Cytoplasmic accumulation of activated leukocyte cell adhesion molecule is a predictor of disease progression and reduced survival in oral cancer patients

Authors

  • Meenakshi Sawhney,

    1. Department of Biochemistry, Institute of Rotary Cancer Hospital, All India Institute for Medical Sciences, Ansari Nagar, New Delhi, India
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    • The first two authors contributed equally to this work.

  • Ajay Matta,

    1. Department of Biochemistry, Institute of Rotary Cancer Hospital, All India Institute for Medical Sciences, Ansari Nagar, New Delhi, India
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    • The first two authors contributed equally to this work.

  • Muzafar A. Macha,

    1. Department of Biochemistry, Institute of Rotary Cancer Hospital, All India Institute for Medical Sciences, Ansari Nagar, New Delhi, India
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  • Jatinder Kaur,

    1. Department of Biochemistry, Institute of Rotary Cancer Hospital, All India Institute for Medical Sciences, Ansari Nagar, New Delhi, India
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  • Siddhartha DattaGupta,

    1. Department of Pathology, Institute of Rotary Cancer Hospital, All India Institute for Medical Sciences, Ansari Nagar, New Delhi, India
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  • Nootan K. Shukla,

    1. Department of Surgical Oncology, Institute of Rotary Cancer Hospital, All India Institute for Medical Sciences, Ansari Nagar, New Delhi, India
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  • Ranju Ralhan

    Corresponding author
    1. Department of Biochemistry, Institute of Rotary Cancer Hospital, All India Institute for Medical Sciences, Ansari Nagar, New Delhi, India
    • Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India
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    • Fax: 91-11- 26588663.


Abstract

Activated leukocyte cell adhesion molecule (ALCAM) has been proposed to function as a cell surface sensor for cell density, controlling the transition between local cell proliferation and tissue invasion in cancer progression. Herein, we determined ALCAM expression in 107 oral squamous cell carcinomas (OSCCs), 78 oral lesions (58 hyperplasias and 20 dysplasias) and 30 histologically normal oral tissues using immunohistochemistry and correlated with clinicopathological parameters. Significant increase in ALCAM immunopositivity was observed from normal oral mucosa, hyperplasia, dysplasia to OSCCs (ptrend < 0.001). Increased ALCAM expression was observed in cytoplasm of epithelial cells as early as in hyperplasia (p = 0.001, OR = 3.8). Sixty-five of 107 (61%) OSCCs showed significant overexpression of ALCAM protein in cytoplasm/membrane of tumor cells (p = 0.043; OR = 3.3) in comparison with the normal oral tissues. Among OSCCs, cytoplasmic ALCAM was associated with advanced tumor size, tumor stage and tobacco consumption. Importantly, cytoplasmic ALCAM was an independent predictor of poor prognosis of OSCCs in multivariate analysis (p = 0.012, OR = 6.2). In an attempt to understand the molecular basis of cytoplasmic localization of ALCAM, 14-3-3ζ and 14-3-3σ were identified as its novel binding partners in oral cancer cells. In conclusion, increased expression of ALCAM is an early event in oral tumorigenesis; its cytoplasmic accumulation in tumor cells is a predictor of poor prognosis of OSCCs, underscoring its potential as a candidate prognostic marker for oral cancer. © 2008 Wiley-Liss, Inc.

Head and neck/oral squamous cell carcinoma (HNOSCC) is the sixth most common cause of cancer deaths in USA and remains a significant cause of cancer morbidity and mortality worldwide.1 Head-and-neck cancer sites are readily amenable to clinical examination, yet a lack of suitable molecular markers for early detection and risk assessment is clearly reflected by the fact that more than 50% of all HNOSCC patients have advanced disease at the time of diagnosis.2–6 Indeed, the 5-year survival rates of HNOSCC patients are in general poor (about 50% overall) and the prognosis of advanced HNOSCC cases has not improved much over the past 3 decades.2, 4 This limits treatment options and renders management of HNOSCC extremely challenging. Recurrence and formation of second primary tumors are frequent (10–25% of cases).7, 8 The clinical course of advanced disease is difficult to predict because the current clinicopathologic prognostic factors are not accurate predictors of clinical outcome. The heterogeneity of clinical outcomes in advanced stage HNOSCC patients emphasizes the need for accurate prognostic factors that can identify patients who are likely to develop recurrent tumors or second primary tumors, and thus might be candidates for novel therapeutics. The limited effectiveness of therapy for patients with advanced stage and recurrent disease is a reflection of an incomplete understanding of the molecular basis of head and neck carcinogenesis.9, 10 Conceivably, improvement in the ability to identify in an early stage and to predict malignant progression of HNOSCC lesions would lead to more effective treatment and reduction of morbidity and mortality.

