Biochemical and Biophysical Research Communications
Ectopic coexpression of keratin 8 and 18 promotes invasion of transformed keratinocytes and is induced in patients with cutaneous squamous cell carcinoma
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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TGF-β Promotes Heterogeneity and Drug Resistance in Squamous Cell Carcinoma
2015, CellCitation Excerpt :By contrast, in TGFβ-CreER-driven YFP+ clones, K10 was rarely detected, and leading edge cells showed reduced K5 (Figure S2E). Conversely, K13 and K18, ectopically induced in skin SCCs (Nischt et al., 1988; Yamashiro et al., 2010), were readily detected in TGFβ-CreER-driven YFP+ clones, but not in most K14-CreER-driven ones nor in unmarked, TGFβ-non-responsive basal tumor cells from TGFβ-CreER animals (Figures S2F and S2G). As shown in Figures 2C and S2H, fewer TGF-β-responding (YFP+) basal cells were in S-phase relative to TGF-β non-responding (YFPneg) basal cells.
Lumican expression, localization and antitumor activity in prostate cancer
2013, Experimental Cell ResearchCitation Excerpt :Keratin 8 and keratin 18, are not expressed in normal keratinocytes, however both forms of keratins are co-expressed in metastatic keratinocytes forming keratin8/18 filaments. The co-expression of keratin 8/18 filaments in normal keratinocytes enables these cells to efficiently invade an artificial basement membrane [58]. Our studies revealed a decrease in both keratin 8 and 18 expressions in prostate cancer cells seeded upon lumican, as well as, a change in the disposition of keratin 8 and 18 from organized filaments extending throughout the cellular protrusions, lamellipodia, to a perinuclear localization.
Cav1 suppresses tumor growth and metastasis in a murine model of cutaneous SCC through modulation of MAPK/AP-1 activation
2013, American Journal of PathologyCitation Excerpt :K8 and K18 have been associated with increased invasion in a variety of cell types.42–45 Recently, Yamashiro et al33 have shown that lymph node metastatic derivatives of PAM212 (LY-1 and 2) overexpress both K8 and K18 compared with parental PAM212 cells. Furthermore, the overexpression of K8 and K18 in parental PAM212 confers a more invasive phenotype.
Modulations of gene expression induced by daily ultraviolet light can be prevented by a broad spectrum sunscreen
2012, Journal of Photochemistry and Photobiology B: BiologyCitation Excerpt :In addition, we found that keratin 18 gene expression was significantly up-regulated following DUVR exposure. Recently Yamashiro et al. showed that co-expression of keratin 18 and 8 promotes invasion of transformed keratinocytes and is up regulated in invasive squamous cell carcinomas [23]. It could be supposed that over expression of this gene by DUVR could have harmful impact on basal cancer cells.
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These two authors contributed equally to this work.