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 mitogen-activated 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=42) compared to HEKs (n=8) revealed that p38α and p38δ isoforms are predominantly expressed in both cell types and that MKK3 is the primary upstream activator expressed. Inhibition of endogenous p38α or p38δ 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. Dominant-negative p38α and p38δ inhibited squamous cell carcinoma (SCC) cell proliferation and inhibition of p38α activity also compromised survival of SCC cells. p38α and p38δ were predominantly expressed in HNSCCs (n=24) and nonneoplastic epithelium in vivo (n=6), with MKK3 being the primary upstream activator. Activation and expression of p38α and p38δ by tumor cells was detected in HNSCCs in vivo (n=16). Adenoviral expression of dominant-negative p38α or p38δ 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 p38α and p38δ 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.

Introduction

Tumor cell invasion and metastasis are hallmarks of cancer, characterized as complex multistage processes, in which malignant cells detach from the primary tumor, degrade components of the extracellular matrix (ECM), invade adjacent tissues and disseminate to distant sites (Hanahan and Weinberg, 2000). Matrix metalloproteases (MMPs) are a family of 23 zinc-dependent neutral endopeptidases capable of degrading virtually all components of the ECM. MMPs contribute to several aspects of tumor development, including tumor growth, angiogenesis, invasion and metastasis (Egeblad and Werb, 2002), and overexpression of several MMPs is associated with tumor aggressiveness and poor prognosis (Vihinen and Kähäri, 2002).

Mitogen-activated protein kinase (MAPK) pathways regulate the expression of MMPs, and in this way control malignant cell invasion (Reddy et al., 2003). Three MAPK pathways have been extensively characterized: the ERK1,2 pathway, the c-jun N-terminal-regulated kinase (JNK) pathway, and the p38 pathway (Garrington and Johnson, 1999). The ERK1,2 signaling cascade (Raf/MEK1,2/ERK1,2) is activated mainly by mitogens, and activation of this pathway promotes cell survival, proliferation and differentiation, whereas JNK and p38 MAPK pathways are stress-activated signaling cascades. The p38 MAPK pathway is activated by proinflammatory cytokines, and by several forms of cellular stress, for example, UV light, arsenite and osmotic shock. Four mammalian p38 isoforms have been characterized: p38α, β, γ and δ. Although these isoforms are 60–70% identical in amino-acid sequence, they differ in their tissue-specific expression and sensitivity to chemical inhibitors (Ono and Han, 2000). It has been proposed that the balance between the ERK1,2 and p38 pathway signaling is pivotal in determining cell fate (Xia et al., 1995; Olson and Hallahan, 2004). In this context, we have previously noted that stress-induced apoptosis in nontransformed cells involves MKK3b → p38α/β-mediated inactivation of the ERK1,2 pathway as a result of MEK1,2 dephosphorylation (Westermarck et al., 2001; Li et al., 2003). In contrast, p38 MAPK-induced dephosphorylation of MEK1,2 was not detected in several malignant cell lines, suggesting that the absence of this p38 MAPK-mediated inhibition of the ERK1,2 pathway provides a survival advantage for cancer cells (Li et al., 2003).

Head and neck squamous cell carcinoma (HNSCC) is characterized by high recurrence and metastasis rate and poor prognosis. Currently, few effective therapies are available for recurrent and advanced disease (Hunter et al., 2005). In this study, we have examined the function of distinct p38 MAPK isoforms in growth and invasion of HNSCCs. We show that the p38-mediated inhibition of the ERK1,2 pathway has been compromised in HNSCC cells, but is functional in normal human keratinocytes. Inhibition of the activity of predominantly expressed p38 MAPK isoforms, p38α and p38δ, effectively reduced expression of collagenase-1 (MMP-1) and collagenase-3 (MMP-13) as well as the invasive capacity of squamous cell carcinoma (SCC) cells. We also demonstrate that p38α activity is required for SCC cell survival, and that p38δ activity is required for cell proliferation. We show that p38α and p38δ are expressed and activated by tumor cells in HNSCCs in vivo. Together, our results provide evidence of a novel role for p38α and p38δ MAPK isoforms in promoting the invasive and proliferative phenotype of HNSCC cells, which suggests isoform-specific inhibition of the p38 MAPK pathway as a relevant approach for cancer therapy.

Results

p38 MAPK mediates inactivation of the ERK1,2 pathway in normal keratinocytes, but not in HNSCC cells

We have previously noted that MKK3b → p38α/β-mediated inactivation of the ERK1,2 pathway as a result of dephosphorylation of MEK1,2 was required for arsenite-induced apoptosis in normal human fibroblasts (Li et al., 2003) Interestingly, this MEK1,2 dephosphorylation was not detected in transformed cell lines studied, suggesting that the absence of this p38 MAPK-mediated negative regulation of the ERK1,2 pathway may provide a survival advantage for malignant cells (Li et al., 2003). To determine whether this cross talk between p38 and ERK1,2 pathways is present in normal epithelial cells, we treated primary human epidermal keratinocytes (HEKs) with arsenite (80 μ M) and monitored the activation of ERK1,2 and p38 pathways by Western blotting with phospho-specific antibodies. Arsenite treatment potently activated the p38 MAPK after 15 min (Figure 1a). This was accompanied by a simultaneous activation of MEK1,2 and ERK1,2, followed by marked dephosphorylation of both at 30 and 60 min time points (Figure 1a). No alterations were detected in total MEK1,2 and ERK2 levels after arsenite treatment (Figure 1a). Importantly, the arsenite-induced MEK1,2 dephosphorylation was dependent on p38 activity, as pretreatment of HEK with specific p38 MAPK inhibitor SB203580 (20 μ M) prevented arsenite-induced MEK1,2 dephosphorylation at 60 min of incubation (Figure 1b). Similar effect was noted with lower concentration (5 μ M) of SB203580 (not shown).

