pubmed.ncbi.nlm.nih.gov

Functional p38 MAPK identified by biomarker profiling of pancreatic cancer restrains growth through JNK inhibition and correlates with improved survival - PubMed

  • ️Wed Jan 01 2014

Functional p38 MAPK identified by biomarker profiling of pancreatic cancer restrains growth through JNK inhibition and correlates with improved survival

Yi Zhong et al. Clin Cancer Res. 2014.

Abstract

Purpose: Numerous biomarkers for pancreatic cancer have been reported. We determined the extent to which such biomarkers are expressed throughout metastatic progression, including those that effectively predict biologic behavior.

Experimental design: Biomarker profiling was performed for 35 oncoproteins in matched primary and metastatic pancreatic cancer tissues from 36 rapid autopsy patients. Proteins of significance were validated by immunolabeling in an independent sample set, and functional studies were performed in vitro and in vivo.

Results: Most biomarkers were similarly expressed or lost in expression in most samples analyzed, and the matched primary and metastases from a specific patient were most similar to each other than to other patients. However, a subset of proteins showed extensive interpatient heterogeneity, one of which was p38 MAPK. Strong positive pp38 MAPK immunolabeling was significantly correlated with improved postresection survival by multivariate analysis (median overall survival 27.9 months, P = 0.041). In pancreatic cancer cells, inhibition of functional p38 by SB202190 increased cell proliferation in vitro in both low-serum and low-oxygen conditions. High functional p38 activity in vitro corresponded to lower levels of pJNK protein expression, and p38 inhibition resulted in increased pJNK and pMKK7 by Western blot analysis. Moreover, JNK inhibition by SP600125 or MKK7 siRNA knockdown antagonized the effects of p38 inhibition by SB202190. In vivo, SP600125 significantly decreased growth rates of xenografts with high p38 activity compared with those without p38 expression.

Conclusions: Functional p38 MAPK activity contributes to overall survival through JNK signaling, thus providing a rationale for JNK inhibition in pancreatic cancer management.

©2014 American Association for Cancer Research.

PubMed Disclaimer

Conflict of interest statement

The authors have no financial conflicts of interest related to this work.

Figures

Figure 1
Figure 1. Biomarker Profiling of a Matched Primary and Metastasis Tissue Microarray

(A). Scanning power view of a hematoxylin and eosin stained section of the TMA constructed for biomarker profiling. An example of cores of the matched primary and liver metastasis from a single patient are shown at 10x magnification. (B) Proteins and evaluated by biomarker profiling. (C) Heatmap generated by unbiased Hierarchical Cluster Analysis of expression data. Dendrograms indicate the presence of three distinct clusters based on patterns of protein expression. The relationship of protein expression to the gross metastatic burdens (MB) of each patient at autopsy are shown to the right of the heat map, with the colors corresponding to no metastases (yellow), 1–10 metastases (green), 11–100 metastases (blue) and >100 metastases (red). For each patient labeling patterns for both the primary carcinoma and matched metastasis are independently plotted on their own row. (D) Box plot illustrating the predicted probability of having a high metastatic burden (>10 metastases). (E) Examples of high (H-score ≥ 150) and low (H-score <150) protein expression for pp38, MUC5AC and S100A2 in the neoplastic epithelium of resected pancreatic cancer tissues. (F) Kaplan Meier survival plot of patients who completed adjuvant chemoradiation with high versus low pp38 immunolabeling of their resected tissues.

Figure 2
Figure 2. Effects of p38 MAPK inhibitor SB202190 on pancreatic cell proliferation

