Histopathology 2014 DOI: 10.1111/his.12544
Differentiation reprogramming in gastric intestinal metaplasia and dysplasia: role of SOX2 and CDX2 V^ ania Camilo,1 M onica Garrido,2 Pedro Valente,2 Sara Ricardo,1 Ana Luısa Amaral,1 Rita Barros,1 Paula Chaves,3 F atima Carneiro,1,2,4 Leonor David1,2 & Raquel Almeida1,2,5 1
Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 2Faculty of Medicine of the University of Porto, Porto, 3Portuguese Oncology Institute Francisco Gentil, E.P.E., Lisbon, 4Pathology Department, Centro Hospitalar S. Jo~ao, and 5Biology Department, Faculty of Sciences of the University of Porto, Porto, Portugal
Date of submission 14 April 2014 Accepted for publication 31 August 2014 Published online Article Accepted 6 September 2014
Camilo V, Garrido M, Valente P, Ricardo S, Amaral A L, Barros R, Chaves P, Carneiro F, David L, Almeida R (2014) Histopathology
Differentiation reprogramming in gastric intestinal metaplasia and dysplasia: role of SOX2 and CDX2 Aims: Intestinal metaplasia (IM), which results from de-novo expression of CDX2, and dysplasia are precursor lesions of gastric cancer that are associated with an increased risk for cancer development. There is much evidence suggesting a role for the transcription factor SOX2 in gastric differentiation. The aim of this study was to attempt to establish the relationship of SOX2 with CDX2 and with the differentiation reprogramming that characterizes gastric carcinogenesis, to assess their involvement in IM and dysplasia. Methods and results: Characterization of gastric (SOX2, MUC5AC, and MUC6) and intestinal (CDX2 and MUC2) markers in normal gastric mucosa, in 55 foci of IM and in 26 foci of dysplasia, was performed
by immunohistochemistry. SOX2 was expressed in the normal gastric mucosa, in the presumptive stem cell compartment, and was maintained in 7% of the complete (MUC5AC-negative) and 85% of the incomplete (MUC5AC-positive) IM subtypes. Twelve per cent of the dysplastic lesions expressed SOX2, and the association with MUC5AC was lost. CDX2 was present in all IMs and dysplastic lesions. Conclusions: SOX2 is associated with gastric differentiation in incomplete IM and is lost in the progression to dysplasia, whereas CDX2 is acquired de novo in IM and maintained in dysplasia. This suggests that the balance between gastric and intestinal differentiation programmes impacts on the gastric carcinogenesis cascade progression.
Keywords: CDX2, dysplasia, gastric cancer, intestinal metaplasia, SOX2
Introduction Gastric cancer is the fifth most common malignancy and the third leading cause of cancer-related deaths worldwide.1 The majority of gastric cancers are diagnosed late, and therefore have a dismal prognosis, which is reflected by a 5-year survival rate of no
Address for correspondence: R Almeida, IPATIMUP, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal. e-mail: [email protected] © 2014 John Wiley & Sons Ltd.
more than 25%.2 Thus, the most promising strategies to control the disease are based on prevention and early diagnosis. Gastric cancer is usually initiated by an inflammatory process associated with infection by Helicobacter pylori, which may lead to multifocal atrophic gastritis, intestinal metaplasia (IM), dysplasia, and finally cancer.3 IM represents a switch from a gastric to an intestinal differentiation profile, and is the most frequent premalignant condition of the stomach, appearing adjacent to >80% of gastric cancers.4 It is a heterogeneous lesion with two subtypes,
