BCR-ABL1 promotes leukemia by converting p27 into a cytoplasmic oncoprotein.

Recent studies have revealed that p27, a nuclear cyclin-dependent kinase (Cdk) inhibitor and tumor suppressor, can acquire oncogenic activities upon mislocalization to the cytoplasm. To understand how these antagonistic activities influence oncogenesis, we dissected the nuclear and cytoplasmic functions of p27 in chronic myeloid leukemia (CML), a well-characterized malignancy caused by the BCR-ABL1 tyrosine kinase. p27 is predominantly cytoplasmic in CML and nuclear in normal cells. BCR-ABL1 regulates nuclear and cytoplasmic p27 abundance by kinase-dependent and -independent mechanisms, respectively. p27 knockdown in CML cell lines with predominantly cytoplasmic p27 induces apoptosis, consistent with a leukemogenic role of cytoplasmic p27. Accordingly, a p27 mutant (p27(CK-)) devoid of Cdk inhibitory nuclear functions enhances leukemogenesis in a murine CML model compared with complete absence of p27. In contrast, p27 mutations that enhance its stability (p27(T187A)) or nuclear retention (p27(S10A)) attenuate leukemogenesis over wild-type p27, validating the tumor-suppressor function of nuclear p27 in CML. We conclude that BCR-ABL1 kinase-dependent and -independent mechanisms convert p27 from a nuclear tumor suppressor to a cytoplasmic oncogene. These findings suggest that cytoplasmic mislocalization of p27 despite BCR-ABL1 inhibition by tyrosine kinase inhibitors may contribute to drug resistance, and effective therapeutic strategies to stabilize nuclear p27 must also prevent cytoplasmic mislocalization.

Separation of telomerase functions by reverse genetics.

The canonical function of the human telomerase protein (hTERT) is to synthesize telomeric DNA, but it has other biological activities, including enhancing cell proliferation, decreasing apoptosis, regulating DNA damage responses, and increasing cellular proliferative lifespan. The mechanistic relationships among these activities are not understood. We previously demonstrated that ectopic hTERT expression in primary human mammary epithelial cells diminishes their requirement for exogenous mitogens, thus giving them a proliferative advantage in a mitogen-depleted environment. Here, we show that this phenotype is caused by a combination of increased cell division and decreased apoptosis. In addition, we use a panel of hTERT mutants to demonstrate that this enhanced cell proliferation can be uncoupled not only from telomere elongation, but also from other telomerase activities, including cellular lifespan extension and regulation of DNA damage responses. We also find that the proliferative function of hTERT, which requires hTERT catalytic activity, is not caused by increased Wnt signaling, but is accompanied by alterations in key cell cycle regulators and is linked to an hTERT-catalyzed decrease in the levels of the RNA component of mitochondrial RNA processing endoribonuclease. Thus, enhanced cell proliferation is an independent function of hTERT that could provide a new target for the development of anti-telomerase cancer therapeutic agents.

Testing the importance of p27 degradation by the SCFskp2 pathway in murine models of lung and colon cancer.

Decreased expression of the CDK inhibitor p27kip1 in human tumors directly correlates with increased resistance to chemotherapies, increased rates of metastasis, and an overall increased rate of patient mortality. It is thought that decreased p27 expression in tumors is caused by increased proteasomal turnover, in particular activation of the pathway governed by the SCFskp2 E3 ubiquitin protein ligase. We have directly tested the importance of the SCFskp-mediated degradation of p27 in tumorigenesis by analyzing the tumor susceptibility of mice that express a form of p27 that cannot be ubiquitinated and degraded by this pathway (p27T187A). In mouse models of both lung and colon cancer down-regulation of p27 promotes tumorigenesis. However, we found that preventing p27 degradation by the SCFskp2 pathway had no impact on tumor incidence or overall survival in either tumor model. Our study unveiled a previously unrecognized role for the control of p27 mRNA abundance in the development of non-small cell lung cancers. In the colon cancer model, the frequency of intestinal adenomas was similarly unaffected by the p27T187A mutation, but, unexpectedly, we found that it inhibited progression of intestinal adenomas to carcinomas. These studies may guide the choice of clinical settings in which pharmacologic inhibitors of the Skp2 pathway might be of therapeutic value.

A mouse model for cyclin E-dependent genetic instability and tumorigenesis.

Ubiquitination of murine cyclin E is triggered by phosphorylation on threonine 393. Cyclin E(T393A) knockin mice exhibited increased cyclin E stability, but no phenotypic abnormalities. Importantly, loss of the p53 pathway exacerbated the effect of the T393A mutation. Thus, in p21(-/-) cells the T393A mutation had an exaggerated effect on cyclin E abundance and its associated kinase activity, which caused abnormal cell cycle progression, and genetic instability involving chromosome breaks and translocations. Moreover, cyclin E(T393A) acted synergistically with p53 deficiency to accelerate tumorigenesis in cyclin E(T393A) p53(-/-) mice; Ras more readily transformed cyclin E(T393A) p53(-/-) cells than p53(-/-) cells in vitro; and cyclin E(T393A) mice had a greatly increased susceptibility to Ras-induced lung cancer.

ERK phosphorylates p66shcA on Ser36 and subsequently regulates p27kip1 expression via the Akt-FOXO3a pathway: implication of p27kip1 in cell response to oxidative stress.

Mice deficient for p66shcA represent an animal model to link oxidative stress and aging. p66shcA is implicated in oxidative stress response and mitogenic signaling. Phosphorylation of p66shcA on Ser36 is critical for its function in oxidative stress response. Here we report the identification of ERK as the kinase phosphorylating p66shcA on Ser36. Activation of ERKs was necessary and sufficient for Ser36 phosphorylation. p66shcA interacted with ERK and was demonstrated to be a substrate for ERK, with Ser36 being the major phosphorylation site. Furthermore, in response to H2O2, inhibition of ERK activation repressed p66shcA-dependent phosphorylation of FOXO3a and the down-regulation of its target gene p27kip1. Down-regulation of p27 might promote cell survival, as p27 played a proapoptotic role in oxidative stress response. As a feedback regulation, Ser36 phosphorylated p66shcA attenuated H2O2-induced ERK activation, whereas p52/46shcA facilitated ERK activation, which required tyrosine phosphorylation of CH1 domain. p66shcA formed a complex with p52/46ShcA, which may provide a platform for efficient signal propagation. Taken together, the data suggest there exists an interplay between ERK and ShcA proteins, which modulates the expression of p27 and cell response to oxidative stress.

Antigen-specific dose-dependent system for the study of an inheritable and reversible phenotype in mouse CD4+ T cells.

The transgenic T-cell receptor in mouse TEa CD4+ lymphocytes recognizes an endogenous peptide, Ealpha52-68, presented in the context of the major histocompatibility complex class II molecule I-Ab. In response to an optimal peptide concentration TEa cells enter the cell cycle and proliferate. However, a single exposure to high doses of the specific peptide diminished cell expansion upon subsequent restimulation. This hyporesponsive, or anergic, phenotype can still be detected after multiple restimulations indicating that the hyporesponsiveness persists despite cell division and it was inherited by daughter cells. Furthermore, we demonstrated that this hypoproliferative response is associated with high p27Kip1 and cyclin E protein levels, and reduced intracellular interleukin-2 (IL-2) expression. Addition of exogenous IL-2 was required to reset p27Kip1 levels in the progeny derived from hyporesponsive TEa cells. Thus, we have established antigen dose-dependent induction of a reversible, inheritable (i.e. epigenetic) phenotype and we have identified at least three components of the network of interactions: p27Kip1 cyclin E, and IL-2 expression.