Research paper
Cancer Biology & Therapy 10:7, 694-699; October 1, 2010; © 2010 Landes Bioscience
CIP2A expression and localization in oral carcinoma and dysplasia Joseph Katz,1,* Andrew Jakymiw,2 Monet K. Ducksworth,2 Carol M. Stewart,1 Indraneel Bhattacharyya,1 Seunghee Cha1 and Edward K.L. Chan2 Department of Oral & Maxillofacial Diagnostic Sciences; University of Florida College of Dentistry; 2Department of Oral Biology; University of Florida College of Dentistry; FL USA
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Key words: oral cancer, EGFR, CIP2A, dysplasia, C MYC
Aims: Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy of the oral cavity resulting in severe morbidity and mortality. To date only few proteins have been suggested as potential biomarkers or targets for this type of cancer. Cancerous inhibitor of PP2A (CIP2A) is a protein expressed in epithelial tissues that stabilizes the oncogene c-Myc and causes cell transformation. This study was designed to investigate the expression of CIP2A in OSCC cell lines and tissues representing human normal, dysplasia and OSCC. Results: CIP2A was significantly increased in the human carcinoma cell lines compared to the primary gingival cell line. CIP2A was overexpressed in the human oral dysplasia and OSCC tissues compared to normal oral tissues. CIP2A was also preferentially localized in the dysplastic and OSCC epithelial areas compared to EGFR that was expressed mainly in areas of relatively normal epithelium and in dysplastic tissues above the basal layers. Methods: Using quantitative real time PCR, mRNA quantification for CIP2A was performed in a primary gingival cell line and OSCCs CAL 27 and SCC-25. Paraffin embedded human specimen classified as normal, dysplastic or OSCC were immunohistochemically stained for CIP2A expression. EGFR and CIP2A were also stained by immunofluorescence for colocalization. Samples of human normal oral tissue and OSCC were studied by PCR for mRNA expression of CIP2A. Conclusions: CIP2A may play a significant role in oral malignant transformation and therefore, it may be a potential target for chemotherapy of OSCC.
Introduction It is estimated that about 35,000 new cases and over 8,000 deaths related to cancer of the oral cavity and pharynx will occur annually in the USA in 2010.1,2 Oral cavity and pharynx cancers are currently ranked as the sixth- eight most prevalent cancers in the world, with squamous cell carcinomas (SCC) of the oral mucosa by far the most common type (83% –90%).3,4 It has been demonstrated that early detection of cancerous lesions generally results in reduced morbidity and mortality for some types of cancer.4 This is important for oral cancers because cases that require substantial resection or radiation treatment can result in significant morbidity.4 Unfortunately, oral precancerous lesions are frequently asymptomatic and can be difficult to detect at the earliest stages using current visual inspection methods.5 Although the WHO classification for epithelial dysplasia is accepted by most of the oral pathologists, there is a great variability in their interpretation of the criteria.6-8 Furthermore, studies have demonstrated significant interexaminer discrepancies in the assessment and grading of the histopathological features of dysplastic lesions.7,8 Keeping this in mind, it is obvious that the adoption of new reliable markers
of epithelial dysplasia may be instrumental in the accurate and reproducible histological diagnosis of oral dysplasia. Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor of the ErbB family, which is expressed in a variety of solid tumors, including oral cancers.9 High EGFR expression has been correlated with tumor size, metastasis and survival.10 In recent years, EGFR has been considered a promising target for monoclonal antibody as well as other therapy.10,11 However, EGFR targeted therapy is not universally applicable for oral cancers and the identification of alternate markers is needed. In 2002, our laboratory reported the cloning and characterization of a novel autoantigen p90 which was shown to be overexpressed in hepatocellular carcinoma and gastric cancer.12 In a subsequent collaborative study, p90 was discovered as protein that binds to and inhibit Protein Phosphatase 2A (PP2A) activity and shown to play a critical role in cancer progression.13 Thus p90 was renamed the cancerous inhibitory protein of PP2A or CIP2A. Most interestingly CIP2A was shown to inhibit of the degradation of c-myc oncoprotein and was reported to be overexpressed in oral cancer in the same study using a Finnish cohort.13 The purpose of the present study was to examine the expression of CIP2A in oral cancer cell lines and oral dysplasia and OSCC
*Correspondence to: Joseph Katz; Email:
[email protected] Submitted: 06/11/10; Revised: 07/05/10; Accepted: 07/06/10 Previously published online: www.landesbioscience.com/journals/cbt/article/12895 DOI: 10.4161/cbt.10.7.12895 694
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research paper
Research paper
Figure 1. p90/CIP2A is overexpressed in oral squamous cell carcinoma cell lines. (A) western blotting analysis of p90/CIP2A protein levels in two cell lines derived from oral squamous cell carcinoma of the tongue (CAL 27 and SCC-25) compared to normal primary gingival epithelial cells (pGEC). Tubulin was detected as a loading control. (B) Real-time quantitative PCR analysis of the comparative levels of p90/CIP2A mRNA levels in CAL 27, SCC-25 and pGEC cells. The results represent the mean ± S.D. of two different experiments.