Intense efforts are being directed toward developing accurate predictors of clinical outcome using high throughput techniques, such as differential display-reverse transcription PCR (DD), cDNA microarrays and proteomics, to assess global gene/protein expression patterns in head and neck cancer.11–14 In search of such novel molecular targets, our laboratory reported increased levels of activated leukocyte cell adhesion molecule (ALCAM) transcripts in oral squamous cell carcinomas (OSCCs), that constitute majority of HNOSCCs in India, using DD in clinical specimens and cell lines.15, 16 Importantly, ALCAM mRNA up-regulation was also observed in cell cultures from a human oral hyperplasia (AMOL-III), exposed to smokeless tobacco extracts (ST) in vitro16, providing the rationale for in-depth investigation of ALCAM expression in different stages of development and progression of OSCC.

ALCAM/melanoma metastasizing clone D (MEMD)/CD166 is a transmembrane glycoprotein of Ig superfamily that mediates cell–cell adhesion through both homophilic (ALCAM-ALCAM) and heterophilic (ALCAM-CD6) interactions.17 ALCAM, first identified as a CD6 ligand,18 is involved in hematopoiesis,19, 20 neurite extension,21 osteogenesis22 and embryonal implantation in the uterus.23 The stage-specific expression of ALCAM during fetal development resembles distinct steps of tumor metastasis. Expression of ALCAM correlates with the aggregation and metastatic potential of few human tumors including melanoma, prostate, ovarian, breast, lung, colorectal, esophageal, pancreatic cancer and hepatocellular carcinoma.24–36 In recent years, ALCAM has become one of the frequently applied cell surface markers to select pluripotent cells from mesenchymal progenitor populations by flow cytometry.30, 32

Herein, we investigated the clinical significance of ALCAM expression in different stages of oral tumorigenesis and determined its correlation with clinicopathologic factors and disease prognosis, with the aim of exploring its association with biological evolution of oral cancer and its utility as a molecular marker of the disease. The expression patterns of ALCAM protein in clinically characterized nonmalignant, premalignant and malignant oral tissues were evaluated using immunohistochemistry. In support of our clinical findings, we attempted to identify its interaction partners in oral cancer cells.

Abbreviations

ALCAM, activated leukocyte cell adhesion molecule; CD, cluster of differentiation; CLSM, confocal laser scan microscopy; DD, differential display-reverse transcription PCR; ESCC, esophageal squamous cell carcinoma; HNOSCC, head and neck/oral squamous cell carcinoma; MDSCC, moderately differentiated squamous cell carcinoma; MEMD, melanoma metastasizing molecule D; PDSCC, poorly differentiated squamous cell carcinoma; ST, smokeless tobacco extract; WDSCC, well differentiated squamous cell carcinoma.

Material and methods

Patients and tissue specimens

Institutional Human Ethics Committee approved this study prior to its commencement. One hundred seven primary OSCC patients (age range, 29–75 years, mean age 40 years) undergoing curative oral cancer surgery, at the Surgical Oncology Unit of Dr. B.R. Ambedkar Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India, were enrolled in this study after obtaining prior written consent of the patients. The clinical and pathological data including clinical TNM staging (tumor, node, metastasis based on Union International Center le Cancer TNM classification), site of the lesion, histopathological differentiation, age, gender and tobacco consumption habits were recorded in a predesigned performa as described previously.37 The diagnosis was based on clinical examination and histopathological analysis of the tissue specimens. The site distribution of OSCC cases was buccal mucosa (36), tongue (35), alveolus (12), lip (6) and other sites (18), including ginigivobuccal sulcus, hard palate, soft palate, retromolar trigone and floor of the mouth. The tumors were histologically graded as well, moderately or poorly differentiated SCCs. Biopsies from oral lesions with histological evidence of hyperplasia (58 cases) and dysplasia (20 cases) were also included in this study. The site distribution of oral lesions was buccal mucosa (53), tongue (12), alveolus (5), lip (6) and ginigivobuccal sulcus (2). Thirty nonmalignant tissues taken from a distant site of OSCCs (with histologically confirmed normal oral epithelium hither to referred to as oral normal tissues) were also evaluated for ALCAM expression. After excision, tissues were immediately snap frozen in liquid N2 and stored at −80°C till further use and one piece was collected in 10% formalin and embedded in paraffin for histopathological and immunohistochemical analyses.

