A study of Smad4, Smad6 and Smad7 in Surgically Resected Samples of Pancreatic Ductal Adenocarcinoma and Their Correlation with Clinicopathological Parameters and Patient Survival
© Singh et al; licensee BioMed Central Ltd. 2011
Received: 24 May 2011
Accepted: 23 December 2011
Published: 23 December 2011
Smad4 is the common mediator of the tumor suppressive functions of TGF-beta. Smad6 and Smad7 are the antagonists of the TGF-beta pathway. This study investigates the differential protein expressions of Smad4, Smad6 and Smad7 in tumor as compared to normal tissue of pancreatic ductal adenocarcinoma (PDAC) and compares them with clinicopathological parameters and patient survival.
There was a significant difference in protein expressions of Smad4 (p = 0.0001), Smad6 (p = 0.0015) and Smad7 (p = 0.0005) protein in tumor as compared to paired normal samples. Loss of Smad7 expression correlated significantly with tumor size (r = 0.421, p < 0.036) and margin status (r = 0.431; p < .032). Patients with moderate to high Smad4 protein expression had a better survival (median survival = 14.600 ± 2.112 months) than patients with absent or weak Smad4 protein expression (median survival = 7.150 ± 0.662). In addition, advanced disease stage correlated significantly with poor prognosis.
Loss of Smad4 significantly correlated with poor survival of PDAC patients. In the cases where Smad4 is expressed, Smad6 inhibition is possibly a novel mechanism for Smad4 inactivation. Smad7 has a role in pathobiology of PDAC. Further investigation in the roles of Smad6 and Smad7 would help in the identification of novel therapeutic targets for PDAC.
KeywordsPancreas pancreatic adenocarcinoma Smad4 Smad6 Smad7 clinicopathological parameters prognosis
Smad proteins are a family of intracellular mediators of the transforming growth factor beta (TGF-β) family of cytokines. On ligand binding, TGF-β Receptor II (TβRII) becomes constitutively active, heterodimerizes with TGF-β Receptor I (TβRI) and transphosphorylates its GS domain resulting in its activation [1, 2]. Once activated, TβRI phosphorylates a class of molecules known as receptor-regulated Smads (R-Smads), Smad2 and Smad3, at an SSXS motif at their C-terminal end . Active, phosphorylated R-Smads heterodimerize with common-Smad (Co-Smad), Smad4, translocate to the nucleus and regulate gene expression [4, 5]. A third class of Smad proteins, the inhibitory Smads (I-Smads), Smad6 and Smad7 act as negative regulators and act by blocking R-Smads' interaction with TβRI, phosphorylation by TβRI or heterodimerization with Smad4 [6, 7].
Smad4 is being explored as one of the major molecular markers in pancreatic ductal adenocarcinoma (PDAC) (as reviewed by ). Although lost in many cancers, loss of Smad4 is more sensitive and specific to pancreatic cancer . Studies have shown SMAD4 gene to be inactivated in 55% of pancreatic cancers [10–12]. The inactivation of SMAD4 gene occurs either by deletion of both alleles (35%) or by intra-genic mutation in one allele coupled with the loss of the other allele (20%) . A number of studies demonstrate the role of Smad4 in pancreatic ductal adenocarcinoma, but only a few studies have explored the roles of inhibitory Smads, Smad6 and Smad7 in this disease (as reviewed by ).
In the present study, we examined the differential protein expressions of Smad4, Smad6 and Smad7 in surgically resected samples of paired tumor tissue of pancreatic ductal adenocarcinoma versus adjacent normal tissue. A combinatorial expression of these three Smads was evaluated to gain an insight into how they influenced one another in pancreatic ductal adenocarcinoma as compared to normal pancreas. Influence of the expression levels of these proteins on clinicopathological parameters and patient survival was studied.
The study was conducted after obtaining a formal approval from the Ethics Committee of the Postgraduate Institute of Medical Education and Research. Informed oral consent was obtained from each patient for participation in the study.
Clinical profile of patients with pancreatic ductal adenocarcinoma (n = 25)
No. of patients
Age (range 28-75)
< 3 cm
Lymph Node status
Surgically resected samples were collected and tumor was confirmed by performing hematoxylin and eosin (H&E) staining on frozen sections taken on autoclaved glass slides. Similarly, the presence of normal pathology in the adjacent normal tissues was also confirmed. Initially, samples from a total of 32 consecutive patients were collected, out of which 25 samples that showed tumor tissue in more than 90% of the area of the section were included for study. Part of the samples to be used for immunohistochemistry were formalin fixed, and part of them were snap frozen and stored at - 80°C for further molecular analysis.
