SI-CLP inhibits the growth of mouse mammary adenocarcinoma by preventing recruitment of tumor-associated macrophages

Chitinase-like proteins (CLP) are chitin-binding proteins that lack chitin hydrolyzing activity, but possess cytokine-like and growth factor-like properties, and play crucial role in intercellular crosstalk. Both human and mice express two members of CLP family: YKL-40 and stabilin-1 interacting chitinase-like protein (SI-CLP). Despite numerous reports indicating the role of YKL-40 in the support of angiogenesis, tumor cell proliferation, invasion and metastasis, the role of its structurally related protein SI-CLP in cancer was not reported. Using gain-of-function approach, we demonstrate in the current study that the expression of recombinant SI-CLP in mouse TS/A mammary adenocarcinoma cells results in significant and persistent inhibition of in vivo tumor growth. Using quantitative immunohistochemistry, we show that on the cellular level this phenomenon is associated with reduced infiltration of tumor-associated macrophages (TAMs), CD4+ and FoxP3+ cells in SI-CLP expressing tumors. Gene expression analysis in TAM isolated from SI-CLP-expressing and control tumors demonstrated that SI-CLP does not affect macrophage phenotype. However, SI-CLP significantly inhibited migration of murine bone-marrow derived macrophages and human primary monocytes toward monocyte-recruiting chemokine CCL2 produced in the tumor microenvironment (TME). Mechanistically, SI-CLP did not affect CCL2/CCR2 interaction, but suppressed cytoskeletal rearrangements in response to CCL2. Altogether, our data indicate that SI-CLP functions as a tumor growth inhibitor in mouse breast cancer by altering cellular composition of TME and blocking cytokine-induced TAM recruitment. Taking into consideration weak to absent expression of SI-CLP in human breast cancer, it can be considered as a therapeutic protein to block TAM-mediated support of breast tumor growth.     

Raf kinase inhibitor protein suppresses nuclear factor-κB-dependent cancer cell invasion through negative regulation of matrix metalloproteinase expression

Accumulating evidence suggests that Raf kinase inhibitor protein (RKIP), which negatively regulates multiple signaling cascades including the Raf and nuclear factor-κB (NF-κB) pathways, functions as a metastasis suppressor. However, the basis for this activity is not clear. We investigated this question in a panel of breast cancer, colon cancer and melanoma cell lines. We found that RKIP negatively regulated the invasion of the different cancer cells through three-dimensional extracellular matrix barriers by controlling the expression of matrix metalloproteinases (MMPs), particularly, MMP-1 and MMP-2. Silencing of RKIP expression resulted in a highly invasive phenotype and dramatically increased levels of MMP-1 and MMP-2 expression, while overexpression of RKIP decreased cancer cell invasion in vitro and metastasis in vivo of murine tumor allografts. Knockdown of MMP-1 or MMP-2 in RKIP-knockdown cells reverted their invasiveness to normal. In contrast, when examining migration of the different cancer cells in a two-dimensional, barrier-less environment, we found that RKIP had either a positive regulatory activity or no activity, but in no case a negative one (as would be expected if RKIP suppressed metastasis at the level of cell migration itself). Therefore, RKIP’s function as a metastasis suppressor appears to arise from its ability to negatively regulate expression of specific MMPs, and thus invasion through barriers, and not from a direct effect on the raw capacity of cells to move. The NF-κB pathway, but not the Raf pathway, appeared to positively control the invasion of breast cancer cells. A regulatory loop involving an opposing relationship between RKIP and the NF-κB pathway may control the level of MMP expression and cell invasion.     

Reduction of Raf-1 kinase inhibitor protein expression correlates with breast cancer metastasis.