Antibodies

Goat polyclonal anti-ALCAM antibody (Clone N-21, sc-8548), rabbit polyclonal 14-3-3ζ antibody (Clone C-16, sc-1019) and goat polyclonal 14-3-3σ antibody (Clone N-14, sc-7681) were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. For colocalization studies, mouse monoclonal anti-ALCAM antibody was purchased from Abcam (Cambridge, MA). Rabbit anti-goat Alexa Fluor® 594 (Cat no A-11080) and goat anti-rabbit Alexa Fluor® 594(Cat no A-11012) was purchased from Invitrogen (Carlsbad, CA). Goat anti-mouse FITC (Cat no. F2012) was purchased from Sigma (Bangalore, India).

Immunohistochemical analysis of ALCAM protein in oral tissues

Paraffin embedded sections (5 μm thickness) of human oral tissue specimens were stained with hematoxylin and eosin for histopathological analysis and immunostaining was done on serial sections as described previously by Verma et al.34 Briefly, the sections were deparaffinized in xylene, hydrated in graded alcohol and incubated with hydrogen peroxide (0.3% v/v) in methanol for 20 min to quench the endogenous peroxidase activity. Slides were washed with Tris-buffer saline (TBS, 0.1 M; pH = 7.4) and pretreated in a microwave oven in citrate buffer (0.01 M, pH = 6.0) for antigen retrieval. Nonspecific binding was blocked with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS, 0.01 M; pH = 7.2) for 1 hr. Thereafter, slides were incubated with anti-ALCAM antibody (Santa Cruz Biotechnology, CA) for 16 hr at 4°C and washed with TBS. The primary antibody was detected using strep-avidin-biotin complex using Dako LSAB plus kit (Dako CYTOMATION, Glostrup, Denmark) and diaminobenzidine as chromogen. All incubations were performed at room temperature in a moist chamber. Slides were washed with TBS (0.1 M, pH = 7.4), 3 times after every step. Finally, the sections were counterstained with Mayer's hematoxylin and mounted with D.P.X mountant. In negative controls, the primary antibody was replaced by nonimmune IgG of the same isotype to ensure specificity. Esophageal squamous cell carcinoma (ESCC) tissue sections with known immunopositivity for ALCAM protein as reported earlier,34 were used as positive control in each batch of sections analyzed.

Positive criterion for immunohistochemical staining

The immunopositive staining was evaluated in randomly selected 5 areas of the tissue section and specific staining in epithelial cells was defined as positive staining. Sections were scored as positive if epithelial cells showed immunopositivity in cytoplasm and/or plasma membrane when observed by 3 of us independently who were blinded to clinical outcome, i.e., the slides were coded and pathologist did not have prior knowledge of the local tumor burden, lymphonodular spread and grading of the tissue samples, while scoring the immunoreactivity.37 Cytoplasmic staining in these sections was scored as follows: 0, <10% cells; 1, 10–30%; 2, 30–50%; 3, 50–70% and 4, >70%, cells showed immunoreactivity. Sections were also scored semi-quantitatively on the basis of intensity as follows: 0, none; 1, mild; 2, moderate; 3, intense. Finally, a total score (ranging from 0 to 7) was obtained by adding the scores of percentage positivity and intensity. The sections were considered immunopositive if the total score was ≥2.

Follow-up study

Seventy of the 107 OSCC patients, who underwent treatment of primary OSCC between 2002-2005, could be followed regularly in the follow-up clinic, while 37 patients were lost to follow-up. Survival status of patients was verified and regularly updated from Tumor Registry records, Institute of Rotary Cancer Hospital, as of May 2008. As per our protocol, OSCC patients with T1 and T2 tumors were treated with radical radiotherapy or surgery alone, whereas majority of patients with T3 and T4 disease were treated using a combination of radical surgery followed by postoperative radical radiotherapy as described.37 The patients were followed up periodically and time to recurrence was recorded. If a patient died during the follow-up, patient survival time was censored at the time of death. Medical history, clinical examination and radiological evaluation were used to determine whether death 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 36/70 (51%) patients monitored in this study. Patients who did not show recurrence were alive till the end of the follow-up period. Among the 37 patients, who were lost to follow-up, the number of deaths could not be ascertained; therefore overall survival could not be considered as a separate parameter in our study. Only disease-free survival of the patients was studied. Disease-free survival was expressed as the number of months from the date of surgery to the loco-regional relapse. Patients were monitored for a period of median 33 months and maximum 70 months.

Statistical analysis

The immunohistochemical data were subjected to statistical analysis using SPSS 10.0 software. The relationships between ALCAM expression and clinicopathological parameters were tested in univariate analysis by chi-square and Fisher's exact test. To determine the independent predictors for tumorigenesis, logistic regression analysis was carried out in stepwise manner for the individual variables, clinicopatholgical parameters and ALCAM expression. Different combinations of variables were generated to assess the association between these combinations and patient prognosis. Follow-up studies were analyzed by Kaplan-Meier and Cox's proportional hazards test. Only disease-free survival was evaluated in the present study, as the number of deaths because of disease progression did not allow a reliable statistical analysis. The association between patient outcome and variables was assessed by log-rank test. Two sided p values were calculated and p ≤ 0.05 was considered to be significant.

