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Purpose: Overexpression of transforming growth factor (TGF)-β has been implicated in promoting immune suppression, tumor angiogenesis, tumor cell migration, and invasion in many cancers, including carcinoma of the breast. Thus, targeted down-regulation of TGF-β1 expression in breast cancer in situ and determination of its implications would provide new treatment approaches for disease management.Experimental Design: Small interfering RNA constructs targeting TGF-β1 were validated and used to develop clonal derivatives of the metastatic breast cancer cell line MDA-MB-435. The cells were used in several in vitro analyses, including migration, invasion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis, and signaling assays. A wound-healing assay was used to determine migration of the cells in culture and a Boyden chamber transwell assay was used for invasion. Further, the clones were used in an in vivo mouse model for the kinetics of tumor growth and gene expression in the primary site and in lungs upon metastasis.Results: Inhibition of TGF-β1 expression in MDA-MB-435 cells showed a 35% decrease in migration and a 55% decrease in invasion in vitro, with a 50% increase in proliferation and no effect on apoptosis. In vivo analysis indicated a 90% decrease in the number of mice bearing macroscopic lung metastases; however, the primary tumors did not show any difference in the growth kinetics when compared with the parental MDA-MB-435 cells. Analysis of TGF-β signaling pathways in the clonal derivatives showed a decrease in Smad2 activation and an increase in AKT and extracellular signal-regulated kinase activation. Interestingly, analysis of TGF-β receptor expression showed a decrease in both receptor I and II expression in TGF-β1 silenced cells. These results suggest that inhibition of TGF-β1 ligand may act as a negative feedback loop to disrupt the function of all TGF-β isoforms.Conclusions: Therapies targeting the TGF-β signaling pathway may be more effective in late-stage disease to prevent organ metastasis but not primary tumor formation and may be combined with other tumor-targeted therapies normally limited by increased circulating TGF-β levels.

Supplementary Data from Silencing of Transforming Growth Factor-β1 In situ by RNA Interference for Breast Cancer: Implications for Proliferation and Migration In vitro and Metastasis In vivo

BACKGROUND Although anti-angiogenic therapy is a promising new line of therapy for prostate cancer, we recently reported that stable expression of endostatin arrested the progression of prostate cancer to poorly differentiated state and distant metastasis in TRAMP mice. However, the same therapy failed to provide any benefit when given either during or after the onset of metastatic switch. The present study determined the possible mechanisms behind the selective advantage of endostatin therapy in early-stage disease. METHODS Angiogenesis-related gene expression analysis was performed to identify target genes and molecular pathways involved in the therapy effects. Based on the results from in vivo studies, and recapitulation of the in vivo data in vitro using tumorigenic and non-tumorigenic human prostate cancer cells that are either androgen-sensitive or androgen-independent, analyses of possible mechanisms of the selective advantage of early treatment were performed using assays for cell proliferation, apoptosis, migration, and cell signaling. The identified mechanisms were further confirmed in vivo. RESULTS Results indicated that cells with high androgen receptor (AR) expression were more sensitive to endostatin treatment than androgen-independent cells with low or no AR expression. Endostatin was found to significantly downregulate the expression of growth factors, receptor tyrosine kinases, proteases, and AR both in vitro and in vivo only when the cells express high-levels of AR. Cell proliferation was not influenced by endostatin treatment but migration was significantly affected only in androgen-sensitive cells. Targeted downregulation of AR prior to endostatin treatment in androgen-sensitive cells and overexpression of AR in androgen-independent cells indicated that the effect of endostatin via AR downregulation is mediated by a non-genotropic mechanism on Ras and RhoA pathways, and independently of AR on MAPK/ERK pathway. CONCLUSIONS These data indicate that systemically stable endostatin expression delays the onset of metastatic switch by acting on multiple pathways involving AR. Prostate 69:1055–1066, 2009. © 2009 Wiley-Liss, Inc.

