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Open Access

Peer-reviewed

Research Article

Expression of c-fos Was Associated with Clinicopathologic Characteristics and Prognosis in Pancreatic Cancer

  • Jun-Chao Guo ,

    Contributed equally to this work with: Jun-Chao Guo, Jian Li

    Affiliation Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing, China

    ⨯
  • Jian Li ,

    Contributed equally to this work with: Jun-Chao Guo, Jian Li

    Affiliation Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing, China

    ⨯
  • Yu-Pei Zhao ,

    zhao8028@263.net

    Affiliation Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing, China

    ⨯
  • Li Zhou,

    Affiliation Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing, China

    ⨯
  • Quan-Cai Cui,

    Affiliation Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing, China

    ⨯
  • Wei-Xun Zhou,

    Affiliation Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing, China

    ⨯
  • Tai-Ping Zhang,

    Affiliation Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing, China

    ⨯
  • Lei You

    Affiliation Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing, China

    ⨯

Abstract

It has long been regarded that pancreatic cancer (PC) is a life-threatening malignant tumor. Thus, much attention has been paid for factors, especially relative molecules, predictive for prognosis of PC. However, c-fos expression in PC was less investigated. In addition, its association with clinicopathologic variables and prognosis remains unknown. In the present study, expression of c-fos was detected by tissue microarray-based immunohistochemical staining in cancer and adjacent tissues from 333 patients with PC. The staining results were correlated with clinicopathologic parameters and overall survival. Furthermore, prognostic significance of c-fos in subsets of PC was also evaluated. It was shown that low expression of c-fos was more often in cancer than in adjacent tissues of PC (P<0.001). Besides, high cancerous c-fos expression was significantly associated with tumor site and T stage, whereas peri-neural invasion was of a borderline significant relevance. Log-rank test revealed that high expression of c-fos in cancer tissues was a significant marker of poor overall survival, accompanied by some conventional clinicopathologic variables, such as sex, grade, peri-neural invasion, T and N stages. More importantly, cancerous c-fos expression was identified as an independent prognosticator in multivariate analysis. Finally, the prognostic implication of c-fos expression was proven in four subsets of patients with PC. These data suggested that c-fos expression was of relationships with progression and dismal prognosis of PC.

Citation: Guo J-C, Li J, Zhao Y-P, Zhou L, Cui Q-C, Zhou W-X, et al. (2015) Expression of c-fos Was Associated with Clinicopathologic Characteristics and Prognosis in Pancreatic Cancer. PLoS ONE 10(3): e0120332. https://doi.org/10.1371/journal.pone.0120332

Academic Editor: William B. Coleman, University of North Carolina School of Medicine, UNITED STATES

Received: October 17, 2014; Accepted: January 30, 2015; Published: March 19, 2015

Copyright: © 2015 Guo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper.

Funding: This work was supported by National High Technology Research and Development Program (863 Program, 2014AA020609, http://program.most.gov.cn/), China. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Pancreatic cancer (PC) is one of most life-threatening solid neoplasms, because this type of malignancy carries the mortality almost equal to its incidence [1]. It was recently summarized that improvement in long-term prognosis of PC, although surgical resection was more and more applied, was not achieved within two decades [2]. Therefore, exploration of prognostic markers of PC has been the hotspot of study. Thus far, many clinical and pathologic ones, such as lymph node status, neural invasion, tumor marker levels and resection margin, were identified [3–8]. On the other hand, impacts of molecules involved in tumor pathogenesis and progression on prognosis of PC were gradually valued and recently reviewed [9,10]. Of course, more molecular prognosticators need to be discovered.

It has been well known that c-fos (the human homolog of the retroviral oncogene v-Fos), one of major components of activator protein (AP)-1 complex, mediates cancer growth, angiogenesis, invasion and metastasis, by regulating expressions of down-stream genes [11]. The findings that c-fos expression was significantly associated with recurrence, metastasis and poor prognosis in squamous lung cancer, breast cancer and osteosarcoma provided its potential as a proto-oncogene [12–14]. However, other data suggested that patients with high c-fos expression carried favorable survival in colorectal, ovarian and gastric cancers [15–18]. Thus, c-fos expression and its biological effects might be tissue-type specific. In PC, quite different positive rates of c-fos and its adverse associations with malignant phenotypes, such as lymph node metastasis and multidrug resistance, were previously reported [19–22]. However, its clinicopathologic and prognostic significances in PC remain unknown.