Cell culture

Human head and neck squamous carcinoma cell lines, HSC2 and SCC4 were grown in monolayer cultures in Dulbecco's modified eagle medium (DMEM/F-12) (Sigma, MO) supplemented with 10% fetal bovine serum (FBS), 2.5mM L-glutamine, 1× sodium pyruvate, 1× vitamins, 1 mM MEM, 100 μg/mL streptomycin and 100 U/mL penicillin in a humidified incubator (5% carbon dioxide, 95% air) at 37°C as described by us previously.16

Coimmunoprecipitation

Coimmunoprecipitation (Co-IP) assays were carried out as described by us previously.37 Briefly, oral cancer cells, HSC2 and SCC4 were rinsed in ice-cold PBS and lyzed in IP 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, Uppsala, Sweden), followed by overnight incubation with polyclonal ALCAM, 14-3-3ζ (Rabbit polyclonal 14-3-3ζ antibody, Clone C-16, sc-1019) and 14-3-3σ antibodies (Goat polyclonal 14-3-3σ antibody, Clone N-14, sc-7681) (Santa Cruz Biotechnology) on a rocker at 4°C. Immunocomplexes were pulled down by incubating with Protein A-Sepharose for 2 hr 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, whereas immunoprecipitates obtained using 14-3-3ζ, 14-3-3σ or ALCAM antibodies were used as positive controls in each Western blot, respectively. Protein A-Sepharose-bound immunocomplexes were then resuspended in Laemelli sample buffer (10 mM Tris, pH = 7.4, 10% v/v glycerol, 2% w/v SDS, 5 mM EDTA, 0.02% bromophenol blue and 6% β-mercaptoethanol), boiled for 5 min and analyzed by Western blotting using specific antibodies. Briefly, the resuspended proteins were resolved on 10% sodium dodecyl sulphate (SDS)-polyacrylamide gels. The proteins were then electro-transferred onto polyvinylidenedifluoride (PVDF) membrane. After blocking with 5% nonfat powdered milk in TBS (0.1 M, pH = 7.4), blots were incubated with respective antibodies (dilution 1:200) at 4°C overnight. Membranes were incubated with HRP-conjugated secondary antibody (Dako CYTOMATION, Denmark), at an appropriate dilution in 1% BSA, for 2 hr at room temperature. After each step, blots were washed thrice with Tween (0.1%)-Tris-buffered saline (TTBS). Protein bands were detected by enhanced chemiluminescence method (ECL, Amersham, Buckinghamshire, UK) on XO-MAT film.

Colocalization studies of ALCAM, 14-3-3ζ and 14-3-3σ in oral cancer cells using confocal laser scan microscopy

Oral cancer cells, SCC4, were grown on coverslips in DMEM/F-12 medium supplemented with 10% FBS at 37°C and processed for confocal laser scan microscopy as described by us previously.38 Cells were rinsed in Dulbecco's PBS (DPBS), fixed in methanol for 5 min at −20°C and incubated with specific primary antibodies, mouse monoclonal anti-ALCAM and goat polyclonal anti-14-3-3σ antibody or mouse monoclonal anti-ALCAM and rabbit polyclonal anti-14-3-3ζ antibody, and incubated as a cocktail at 4°C overnight. After rinsing in phosphate buffer saline-0.1% Tween (PBST, 1X) the coverslips were incubated with either anti-mouse FITC conjugated/goat anti-rabbit Alexa Fluor® 594 conjugated and goat anti-mouse FITC conjugated/rabbit anti-goat Alexa Fluor® 594 conjugated secondary antibody for 45 min at 37°C in the dark. Coverslips were washed and counterstained with DAPI for 30 sec. In negative controls, the primary antibodies were replaced by nonimmune mouse IgG of the same isotype to ensure specificity. Thereafter, the slides were rinsed and mounted in fluorescence mounting medium and examined with Lieca TCS SP2 confocal laser scanning microscopy (CLSM).

Results

Immunohistochemical analysis of ALCAM in oral normal tissues, hyperplasia, dysplasia and OSCCs

The results of immunohistochemical analysis of ALCAM in normal oral tissues, hyperplasia, dysplasia and OSCCs are summarized in Table I. No detectable immunostaining (cytoplasmic/membranous) of ALCAM was observed in 60% of normal oral tissues analyzed (Fig. 1a). Ten of the thirty (33%) normal tissues showed membranous localization of ALCAM in the epithelial cells (Fig. 1b) and the remaining 2 of 30 (7%) tissues showed faint cytoplasmic immunopositivity also.