2 V Camilo et al.
complete and incomplete, both characterized by the presence of intestinal markers such as the transcription factor CDX2 and the mucin MUC2. The complete IM subtype is defined by the absence of gastric differentiation markers, such as mucins MUC5AC and MUC6, whereas in the incomplete IM subtype there is concomitant expression of gastric and intestinal markers.5 This classification has clinical implications, because the incomplete subtype is more frequently associated with gastric cancer and shows an increased risk for cancer progression.4,6 CDX2 is a homeobox transcription factor that is critical for intestinal differentiation,7,8 and is a specific biomarker of the early steps of the gastric carcinogenic cascade, driving the onset of IM.9,10 The key role of CDX2 in the metaplastic transformation of the gastric mucosa was categorically demonstrated by the use of two transgenic mouse models with ectopic expression of CDX2 in the gastric epithelium and subsequent development of IM with absorptive, goblet and enteroendocrine cell types.11,12 On the other hand, an increasing amount of evidence supports the involvement of the transcription factor SOX2 in gastric differentiation. SOX2 is a member of the high mobility group domain proteins, and is essential for maintaining pluripotency in embryonic stem cells and also for reprogramming fibroblasts into induced pluripotent stem cells.13,14 More recently, it has also been identified as an adult stem cell marker in mice.15 Furthermore, modulating SOX2 expression in mice demonstrated the importance of this protein in the differentiation of the oesophageal epithelium and normal morphogenesis of the oesophagus, trachea, and lung.16,17 In the digestive tract, it was observed in chicks and mice that SOX2 expression
and CDX2 expression are mutually exclusive.18,19 Invitro studies showed that SOX2 negatively regulated the CDX2 promoter by hampering the action of other transcription factors in an intestinal context, and that SOX2 down-regulation led to CDX2 overexpression in a gastric context.20,21 In accordance with these observations, we have demonstrated that SOX2 and CDX2 can be inversely regulated by the bone morphogenetic protein pathway and H. pylori.22,23 Moreover, two mouse models with conditional CDX2 loss of function showed increased SOX2 expression in the regions where CDX2 was abrogated, with a consequent shift from intestinal to a gastric/oesophageal differentiation.24,25 The same was observed when forced expression of SOX2 was induced in the intestine,26,27 suggesting that the interplay of these two transcription factors is critical for the balance between gastric and intestinal differentiation. Here, we sought to characterize SOX2 expression and its relationship with CDX2 in gastric cancer precursor lesions, in an attempt to establish its involvement in the differentiation reprogramming that characterizes gastric carcinogenesis.
Materials and methods MATERIAL
Normal gastric mucosa was studied in 10 biopsies representative of the body (n = 5) and the antral (n = 5) regions obtained from individuals with no gastric pathologies. IM (55 foci) and the adjacent gastric mucosa were studied in 11 surgical specimens obtained from patients with gastric carcinoma undergoing surgery at Centro Hospitalar S. Jo~ ao, Porto,
Table 1. Primary antibodies and immunohistochemistry conditions used in this study Antigen retrieval Antigen retrieval buffer conditions Dilution
Incubation time (min)
Antibody
Clone
Localization
CDX2
CDX2-88 Citrate buffer, 10 mM, pH 6.0
40 min at 98°C
1:50
MUC2
PMH1
0.1 U/ml neuraminidase*
2 h at 37°C
Undiluted Overnight (4°C) Cytoplasmic Supernatant28
MUC5AC CLH2
None
None
1:10
Overnight (4°C) Cytoplasmic Supernatant28
MUC6
CLH5
None
None
1:10
Overnight (4°C) Cytoplasmic Supernatant28
SOX2
SP-76
EDTA, 10 mM, pH 8.0
40 min at 98°C
1:50
1 h (room temperature)
Overnight (4°C) Nuclear
Nuclear
Source Biogenex, San Ramon, CA, USA
Cell Marque, Rockling, CA, USA
*Neuraminidase from Clostridium perfringens type VI (Sigma) was diluted in sodium acetate buffer (pH 5.5). © 2014 John Wiley & Sons Ltd, Histopathology
SOX2 and CDX2 expression in gastric carcinogenesis
A
B
C
3
D
Figure 1. SOX2 expression in the normal gastric mucosa. A,C, Haematoxylin and eosin staining of gastric mucosa from incisura and antrum, respectively. B,D, Immunodetection of SOX2 (brown) in the same regions of the gastric mucosa.
