Cancer arises when a cell evades normal proliferative controls, often by mutations in genes that control cell growth and division. Various checkpoints exist to ensure that cells replicate without genetic errors and repair damaged DNA, to avoid both the uncoupling of replication from cell cycle control as well as to avoid the transmission of genetic mutations. Recent evidence demonstrates that DNA damage responses are activated in early premalignant tissue but not in normal tissue. Checkpoints are often the targets for oncogenic mutation, thereby uncoupling proliferation from apoptosis while enhancing proliferation itself during transformation and tumorigenesis. In addition to evasion of checkpoints, cancer cells must also inactivate the apoptotic pathways. Apoptotic mechanisms exist to protect cells against the loss of checkpoints, irreparable DNA damage and sustained oncogenic stimuli.

I have investigated the role of Spy1 expression in apoptosis and checkpoint activation to begin to understand the molecular mechanisms by which Spy1 may contribute to oncogenesis. A correlation between Spy1 and breast cancer recently published ( Zucchi et al., PNAS, 2004), examined the altered regulation of genes in nodal metastatic and invasive ductal breast carcinomas, identifying Spy1 as one of the fifty most up-regulated genes. These data suggest that deregulation of Spy1 expression plays a key role in oncogenesis.

Spy1 is the originally identified member of the Speedy/Ringo family of vertebrate cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin dependent kinases. In our investigations we show that Spy1 expression enhances cell survival in response to UV irradiation by preventing the activation of caspases and apoptosis in a U2OS osteosarcoma cell line. Using an inducible system allowing for regulated expression of Spy1, we show that Spy1 expression prevents activation of caspase-3 and suppresses apoptosis in response to UV irradiation. Spy1expression also allows for UV irradiation-resistant DNA synthesis and permits cells to progress into mitosis, as demonstrated by phosphorylation on histone H3, indicating that Spy1 expression can inhibit the S-phase/replication and G2/M checkpoints. We demonstrate that Spy1 expression inhibits phosphorylation of Chk1, RPA, and histone H2A.X, which may directly contribute to the decrease in apoptosis and checkpoint bypass. Furthermore, mutation of the conserved Speedy/Ringo box, known to mediate interaction with CDK2, abrogates the ability of Spy1 to inhibit apoptosis and the phosphorylation of Chk1 and RPA. The data presented indicate that Spy1 expression allows cells to evade checkpoints and apoptosis and suggest that Spy1 regulation of CDK2 is important for the response to DNA damage.

Spy1 expression prevents activation of apoptotic machinery and, importantly, prevents activation of both the S-phase/replication checkpoint and the G2/M checkpoint. Spy1 expression suppresses signaling to mediators of the checkpoint response, which are specific for apoptosis caspase-3), checkpoint activation/DNA repair (H2A.X and RPA), or are common to both pathways (Chk1). Furthermore, we show that the interaction of Spy1 with CDK2 is required for these effects, suggesting that Spy1 association with CDKs may play a prominent role in abnormal cell cycle events, such as the DNA damage response, checkpoint signaling, and apoptosis. The evasion of checkpoints and apoptosis are both traits selected by cancer cells. These findings are relevant to the role of Spy1 overexpression reported in invasive breast carcinomas.

PUBLICATIONS (resulting from this training)

Meyer AN, RF Gastwirt, DD Schlaepfer, DJ Donoghue. (2004) The cytoplasmic tyrosine kinase Pyk2 as a novel effector of fibroblast growth factor receptor 3 activation. J Biol. Chem. 279:28450-7.

Gastwirt RF, DA Slavin, CW McAndrew and DJ Donoghue. (2006) Spy1 Expression Prevents Normal Cellular Responses to DNA Damage: Inhibition of Apoptosis and Checkpoint Activation. J Biol. Chem. 281:35423-35.

Gastwirt RF, CW McAndrew and DJ Donoghue. (2007) Speedy/RINGO Regulation of CDKs in Cell Cycle, Checkpoint Activation and Apoptosis. Cell Cycle, 6:1188-1193.

McAndrew CW, RF Gastwirt, AN Meyer, LA Porter, and DJ Donoghue. (2007) Spy1 Enhances Phosphorylation and Degradation of the Cell Cycle Inhibitor p27. Cell Cycle, 6:1937-1945.

McAndrew, CW, RF Gastwirt and DJ Donoghue (2009) Regulation of the Cell Cycle. In "Wiley Encyclopedia of Chemical Biology" (N R Civjan, Ed.) John Wiley & Sons, Inc. (Invited Review, in press).