Figure 1.

Immunohistochemical analysis of ALCAM in oral tissues. Paraffin embedded oral tissue sections were stained using anti-ALCAM antibody as described in Material and methods section. (a) Normal oral mucosa showing no detectable immunostaining of ALCAM; (b) Normal oral mucosa showing membranous staining of ALCAM; (c) Oral hyperplasia showing cytoplasmic immunostaining of ALCAM; (d) Dysplastic lesion showing cytoplasmic immunostaining in epithelial cells; (e) OSCC showing cytoplasmic ALCAM staining in tumor cells; (f) Negative control showing no immunostaining of ALCAM protein in epithelial cells (af, Original magnification 400×). Arrow shows membranous (M) and cytoplasmic (C) staining in (b), (c) and (d), respectively. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Table I. ANALYSIS OF ALCAM IN NORMAL ORAL TISSUE, HYPERPLASIA, DYSPLASIA AND OSCCs
Clinicopathological featuresTotal casesALCAM overall positivityALCAM cytoplasmic positivityALCAM membrane positivity
Nn (%)n (%)n (%)
  • 1

    Normal vs. hyperplasia.

  • 2

    Normal vs. dysplasia.

  • 3

    Normal vs. OSCCs.

  • 4

    WDSCC vs. MDSCC + PDSCC.

  • 5

    (T1 + T2) vs. (T3 + T4).

  • 6

    Stage I/II vs. III/IVa.

  • 7

    Tobacco nonconsumer vs. consumer.

Normal3012 (40)2 (7)10 (33)
Hyperplasia5834 (59)23 (40)11 (19)
  p = 0.0011 
Dysplasia2014 (70)10 (50)4 (20)
 p = 0.0382p = 0.0012 
OSCC10765 (61)53 (50)12 (11)
 p = 0.0433p < 0.0013 
Age
 <40129 (75)8 (67)1 (8)
 ≥409556 (59)45 (47)11 (12)
Gender
 Male8555 (65)46 (54)9 (11)
 Female2210 (45)7 (32)3 (14)
Differentiation
 WDSCC6345 (71)36 (57)9 (14)
 MDSCC + PDSCC4420 (45)17 (39)3 (9)
 p = 0.0074  
Tumor stage
 T1 + T25027 (54)19 (38)8 (16)
 T3 + T45738 (67)34 (60)4 (7)
  p = 0.0255 
Nodal status
 N06133 (54)26 (43)7 (12)
 N1–44632 (70)27 (59)5 (11)
Stage
 I + II4017 (40)11 (28)6 (15)
 III + IVa6748 (69)42 (63)6 (9)
 p = 0.0036p = 0.0016 
Tobbaco habits
 Nonconsumer196 (26)4 (21)2 (10)
 Tobacco consumer8859 (65)49 (56)10 (11)
 p = 0.0047p = 0.017 

Eleven of 58 (19%) hyperplastic lesions showed only membranous ALCAM expression. Importantly, 23 of 58 (40%) hyperplasias showed loss of membranous ALCAM and significant increase in its cytoplasmic accumulation (Fig. 1c, p = 0.001, OR = 3.8, 95% CI = 1.9–42.2). Among the dysplasias analyzed (n = 20), 10 cases showed increased cytoplasmic accumulation of ALCAM (Fig. 1d). Chi-square trend analysis showed significant increase in ALCAM expression in different stages of oral tumorigenesis (normal to hyperplasia, dysplasia, OSCCs [ptrend < 0.001, Table I]).

Among the OSCCs, 65/107 (61%) cases showed overall (membranous or cytoplasmic) increased expression of ALCAM in tumor cells, with 50% of tumor tissues showing cytoplasmic accumulation only (Fig. 1e). Membranous localization of ALCAM protein was observed less frequently (11%) in OSCCs in comparison with the hyperplastic or dysplastic lesions. No immunostaining was observed in tissue sections used as negative controls where the primary antibody was replaced with isotype specific IgG (Fig. 1f), while the positive control showed increased cytoplasmic expression (data not shown).

Correlation of ALCAM expression in OSCCs with clinicopathologic parameters

Significant reduction in overall ALCAM immunopositivity was observed with de-differentiation in OSCCs; 45/63 (71%) well-differentiated SCCs showed strong immunostaining of ALCAM in comparison with 45% immunopositivity in the moderately and poorly differentiated SCCs (p = 0.007, OR = 0.33, 95% CI = 0.1–0.7). Further, increased cytoplasmic accumulation of ALCAM protein was associated with increased tumor size (p = 0.025, OR = 2.4, 95% CI = 1.1–5.2), late clinical stage (p = 0.001, OR = 4.4, 95% CI = 1.8–10.3) and tobacco consumption (p = 0.010, OR = 4.7, 95% CI = 1.4–15.3). These clinical studies suggested an association of cytoplasmic ALCAM expression with aggressive biological behavior of OSCCs. Therefore, we hypothesized that cytoplasmic ALCAM expression may serve as a marker of clinical outcome in OSCC patients. To test this hypothesis, we determined the relationship between ALCAM immunopositivity and disease prognosis.