Figure 1
figure1

Activation of p38 MAPK results in MEK1,2 dephosphorylation in normal human keratinocytes, but not in HNSCC cells. (a) Primary HEKs were treated with arsenite (80 μ M) for the indicated periods of time, after which cell lysates were analysed by Western blotting with the antibodies specific for phosphorylated p38 (p-p38), MEK1,2 (p-MEK1,2) and ERK1,2 (p-ERK1,2), and for total p38, MEK1,2 and ERK2. (b) HEK were pretreated for 1 h with p38 MAPK inhibitor SB203580 (20 μ M), as indicated, followed by treatment with arsenite (80 μ M) for 15 and 60 min. Cell lysates were then analysed by Western blotting with the antibodies specific for p-p38, p-MEK1,2 and total MEK1,2. (c) Serum-starved UT-SCC-2, -7, -8, -9 and -33 were treated with arsenite (80 μ M) for the indicated periods of time. Cell lysates were then analysed by Western blotting with the antibodies specific for p-p38, p-MEK1,2, p-ERK1,2 and total MEK1,2. (d) HEK, UT-SCC-2, -7, -8 were cultured in KBM, after which cell lysates were analysed by immunoblotting with antibodies specific for phosphorylated p38 (p-p38) and total p38. Levels of p38 MAPK phosphorylation were quantitated relative to total p38. (e) HNSCC cells (UT-SCC2, -7 and -8) were infected with adenovirus for constitutively active MKK3b (MKK3bE) and control adenovirus (LacZ) and incubated for 24 h. Cell lysates were then analysed by Western blotting with the antibodies specific for p-p38, p-MEK1,2 and p-ERK1,2. Equal loading was controlled by antibody specific for β-actin.

In a panel of five HNSCC cell lines examined, p38 MAPK was activated by arsenite at 15 or 30 min of incubation, and this was associated with sustained activation of MEK1,2, with the exception of UT-SCC-8, in which high basal MEK1,2 activation was noted before addition of arsenite (Figure 1c). ERK1,2 was also activated by arsenite treatment in two SCC cells, but not in cell lines with high basal ERK1,2 activation (UT-SCC-8, UT-SCC-9 and UT-SCC-33). These differences in MEK1,2 and ERK1,2 activation are unclear, although they could be indicative of the variability between cell lines even when established from the same type of malignant tumor. Exposure of SCC cells to higher arsenite concentration (250 μ M) resulted in more rapid p38 MAPK activation, but did not induce MEK1,2 dephosphorylation (not shown). To assess whether lower basal p38 phosphorylation of HNSCC could explain the lack of p38-mediated MEK1,2 dephosphorylation, HEK and HNSCC cells were cultured in identical conditions and evaluated for p38 phosphorylation by Western blotting (Figure 1d). When p38 phosphorylation levels were quantitated relative to total p38 protein, the basal levels of p38 phosphorylation were variable but comparable between HEKs and HNSCCs. In addition, activation of p38 by infection of HNSCC cells with adenovirus for constitutively active MKK3b (MKK3bE) did not result in dephosphorylation of MEK1,2 (Figure 1e).

p38α and p38δ and MKK3 are the predominant p38 MAPK pathway components expressed by normal keratinocytes and HNSCC cells

Recent studies indicate that p38 MAPK-mediated cellular response to stress is determined by the expression pattern of distinct p38 isoforms (Enslen et al., 1998; Wang et al., 1998; Pramanik et al., 2003). To determine whether lack of arsenite-induced dephosphorylation of MEK1,2 in HNSCC cells could be owing to selective loss of one or several of the components of the p38 MAPK pathway, 42 recently established HNSCC cells lines of the UT-SCC series (Lansdorf et al., 1999) and eight primary HEKs were analysed for the expression of p38α, p38β, p38γ and p38δ isoforms by real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR). The results showed that p38α and p38δ were the predominant p38 isoforms expressed in both HEKs and HNSCC cells (Figure 2a). The expression of p38α appeared higher in HNSCC cells, but no statistically significant differences were detected between the cell types (Figure 2a).

Figure 2
figure2

Expression of p38 MAPK pathway components and MMP-1, -9 and -13 in HEKs and HNSCC cells. Total RNA from HNSCC cell lines (n=42) and HEKs (n=8) were analysed for mRNA expression of (a) p38 isoforms p38α, p38β, p38γ and p38δ. (b) p38 upstream activator kinases, MKK3 and MKK6, and (e) for collagenase-1 (MMP-1), gelatinase-B (MMP-9) and collagenase-3 (MMP-13) by quantitative real-time RT–PCR. β-Actin mRNA expression was used as internal control to normalize all determined mRNA levels. S.d.s of duplicate analyses were <5% of the means. Means are indicated when levels were >0. Statistical significance, as determined by Mann–Whitney test, HNSCC compared to HEK: *P<0.001. (c) The expression of p38α and p38δ was determined in lysates of HEK and HNSCC cells (UT-SCC-2, -7 and -8) by Western blot analysis. (d) HNSCC cells (UT-SCC-2) were infected with adenoviruses encoding p38αFLAG and p38δFLAG for 24 h and incubated for additional 24 h. FLAG-tagged p38α and p38δ were immunoprecipitated from cell lysates and analysed by Western blotting for activation using phospho-p38 (p-p38)-specific antibody. Equal loading was controlled by FLAG-specific antibody.