In the present study, the authors aimed to address the issues, based on a large Chinese cohort with PC.

Materials and Methods

Patients

The study cohort consisted of 333 patients with PC, including 206 males and 127 females. Ages ranged from 34 to 85 (median: 60) years. Patient selection criteria: (1) Histologically proven adenocarcinoma; (2) Surgically resected specimens available; (3) Clinicopathologic data available. Table 1 summarizes the clinicopathologic characteristics of the patients. The paired formalin-fixed paraffin-embedded cancer and non-cancer tissues for patients were used. The written informed consent of all patients was obtained. The project (including its consent procedure) was approved by the Ethics Committee of Peking Union Medical College Hospital.

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Table 1. The criteria on the grades integrating positive ratio and intensity.

https://doi.org/10.1371/journal.pone.0120332.t001

Construction of Tissue Microarray (TMA)

Tissue microarray construction was performed according to methods and standards same with those previously reported [23].

Immunohistochemical Staining

A rabbit anti-human polyclonal antibody for c-fos (sc-52, dilution: 1: 50, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a two-step immunostaining kit (EnVisionTM+kit, Dako, Denmark) were used for staining. The blocking peptide (Santa Cruz) was adopted for specificity validation of the primary antibody. Slides were stained in accordance with the method that was previously applied [23]. Non-immune rabbit serum at the same dilution was adopted as the negative control.

Evaluation of Staining Results

Two independent pathologists who were blinded to the clinicopathologic and survival data (Q.C. C. and W.X. Z.) evaluated staining results. The brown nuclear and/or cytoplasm coloration was defined as the positive signal. According to previous suggested [24], the ratio of positive cells was divided into three grades (0–10%, grade 1; >10-≥50%, grade 2; >50%, grade 3). On the other hand, staining intensity was also scored to three groups (faint, score 1; moderate, score 2; intensive, score 3). The final grade was obtained by multiplication of positive ratio grade and intensity score (shown in Table 1), and separated into low (final grade <6) and high expressions (final grade ≥6).

Follow-Up

Follow-up (median: 14 months; range: 2 to 87 months) was performed for 229 patients, mainly through outpatient clinic, telephone and mail. During the follow-up term, 170 patients have died.

Statistical Analysis

McNemar's test and Pearson’s correlation analysis were used to compare c-fos expression between cancer and adjacent non-cancer tissues, and to show the correlation between them. The association between c-fos expression and clinicopathologic characteristics was revealed by Chi-square test. Kaplan-Meier method and log-rank test were applied to estimate overall survival and their differences. Multivariate Cox regression (Proportional hazard model) analysis was performed for identification of independent prognostic factors. All the analyses were finished by statistical software package SPSS11.5 (SPSS Inc, Chicago, IL). A P value less than 0.05 was defined as statistically significant.

Results

Expression of c-fos and Its Association with Clinicopathologic Characteristics in PC

It was shown that the positive signal mainly located in cell nucleus, and being also seen in cytoplasm (Fig. 1). After treatment with the blocking peptide, the finding that positive signal disappeared (S1 Fig.) validated the specificity of the primary antibody. Low and high expressions were found in cancer tissues from 167 and 166 patients with PC, and adjacent non-cancer tissues from 63 and 270 individuals. Low c-fos expression was more common in cancer than in adjacent tissues (P<0.001, McNemar's test). There were significant correlation between c-fos expression in cancer tissues and that in adjacent non-cancer tissues (P<0.001, Pearson’s correlation analysis). Chi-square analysis showed that high cancerous c-fos expression was significantly frequent in non-head and T3 tumors, compared with head and T1–2 ones, respectively (Table 2, P = 0.013 and 0.017). Moreover, c-fos expression in cancer tissues was of marginally significant relationship with perineural invasion (Table 2, P = 0.069). Expression of c-fos in adjacent tissues was not related to clinicopathologic parameters included (data not shown).