Portugal. IM foci were classified as complete type (n = 29) or incomplete type (n = 26) according to the pattern of mucin expression.5 Twenty-six foci of dysplasia were studied in 22 samples obtained by endoscopic submucosal dissection at the same hospital. Foci were considered to be distinct when they were separated by a stretch of gastric glands. The use of retrospective samples for which informed consent cannot be obtained is authorized for research studies by Portuguese law.
were stained,28 with a consensus of three observers (V.C., L.D., and R.A.).
IMMUNOHISTOCHEMISTRY
Results
Paraffin-embedded specimens were subjected to immunohistochemistry for SOX2, CDX2, MUC5AC, MUC6, and MUC2, using the antibodies described in Table 1. Detection of CDX2, MUC5AC and MUC2 was performed by incubation with a biotin-labelled rabbit anti-mouse secondary antibody (1:100; Dako, Glostrup, Denmark) followed by incubation with an avidin/biotin detection system (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) and development with 3,30 -diaminobenzidine. Detection of SOX2 was performed using the REAL Envision Detection System Peroxidase/DAB+ (Dako), according to the manufacturer’s instructions. For double staining (SOX2/MUC5AC or SOX2/ MUC6), immunohistochemistry was first performed for SOX2 using the REAL Envision Detection System Peroxidase/DAB+, and this was immediately followed by immunohistochemistry for MUC5AC or MUC6 using the Envision G2 System/AP (Permanent Red) (Dako), according to the manufacturer’s instructions. Tissue counterstaining was performed using Mayer haematoxylin. Mucin expression in dysplastic lesions was considered to be positive when >5% of the cells © 2014 John Wiley & Sons Ltd, Histopathology
STATISTICAL ANALYSIS
Statistical analysis was performed with STATVIEW v 5.0 (SAS Institute Inc., Cary, North Carolina, USA). Distributions were compared by use of the v2 test and Student’s t-test, and significance was assumed whenever P-values were
Differentiation reprogramming in gastric intestinal metaplasia and dysplasia: role of SOX2 and CDX2 V^ ania Camilo,1 M onica Garrido,2 Pedro Valente,2 Sara Ricardo,1 Ana Luısa Amaral,1 Rita Barros,1 Paula Chaves,3 F atima Carneiro,1,2,4 Leonor David1,2 & Raquel Almeida1,2,5 1
Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 2Faculty of Medicine of the University of Porto, Porto, 3Portuguese Oncology Institute Francisco Gentil, E.P.E., Lisbon, 4Pathology Department, Centro Hospitalar S. Jo~ao, and 5Biology Department, Faculty of Sciences of the University of Porto, Porto, Portugal
Date of submission 14 April 2014 Accepted for publication 31 August 2014 Published online Article Accepted 6 September 2014
Camilo V, Garrido M, Valente P, Ricardo S, Amaral A L, Barros R, Chaves P, Carneiro F, David L, Almeida R (2014) Histopathology
Differentiation reprogramming in gastric intestinal metaplasia and dysplasia: role of SOX2 and CDX2 Aims: Intestinal metaplasia (IM), which results from de-novo expression of CDX2, and dysplasia are precursor lesions of gastric cancer that are associated with an increased risk for cancer development. There is much evidence suggesting a role for the transcription factor SOX2 in gastric differentiation. The aim of this study was to attempt to establish the relationship of SOX2 with CDX2 and with the differentiation reprogramming that characterizes gastric carcinogenesis, to assess their involvement in IM and dysplasia. Methods and results: Characterization of gastric (SOX2, MUC5AC, and MUC6) and intestinal (CDX2 and MUC2) markers in normal gastric mucosa, in 55 foci of IM and in 26 foci of dysplasia, was performed
by immunohistochemistry. SOX2 was expressed in the normal gastric mucosa, in the presumptive stem cell compartment, and was maintained in 7% of the complete (MUC5AC-negative) and 85% of the incomplete (MUC5AC-positive) IM subtypes. Twelve per cent of the dysplastic lesions expressed SOX2, and the association with MUC5AC was lost. CDX2 was present in all IMs and dysplastic lesions. Conclusions: SOX2 is associated with gastric differentiation in incomplete IM and is lost in the progression to dysplasia, whereas CDX2 is acquired de novo in IM and maintained in dysplasia. This suggests that the balance between gastric and intestinal differentiation programmes impacts on the gastric carcinogenesis cascade progression.
