Increased EMMPRIN (CD 147) expression during oral carcinogenesis

https://doi.org/10.1016/j.yexmp.2005.09.011Get rights and content

Abstract

Gene expression profiling of oral premalignant (OPM) cells and normal oral epithelial (NOR) cells showed that EMMPRIN expression was markedly upregulated in OPM cells compared to NOR cells. We used an oral squamous cell carcinoma (OSCC) progression model composed of cell lines, organotypic cultures and tissue specimens to characterize EMMPRIN expression patterns by microarray analysis, qRT-PCR, Western blotting and immunohistochemistry. EMMPRIN levels are elevated in OPM and primary and metastatic OSCC cells as compared to NOR. EMMPRIN was detected as high and low glycosylated forms in the OPM and OSCC cellular extracts and was released in the media by OSCC cells but not by OPM cells. EMMPRIN expression in an organotypic culture model of normal and OPM mucosae mirrored the expression patterns in the respective tissues in vivo. EMMPRIN expression was limited to basal cells of normal, benign hyperkeratotic and inflammatory (lichen planus) oral mucosa. EMMPRIN expression is increased in dysplastic leukoplakias spreading to more superficial layers, and its expression levels correlated significantly with the degree of dysplasia. Primary and metastatic OSCC showed strong cell surface expression of EMMPRIN. These results suggest that EMMPRIN overexpression occurs at a very early stage of oral carcinogenesis and plays a contributing role in OSCC tumorigenesis.

Introduction

Oral squamous cell carcinoma (OSCC) is the most common cancer detected in the head and neck region and ranks among the top ten most frequently diagnosed cancers worldwide with an estimated 500,000 patients affected annually worldwide (Parkin et al., 1999, Pisani et al., 1999). Moreover, OSCC incidence is increasing in most developing countries, especially among younger persons (Macfarlane et al., 1994). Despite remarkable recent advances in cancer treatment, the long-term survival of patients with OSCC has only marginally improved over the past three decades, with only 50% of OSCC patients surviving 5 years after diagnosis (Forastiere et al., 2001). Early diagnosis and treatment appear to be the most powerful means by which to improve prognosis, especially if the diagnosis can be made in the preinvasive, intraepithelial stage (Boyle et al., 1993, O’Shaughnessy et al., 2002, Lippman et al., 2005).

Development of OSCC is a multistep process of genetic and epigenetic changes that typically manifest clinically in two stages: (1) an oral premalignant (OPM) lesion and (2) progression or transformation to OSCC (Braakhuis et al., 2004, Kim and Califano, 2004, Lippman et al., 2005). OPM lesions are readily identifiable clinically and usually present as white patches (leukoplakia) or as red patch (erythroplakia) (Neville et al., 2002). Leukoplakia is the most common, representing 85% of such lesions and showing a prevalence rate of 2–8% in people over the age of 70 (Neville et al., 2002, Neville and Day, 2002). Since only up to 20% of oral leukoplakias progress to malignancy (Neville et al., 2002), a major challenge is to accurately measure risk of OSCC development in individual leukoplakias so that they may be treated accordingly. If the disease can be halted at this stage, other benefits accrue. Patients with OSCC have a significantly increased risk of developing subsequent second primary or recurrent OSCC (Kopelovich et al., 1999). Recent genetic studies indicate that the second primary OSCC shows similar genetic alterations as the original tumor and thus appears to be derived from the spread of the original OPM clone (Braakhuis et al., 2003), perhaps because a ‘field’ of premalignant clones remained after resection of the primary tumor (Tabor et al., 2001). Along a similar fashion, OPM lesions which have the potential to develop into new tumors persist in one fourth of the OSCC patients with cancer-free surgical margins (Weijers et al., 2002). It would be ideal to evaluate these for markers of high risk since so many of these lesions have extensive involvement.

Malignancy risk of oral leukoplakia is currently assessed by the presence and degree of oral epithelial dysplasia (OED). Histologic grading classifies oral leukoplakias into stages with increasing risk of malignant transformation, namely: epithelial hyperkeratosis and hyperplasia; mild, moderate and severe OED; and carcinoma-in-situ. Among the leukoplakias with OED, a substantial portion (15–20%) progress to OSCC with a constant rate of 2–3% per year (Lumerman et al., 1995, Schepman et al., 1999).

