p38 MAPK Mediates Epithelial-Mesenchymal Transition by Regulating p38IP and Snail in Head and Neck Squamous Cell Carcinoma.

1Lung Cancer Research Program of the Jonsson Comprehensive Cancer Center; 2Division of Pulmonary and Critical Care Medicine, Department of Medicine; 3Department of Head and Neck Surgery; 4Department of Pathology and Laboratory Medicine; 5Jonsson

Comprehensive Cancer Center; 6UCLA Head and Neck Cancer Program; and 7Department of Biostatistics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095; 7Veterans’ Affairs Greater Los Angeles Healthcare System, Los Angeles, CA 90073.

Ds Ac

Conclusion Ø  Abrogation of p38 results in downregulation of E-cadherin and other

markers of EMT in HNSCC

Ø  P38 IP is dysregulated in HNSCC

Ø  Targetting the p38-p38IP axis may be a useful therapeutic strategy to block EMT and metastatic behavior of EMT

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Yuan Lin, PhD 1,2; Jon Mallen-St. Clair, MD PhD3; Edward C. Kuan MD3; Guanyu Wang, MD PhD 1,2; Jie Luo MS1,2 ; Fernando Palma-Diaz MD4; Chi Lai MD4; David A. Elashoff PhD 6; Sherven Sharma PhD

1,2,4,5,7; Steven M. Dubinett MD 1,2,4,5,7; and Maie A. St. John MD PhD3,5

Figure 1. (A) p38 was knocked down by ShRNA in Tu686 and Tu212 cells. Protein from whole cell lysates was analyzed for E-cadherin and Snail expression by western blot. p38 knockdown up-regulates E-cadherin and down-regulates Snail expression in HNSCC cell lines. (C) p38 knockdown up regulates expression of E-cadherin in HNSCC cell lines by Immunofluorescence. E-cadherin immunofluorescence staining of Tu212 cells demonstrates strong staining in p38shRNA Tu212 cells as compared to NS Tu212. (D) p38 is required for robust IL-1β induced Vimentin upregulation. Knockdown of p38 expression in the Tu686 line completely diminishes Vimentin expression. Even Il-1β stimulation fails to allow for detectable vimentin expression. (E) p38 Knockdown affects phenotype in Spheroid Model After 4 days in spheroid culture. Tu212 cells demonstrate a grape-like to stellate morphology. Tu212 p38 shRNA cells organize into a round or mass-like configuration. (F) IL-1β induces the phosphorylation of p38 in a time-dependent manner (left). IL-1β has a rapid and robust effect on activation of p38 in snail over expressing cell lines (right).

References

Educational Objective At the conclusion of this presentation, the participants should be able to recognize the role of p38-p38IP signaling in the inflammation-induced promotion of epithelial-to-mesenchymal transition (EMT) in head and neck squamous cell carcinoma (HNSCC).

Figure 1. p38 activity is required for E-Cadherin down regulation and snail upregulation.

1. Elloul S, S.I., Tropé CG, Benshushan A, Davidson B, Reich R. , Expression of E-cadherin transcriptional regulators in ovarian carcinoma. Virchows Arch 2006: 2006(449). 2. Liu X, X.W., Wang XD, Li YF, Han J, Li Y. , The p38-Interacting Protein (p38IP) Regulates G2/M Progression by Promoting α-tubulin Acetylation via Inhibiting Ubiquitination-induced Degradation of the Acetyltransferase GCN5. J Biol Chem, 2013: 288(51):36648-61. 3. Zohn IE, L.Y., Skolnik EY, Anderson KV, Han J, Niswander L. , p38 and a p38-interacting protein are critical for downregulation of E-cadherin during mouse gastrulation. Cell Cycle 2006: 125: 957-969.

Discussion

Figures

Contact info: jmallenstclair@gmail.com

Background In the present study, we investigated the role of p38-p38IP signaling in the inflammation-induced promotion of epithelial-to-mesenchymal transition EMT in HNSCC

Study Design & Methods Quantitative RT-PCR, western blot analysis, spheroid modeling and immunohistochemical staining of human HNSCC tissue sections were used.

Results p38 inhibitor treated and p38 shRNA HNSCC cell lines demonstrate a significant upregulation in E-cadherin mRNA and a decrease in the mRNA expression of Snail. p38 binds to and stabilizes p38IP, a subunit of histone SPT3-TAF9-GCN5 acetyltransferase (STAGA), resulting in enhanced transcription of Snail. p38 shRNA HNSCC cell lines show a less invasive phenotype in a spheroid model. In clinical HNSCC samples, p38 interacting protein (p38IP) is significantly increased compared to adjacent normal tissue. An inverse relationship between p38, p38IP and E-cadherin is demonstrated.

Figure 2. Immunohistochemistry patterns of p38, E-cadherin and Snail expression in early and late stage HNSCC patient specimens.

Figure 3. p38IP upregulation in HNSCC specimens and cell lines.

Figure 2. Reciprocal expression of E-cadherin and p38; E-cadherin and Snail; and co expression of p38 and Snail is evident in HNSCC cells in situ. (A) E-cadherin immunohistochemical staining of a well differentiated stage I squamous cell carcinoma (original magnification, 100x) demonstrates strong membranous staining, especially as the cells become more squamoid. (B) Snail immunohistochemical staining of corresponding section of previously shown well-differentiated squamous cell carcinoma (original magnification, 100x) exhibits faint, nonspecific staining. (C) p38 immunohistochemical staining of corresponding section of previously shown well-differentiated squamous cell carcinoma (original magnification, 100x) exhibits faint, nonspecific staining. (D) E-cadherin immunohistochemical staining of a poorly differentiated stage IV squamous cell carcinoma (original magnification, 100x) shows no staining of tumor cells. (E) Snail immunohistochemical staining of corresponding section of previously shown poorly differentiated squamous cell carcinoma (original magnification, 100x) reveals stronger staining of tumor cells. (F) p38 immunohistochemical staining of corresponding section of previously shown poorly differentiated squamous cell carcinoma (original magnification, 100x) reveals stronger staining of tumor cells. (G-L) Higher magnification immunohistochemical staining (2000x) of E-cadherin, Snail and p38 from previously shown sections A-F.

