Smad4-expression is decreased in breast cancer tissues: a retrospective study
© Stuelten et al; licensee BioMed Central Ltd. 2006
Received: 30 October 2005
Accepted: 26 January 2006
Published: 26 January 2006
Although transforming growth factor β (TGF-β) typically inhibits proliferation of epithelial cells, consistent with a tumor suppressor activity, it paradoxically also exhibits pro-metastatic activity in the later stages of carcinogenesis. Since tumors often display altered TGF-β signaling, particularly involving the Smad-pathway, we investigated the role of Smad4-expression in breast cancer.
Smad4 expression was investigated by immunohistochemistry in formalin-fixed, paraffin-embedded tissue from 197 samples of primary breast cancer obtained between 1986 and 1998. The prognostic value of Smad4-expression was analyzed.
Smad4 expression was found to be reduced in lobular and ductal breast carcinoma as compared to surrounding uninvolved lobular and ductal breast epithelia (p < 0.001, n = 50). Smad4-expression correlated positively with expression of TGF-β-receptor I (p < 0.001, n = 197) and TGF-β-receptor II (p < 0.001, n = 197), but showed no significant correlation with tumor size, metastases, nodal status, histological grade, histological type, or estrogen receptor expression. While not achieving statistical significance, there was a trend towards longer survival times in patients with Smad4 negative tumors.
According to the suggested role of Smad4 as a tumor suppressor we observed that expression of Smad4 is lower in human breast cancer than in surrounding breast epithelium. However, we also observed a trend towards longer survival times in Smad4-negative patients, indicating the complex role of TGF-β signaling in tumor progression.
Transforming growth factor beta (TGF-β) is an important regulator of epithelial cell growth. Conflicting data exist about the influence of TGF-β on the development and progression of breast cancer. The growth of many human breast cancer cell lines is inhibited by TGF-β [1, 2] due to an inhibition of cell division and an induction of apoptosis. This is consistent with a tumor suppressor effect in well-differentiated tumors [3, 4]. On the other hand, certain highly aggressive breast cancer cell lines are refractory to suppressive effects of TGF-β on cell growth and may acquire sensitivity to pro-metastatic effects of TGF-β in later stages of tumorigenesis [5–8].
Smad proteins are the principal transducers of signals from TGF-β. TGF-β binds to homodimers of the TGF-β type II receptor (TβRII) which recruits and activates homodimers of TGF-β type I receptor (TβRI) serine/threonine kinase. Activated TβRI phosphorylates Smad2 or Smad3 [9, 10] which heterodimerize with Smad4. These heterocomplexes translocate into the nucleus where they bind DNA and regulate TGF-β dependent gene expression . TGF-β signaling is altered in various tumors. We have recently shown that loss of TβRII expression correlated significantly with better prognosis in estrogen receptor negative breast cancer patients , but little is known about the influence of the various downstream TGF-β signal transduction pathways on breast cancer prognosis. Deletions or mutations of Smad4 (also known as DPC4, deleted in pancreatic cancer), a tumor suppressor  and the only known co-Smad protein in TGF-β-, activin- and bone morphogenetic protein-signaling, are found in various epithelial tumors, including pancreatic carcinoma [14, 15], esophageal carcinoma , colorectal carcinoma , renal cell carcinoma , as well as breast carcinoma [19, 20]. However, little is known about the expression level of Smad4 or its prognostic significance in breast cancer.
In order to gain further insight into the status of Smad signal transduction in breast cancer progression, we used immunohistochemistry to analyze the expression of Smad4 in archival tissues of 197 breast cancer patients. Smad4 expression in tumor tissues was compared to that of surrounding normal tissues and correlated to established prognostic markers for breast cancer as well as overall survival.
Formalin-fixed, paraffin-embedded invasive breast carcinomas collected from 1987 to 1998 were retrieved from the archives of pathology of the Robert Bosch Hospital Stuttgart. Clinical and pathological data were retrieved from clinical databases as well as from the archives of pathology and included estrogen receptor, progesterone receptor, TGF-β-receptor I, TGF-β-receptor II, tumor size, nodal metastases, metastases, histological grade, tumor stage, histological type, age, menopausal status, therapy, and survival time of patients. Cases with incomplete clinical data sets or without sufficient archived tissues were excluded from the study (number of included cases: 197, number of excluded cases: 69, drop out rate: 25.9 %).
MCF-7 and MDA468 cells were grown in Dulbecco's modified Eagles medium (DMEM, Invitrogen, Carlsbad, USA) supplemented with 10% fetal calf serum (Invitrogen). Culture of MCF10CA1a, MCF10CA1h, MCF10A1k.cl2, and MCF10A cells is described elsewhere .