Clinical and pathological data were obtained from the patients' medical records. Clinical and pathological variables included age, gender, tumor size, margin status, stage, grade, and survival.
Antibodies used in the study
Primary antibody incubation
Pressure cooker for 20 min
2 h at room temperature
2 μg/ml (1:100)
Microwaving:3 Cycles 3 min +1 Cycle 1 min
Overnight at 4°C
4 μg/ml (1:50)
Microwaving: 3 Cycles 3 min +1 Cycle 1 min
Overnight at 4°C
4 μg/ml (1:50)
Immunohistochemical scoring was done independently by two senior cytopathologists (BDR and RS) and only samples with complete concordance in staining and histopathology were included in the study. The slides were scored as follows: 0 (no staining), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining), a scoring system previously described by Hua et al . Paired adjacent normal tissue samples served as positive controls for each of the cases. There was a complete concordance in all the cases except one, where high and moderate expression of Smad4 for the same normal tissue sample was respectively reported. Re-evaluation, however, eliminated the discrepancy.
Fisher's exact test was used to compare Smad4, Smad6 and Smad7 protein expression in normal and tumor tissue. Spearman rank correlation test was used to correlate Smad4, Smad6 and Smad7 protein expression in tumor tissue with clinicopathological parameters. Kaplan-Meier survival analysis was used to analyse the influence of Smad4, Smad6 and Smad7 protein expression in tumor tissue and clinicopathological parameters on survival. A probability value of less than 0.05 was considered to be significant.
Immunohistochemical expression of Smad4, Smad6 and Smad7
Immunohistochemical expression of Smad4, Smad6 & Smad7 in paired samples of Normal pancreas and Pancreatic ductal adenocarcinoma (n = 25)
Smad4 showed cytoplasmic as well as nuclear staining, which was both diffuse and focal (Figure 1a, b). Most normal tissue showed strong to moderate Smad4 immunoreactivity (24/25, 96%), whereas most tumor tissue showed absent (10/25, 40%) or weak (10/25, 40%) immunoreactivity for Smad4. Strong to moderate expression was seen only in 20% (5/25) of tumor samples. The difference in Smad4 protein levels in tumor tissue as compared to normal pancreatic tissue was highly significant on Fisher's exact test (two tailed p value = 0.0001).
The protein immunoreactivity was predominantly cytoplasmic (Figure 1c, d). Here also, most of the normal tissues showed strong (18/25, 72%) to moderate (5/25, 20%) immunopositivity. However, A good number (12/25, 48%) of tumor tissues showed moderate levels of Smad6 protein expression, in contrast to Smad4 where most of the cases were weakly positive or absent. Complete loss of Smad6 expression was seen only in 28%(7/25) of tumor cases. The difference in Smad6 protein levels in tumor tissue as compared to normal pancreatic tissue was highly significant by Fisher's exact test (p = 0.0015).
The immunoreactivity was predominantly cytoplasmic, although occasional nuclear positivity was obtained in some normal pancreatic ducts (Figure 1e, f). Similar to Smad4, normal pancreatic tissue showed moderate to high levels of Smad7 expression in most of the samples (18/25, 72%), whereas, more than half of tumor tissue showed complete loss of protein expression (4/25, 56%), and another 24%(6/25) of cases showed low expression. The difference in the expression levels of Smad7 in normal pancreatic samples as compared to tumor samples was highly significant by Fisher's exact test (p = 0.0005).
Co-expression of Smad4 with inhibitory Smads
Combinatorial expression of Smad4, Smad6 & Smad7 in 25 tumor samples of pancreatic ductal adenocarcinoma patients
(n = 18, 72.0%)
(n = 11, 44.0%)
Smad6 or Smad7
(n = 21, 84.05)
Smad6 and Smad7
(n = 9, 36%)
Smad4 positive cases (n = 15)
Smad4 negative cases (n = 10)
Correlation of Smad4, Smad6 and Smad7 protein expression with clinocopathological parameters
Correlations of the expression of Smad4, Smad6 and Smad7 with clinicopathological parameters in 25 patients of pancreatic ductal adenocarcinoma
Loss/low expression of Smad4 (%)
Loss/low expression of Smad 6 (%)
Loss/low expression of Smad7(%)
< 3 cm
Univariate analysis for survival
Summary of results of survival analysis by Kaplan-Meier test
No. of patients
95% Confidence Interval(CI)
5.000 ± 2.191
9.000 ± 2.225
10.000 ± 1.754
6.000 ± 1.732
7.000 ± 1.198
10.000 ± 1.125
10.000 ± 2.066
7.000 ± 0.949
< 3 cm
9.000 ± 0.933
≥ 3 cm
9.000 ± 1.038
6.000 ± 0.745
13.000 ± 2.191
7.000 ± 0.926
10.000 ± 1.477
7.000 ± 2.236
10.000 ± 3.286
Smad4 is the common mediator of the tumor suppressive functions of TGF-beta. Smad6 and Smad7 are antagonists of the TGF-beta pathway. In this work, we further establish the role of Smad4 as a potential prognostic marker for pancreatic ductal adenocarcinoma. We also identified different roles for Smad6 and Smad7 in influencing pancreatic cancer biology.