Purpose: Overexpression of transforming growth factor (TGF)-β has been implicated in promoting immune suppression, tumor angiogenesis, tumor cell migration, and invasion in many cancers, including carcinoma of the breast. Thus, targeted down-regulation of TGF-β1 expression in breast cancer in situ and determination of its implications would provide new treatment approaches for disease management. Experimental Design: Small interfering RNA constructs targeting TGF-β1 were validated and used to develop clonal derivatives of the metastatic breast cancer cell line MDA-MB-435. The cells were used in several in vitro analyses, including migration, invasion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis, and signaling assays. A wound-healing assay was used to determine migration of the cells in culture and a Boyden chamber transwell assay was used for invasion. Further, the clones were used in an in vivo mouse model for the kinetics of tumor growth and gene expression in the primary site and in lungs upon metastasis. Results: Inhibition of TGF-β1 expression in MDA-MB-435 cells showed a 35% decrease in migration and a 55% decrease in invasion in vitro, with a 50% increase in proliferation and no effect on apoptosis. In vivo analysis indicated a 90% decrease in the number of mice bearing macroscopic lung metastases; however, the primary tumors did not show any difference in the growth kinetics when compared with the parental MDA-MB-435 cells. Analysis of TGF-β signaling pathways in the clonal derivatives showed a decrease in Smad2 activation and an increase in AKT and extracellular signal-regulated kinase activation. Interestingly, analysis of TGF-β receptor expression showed a decrease in both receptor I and II expression in TGF-β1 silenced cells. These results suggest that inhibition of TGF-β1 ligand may act as a negative feedback loop to disrupt the function of all TGF-β isoforms. Conclusions: Therapies targeting the TGF-β signaling pathway may be more effective in late-stage disease to prevent organ metastasis but not primary tumor formation and may be combined with other tumor-targeted therapies normally limited by increased circulating TGF-β levels.

We recently reported that stable expression of endostatin and angiostatin arrested the progression of well differentiated prostate carcinoma to poorly-differentiated state and distant metastasis in TRAMP mice when given prior to the onset of metastasis switch. Analysis of the expression of angiogenesis-related genes in the prostate tissue of endostatin treated mice indicated a significant downregulation of genes involved in cell motility, proliferation, metastasis and angiogenesis when therapy was initiated at early stage-disease. In vitro experiments demonstrated that recombinant endostatin treatment significantly downregulated the expression of growth factors, receptor tyrosine kinases and proteases in androgen sensitive cells. A similar pattern was confirmed in prostate tissues obtained from in vivo studies. Targeted downregulation of androgen receptor (AR) prior to endostatin treatment indicated that the effect of endostatin through AR downregulation is by a non-genotypic mechanism on MAPK/ERK pathway, and independently of Src kinase. Further analysis indicated the kinetics of endostatin internalization for androgen sensitive prostate cancer cells to be much higher than in androgen independent cells. Co-immunoprecipitation studies in human prostate cancer cells, and yeast two-hybrid studies with endostatin and AR demonstrated a direct binding of endostatin protein with AR. Based on the results so far, we predict that direct interaction of endostatin with AR possibly eliminates ligand binding during early stages of prostate cancer arresting and delaying the progression of the disease. However, once metastatic switch occurs, when the cells grow in androgen independent manner, a limitation in endostatin internalization or binding of non-androgen ligands at other domains in AR impairs the tumoristatic ability of endostatin. Ongoing studies are aimed in identifying interaction domains of endostatin with AR by NMR as well as testing the combination of endostatin therapy with therapies targeting AR signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1246.