Detection of Smad4
3 μm paraffin sections were dewaxed by routine techniques. Immunohistochemistry was performed by the EnVision-technique (DAKO, Hamburg, Germany) using an Autostainer (DAKO). In brief, slides were soaked with antigen retrieval buffer (DAKO) and microwave-treated (3 × 5 min, 600 W) followed by treatment with blocking solution (methanol/H2O2) to block endogenous peroxidase. Slides were incubated at room temperature for 30 min with a monoclonal antibody to Smad4 (clone B-8, Santa Cruz Biotechnology Inc, Heidelberg, Germany) diluted 1:50 with antibody dilution medium (DAKO), and for 30 min with peroxidase-labeled polymer (DAKO). Anti-Smad4-antibody was detected by 3,3'-diaminobenzidine/ H2O2 containing staining solution. Sections were counterstained with hematoxylin. Negative controls were set up by omitting the primary antibody or the peroxidase labeled polymer. As a positive control specimens were stained with a different primary antibody to Smad4 (clone DCS-46, acris, Bad Nauheim, Germany) which yielded results identical to clone B-8 (data not shown). Immunostaining using an antibody to cytokeratin 18 (DAKO) was used as an additional control for the staining method.
To obtain specimens for immunocytochemistry, cells were grown on sterile coverslips to 70% to 80% confluence and fixed in acetone (-20°C, 5 min). For detection of Smad4 the protocol described above was slightly modified: No antigen retrieval was performed and specimens were incubated with the primary antibody (anti-Smad4, 1:50 in TBST) at room temperature overnight.
Immunohistological labeling was evaluated for tumor tissues as well as for adjacent, unaffected breast epithelia (i.e. breast epithelia not containing DCIS, LCIS or atypical ductal hyperplasia) by scoring staining intensity as: 0 (negative), 1 (weak), 2 (moderate), 3 (strong) as well as by scoring the fraction of Smad4-positive tumors cells: 1 (0% to 20%), 2 (21% to 50%), 3 (51% to 80%), 4 (81% to 100%). An Immunoreactive Score (IRS) was calculated by multiplying "staining intensity score" by "fraction of positive cells". IRS = 0 was defined as Smad4 – negative, an IRS between 1 and 12 (1≤IRS≤12) as Smad4-positive. Specimens were scored by 2 investigators and in cases of conflict, specimens were re-evaluated together.
Detection of TGF-β receptors
Immunostaining of TβRI and TβRII was described earlier .
For Western blots cells were grown to 80% – 90% subconfluent monolayers and processed as described elsewhere  using a monoclonal antibody against Smad4 (clone B-8, Santa Cruz Biotechnology Inc, Santa Cruz, California, USA), or a monoclonal antibody against α-tubulin (Zymed Laboratories Inc, San Francisco, California, USA).
For dot blots tumor tissues were snap frozen, homogenized by use of a Mikro Dissmembrator S (Braun Biotech International, Melsungen Germany), and incubated with lysis buffer (50 mM Tris/HCl pH7.6, 250 mM NaCl, 5 mM EDTA, 0.1% (v/v) Triton X-100) supplemented with the protease inhibitor complete (Roche Applied Science, Mannheim, Germany). Insoluble components were pelleted (12000 rpm, 4°C, 15 min) and protein concentrations were determined by the Bradford protein assay (Bio-Rad Laboratories GmbH, Munich, Germany). Samples were dotted onto a nitrocellulose membrane (Schleicher&Schuell, Dassel, Germany). Dot blots were probed with anti Smad4 – antibody (clone B-8, Santa Cruz Biotechnology Inc), detected with alkaline phosphatase-conjugated secondary antibody (DAKO, Hamburg, Germany) and developed using BCIP/NBT (Roche Applied Science) solution (100 mM Tris/HCl pH9.5, 100 mM NaCl, 50 mM MgCl2, NBT 100 μl/100 ml, BCIP 75 μl/100 ml)
IRS values calculated for breast cancer and adjacent normal breast epithelia originating from the same slide were compared by paired two-sample sign test. Cross tabulations were analyzed by Spearman's rank correlation test. Survival time analysis was done by the Kaplan-Meier estimator  and the log-rank test . p-values < 0.05 were considered to be significant. No p-value was adjusted for possible multiple testing. Analysis was performed using SPSS version 10.0 for both, patient with "unknown cause of death" classified as "death due to cancer" and as "death not due to cancer", and similar results were obtained for both analyses; results shown in this work were obtained by the analyses classifying "unknown cause of death" as "death not due to cancer".