In this study, Smad4 was expressed in most of the normal samples (96%) but lost in 40% of tumor samples. In tumor samples, even where it was expressed, there was weak expression in the majority of cases. Kaplan-Meier analysis for survival demonstrated that patients with moderate Smad4 protein expression had a better survival than patients with weak or negative Smad4 protein expression. Despite one report of Smad4 expression to be inversely related to survival in surgically resected pancreatic ductal adenocarcinoma patients , there is growing evidence for the correlation of Smad4 status to patient survival in this disease [16, 18]. One study also correlated the Smad4 expression with the pattern of disease progression (local v distant dominant) and proposed to further explore its role as a predictive biomarker for personalized treatment strategies . Our observations further adds to preexisting data and establish Smad4 as a potential prognostic marker for pancreatic ductal adenocarcinoma. However, a recent meta-analysis analyzing 5 studies evaluating Smad4 could not find any significant overall association between Smad4 expression and survival . This indicates difficulty in making a reliable conclusion regarding the relative prognostic value of immunohistochemical markers when analyzed in a limited patient series.
For Smad6 and Smad7, although occasional samples showed nuclear staining, cytoplasmic staining was predominant. Previous reports have shown that while Smad7 appears to reside predominantly in the nucleus at basal state, it translocates to the cytoplasm upon TGF-β stimulation . The cytoplasmic staining of Smad6 and Smad7 in most samples implies that these two inhibitory Smads were in their activated states in most tumor samples.
There are just two reports on Smad6 expression in pancreatic cancer till date. One of them conducted in pancreatic cancer cell line, found Smad6 and Smad7 levels to be elevated in pancreatic cancer . The second study, conducted on patient samples, contradicts this and demonstrates that the increased expressions of either Smad6 or Smad7 are infrequent in tumor compared to normal samples . Our study goes a step further, and shows that Smad6 as well as Smad7 are lost in tumor as compared to normal samples. We showed a loss of expression of Smad6 in 28% tumor samples as compared to 8% loss in normal samples. In cases where Smad6 was expressed, the expression was mostly moderate to high. Its cytoplasmic staining, along with its co expression with Smad4 in 14 out of 15 Smad4 positive cases suggests that Smad6 can be one of the possible inhibitory mechanisms for Smad4 inactivation. Thus, in pancreatic ductal adenocarcinoma cases where Smad4 itself is not lost, we illustrate a novel mechanism for its inhibition. Our study, for the first time ascribes Smad6 a role in pancreatic cancer biology, which can be further explored for the development of novel therapeutic target.
Previous studies have shown Smad7 overexpression in pancreatic cancer cell lines [22–24]. Similar to our study, few other studies have shown a loss of Smad7 in patient samples [24, 25]. This difference in expression in cell lines as compared to tissue samples might be because of a possible reversal of phenotype in artificial tissue culture systems. In our study, loss of Smad7 expression surpassed that of Smad4 and was absent in 56% of tumor samples, which is quite close to what has been reported by Guo et al . Amongst clinicopathological parameters, loss of Smad7 significantly correlated with both tumor size as well as margin status. On similar lines, Wang et al showed a significant correlation between the low Smad7 expression and lymph node metastasis . These observations, put together, indicate a role for Smad7 in the aggressiveness of this disease. In fact, different studies have isolated different molecules, like KLF11, retinoblastoma, thioredoxin, which are involved in Smad7 dependent aggressiveness of pancreatic cancer [23, 27, 28]. However, unlike Wang et al, we did not find a significant correlation between loss of Smad7 and patient survival.
The smaller sample size in the study is acknowledged, the reasons being i) choice of prospective samples for the study, ii) low incidence of pancreatic cancer in Indian population: 0.5-2.4 per 100000 men and 0.2-1.8 per 100000 women , iii) limited time for the collection of patient samples, iv) exclusion of archival samples due to poor and unreliable staining.
The present study strongly substantiates the previous reports in further establishing the role of Smad4 as a prognostic marker. It also suggests that a further exploration into the newly found roles for Smad6 and Smad7 in PDAC biology, with a larger sample size, may help discern some novel therapeutic targets for this disease, subsequently contributing to the improvement in therapeutic strategies and better disease management for PDAC patients.
