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Disruption Of Tgf-Beta/Smad Signaling Events For The Therapy Of Breast Cancer Metastasis
Investigator Initiated Research
Background & Rationale: Several studies have established that transforming growth factor-beta (TGF-beta) plays a dual role during breast carcinogenesis; an early tumor suppressive and a late-stage pro-oncogenic role concomitant with a progressive increase in locally secreted TGF-beta levels. It has also been shown that high incidence of breast cancer metastasis is associated with increase in phoshorylated Smad2 in the nucleus. These observations suggest that the TGF beta - TGF beta R - Smad2 signaling axis is involved in breast cancer metastasis and it is a legitimate target for therapeutic intervention. One of the clues supporting this rationale is the fact that there was significant degree of suppression of the in vivo incidence of macrometastases in mouse models for breast cancer upon overexpression of the inhibitory Smad7. We propose that the molecular effectors downstream of the anti-metastatic effect of Smad7 and the small peptides that confer the anti-metastatic effects of Smad7 are legitimate therapeutic agents for discovery and further development.
Objective/Hypothesis and Expected Outcome: The overall objective of this proposal is to elucidate the molecular events/factors that are responsible for the metastatic inhibitory effect of Smad7 in breast cancer and design short peptides that mimic this phenomenon and identify the Smad7 downstream factors that mediate the anti-metastatic effects as potentially novel therapeutic agents. We hypothesize that Smad7 overexpression suppresses metastatic phenotype of breast cancer cells by promoting the re-establishment of epithelial phenotype and by suppression of invasive properties. The studies proposed here will aid in the identification of the molecular biomarkers/effector genes responsible for the anti-metastatic effect of Smad7 and identify small peptides that retain the anti-metastatic effects of full-length Smad7. These factors/molecules are legitimate candidates for further development as therapeutic agents for metastatic breast cancer.
Specific Aims: 1) To establish breast cancer cell culture model systems and define the active inhibitory domain of Smad7; 2) To elucidate the molecular mechanisms through which Smad7 exerts its suppressive effects on the invasive/metastatic properties of breast cancer cells; 3) To design and screen for short peptides that mimic Smad7 in preventing interaction between TGF- beta receptor I and the R-Smads using cell culture models; and 4) To test the effect of Smad7 and Smad7 mimetic peptides in inhibiting metastatic breast tumor in vivo and the identification of effector gene(s) downstream of the anti-metastatic effect of Smad7.
Study Design: In this study, we propose a strategy to elucidate the molecular basis for the anti-metastatic effect of Smad7 by uncovering novel strategies/agents for breast cancer therapy. Breast cancer cell line model systems will be established to investigate the inhibitory role of Smad7 overexpression on invasiveness and metastasis of breast cancer cells. Deletion mutants of Smad7 will be generated to map the domains critical for the inhibitory effect. Short peptides corresponding to the inhibitory domain of Smad7 will be designed and screened using peptide competition assays for their ability to mimic the inhibitory function of Smad7. Gene expression profiling of orthotopic xenografts in mouse models exhibiting distal metastasis and inhibition of metastatic breast tumor formation due to Smad7 expression and/or Smad7 mimetic peptide treatment will be carried out using microarrays to uncover the molecular players underlying the metastasis inhibition phenomenon. Selected genes from these analyses will be functionally tested to confirm their involvement in the inhibition of metastatic breast cancer progression as potentially novel therapeutic agents for future development. These studies will also validate Smad7 derived small peptide(s) that retain the anti-metastatic effects.
Innovation: Metastasis is the major cause of the death among breast cancer patients. Therefore, breakthroughs in the discovery of drugs for the prevention of breast cancer invasion and metastasis are urgently needed. The fact that the TGF-beta levels are increased locally and systemically in advanced breast tumors which positively correlates with metastasis warrants innovative approaches to block the TGF- beta downstream effects for therapy. The current proposal is aimed at filling the gap in knowledge by unraveling the molecular mechanisms underlying the Smad7 mediated inhibition of the breast cancer metastatic properties. These studies may unravel novel factors that can be developed as ?smart drugs? in the future for therapeutic applications as well as help to design peptides that mimic the metastasis inhibitory function of Smad7 which could also become novel therapeutic agent(s) for breast cancer metastasis.
Impact/Relevance: This proposal directly addresses the causes of in vivo breast cancer progression to the advanced metastatic stage and explores avenues/agents for therapy. The outcome of this study is the elucidation of the effects of hyperactive TGF-beta signaling in breast cancer metastasis to reveal novel gene products/agents that are amenable for therapeutic applications. Furthermore, in the short-term, the designing of the small molecules of peptides based on Smad7 function that elicit similar anti-metastatic effects could provide novel therapeutic agents for testing in clinical trials for breast cancer.
The majority of breast cancer patient deaths are due to formation of metastases in distal tissues, predominantly in bone and lung. One of the most critical initial steps for the breast cancer metastasis is the entry of cells into the blood circulation in a process known as intravasation enabling them to travel to distal parts of the body. These cancer cells in the circulation extravasate and act as the ?seeds? to form the new metastatic tumor masses. During the intravasation step, breast cancer cells change their shape, from epithelial (well structured, adhesive and tightly packed) to mesenchymal (more elongated, spindle-shaped, poorly adhesive) via a process called epithelial to mesenchymal transition (EMT), to facilitate the mobility and entry of the cancer cells into the circulation.
Transforming growth factor-beta is a small peptide molecule that binds to specific receptors and activates a signaling cascade which may involve a class of protein mediators known as the receptor restricted (R-Smads) and the common co-mediator Smad (Co-Smad: Smad4). Overactivation of this signaling cascade mediated by increased levels of TGF-beta has been implicated with high incidence of breast cancer metastases. TGF-beta has been shown to induce EMT in breast cancers, thus promoting cell motility and invasiveness that facilitate the process of metastasis. Interestingly, recent evidence has shown that overexpression of the Smad7, can significantly decrease the incidence of breast cancer metastases in mouse models. However, there is a gap in knowledge as to the molecular events underlying this important phenomenon.
We hypothesize that a better understanding of the molecular basis of this effect could help to unravel critical effectors of the Smad7-mediated inhibition of breast cancer metastasis which have the potential to be developed as novel therapeutic agents. Furthermore, small peptides mimicking the Smad7 function could also be effective as therapeutic agents that could block the chain of events that occur during the metastatic progression of breast cancer.
In order to accomplish these tasks, we propose to develop and test human breast cancer model cell lines representing controls that are proficient for metastasis and the corresponding Smad7 overexpression mediated metastasis inhibited breast cancer cells with accompanying suppression of invasiveness and motility and the reversal of EMT. In order to make these anti-angiogenic/metastatic effects of Smad7 amenable to therapy, we are planning to dissect the various domains of Smad7 to determine the amino acid sequences primarily responsible and sufficient to cause these effects. Based on these results, we hope to design and test short peptides that are able to penetrate into the cells and mimic Smad7 function to revert the invasive/metastatic properties.
Furthermore, we plan to employ genome-wide gene expression profiling of tumors derived from the model cell lines that produce metastatic tumors when orthotopically implanted in nude mice and inhibited for metastasis by Smad7/Smad7 mimetic peptides using microarrays, to uncover the critical effector genes that are responsible for the Smad7-mediated metastasis suppressive effects for development in the future as novel therapeutic agents. The outcome of this study would be of great importance, since it is likely to elucidate novel agents for metastatic breast cancer therapy.