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Fig 1. Expression of c-fos in pancreatic cancer.

(A) High expression in cancer tissue (original magnification ×200). (B) High expression in adjacent non-cancer tissue (original magnification ×200).

https://doi.org/10.1371/journal.pone.0120332.g001

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Table 2. Associations of cancerous c-fos expression with clinicopathologic characteristics of PC.

https://doi.org/10.1371/journal.pone.0120332.t002

Prognostic Significance of c-fos in the Whole Cohort of PC

Univariate analysis in the whole cohort of PC showed that high cancerous c-fos expression predicted shortened overall survival (P = 0.020; Fig. 2 and Table 3), in addition to sex, histological grade, perineural invasion, T and N stages (P<0.05; Table 3). Multivariate Cox regression defined c-fos expression as one of independent prognostic indicators for overall survival of PC (P = 0.022; Table 3).

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Fig 2. Post-surgical overall survival of pancreatic cancer according to c-fos expression in cancer tissues (Log-rank test; P = 0.020).

https://doi.org/10.1371/journal.pone.0120332.g002

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Table 3. Prognostic factors of PC after resection (the whole cohort).

https://doi.org/10.1371/journal.pone.0120332.t003

Influences of c-fos Expression on Overall Survival in Subsets of PC

Because the present study evaluated eight clinicopathologic variables, patients were divided into sixteen subsets. Univariate analysis defined cancerous c-fos expression as a significant predictor for overall survival in four ones, i.e. male patients, tumors ≤4cm, N0 tumors and those without perineural invasion (P<0.05; Fig. 3). Cox regression analysis established that c-fos expression in cancer tissues was of independent prognostic implication in males, N0 tumors and those without perineural invasion (P<0.05; Table 4).

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Fig 3. Prognostic significances of cancerous c-fos expression in subsets of PC after resection.

(A) Male patients (P = 0.006). (B) Tumors ≤4cm (P = 0.026). (C) Tumors without peri-neural invasion (P = 0.039). (D) N0 tumors (P = 0.008). N, lymph node.

https://doi.org/10.1371/journal.pone.0120332.g003

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Table 4. Multivariate Cox regression analysis for prognostic significance of cancerous c-fos expression in some subsets of PC after resection.

https://doi.org/10.1371/journal.pone.0120332.t004

Discussion

As an important member of the fos family of transcription factors, c-fos was demonstrated to be a key regulator of proliferation, differentiation, angiogenesis, invasion and metastasis of cancer, through AP-1-related mechanisms [11]. However, it is interesting that c-fos plays different, even opposite roles in different cancers. In breast and bladder cancers as well as glioma cells [25–28], c-fos seems to have pro-oncogenic properties, unlike in ovarian cancer [29]. Although in a same kind of malignancy, PC, results concerning expression and phenotypes of c-fos remain to be inconsistent. Using immunohistochemistry for a few individuals, Lee et al. [19] found that c-fos was highly expressed in PC tissues, but only weak expression was observed in a small number of patients enrolled in another study [20]. Furthermore, the findings that c-fos was down-regulated in PC with lymph node metastasis and increased c-fos expression induced multidrug resistance 1 (MDR1) in PC cells indicate the complex roles of the molecule in PC [21,22]. Other investigations that showed that c-fos contributes to cell motility [30] and functions as one of targets of some tumor suppressor genes [31,32], support its oncogenic potential in PC. In the current investigation, expression of c-fos was first detected for more than 300 patients with PC. It is unexpected that low c-fos expression is more frequent in cancer than in adjacent non-cancer tissues (McNemar's test). Then, the positive/potentially positive associations between high c-fos expression and later T stage as well as presence of perineural invasion were shown. The results indicate that c-fos might play crucial roles in both initiation and progression of PC. Previous work found that c-fos induced inflammation-mediated tumorigenesis [33]. In addition, chronic pancreatitis (CP) was well known as a significant etiological factor of PC [34]. Therefore, whether c-fos plays an important role in the process from CP to PC might be of interest. It was suggested that K-ras, a crucial mutated gene in both CP and PC, activated Raf/MEK/extracellular signal-regulated kinase (ERK) pathway through a complex cascade [34], whereas ERK, a kind of mitogen-activated protein kinase (MAPK), was involved in activation of c-fos [35]. Thus, high c-fos expression in adjacent non-cancer tissues in the current study might provide a preliminary clue of its impacts on conversion from inflammation to malignancy in the pancreas. On the other hand, cancerous c-fos expression was revealed to be significantly and marginally significantly associated with T stage and perineural invasion, indicating that this transcriptional factor might be a promoter of PC progression. Taken together, our data supported that c-fos serves as a proto-oncogene in the course from malignant transformation to progression in PC, different with previous one showing down-regulation of c-fos in PC with lymph node metastasis [21]. In the future, detailed explorations for phenotypes and relative mechanisms worth to be done.