Association of ALCAM expression with disease outcome

Seventy of the 107 OSCC patients could be followed up for a maximum period of 70 months. Kaplan-Meier survival analysis revealed shorter median disease-free survival (18 months) in OSCC patients having increased ALCAM expression (cytoplasmic/membranous) as compared with patients who did not show detectable immunostaining of ALCAM protein in tumor cells (66 months, p = 0.02; HR = 2.3; 95% CI = 1.1–5.1) as shown in Figure 2a. Importantly, markedly reduced disease-free survival of OSCCs was observed in patients showing increased cytoplasmic accumulation of ALCAM in tumor cells (16 months, p = 0.003; HR = 2.8; 95% CI = 1.3–5.7) underscoring the utility of ALCAM protein as an adverse prognostic marker for OSCCs. In multivariate analysis, the parameters—ALCAM overexpression, tumor stage (T1-T4), lymph node status (N0-4) and clinical stage (I-IVa) were analyzed; cytoplasmic accumulation of ALCAM (p = 0.012) with a relative risk of 6.2 and tumor stage (p = 0.041) with a relative risk of 3.7 emerged as the independent significant prognostic factors for OSCCs.

Figure 2.

Kaplan-Meier survival analysis in OSCC patients (a) OSCCs showing increased expression of ALCAM in cytoplasm/membrane showed significantly reduced disease-free survival time of 18 months (p = 0.02); (b) Median time for disease-free survival (no recurrence/metastasis) in tumors showing cytoplasmic accumulation of ALCAM was 14 months (p = 0.003). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

In an effort to understand the mechanism responsible for the cytoplasmic accumulation of ALCAM, we made an attempt to identify its binding partners in oral cancer cells using immunoprecipitation assays.

ALCAM interacts with 14-3-3ζ and 14-3-3σ in oral cancer cells

The 14-3-3 proteins are phosphoserine/threonine binding proteins. Various studies revealed optimal binding motifs of 14-3-3s correspond to mode-1 (RSXpSXP) and mode-2 (RXF/YXpSXP), where pS denotes phosphoserine or phosphothreonine sequences that are recognized by all 14-3-3 isoforms.39, 40 In addition, Coblitz et al.41 using a genetic screen, identified the C-terminal sequence SWpTX as a “mode-3” 14-3-3-binding motif. Using motif scan software, we observed 3 binding sites of 14-3-3 Mode 1 on ALCAM protein shown as pST bind (Fig. 3a). Further, coimmunoprecipitation assays were carried out using specific antibodies for ALCAM, 14-3-3ζ and 14-3-3σ in oral cancer cells, HSC2/SCC4. Immunocomplexes of 14-3-3ζ and 14-3-3σ were precipitated from HSC2/SCC4 cells using specific antibodies. The immunocomplexes were resolved using SDS-PAGE and Western blot analysis using ALCAM antibody showed the presence of ALCAM in immunoprecipitates of both 14-3-3ζ and 14-3-3σ isoforms in HSC2/ SCC4 cells [Fig. 3b(i)]. The results were further confirmed by reverse immunoprecipitation assays, wherein the ALCAM immunoprecipitates showed the presence of 14-3-3ζ and 14-3-3σ proteins [Fig. 3b(ii) and 3b(iii)].

Figure 3.

(a) Motif scans of ALCAM protein. Using Scansite software (www.scansite.mit.edu) available online, motif search for presence of 14-3-3 binding motif, Mode 1 (RSXpSXP, where pS denotes phosphoserine or phosphothreonine) in ALCAM protein. Figure 3a shows presence of 3 binding motifs, Mode 1 (shown as pST bind) in addition to immunoglobulin domain (ig) and cluster of differentiation 34 antigen (CD34 antigen) domain in ALCAM protein. (b) Immunoprecipitation assays of ALCAM, 14-3-3ζ and 14-3-3σ protein in oral cancer cells, HSC2 and SCC4. Immunoprecipitation assays were carried out using specific antibodies for ALCAM, 14-3-3ζ and 14-3-3σ proteins in whole cell lysates of oral cancer cells and analyzed by Western blotting as described in Material and methods. Panel (i) shows Western blot analysis of ALCAM, demonstrating the binding of ALCAM protein with 14-3-3ζ and 14-3-3σ isoforms in oral cancer cells HSC2/SCC4. Similarly, using reverse immunoprecipitation assays, Western blot analysis of (ii) 14-3-3ζ and (iii) 14-3-3σ protein confirmed the binding of ALCAM protein with 14-3-3ζ and 14-3-3σ isoforms. In each Western blot, immunoprecipitates obtained using specific antibodies for ALCAM, 14-3-3ζ and 14-3-3σ showing an intense band were used as positive controls, whereas negative control in which primary antibody was replaced by isotype IgG, no band was observed. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Colocalization studies using CLSM, clearly showed the cytoplasmic colocalization of ALCAM with 14-3-3ζ and 14-3-3σ in oral cancer cells (Figs. 4a and 4b), suggesting interactions between these proteins. Negative controls did not show any nonspecific fluorescence (data not shown). These results confirm our findings of coimmunoprecipiation assay. Together, these results indicated that 14-3-3s may account for the cytoplasmic accumulation of ALCAM protein in oral cancer cells.

