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Smad4 loss synergizes with TGFα overexpression in promoting pancreatic metaplasia, PanIN development, and fibrosis.

Garcia-Carracedo D, Yu CC, Akhavan N, Fine SA, Schönleben F, Maehara N, Karg DC, Xie C, Qiu W, Fine RL, Remotti HE, Su GH - PLoS ONE (2015)

Bottom Line: PanIN lesions and number of ducts were counted, and proliferation was measured by Ki67 immunohistochemistry (IHC).Expression analyses of fibrosis, pancreatitis, or desmoplasia associated markers (α-SMA, Shh, COX-2, Muc6, Col1a1, and Ctgf) were performed by IHC and/or qRT-PCR.The inactivation of Smad4 in the exocrine compartment was responsible for both the enhanced PanIN formation and fibrosis in the pancreas.

View Article: PubMed Central - PubMed

Affiliation: The Department of Pathology, Columbia University Medical Center, New York, New York, United States of America; Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York, United States of America.

ABSTRACT

Aims: While overexpression of TGFα has been reported in human pancreatic ductal adenocarcinoma (PDAC), mice with overexpressed TGFα develop premalignant pancreatic acinar-to-ductal metaplasia (ADM) but not PDAC. TGF-β signaling pathway is pivotal to the development of PDAC and tissue fibrosis. Here we sought to investigate the interplay between TGFα and TGF-β signaling in pancreatic tumorigenesis and fibrosis, namely via Smad4 inactivation.

Methods: The MT-TGFα mouse was crossed with a new Smad4 conditional knock-out mouse (Smad4flox/flox;p48-Cre or S4) to generate Smad4flox/flox;MT-TGFα;p48-Cre (STP). After TGFα overexpression was induced with zinc sulfate water for eight months, the pancreata of the STP, MT-TGFα, and S4 mice were examined for tumor development and fibrotic responses. PanIN lesions and number of ducts were counted, and proliferation was measured by Ki67 immunohistochemistry (IHC). Qualitative analysis of fibrosis was analyzed by Trichrome Masson and Sirius Red staining, while vimentin was used for quantification. Expression analyses of fibrosis, pancreatitis, or desmoplasia associated markers (α-SMA, Shh, COX-2, Muc6, Col1a1, and Ctgf) were performed by IHC and/or qRT-PCR.

Results: Our STP mice exhibited advanced ADM, increased fibrosis, increased numbers of PanIN lesions, overexpression of chronic pancreatitis-related marker Muc6, and elevated expression of desmoplasia-associated marker Col1A1, compared to the MT-TGFα mice. The inactivation of Smad4 in the exocrine compartment was responsible for both the enhanced PanIN formation and fibrosis in the pancreas. The phenotype of the STP mice represents a transient state from ADMs to PanINs, closely mimicking the interface area seen in human chronic pancreatitis associated with PDAC.

Conclusion: We have documented a novel mouse model, the STP mice, which displayed histologic presentations reminiscent to those of human chronic pancreatitis with signs of early tumorigenesis. The STP mice could be a suitable animal model for interrogating the transition of chronic pancreatitis to pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus

Significant increased number of PanIN lesions observed in the STP mice.(A) Pancreas specimens from 4 and 8-months zinc sulfate treated MT-TGFα (panels i, iii, v, vii) and STP (panels ii, iv, vi, viii) were H&E stained (panels i-iv), co-immunolabeled (co-IHC) with antibodies against cytokeratin-19 (brown) and amylase (pink) (panels v, vi), or Alcian blue stained (panels vii, viii). MT-TGFα mice display ductal proliferation with increased ADM at both 4 and 8 months of treatment (panels i, iii). STP mice showed progression from ADM to PanIN-1 & -2 lesions (panels ii, iv). Arrows indicate ADM lesions and plus signs denote PanIN-1 lesions. The occurrence of ADMs in both models was demonstrated by co-IHC of Ck19 and amylase (panels v, vi). Alcian blue indicated vastly increased mucin contents within ducts in the STP mice (panel viii) over the MT-TGFα mice (panel vii). (B) Morphometric analysis of pancreata from MT-TGFα and STP mice treated with 8-months of zinc sulfate. Four mice per genotype were analyzed with a minimum of five fields counted per mouse (100x). (Upper panel): The graph depicts the average number of ducts per 200x field. There was no significant difference in the number of total ductal structures counted in both models. (Middle panel): The graph depicts the average number of PanIN-1 & -2 lesions per 200x field. The STP mice exhibited marked increase of PanIN-1 & -2 lesions than the MT-TGFα mice. (Lower panel): Percentage of Alcian blue positive area per 100x field was presented. The increased PanIN lesions were consistent to the enhanced Alcian blue positivity in the STP mice. Values are presented as mean ±SEM. (Student’s t-test, ** p < 0.05). Magnifications: panels 100X; insets 400x.
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pone.0120851.g003: Significant increased number of PanIN lesions observed in the STP mice.(A) Pancreas specimens from 4 and 8-months zinc sulfate treated MT-TGFα (panels i, iii, v, vii) and STP (panels ii, iv, vi, viii) were H&E stained (panels i-iv), co-immunolabeled (co-IHC) with antibodies against cytokeratin-19 (brown) and amylase (pink) (panels v, vi), or Alcian blue stained (panels vii, viii). MT-TGFα mice display ductal proliferation with increased ADM at both 4 and 8 months of treatment (panels i, iii). STP mice showed progression from ADM to PanIN-1 & -2 lesions (panels ii, iv). Arrows indicate ADM lesions and plus signs denote PanIN-1 lesions. The occurrence of ADMs in both models was demonstrated by co-IHC of Ck19 and amylase (panels v, vi). Alcian blue indicated vastly increased mucin contents within ducts in the STP mice (panel viii) over the MT-TGFα mice (panel vii). (B) Morphometric analysis of pancreata from MT-TGFα and STP mice treated with 8-months of zinc sulfate. Four mice per genotype were analyzed with a minimum of five fields counted per mouse (100x). (Upper panel): The graph depicts the average number of ducts per 200x field. There was no significant difference in the number of total ductal structures counted in both models. (Middle panel): The graph depicts the average number of PanIN-1 & -2 lesions per 200x field. The STP mice exhibited marked increase of PanIN-1 & -2 lesions than the MT-TGFα mice. (Lower panel): Percentage of Alcian blue positive area per 100x field was presented. The increased PanIN lesions were consistent to the enhanced Alcian blue positivity in the STP mice. Values are presented as mean ±SEM. (Student’s t-test, ** p < 0.05). Magnifications: panels 100X; insets 400x.

Mentions: Mice with homozygous deletion of Smad4 in the pancreas showed no evidence of any gross anatomic or physiological abnormalities, and exhibited normal pancreatic cytoarchitecture (Fig. 1B, Fig. 2i, & S2 Fig.). As previously reported, pancreata of the MT-TGFα mice underwent a progressive histologic transformation, characterized by diffuse fibrosis and altered acinar cell structure, ADM, ductal proliferation and dilatation [9, 10] (Fig. 2iii & Fig. 3A-i, iii). The STP mice showed more pronounced interlobular fibrosis, tubular metaplasia, islands of proliferating cells within the tubules and ADM that involved progressive dilatation of the acinar lumen (Fig. 2v & Fig. 3A-ii, iv). With the decrease in the height of acinar cells, we also observed a cuboidal epithelium morphologically simulating metaplastic ducts and/or PanIN-1 in the STP mice (Fig. 3A-iv). Both the MT-TGFα mice and STP mice presented disruption of islet cells due to ductal proliferation and increased fibrosis as shown (S2v-vii Fig., S2ix-xi Fig.). The diminished exocrine compartment observed was likely due to ADM as demonstrated by co-IHC of amylase and CK19 (Fig. 3A-v, vi & S2iv, iii, xii Fig.).


