headand-neck carcinogenesis. We speculate that targeting the stratifin-YWHAZ heterodimer, using a small-molecule modulator/peptide inhibitor that intervenes with 14-3-3 client protein interactions, may serve as a plausible therapeutic strategy for head-and-neck cancer.

Acknowledgment. This work was supported by a grant from the Department of Biotechnology, New Delhi, India. R.R. gratefully acknowledges support from the International Union Against Cancer (UICC), in the form of an NCI-Novartis Translational Cancer Research Fellowship, and from the Ontario Institute for Cancer Research (OICR) that enable her to conduct the proteomics study in K.W.M.S.’s laboratory. A.M. thanks the Council of Scientific and Industrial Research, Government of India, for award of a Senior Research Fellowship. The authors gratefully acknowledge the support and cooperation of their pathology collaborator, Dr. Terence J. Colgan, Pathology and Laboratory Medicine, Mount Sinai Hospital, and the technical assistance of Muntajib Alhaq and Maria Mendes (Mount Sinai Hospital). We thank Applied

Biosystems for reagent support and collaboration. K.W.M.S. acknowledges infrastructural support from the Ontario Research and Development Challenge Fund and Applied Biosystems.

Supporting Information Available: Table summarizing the results of the immunohistochemical analysis of stratifin expression in HNOSCCs and nonmalignant mucosa, and the relationship with clinicopathological parameters. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Cho, W. C. Contribution of oncoproteomics to cancer biomarker discovery. Mol. Cancer 2007, 6, 25. (2) Tanke, H. J. Genomics and proteomics: the potential role of oral diagnostics. Ann. N.Y. Acad. Sci. 2007, 1098, 330–4. (3) Najam-ul-Haq, M.; Rainer, M.; Trojer, L.; Feuerstein, I.; Vallant, R. M.; Huck, C. W.; Bakry, R.; Bonn, G. K. Alternative profiling platform based on MELDI and its applicability in clinical proteomics. Expert Rev. Proteomics 2007, 4, 447–52. (4) VanMeter, A.; Signore, M.; Pierobon, M.; Espina, V.; Liotta, L. A.; Petricoin, E. F. Reverse-phase protein microarrays: application to

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Prognostic Significance of Head-and-Neck Cancer Biomarkers

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