PURPOSE: Raf-1 kinase inhibitor protein (RKIP) was originally identified as the first physiologic inhibitor of the Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) pathway. This pathway regulates fundamental cellular functions, including those that are subverted in cancer cells, such as proliferation, transformation, survival, and metastasis. Recently, RKIP has been recognized as a strong candidate for a metastasis suppressor gene in cell and animal model systems. Therefore, we investigated whether RKIP expression is altered in clinical specimens of human primary breast cancers and their lymph node metastases. EXPERIMENTAL DESIGN: Paraffin-embedded tumor samples from 103 breast cancer patients were examined immunohistochemically for the expression of RKIP, activated ERK, and apoptosis. The specificity of the antibodies used was validated by competition experiments with purified recombinant RKIP protein. RESULTS: RKIP expression was high in breast duct epithelia and retained to varying degrees in primary breast tumors. However, in lymph node metastases, RKIP expression was highly significantly reduced or lost (P = 0.000003). No significant correlations were observed between RKIP expression and histologic type, tumor differentiation grade, size, or estrogen receptor status. CONCLUSION: This is the first study of RKIP expression in a large clinical cohort. It confirms the results of cell culture and animal studies, suggesting that in human breast cancer, RKIP is a metastasis suppressor gene whose expression must be down-regulated for metastases to develop. RKIP expression is independent of other markers for breast cancer progression and prognosis.     

KRAS promotes tumor metastasis and chemoresistance by repressing RKIP via the MAPK–ERK pathway in pancreatic cancer

Oncogenic KRAS plays a crucial role in pancreatic ductal adenocarcinoma (PDAC) development and progression. However, the mechanism has not been clearly elucidated. RKIP is a tumor repressor, and loss of RKIP has been shown in PDAC. Here, we found that KRAS expression was inversely correlated with RKIP expression in PDAC fresh tissue regardless of the KRAS mutant status. The negative correlation between KRAS and RKIP was further confirmed in our PDAC tissue microarray. KRAS overexpression and RKIP downregulation were associated with poor clinical outcomes. Knockdown or overexpression of KRAS in PDAC cell lines robustly increased or decreased, respectively, RKIP protein and mRNA levels. Furthermore, the MAPK–ERK pathway was involved in the regulation of RKIP. KRAS‐regulated RKIP expression, which in turn affected the expression of pivotal epithelial–mesenchymal transition (EMT) and apoptosis factors. The biological function of the KRAS–RKIP axis was demonstrated in human pancreatic cancer cells in vitro and in vivo. KRAS knockdown increased RKIP expression and inhibited metastasis and chemoresistance. Moreover, the feature of metastasis and chemoresistance was rescued in the KRAS‐knockdown cells through the inhibition of RKIP by RNA interference. In conclusion, our studies demonstrate how KRAS inhibits the tumor suppressor RKIP, thus offering novel justification for targeting RKIP as a strategy to overcome KRAS‐induced tumor metastasis and chemoresistance in PDAC.     

PTEN inhibits macrophage polarization from M1 to M2 through CCL2 and VEGF-A reduction and NHERF-1 synergism

PTEN has been studied in several tumor models as a tumor suppressor. In this study, we explored the role of PTEN in the inhibition state of polarized M2 subtype of macrophage in tumor microenvironment (TME) and the underlying mechanisms. To elucidate the potential effect in TME, RAW 264.7 macrophages and 4T1 mouse breast cancer cells were co-cultured to reconstruct tumor microenvironment. After PTEN was down-regulated with shRNA, the expression of CCL2 and VEGF-A, which are definited to promote the formation of M2 macrophages, have a dramatically increase on the level of both gene and protein in co-cultured RAW 264.7 macrophages. And at the same time, NHERF-1 (Na⁺/H⁺ exchanger regulating factor-1), another tumor suppressor has a similar tendency to PTEN. Q-PCR and WB results suggested that PTEN and NHERF-1 were consistent with one another no matter at mRNA or protein level when exposed to the same stimulus. Coimmunoprecipitation and immunofluorescence techniques confirmed that PTEN and NHERF-1 were coprecipitated, and NHERF-1 protein expression was properly reduced with rCCL2 effect. In addition, cell immunofluorescence images revealed a profound transferance, in co-cultured RAW 264.7 macrophages, an up-regulation of NHERF-1 could promote the PTEN marked expression on the cell membrane, and this form for the interaction was not negligible. These observations illustrate PTEN with a certain synergy of NHERF-1, as well as down-regulation of CCL2 suppressing M2 macrophage transformation pathway. The results suggest that the activation of PTEN and NHERF-1 may impede the evolution of macrophages beyond the M1 into M2 phenotype in tumor microenvironment.     