The upstream activators of the p38 MAPK isoforms, MKK3 and MKK6, are thought to have nonredundant functions and thereby may also mediate specific p38 MAPK responses (Enslen et al., 1998, 2000). In this context, we also quantitated the expression of MKK3 and MKK6 mRNAs in the same samples by quantitative RT–PCR. MKK3 was found to be the main upstream activator of p38 expressed at high levels in both cell types, whereas MKK6 mRNA was barely detectable (Figure 2b). In HEKs, MKK6 mRNA levels were less than 0.05% of β-actin mRNA, whereas the average level for MKK3 mRNA was 7% of β-actin mRNA. Similarly, in SCC cells, MKK6 mRNA levels were very low (0.13% of β-actin mRNA), although MKK3 mRNA expression was high (8% of β-actin mRNA) (Figure 2b). Western blot analysis of HEK and HNSCC cell lysates revealed that p38α and p38δ were expressed by both types of cells at comparable levels (Figure 2c), whereas we were unable to detect either p38β or p38γ in these cells (data not shown).

To determine whether the predominant p38 MAPK isoforms, p38α and p38δ, were activated in SCC cells, HNSCC cell cultures were transduced with adenoviruses encoding wild-type p38α-FLAG and p38δ-FLAG for 24 h. Western blot analysis of immunoprecipitated Flag-tagged p38α and p38δ with antibody specific for phosphorylated p38 revealed that adenovirally expressed p38α and p38δ were basally activated in SCC cells (Figure 2d).

We also analysed the expression of p38-dependent invasion proteinases collagenase-1 (MMP-1), gelatinase-B (MMP-9) and collagenase-3 (MMP-13) mRNAs in the same HNSCC cells and normal epidermal keratinocytes. MMP-1 mRNA expression was threefold higher in HNSCC cells than in HEKs (73 and 27% of β-actin mRNA, respectively), but the difference was not statistically significant (Figure 2d). MMP-9 was expressed at low levels in both HNSCC cells and HEKs (0.2% of β-actin mRNA for both cell types). However, MMP-13 was overexpressed in HNSCC cell lines (P<0.0001). The average MMP-13 mRNA expression in HEK was 0.003%, compared to an average of 25% of β-actin mRNA in SCCs (Figure 2e).

Inhibition of p38α and p38δ reduces MMP-1 and MMP-13 expressions by HNSCC cells

To investigate the specific role of p38α and p38δ in the invasion capacity of the SCC cells, we first treated UT-SCC-2, -7, -8 and -9 cells with the p38 inhibitor, SB203580 (20 μm) for 24 h and quantitated the expression of MMP-1 and MMP-13 mRNAs by RT–PCR. MMP-13 mRNA levels were potently (up to 90%) downregulated by SB203580 in all SCC cell lines examined, as compared to the untreated controls (P<0.05 for all comparisons) (Figure 3a). MMP-1 mRNA levels were also reduced by SB203580 up to 91% in the same SCC cell lines (Figure 3a).

Figure 3
figure3

Inhibition of p38α and p38δ reduces MMP-1 and MMP-13 expression and invasion of SCC cells. (a) Serum-starved UT-SCC-2, -7, -8 and -9 were treated with p38 MAPK inhibitor SB203580 (20 μ M) for 24 h and (b), UT-SCC-2 cells were infected with adenoviruses for dominant-negative p38α (p38αAF), dominant-negative p38δ (p38δAF) dominant negative MKK3b (MKK3bA) or LacZ (control virus) and incubated for 24 h. Total RNA was isolated and analysed by real-time quantitative RT–PCR for expression of MMP-1 and MMP-13 mRNAs. β-Actin mRNA expression was used as internal control to normalize all determined mRNA levels. S.d.s of duplicate analyses were <5% of the means. Values are shown relative to those in (a) untreated controls, or (b) control virus infected cells. (c) HNSCC cells (UT-SCC-7 and -8) were infected with adenoviruses for dominant-negative p38α (p38αAF), dominant-negative p38δ (p38δAF) or LacZ (control virus) and incubated for 96 h. Media were then collected and analysed for MMP-1, MMP-13 and TIMP-1 levels by Western blotting. (d) HNSCC cells (UT-SCC-2, -7 and -8) were infected with adenoviruses for dominant-negative p38α (p38αAF), dominant-negative p38δ (p38δAF) or LacZ (control virus) and incubated for 24 h. Subsequently, an equal number of cells (105) were seeded onto cell culture inserts coated with 1 mm of type I collagen and allowed to invade for 24 h, and the number of invaded cells was counted. Statistical significance as determined by Mann–Whitney test, p38αAF and p38δAF compared to LacZ infected cells: *P <0.05. (e) A total of 50–70% confluent cells in serum-free medium were transfected with siRNA duplexes for p38α, p38δ and scrambled control (scrm). After 6 h, the medium was equilibrated to 10% FCS, incubated for 24 h and used for invasion assays as in (d) (upper panel). Statistical significance as determined by Mann–Whitney test, p38α and p38δ siRNA compared to control siRNA-transfected cells: **P<0.01. The levels of p38α and p38δ in cell lysates were determined by Western blot analysis (lower panel).

As SB203580 only inhibits the activity of p38α and p38β isoforms (Ono and Han, 2000), we next studied the specific role of the two predominant p38 isoforms present in SCC cell lines, p38α and p38δ, in regulating MMP expression. To this end, we infected UT-SCC-2 cells with adenoviruses encoding dominant-negative p38α (p38αAF), p38δ (p38δAF) and MKK3b (MKK3bA), and quantitated the mRNA levels of MMP-1 and MMP-13 after 24 h. Expression of dominant-negative p38α reduced basal MMP-13 mRNA expression by 96% and MMP-1 expression by 73%, as compared to LacZ adenovirus-infected control cultures (Figure 3b). In parallel cultures, p38δAF also reduced both MMP-13 and MMP-1 mRNA expressions by 35 and 60%, respectively. Furthermore, expression of dominant-negative MKK3b potently reduced MMP-1 and MMP-13 expressions in SCC cells (by 40 and 80%, respectively) (Figure 3b).

