LuZhe Sun

Demonstration That Mutation of the Type II Transforming Growth Factor β Receptor Inactivates Its Tumor Suppressor Activity in Replication Error-positive Colon Carcinoma Cells

Escape from negative growth regulation by transforming growth factor β (TGF-β) as a result of the loss of TGF-β type II receptor (RII) expression has been found to be associated with the replication error (RER) colorectal cancer genotype, which is characteristic of hereditary nonpolyposis colorectal cancers. The RER-positive HCT 116 colon carcinoma cell line was examined for RII mutations. A 1-base deletion was found within a sequence of 10 repeating adenines (nucleotides 709-718), which resulted in a frameshift mutation. Although it is reasonable to predict that the loss of RII function would be an important determinant of malignancy, the large number of potential mutations in cells of this phenotype raises the possibility that an RII mutation may not be a key event in the tumorigenic phenotype of these cells. One way to test directly the importance of RII mutations in determining the malignant phenotype would be to restore its expression. If restoration of expression leads to diminished tumorigenicity, it would indicate that RII mutation is an important determinant of malignancy in the RER phenotype. To determine whether restoration of RII would lead to reversal of malignancy in RER colon cancers, an RII expression vector was transfected into the HCT 116 cell line. RII stable clones showed mRNA and protein expression of transfected RII. The fibronectin mRNA level was increased by exogenous TGF-β treatment in a dose-dependent manner in RII-positive clones, whereas the control cells remained insensitive. The RII transfectants showed reduced clonogenicity in both monolayer culture and soft agarose. They were growth arrested at a lower saturation density than control cells. TGF-β-neutralizing antibody stimulated the proliferation of RII-transfected but not control cells, indicating that the alterations in the growth parameters of the transfected cells were due to the acquisition of autocrine-negative activity. Tumorigenicity in athymic mice was reduced and delayed in RII transfectants. These results indicate that reconstitution of TGF-β autocrine activity by reexpression of RII can reverse malignancy in RER colon cancers, thus verifying that the malignancy of hereditary nonpolyposis colorectal cancer can be directly associated with the loss of RII expression.

Secreted Frizzled-Related Protein 1 Loss Contributes to Tumor Phenotype of Clear Cell Renal Cell Carcinoma

Purpose: Incidence and mortality rates for renal cell carcinoma (RCC) have been rising for decades. Unfortunately, the molecular events that support RCC carcinogenesis remain poorly understood. In an effort to gain a better understanding of signaling events in clear cell RCC (cRCC), we investigated the antitumor activity of secreted frizzled-related protein 1 (sFRP1), a negative regulator of Wnt signaling. Experimental Design: Genomic profiling of cRCC tumors and patient-matched normal tissues was done and confirmed using quantitative PCR and immunohistochemistry. Methylation-specific PCR was done on patient samples to evaluate the mechanism responsible for sFRP1 loss. sFRP1 expression was restored in cRCC cells and the effects on tumor phenotype were characterized. Results: Genomic profiling, quantitative PCR, and immunohistochemistry indicated that loss of sFRP1 occurred in cRCC and papillary RCC patient tissues. Twelve Wnt-regulated genes were up-regulated in cRCC tissues, including c-myc and cyclin D1, potentiators of cell proliferation and survival. Methylation of the sFRP1 gene was one mechanism identified for attenuation of sFRP1 mRNA. Stable reexpression of sFRP1 in cRCC cells resulted in decreased expression of Wnt target genes, decreased growth in cell culture, inhibition of anchorage-independent growth, and decreased tumor growth in athymic nude mice. Conclusions: To our knowledge, this is the first report to show that stable restoration of sFRP1 expression in cRCC cells attenuates the cRCC tumor phenotype. Our data support a role for sFRP1 as a tumor suppressor in cRCC and that perhaps loss of sFRP1 is an early, aberrant molecular event in renal cell carcinogenesis.