Immunohistochemical detection of the Raf kinase inhibitor protein in nonneoplastic gastric tissue and gastric cancer tissue

Expression of the Raf kinase inhibitor protein (RKIP), a metastasis suppressor, is high in normal tissues, low in primary cancers, and lowest or absent in metastatic cancers. Here, we studied the expression of RKIP in nonneoplastic gastric tissue and gastric cancer tissue by immunohistochemistry (IHC) and evaluated its role in the genesis and metastasis of gastric cancer. RKIP immunoreactivity was evaluated in 40 samples of nonneoplastic gastric tissues and 75 samples of gastric cancer tissues. Among the 40 samples of nonneoplastic gastric tissue, 35 (87.5%) were positive for RKIP expression and 5 (12.5%) were negative; in the 75 samples of primary gastric cancer tissue, 39 (52%) were positive for RKIP expression and 36 (48%) were negative. Among 26 samples of metastatic lymph node tissues, 5 (19.2%) were positive for RKIP expression and 21 (80.8%) were negative. RKIP expression level was highest in nonneoplastic gastric tissue, low in primary gastric cancer tissue, and lowest or undetectable in metastatic gastric cancer tissue. Our data suggest that RKIP may play a role in the genesis and metastasis of gastric cancer.     

Tumor-derived CCL5 does not contribute to breast cancer progression

Besides functioning as a chemotactic factor, CCL5 has been associated with progression of disease in women with breast cancer, immune modulation and metastasis. Here we asked whether CCL5 produced by tumor cells contributed to growth or metastasis of breast cancer. For this purpose, we used two murine mammary carcinomas, the 4T1 tumor which is metastatic and constitutively expresses CCL5, and the 168 tumor which is not metastatic and does not constitutively express CCL5. RNA interference was used to inhibit CCL5 expression from the 4T1 tumor, and a CCL5 transgene was used to express CCL5 by the 168 tumor. Six different clones of 4T1 that exhibited stable reduction in CCL5 expression, and three different clones of 168 that exhibited stable CCL5 expression were compared to the parental tumors and vector transfected controls. Significantly, in both models, tumor-derived CCL5 expression did not correlate with MHC expression, growth rate, or metastatic ability of the tumors. These results show that tumor-derived CCL5 expression alone does not make a significant contribution to breast cancer progression.     

乳腺癌微环境中趋化因子CCL20作用的研究进展

趋化因子是一类低分子量细胞因子,在调节肿瘤微环境中发挥重要作用。肿瘤微环境中的趋化因子不仅能够趋化白细胞,也能诱导附近的反应细胞影响肿瘤生长、侵袭、转移和血管生成。趋化因子与炎症和肿瘤疾病的发生发展密切相关,其中CC家族中的趋化因子CCL20在多种恶性肿瘤的侵袭和转移中发挥重要作用。近年发现,趋化因子CCL20在乳腺癌免疫抑制、血管生成及诱导上皮间质转化和肿瘤侵袭转移中起重要的调控作用。然而,CCL20在乳腺癌中的作用机制尚未明确。本文重点阐述趋化因子CC亚家族成员CCL20在乳腺癌微环境中的功能,探讨CCL20与乳腺癌微环境之间的关系,以期为乳腺癌靶向治疗提供新的思路。     

Clinical implications for loss or diminution of expression of Raf-1 kinase inhibitory protein and its phosphorylated form in ductal breast cancer

Raf Kinase inhibitory protein (RKIP) is a well-established metastasis suppressor that is frequently downregulated in aggressive cancers. The impact of RKIP and its phosphorylated form on disease-free survival (DFS) and other clinicopathological parameters in breast cancer is yet to be discovered. To this end, we examined RKIP expression in 3 independent breast cancer cohorts. At the Protein level, loss or reduced total RKIP expression was associated with large-sized tumors characterized by high proliferative index, high-grade and diminished estrogen (ER) and progesterone receptor expression. Loss or diminution of RKIP expression was significantly associated with shorter DFS in all cohorts. Moreover, the complete loss of p-RKIP was an independent prognostic factor using multivariate analysis in operable invasive ductal breast cancer. We show for the first time that ER, partly, drives RKIP expression through MTA3-Snail axis. Consistent with this finding, we found that, at the mRNA level, RKIP expression varied significantly across the different molecular subtypes of breast cancer with the Luminal (ER+) subtype expressing high levels of RKIP and the more aggressive Claudin-low (ER-) subtype, which depicted the highest epithelial to mesenchymal transition (EMT) registered the lowest RKIP expression levels. In conclusion, loss of expression/diminution of RKIP or its phosphorylated form is associated with poor diseases-free survival in breast cancer. Determining the expression of RKIP and p-RKIP adds significant prognostic value to the management and subtyping of this disease.     