Keywords: CDX2, dysplasia, gastric cancer, intestinal metaplasia, SOX2
Introduction Gastric cancer is the fifth most common malignancy and the third leading cause of cancer-related deaths worldwide.1 The majority of gastric cancers are diagnosed late, and therefore have a dismal prognosis, which is reflected by a 5-year survival rate of no
Address for correspondence: R Almeida, IPATIMUP, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal. e-mail: [email protected] © 2014 John Wiley & Sons Ltd.
more than 25%.2 Thus, the most promising strategies to control the disease are based on prevention and early diagnosis. Gastric cancer is usually initiated by an inflammatory process associated with infection by Helicobacter pylori, which may lead to multifocal atrophic gastritis, intestinal metaplasia (IM), dysplasia, and finally cancer.3 IM represents a switch from a gastric to an intestinal differentiation profile, and is the most frequent premalignant condition of the stomach, appearing adjacent to >80% of gastric cancers.4 It is a heterogeneous lesion with two subtypes,
2 V Camilo et al.
complete and incomplete, both characterized by the presence of intestinal markers such as the transcription factor CDX2 and the mucin MUC2. The complete IM subtype is defined by the absence of gastric differentiation markers, such as mucins MUC5AC and MUC6, whereas in the incomplete IM subtype there is concomitant expression of gastric and intestinal markers.5 This classification has clinical implications, because the incomplete subtype is more frequently associated with gastric cancer and shows an increased risk for cancer progression.4,6 CDX2 is a homeobox transcription factor that is critical for intestinal differentiation,7,8 and is a specific biomarker of the early steps of the gastric carcinogenic cascade, driving the onset of IM.9,10 The key role of CDX2 in the metaplastic transformation of the gastric mucosa was categorically demonstrated by the use of two transgenic mouse models with ectopic expression of CDX2 in the gastric epithelium and subsequent development of IM with absorptive, goblet and enteroendocrine cell types.11,12 On the other hand, an increasing amount of evidence supports the involvement of the transcription factor SOX2 in gastric differentiation. SOX2 is a member of the high mobility group domain proteins, and is essential for maintaining pluripotency in embryonic stem cells and also for reprogramming fibroblasts into induced pluripotent stem cells.13,14 More recently, it has also been identified as an adult stem cell marker in mice.15 Furthermore, modulating SOX2 expression in mice demonstrated the importance of this protein in the differentiation of the oesophageal epithelium and normal morphogenesis of the oesophagus, trachea, and lung.16,17 In the digestive tract, it was observed in chicks and mice that SOX2 expression
and CDX2 expression are mutually exclusive.18,19 Invitro studies showed that SOX2 negatively regulated the CDX2 promoter by hampering the action of other transcription factors in an intestinal context, and that SOX2 down-regulation led to CDX2 overexpression in a gastric context.20,21 In accordance with these observations, we have demonstrated that SOX2 and CDX2 can be inversely regulated by the bone morphogenetic protein pathway and H. pylori.22,23 Moreover, two mouse models with conditional CDX2 loss of function showed increased SOX2 expression in the regions where CDX2 was abrogated, with a consequent shift from intestinal to a gastric/oesophageal differentiation.24,25 The same was observed when forced expression of SOX2 was induced in the intestine,26,27 suggesting that the interplay of these two transcription factors is critical for the balance between gastric and intestinal differentiation. Here, we sought to characterize SOX2 expression and its relationship with CDX2 in gastric cancer precursor lesions, in an attempt to establish its involvement in the differentiation reprogramming that characterizes gastric carcinogenesis.