Recent studies indicated that the tumor microenvironment plays an important role in tumor growth. Both tumor cells and stromal cells produce TGF-β1, which contributes to autocrine and paracrine functions in the tumor microenvironment. During cell transformation, TGF-β induces angiogenesis, produces reactive stroma, and tumor invasiveness suggesting that crosstalk between the tumor and the stroma is very important in the progression of metastatic disease. The present study examined the effects of TGF-β1 crosstalk in the tumor microenvironment and its role in mediating tumor formation and tumor development by targeted abrogation of TGF-β expression in the tumor cells in situ. Athymic female mice were injected subcutaneously with either normal or tumor derived fibroblast, unmodified MDA-MB 435 breast cancer cells, MDA-MB 435 clones containing TGF-β siRNA either alone or in combination with fibroblasts. Implantation of the cells subcutaneously showed significant difference in tumor formation between the groups. Control animals injected with fibroblast only developed no tumors. In animals injected with TGF-β-silenced MDA-MB 435 clones there was 12.5% tumor incidence. When these same cells were injected in combination with fibroblast the tumor incidence increased to 57%. Unmodified MDA-MB 435 cells showed a tumor incidence of 85% when injected alone and 100% in the presence of fibroblasts. This data suggests that when TGF-β is inhibited in the tumor cells the surrounding stroma can provide growth factors that continue to support tumor formation. In addition to measuring the tumor incidence, tumor growth was assessed every two days over the course of 6 weeks or until tumor was removed due to necrosis. Control animals injected with fibroblasts alone showed no tumor growth. The tumors formed from TGF-β-silenced MDA-MB 435 clones display an average tumor volume of 11.2 mm3. When these cells were co-injected with fibroblasts, the average tumor volume increased to 76 mm3. Unmodified MDA-MB 435 cells produced an average tumor volume of 164.3 mm3 but when injected with fibroblast the volume increased to 229.1 mm3. This data suggests that signaling from the tumor microenvironment can promote tumor progression through TGF-β signaling pathway. Thus, understanding the role of TGF-β1 expression in the tumor microenvironment will help to develop targeted therapies for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 541.

Transforming Growth Factor-|[beta]| (TGF-|[beta]|) has been shown to perform a dual function as both a tumor suppressor in early stage carcinogenesis and a pro-metastatic factor in late-stage tumor progression. Early studies have shown that inhibition of TGF-|[beta]| signaling pathway can alter various aspects of tumor metastasis such as migration, invasion, angiogenesis and immune suppression. While these methods have shown modest success, several limitations prevent the wide spread use of these inhibitors as an anti-metastatic therapy. RNA interference (RNAi) has emerged as an effective tool for gene specific silencing in vitro and in vivo. The central hypothesis of the proposed studies is that adeno-associated virus (AAV) mediated deliver of siRNA sequences targeting TGF-|[beta]|1 in MDA- MB 435 breast cancer cells can alter the metastatic phenotype at defined time-points of tumor progression and/or act as an adjuvant to primary chemotherapies. Several siRNA target sequences have been screened to determine silencing capacity and two specific targets, siRNA4 and siRNA7 showed |[sim]|90% inhibition of TGF|[beta]|1 transcripts by Northern blot and at least a 5-6 fold reduction by RT-PCR analysis. The two siRNA sequences, along with a scrambled sequence, were cloned into a tet-inducible expression vector and used to generate a stable cell line that constitutively expresses this construct. This cell line is being using in various in vitro assays such as migration, invasion and gene expression, to determine the effects of TGF-|[beta]| silencing. This cell line will later be used in an orthotopic breast cancer model to determine the exact time-point at which TGF-|[beta]| silencing is most effective. A second set of studies are proceeding in parallel to determine the transduction of AAV serotypes 1-6 both in vitro and in vivo. Initial in vitro data has shown that AAV2 and AAV6 show transduction efficiencies of 40% |[ndash]| 60% in MDA-MB 435 and MDA-MB 231 cells. In vivo transduction studies, of the primary tumor mass, are ongoing using AAV-|[beta]|gal, Ad-GFP and AAV-GFP. Ongoing studies will determine the effects of inhibition on SMAD-independent signaling pathways that can play a role in detachment, migration, immune-suppression and angiogenesis. This inducible siRNA will allow better insight into the TGF-|[beta]| |[ldquo]|switch|[rdquo]| from tumor suppressor to pro-metastatic factor along disease progression and possible development of a TGF-|[beta]| targeted anti-cancer therapy. If promising, this therapy may also be used in combination with current chemotherapies to provide a synergistic effect against breast cancer metastasis.