Figure 4.

Colocalization of ALCAM, 14-3-3ζ and 14-3-3σ proteins in oral cancer cells. SCC4 cells were processed for colocalization experiments as described in Material and methods section. Cells were incubated with mouse monoclonal anti-ALCAM/rabbit polyclonal anti-14-3-3ζ antibody or mouse monoclonal anti-ALCAM/goat polyclonal anti-14-3-3σ antibody overnight at 4°C. Panel (a) shows (i) DAPI stained nuclei in blue color, (ii) Cytoplasmic expression of ALCAM (green color of FITC) in SCC4 cells, (iii) Cytoplasmic expression of 14-3-3ζ protein (red in color) and (iv) Merged picture of (ii) and (iii) showing colocalization of ALCAM and 14-3-3ζ in SCC4 cells. Similarly Panel (b) shows (i) DAPI stained nuclei in blue color, (ii) Cytoplasmic expression of ALCAM (green color of FITC) in SCC4 cells, (iii) Cytoplasmic expression of 14-3-3σ protein (red in color) and (iv) Merged picture of (ii) and (iii) showing colocalization of ALCAM and 14-3-3σ in SCC4 cells. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Discussion

To our knowledge the present study is the first to demonstrate alterations in ALCAM protein expression in different stages of development and progression of oral cancer. The salient findings of the study are (i) overexpression of ALCAM is an early event in the development of oral cancer, occurring in preneoplastic stages and is sustained during disease progression; (ii) significant increase in cytoplasmic accumulation of ALCAM is observed during development of OSCC (normal, hyperplasia, dysplasia and cancer); (iii) overall ALCAM expression is associated with differentiation status of the tumors; (iv) cytoplasmic ALCAM accumulation is associated with advanced tumor size, tumor stage and tobacco consumption; (v) cytoplasmic ALCAM is a marker of poor clinical outcome in OSCCs patients and may help to identify patients who could benefit from more frequent follow-up or alternative therapeutic modalities; (vi) binding of ALCAM to 14-3-3ζ/14-3-3σ may account for its cytoplasmic accumulation in oral cancer cells.

Using differential display, our previous studies showed increased levels of ALCAM transcripts in clinical samples of early premalignant stages of oral carcinogenesis and OSCCs in comparison with normal oral tissues, as well as in studies on oral cancer cell lines and premalignant cell cultures exposed to ST in vitro.15, 16 Herein, we demonstrate overexpression of ALCAM protein and change in its subcellular localization from the plasma membrane to cytoplasm of epithelial cells in different stages of development and progression of oral cancer. Taken together, our studies suggest that regulation of ALCAM expression is likely to occur at the transcription level. Furthermore, in an earlier independent study in our laboratory, Verma et al.34 observed increased expression of ALCAM protein in esophageal dysplasias and squamous cell carcinomas supporting the findings that altered ALCAM expression is observed in early stages of development of squamous cell carcinomas of the upper aerodigestive tract. Similar up-regulation of ALCAM was also observed in gastrointestinal adenocarcinmomas42 and in colon carcinogenesis, where it was speculated to be an early and important event in malignant cell transformation and an independent prognostic marker.31 Importantly, cytoplasmic ALCAM emerged as a marker of poor clinical outcome in OSCCs patients in our study. In support of our findings, cytoplasmic overexpression of ALCAM has been demonstrated to be a prognostic marker of disease progression in ovarian28 and breast cancer.29, 43 These clinical studies emphasize the importance of ALCAM in human cancers and underscore the need to gain insight into its mechanism of action in cancer cells.