Inhibition of Pulmonary and Skeletal Metastasis by a Transforming Growth Factor-β Type I Receptor Kinase Inhibitor

Transforming growth factor-beta (TGF-beta) signaling has been shown to promote invasion and metastasis in various models of human cancers. In this study, we investigated the efficacy of a TGF-beta type I receptor kinase inhibitor (TbetaRI-I) to limit early systemic metastases in an orthotopic xenograft model of lung metastasis and in an intracardiac injection model of experimental bone and lung metastasis using human breast carcinoma MDA-MB-435-F-L cells, a highly metastatic variant of human breast cancer MDA-MB-435 cells, expressing the enhanced green fluorescent protein (EGFP). Treatment of the cells with the TbetaRI-I had no effect on their growth but blocked TGF-beta-stimulated expression of integrin alpha(v)beta(3) and cell migration in vitro. Systemic administration of the TbetaRI-I via i.p. injection effectively reduced the number and size of the lung metastasis in both orthotopic xenograft and experimental metastasis models with no effects on primary tumor growth rate compared with controls. TbetaRI-I treatment also reduced the incidence of widespread early skeletal metastases in the femur, tibia, mandible, and spine detected by whole-body EGFP fluorescence imaging. Tumor burden in femora and tibiae was also reduced after TbetaRI-I treatment as detected by histomorphometry analysis compared with the placebo controls. Our results indicate for the first time that abrogation of TGF-beta signaling by systemic administration of the TbetaRI-I can inhibit both early lung and bone metastasis in animal model systems and suggest antimetastatic therapeutic potential of the TbetaRI-I.

Reduced Expression of Transforming Growth Factor β Type I Receptor Contributes to the Malignancy of Human Colon Carcinoma Cells

Transforming growth factor β (TGFβ) type I (RI) and type II (RII) receptors are essential for TGFβ signal transduction. A human colon carcinoma cell line, designated GEO, is marginally responsive to TGFβ and expresses a low level of RI mRNA relative to colon carcinoma cells, which are highly responsive to TGFβ. Hence, the role of RI as a limiting factor for TGFβ sensitivity and the contribution of low RI levels to the malignant phenotype of GEO cells were examined. Stable transfection of a tetracycline-regulatable rat RI cDNA increased TGFβ1 binding to RI and resulted in increased growth inhibition by exogenous TGFβ1. In contrast, although stable transfection of an RII expression vector into the same GEO cells increased TGFβ1 binding to RII, growth inhibition by exogenous TGFβ1 was not altered. This indicated that the low level of RI is a limiting factor for the growth-inhibitory effects of TGFβ in GEO cells. RI-transfected cells were growth-arrested at a lower saturation density than GEO control cells. They also showed reduced growth and clonogenicity in plating efficiency and soft agarose assays, whereas RII-transfected cells did not show any differences from the NEO control cells in these assays. Tetracycline repressed RI expression in transfected cells and reversed the reduction in plating efficiency of RI-transfected clones, confirming that growth effects were due to increased RI expression in transfected cells. TGFβ1 neutralizing antibody stimulated the proliferation of RI-transfected cells but had little effect on GEO control cells, indicating that increased autocrine-negative TGFβ activity also resulted from increased RI expression. Tumorigenicity in athymic nude mice was significantly delayed in RI-transfected cells. These results indicate that low RI expression can be a limiting factor for response to exogenous TGFβ, as well as TGFβ autocrine-negative activity, and that reduction of RI expression can contribute to malignant progression.

Extracellular domain of TGFβ type III receptor inhibits angiogenesis and tumor growth in human cancer cells

TGFβ overexpression in human cancer cells has been shown to promote tumor progression. In the present study, we sought to determine whether sequestration of endogenous TGFβ by the expression of a soluble TGFβ type III receptor (sRIII), can reduce malignancy in human carcinoma cells and whether the tumor-suppressive activity of sRIII is associated with the inhibition of angiogenesis. Ectopic expression of sRIII significantly inhibited the growth of tumors formed by human colon carcinoma HCT116 and breast carcinoma MDA-MB-435 cells in nude mice. It also reduced the metastatic potential of the MDA-MB-435 cells. Thus, endogenous TGFβ appears to be necessary for the progression of these two carcinomas. Furthermore, when the tumor cells were mixed with Matrigel and embedded subcutaneously in nude mice, the blood volume in Matrigel plugs containing sRIII-expressing cells as indicated by hemoglobin levels was significantly lower than that in Matrigel plugs containing the respective control cells. Blood vessel counts in paraffin sections of the Matrigel plugs containing sRIII-expressing cells were also significantly lower than those in paraffin sections of the Matrigel plugs containing control cells. Treatment of human endothelial cells with a recombinant sRIII significantly inhibited their ability to form a capillary web structure on Matrigel. These results for the first time indicate that the sRIII-induced tumor suppression appears to be in part due to the inhibition of angiogenesis.

