TGF-β Signaling in Breast Cancer Cell Invasion and Bone Metastasis

The contribution of transforming growth factor β (TGF-β) signaling to breast cancer has been studied for more than two decades. In an early phase TGF-β may act as a tumour suppressor, while later, when cells have become resistant to its anti-mitogenic effects, the role of TGF-β switches towards malignant conversion and progression. TGF-β stimulates cell invasion and modifies the microenvironment to the advantage of cancer cells. Studies have shown that TGF-β promotes bone and lung metastasis via different mechanisms. The therapeutic strategies to target the TGF-β pathway in breast cancer are becoming increasingly clear. This review will focus on the role TGF-β in breast cancer invasion and metastasis

USP4 is regulated by Akt phosphorylation and deubiquitylates TGF-beta type I receptor

Stability and membrane localization of Transforming growth factor-β (TGF-β) type I receptor (TβRI) is essential for controlling TGF-β signaling. TβRI is targeted for ubiquitination-mediated degradation by Smad7/Smurf2 complex. However, it is unclear whether polyubiquitin modified TβRI can be reversed. Here we performed a genome-wide gain of function screen and identified ubiquitin-specific protease (USP) 4 as a strong inducer of TGF-β signaling. Putative oncogenic USP4 was found to interact with TβRI as deubiquitinating enzyme thus maintains TβR1 levels at the plasma membrane. Depletion of USP4 mitigates TGF-β-induced breast cancer cell migration, epithelial to mesenchymal transition and metastasis. Importantly, Akt/Protein kinase B (PKB), which has been associated with poor prognosis in breast cancer, associates with and phosphorylates USP4. Akt mediated phosphorylation relocates USP4 to cytoplasm and membrane and is required for maintaining its protein stability. Moreover, Akt-induced breast cancer cell migration was inhibited by USP4 depletion and TβRI kinase inhibition. Our results identified USP4 as an important determinant for crosstalk between TGF-β and Akt, which provides new opportunities for cancer treatment

Estrogen receptor-α recruits P-TEFb to overcome transcriptional pausing in intron 1 of the MYB gene

The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα +) breast tumors and cell lines. Expression of MYB is controlled, in breast cancer and other cell types, by a transcriptional pausing mechanism involving an attenuation site located ∼1.7kb downstream from the transcription start site. In breast cancer cells, ligand-bound ERα binds close to, and drives transcription beyond this attenuation site, allowing synthesis of complete transcripts. However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation. Using cyclin dependent kinase 9 (CDK9) inhibitors, reporter gene assays and measurements of total and intronic MYB transcription, we show that functionally active CDK9 is required for estrogen-dependent transcriptional elongation. We further show by ChIP and co-immunoprecipitation studies that the P-TEFb complex (CDK9/CyclinT1) is recruited to the attenuation region by ligand-bound ERα, resulting in increased RNA polymerase II Ser-2 phosphorylation. These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy

Robust, reversible gene knockdown using a single lentiviral short hairpin RNA vector

Manipulation of gene expression is an invaluable tool to study gene function in vitro and in vivo. The application of small inhibitory RNAs to knock down gene expression provides a relatively simple, elegant, but transient approach to study gene function in many cell types as well as in whole animals. Short hairpin structures (shRNAs) are a logical advance as they can be expressed continuously and are hence suitable for stable gene knockdown. Drug-inducible systems have now been developed; however, application of the technology has been hampered by persistent problems with low or transient expression, leakiness or poor inducibility of the short hairpin, and lack of reversibility. We have developed a robust, versatile, single lentiviral vector tool that delivers tightly regulated, fully reversible, doxycycline-responsive knockdown of target genes (FOXP3 and MYB), using single short hairpin RNAs. To demonstrate the capabilities of the vector we targeted FOXP3 because it plays a critical role in the development and function of regulatory T cells. We also targeted MYB because of its essential role in hematopoiesis and implication in breast cancer progression. The versatility of this vector is hence demonstrated by knockdown of distinct genes in two biologically separate systems.Cheryl Y. Brown, Timothy Sadlon, Tessa Gargett, Elizabeth Melville, Rui Zhang, Yvette Drabsch, Michael Ling, Craig A. Strathdee, Thomas J. Gonda, and Simon C. Barr