It was shown that c-fos was linked to poor or good prognosis in many types of cancers [12–18]. However, its impact on outcome of PC has not been elucidated. Here, we report that high c-fos expression in cancer tissues was associated with unsatisfactory overall survival in both univariate and multivariate analyses, presenting its power as a strong predictor of gloomy prognosis in PC. The results that c-fos was of independent prognostic significance in some subsets of PC strengthen its prognostic role. These findings, supported by the positive relationship between c-fos expression and malignant phenotypes of PC established in the present study, are consistent with those derived from osteosarcoma, lung and breast cancers [12–14]. No doubt these survival analysis results also indicate the function of c-fos as a proto-oncogene.

In conclusion, our data demonstrated that c-fos expression is related to progression and dismal prognosis of PC. Thus, this protein might be a novel molecular maker of PC.

Supporting Information

S1 Fig. Specificity validation of the primary antibody against c-fos in pancreatic cancer.

(A) With blocking peptide (original magnification ×200). (B) Without blocking peptide (original magnification ×200).

https://doi.org/10.1371/journal.pone.0120332.s001

(TIF)

Author Contributions

Conceived and designed the experiments: YPZ JCG. Performed the experiments: JCG JL. Analyzed the data: JCG LZ QCC WXZ. Contributed reagents/materials/analysis tools: TPZ LY. Wrote the paper: JCG LZ.