In further experiments, we infected SCC cells with adenoviruses for dominant-negative p38α and p38δ, incubated the cells for 96 h and analysed the accumulation of MMP-1 and MMP-13 in the culture medium. Both UT-SCC-7 and -8 cells infected with dominant-negative p38α and p38δ demonstrated reduced production of MMP-1 and MMP-13 when compared to LacZ-infected controls (Figure 3c).

Adenoviral expression of dominant-negative p38α resulted in marked reduction in invasion of UT-SCC-2, -7 and -8 cells through collagen (100, 94 and 97%, respectively; P<0.05 for all comparisons; Figure 3d). Adenoviral expression of dominant-negative p38δ also reduced the invasive capacity of UT-SCC-2, -7 and -8 cells by 41, 58 and 82%, respectively (P<0.05 for all comparisons; Figure 3d). In addition, specific inhibition of p38α and p38δ expressions by corresponding specific siRNAs (Figure 3e, lower panel) resulted in potent inhibition of the invasion capacity of UT-SCC-2, -7 and -8 cells (Figure 3e, upper panel, P<0.01 for all comparisons).

Inhibition of p38α and p38δ activity reduces the proliferation of HNSCC cells

Inhibition of endogenous p38 MAPK activity with SB203580 treatment significantly reduced the cell number in cultures of UT-SCC-7 and -8 cells (P<0.05 for both), as compared to control cultures after 4 days (Figure 4a). Adenoviral expression of dominant-negative p38α (p38αAF) resulted in a statistically significant reduction in cell number in all three SCC cell lines tested, as compared to control virus-infected cells at 4-day timepoint (P<0.05 for all comparisons; Figure 4b). Expression of dominant-negative p38δ (p38δAF) also resulted in a significant (P<0.05) reduction in cell number, as compared to LacZ virus-infected control cultures, but the effect was not as pronounced as that noted with dominant-negative p38α (Figure 4a).

Figure 4
figure4

Inhibition of p38α and p38δ activity reduces the proliferation and survival of HNSCC cells. (a) An equal number of HNSCC cells (UT-SCC-7 and -8) were seeded and allowed to attach overnight. Cells were incubated with DMEM with 0.5% FCS supplemented with p38 inhibitor SB203580 (20 μ M). The medium was refreshed every 24 h and the cells were counted at the indicated time points. Statistical significance, as determined by Mann–Whitney test, when compared to untreated cells: *P<0.05. (b) UT-SCC-2, -7 and -8 cells were infected with adenoviruses for dominant-negative p38α (p38αAF), dominant-negative p38δ (p38δAF) or LacZ (control adenovirus). Cells were counted on the days indicated. Statistical significance, as determined by Mann–Whitney analysis, compared to LacZ infected cells: *P<0.05. (c) HNSCC cells (UT-SCC-2, -7 and -8) were infected with adenoviruses for dominant-negative p38α (p38αAF), dominant-negative p38δ (p38δAF), dominant-negative MKK3b (MKK3bA) and LacZ (control adenovirus) for 48 h. Subsequently, cells were analysed for viability after incubation with PI by FACS. Results are shown as percentage of PI positive cells. (d) HNSCC cells (UT-SCC-2 and -8) were infected with p38αAF, p38δAF, LacZ adenoviruses or left uninfected and incubated for 48 h. Lysates were then analysed by Western blotting for cleavage of PARP. The upper band represents uncleaved form of PARP, and the lower band (*) the proteolytically processed form of PARP. The values below panels represent the levels of uncleaved PARP as compared to the levels in control cultures (1).

p38α promotes survival of HNSCC cells

Next, we examined the specific roles of p38α and p38δ on SCC cell survival following adenoviral expression of dominant-negative p38α, p38δ and MKK3b for 48 h. Inhibition of p38α activity resulted in the most marked increase in cell death for all three cell lines tested (Figure 4c). Expression of dominant-negative MKK3b also resulted in an increase in cell death, although less than p38αAF. Interestingly, p38δ inhibition also caused cell death, although considerably less potently than both p38αAF and MKK3bA expressing cells (Figure 4c).

To study whether the increase in cell death was the result of apoptosis, we analysed lysates of cells expressing dominant-negative p38α and p38δ for cleavage of the apoptosis marker, poly(ADP-ribose)polymerase (PARP), by Western blotting. After 48 h, reduction of full-length PARP was evident in p38αAF expressing SCC cells, but not in LacZ control or the p38δAF adenovirus-infected cells (Figure 4d). Quantitation of full-length PARP demonstrated a 97 (UT-SCC-2) and 51% (UT-SCC-8) reduction in cells expressing p38αAF as compared to control cells.

p38α, p38δ and MKK3 are the predominant p38 MAPK pathway components expressed in normal epithelium and HNSCCs in vivo

The expression profile of the components of the p38 MAPK pathway in vivo was analysed in 24 HNSCC tumor samples and six nonneoplastic uvular epithelium samples by quantitative RT–PCR. Consistent with the HNSCC cell-line data above, p38α and p38δ were the predominant isoforms expressed in both HNSCC tumors and in normal epithelium (Figure 5a). Interestingly, the average expression level of p38δ mRNA was somewhat lower in HNSCC tumors than in normal tissue. The expression of p38β mRNA was detectable, but low in HNSCC and normal epithelium. MKK3 mRNA expression was high (29 and 38% of β-actin mRNA) in both SCC tumors and normal epithelium, respectively, whereas the average MKK6 mRNA expression was very low (0.4 and 0.2% of β-actin mRNA) in the HNSCC tumors and normal epithelium (Figure 5b).