Association of Smad4-expression and breast cancer characteristics (n = 197). In case of histological type carcinoma other than ductal invasive or lobular invasive were excluded from statistical analysis (resulting n = 178). Data were calculated by Spearman's rank correlation test.
rhos (95% confidence interval)
Immunostaining of Smad4 can be used to determine Smad4 expression
Smad4 expression in tumor tissues and surrounding normal tissues
Correlation of the expression of Smad4 and TGF-β-receptors
Since Smad4 is a key component of the TGF-β-signaling pathway, we investigated its expression compared to that of the TGF-β-receptors. Smad4-expression correlated significantly with the expression of TβRI (ρS = 0.366, p < 0.001, Spearman's rank correlation test; Fig. 2b, upper panel) and TβRII (ρS = 0.286, p < 0.001, Spearman's rank correlation test; Fig. 2b, lower panel) as well as with the coexpression of TβRI and TβRII (ρS = 0.345, p < 0.001, Spearman's rank correlation test, data not shown).
Correlation of Smad4-expression with prognostic parameters and influence of Smad4 on overall survival time
Analysis of survival time of Smad4-positive and Smad4-negative patients stratified with regard to tumor stage, ER-status, TβRI-status, and TβRII-status. 5-year survival rates were computed by the Kaplan-Meier estimator. Survival curves were compared by log-rank test (n = 197).
5 year survival
Log rank test
Because of the central role of Smad4 in TGF-β signaling and the well-described cross-talk of TGF-β and estrogen signaling [30–32], survival curve analysis was performed after stratifying the collective with regard to ER, TβRI, and TβRII (Table 2). Although no significant influence of Smad4 expression on survival curves could be shown for any group, nevertheless there was a trend towards higher 5-year survival rate in TβRII negative, Smad4 negative patients (5-year survival rate: 100%, n = 19) than in TβRII negative, Smad4 positive patients (5-year survival rate: 80%, n = 39), or TβRII positive, Smad4 negative patients (5-year survival rate: 85%, n = 25,) and TβRII positive, Smad4 positive patients (5-year survival rate: 72.8%, n = 114). Taken together, patients with a "double hit" in the TGF-β pathway had the highest 5-year survival rate, followed by patients with a "single hit", whereas those with intact signaling showed the worst survival. There was no survival effect for the presence or absence of TβRI, which is much less prevalently altered in cancer than the ligand-binding TβRII, whereas the effect of Smad4 could still be seen in this stratification. Stratifying for ER again revealed a trend for longer 5-year survival rate in Smad4 negative patients than in Smad4 positive patients, as well as a trend towards a longer 5 year survival rate in Smad4 positive, ER positive patients (78.9%) as compared to Smad4 positive, ER negative patients (66.5%; Table 2), consistent with the known effect of the ER status on survival.
We have used immunohistochemical staining to show that Smad4 expression is markedly decreased in breast cancer compared to surrounding normal breast epithelium. Smad4 staining correlated with the expression of TβRI and TβRII. In Smad4 negative patients of stage III breast cancer, i.e. in patients with positive lymph nodes at primary diagnosis, a trend for an increased 5-year survival was observed.
Although the expression of Smad4 in breast cancer tissues as well as in normal epithelia varied greatly between specimens from individual patients, Smad4 expression was significantly reduced in tumor tissues as compared to the surrounding normal epithelia within the same specimen. Overall, we found that 23% of the specimens were Smad4 negative and that a further 41% stained weakly. In contrast, a tissue microarray analysis of Smad4 expression in 456 cases of breast carcinomas by Xie et al. , reported that only 2% breast cancer tissues were Smad4-negative. This difference might be due to the different age of the specimens, which was up to 70 years in the set used by Xie et al. , and to staining methods employing different secondary detection methods, as well as to the different analysis of the specimens. Another recently published paper  showed that Smad4 mRNA expression is reduced in ductal carcinoma as compared to normal tissues. At the protein level, we have shown that in the MCF10-system of genetically related cell lines of differing degrees of malignancy, Smad4 levels were higher in non-malignant MCF10A cells than in the increasingly malignant cell lines MCF10At1k.cl2, MCF10CA1h, and MCF10CA1a, indicating that decreased Smad4 protein expression might accompany tumor progression from early stages on in-situ and in-vivo. The reduced expression of Smad4 observed in this study could have resulted either from decreased transcription rates from the Smad4 gene or from increased degradation of the Smad4 protein. The latter appears more probable as various studies have shown that the steady state level of Smad4 is tightly regulated by ubiquitinylation [35, 36] and sumoylation [37, 38].
