Ectodomain Shedding of TGF-beta Receptors: Role in Signaling and Breast Cancer Biology

<p>The transforming growth factor beta (TGF-beta) signaling pathway is a critical regulator of multiple biological processes that are involved in cancer progression, such as proliferation, migration, invasion and metastasis. TGF-beta ligands bind to multiple high-affinity receptors (TbetaRI, TbetaRII, TbetaRIII), whose expression on the cell surface, and subsequent ability to transduce signaling, can be modulated by ectodomain shedding. </p><p> TbetaRIII, also known as betaglycan, is the most abundantly expressed TGF-beta receptor. TbetaRIII suppresses breast cancer progression through inhibiting migration, invasion, metastasis, and angiogenesis. TbetaRIII binds TGF-beta ligands, with membrane-bound TbetaRIII presenting ligand to enhance TGF-beta signaling. However, TbetaRIII can also undergo ectodomain shedding, releasing soluble TbetaRIII, which binds and sequesters ligand to inhibit downstream signaling. To investigate the relative contributions of soluble and membrane-bound TbetaRIII on TGF-beta signaling and breast cancer biology, here I describe TbetaRIII mutants with impaired (Delta-Shed-TbetaRIII) or enhanced ectodomain shedding (SS-TbetaRIII). Relative to wild-type (WT)-TbetaRIII, Delta-Shed-TbetaRIII increased TGF-beta signaling and blocked TbetaRIII's ability to inhibit breast cancer cell migration and invasion. Conversely, SS-TbetaRIII, which increased soluble TbetaRIII production, decreased TGF-beta signaling and increased TbetaRIII-mediated inhibition of breast cancer cell migration and invasion. </p><p> TbetaRI is released from the cell surface by a common sheddase of the A disintegrin and metalloproteinase (ADAM) family, ADAM17. This shedding event results in a downregulation of TGF-beta signaling. Here, I present evidence that a closely related protease, ADAM10, may be a novel sheddase for TbetaRI. A specific ADAM10 inhibitor was able to increase cell surface expression of TbetaRI, and decrease levels of circulating soluble TbetaRI in vivo. Interestingly, inhibition of ADAM10 concurrently increased shedding of TbetaRIII, and was able to alter TGF-beta signaling in a TbetaRIII-dependent manner. </p><p> </p><p> Together, these studies suggest that ectodomain shedding of TGF-beta receptors is an important determinant for regulation of TGF-beta-mediated signaling and biology.</p>Dissertatio

Digital Microfluidic Platform to Maximize Diagnostic Tests with Low Sample Volumes from Newborns and Pediatric Patients

&ldquo;Children are not tiny adults&rdquo; is an adage commonly used in pediatrics to emphasize the fact that children often have different physiological responses to sickness and trauma compared to adults. However, despite widespread acceptance of this concept, diagnostic blood testing is an excellent example of clinical care that is not yet customized to the needs of children, especially newborns. Cumulative blood loss resulting from clinical testing does not typically impact critically ill adult patients, but can quickly escalate in children, leading to iatrogenic anemia and related comorbidities. Moreover, the tests prioritized for rapid, near-patient testing in adults are not always the most clinically relevant tests for children or newborns. This report describes the development of a digital microfluidic testing platform and associated clinical assays purposely curated to address current shortcomings in pediatric laboratory testing by using microliter volumes (&lt;50 &micro;L) of samples. The automated platform consists of a small instrument and single-use cartridges, which contain all reagents necessary to prepare the sample and perform the assay. Electrowetting technology is used to precisely manipulate nanoliter-sized droplets of samples and reagents inside the cartridge. To date, we have automated three disparate types of assays (biochemical assays, immunoassays, and molecular assays) on the platform and have developed over two dozen unique tests, each with important clinical application to newborns and pediatric patients. Cell lysis, plasma preparation, magnetic bead washing, thermocycling, incubation, and many other essential functions were all performed on the cartridge without any user intervention. The resulting assays demonstrate performance comparable to standard clinical laboratory assays and are economical due to the reduced hands-on effort required for each assay and lower overall reagent consumption. These capabilities allow a wide range of assays to be run simultaneously on the same cartridge using significantly reduced sample volumes with results in minutes