Analysis of Axin2 expression and function in murine models for pancreatic cancer
© The Author(s) 2016
Received: 11 July 2016
Accepted: 14 July 2016
Published: 22 August 2016
The involvement of Wnt in carcinogenesis and progression of pancreatic cancer is currently intensely discussed. We evaluated activation of the Wnt signaling pathway by using a Wnt reporter mouse strain expressing β-galactosidase under the control of the Axin2 promotor during pancreatitis induced formation of precancerous lesions. We also evaluated activation of Wnt signaling during interaction of pancreatic cancer with the tumor stroma.
Activation of Wnt signaling was observed during acinar-to-ductal metaplasia after chronic as well as acute pancreatitis. Activation of Wnt signaling was also noticed during growth of pancreatic cancer in an orthotopic syngeneic pancreas cancer model. Activation of Wnt signaling was, however, not observed in carcinoma associated fibroblasts, but was detected in few cell clusters inside the tumor. Genetic ablation of Axin2 significantly reduced body weight without having a major impact on blood glucose concentration. However, ablation of Axin2 had no influence on the observed β-galactosidase positive cell clusters or on tumor weight.
These data demonstrate that the Wnt signaling pathway is activated during acinar-to-ductal metaplasia after injury to the pancreas. However these data do not support a major role of Wnt signaling or of Axin2 in carcinoma associated fibroblasts and tumor growth.
KeywordsSolid-pseudopapillary neoplasms Acinar cell carcinoma Founder mutations Cancer immunology Diabetes Hyperglycemia
The expression of Axin2 is induced by canonical Wnt signaling during cancerogenesis but also during many other Wnt-regulated physiological and pathophysiological processes . The introduction of the β-galactosidase gene into the locus of Axin2 in mice, therefore, generated a reporter strain (Axin2+/lacZ), which reliably expresses β-galactosidase in cells where Wnt signaling is activated . Axin2 functions as a scaffold protein that facilitates the phosphorylation and thereby the degradation of β-catenin, which is the key protein necessary for canonical Wnt signaling . Thus, Axin2 inhibits the canonical Wnt signaling pathway in form of a negative feedback loop . In Axin2lacZ/lacZ mice, both Axin2 alleles are replaced by β-galactosidase. This can result in prolonged Wnt signaling in vivo .
It is well established that Wnt signaling is involved in the cancerogenesis of multiple gastrointestinal carcinomas . In colorectal carcinoma 90 % of all tumors have a mutation in a gene coding for regulatory components of the canonical Wnt signaling pathway such as CTNNB1 (β-catenin) or adenomatous polyposis coli (APC). These mutations result in activation of the canonical Wnt signaling pathway . In hepatocellular carcinoma 3 to 44 % of tumors contain mutations of CTNNB1 and 5 to 25 % contain mutations in AXIN1 resulting in activation of Wnt signaling . Loss of function mutation of AXIN2 can also be found in hepatocellular and colorectal carcinoma .
However, it is still controversial how important this signaling pathway is during carcinogenesis of pancreatic cancer . About 24 % of acinar cell carcinomas (ACC) have molecular alterations in the canonical Wnt signaling pathway. Similarly, solid-pseudopapillary neoplasms (SPNs) in the pancreas usually harbor mutations in CTNNB1 . The prognosis of this rare neoplasm is excellent and most patients are cured by surgical resection . To the contrary, the prognosis of pancreatic ductal adenocarcinoma (PDA) is very dismal. Dependent on the study CTNNB1 mutations have been identified in none or very few PDAs [6, 7]. Nevertheless, recent publications suggest that activation of canonical Wnt signaling via alternative mechanisms might contribute to the carcinogenesis of PDA [6, 7]. For example, mutations of RNF43, which can regulate Wnt signaling, were detected in 6–10 % of PDAs [6, 7].
Only few publications exist that suggest that Wnt signaling might also contribute to tumor stroma interaction [8, 9]. This interaction can be based on the expression of distinct Wnts in cancer associated fibroblasts, which promotes tumor progression . Alternatively, expression of Wnts in carcinoma cells can induce Wnt signaling in the desmoplastic reaction, which indirectly promotes tumor aggressiveness .