MYB suppresses differentiation and apoptosis of human breast cancer cells

Introduction: MYB is highly expressed in estrogen receptor positive (ER + ve) breast tumours and tumour cell lines. We recently demonstrated that MYB is essential for the proliferation of ER + ve breast cancer cells, and have now investigated its role in mammary epithelial differentiation.Methods: MCF-7 breast cancer cells were treated with sodium butyrate, vitamin E succinate or 12-O-tetradecanoylphorbol-13-acetate to induce differentiation as measured by Nile Red staining of lipid droplets and β-casein expression. The non-tumorigenic murine mammary epithelial cell (MEC) line, HC11, was induced to differentiate with lactogenic hormones. MYB levels were manipulated by inducible lentiviral shRNA-mediated knockdown and retroviral overexpression.Results: We found that MYB expression decreases following chemically-induced differentiation of the human breast cancer cell line MCF-7, and hormonally-induced differentiation of a non-tumorigenic murine mammary epithelial cell (MEC) line, HC11. We also found that shRNA-mediated MYB knockdown initiated differentiation of breast cancer cells, and greatly sensitised them to the differentiative and pro-apoptotic effects of differentiation-inducing agents (DIAs). Sensitisation to the pro-apoptotic effects DIAs is mediated by decreased expression of BCL2, which we show here is a direct MYB target in breast cancer cells. Conversely, enforced expression of MYB resulted in the cells remaining in an undifferentiated state, with concomitant suppression of apoptosis, in the presence of DIAs.Conclusions: Taken together, these data imply that MYB function is critical in regulating the balance between proliferation, differentiation, and apoptosis in MECs. Moreover, our findings suggest MYB may be a viable therapeutic target in breast cancer and suggest specific approaches for exploiting this possibility

The Role and Function of MYB in Human Breast Cancer

Breast cancer is one of the most common malignancies in women. It continues to be a major burden and cause of death among women worldwide. This project investigated the role and function of the oncogene, MYB, in human breast cancer. Even though there are only a few studies that have investigated MYB in breast cancer, they have provided a series of observations that make a strong prima facie case for the causative involvement of MYB in breast cancer. Further investigation was needed to move from the correlative and circumstantial evidence that surrounds this oncogene in breast cancer. Based on the evidence surrounding the potential role of MYB in breast cancer, this project examined the hypothesis that MYB contributes to mammary carcinoma. This project hypothesized that in breast cancer cells, transcriptional attenuation occurs within the first intron of MYB. It was also hypothesized that the estrogen receptor (ER) is capable of overcoming the transcriptional attenuation. It is shown that the estrogen-mediated control of MYB is via a transcriptional attenuation site within the first intron. Furthermore, it is shown that estrogen leads to binding of ER within a region adjacent to the transcriptional attenuation motif and overcomes the elongation block. The second aim was to determine whether MYB is required for the growth of ER positive tumour cells in vitro and in vivo. By using antisense oligonucleotides and RNA interference to block MYB expression it was discovered that MYB was required for the proliferation of ER positive breast cancer cells. This provided the first functional evidence of a role for MYB in breast cancer. Furthermore, xenograft experiments were used to study the effect of MYB inhibition on tumour growth. The data from the xenografted tumours indicated that MYB knockdown leads to a significant decrease in tumour growth as compared to the control xenografts. As part of the second aim, the pattern of MYB expression in breast cells undergoing differentiation was examined. To further understand the function of MYB in breast cancer and in mammary epithelial cells (MECs) generally, its role in the differentiation of mammary carcinoma cells and MECs were examined. Differentiation of human breast cancer cell lines and non-tumorigenic MECs can be induced by chemical agents or lactogenic hormones, respectively. It was confirmed that, like some other cancer cells (such as haematopoietic cells, and the epithelial colon cells), that MYB expression decreases during the process of differentiation in mammary carcinoma cells and MECs. A functional role for MYB in MEC differentiation was further implied by the effects of MYB knockdown on breast cancer cell lines. While a small degree of differentiation was observed with an inducible MYB shRNA alone, a more dramatic effect was apparent with MYB knockdown and (otherwise) marginally-effective concentrations of differentiation inducing agents (DIAs), which resulted in essentially complete differentiation. Conversely, enforced MYB expression was able to block differentiation of both carcinoma cells and non-tumorigenic MECs, again strikingly paralleling MYB’s activities in other cell systems. Finally, the third aim was to ascertain if high levels of MYB expression can lead to breast tumours. This required the generation of transgenic mice which overexpressed Myb specifically in the mammary gland. Given the importance of estrogen and its receptor in the pathogenesis, as well as treatment, of this disease, a transgenic mouse model which would allow the development of ER+ tumours was preferred. The neu-related lipocalin (NRL) promoter has been shown previously to develop mammary adenocarcinomas that are ER α positive and negative, similar to human disease. As such, the NRL promoter was used to study the interaction between Myb and estrogen in disease development and progression. Preliminary investigation on the founder mice showed promising results, but, further investigation are necessary before any conclusions may be drawn. This project has shown that MYB is an effector of estrogen/ER signalling and this has provided the first demonstration of a functional role of MYB in human breast cancer. Also, it has been shown that MYB plays an important role in regulating the balance between proliferation, differentiation, and apoptosis in mammary epithelial cells