TGF-β inhibits cell proliferation in breast epithelial cells and many breast cancer cell lines . In our specimens, Smad4 expression in tumor tissue was lower than in surrounding epithelia, indicating impaired TGF-β signaling and possible escape from TGF-β-dependent growth inhibition. Given the central role of Smad4 in the signaling of all TGF-β-related superfamily members, reduced or absent Smad4-expression as described here would be expected to alter the signaling not only of TGF-β, but also of the BMPs and activins, which also can inhibit proliferation of breast cancer cells [39–41]. Of these proteins, TGF-β, that additionally signals via MAPK-pathways  and interacts with ER signaling , paradoxically also exhibits pro-metastatic activity in later stages of cancer progression [7, 42]. Consistent with this pro-metastatic role of TGF-β we have previously shown that loss of TβRII expression correlated significantly with better prognosis in estrogen receptor negative breast cancer patients . In this work we show that loss of Smad4 expression as a trend correlates with increased survival times. Although not reaching significance, this effect was found in all subgroups. Recent data showing that growth of primary xenografted tumors of MDA-MB-231 human breast cancer cells in mice is not affected by the Smad4 status, whereas metastasis of these cells to bone is Smad4-dependent, provide a basis for the trend we have observed of increased survival times in Smad4 negative patients. This trend showing an effect of Smad4 on survival time was most prominent in TβRII negative patients indicating that silencing of TGF-β signal transduction via the Smad pathway might improve patient survival. Similarly, the trend towards higher 5-year survival rate of Smad4 positive, ER positive patients as compared to Smad4 positive, ER negative patients might be due to interaction of ER and Smad3 that is known to inhibit TGF-β signal transduction , again presumably reducing the pro-metastatic effect of TGF-β.
Survival in breast cancer patients that undergo surgery of the primary tumor greatly depends on the systemic spread of the tumor and complications caused by metastasis. In our collective, loss of Smad4 showed a trend towards improved survival of patients with advanced disease (WHO stage 3/4), and was particularly striking for WHO stage 3, whereas the data of patients at stage 4 were weak due to the low case number. This effect was likely due to the benefit of loss of Smad4 in patients with positive nodal status, as Smad4 negative patients staged N1 had a 50% increased 5-year survival rate as compared to Smad4 positive patients. Similarly, biallelic loss of TβRII expression due to microsatellite instability has previously been reported to positively influence the prognosis particularly of node positive patients suffering from colon cancer .
In our studies loss of Smad4 and TGFβRII showed a trend for longer survival times in subgroups (N1 or ER negative, respectively), but loss of Smad2-phosphorylation was previously correlated with a worse all-over survival in node positive breast cancer . On the other hand, in-vivo data obtained by employing different mouse models of breast cancer [8, 21, 23, 45] indicate that reducing TGF-β-signaling by impairing TβRII, TβRI kinase activity or Smad3-phosphorylation enhances development of the primary lesion but reduces metastasis whereas constitutive activation of TβRI has opposite effects. These seemingly contradictory results might be due, at least in part, to the distinct roles of Smad2, Smad3, and Smad4 in TGF-β signaling in the changing signaling context of cancer progression, resulting in altered target gene expression and ultimately different biological effects. TGF-β signaling via canonical and non-canonical pathways is complex, and the all-over effect of TGF-β on cell behavior is context dependent. Based on this complexity, the power of the exploratory study presented here is limited, although the results are consistent with the current literature. It will be the goal of future work to further elucidate the role of TGF-β and the proteins involved in TGF-β signal transduction in progression of breast cancer.
Our data show that Smad4 expression in breast cancer is lower than in normal adjacent breast epithelial tissue and imply that impairment of TGF-β/Smad-signaling because of loss of TβRII or Smad4 might improve 5-year survival by possibly slowing down metastases. Our data from human tumors, although not achieving statistical significance, are in agreement with results obtained in vitro and in animal models, where reduced TGF-β signaling provides a survival benefit in advanced tumor stages due to decreased metastases.
List of abbreviations
bone morphogenetic protein
mitogen activated protein kinase
TGF-β receptor 1
TGF-β receptor II
transforming growth factor β
We thank Kerstin Gawronski, Petra Hauptvogel, and Monika McClellan for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (grant Kn 228/2-1/2) and the Robert Bosch Foundation.
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