Stat3 orchestrates interaction between endothelial and tumor cells and inhibition of Stat3 suppresses brain metastasis of breast cancer cells

Brain metastasis is a major cause of morbidity and mortality in patients with breast cancer. Our previous studies indicated that Stat3 plays an important role in brain metastasis. Here, we present evidence that Stat3 functions at the level of the microenvironment of brain metastases. Stat3 controlled constitutive and inducible VEGFR2 expression in tumor-associated brain endothelial cells. Furthermore, inhibition of Stat3 by WP1066 decreased the incidence of brain metastases and increased survival in a preclinical model of breast cancer brain metastasis. WP1066 inhibited Stat3 activation in tumor-associated endothelial cells, reducing their infiltration and angiogenesis. WP1066 also inhibited breast cancer cell invasion. Our results indicate that WP1066 can inhibit tumor angiogenesis and brain metastasis mediated by Stat3 in endothelial and tumor cells.     

A mutual activation loop between breast cancer cells and myeloid-derived suppressor cells facilitates spontaneous metastasis through IL-6 trans-signaling in a murine model

Introduction

Tumor cell interactions with the microenvironment, especially those of bone-marrow-derived myeloid cells, are important in various aspects of tumor metastasis. Myeloid-derived suppressor cells (MDSCs) have been suggested to constitute tumor-favoring microenvironments. In this study, we elucidated a novel mechanism by which the MDSCs can mediate spontaneous distant metastasis of breast cancer cells.

Methods

Murine breast cancer cells, 4T1 and EMT6, were orthotopically grafted into the mammary fat pads of syngeneic BALB/c mice. CD11b⁺Gr-1⁺ MDSCs in the spleen, liver, lung and primary tumor mass were analyzed. To evaluate the role of MDSCs in the distant metastasis, MDSCs were depleted or reconstituted in tumor-bearing mice. To evaluate whether MDSCs in the metastasizing tumor microenvironment affect breast cancer cell behavior, MDSCs and cancer cells were co-cultivated. To investigate the role of MDSCs in in vivo metastasis, we blocked the interactions between MDSCs and cancer cells.

Results

Using a murine breast cancer cell model, we showed that murine breast cancer cells with high IL-6 expression recruited more MDSCs and that the metastasizing capacity of cancer cells paralleled MDSC recruitment in tumor-bearing mice. Metastasizing, but not non-metastasizing, tumor-derived factors induced MDSCs to increase IL-6 production and full activation of recruited MDSCs occurred in the primary tumor site and metastatic organ in the vicinity of metastasizing cancer cells, but not in lymphoid organs. In addition, tumor-expanded MDSCs expressed Adam-family proteases, which facilitated shedding of IL-6 receptor, thereby contributing to breast cancer cell invasiveness and distant metastasis through IL-6 trans-signaling. The critical role of IL-6 trans-signaling was confirmed in both the afferent and efferent pathways of metastasis.

Conclusion

In this study, we showed that metastasizing cancer cells induced higher MDSCs infiltration and prompted them to secret exaggerated IL-6 as well as soluble IL-6Rα, which, in turn, triggered a persistent increase of pSTAT3 in tumor cells. This potential tumor-MDSC axis involving IL-6 trans-signaling directly affected breast cancer cell aggressiveness, leading to spontaneous metastasis.     

Reduced expression of Raf kinase inhibitor protein correlates with poor prognosis in pancreatic cancer

Aim

Raf kinase inhibitory protein (RKIP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. But its function in pancreatic cancer was not yet clarified completely. The aim of this study was to investigate the involvement of RKIP in pancreatic cancer.

Methods

RKIP expression was investigated retrospectively by immunohistochemistry in paraffin-embedded primary tumor tissue samples from a series (n?=?99) of consecutive patients with pancreatic cancer. Survival was calculated using Kaplan?CMeier curves. Parameters found to be of prognostic significance in univariate analysis were verified in a multivariate Cox regression model.