Although the severity of OED is known to be proportional to the risk of subsequent OSCC development, histologic grading of OED is an imprecise art, and considerable improvement is needed for predicting OSCC risk for the individual lesions (Sudbo et al., 2001a, Sudbo et al., 2001b). The ability to change our current paradigms of diagnosis and treatment of oral leukoplakias depends in large part on the development of refined and clinically significant molecular risk markers that can assist the microscopic grading of OED (O’Shaughnessy et al., 2002, Lippman et al., 2005). In order to identify gene products that can be used as molecular risk markers for OPM lesions, we used oligonucleotide microarrays for global gene expression profiling of normal oral epithelial cells and OPM cell lines. It should be noted that these OPM cell lines were derived from leukoplakias adjacent to existing OSCC and therefore have proven malignancy risk (Bouquot, 1999). Our data showed that expression of EMMPRIN (extracellular matrix metalloproteinase inducer) was markedly upregulated in OPM-derived cells compared to NOR. EMMPRIN (also known as CD147 and M6 in humans; basigin and gp42 in mouse) was originally isolated from the plasma membranes of malignant tumor cells (Biswas, 1982, Biswas, 1984). EMMPRIN has recently been recognized as an important modulator of tumor–stroma cross talk and mediates a wide range of tumor-promoting molecular events that include acquisition of anchorage-independent growth and invasive phenotype by tumor cells, as well as tumor-cell-induced angiogenesis (Yan et al., 2005). This critical role during early tumorigenesis prompted us to focus on EMMPRIN as potential molecular diagnostic marker and therapeutic target for early diagnosis and treatment of OPM lesions.

We used microarray RNA expression profiling, qRT-PCR, Western blotting and immunohistochemistry to characterize EMMPRIN expression patterns in an OSCC tumor progression model composed of differently staged cell lines, in vitro 3-dimensional organotypic culture models of normal oral mucosa and OPM lesion, and archival tissue specimens.

Our data show that EMMPRIN transcript levels are markedly upregulated in cell lines derived from OED, primary OSCC and metastatic OSCC compared to normal human oral epithelial and epidermal keratinocytes. Our data show that upregulation of EMMPRIN expression occurs during the early stage of OPM lesions and appears to be a critical phenotypic alteration that precedes the development of OSCC.

Section snippets

Cell lines, organotypic culture and tissue specimens

All protocols for the use of human cell lines and tissue specimens in this work were approved by the Institutional Review Boards of The University of Louisville and the University of Texas at Houston. Primary cultures of NOR were prepared from discarded human gingival tissue. Normal human epidermal keratinocytes (NHEK) were obtained from Cambrex BioScience (Walkersville, MD). The Leuk1 cell line was derived from a dysplastic leukoplakia adjacent an early invasive OSCC (T1N0M0) involving the

Global gene expression analysis revealed elevated expression of EMMPRIN in OED and OSCC cell lines

We performed a global analysis of gene expression of 22,500 genes in a panel of cell lines to identify genes that are differentially expressed during premalignant transformation of oral epithelial cells. These cell lines represented different phenotypes ranging from normal (NOR, NHEK) to premalignant (Leuk1, Leuk2) to primary OSCC (686Tu, 1386Tu) and to metastatic OSCC (686Ln, 1386Ln). While examining the gene expression profiles of these cells, we detected markedly upregulated expression of

EMMPRIN: a potential molecular risk marker for OSCC development in OPM lesions

OPM lesions, especially leukoplakia, represent an excellent model system to study molecular markers of cancer risk assessment and response to therapy for the following reasons: (1) these lesions are easy to monitor and biopsy compared to the precancers of most other anatomic sites; (2) several follow-up studies have documented the risk of subsequent OSCC development in these lesions; (3) most of these lesions are clearly related to tobacco exposure, which is the primary risk factor for OSCC

Conclusion

In vivo expression patterns of EMMPRIN in oral premalignant and malignant lesions mirror the expression levels observed in vitro with cell lines and organotypic cultures established from cells derived from normal oral mucosa, premalignant leukoplakia and primary and metastatic OSCC. Upregulation of EMMPRIN expression occurs at a very early stage as OPM lesions progress through hyperplasia/hyperkeratosis to dysplasia and to invasive OSCC. Thus, upregulated expression of EMMPRIN seems to be an

Acknowledgments

This work was supported by NIH/NIDCR grants DE13150 (W.Z.) and DE14395 (P.G. S.), a Philip Morris USA Inc. and Philip Morris International External Research Program grant (W.Z) and by a Fleming and Davenport Award, Texas Medical Center (N.V.).

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