Dual role of SnoN in mammalian tumorigenesis

ABSTRACT SnoN is an important negative regulator of transforming growth factor β signaling through its ability to interact with and repress the activity of Smad proteins. It was originally identified as an oncoprotein based on its ability to induce anchorage-independent growth in chicken embryo fibroblasts. However, the roles of SnoN in mammalian epithelial carcinogenesis have not been well defined. Here we show for the first time that SnoN plays an important but complex role in human cancer. SnoN expression is highly elevated in many human cancer cell lines, and this high level of SnoN promotes mitogenic transformation of breast and lung cancer cell lines in vitro and tumor growth in vivo, consistent with its proposed prooncogenic role. However, this high level of SnoN expression also inhibits epithelial-to-mesenchymal transdifferentiation. Breast and lung cancer cells expressing the shRNA for SnoN exhibited an increase in cell motility, actin stress fiber formation, metalloprotease activity, and extracellular matrix production as well as a reduction in adherens junction proteins. Supporting this observation, in an in vivo breast cancer metastasis model, reducing SnoN expression was found to moderately enhance metastasis of human breast cancer cells to bone and lung. Thus, SnoN plays both protumorigenic and antitumorigenic roles at different stages of mammalian malignant progression. The growth-promoting activity of SnoN appears to require its ability to bind to and repress the Smad proteins, while the antitumorigenic activity can be mediated by both Smad-dependent and Smad-independent pathways and requires the activity of small GTPase RhoA. Our study has established the importance of SnoN in mammalian epithelial carcinogenesis and revealed a novel aspect of SnoN function in malignant progression.

Transforming growth factor-β suppresses the ability of ski to inhibit tumor metastasis by inducing its degradation

c-Ski is an important corepressor of transforming growth factor-beta (TGF-beta) signaling through its ability to bind to and repress the activity of the Smad proteins. It was initially identified as an oncogene that promotes anchorage-independent growth of chicken and quail embryo fibroblasts when overexpressed. Although increased Ski expression is detected in many human cancer cells, the roles of Ski in mammalian carcinogenesis have yet to be defined. Here, we report that reducing Ski expression in breast and lung cancer cells does not affect tumor growth but enhances tumor metastasis in vivo. Thus, in these cells, Ski plays an antitumorigenic role. We also showed that TGF-beta, a cytokine that is often highly expressed in metastatic tumors, induces Ski degradation through the ubiquitin-dependent proteasome in malignant human cancer cells. On TGF-beta treatment, the E3 ubiquitin ligase Arkadia mediates degradation of Ski in a Smad-dependent manner. Although Arkadia interacts with Ski in the absence of TGF-beta, binding of phosphorylated Smad2 or Smad3 to Ski is required to induce efficient degradation of Ski by Arkadia. Our results suggest that the ability of TGF-beta to induce degradation of Ski could be an additional mechanism contributing to its protumorigenic activity.

Induction of Transforming Growth Factor-β Receptor Type II Expression in Estrogen Receptor-positive Breast Cancer Cells through SP1 Activation by 5-Aza-2′-deoxycytidine