Smad4 loss synergizes with TGFα overexpression in promoting pancreatic metaplasia, PanIN development, and fibrosis.

Garcia-Carracedo D, Yu CC, Akhavan N, Fine SA, Schönleben F, Maehara N, Karg DC, Xie C, Qiu W, Fine RL, Remotti HE, Su GH - PLoS ONE (2015)

Significant increased number of PanIN lesions observed in the STP mice.(A) Pancreas specimens from 4 and 8-months zinc sulfate treated MT-TGFα (panels i, iii, v, vii) and STP (panels ii, iv, vi, viii) were H&E stained (panels i-iv), co-immunolabeled (co-IHC) with antibodies against cytokeratin-19 (brown) and amylase (pink) (panels v, vi), or Alcian blue stained (panels vii, viii). MT-TGFα mice display ductal proliferation with increased ADM at both 4 and 8 months of treatment (panels i, iii). STP mice showed progression from ADM to PanIN-1 & -2 lesions (panels ii, iv). Arrows indicate ADM lesions and plus signs denote PanIN-1 lesions. The occurrence of ADMs in both models was demonstrated by co-IHC of Ck19 and amylase (panels v, vi). Alcian blue indicated vastly increased mucin contents within ducts in the STP mice (panel viii) over the MT-TGFα mice (panel vii). (B) Morphometric analysis of pancreata from MT-TGFα and STP mice treated with 8-months of zinc sulfate. Four mice per genotype were analyzed with a minimum of five fields counted per mouse (100x). (Upper panel): The graph depicts the average number of ducts per 200x field. There was no significant difference in the number of total ductal structures counted in both models. (Middle panel): The graph depicts the average number of PanIN-1 & -2 lesions per 200x field. The STP mice exhibited marked increase of PanIN-1 & -2 lesions than the MT-TGFα mice. (Lower panel): Percentage of Alcian blue positive area per 100x field was presented. The increased PanIN lesions were consistent to the enhanced Alcian blue positivity in the STP mice. Values are presented as mean ±SEM. (Student’s t-test, ** p < 0.05). Magnifications: panels 100X; insets 400x.
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Related In: Results  -  Collection

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pone.0120851.g003: Significant increased number of PanIN lesions observed in the STP mice.(A) Pancreas specimens from 4 and 8-months zinc sulfate treated MT-TGFα (panels i, iii, v, vii) and STP (panels ii, iv, vi, viii) were H&E stained (panels i-iv), co-immunolabeled (co-IHC) with antibodies against cytokeratin-19 (brown) and amylase (pink) (panels v, vi), or Alcian blue stained (panels vii, viii). MT-TGFα mice display ductal proliferation with increased ADM at both 4 and 8 months of treatment (panels i, iii). STP mice showed progression from ADM to PanIN-1 & -2 lesions (panels ii, iv). Arrows indicate ADM lesions and plus signs denote PanIN-1 lesions. The occurrence of ADMs in both models was demonstrated by co-IHC of Ck19 and amylase (panels v, vi). Alcian blue indicated vastly increased mucin contents within ducts in the STP mice (panel viii) over the MT-TGFα mice (panel vii). (B) Morphometric analysis of pancreata from MT-TGFα and STP mice treated with 8-months of zinc sulfate. Four mice per genotype were analyzed with a minimum of five fields counted per mouse (100x). (Upper panel): The graph depicts the average number of ducts per 200x field. There was no significant difference in the number of total ductal structures counted in both models. (Middle panel): The graph depicts the average number of PanIN-1 & -2 lesions per 200x field. The STP mice exhibited marked increase of PanIN-1 & -2 lesions than the MT-TGFα mice. (Lower panel): Percentage of Alcian blue positive area per 100x field was presented. The increased PanIN lesions were consistent to the enhanced Alcian blue positivity in the STP mice. Values are presented as mean ±SEM. (Student’s t-test, ** p < 0.05). Magnifications: panels 100X; insets 400x.
Mentions: Mice with homozygous deletion of Smad4 in the pancreas showed no evidence of any gross anatomic or physiological abnormalities, and exhibited normal pancreatic cytoarchitecture (Fig. 1B, Fig. 2i, & S2 Fig.). As previously reported, pancreata of the MT-TGFα mice underwent a progressive histologic transformation, characterized by diffuse fibrosis and altered acinar cell structure, ADM, ductal proliferation and dilatation [9, 10] (Fig. 2iii & Fig. 3A-i, iii). The STP mice showed more pronounced interlobular fibrosis, tubular metaplasia, islands of proliferating cells within the tubules and ADM that involved progressive dilatation of the acinar lumen (Fig. 2v & Fig. 3A-ii, iv). With the decrease in the height of acinar cells, we also observed a cuboidal epithelium morphologically simulating metaplastic ducts and/or PanIN-1 in the STP mice (Fig. 3A-iv). Both the MT-TGFα mice and STP mice presented disruption of islet cells due to ductal proliferation and increased fibrosis as shown (S2v-vii Fig., S2ix-xi Fig.). The diminished exocrine compartment observed was likely due to ADM as demonstrated by co-IHC of amylase and CK19 (Fig. 3A-v, vi & S2iv, iii, xii Fig.).