Results

RKIP expression was high in normal pancreatic epithelium and retained to varying degrees in pancreatic cancer tissues. However, in tumor tissues with lymph node metastasis (P?=?0.008) and high UICC stage (P?=?0.006), RKIP expression was highly significantly reduced or lost. Furthermore, the reduced expression of RKIP significantly correlated with both poor overall and disease-free survival (P?=?0.008 and 0.01, respectively). Multivariate analyses revealed RKIP to be an independent prognosticator.

Conclusion

These findings suggest that RKIP could be a promising marker for predicting a better prognosis in pancreatic cancer.     

The CC chemokine MCP-1/CCL2 in pancreatic cancer progression: regulation of expression and potential mechanisms of antimalignant activity

The aim of this study was to discover whether MCP-1/CCL2, a CC chemokine able to attract macrophages, is expressed in human pancreatic cancer and how it modulates cancer progression. All primary tumors were tested, and 6 of 14 pancreatic cancer cell lines were constitutively secreted CCL2. Analysis of the regulation demonstrated that the expression of CCL2 was significantly elevated and in a synergistic manner by IFN-gamma, tumor necrosis factor alpha, and interleukin 1beta. By immunohistochemistry and in situ hybridization, CCL2 production was confirmed in neoplastic ducts from surgical specimens. Serum levels of CCL2 in pancreatic cancer patients were significantly higher than in normal healthy subjects (P < 0.0001). Patients with high circulating levels of CCL2 had significantly higher survival rate than low CCL2 producers. Serum CCL2 levels positively correlated with tumor macrophage infiltration and inversely correlated with tumor proliferative activity (Ki67 expression). A direct effect of CCL2 on tumor cells is to be excluded, either because primary tumors as well as cell lines have no detectable CCL2 receptor (CCR2) and because addition of CCL2 on tumor cells in vitro did not modify cell cycle progression or apoptosis. In vitro, a model of tumor microenvironment showed a direct antiproliferative and proapoptotic activity of monocytes toward pancreatic cancer cell, which is mediated at least in part by interleukin 1beta. Moreover, other proinflammatory cytokines such as tumor necrosis factor alpha and IFN-gamma appeared able to induce apoptosis and to reduce the proliferative rate of pancreatic cancer. On the whole, the results presented in our investigation suggest that CCL2 could be a relevant negative regulator of pancreatic cancer progression.     

Clinical significance of RKIP mRNA expression in non-small cell lung cancer

Raf-1 kinase inhibitor protein (RKIP) expression was associated with the onset, development, invasion, and metastasis of numerous tumor types including prostate cancer, melanoma, colorectal cancer, liver cancer, and breast cancer. However, RKIP mRNA expression and the clinical significance in non-small cell lung cancers (NSCLC) remain unresolved. Real-time PCR was performed to detect the expression of RKIP mRNA in 126 pairs of lung tumor tissues (TT) and surrounding normal tissues (sNT). Correlations between RKIP mRNA expression and clinicopathological features were evaluated by statistical analysis. In the 126 patients examined, RKIP mRNA expression was significantly lower in lung TT than the sNT (p?<?0.05). Our results indicated that downregulation of RKIP mRNA expression was associated with a poorer N-stage (p?=?0.019) and poorer pathological TNM stage (p?=?0.015). However, no significant association was observed between the expression status of RKIP mRNA and clinicopathologic factors, such as gender, age, histological type, and the size of the tumor (p?>?0.05). The level of RKIP mRNA expression was found to be significantly downregulated in NSCLC, and the lower mRNA levels correlated with poorer differentiation, advanced pathologic TNM stage in patients with NSCLC.     

