Project 4 Title : RND1-mediated Breast Cancer Suppression Leader: Filippo G. Giancotti, M.D., Ph.D. Key Personnel: Young-Mi Kim, Ph.D, Postdoctoral Research Scholar Surajit Sinha, Ph.D., Postdoctoral Research Scholar PROJECT SUMMARY: We have identified RND1, encoding a Rho family GTPase, as a tumor suppressor inactivated in aggressive breast cancers. Upon silencing of Rnd1, mammary epithelial cells underwent an Epithelial-to-Mesenchymal Transformation (EMT) and became senescent, implying that they had suffered an oncogenic insult. Expression of c-Myc rescued Rnd1-depleted cells from senescence and enabled them to form invasive, multi-acinar structures in 3D Matrigel. Moreover, silencing of Rnd1 conferred tumorigenic and invasive properties upon the stem-like Comma-1D cells. Conversely, expression of Rnd1 suppressed the ability of the basal-like 4T1 cells to colonize the lung upon tail-vein injection. Intriguingly, depletion of Rnd1 potently activated oncogenic Ras signaling and mechanistic studies indicated that Rnd1 suppresses Ras by binding to and activating the atypical Ras-GAP domain of Plexin B1. Analysis of clinical samples revealed that genetic and epigenetic mechanisms contribute to inactivate RND1 and thereby activate Ras signaling in a substantial fraction of basal-like and Triple Negative (TN) breast cancers. Based on these new findings, we propose to further examine the tumor suppressor function of Rnd1 in cultured cells and mouse models and to develop strategies for the treatment of Rnd1-deficient breast cancers.
In Specific Aim 1, we will examine the molecular mechanisms through which Rnd1 suppresses Ras signaling, EMT, and oncogenesis. Our Preliminary Studies indicate that Rnd1 suppresses Ras signaling and EMT by binding to the insert segment of the split Ras GAP domain of Plexin B1, which acts selectively on Rap1. To examine the mechanism by which Rnd1 suppresses Ras signaling, EMT, and oncogenesis, we will use genetic reconstitution with wild type and mutant forms of various signaling proteins followed by biochemical, phenotypic and functional analysis. Based on the known functions of Rap1, we will examine if Rap1 promotes Ras signaling by inactivating p120 Ras-GAP as well as by activating BRAF. In addition, we will explore why loss of Rnd1 produces a dominant effect in mammary epithelial cells expressing additional Ras-GAP proteins. These studies will be conducted in collaboration with Neal Rosen (Project 3).
In Specific Aim 2, we will examine if genetic inactivation of Rnd1 contributes to initiate and maintain mammary tumorigenesis and promotes metastasis in mice. Our Preliminary Studies indicate that inactivation of Rnd1 confers tumorigenic and invasive properties upon Comma-1D cells. To examine if inactivation of Rnd1 is sufficient to initiate and is necessary to maintain mammary tumorigenesis in vivo or it cooperates with other oncogenic lesions to mediate these effects, we will make use of a new technology developed by the Lowe laboratory (Core B: Speedy Mice). Tet-inducible shRNAs will be introduced in ES cells at the Col1 locus by using a cassette exchange strategy and in then in mice by using tetraploid complementation. Mice carrying these alleles will be crossed to MMTV-rtTTA mice and the compound mice crossed to MMTV-Myc mice as well as Tp53+/- mice. In vivo and ex vivo studies will be conducted to study the effect of ablation of Rnd1, alone or in combination with overexpression of Myc or inactivation of p53 on mammary tumor initiation, maintenance, and metastasis. In this Aim, we will also examine the role of Id1 as an additional cooperating oncogenic alteration. These studies will be performed in collaboration with Robert Benezra (Project 2).
In Specific Aim 3, we will develop pharmacological strategies for the treatment of Rnd1-deficient breast cancers. Our Preliminary Studies indicate that inhibition of MEK suppresses the proliferation, invasion, and anchorage-independent growth of MCF-10A cells transformed by loss of Rnd1. To examine if inhibition of MEK is a potentially effective therapeutic strategy for Rnd1-deficient breast cancers, we will conduct preclinical studies on multiple Rnd1- deficient breast cancer cells. In collaboration with Neil Rosen and Jos? Baselga (Project 3), we will examine the preclinical efficacy of two novel allosteric MEK inhibitors. Since the effect of pharmacological inhibition of the Ras-ERK pathway is often limited by release of negative feed back loops, which operate to restrain its activation, or other resistance mechanisms, we will identify the specific mechanism of resistance operating in Rnd1-deficient cancer cells and develop pharmacological combinations designed to improve the effect of MEK inhibitors. Potentially effective drug combinations will undergo testing in xenograft models.
The oncogenic alterations leading to activation of Ras in basal-like and TN breast cancers are poorly defined. Our studies promise to fill a gap in our understanding of the oncogenic pathways operating in these cancers. Furthermore, they may open up sorely needed new therapeutic avenues for this sizeable subset of aggressive breast cancers.
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