Purpose: To investigate the clinical relevance of the recently characterized human oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A) in human breast cancer. Experimental Design: CIP2A expression (mRNA and protein) was measured in three different sets of human mammary tumors and compared with clinicopathologic variables. The functional role of CIP2A in breast cancer cells was evaluated by small interfering RNA-mediated depletion of the protein followed by an analysis of cell proliferation, migration, anchorage-independent growth, and xenograft growth. Results: CIP2A mRNA is overexpressed (n = 159) and correlates with higher ScarffBloom-Richardson grades (n = 251) in samples from two independent human breast cancer patients. CIP2A protein was found to be overexpressed in 39% of 33 human breast cancer samples. Furthermore, CIP2A mRNA expression positively correlated with lymph node positivity of the patients and with the expression of proliferation markers and p53 mutations in the tumor samples. Moreover, CIP2A protein expression was induced in breast cancer mouse models presenting mammary gland-specific depletion of p53 and either BRCA1 or BRCA2. Functionally, CIP2A depletion was shown to inhibit the expression of its target protein c-Myc. Loss of CIP2A also inhibited anchorageindependent growth in breast cancer cells. Breast cancer is the most common malignancy that affects women, with >1 million cases occurring worldwide annually. Further, breast cancer is the most important cause of cancerrelated deaths in women. However, the understanding of the molecular mechanisms that maintain the malignant growth of breast cancer cells remains incomplete (1).The oncogenic transformation of human cells requires the perturbation of a distinct set of oncogenes and tumor suppressors (2). It was recently shown that the tumor suppressor activity of protein phosphatase 2A (PP2A) prevents the transformation of human breast epithelial cells (3). The role of PP2A as a relevant breast cancer tumor suppressor was further strengthened by a recent study showing that somatic mutations occurred in one of the subunits of the functional PP2A trimer (PP2A Aβ) in 13% of human breast cancers and that PP2A trimers containing this mutation fail to suppress the oncogenic activity of RalA (4, 5). In

Senescence induction contributes to cancer therapy responses and is crucial for p53-mediated tumor suppression. However, whether p53 inactivation actively suppresses senescence induction has been unclear. Here, we show that E2F1 overexpression, due to p53 or p21 inactivation, promotes expression of human oncoprotein CIP2A, which in turn, by inhibiting PP2A activity, increases stabilizing serine 364 phosphorylation of E2F1. Several lines of evidence show that increased activity of E2F1-CIP2A feedback renders breast cancer cells resistant to senescence induction. Importantly, mammary tumorigenesis is impaired in a CIP2A-defi cient mouse model, and CIP2A-defi cient tumors display markers of senescence induction. Moreover, high CIP2A expression predicts for poor prognosis in a subgroup of patients with breast cancer treated with senescence-inducing chemotherapy. Together, these results implicate the E2F1-CIP2A feedback loop as a key determinant of breast cancer cell sensitivity to senescence induction. This feedback loop also constitutes a promising prosenescence target for therapy of cancers with an inactivated p53-p21 pathway. SIGNIFICANCE:It has been recently realized that most currently used chemotherapies exert their therapeutic effect at least partly by induction of terminal cell arrest, senescence. However, the mechanisms by which cell-intrinsic senescence sensitivity is determined are poorly understood. Results of this study identify the E2F1-CIP2A positive feedback loop as a key determinant of breast cancer cell sensitivity to senescence and growth arrest induction. Our data also indicate that this newly characterized interplay between 2 frequently overexpressed oncoproteins constitutes a promising prosenescence target for therapy of cancers with inactivated p53 and p21. Finally, these results may also facilitate novel stratifi cation strategies for selection of patients to receive senescence-inducing cancer therapies. Cancer Discov; 3(2);

Apoptosis as an active, targeted process provides a potential new therapeutic approach for treating breast cancer.

SUMMARY Understanding of the mechanisms determining MYC’s transcriptional -and proliferation promoting activities in vivo could facilitate approaches for MYC targeting. However, post-translational mechanisms that control MYC function in vivo are poorly understood. Here we demonstrate that MYC phosphorylation at serine 62 enhances MYC accumulation on Lamin A/C-associated nuclear structures and the Protein Phosphatase 2A (PP2A) inhibitor protein CIP2A is required for this process. CIP2A is also critical for serum induced MYC phosphorylation, and for MYC-elicited proliferation induction in vitro. Complementary transgenic approaches and an intestinal regeneration model further demonstrated the in vivo importance of CIP2A and serine 62 phosphorylation for MYC activity upon DNA damage. However, targeting of CIP2A did not influence normal function of intestinal crypt cells. The data underline the importance of nuclear organization in the regulation of MYC phosphorylation, leading to in vivo demonstration of a strategy for inhibiting MYC activity without detrimental physiological effects.