Figure 5
figure5

Expression of p38 MAPK pathway components and MMP-1, -9 and -13 in HNSCC tumors and normal uvular epithelium in vivo. Total RNA from HNSCC tumors (n=24) and 6 uvular biopsies (n=6) were analysed for mRNA expression of (a) p38 isoforms p38α, p38β, p38γ and δ, (b) p38 upstream activator kinases, MKK3 and MKK6 and (c) for collagenase-1 (MMP-1), gelatinase-B (MMP-9) and collagenase-3 (MMP-13) by quantitative real-time RT–PCR. β-Actin mRNA expression was used as internal control to normalize all determined mRNA expression. S.d.s of duplicate analyses were <5% of the means (Mann). Means are indicated when >0. Statistical significance, as determined by Mann–Whitney test, HNSCC compared to control: *P<0.05; **P<0.001.

MMP-1, MMP-9 and MMP-13 are overexpressed in HNSCCs in vivo

The expression of p38 MAPK-regulated invasion proteinases, MMP-1, MMP-9 and MMP-13 mRNA levels was analysed in the 24 tumor biopsies compared to six nonneoplastic uvular epithelium. MMP-1 mRNA was expressed on an average of 0.1% in normal tissue biopsies, compared with an average mRNA of 546% of β-actin mRNA in SCC tumors (P<0.0001; Figure 5c). MMP-9 mRNA was overexpressed in HNSCC tumors with 16 and 0.2% of β-actin mRNA in normal tissues (P<0.0001). MMP-13 mRNA was virtually undetectable in normal tissue, demonstrated by an average mRNA expression of 0.07% of β-actin mRNA levels, whereas HNSCC tumors had an average MMP-13 mRNA levels 106% of β-actin mRNA (P<0.001).

Expression and activation of p38α and p38δ in HNSCC tumors in vivo

Sixteen paraffin-embedded HNSCC tumor sections were immunostained using an antibody specific for the phosphorylated p38 MAPK isoforms. Positive nuclear staining for active p38 was observed in tumor cells in all 16 tumors in addition to cytoplasmic staining (not shown). Staining of parallel sections of tumors for proliferation marker Ki67 revealed that the tumor cells were also proliferatively active. Tumor cells in all tumors were positive for p38α, which was detected exclusively in cytoplasm in six tumors. In addition, p38δ-positive tumor cells were detected in 15 out of 16 HNSCC tumor sections. The staining for p38δ was noted only in cytoplasm of tumor cells in 10 tumors. The average relative number of phospho-p38 positive cells in SCC tumors was 69±24. A total of 64±21% of tumor cells were positive for p38α and 21±16% were positive for p38δ.

Inhibition of p38 signaling suppresses growth of SCCs in vivo

The role of p38 isoforms in the implantation and subsequent growth of SCC tumors in vivo was examined in severe combined immunodeficiency (SCID) mice. Cutaneous SCC cells (UT-SCC-7) were infected with recombinant adenoviruses for dominant-negative p38α and p38δ, and LacZ-control virus for 6 h, and further incubated for 18 h to obtain overexpression of dominant-negative p38α or p38δ. Thereafter, the infected cells were injected simultaneously in the back of SCID mice. Interestingly, the growth of SCC xenografts in SCID mice was significantly suppressed by adenoviral overexpression of dominant-negative p38α of p38δ, as compared to LacZ adenovirus-transduced control cells. The average tumor volume in p38αAF group was 49% smaller than in LacZ control group and the average tumor volume in p38δAF group was 70% smaller than in control group at the end of the experiment, that is 27 days after injection of SCC cells (Figure 6a).

Figure 6
figure6

Inhibition of p38 signaling by dominant-negative p38α and p38δ isoforms suppresses the growth of SCCs in vivo. (a) Human metastatic cutaneous SCC cells (UT-SCC-7) in culture were infected with recombinant adenoviruses for dominant-negative p38α (RAdp38αAF), dominant-negative p38δ (RAdp38δAF) or LacZ control virus (RAdLacZ) at MOI 700 for 6 h. After additional 18 h incubation, cells (5 × 106) were implanted subcutaneously in the back of SCID/SCID mice and tumor growth was measured twice a week. Statistical significance between LacZ and p38αAF or p38δAF groups were determined by Student's t-test: *P<0.05; **P<0.02 (n=6). (b) The proliferating cells in SCC xenografts established from UT-SCC-7 cells were identified by immunohistochemistry using Ki67 as a marker. Mayer's hematoxylin was used as counterstain. Stainings from each group are shown. Magnification × 10. (c) The relative area of Ki67-positive cells was determined in four distinct fields at × 20 magnification from all tumor sections using digital imaging. The average number of proliferating cells is shown as percentage of the tumor area. Statistical significance between LacZ and p38αAF or p38δAF groups was determined by Student's t-test: P<0.01.

The Ki67-positive tumor cells were detected in the vicinity of the tumor margins in xenografts established with RAdLacZ-, RAdp38αAF- and RAdp38δAF-infected UT-SCC-7 cells (Figure 6b). However, the number of Ki67 positive cells was clearly lower in RAdp38αAF and RAdp38δAF xenografts than in RAdLacZ xenografts (Figure 6b). Quantitation of the relative Ki67-positive area at the tumor margin revealed that the number of proliferating cells was 49% lower in RAdp38αAF and 54% lower in RAdp38δAF-infected SCC xenografts than in RAdLacZ-infected control SCC xenografts (Figure 6c). In addition, a correlation was noted between the relative number of proliferating cells and the volume of the tumors at the end of the experiment (Figure 6c).

