Indian Journal of Cancer Medknow Publications on behalf of Indian Cancer Society ISSN: 0019-509X EISSN: 1998-4774 Vol. 48, Num. 2, 2011, pp. 170-174

Indian Journal of Cancer, Vol. 48, No. 2, April-June, 2011, pp. 170-174

Original Article

A comprehensive examination of Smad4, Smad6 and Smad7 mRNA expression in pancreatic ductal adenocarcinoma

P Singh¹, JD Wig², R Srinivasan³, BD Radotra³

¹ School of Molecular and Microbial Biosciences, The University of Sydney, NSW, 2006, Australia
² Department of General Surgery, Government Medical College and Hospital, Sector-32, Chandigarh, Punjab, India
³ Department of Cytology and Gynaecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, Punjab, India

Correspondence Address: P Singh School of Molecular and Microbial Biosciences, The University of Sydney, NSW, 2006 Australia puneetsingh_bio@y7mail.com

DOI: 10.4103/0019-509X.82876

Abstract

Keywords: Clinicopathological parameters, prognosis, Smad4, Smad6, Smad7, pancreatic ductal adenocarcinoma

Introduction

Smad4, Smad6 and Smad7 are important molecules in pancreatic ductal adenocarcinoma (PDAC) biology. Only a few studies have examined the expression profiles of Smad4, Smad6 and Smad7 transcripts in PDAC patient samples and only one study has attempted to understand the relationship with Smad protein expression. Results from these studies suggest loss of Smad4 mRNA expression, ^[1],[2] increased Smad7 mRNA expression, ^[3] and a significant positive correlation of Smad4 mRNA expression and Smad4 protein levels ^[4] in PDAC.

In this report, we present data examining the expression profiles of Smad4, Smad6 and Smad7 mRNA in patient samples of PDAC and their relationship to Smad protein expression, SMAD4 genetic alterations, clinicopathological parameters and patient survival. These data were obtained during a comprehensive study of Smad4, Smad6 and Smad7 in PDAC, which examined the relationship of molecular parameters with clinicopathological parameters and patient survival.

Materials and Methods

Twenty-five consecutive patients of PDAC were included in the study. Approval for the study was obtained from the Ethics Committee of the Postgraduate Institute of Medical Education and Research. Informed consent was obtained from each patient for participation in the study.

Surgically resected samples of paired tumor and adjacent normal tissue were collected. Tumor tissue was confirmed by taking frozen sections on autoclaved glass slides and staining them with Hematoxylin and Eosin. Only the samples with tumor in >90% of the area of the section were included in the study.

Immunohistochemical labeling was done on 4 μm tissue sections mounted on slides coated with poly-l-lysine (Sigma, St. Louis, Missouri, USA) using the routine Streptavidin-Biotin immunoperoxidase technique. Sections were deparaffinized in xylene, and rehydrated through a series of graded alcohols to distilled water and microwaved in buffered sodium citrate. Endogenous peroxidase was blocked by incubating in hydrogen peroxidase with methanol, followed by overnight incubation with monoclonal antibodies [Table - 1]. Novastatin Universal Detection kit (Ready to use, Novacastra Laboratories Ltd., Newcastle, UK) containing biotinylated secondary antibody was applied and staining was visualized using3,3′-Diaminobenzidinetetrahydrochloride (Sigma Chemical Co., St. Louis, MO, USA) solution as the chromogen. The sections were counterstained in Mayer′s hematoxylin, rinsed in water, and mounted in Di-N-Butyle Phthalate in Xylene (DPX). The brown product obtained was visualized and scored under light microscopy. Immunohistochemical scoring was done independently by two pathologists and in all the cases the results were found concordant. The slides were scored as follows: 0 (no staining), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining). ^[5]

mRNA was isolated from tissue samples using the Micro to Midi Kit (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA was reverse transcribed into 20 μl of cDNA with 100 nanograms of random hexamers and 200 U of SuperScript II (Invitrogen) at 42°C for 1 hour. Polymerase chain reaction (PCR) was carried out on cDNA for Smad4, Smad6 and Smad7 transcripts using sequence-specific primers that have been previously described. ^[6] The primer sequences and the PCR program for each of the three Smads and β-actin are shown in [Table - 2].

Each PCR reaction (25 μl) included 2 μl of the cDNA synthesis product, 100 ng of each primer, 1.0 units of Taq polymerase (Boehringer Mannheim, Indianapolis, IN, USA), and 2 mmol/l dNTPs (Boehringer Mannheim). A no-RT control that would yield a PCR product in case of genomic DNA contamination was included for each of the reaction cycles. PCR products were resolved onto 2.5% agarose gels, and the band intensities for the relative levels of Smad4, Smad6 and Smad7 against the housekeeping gene β-actin were measured using Bio-Rad′s Quantity One software.