Thus, the purpose of this study was to evaluate the activation of the Wnt signaling pathway in precancerous lesions such as tubular complexes after acinar-to-ductal metaplasia (ADM) and during the tumor stroma interaction of fully established PDA.
Wnt signaling in tubular complexes
Quantification of β-galactosidase+ tubular complexes during chronic pancreatitis on day 40
Number of β-galactosidase+ tubular complexes
Number of tubular complexes observed
Number of mice analyzed
Quantification of β-galactosidase+ tubular complexes after acute pancreatitis on day 28
Number of β-galactosidase+ tubular complexes
Number of tubular complexes observed
Number of mice analyzed
Thus, Wnt signaling is activated in assumed precancerous lesions such as tubular complexes by pancreatitis, which is a well defined risk factor for the development of pancreatic cancer. This observed activation of canonical Wnt signaling upon tissue damage is consistent with previous publications. For example, it was suggested that the observed increase in β-catenin mRNA level  or β-catenin protein concentration  after tissue damage in the pancreas indicates activation of Wnt signaling. In addition, tissue damage induced the expression of Axin2 as demonstrated by quantitative PCR  and the activation of Wnt signaling in ductal cells was also observed after partial pancreatic duct ligation .
The following three hypotheses can be defined when describing the observed activation of Wnt signaling during ADM in respect to cancerogenesis: (a) Activation of Wnt signaling has no effect on cancerogenesis, but is a concomitant phenomenon when cells are stuck in dedifferentiation, (b) Wnt signaling inhibits cancerogenesis, (c) Wnt signaling promotes cancerogenesis. Possibly, the function of Wnt signaling is completely different during cancerogenesis leading to PDA or other subtypes of pancreatic cancer.
In respect to cancerogenesis of PDA most data support the first or second hypothesis and disagree with the third hypothesis. For example, activation of Wnt signaling does not cooperate with oncogenic Ras to induce PDA formation in mice . Moreover, only few mutations have been found in human PDAs that activate Wnt signaling suggesting that these mutations are not founder mutations of the carcinoma [6, 7].
In respect to cancerogenesis of SPNs or ACCs most data disagree with the first and second hypothesis and support the third hypothesis. For example, activation of Wnt signaling in mice results in the formation of tumors resembling human SPNs , whereas activation of Wnt signaling in p53 deficient mice results in the formation of tumors resembling human ACC . Moreover, human SPNs usually harbor mutations in CTNNB1 that activate Wnt signaling and about 24 % of ACCs in patients show molecular changes in key proteins of the canonical Wnt signaling pathway . Thus, permanent activation of canonical Wnt signaling may promote cancerogenesis of SPNs and ACCs, but not of PDA.
Wnt signaling during tumor stroma interaction
acinar cell carcinoma
pancreatic ductal adenocarcinoma
DZ carried out the study design, the interpretation of data, the cell injections into mice, histochemical evaluations, and drafted the manuscript. All other authors participated in the study (TK: Figs. 1, 2; Tables 1, 2; ACA: Figs. 3c, and 4; MS: Fig. 4, TR: Fig. 3b), carried out the analysis and interpretation of data, and revised the manuscript. BV made substantial contributions to the design of the study and revision of the manuscript. All authors read and approved the final manuscript.
We thank Berit Blendow, Dorothea Frenz, Eva Lorbeer-Rehfeldt, and Maren Nerowski (Institute for Experimental Surgery, Rostock University Medical Center) for excellent technical assistance. The Axin2tm1Wbm mouse strain was a generous gift from Prof. Walter Birchmeier (Berlin, Germany). Funding acknowledgement: Supported by the Forschungsförderung der Medizinischen Fakultät der Rostocker Universität (FORUN) (project 889017).
The authors declare that they have no competing interests.
Availability of data
All data generated or analyzed during this study are included in this published article and its Additional file 1.
All experiments on mice were executed in accordance with German legislation and the EU-directive 2010/63/EU and were approved by the Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern.
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