Discussion

The results of the present study indicate that stress-induced activation of p38 signaling in normal squamous epithelial cells, that is, primary HEKs causes attenuation of MEK1,2 phosphorylation. Functionally, p38-mediated MEK1,2 dephosphorylation has been shown to be required for stress-induced apoptosis in both normal human skin fibroblasts and in cardiac ventricular myocytes (Li et al., 2003; Liu and Hofmann, 2004). Interestingly, recent studies have also shown that cellular transformation by constitutively activated Raf-1 does not take place without simultaneous suppression of p38 activity (Pruitt et al., 2002). Taken together, these observations suggest that inhibition of p38-mediated MEK1,2 dephosphorylation is beneficial for cell survival and may be an early event required for cellular transformation. In this context, our previous work indicated that p38-mediated MEK1,2 dephosphorylation is absent in transformed cell lines (Liu and Hofmann, 2004). In this study, we have extended these findings and provided evidence that p38-mediated dephosphorylation of MEK1,2 is absent also in cell lines established from HNSCC tumors.

It could be proposed that selective reduction in the cellular expression levels of p38 MAPK pathway components could account for the lack of p38-mediated MEK1,2 dephosphorylation in HNSCC cells. To study this possibility, we performed quantitative expression analysis of the components of the p38 MAPK pathway in a panel of HNSCC cell lines and in vivo tumor material. The results indicate that the expression profile between the HNSCC cells and normal HEKs in culture is very similar. A similar expression pattern was also observed in the in vivo material of HNSCC tumors and normal epithelium. Interestingly, we noted that the predominant p38 isoforms expressed in both HNSCC cells and normal epithelial cells in culture and in vivo are p38α and p38δ. Importantly, we also noted that HNSCC cells in culture, as well as HNSCC tumors in vivo demonstrated activation of p38α and p38δ. Interestingly, MKK6 gene expression was almost undetectable, whereas MKK3 was abundantly expressed both in vitro and in vivo for both cell types. Differential expression of MKK6 and MKK3 could be explained by negative feedback regulation of MKK6 mRNA stability through MKK3 → p38α pathway (Ambrosino et al., 2003). In this context, it is also important to note that both p38α and p38δ can be activated by MKK3 (Keesler et al., 1998; Enslen et al., 2000).

Previous studies have indicated that p38 pathway may also serve as a tumor suppressor, as high p38/ERK1,2 ratio promotes tumor growth arrest and dormancy in vivo (Aguirre-Ghiso et al., 2001). In addition, inactivation of p38 enhances Ras-mediated tumor formation (Bulavin et al., 1999, 2002). The tumor-suppressor role of p38 appears to be mediated by p53 signaling (Bulavin et al., 1999). Interestingly, majority of HNSCCs carry inactivating p53 mutations, which would therefore allow elevated constitutive p38 activity without compromising cell survival (Brachman et al., 1992; Somers et al., 1992). In such case, p38 activity can serve to benefit tumor progression and even promote invasion. Interestingly, p38δ activation causes a simultaneous inactivation of ERK1,2 during epidermal keratinocyte differentiation (Efimova et al., 2003). However, the uncoupling of the p38 MAPK pathway and the ERK1,2 pathway, as demonstrated here, allows HNSCC cells to benefit from the growth-promoting activity of both signaling cascades.

It is tempting to speculate that inhibition of p38-mediated MEK1,2 dephosphorylation allows the malignant cells to endure the stressful conditions of the tumor environment without inhibiting the growth-promoting effects of ERK1,2 pathway. However, constitutive p38 MAPK activity observed in SCC cells would only benefit the tumor if the p38 activity is linked with its cancer-promoting characteristics, such as regulating the expression of invasion proteinases (Simon et al., 1998; Huang et al., 2000; Johansson et al., 2000). Indeed, the expression of both MMP-1 and MMP-13 in HNSCC cell lines was potently reduced by inhibiting the endogenous p38 activity by chemical inhibitor or by expression of the dominant-negative mutants of p38α and p38δ. These results extend previous observations demonstrating a positive role for p38 signaling in both transcriptional and post-transcriptional regulation of MMP-1 and MMP-13 expressions in SCC cells and fibroblasts (Ravanti et al., 1999; Westermarck et al., 2000, 2001; Reunanen et al., 2002) by identifying p38α and p38δ as the p38 isoforms regulating the expression of these invasion proteinases by HNSCC cells.

Quantitation of the expression of p38 MAPK-regulated MMPs in a panel of HNSCC cell lines and tumor samples indicated that MMP-13 mRNA was significantly overexpressed in the HNSCC cell lines and tumors, as compared to normal keratinocytes and uvular epithelium. These findings are in accordance with our previous observations indicating that MMP-13 is specifically overexpressed by SCC cells in culture and in vivo (Johansson et al., 1997). The expression of MMP-1 was also high in HNSCC cells, as compared to normal HEKs, but the difference was not statistically significant. Interestingly, the expression of MMP-1 mRNA was significantly higher in HNSCC tumors, as compared to normal epithelial tissue. Similarly, MMP-9 mRNA levels were significantly higher in HNSCC tumor samples. These results indicate that the expression of these p38-regulated invasion proteinases is significantly upregulated in vivo in HNSCCs showing activation of p38.