The role of ALCAM in cancer has been investigated in-depth using melanoma as a model system. Expression of an NH2-terminally truncated, transmembrane variant of ALCAM (ΔN-ALCAM) leads to tumor cell migration and transition from primary tumor growth to tissue invasion.17 ALCAM-ALCAM homophilic interactions have been proposed to transduce signals in response to local cell saturation for the activation of proteolytic cascades (MMP-2) and consequent truncation of ALCAM; loss of ALCAM homophilic interactions may be a prerequisite for transformed cells to separate from the cell cluster and may account for the enhanced metastatic properties of melanoma.44 In contrast to these previously reported promotive effects of ΔN-ALCAM, recently, a secreted NH2-terminal fragment of ALCAM, sALCAM, has been shown to impair the migratory capacity of transfected melanoma cells in vitro, and reduce the basement membrane penetration in reconstituted human skin equivalents. Further, sALCAM also diminished the metastatic capacity of melanoma in nude mice. Importantly, L1 neuronal cell adhesion molecule (L1CAM/CD171), another progression marker of several cancers including melanoma, was also suppressed on sALCAM overexpression; in contrast, it was up-regulated by ΔN-ALCAM. These opposite effects induced by alternative strategies targeting ALCAM functions suggest important role of ALCAM in orchestrating cell adhesion, growth, invasion and proteolysis in the tumor tissue microenvironment.45 However, whether these opposing effects are also observed in other cancers including OSCCs remains to be determined. Our study showed the progressive loss of membranous ALCAM and its accumulation in the cytoplasm in hyperplasia, dysplasia and sustaining these alterations in subcellular localization of ALCAM in invasive tumors support the proposed roles of ALCAM in melanoma. It will be interesting to determine if the secreted ALCAM can be detected in body fluids such as blood or saliva of patients with oral lesions and OSCCs in future studies.

A unique finding of our study is that alteration in expression of ALCAM occurs as early as in hyperplasia, whereas the increase in cytoplasmic expression is more marked in dysplasia during the development of oral cancer. A majority of oral lesions in the Indian patients are observed in chronic consumers of tobacco, especially the smokeless form of tobacco. These patients often have chronic inflammation and we are investigating the link between chronic inflammation and cancer. In our previous studies, we have shown increased ALCAM expression by differential display and activation of NFκB in oral premalignant and cancer cells exposed to ST or TNFα.15, 16, 38 Further, Ofori-Acquah et al.24 recently proposed the presence of consensus DNA binding sequences for NFκB and AP-1 in ALCAM promoter. DNA-binding and reporter gene experiments indicate that NFκB element is functional and it is likely to be involved in increasing expression of ALCAM in tumors. However, the mechanism by which ST affects ALCAM expression and its localization requires further investigation. Nevertheless, our observations suggest that ALCAM is an important candidate biological target in oral tumorigenesis, warranting in-depth study of its role in development of oral cancer.

In this context, it is important to note that Nellisen et al.46 observed ALCAM-mediated adhesion to be induced when the actin cytoskeleton was chemically disrupted by low concentrations of cytochalasin D (CytD). Further, Zimmerman et al.47 proposed PKCα to be an important player in the induction of cytoskeleton-dependent homotypic ALCAM-mediated adhesion. However, the cytoplasmic domain of ALCAM is not a direct target for PKCα. Two actin bundling proteins, fascin and filamin, are substrates for PKCα and are candidates in the cytoskeleton-dependent regulation of ALCAM-mediated adhesion. Tomita et al.48 suggested that α-catenin is another potential candidate linking the ALCAM-cytoplasmic domain to the actin cytoskeleton. In search of the novel binding proteins that may serve as link in ALCAM-mediated cellular effects in oral tumors, we have shown the presence of 14-3-3 mode-1, in ALCAM protein using Scansite software, following bioinformatics approach. Our in vitro experimental data supported the in silico findings, wherein we have shown that both 14-3-3ζ and 14-3-3σ coimmunoprecipitated with ALCAM. Further, the reverse immunoprecipitation and immunofluorescence assays showing cytoplasmic colocalization of ALCAM with 14-3-3ζ and 14-3-3σ proteins confirmed our coimmunoprecipitation findings, suggesting that these 14-3-3 proteins may be involved in the cytoplasmic accumulation of ALCAM in oral cancer cells. Furthermore, the role of 14-3-3s in cell adhesion and integrin-mediated signal transduction has recently been well established.49, 50

In conclusion, our study demonstrated increased expression of ALCAM protein to be an early event in the development of oral cancer and its progressive cytoplasmic accumulation with disease progression. Among OSCCs, cytoplasmic ALCAM accumulation emerged as an independent predictor of poor prognosis in multivariate analysis. Our in vitro findings suggested that ALCAM binding to 14-3-3ζ and 14-3-3σ in oral cancer cells may be responsible for its accumulation in cytoplasm warranting an in-depth functional analysis of these interactions in oral cancer.

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