References

  1. 1. Hidalgo M. Pancreatic cancer. N Engl J Med. 2010; 362: 1605–1617. pmid:20427809
  2. 2. Witkowski ER, Smith JK, Tseng JF. Outcomes following resection of pancreatic cancer. J Surg Oncol. 2013; 107: 97–103. pmid:22991309
  3. 3. Murakami Y, Uemura K, Sudo T, Hashimoto Y, Yuasa Y, Sueda T. Prognostic impact of para-aortic lymph node metastasis in pancreatic ductal adenocarcinoma. World J Surg. 2010; 34: 1900–1907. pmid:20376442
  4. 4. La Torre M, Cavallini M, Ramacciato G, Cosenza G, Rossi Del Monte S, Nigri G, et al. Role of the lymph node ratio in pancreatic ductal adenocarcinoma. Impact on patient stratification and prognosis. J Surg Oncol. 2011; 104: 629–633. pmid:21713779
  5. 5. Chatterjee D, Rashid A, Wang H, Katz MH, Wolff RA, Varadhachary GR, et al. Tumor invasion of muscular vessels predicts poor prognosis in patients with pancreatic ductal adenocarcinoma who have received neoadjuvant therapy and pancreaticoduodenectomy. Am J Surg Pathol. 2012; 36: 552–559. pmid:22301496
  6. 6. Chatterjee D, Katz MH, Rashid A, Wang H, Iuga AC, Varadhachary GR, et al. Perineural and intraneural invasion in posttherapy pancreaticoduodenectomy specimens predicts poor prognosis in patients with pancreatic ductal adenocarcinoma. Am J Surg Pathol. 2012; 36: 409–417. pmid:22301497
  7. 7. Distler M, Pilarsky E, Kersting S, Grützmann R. Preoperative CEA and CA 19-9 are prognostic markers for survival after curative resection for ductal adenocarcinoma of the pancreas—a retrospective tumor marker prognostic study. Int J Surg. 2013; 11: 1067–1072. pmid:24161419
  8. 8. Zhang Y, Frampton AE, Cohen P, Kyriakides C, Bong JJ, Habib NA, et al. Tumor infiltration in the medial resection margin predicts survival after pancreaticoduodenectomy for pancreatic ductal adenocarcinoma. J Gastrointest Surg. 2012; 16: 1875–1882. pmid:22878786
  9. 9. Ansari D, Rosendahl A, Elebro J, Andersson R. Systematic review of immunohistochemical biomarkers to identify prognostic subgroups of patients with pancreatic cancer. Br J Surg. 2011; 98: 1041–1055. pmid:21644238
  10. 10. Winter JM, Yeo CJ, Brody JR. Diagnostic, prognostic, and predictive biomarkers in pancreatic cancer. J Surg Oncol. 2013; 107: 15–22. pmid:22729569
  11. 11. Milde-Langosch K. The Fos family of transcription factors and their role in tumourigenesis. Eur J Cancer. 2005; 41: 2449–2461. pmid:16199154
  12. 12. Volm M, Drings P, Wodrich W. Prognostic significance of the expression of c-fos, c-jun and c-erbB-1 oncogene products in human squamous cell lung carcinomas. J Cancer Res Clin Oncol. 1993; 119: 507–510. pmid:8100821
  13. 13. Bland KI, Konstadoulakis MM, Vezeridis MP, Wanebo HJ. Oncogene protein co-expression. Value of Ha-ras, c-myc, c-fos, and p53 as prognostic discriminants for breast carcinoma. Ann Surg. 1995; 221: 706–718. pmid:7794075
  14. 14. Gamberi G, Benassi MS, Bohling T, Ragazzini P, Molendini L, Sollazzo MR, et al. C-myc and c-fos in human osteosarcoma: prognostic value of mRNA and protein expression. Oncology. 1998; 55: 556–563. pmid:9778623
  15. 15. Singh A, Tong A, Ognoskie N, Meyer W, Nemunaitis J. Improved survival in patients with advanced colorectal carcinoma failing 5-fluorouracil who received irinotecan hydrochloride and have high intratumor C-fos expression. Am J Clin Oncol. 1998; 21: 466–469. pmid:9781601
  16. 16. Mahner S, Baasch C, Schwarz J, Hein S, Wölber L, Jänicke F, et al. C-Fos expression is a molecular predictor of progression and survival in epithelial ovarian carcinoma. Br J Cancer. 2008; 99: 1269–1275. pmid:18854825
  17. 17. Jin SP, Kim JH, Kim MA, Yang HK, Lee HE, Lee HS, et al. Prognostic significance of loss of c-fos protein in gastric carcinoma. Pathol Oncol Res. 2007; 13: 284–289. pmid:18158562