Figure 3. p38IP mRNA level was increased in HNSCC cells (Tu212, Tu686), compared to normal epithelial cells (HOK, OKF) (p<0.05). (B). p38IP protein was increased in HNSCC cells, compared to normal epithelial cells. (C). p38IP protein was increased in HNSCC patient tumors (T), compared to adjacent normal tissue (N). (D) p38IP mRNA level was increased in HNSCC patient tumors, compared to adjacent normal tissue(p<0.05). (E). p38IP mRNA was measured in an independent HNSCC patient microarray database (NCBI GeoDataset GSE9638) (p<0.05).

Metastasis is a primary cause of mortality in many cancers, including HNSCC. Understanding the molecular mechanisms that mediate EMT may enable identification of novel therapeutic targets. Previous work has demonstrated that loss of E-cadherin gene expression, which is the hallmark of EMT, is mainly due to an up-regulation of Snail and other transcription factors that directly repress E-cadherin expression[1]. To determine the role of p38 in EMT in HNSCC we inhibited p38, and knocked down expression of p38 using shRNA in HNSCC cell lines. p38 kinase inhibitor SB203580 treated, and p38 shRNA HNSCC cell lines demonstrated a significant up regulation in E-cadherin mRNA and a decrease in the mRNA expression of the transcriptional repressor Snail. The addition of SB203580 in HNSCC cell lines also led to an up regulation of other epithelial markers (β-catenin), and a down regulation in mesenchymal markers (vimentin). Interestingly, p38 shRNA HNSCC cell lines show a less invasive and more epithelial phenotype in a 3-dimensional spheroid model. Intriguingly, we found that p38IP, a histone acetyltransferase subunit downstream of p38 signaling, is dysregulated in HNSCC cells and patient samples. p38IP has been reported to promote the function of the acetyltransferase enzyme GCN5 in the STAGA complex in vitro [2]. Combining our data herein with the existing literature [3] it is plausible that upon activation, p38 binds to p38IP and stabilizes p38IP to enhance its function. We demonstrate for the first time herein that in HNSCC: p38IP enhances transcription of STAGA targeted genes by interacting with promoter sequences. Additionally, we observed increased expression of the mesenchymal marker Snail and decreased expression of epithelial marker E-cadherin in p38IP over expressing cells (Figure 6E). Taken together, these data indicate that that p38MAPK-p38IP-GCN5 axis is important for the Snail-induced E-cadherin down-regulation and cell invasion. Here we document p38-dependent transcriptional regulation of E-cadherin in HNSCC. Furthermore, in human oral squamous cell carcinoma we confirm a reciprocal relationship between p38 and E-cadherin. These findings suggest that therapies targeting the p38-p38IP pathway may diminish the propensity for tumor metastasis in HNSCC by blocking the subsequent activation of EMT. This newly defined pathway for transcriptional regulation of E-cadherin in HNSCC may have important implications for chemoprevention as well as therapies utilizing p38 inhibitors in combination with other agents.

Figure 4. Immunohistochemical staining of HNSCC patient specimens and adjacent normal tissues.

Figure 4. (Left). H&E staining and immunohistochemical staining of p38IP and p-p38 in consecutive HNSCC patient specimens and adjacent normal tongue epithelial tissues (original magnification, 100x) showed stronger staining of p-p38 and p38IP in tumor sites. (Right). H&E, p38IP and p-p38 staining (high magnification, 2000x) in previous shown HNSCC patient specimens and adjacent normal tongue epithelial tissues confirmed stronger staining of p-p38 and p38IP in tumor sites.

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Figure 5. p38 mediated activation of p38IP in inflammation.

Figure 5. (A). Inflammatory cytokines increase p38IP levels in HNSCC cells. Tu212 and Tu686 cells were harvested 0, 5 and 30 minutes after TNF-α treatment (B). Hydrogen peroxide increases p38IP levels in HNSCC cells. Tu212 and Tu686 cells were harvested 0, 5, 15, 30 and 60 minutes after 100 µM hydrogen peroxide treatment. (C-D). The induction of p38IP is absent when p38 was knocked-down using shRNA (C) or inhibited by SB203580 (D) in Tu212 cells.

Figure 6. p38IP promoted snail expression.

Figure 6. (A) Structure analysis revealed sequence similarity of p38IP and yeast SPT20. (B). Co-immunoprecipitation revealed that p38IP binds to GCN5 of STAGA complex. 293T cells were co-transfected with HA-p38IP and FLAG-GCN5. Cell lysate was precipitated and blotted with respective antibodies. (C). Real-time PCR demonstrated that p38IP over expression led to increases in STAGA targeted gene GTF2H4 and c-Myc target gene TERT expression levels (p<0.05). (D). Chromatin precipitation showed enhanced association of p38IP and STAGA-targeting promoters upon activation(p<0.05). Genomic DNA in HA-p38IP over expressing 293T cells was precipitated using HA antibody. STAGA specific promoter primers V1 V2 V3 V4 were used to identify p38IP binding in quantitative PCR. (E). p38IP over expression increased transcription of Snail (p<0.05). mRNA levels of Snail and E-cadherin were measured by real-time PCR. (F). p38 MAPK--p38IP drive Snail-induced EMT and cell invasion.