To compare protein and transcript expression levels between normal and tumor groups, Student′s t-test and Pearson′s chi-square test were applied. The mean and median levels of expression of each molecule investigated in this study were calculated. The analysis of interrelationship and the correlation of expression between different proteins were done by employing Spearman′s Rank Correlation test. The correlation of expression between the protein and transcript levels was done by Pearson′s Correlation test. Survival analysis was done using Kaplan-Meier Logrank test. A probability value of <0.05 was considered to be significant.

Results

A total of 25 subjects (13 males, 12 females) with a mean age of 54.63 (range 28-75) years, of histopathologically proven PDAC were included. The cases were collected over a period of 36 months. The patients were staged according to the Tumor, Node and Metastasis (TNM) classification of the International Union against Cancer. ^[7] Clinicopathological profile of the patients is given in [Table - 3].

As compared to normal controls, tumor tissues showed significantly low protein expression for Smad4 (P < 0.001), Smad6 (P = 0.027) and Smad7 (P < 0.0001). Complete loss of expression was observed in 40, 21.87 and 57.89% of cases for Smad4, Smad6 and Smad7, respectively. Moreover, Smad4 and Smad6 co-expressed in all except one case (unpublished data).

There was a decrease in the expression levels of transcript for all the three Smads in tumor samples as compared to normal control samples [Table - 4], [Figure 1]. However, this difference was significant only in case of Smad4 (Student′s t-test, P = 0.036).

A significant positive correlation between Smad4 and Smad7 transcripts was observed in normal samples (Pearson′s correlation, r = 0.642, P < 0.0001), whereas no significant correlation was seen in the other two combinations, i.e. between Smad4 and Smad6, or Smad6 and Smad7 transcripts. .

In tumor samples, a significant positive correlation was found between Smad4 and Smad6 (r = 0.777, P < 0.000), Smad4 and Smad7 (r = 0.689, P < 0.0001), and Smad6 and Smad7 (r = 0.588, P < 0.0001) transcript levels.

No significant correlation between mRNA and protein expression levels for any of the three Smads for either normal or tumor samples was found.

There was a total absence of mRNA expression in seven samples, out of which, mutations/deletions were present in 5 samples. [Table - 5]. In three cases, Smad4 mRNA expression was present in the presence of SMAD4 gene mutations, and in all these cases Smad4 protein was expressed. Genetic alterations significantly negatively correlated with Smad4 mRNA expression (r = −0.511, P = 0.015).

No significant relationship was found between Smad4, Smad6 and Smad7 mRNA expression and clinicopathological parameters and patient survival.

Discussion

Complete loss of expression of Smad4, Smad6 and Smad7 proteins was observed in 40, 21.87 and 57.89% of cases, respectively. Thus, Smad4 and Smad7 levels were markedly and Smad6 levels were moderately decreased in tumor samples as compared to normal controls. mRNA expressions of Smad4, Smad6 and Smad7 were decreased in tumor samples compared to normal controls, but the values were significant only for Smad4 mRNA (P = 0.036). This finding for Smad4 mRNA expression is similar to those of earlier studies ^[2],[8] However, our findings for Smad6 and Smad7 mRNA expression are in contrast to previous reports where no change was seen in pancreatic cancer as compared to normal samples. ^[1] There is only one study that reports an overexpression of Smad7 mRNA in tissue samples of PDAC. ^[3]

In tumor samples, a significant association of expression was found between Smad4 and Smad6 (P < 0.000), Smad4 and Smad7 (P < 0.0001) and Smad6 and Smad7 (P < 0.0001). This suggests that these molecules are regulated similarly. Previous reports have demonstrated that Smad4 protein acts as a transcriptional regulator for SMAD6^[9] and SMAD7^[10],[11] genes, thus corroborating our in vivo findings. Furthermore, co-expression of Smad4 and Smad6 proteins in tumor samples, high levels of Smad6 protein expression in comparison to Smad7 protein expression, and cytoplasmic location of Smad6 (indicating that it is functionally active) ^[12] suggest that Smad6 plays a major role in inhibiting Smad4 in pancreatic cancers.

The absence of correlation of mRNA and protein expression for either Smad4, Smad6 or Smad7 and the fact that unlike protein expression, most samples were positive for mRNA expression suggests the involvement of post-transcriptional regulatory mechanisms. A similar observation has been made for Smad4 in an earlier study on invasive ductal carcinoma (IDC) of the pancreas. ^[4] Suggested post-transcriptional regulatory mechanisms may include the involvement of other pathways in protein stability or the presence of mutations at the gene locus leading to immediate degradation of either the mRNA or the translated protein. ^{[13],[14],[15],[16],[17]}

The absence of concordance between SMAD4 gene status, mRNA expression and Smad4 protein expression also suggests the presence of other regulatory mechanisms in Smad4 transcription and translation.

Acknowledgments

We are thankful to Indian Council of Medical Research, India for providing PhD stipend for PS to carry out this study and Post Graduate Institute of Medical Education and Research, India, for providing the infrastructure and funding this project.

References

Copyright 2011 - Indian Journal of Cancer

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