三阴性乳腺癌肿瘤微环境特征免疫相关生物学标志物筛选及功能预测分析

目的  通过研究三阴性乳腺癌（TNBC）患者中的免疫微环境细胞浸润情况,从免疫微环境层面对TNBC 患者的预后进行分析,提出新的TNBC 免疫预后相关生物学标志物并描述其在TNBC中的生物学过程,为临床治疗TNBC 提供新的治疗靶点。方法  检索GEO TNBC 相关公开数据库并下载样本的转录组学信息,通过CIBERSORT 分析TNBC 患者的免疫细胞成分,再通过无监督聚类对TNBC患者的免疫细胞成分进行聚类,寻找最佳的分类类型。采用差异基因分析探索TNBC 中表达量异常的基因;通过lasso-logistic 模型进行降维筛选免疫亚型的生物标志物;通过WGCNA进行基因共表达分析,并通过GSEA 分析基因模块的主要富集的通路注释其参与的生物学过程。结果  通过整理下载的GSE103091 数据,共纳入包含mRNA 数据和完整总生存数据TNBC 患者100 例,其免疫细胞成分可分两类：低免疫浸润组43 例,其中转移19 例（44．2％）;高免疫浸润组57 例,其中转移11 例（19．3％）,两组转移患者比例差异有统计学意义（χ² ＝5．09,P ＝ 0．01）。低免疫浸润主要受到正向免疫调节相关通路和细胞刺激因子相关通路的调控,患者总生存较低,预后不良。通过Lasso-logistic 迭代特征选取算法分析发现,FOLH1、WDR18、LINC00638、OAS3、SETDB2可以很好地代表TNBC 的免疫亚型,并且发现FOLH1、WDR18、LINC00638、OAS3、SETDB2可以作为预测TNBC 患者预后的关键基因。结论  在TNBC 患者中FOLH1、WDR18、LINC00638、OAS3、SETDB2是重要的预后相关基因,并且通过T 细胞激活等免疫调节相关通路影响患者预后。     

Raf kinase inhibitor protein: a prostate cancer metastasis suppressor gene

Defining the mechanisms that confer metastatic ability on cancer cells is an important goal towards prevention of metastasis. A gene array screen between a non-metastatic prostate cancer cell and its metastatic derivative line revealed decreased expression of Raf kinase inhibitor protein (RKIP) in the metastatic cell line. This finding is consistent with the possibility that loss of RKIP is associated with metastasis. RKIP is expressed in many tissues including brain, lung, and liver. RKIP blocks Raf-induced phosphorylation of MEK. In addition to its modulation of Raf signaling, RKIP modulates both G-protein signaling and NF-kappaB activity. The impact that RKIP has on multiple signaling pathways grants it the ability to play a role in several cellular functions including membrane biosynthesis, spermatogenesis, and neural signaling. Novel cellular functions for RKIP continue to be identified, several of which contribute to cancer biology. For example, RKIP promotes apoptosis of cancer cells, which suggests that loss of RKIP in cancer will protect cancer cells against cell death. Additionally, restoration of RKIP expression ina metastatic prostate cancer cell line does not effect primary tumor growth, but it does inhibit prostate cancer metastasis. These parameters identify RKIP as a metastasis suppressor gene, which suggest that it or proteins it interacts with are putative molecular targets to control metastasis. These findings are supported by the observation that RKIP expression is decreased in metastases of prostate cancer patients, compared to normal prostate or the primary prostate tumor. In this review, RKIP biology and its role in cancer will be described.     

The tumor suppressor Smad4 is required for transforming growth factor beta-induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells

Transforming growth factor beta (TGF-beta) can act as suppressor and promoter of cancer progression. Intracellular Smad proteins (i.e., receptor regulated Smads and common mediator Smad4) play a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-beta, but their function in TGF-beta-induced invasion and metastasis is unclear. Here, we have investigated the role of Smad4 in a cellular and mouse model for TGF-beta-induced breast cancer progression. Consistent with its tumor suppressor function, specific silencing of Smad4 in NMuMG mammary gland epithelial cells using small hairpin RNA (shRNA)-expressing RNAi vectors strongly mitigated TGF-beta-induced growth inhibition and apoptosis. Smad4 knockdown also potently inhibited TGF-beta-induced epithelial to mesenchymal transition of NMuMG cells as measured by morphologic transformation from epithelial to fibroblast-like cells, formation of stress fibers, inhibition of E-cadherin expression, and gain of expression of various mesenchymal markers. Furthermore, we show that knockdown of Smad4 in MDA-MB-231 breast cancer cells strongly inhibited the frequency of bone metastasis in nude mice by 75% and significantly increased metastasis-free survival. Communication of MDA-MB-231 cells with the bone microenvironment, which is needed for optimal tumor cell growth and metastasis, may be affected in Smad4 knockdown cells as TGF-beta-induced expression of interleukin 11 was attenuated on Smad4 knockdown. Taken together, our results show that Smad4 plays an important role in both tumor suppression and progression of breast cancer cells.