p38α and p38δ mitogen-activated protein kinase isoforms regulate invasion and growth of head and neck squamous carcinoma cells

Abstract

Recent studies indicate that the specificity of p38 mitogenactivated protein kinase (MAPK)-mediated cellular stress responses is determined by the expression pattern of the distinct p38 isoforms. Here, we have analysed the function of distinct p38 isoforms in the growth and invasion of head and neck squamous cell carcinomas (HNSCCs). Activation of p38 MAPK by arsenite resulted in inactivation of the ERK1,2 signaling pathway by dephosphorylation of MEK1,2 in primary human epidermal keratinocytes (HEKs), whereas in HNSCC cells this p38-mediated inhibition of the ERK1,2 pathway was absent. Quantitation of p38 pathway component mRNA expression in HNSCC cell lines (n ¼ 42) compared to HEKs (n ¼ 8) revealed that p38a and p38d isoforms are predominantly expressed in both cell types and that MKK3 is the primary upstream activator expressed. Inhibition of endogenous p38a or p38d activity by adenoviral delivery of corresponding dominant-negative p38 isoforms potently reduced MMP-13 and MMP-1 expressions, and suppressed the invasion of HNSCC cells through collagen. Dominantnegative p38a and p38d inhibited squamous cell carcinoma (SCC) cell proliferation and inhibition of p38a activity also compromised survival of SCC cells. p38a and p38d were predominantly expressed in HNSCCs (n ¼ 24) and nonneoplastic epithelium in vivo (n ¼ 6), with MKK3 being the primary upstream activator. Activation and expression of p38a and p38d by tumor cells was detected in HNSCCs in vivo (n ¼ 16). Adenoviral expression of dominantnegative p38a or p38d in cutaneous SCC cells potently inhibited their implantation in skin of severe combined immunodeficiency mice and growth of xenografts in vivo. Our results indicate that p38a and p38d specifically promote the malignant phenotype of SCC cells by regulating cell survival, proliferation and invasion, suggesting these p38 MAPK isoforms as potential therapeutic targets in HNSCCs.

Phenotype-assisted transcriptome analysis identifies FOXM1 downstream from Ras–MKK3–p38 to regulate in vitro cellular invasion

“…Of note, we found MKK3 expression to be induced in Ha-Ras-transformed NIH3T3 cells, suggesting this to be a critical event toward the development of a malignant phenotype brought about by Ha-Ras. Our data support the findings of other investigators, that activation of the MKK3/6-p38 pathway is sufficient to induce in vitro invasion (Reunanen et al, 2002;Shin et al, 2005;Song et al, 2006;Junttila et al, 2007). Our findings exceed current knowledge in that we show that activation of this pathway is also sufficient to induce anchorageindependent growth, a hallmark of the malignant phenotype.…”

Serpin Peptidase Inhibitor Clade A Member 1 (SerpinA1) Is a Novel Biomarker for Progression of Cutaneous Squamous Cell Carcinoma

“…19 -21 SCC cells were cultured in Dulbecco's modified Eagle's medium supplemented with 6 mmol/L glutamine, nonessential amino acids, and 10% fetal calf serum. 20,21 HaCaT, a spontaneously immortalized, nontumorigenic human epidermal keratinocyte-derived cell line, 22 and the Ha-rastransformed tumorigenic HaCaT cell lines A5, II4, and RT3 23,24 (a gift from Dr. Norbert E. Fusenig, German Cancer Research Center, Heidelberg, Germany) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. G418 (200 g/mL) was added to the medium of the Ha-ras-transformed HaCaT cell lines.…”

“…Keratinocytes were cultured in keratinocyte basal medium 2, supplemented with SingleQuots (Cambrex Bioscience, Walkersville, MD), as previously described. 20,21 …”

“…cDNA was reverse transcribed from 1 g of RNA using reverse transcriptase M-MLV RNase H without reverse transcriptase and random hexamers (Promega, Helsinki, Finland). 20,21 …”

“…20,21 The levels of ␤-actin and glyceraldehyde-3-phosphate dehydrogenase mRNA (as housekeeping genes) were determined as described previously. 21 …”