Previous studies suggest that estrogen receptor-positive (ER+) breast cancer cells acquire resistance to transforming growth factor-β (TGF-β) because of reduced expression levels of TGF-β receptor type II (RII). We now report that treatment of ER+ breast cancer cells with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-2′-dC) leads to accumulation of RII transcript and protein in three different cell lines. RII induction restored TGF-β response in MCF-7L breast cancer cells as indicated by the enhanced activity of a TGF-β responsive promoter-reporter construct (p3TP-Lux). A transiently transfected RII promoter-reporter element (RII-chloramphenicol acetyltransferase) showed an increase in activity in the 5-aza-2′-dC-treated MCF-7L cells compared with untreated cells, suggesting the activation of a transactivator of RII transcription. Using electrophoretic mobility shift assays, the enhanced binding of proteins from 5-aza-2′-dC-treated MCF-7L nuclear extracts to radiolabeled Sp1 oligonucleotides was demonstrated. An RII promoter-chloramphenicol acetyltransferase construct containing a mutation in the Sp1 site was not expressed in the 5-aza-2′-dC-treated MCF-7L cells, further demonstrating that induction of Sp1 activity by 5-aza-2′-dC in the MCF-7L cells was critical to RII expression. Northern analysis indicated that 5-aza-2′-dC treatment did not affect the Sp1 transcript levels. Western blot analysis revealed an increase of Sp1 protein in the 5-aza-2′-dC-treated MCF-7L cells, but there was no change in the c-Jun levels. Studies after cyclohexamide treatment suggested an increase in the Sp1 protein stability from the 5-aza-2′-dC-treated MCF-7L extracts compared with untreated control extracts. These results indicate that the transcriptional repression of RII in the ER+ breast cancer cells is caused by suboptimal activity of Sp1, whereas treatment with 5-aza-2′-dC stabilizes the protein thus increasing steady-state Sp1 levels and thereby leads to enhanced RII transcription and subsequent restoration of TGF-β sensitivity.

Expression of Transforming Growth Factor β Type III Receptor Suppresses Tumorigenicity of Human Breast Cancer MDA-MB-231 Cells

Transforming growth factor β (TGF-β) promotes tumor progression in some model systems including human breast cancer cells. In this study, we report that human breast cancer cell lines express reduced amounts of TGF-β type III receptor (RIII) when compared with untransformed human mammary epithelial cells. Consequently, we examined whether expression of RIII in human breast cancer MDA-MB-231 cells could reduce TGF-β’s tumor promoting activity by sequestering active TGF-β isoforms produced by the cells. A tetracycline-repressible human RIII expression vector was stably transfected into the cell line. RIII expression in a pool of transfected clones and a single clone was found to be reversibly repressed by tetracycline treatment. Expression of RIII reduced the amount of active TGF-β1 and TGF-β2 in the conditioned medium. The medium conditioned by control cells showed a significantly higher growth inhibitory effect than that conditioned by RIII-transfected cells on the growth of the mink lung epithelial CCL64 cells. A conditioned medium collected from RIII-transfected cells treated with tetracycline significantly increased its growth inhibitory activity to that of control cells. Expression of RIII also reduced tumor incidence and growth rate in two separate experiments when the cells were inoculated in athymic nude mice. Treatment of the mice with tetracycline repressed RIII expression in the tumors generated by RIII-transfected cells and increased tumor incidence and growth rate. These results suggest that TGF-β RIII can reduce tumorigenicity of MDA-MB-231 cells apparently by sequestering TGF-β isoforms produced by these cells.

Autocrine TGFβ supports growth and survival of human breast cancer MDA-MB-231 cells

Using a cell model system established by ectopic expression of a soluble TGFβ type III receptor (sRIII) containing the whole extracellular domain of the type III receptor in human breast cancer MDA-MB-231 cells, we observed that the expression of sRIII antagonized TGFβ activity and inhibited both anchorage-dependent and anchorage-independent cell growth. Further studies revealed that sRIII expression induced apoptosis both in vitro and in vivo. Treatment with TGFβ neutralizing antibodies or a recombinant human sRIII also induced apoptosis in the MDA-MB-231 parental cells, suggesting that the increased apoptosis after sRIII expression was specifically due to antagonization of autocrine TGFβ signaling. Western blotting showed that sRIII clones had a higher PTEN expression level than the control cells did. Treatment with TGFβ1 decreased PTEN and inhibited apoptosis in sRIII cells to a level similar to that in the control cells. sRIII clones also showed a lower level of phosphorylated-Akt than the control cells, consistent with the inhibitory activity of PTEN on Akt activation. Treatment with LY294002, a specific inhibitor of Akt activator, phosphatidylinositol 3-kinase, also induced apoptosis in a dose dependent manner in the control cells. Our results suggest that autocrine TGFβ signaling is necessary for the growth and survival of MDA-MB-231 cells.