  18. 18. Zhou L, Zhang JS, Yu JC, Cui QC, Zhou WX, Kang WM, et al. Negative association of c-fos expression as a favorable prognostic indicator in gastric cancer. Arch Med Res. 2010; 41: 201–206. pmid:20682178
  19. 19. Lee CS, Charalambous D. Immunohistochemical localisation of the c-fos oncoprotein in pancreatic cancers. Zentralbl Pathol. 1994; 140: 271–275. pmid:7947635
  20. 20. Sakorafas GH, Lazaris A, Tsiotou AG, Koullias G, Glinatsis MT, Golematis BC. Oncogenes in cancer of the pancreas. Eur J Surg Oncol. 1995; 21: 251–253. pmid:7781791
  21. 21. Kim JH, Lee JY, Lee KT, Lee JK, Lee KH, Jang KT, et al. RGS16 and FosB underexpressed in pancreatic cancer with lymph node metastasis promote tumor progression. Tumour Biol. 2010; 31: 541–548. pmid:20571966
  22. 22. Song B, Liu XS, Rice SJ, Kuang S, Elzey BD, Konieczny SF, et al. Plk1 phosphorylation of orc2 and hbo1 contributes to gemcitabine resistance in pancreatic cancer. Mol Cancer Ther. 2013; 12: 58–68. pmid:23188630
  23. 23. Guo JC, Li J, Zhao YP, Zhou L, Cui QC, Zhou WX, et al. N-WASP in pancreatic ductal adenocarcinoma: associations with perineural invasion and poor prognosis. World J Surg. 2014; 38: 2126–2131. pmid:24718883
  24. 24. Volm M, Rittgen W, Drings P. Prognostic value of ERBB-1, VEGF, cyclin A, FOS, JUN and MYC in patients with squamous cell lung carcinomas. Br J Cancer. 1998; 77: 663–669. pmid:9484827
  25. 25. Andersen H, Mahmood S, Tkach V, Cohn M, Kustikova O, Grigorian M, et al. The ability of Fos family members to produce phenotypic changes in epitheloid cells is not directly linked to their transactivation potentials. Oncogene. 2002; 21: 4843–4848. pmid:12101423
  26. 26. Shi R, Peng H, Yuan X, Zhang X, Zhang Y, Fan D, et al. Down-regulation of c-fos by shRNA sensitizes adriamycin-resistant MCF-7/ADR cells to chemotherapeutic agents via P-glycoprotein inhibition and apoptosis augmentation. J Cell Biochem. 2013; 114: 1890–1900. pmid:23494858
  27. 27. Li S, Xu X, Xu X, Hu Z, Wu J, Zhu Y, et al. MicroRNA-490-5p inhibits proliferation of bladder cancer by targeting c-Fos. Biochem Biophys Res Commun. 2013; 441: 976–981. pmid:24220339
  28. 28. Tao T, Wang Y, Luo H, Yao L, Wang L, Wang J, et al. Involvement of FOS-mediated miR-181b/miR-21 signalling in the progression of malignant gliomas. Eur J Cancer. 2013; 49: 3055–3063. pmid:23810250
  29. 29. Oliveira-Ferrer L, Rößler K, Haustein V, Schröder C, Wicklein D, Maltseva D, et al. c-FOS suppresses ovarian cancer progression by changing adhesion. Br J Cancer. 2014; 110: 753–763. pmid:24322891
  30. 30. Akagi J, Egami H, Kurizaki T, Ohmachi H, Ogawa M. Signal transduction pathway of the induction of cell motility in hamster pancreatic ductal adenocarcinoma cell. Invasion Metastasis. 1997; 17: 16–25. pmid:9425321
  31. 31. Li B, Wan X, Zhu Q, Li L, Zeng Y, Hu D, et al. Net expression inhibits the growth of pancreatic ductal adenocarcinoma cell PL45 in vitro and in vivo. PLoS One. 2013; 8: e57818. pmid:23469073
  32. 32. Poi MJ, Knobloch TJ, Yuan C, Tsai MD, Weghorst CM, Li J. Evidence that P12, a specific variant of P16(INK4A), plays a suppressive role in human pancreatic carcinogenesis. Biochem Biophys Res Commun. 2013; 436: 217–222. pmid:23727582
  33. 33. Briso EM, Guinea-Viniegra J, Bakiri L, Rogon Z, Petzelbauer P, Eils R, et al. Inflammation-mediated skin tumorigenesis induced by epidermal c-Fos. Genes Dev. 2013; 27: 1959–1973. pmid:24029918
  34. 34. Ling S, Feng T, Jia K, Tian Y, Li Y. Inflammation to cancer: The molecular biology in the pancreas (Review). Oncol Lett. 2014; 7: 1747–1754. pmid:24932227
  35. 35. O'Donnell A, Odrowaz Z, Sharrocks AD. Immediate-early gene activation by the MAPK pathways: what do and don't we know? Biochem Soc Trans. 2012; 40: 58–66. pmid:22260666