Inhibition of both p38α and p38δ activity also reduced SCC cell number, indicating another mechanism by which the endogenous p38 activity contributes to malignant phenotype of HNSCC cells. Importantly, SCC cell invasion was also drastically impaired by inhibiting the endogenous p38 activity, effectively demonstrating the advantage of this pathway in regulating the HNSCC cell phenotype. On further investigation, we discovered that p38α activity is also required for SCC cell survival, as inhibition of endogenous p38α activity resulted in apoptotic cell death not observed with p38δ inhibition. It is conceivable that application of p38 MAPK inhibition for the treatment of cancer would be relevant for malignant tumors that demonstrate activation of p38 MAPK and are dependent on p38 activity for survival. Activation of p38 MAPK in vivo was detected in all HNSCC tumors analysed, suggesting p38α and p38δ isoforms as specific therapeutic targets in HNSCCs.

The results using an in vivo model of cancer cell invasion showed that cutaneous SCC cell implantation and subsequent growth of xenografts in SCID mice was potently suppressed by adenoviral expression of dominant-negative mutants of p38α and p38δ, as compared to control adenovirus-transduced SCC cells. It is conceivable that the inhibition of growth of tumors established by p38α and p38δ transduced cells was associated with reduced MMP-13 expression, in accordance with our previous observations that specific inhibition of MMP-13 expression inhibits the implantation and growth of cutaneous SCC xenografts (Ala-aho et al., 2004). However, as inhibition of p38α and p38δ signaling also suppressed MMP-1 production, it is likely that MMP-1 also contributes to implantation and subsequent growth of SCC xenografts. Furthermore, the amount of proliferating cells was reduced in tumors derived from dominant-negative p38α- and p38δ-transduced UT-SCC-7 cells. As p38α and p38δ signaling promotes proliferation of SCC cells, and p38α also promotes SCC cell survival, it is likely that the effect of dominant-negative p38α and p38δ on SCC growth is also mediated via suppression of tumor cell proliferation at the time of implantation, resulting in impaired ability of SCC cells to initiate tumor growth.

In conclusion, our results indicate that in HNSCC cells the activity of p38 MAPK to assist in the induction of apoptosis has been compromised, whereas its effect on MMP expression important for cell invasion has been preserved. This fundamental alteration in p38 MAPK pathway function may provide an effective therapeutic opportunity for the treatment of HNSCC by targeting specific p38 MAPK isoforms, p38α and p38δ, which promote the malignant phenotype of HNSCC cells.

Materials and methods

Tumor samples

HNSCC tumor samples (n=24) were collected from surgically removed tumors between years 1990 and 2002 in Turku University Central Hospital from both genders ranging in age from 29 to 87 years. Nonneoplastic tissue samples of uvular and soft palate mucosa of six patients were removed during surgery for sleep apnea. All studies were approved by the Joint Ethical Committee of the University of Turku and Turku University Central Hospital. Participants gave their informed consent, and the study was conducted according to declaration of Helsinki.

Cell cultures

Human HNSCC cell lines (n=42) were established at the time of operation from HNSCCs (Lansdorf et al., 1999). The cell lines used for functional studies were established from primary SCC of oral cavity (UT-SCC-2), gingiva (UT-SCC-33), larynx (UT-SCC-8), skin (UT-SCC-9) and from metastasis of cutaneous SCC (UT-SCC-7) (Lansdorf et al., 1999). Cells were cultured in DMEM supplemented with 6 mmol/l glutamine, nonessential amino acids and 10% fetal calf serum (FCS). Normal HEKs (n=8) were established from skin samples by a modification of a previously described method (Boyce and Ham, 1983) and cultured in Keratinocyte Basal Medium 2 (KBM-2, Cambrex, NJ, USA), supplemented with SingleQuots (Cambrex Bioscience; Walkersville, MD, USA).

Quantitative reverse transcription-PCR analysis

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). From tumor samples, RNA was extracted using acid-guanidium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). cDNA was synthetized using 1 μg of DNAse I (Life Technologies, Carlsbad, CA, USA)-treated RNA using M-MLV Reverse Transcriptase, RNase H Minus (Promega) and random hexamer primers (Promega, Madison, WI, USA). Quantitative real-time RT–PCR analysis of cDNA samples was performed with specific primers and fluorescent probes designed, using Primer Express software (PE Biosystems), to quantitate specifically the levels of p38α, p38β, p38γ, p38δ, MKK3, MKK6, MMP-1, MMP-9 and MMP-13 (Table 1) (Hale et al., 1999). Samples were analysed in duplicate, and in each measurement range of the threshold cycle values was less than 5% of the mean. Expression of each transcript was presented as the percentage of p38 isoform or MMP mRNA expression relative to the control β-actin mRNA expression.

Table 1 Sequences of primers and probes used for quantitative real-time RT–PCR

Adenoviral cell infections

HNSCC cells were infected at multiplicity of infection (MOI) 700 for UT-SCC-7, MOI 400 for UT-SCC-2 and UT-SCC-8, incubated for 6 h in DMEM with 0.5% FCS as described previously (Ala-aho et al., 2002, 2004). The medium was changed and the incubations continued for 24 h before the invasion assay, or 24–96 h for the proliferation assays. Invasion assays were performed as described previously (Ala-aho et al., 2004). For proliferation assays, cells were trypsinized and counted with a hemocytometer. Recombinant adenoviruses for Flag-tagged p38α (RAdp38α-Flag) and p38δ (RAdp38δ-Flag), dominant-negative mutants of p38α (RAdp38αAF) and p38δ (RAdp38δAF) and constitutively active MKK3b (RAdMKK3bE) (Wang et al., 1998) were kindly provided by Dr Jiahuai Han, Scripps Research Institute (La Jolla, CA, USA). Adenovirus RAdLacZ, harboring the Escherichia coli β-galactosidase gene under the control of the CMV IE promoter (Wilkinson and Akrigg, 1992), was kindly provided by Dr Gavin WG Wilkinson (University of Cardiff, UK).

siRNA

The HP-validated siRNA duplexes were obtained from Qiagen: p38α (catalog number SI00605157), p38δ (catalog number: SI02222941) and scrambled control (catalog number 1022076). A total of 50–70% confluent cells in serum-free medium were infected with siRNA duplexes using Oligofectamine reagent (Invitrogen). After 6 h, the medium was equilibrated to 10% FCS, incubated for 24 h and used for invasion assays.