Acknowledgements and funding
We are grateful to Indian Council of Medical Education and Research for providing the stipend (No. 3/1/3/2002-MPD-JRF) for PS to carry out this study as a part of her PhD work. We are thankful for Postgraduate Institute of Medical Education and Research for funding the study and providing the infrastructure.
- Wrana JL, Attisano L, Wieser R, Ventura F, Massague J: Mechanism of activation of the TGF-beta receptor. Nature. 1994, 370: 341-347. 10.1038/370341a0.PubMedView Article
- Massague J: TGF-beta signal transduction. Annu Rev Biochem. 1998, 67: 753-791. 10.1146/annurev.biochem.67.1.753.PubMedView Article
- Souchelnytskyi S, Tamaki K, Engstrom U, Wernstedt C, ten Dijke P, Heldin CH: Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-beta signaling. J Biol Chem. 1997, 272: 28107-28115. 10.1074/jbc.272.44.28107.PubMedView Article
- de Caestecker MP, Yahata T, Wang D, Parks WT, Huang S, Hill CS, Shioda T, Roberts AB, Lechleider RJ: The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain. J Biol Chem. 2000, 275: 2115-2122. 10.1074/jbc.275.3.2115.PubMedView Article
- de Winter JP, Roelen BA, ten Dijke P, van der Burg B, van den Eijnden-van Raaij AJ: DPC4 (SMAD4) mediates transforming growth factor-beta1 (TGF-beta1) induced growth inhibition and transcriptional response in breast tumour cells. Oncogene. 1997, 14: 1891-1899. 10.1038/sj.onc.1201017.PubMedView Article
- Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone MA, Wrana JL, Falb D: The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. Cell. 1997, 89: 1165-1173. 10.1016/S0092-8674(00)80303-7.PubMedView Article
- Imamura T, Takase M, Nishihara A, Oeda E, Hanai J, Kawabata M, Miyazono K: Smad6 inhibits signalling by the TGF-beta superfamily. Nature. 1997, 389: 622-626. 10.1038/39355.PubMedView Article
- Singh P, Srinivasan R, Wig JD: Major molecular markers in pancreatic ductal adenocarcinoma and their roles in screening, diagnosis, prognosis, and treatment. Pancreas. 2011, 40: 644-652. 10.1097/MPA.0b013e31821ff741.PubMedView Article
- van Heek T, Rader AE, Offerhaus GJ, McCarthy DM, Goggins M, Hruban RH, Wilentz RE: K-ras, p53, and DPC4 (MAD4) alterations in fine-needle aspirates of the pancreas: a molecular panel correlates with and supplements cytologic diagnosis. Am J Clin Pathol. 2002, 117: 755-765. 10.1309/5RQ0-JCQU-5XF2-51LQ.PubMedView Article
- Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern SE: DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science. 1996, 271: 350-353. 10.1126/science.271.5247.350.PubMedView Article
- Schutte M, Hruban RH, Hedrick L, Cho KR, Nadasdy GM, Weinstein CL, Bova GS, Isaacs WB, Cairns P, Nawroz H: DPC4 gene in various tumor types. Cancer Res. 1996, 56: 2527-2530.PubMed
- Hruban RH, Goggins M, Kern SE: Molecular genetics and related developments in pancreatic cancer. Curr Opin Gastroenterol. 1999, 15: 404-409. 10.1097/00001574-199909000-00006.PubMedView Article
- Hruban RH, Offerhaus GJ, Kern SE, Goggins M, Wilentz RE, Yeo CJ: Tumor-suppressor genes in pancreatic cancer. J Hepatobiliary Pancreat Surg. 1998, 5: 383-391. 10.1007/s005340050062.PubMedView Article
- Singh P, Wig JD, Srinivasan R: The Smad family and its role in pancreatic cancer. Indian J Cancer. 2011, 48: 351-360. 10.4103/0019-509X.84939.PubMedView Article
- Katz MH, Hwang R, Fleming JB, Evans DB: Tumor-node-metastasis staging of pancreatic adenocarcinoma. CA Cancer J Clin. 2008, 58: 111-125. 10.3322/CA.2007.0012.PubMedView Article
- Hua Z, Zhang YC, Hu XM, Jia ZG: Loss of DPC4 expression and its correlation with clinicopathological parameters in pancreatic carcinoma. World J Gastroenterol. 2003, 9: 2764-2767.PubMedPubMed CentralView Article