(A) Western blotting for total and phospho-p38 (pp38) MAPK expression in immortalized normal (HPNE, HPDE) and pancreatic cancer cell lines. Expression of actin was used as a loading control. The ratio of phosphorylated to total p38 is shown for each cell line, indicating high levels of phosphorylation for Panc5.04, A10.7 and A38.44 cells. Functional p38 MAPK activity as determined by immunoprecipitation of phosphorylated ATF2, a known protein target of p38, is also shown. (B) Immunohistochemical staining for pp38 MAPK in cancer tissues of patients A2 (source of cell line A2.1) and A6 (source of cell line A6L) indicating low levels of pp38 expression. Cancer cells are indicated by arrowheads and scale bars correspond to 50 microns. (C) Cell lines Panc5.04, A10.7 and A38.44 with high p38 MAPK activity were treated with 10 µM of the p38 MAPK inhibitor SB202190 and subjected to cell proliferation assay. (D) Functional p38 MAPK activity levels as reflected by pATF2 levels in Panc5.04, A10.7 and A38.44 cells after treatment with increasing concentrations of SB202190. (E) A2.1 and A6L cells with low levels of p38 MAPK activity were treated with 10 µM of SB202190 and subjected to cell proliferation assay. 0.1% (v/v) of DMSO was used as a vehicle treated control. *, p<0.05; **, p<0.01; ***, p<0.001 (treated group versus untreated control).

Figure 3
Figure 3. Effects of functional p38 MAPK on cell proliferation through the JNK pathway in pancreatic cancer cells

(A) Western blotting for total and phosphorylated JNK and AKT expression in immortalized normal and pancreatic cancer cells. (B) Western blotting for total and phosphorylated JNK, MKK4 and MKK7 expression levels in Panc5.04, A10.7 and A38.44 cells after treatment with 10 µM of SB202190 for up to 2 days. Quantification of pJNK, pMKK4 and pMKK7 expression was shown as a ratio of normalized phosphorylated protein level in the SB202190 treated group to that of vehicle control. (C) Cell proliferation assays of Panc5.04, A10.7 and A38.44 cells treated with 10 µM of p38 MAPK inhibitor SB202190 alone or in combination with 10 µM of the JNK inhibitor SP600125. 0.1% (v/v) of DMSO was used as a vehicle treated control. *, p<0.05; **, p<0.01; ***, p<0.001 (treated group versus untreated control).

Figure 4
Figure 4. Effects of p38 and JNK inhibition on pancreatic cancer cells in vivo

(A) Representative subcutaneous xenografts of Panc 5.04, A10.7, A2.1 or A6L cell lines harvested from mice treated with vehicle (Veh), SB202190 or SP600125 for three weeks. (B) Summary data of xenograft tumor volumes at the end of the experiment. Values shown are the mean ± S.D. of six unique xenografts per cell line per condition. * p<0.05 (C). Western blots of total protein extracted from A10.7 xenografts after treatment with vehicle, SB202190 or SP600125. Quantification of pJNK, and p-p38 MAPK expression was shown as a ratio of normalized phosphorylated protein in the treated group to that of the vehicle control.

Figure 5
Figure 5. Histologic features of treated xenografts

Hematoxylin and eosin or pJNK immunolabeling of representative xenografts from Panc 5.04, A10.7, A2.1 and A6L cells after treatment with vehicle or SB202190 or SP600125. Positive nuclear labeling is seen in Panc 5.04 and A10.7 cells treated with vehicle or SB202190 (images 600x). By contrast, abundant inflammation was seen in SP600125 treated Panc 5.04 cells (100x), and loss of nuclear labeling was seen in A10.7 (600x) Minimal to no pJNK expression was seen in A2.1 and A6L cells (400x). However, arrows in A2.1 indicate rare pJNK positive labeling nuclei.

Comment in

Similar articles

Cited by

References

    1. Stathis A, Moore MJ. Advanced pancreatic carcinoma: current treatment and future challenges. Nat Rev Clin Oncol. 2010;7:163–172. - PubMed
    1. Hidalgo M. Pancreatic cancer. N Engl J Med. 2010;362:1605–1617. - PubMed
    1. Fong ZV, Winter JM. Biomarkers in pancreatic cancer: diagnostic, prognostic, and predictive. Cancer J. 2012;18:530–538. - PubMed
    1. Winter JM, Tang LH, Klimstra DS, Brennan MF, Brody JR, Rocha FG, et al. A novel survival-based tissue microarray of pancreatic cancer validates MUC1 and mesothelin as biomarkers. PLoS ONE. 2012;7:e40157. - PMC - PubMed
    1. Kitamoto S, Yokoyama S, Higashi M, Yamada N, Takao S, Yonezawa S. MUC1 enhances hypoxia-driven angiogenesis through the regulation of multiple proangiogenic factors. Oncogene. 2012 - PubMed

Publication types

MeSH terms

Substances