tissues and determine their distribution and c o-localization in the different components of epithelial cells in relation to EGFR staining. The data generated by this study may be instrumental in developing strategies for early detection of premalignant and malignant oral lesions and identifying new attractive targeted therapies for oral cancer. Results Up-regulation of CIP2A expression in oral cancer cell lines. CIP2A protein and mRNA expression levels in two oral cancer cell lines, SCC-25 and CAL 27, were examined and compared to normal primary gingival epithelial cells (pGECs) by Western blot analysis and quantitative real-time PCR, respectively (Fig. 1). Using tubulin as a loading control, the CIP2A protein levels in both SCC-25 and CAL 27 cells were clearly demonstrated to be up-regulated compared to pGECs (Fig. 1A). Moreover, upon normalizing to 18S rRNA levels, CIP2A mRNA levels were found to be approximately 6 and 4-fold higher in CAL 27 and SCC-25 cells, respectively, compared to pGECs (Fig. 1B). Up-regulation of CIP2A expression in oral cancer tissues. CIP2A and EGFR expression were analyzed in oral squamous cell carcinomas by IIF analysis (Figs. 2A and 2B). As shown in Figure 2B, CIP2A expression in OSCC tissue exhibited a cytoplasmic staining pattern as previously reported.12 Pre-immune rabbit serum yielded no staining of OSCC tissues (data not shown), where as rabbit anti-CIP2A serum yielded strong staining of OSCC tissues. To further demonstrate the specificity of the antiCIP2A antibodies, we pre-incubated the antibodies with either rCIP2A or rGiantin proteins for one hour, and then performed IIF analysis. Figure 2C demonstrated that after pre-incubation with rCIP2A, CIP2A staining was abolished compared to antiCIP2A antibodies pre-incubated with rGiantin. This data also
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Figure 2. CIP2A/EGFR expression and localization in oral squamous cell carcinoma. CIP2A expression by immunofluorescence is highest in differentiated apical cell layers while EGFR expression is highest in basal cell layers. Serial tissue sections were co-stained with rabbit anti-CIP2A (green) and mouse anti-EGFR antibodies (red). Nuclei were counterstained with DAPI (blue). 100X (A) and 400X (B) magnification shown. (C) Specificity of anti-CIP2A staining demonstrated by absorption with recombinant CIP2A protein. Absorption was performed by pre-incubating rabbit anti-CIP2A antibodies with recombinant CIP2A or an unrelated protein giantin as a control. Immunofluorescence was then performed on serial tissue sections. Arrows indicate basement membrane. 100X magnification shown.