- Biankin AV, Morey AL, Lee CS, Kench JG, Biankin SA, Hook HC, Head DR, Hugh TB, Sutherland RL, Henshall SM: DPC4/Smad4 expression and outcome in pancreatic ductal adenocarcinoma. J Clin Oncol. 2002, 20: 4531-4542. 10.1200/JCO.2002.12.063.PubMedView Article
- Tascilar M, Offerhaus GJ, Altink R, Argani P, Sohn TA, Yeo CJ, Cameron JL, Goggins M, Hruban RH, Wilentz RE: Immunohistochemical labeling for the Dpc4 gene product is a specific marker for adenocarcinoma in biopsy specimens of the pancreas and bile duct. Am J Clin Pathol. 2001, 116: 831-837. 10.1309/WF03-NFCE-7BRH-7C26.PubMedView Article
- Crane CH, Varadhachary GR, Yordy JS, Staerkel GA, Javle MM, Safran H, Haque W, Hobbs BD, Krishnan S, Fleming JB: Phase II trial of cetuximab, gemcitabine, and oxaliplatin followed by chemoradiation with cetuximab for locally advanced (T4) pancreatic adenocarcinoma: correlation of Smad4(Dpc4) immunostaining with pattern of disease progression. J Clin Oncol. 2011, 29: 3037-3043. 10.1200/JCO.2010.33.8038.PubMedPubMed CentralView Article
- Smith RA, Tang J, Tudur-Smith C, Neoptolemos JP, Ghaneh P: Meta-analysis of immunohistochemical prognostic markers in resected pancreatic cancer. Br J Cancer. 2011, 104: 1440-1451. 10.1038/bjc.2011.110.PubMedPubMed CentralView Article
- Itoh S, Landstrom M, Hermansson A, Itoh F, Heldin CH, Heldin NE, ten Dijke P: Transforming growth factor beta1 induces nuclear export of inhibitory Smad7. J Biol Chem. 1998, 273: 29195-29201. 10.1074/jbc.273.44.29195.PubMedView Article
- Kleeff J, Ishiwata T, Maruyama H, Friess H, Truong P, Buchler MW, Falb D, Korc M: The TGF-beta signaling inhibitor Smad7 enhances tumorigenicity in pancreatic cancer. Oncogene. 1999, 18: 5363-5372. 10.1038/sj.onc.1202909.PubMedView Article
- Arnold NB, Ketterer K, Kleeff J, Friess H, Buchler MW, Korc M: Thioredoxin is downstream of Smad7 in a pathway that promotes growth and suppresses cisplatin-induced apoptosis in pancreatic cancer. Cancer Res. 2004, 64: 3599-3606. 10.1158/0008-5472.CAN-03-2999.PubMedView Article
- Guo J, Kleeff J, Zhao Y, Li J, Giese T, Esposito I, Buchler MW, Korc M, Friess H: Yes-associated protein (YAP65) in relation to Smad7 expression in human pancreatic ductal adenocarcinoma. Int J Mol Med. 2006, 17: 761-767.PubMed
- Kim YH, Lee HS, Lee HJ, Hur K, Kim WH, Bang YJ, Kim SJ, Lee KU, Choe KJ, Yang HK: Prognostic significance of the expression of Smad4 and Smad7 in human gastric carcinomas. Ann Oncol. 2004, 15: 574-580. 10.1093/annonc/mdh131.PubMedView Article
- Wang P, Fan J, Chen Z, Meng ZQ, Luo JM, Lin JH, Zhou ZH, Chen H, Wang K, Xu ZD, Liu LM: Low-level expression of Smad7 correlates with lymph node metastasis and poor prognosis in patients with pancreatic cancer. Ann Surg Oncol. 2009, 16: 826-835. 10.1245/s10434-008-0284-5.PubMedView Article
- Ellenrieder V, Buck A, Harth A, Jungert K, Buchholz M, Adler G, Urrutia R, Gress TM: KLF11 mediates a critical mechanism in TGF-beta signaling that is inactivated by Erk-MAPK in pancreatic cancer cells. Gastroenterology. 2004, 127: 607-620. 10.1053/j.gastro.2004.05.018.PubMedView Article
- Boyer Arnold N, Korc M: Smad7 abrogates transforming growth factor-beta1-mediated growth inhibition in COLO-357 cells through functional inactivation of the retinoblastoma protein. J Biol Chem. 2005, 280: 21858-21866. 10.1074/jbc.M500583200.PubMedView Article
- Gajalakshmi CK, Swaminathan R, Shanta V: A study on pancreatic cancer in Chennai (Madras), India. Cancer Lett. 1998, 122: 221-226. 10.1016/S0304-3835(97)00392-3.PubMedView Article
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.