The Role of Spy1 Protein Regulation In Breast Cancer

Cell growth and proliferation are tightly controlled by the cyclic regulation of the cyclin dependent kinases (Cdks). Cdks are positively regulated through interactions with regulatory Cyclin partners as well as being negatively regulated through interactions with families of Cdk inhibitors (CKIs). The Spy1/RINGO family of proteins have emerged as a unique class of Cyclin-like proteins capable of directly binding both to the Cdks, as a positive regulatory partner, as well as to at least one member of the CKI\u27s, p27Kip1, as a negative regulator. Abnormally elevated levels of Spy1 promote cell proliferation, nhibit apoptosis and are implicated in aggressive tumorgenesis in all cell/tissue types studied to date. Understanding how Spy1 protein is regulated is essential in resolving how it contributes to normal and abnormal growth processes. Herein, we demonstrate that Spy1 is degraded in a cell-cycle-dependent manner via the ubiquitin-proteasome system. We have resolved the E3 ligase and essential phosphorylation sites mediating Spy1A degradation. Furthermore, we show that Spy1 protein is stabilized in subsets of human breast cancer samples. Using a stable mutant of Spy1 we demonstrate that this represents an oncogenic modification in vitro and accelerates tumor formation in vivo. We further show that these oncogenic properties are largely dependent upon the unique activation of Cdk1 and the subsequent inhibition of the anti-apoptotic regulator FOXO1. Utilizing Spy1 mutants unable to bind to the primary effectors p27 and Cdk2, we have found that Spy1-mediated effects in the breast rely on direct interactions with each of these effectors via separable functional mechanisms. This work reveals novel mechanisms regulating the progression, and potentially the etiology, of human breast cancers and may be of considerable therapeutic relevance

The Cyclin-dependent Kinase Activator, Spy1A, Is Targeted for Degradation by the Ubiquitin Ligase NEDD4

Spy1A is a cyclin-like protein required for progression through the G(1)/S phase of the cell cycle. Elevated Spy1A protein levels have been implicated in tumorigenesis and are attributed to overriding the DNA damage response and enhancing cell proliferation. Understanding how Spy1A is produced and degraded is essential in resolving how it contributes to normal and abnormal growth processes. Herein, we demonstrate that Spy1A is degraded in a cell cycle-dependent manner during mitosis via the ubiquitin-proteasome system. We have resolved the E3 ligase and essential phosphorylation sites mediating Spy1A degradation. Furthermore, we have determined that non-degradable forms of Spy1A do not trigger cell cycle arrest but, rather, contribute to uncontrolled cell growth. Further investigation into the regulation of Spy1A may reveal novel strategies for understanding the etiology and progression of specific growth disorders

The cyclin-like protein Spy1/RINGO promotes mammary transformation and is elevated in human breast cancer

Abstract Background Spy1 is a novel \u27cyclin-like\u27 activator of the G1/S transition capable of enhancing cell proliferation as well as inhibiting apoptosis. Spy1 protein levels are tightly regulated during normal mammary development and forced overexpression in mammary mouse models accelerates mammary tumorigenesis. Methods Using human tissue samples, cell culture models and in vivo analysis we study the implications of Spy1 as a mediator of mammary transformation and breast cancer proliferation. Results We demonstrate that this protein can facilitate transformation in a manner dependent upon the activation of the G2/M Cdk, Cdk1, and the subsequent inhibition of the anti-apoptotic regulator FOXO1. Importantly, we show for the first time that enhanced levels of Spy1 protein are found in a large number of human breast cancers and that knockdown of Spy1 impairs breast cancer cell proliferation. Conclusions Collectively, this work supports that Spy1 is a unique activator of Cdk1 in breast cancer cells and may represent a valuable drug target and/or a prognostic marker for subsets of breast cancers

Direct interactions with both p27 and Cdk2 regulate Spy1-mediated proliferation <i>in vivo</i> and <i>in vitro</i>

<p>Families of cyclin-like proteins have emerged that bind and activate cyclin dependent kinases (Cdk)s, directing the phosphorylation of noncanonical Cdk substrates. One of these proteins, Spy1, has demonstrated the unique ability to directly bind and activate both Cdk1 and Cdk2, as well as binding and promoting the degradation of at least one Cdk inhibitor, p27^Kip1. Spy1 accelerates somatic cell growth and proliferation and is implicated in a number of human cancers including the breast, brain and liver. Herein we isolate key residues mediating the direct interaction with p27. We use mutants of Spy1 to determine the physiological role of direct interactions with distinct binding partners Cdk2 and p27. We demonstrate that disrupting the direct interaction with either Spy1 binding partner decreased endogenous activity of Cdk2, as well as Spy1-mediated proliferation. However, only the direct interaction with p27 was essential for Spy1-mediated effects on p27 stability. <i>In vivo</i> neither mutation completely prevented tumorigenesis, although each mutation slowed the rate of Spy1-mediated tumorigenesis and decreased overall tumor volumes. This work supports the conclusion that direct interaction with both p27 and Cdk2 contribute to Spy1-mediated effects on cell growth. It is important to elucidate the dynamics of these interactions and to consider these data when assessing functional outcomes.</p