SMAD4May Be An Independent Molecular Prognostic Markerof Differentiation And Prognosis In Ductal Breast Carcinoma

Human breast cancers are the most frequent carcinomas in females and thesecond most common cause of cancer-related mortality in women. In recent years, thebreast cancer incidence rate has increased globally; however, mortality rates havedecreased, which has been attributed to increased understanding of underlyingmolecular pathology. Molecular medicine has facilitated the identification of differenttumor markers, which may increase the accuracy of classifying and grading tumors, aswell as assessing prognosis. Such an extensive understanding can assist in clinicaltherapeutic decisions in order to provide the optimal treatment.Nearly80%of all diagnosed preinvasive and invasive breast cancers worldwideare of the ductal subtype. Ductal breast carcinoma is characterized by profoundheterogeneity. This complexity increases the difficulty of accurate histologicassessment and grading, which ultimately affects the choice of treatment regimens.Thus, identifying a molecular marker specific for ductal breast carcinoma, which alsoprovides insight regarding tumor grade and prognosis, is of clinical importance.Scholars has been recently concerned about relationship between SMAD4gene andtumor progression.SMAD4is a downstream mediator of transforming growth factorbeta (TGF-β), an important multifunctional cytokine that regulates cell proliferation,differentiation and extracellular matrix production. As a tumor suppressor, TGF-βinhibits cell growth predominantly by signaling via the SMAD pathway. AlthoughSMAD4is not required for translocation into the nucleus, it is required for the SMADcomplex to act as a transcription factor.The relationship between SMAD4gene inactivation and tumorigenesis has beenextensively studied, especially in pancreatic cancer and colorectal cancer, but thereare limited data evaluating the relationship between SMAD4expression and tumorprogression and very little data in breast cancer. Given the central role played byTGF-β in breast cancer development and progression, studies of SMAD4expressionare potentially of great interest. Similar to TGF-β, SMAD4may play a dual role in breast tumorigenesis with varyingroles during the development and progression of breast cancer. Many previous studieshighlight the complexity of SMAD4interactions and functional activity in breastcancer tissues and cells. TGF-β/SMAD signal may also interact with other signal.TGF-β signaling is both enhanced by, and runs in parallel to the mitogen-activatedprotein kinase (MAPK) signaling.Currently, a definitive diagnosis of most tumors still depends on clinical biopsy.Immunohistochemical (IHC) analysis is a less complicated technique and morewidely available in routine clinical laboratories than genetic analysis. Therefore,immunolabeling for SMAD4is an ideal tool for examining the SMAD4expression inbreast carcinomas and could potentially be used as a diagnostic and prognosticindicator of disease type and/or progression. In this study, we used IHC to investigatethe level of expression of SMAD4in136tumor samples and70cases of tumorsamples from IMGENEX tissue arrays. Moreover, we have tested TGF-β signaling inMDA-MB-468cells with SMAD4and without SMAD4in responding to TGF-β1stimulation. We also have evaluated a potential role of SMAD4in breast carcinomadevelopment.Methods:1. Expression of SMAD4was detected in136ductal breast cancer tissues and120adjacent tissues by immunohistochemical assay.2. IHC for tissues array with70ductal breast cancer patients was carried out forSMAD4expression with the same IHC procedure as for our clinical samples.3Semi-quantitative analysis and quantitative real-time PCR assay of the expression ofSMAD4/SMD4at the level of mRNA in the tissues of136ductal breast carcinomasand120surrounding normal breast epithelial tissues were performed, and theexpression of SMAD4at the level of protein was detected in two kinds of tissues.4Non-malignant breast epithelial MCF10A cells and breast cancer MDA-MB-468,MCF-7, T47D cells were cultured. Western blot was used to detect SMAD4expression of above four kinds of cells at protein level.5. SMAD4was restored in SMAD4mutation of MDA-MB-468cells by using aretrovirus Plasmids pBabe SMAD4. Stable SMAD4expressing MDA-MB-468cellsand SMAD4-null expressing pBabe vector control MDA-MB-468cells weresuccessfully established, which was verified by Western blot assay. 6. TGF-β/SMAD signal pathway members such as p-SMAD2protein expression, aswell as members of the MAPK pathway such as p-ERK and p-P38protein expressionchange were detected by Western blot assay in SMAD4-over expressingMDA-MB-468cells and a SMAD4-null expressing pBabe vector controlMDA-MB-468cells induced by TGF-β1Results:1The expression of SMAD4in136cases of ductal breast cancer tissues wassignificantly lower than in normal breast epithelial tissues, and then there wasnegative correlation between SMAD4expression and histological grade of tumor.2. According to tissues arrays results, SMAD4expression pattern in70cases of ductalbreast cancer tissues samples was identical to in our previous tissues samples.Thesesupported our findings, that is to say, the expression of SMAD4in ductal breastcancer tissues compared to adjacent normal breast epithelial tissues was decreased.3. Western blot results also showed that the expression of SMAD4in breastsurrounding normal epithelial tissues was significantly higher than in ductal breastcancer tissues. This result was also confirmed by quantitative real-time PCR assay andsemi-quantitative RT-PCR assay.4Western blot was used to detect the expression of SMAD4in MDA-MB-468,MCF-7， T47D cells and nonmalignant breast epithelial MCF10A cells. The SMAD4expression level of MCF10A cells was the highest, while the expression ofMDA-MB-468cells was lost.5Stable SMAD4expressing MDA-MB-468cell line and SMAD4-null expressingpBabe vector control MDA-MB-468cell line were successfully established.6SMAD4restoration in MDA-MB-468cells did not interfere a response to canonicTGF-β/SMAD signaling after exposure to TGF-β1. Significantly different betweenthe two cell lines was the non-SMAD signal pathway member induction in responseto TGF-β stimulation, such as p-ERK and p-P38.In conclusion, our data show that SMAD4expression in breast cancer issignificantly lower than that in normal adjacent breast epithelial tissues and thatSMAD4expression levels were inversely related to histologic grade. SMAD4expression level in breast cancer cells played a role in responding non-SMADsignaling but not the canonic SMAD signaling. Our data provide evidence that thereduced expression of SMAD4may play an important role during the development of ductal breast carcinoma and may be a potential molecularmarker for assessing thedegree of differentiation of ductal breast carcinoma. SMAD4may be an independentpredictor of differentiation and prognosis in ductal breast carcinoma.