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Figure 3. CIP2A expression by immunohistochemistry of oral dysplasia. Photomicrograph part for epithelial dysplasia: Figure A. Control specimen representing oral hyperkeratosis sample demonstrating only sporadic positive cells (magnification x10). Figure B. Strong reactivity for CIP2A noted in the basal and parabasal cells (black arrows) in sample from mild to moderate epithelial dysplasia (magnification X10). Figure C. Another sample of mild to moderate epithelial dysplasia displaying with numerous CIP2A positive cells at the basal and parabasal levels (magnification x20) (black arrows) Figure D. Higher magnification (x40) from previous case (C) exhibiting strongly positive CIP2A cells with prominent nuclear and lighter cytoplasmic staining (black arrows).
showed that this rabbit anti-CIP2A serum specifically recognized CIP2A protein. Co-staining of EGFR and CIP2A in the oral squamous cell carcinomas demonstrated interestingly similar staining patterns, but also clearly distinguishing characteristics (Figs. 2A and 2B). CIP2A expression was generally found higher in differentiated epithelium compared to the undifferentiated basal layer (Figs. 2A and 2B). In contrast, EGFR staining was strongest in basal layers (Figs. 2A and 2B). Overlapping CIP2A and EGFR expression was clearly observed in somel samples (Figs. 2A and 2B). CIP2A and EGFR staining were negative in a normal human epithelial control tissue (data not shown). Immunohistological analysis of CIP2A expression in oral cancer tissues. In addition to the IIF analyses above, standard immunohistological methods were also performed. Figures 3 and 4 show representative CIP2A staining of oral dysplasias and OSCCs, respectively. Strong CIP2A staining was noted in the
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basal and parabasal cells in mild to moderate epithelial dysplasias (Fig 3). In OSCCs, strong scattered CIP2A reactivity was noted in groups of malignant epithelial cells. Well differentiated OSCCs often displayed clusters of CIP2A positive cells. Some of the lymphocytic infiltrates also demonstrated CIP2A staining. Additionally, some CIP2A staining was also detected in keratin pearls and the squamous eddies often observed in carcinoma biopsies (Fig 4). Table 1 summarizes the CIP2A protein staining intensity data from a total of 30 human tissues. Eight out of eight cases of OSCCs were graded for a maximal score of 3 per each slide, where as seven out of the eight severe dysplasia cases were graded as 2 and one as 1. From the moderate dysplasias, four out of six were graded 1 and two scored 0. All eight sections from normal oral tissues were scored 0. Moreover, the four tissue types were collapsed into “normal” and “abnormal” categories for further analysis. Results of a Mann-Whitney U test indicated that graded staining
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Figure 4. CIP2A expression by immunohistochemistry of oral squamous cell carcinoma. Photomicrograph part for oral squamous cell carcinoma: Figure A. Control specimen representing oral hyperkeratosis sample demonstrating total lack of any CIP2A expression (magnification x20) Figure B. Strong scattered reactivity for CIP2A noted in groups of malignant epithelial cells (yellow arrows). Some stain is also picked up by keratin pearls and the squamous eddies often seen in carcinoma biopsies (magnification x10) Figure C. A second sample of well differentiated squamous cell carcinoma displaying numerous clusters of CIP2A positive cells. Some of the lymphocytic infiltrate also demonstrates staining (magnification x20) (yellow arrows) Figure D. Another carcinoma sample exhibiting single malignant cells and occasional groups staining positively with CIP2A (magnification x40) (yellow arrows). Again the staining appears localized more to the nuclei and fainter in the cytoplasm. Table 1. Graded staining intensities of CIP2A for each of the three abnormal tissue types were significantly higher than those for normal tissue
Discussion
Staining intensity grade (n)
In the present study we have demonstrated that CIP2A is abundantly expressed in OSCC cell lines as well as dysplastic and Normal Oral Tissue 8 8 0 0 0 malignant human oral epithelium tissues. Moderate Dysplasia 6 2 4 0 0 These data confirms similar findings by Severe Dysplasia 8 0 1 7 0 Juntilla et al.13 who reported high expresOral Squamous Cell Carcinoma 8 0 0 0 8 sion of CIP2A protein in 11 out of 14 head Moderate dysplasia, p < 0.01; severe dysplasia, p < 0.001; oral squamous cell carcinoma, p < 0.001. and neck cancer samples, whereas 9 normal non-malignant tissues from the oral intensity was significantly higher for abnormal than for normal cavity were all negative for CIP2A. tissue samples (p