Flow cytometry

For determining the number of dead cells, SCC cell cultures were trypsinized following treatment, rinsed with PBS and resuspended in culture medium with or without propidium iodide (PI) (Sigma, St Louis, MO, USA). PI-positive cells were then detected using BD FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA) and analysed with Cell Quest Pro software (BD Biosciences).

Statistical analysis

Mann–Whitney U-test was used to determine statistical significance for the difference between two non-normally distributed independent sample groups. For experimental settings, first Kruskall–Wallis test was carried out to evaluate the overall difference between all groups and if the P-value was significant, pairwise comparisons were carried out between the control cells and the treatments using Mann–Whitney U-test (SPSS for Windows, SPSS Inc., Chicago, IL, USA). All P-values are two-tailed.

Western blot analysis

Following protein fractionation by SDS–PAGE, the following antibodies were used for Western blot analysis: mouse monoclonal anti-human MMP-13 antibody (181-15A12) (Calbiochem, San Diego, CA, USA), polyclonal rabbit antiserum against human MMP-1 (a kind gift from Dr Henning Birkedal-Hansen, NIDCR, Bethesda, MD, USA), polyclonal anti-TIMP-1 from Chemicon International Inc. (Temecula, CA, USA), specific antibodies against phospho-p38, phospho-MEK1,2, phospho-ERK1,2, MEK1,2 (all from Cell Signaling Technology, Beverly, MA, USA), PARP and FlagM2 (both from Sigma, St Louis, MO, USA), p38α and p38γ (both from Upstate, Waltham, MA, USA) and ERK2, p38β, p38δ/SAPK4 (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies against anti-mouse IgG Horseradish Peroxidase (HRP)-linked whole antibody (from sheep) (Amersham Biosciences, Piscataway, NJ, USA), rabbit anti-goat immunoglobulins HRP and swine anti-rabbit immunoglobulins (both from DakoCytomation, Glostrup, Denmark) were used and visualized by enhanced chemiluminescence (ECL; Amersham Biosciences, Piscataway, NJ, USA) (Ala-aho et al., 2002).

Growth of human SCC xenografts in SCID mice

All experiments with mice were performed according to institutional guidelines and with permission of the animal test review board of the University of Turku, Finland. Eight-week old SCID/SCID male mice were used to test the growth of human SCC xenografts. UT-SCC-7 cells were infected with adenoviruses RadLacZ, dominant negative p38α (RAdp38αAF) and dominant negative p38δ (RAdp38δAF) as described above at MOI 700 for 6 h, washed with PBS, incubated in serum-free medium for 18 h and detached with trypsin. Cells (5 × 106) in 100 μl of PBS were injected subcutaneously in the back of SCID mice. Each experimental group contained six mice. Tumor size was measured twice a week and calculated as width2 × length × (π/6). Tumors were fixed overnight in phosphate buffered 10% formalin and embedded in paraffin. Serial sections (5 μm) were deparaffinized and processed for immunohistochemistry with citrate buffer in microwave oven. Proliferating cells were detected by Ki67 immunostaining as described below (Ala-aho et al., 2004). The relative area of Ki67-positive cells was determined in four distinct fields at × 20 magnification from all tumor sections using digital imaging. The average number of proliferating cells is shown as percentage of the tumor area.

Immunohistochemistry

Immunostaining of paraffin-embedded HNSCC tumor sections (n=16) was performed using antibodies specific for p38α (1:100) (Upstate, Waltham, MA, USA), p38δ (1:100) (SAPK4, Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-p38 MAPK (1:50) (IHC-specific monoclonal rabbit; Cell Signaling) and Ki67 (1:50) (DaKoCytomation) diluted in PBS. All immunostainings were performed with avidin–biotin–peroxidase complex technique (VectaStain) in combination with diaminobenzidine (DAB), and counterstained with hematoxylin. Negative controls performed with a blocking peptide for phospho-p38 MAPK antibody and with mouse or goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The relative number of phospho-p38-, p38α- and p38δ-positive tumor cells was determined in three distinct fields at × 20 magnification from 10 tumor sections by digital imaging using Soft Imaging System's analySIS program (Lakewood, CO, USA).

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Acknowledgements

We acknowledge the expert technical assistance of Sari Pitkänen, Johanna Markola and Marjo Hakkarainen. This study was supported by grants from the Academy of Finland (projects 203421, 114405 and 8212695), Sigrid Jusélius Foundation, the Cancer Research Foundation of Finland, Turku University Central Hospital (project 13336) and by European Union Framework Programme 6 (LSHC-CT-2003-503297; CANCERDEGRADOME).

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Correspondence to V-M Kähäri.

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Junttila, M., Ala-aho, R., Jokilehto, T. et al. p38α and p38δ mitogen-activated protein kinase isoforms regulate invasion and growth of head and neck squamous carcinoma cells. Oncogene 26, 5267–5279 (2007). https://doi.org/10.1038/sj.onc.1210332

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Keywords

  • p38
  • mitogen-activated protein kinase
  • matrix metalloproteinase
  • squamous cell carcinoma

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