Checkpoint kinase Chk1 is constitutively active in many cancer cell types and new generation Chk1 inhibitors show marked antitumor activity as single agents. Here we present a hitherto unrecognized mechanism that contributes to the response of cancer cells to Chk1 targeted therapy. Inhibiting chronic Chk1 activity in cancer cells induced the tumor suppressor activity of protein phosphatase PP2A, which by dephosphorylating MYC serine 62, inhibited MYC activity and impaired cancer cell survival. Mechanistic investigations revealed that Chk1 inhibition activated PP2A by decreasing the transcription of CIP2A, a chief inhibitor of PP2A activity. Inhibition of cancer cell clonogenicity by Chk1 inhibition could be rescued in vitro either by exogenous expression of CIP2A or by blocking the CIP2A-regulated PP2A complex. Chk1-mediated CIP2A regulation was extended in tumor models dependent on either Chk1 or CIP2A. The clinical relevance of CIP2A as a Chk1 effector protein was validated in several human cancer types, including neuroblastoma where CIP2A was identified as a NMYC-independent prognostic factor. Since the Chk1-CIP2A-PP2A pathway is driven by DNA-PK activity, functioning regardless of p53 or ATM/ATR status, our results offer explanative power for understand how Chk1 inhibitors mediate single-agent anticancer efficacy. Further, they define CIP2A-PP2A status in cancer cells as a pharmacodynamic marker for their response to Chk1-targeted therapy.

Hypocoagulation shown by ROTEM® was associated with bleeding complications in patients with mechanical circulatory support. In contrast, hypercoagulation did not correlate with clinical thrombosis.

Neuroblastoma is a neural crest-derived childhood tumor of the peripheral nervous system in which MycN amplification is a hallmark of poor prognosis. Here we show that is expressed together with phosphorylation-stabilizing factor in regions of the neural plate destined to form the CNS, but MycN is excluded from the neighboring neural crest stem cell domain. Interestingly, ectopic expression of MycN or CIP2A in the neural crest domain biases cells toward CNS-like neural stem cells that express Sox2. Consistent with this, some forms of neuroblastoma have been shown to share transcriptional resemblance with CNS neural stem cells. As high levels correlate with poor prognosis, we posit that a MycN/CIP2A-mediated cell-fate bias may reflect a possible mechanism underlying early priming of some aggressive forms of neuroblastoma. In contrast to, its paralogue is normally expressed in the neural crest stem cell domain and typically is associated with better overall survival in clinical neuroblastoma, perhaps reflecting a more "normal" neural crest-like state. These data suggest that priming for some forms of aggressive neuroblastoma may occur before neural crest emigration from the CNS and well before sympathoadrenal specification.

All mammalian cells absolutely require polyamines (putrescine, spermidine, and spermine) for growth. Here we show that the overexpression of cDNA for S-adenosylmethionine decarboxylase (AdoMetDC), the main regulatory enzyme in the biosynthesis of higher polyamines, induces transformation of rodent fibroblasts when expressed in the sense or the antisense orientation. Both transformants were able to induce invasive tumors in nude mice. Neither transformation was associated with activation of the mitogen-activated protein kinases Erk1 and Erk2. Instead, the AdoMet DC sense, but not antisense, transformants displayed constitutive activation of the c-Jun NH2-terminal kinase (JNK) pathway. However, both transformations converged on persistent phosphorylation of endogenous c-Jun at Ser73. The phenotype of the AdoMetDC sense transformants was reversed by expression of dominant-negative mutants of SEK1 (MKK4), JNK1, and c-Jun (TAM-67), which were also found to impair cytokinesis. Similarly, TAM-67 reverted the morphology of the AdoMetDC-antisense expressors. This report is the first demonstration of a protein whose overexpression or block of synthesis can induce cell transformation. In addition, we show that the polyamine biosynthetic enzymes require c-Jun activation for eliciting their biological effects.