Thursday, November 21, 2024
11/21/2024
The majority of recurrent ovarian cancers spread to the peritoneal cavity in the form of ascites, where tumor cells grow as spheroids, and metastasize to other organs causing morbidity and death. The overall goal of this proposal is to define the mechanisms by which protein kinases control spheroid growth and metastasis of ovarian cancer cells, and use this information to develop kinase inhibitor therapies for the treatment of recurrent ovarian cancer. The Cdc42 downstream effector protein kinases MRCKα and MRCKβ (MRCKs) are highly expressed in ovarian cancer and have been implicated in cell migration, invasion and metastasis. Strikingly, we recently discovered that inhibition of MRCK impaired actin cytoskeleton remodeling and focal adhesion assembly blocking cell migration and spheroid growth and invasion of ovarian cancer cells, revealing MRCK as a new and untapped target for the treatment of recurrent ovarian cancer. Moreover, our most recent findings showed inhibition of MRCK blocked tumor growth and induced tumor apoptosis in ovarian cancer xenografts and ascites models. Nevertheless, the mechanistic basis for MRCK support of ovarian cancer pathogenesis remains poorly understood. Our overall hypothesis is that MRCKs control spheroid growth and invasion of ovarian cancer cells through the regulation of actin cytoskeleton remodeling that involves the phosphorylation and/or interaction with proteins associated with actin and myosin signaling. To test this hypothesis, we have designed two Specific Aims, Aim 1: Identify MRCK protein interactors/substrates that contribute to ovarian cancer using proteomics strategies and Aim 2: Determine the effect of MRCK inhibition on spheroid viability and invasion using in vitro and in vivo ovarian cancer models. In Aim 1, we will perform a focused analysis of our current MRCK substrate candidates using phosphoantibodies and mass spectrometry in 2D and 3D cultures, and will decipher the function of MRCK phosphorylation events using site-directed mutagenesis. Next, we will apply unbiased proteomics methods to define additional interacting partners, substrates and signaling pathways controlled by MRCK. Together, we will establish bona fide MRCK substrates and will define the mechanisms by which MRCK promote spheroid proliferation, survival and invasion in ovarian cancer. In Aim 2, we will inhibit MRCK and monitor viability, invasion and actin remodeling of ovarian cancer spheroids, organoids and ascites isolated from patients with metastatic disease. Next, using a panel of orthotopic ovarian cancer patient-derived xenografts, we will inhibit MRCK and measure in vivo tumor growth, metastasis and ascites. Together, the work proposed here will define the mechanisms by which MRCKs promote ovarian cancer growth and metastasis and establish MRCK as a new target in recurrent ovarian cancer.
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