Materials and methods MATERIAL
Normal gastric mucosa was studied in 10 biopsies representative of the body (n = 5) and the antral (n = 5) regions obtained from individuals with no gastric pathologies. IM (55 foci) and the adjacent gastric mucosa were studied in 11 surgical specimens obtained from patients with gastric carcinoma undergoing surgery at Centro Hospitalar S. Jo~ ao, Porto,
Table 1. Primary antibodies and immunohistochemistry conditions used in this study Antigen retrieval Antigen retrieval buffer conditions Dilution
Incubation time (min)
Antibody
Clone
Localization
CDX2
CDX2-88 Citrate buffer, 10 mM, pH 6.0
40 min at 98°C
1:50
MUC2
PMH1
0.1 U/ml neuraminidase*
2 h at 37°C
Undiluted Overnight (4°C) Cytoplasmic Supernatant28
MUC5AC CLH2
None
None
1:10
Overnight (4°C) Cytoplasmic Supernatant28
MUC6
CLH5
None
None
1:10
Overnight (4°C) Cytoplasmic Supernatant28
SOX2
SP-76
EDTA, 10 mM, pH 8.0
40 min at 98°C
1:50
1 h (room temperature)
Overnight (4°C) Nuclear
Nuclear
Source Biogenex, San Ramon, CA, USA
Cell Marque, Rockling, CA, USA
*Neuraminidase from Clostridium perfringens type VI (Sigma) was diluted in sodium acetate buffer (pH 5.5). © 2014 John Wiley & Sons Ltd, Histopathology
SOX2 and CDX2 expression in gastric carcinogenesis
A
B
C
3
D
Figure 1. SOX2 expression in the normal gastric mucosa. A,C, Haematoxylin and eosin staining of gastric mucosa from incisura and antrum, respectively. B,D, Immunodetection of SOX2 (brown) in the same regions of the gastric mucosa.
Portugal. IM foci were classified as complete type (n = 29) or incomplete type (n = 26) according to the pattern of mucin expression.5 Twenty-six foci of dysplasia were studied in 22 samples obtained by endoscopic submucosal dissection at the same hospital. Foci were considered to be distinct when they were separated by a stretch of gastric glands. The use of retrospective samples for which informed consent cannot be obtained is authorized for research studies by Portuguese law.
were stained,28 with a consensus of three observers (V.C., L.D., and R.A.).
IMMUNOHISTOCHEMISTRY
Results
Paraffin-embedded specimens were subjected to immunohistochemistry for SOX2, CDX2, MUC5AC, MUC6, and MUC2, using the antibodies described in Table 1. Detection of CDX2, MUC5AC and MUC2 was performed by incubation with a biotin-labelled rabbit anti-mouse secondary antibody (1:100; Dako, Glostrup, Denmark) followed by incubation with an avidin/biotin detection system (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA) and development with 3,30 -diaminobenzidine. Detection of SOX2 was performed using the REAL Envision Detection System Peroxidase/DAB+ (Dako), according to the manufacturer’s instructions. For double staining (SOX2/MUC5AC or SOX2/ MUC6), immunohistochemistry was first performed for SOX2 using the REAL Envision Detection System Peroxidase/DAB+, and this was immediately followed by immunohistochemistry for MUC5AC or MUC6 using the Envision G2 System/AP (Permanent Red) (Dako), according to the manufacturer’s instructions. Tissue counterstaining was performed using Mayer haematoxylin. Mucin expression in dysplastic lesions was considered to be positive when >5% of the cells © 2014 John Wiley & Sons Ltd, Histopathology
STATISTICAL ANALYSIS
Statistical analysis was performed with STATVIEW v 5.0 (SAS Institute Inc., Cary, North Carolina, USA). Distributions were compared by use of the v2 test and Student’s t-test, and significance was assumed whenever P-values were