Bottom Line: PanIN lesions and number of ducts were counted, and proliferation was measured by Ki67 immunohistochemistry (IHC).Expression analyses of fibrosis, pancreatitis, or desmoplasia associated markers (α-SMA, Shh, COX-2, Muc6, Col1a1, and Ctgf) were performed by IHC and/or qRT-PCR.The inactivation of Smad4 in the exocrine compartment was responsible for both the enhanced PanIN formation and fibrosis in the pancreas.

View Article: PubMed Central - PubMed

Affiliation: The Department of Pathology, Columbia University Medical Center, New York, New York, United States of America; Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York, United States of America.

ABSTRACT

Aims: While overexpression of TGFα has been reported in human pancreatic ductal adenocarcinoma (PDAC), mice with overexpressed TGFα develop premalignant pancreatic acinar-to-ductal metaplasia (ADM) but not PDAC. TGF-β signaling pathway is pivotal to the development of PDAC and tissue fibrosis. Here we sought to investigate the interplay between TGFα and TGF-β signaling in pancreatic tumorigenesis and fibrosis, namely via Smad4 inactivation.

Methods: The MT-TGFα mouse was crossed with a new Smad4 conditional knock-out mouse (Smad4flox/flox;p48-Cre or S4) to generate Smad4flox/flox;MT-TGFα;p48-Cre (STP). After TGFα overexpression was induced with zinc sulfate water for eight months, the pancreata of the STP, MT-TGFα, and S4 mice were examined for tumor development and fibrotic responses. PanIN lesions and number of ducts were counted, and proliferation was measured by Ki67 immunohistochemistry (IHC). Qualitative analysis of fibrosis was analyzed by Trichrome Masson and Sirius Red staining, while vimentin was used for quantification. Expression analyses of fibrosis, pancreatitis, or desmoplasia associated markers (α-SMA, Shh, COX-2, Muc6, Col1a1, and Ctgf) were performed by IHC and/or qRT-PCR.

Results: Our STP mice exhibited advanced ADM, increased fibrosis, increased numbers of PanIN lesions, overexpression of chronic pancreatitis-related marker Muc6, and elevated expression of desmoplasia-associated marker Col1A1, compared to the MT-TGFα mice. The inactivation of Smad4 in the exocrine compartment was responsible for both the enhanced PanIN formation and fibrosis in the pancreas. The phenotype of the STP mice represents a transient state from ADMs to PanINs, closely mimicking the interface area seen in human chronic pancreatitis associated with PDAC.

Conclusion: We have documented a novel mouse model, the STP mice, which displayed histologic presentations reminiscent to those of human chronic pancreatitis with signs of early tumorigenesis. The STP mice could be a suitable animal model for interrogating the transition of chronic pancreatitis to pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus