Ectopic coexpression of keratin 8 and 18 promotes invasion of transformed keratinocytes and is induced in patients with cutaneous squamous cell carcinoma

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Abstract

Cutaneous squamous cell carcinoma (cSCC) results from transformation of epidermal keratinocytes. Invasion of transformed keratinocytes through the basement membrane into the dermis results in invasive cSCC with substantial metastatic potential. To better understand the mechanisms for invasion and metastasis, we compared the protein expression profiles of a non-metastatic transformed mouse keratinocyte line and its metastatic derivative. Keratin 8 (Krt8) and Krt18, not seen in normal keratinocytes, were coexpressed and formed Krt8/18 filaments in the metastatic line. The metastatic line efficiently invaded an artificial basement membrane in vitro owing to the Krt8/18-coexpression, since coexpression of exogenous Krt8/18 in the non-invasive parental line conferred invasiveness. To test whether the Krt8/18-coexpression is induced and is involved in cSCC invasion, we examined specimens from 21 pre-invasive and 24 invasive cSCC patients by immunohistochemistry, and the ectopic Krt8/18-coexpression was almost exclusively found in invasive cSCC. Further studies are needed to examine the clinical significance of ectopic Krt8/18-coexpression in cSCC.

Research highlights

Keratin 8 and 18 were ectopically coexpressed in metastatic mouse keratinocyte lines. ► Metastatic lines were invasive in vitro, but the isogenic non-metastatic line was not. ► Coexpression of exogenous keratin 8 and 18 rendered the non-metastatic line invasive. ► Keratin 8/18 in human cutaneous squamous cell carcinoma was examined systematically. ► Keratin 8/18 was almost exclusively found in invasive but not pre-invasive carcinoma.

Introduction

Cutaneous squamous cell carcinoma (cSCC) arises from transformed intra-epidermal keratinocytes [1]. Invasive cSCC, the growth of transformed keratinocytes through the basement membrane into the dermis, is associated with significant risk of metastasis, unlike its pre-invasive intra-epidermal stages such as actinic keratosis (AK, partial-thickness epidermal involvement) and Bowen’s disease (BD, full-thickness) [1]. When untreated, 5–8% of them advance to invasive cSCC [2], [3], but the mechanisms leading to basement membrane invasion and metastasis are not completely understood.

Pam212 is a spontaneously transformed mouse epidermal keratinocyte line [4]. Pam212 cells form SCC in mice but are not metastatic. However, derivatives of Pam212 cells were established from rare lymph node metastases (LY lines), and they were found highly metastatic [5]. As Pam212 and LY lines share the same genetic background, they are well suited to identify genes involved in metastasis. Indeed, a previous study compared their mRNA expression profiles and identified candidates: these genes regulated growth, apoptosis, inflammation and angiogenesis [6]. However, mRNA expression does not necessarily correlate with protein expression. Therefore, we compared their protein expression profiles using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS).

LY lines, but not Pam212, expressed keratin 8 (Krt8) and Krt18, both of which are members of “simple epithelial keratins” (Krt7, 8, 18, 19, and 20) present in non-stratified (simple) epithelia, but not keratinocytes [7]. Such ectopic Krt8/18-coexpression was previously associated with invasion and metastasis of some cancer cells [8], [9]. Here we found that LY cells possess a highly invasive potential in vitro, in addition to their known metastatic potential. Further, coexpression of exogenous Krt8 and Krt18 in non-invasive Pam212 cells conferred invasiveness. Finally, induction of Krt8/18-coexpression was found when specimens from invasive cSCC patients were examined immunohistochemically.

Section snippets

Cell lines

Pam212 and LY lines (LY-1 and LY-2) were gifts from Drs. Stuart H. Yuspa and Zhong Chen, respectively (National Institutes of Health, Bethesda, MD). Plasmids pEGFP-Krt8 and -Krt18 [10] encoding enhanced green fluorescent protein (EGFP)-tagged human Krt8 and Krt18, respectively, were provided by Dr. Keiichi I. Nakayama (Kyushu University, Fukuoka, Japan). The inserts were subcloned into the retroviral expression plasmid pQCXIP (Clontech) and the resultant plasmids were cotransfected into HEK293T

Ectopic coexpression of Krt8/18 in metastatic keratinocytes

2D-DIGE comparison of Pam212 and LY-1 cells revealed 7 up-regulated protein spots (spots #1–7) and 7 down-regulated spots (spots #8–14) in LY-1 cells (Fig. 1A, Table 1). PMF analyses of these and nearby spots indicated that Krt8 (spots #1–3) and Krt18 (spots #4, 5) were the most up-regulated proteins (>42-fold and > 12-fold, respectively), while Krt14 (spots #8, 9) was the most down-regulated protein (>14-fold). Immunoblotting indicated that Krt8 and Krt18 were up-regulated to similar levels in

Discussion

We carried out the first proteomic comparison between non-metastatic and metastatic transformed keratinocytes. We found that Krt8 and Krt18 were expressed at high levels and formed Krt8/18-filaments in metastatic cells. Using an in vitro assay, we also showed that metastatic cells were highly invasive in vitro, while non-metastatic cells were non-invasive. Finally, the causal role of Krt8/18-coexpression in keratinocyte invasion was demonstrated for the first time by coexpressing exogenous

Acknowledgments

We thank Drs. Yuspa, Chen and Nakayama for materials. This work was supported by Grants-in-Aid for Young Scientists (21791085) and Grants-in-Aid for Scientific Research (20590311) from the Japan Society for the Promotion of Science.

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    These two authors contributed equally to this work.

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