CIP2A Interacts with TopBP1 and Drives Basal-Like Breast Cancer Tumorigenesis

Basal-like breast cancers (BLBC) are characterized by defects in homologous recombination (HR), deficient mitotic checkpoint, and high-proliferation activity. Here, we discover CIP2A as a candidate driver of BLBC. CIP2A was essential for DNA damage-induced initiation of mouse BLBC-like mammary tumors and for survival of HR-defective BLBC cells. CIP2A was dispensable for normal mammary gland development and for unperturbed mitosis, but selectively essential for mitotic progression of DNA damaged cells. A direct interaction between CIP2A and a DNA repair scaffold protein TopBP1 was identified, and CIP2A inhibition resulted in enhanced DNA damage-induced TopBP1 and RAD51 recruitment to chromatin in mammary epithelial cells. In addition to its role in tumor initiation, and survival of BRCA-deficient cells, CIP2A also drove proliferative MYC and E2F1 signaling in basal-like triple-negative breast cancer (BL-TNBC) cells. Clinically, high CIP2A expression was associated with poor patient prognosis in BL-TNBCs but not in other breast cancer subtypes. Small-molecule reactivators of PP2A (SMAP) inhibited CIP2A transcription, phenocopied the CIP2A-deficient DNA damage response (DDR), and inhibited growth of patient-derived BLBC xenograft. In summary, these results demonstrate that CIP2A directly interacts with TopBP1 and coordinates DNAdamage-induced mitotic checkpoint and proliferation, thereby driving BLBC initiation and progression. SMAPs could serve as a surrogate therapeutic strategy to inhibit the oncogenic activity of CIP2A in BLBCs. Significance: These results identify CIP2A as a nongenetic driver and therapeutic target in basal-like breast cancer that regulates DNA damage-induced G2-M checkpoint and proliferative signaling.Peer reviewe

CIP2A Promotes T-Cell Activation and Immune Response to Listeria monocytogenes Infection

The oncoprotein Cancerous Inhibitor of Protein Phosphatase 2A ( CIP2A) is overexpressed in most malignancies and is an obvious candidate target protein for future cancer therapies. However, the physiological importance of CIP2A-mediated PP2A inhibition is largely unknown. As PP2A regulates immune responses, we investigated the role of CIP2A in normal immune system development and during immune response in vivo. We show that CIP2A-deficient mice (CIP2A(HOZ)) present a normal immune system development and function in unchallenged conditions. However when challenged with Listeria monocytogenes, CIP2A(HOZ) mice display an impaired adaptive immune response that is combined with decreased frequency of both CD4(+) T-cells and CD8(+) effector T-cells. Importantly, the cell autonomous effect of CIP2A deficiency for T-cell activation was confirmed. Induction of CIP2A expression during T-cell activation was dependent on Zap70 activity. Thus, we reveal CIP2A as a hitherto unrecognized mediator of T-cell activation during adaptive immune response. These results also reveal CIP2A(HOZ) as a possible novel mouse model for studying the role of PP2A activity in immune regulation. On the other hand, the results also indicate that CIP2A targeting cancer therapies would not cause serious immunological side-effects.Peer reviewe

CIP2A interacts with TopBP1 and drives basal-like breast cancer tumorigenesis

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Ovarian Cancers with Low CIP2A Tumor Expression Constitute an APR-246-Sensitive Disease Subtype

Publisher Copyright: © 2022 American Association for Cancer Research.Identification of ovarian cancer patient subpopulations with increased sensitivity to targeted therapies could offer significant clinical benefit. We report that 22% of the high-grade ovarian cancer tumors at diagnosis express CIP2A oncoprotein at low levels. Furthermore, regardless of their significantly lower likelihood of disease relapse after standard chemotherapy, a portion of relapsed tumors retain their CIP2A-deficient phenotype. Through a screen for therapeutics that would preferentially kill CIP2A-deficient ovarian cancer cells, we identified reactive oxygen species inducer APR-246, tested previously in ovarian cancer clinical trials. Consistent with CIP2A-deficient ovarian cancer subtype in humans, CIP2A is dispensable for development of MISIIR-Tag-driven mouse ovarian cancer tumors. Nevertheless, CIP2A-null ovarian cancer tumor cells from MISIIR-Tag mice displayed APR-246 hypersensitivity both in vitro and in vivo. Mechanistically, the lack of CIP2A expression hypersensitizes the ovarian cancer cells to APR-246 by inhibition of NF-κB activity. Accordingly, combination of APR-246 and NF-kB inhibitor compounds strongly synergized in killing of CIP2A-positive ovarian cancer cells. Collectively, the results warrant consideration of clinical testing of APR-246 for CIP2A-deficient ovarian cancer tumor subtype patients. Results also reveal CIP2A as a candidate APR-246 combination therapy target for ovarian cancer.Peer reviewe

KSHV viral cyclin interferes with T-cell development and induces lymphoma through Cdk6 and Notch activation in vivo

<div><p>Kaposi's sarcoma herpesvirus (KSHV)-encoded v-cyclin, a homolog of cellular cyclin D2, activates cellular CDK6, promotes G1-S transition of the cell cycle, induces DNA damage, apoptosis, autophagy and is reported to have oncogenic potential. Here we show that <i>in vivo</i> expression of v-cyclin in the B- and T-cell lymphocyte compartments results in a markedly low survival due to high penetrance of early-onset T-cell lymphoma and pancarditis. The v-cyclin transgenic mice have smaller pre-tumorigenic lymphoid organs, showing decreased cellularity, and increased proliferation and apoptosis. Furthermore, v-cyclin expression resulted in decreased amounts of CD3-expressing mature T-cells in the secondary lymphoid organs concurrent with alterations in the T-cell subpopulations of the thymus. This suggests that v-cyclin interferes with normal T-cell development. As the Notch pathway is recognized for its role in both T-cell development and lymphoma initiation, we addressed the role of Notch in the v-cyclin-induced alterations. Fittingly, we demonstrate induction of Notch3 and Hes1 in the pre-tumorigenic thymi and lymphomas of v-cyclin expressing mice, and show that lymphoma growth and viability are dependent on activated Notch signaling. Notch3 transcription and growth of the lymphomas was dependent on CDK6, as determined by silencing of CDK6 expression or chemical inhibition, respectively. Our work here reveals a viral cyclin-CDK6 complex as an upstream regulator of Notch receptor, suggesting that cyclins can play a role in the initiation of Notch-dependent lymphomagenesis.</p></div

Cell autonomous function for CIP2A in T-cell activation.

<p>(A) CIP2A protein expression from activated WT or CIP2A^HOZ CD8 T-cells. Mean + S.E.M of CIP2A protein expression using β-actin as a normalization control is shown. (B) CD4⁺ cells from Zap70^+/- or ATP analogue HXJ2-sensitive Zap70^(AS) mice were stimulated <i>in vitro</i> with plate-bound anti-CD3 and anti-CD28 antibodies in the presence or absence of HXJ42 (1 μM). Cells were harvested at indicated time-points and shown is real-time PCR analysis of CIP2A transcript levels relative to actin as normalized to the Zap70^+/- unstimulated sample. Shown is a representative of two independent experiments with identical results. (C) Cell surface staining of CD69 from CD4⁺CD62L⁺ T-cells isolated from WT or CIP2A^HOZ mice stimulated with anti-CD3 and anti-CD28 for 24h. The mean + S.E.M. of three independent experiments is shown. Student's t test. (D) Number of viable splenocytes determined by CellTiter-Glo Assay 7 days post-stimulation with IL-2 (20U/ml) and anti-CD3 (1.25, 2.5 or 5 μg/ml). Blue bars indicate medians, circle individual data points (n = 6 for WT & CIP2A^HOZ cells). * p<0.05, ** p<0.01, Student’s t-test. (E) Human CD4⁺ T-cells isolated from umbilical cord blood pooled from 5–6 individuals were nucleofected with scramble nontargeting siRNA or CIP2A siRNA. Cells were rested for 48hrs and activated with anti-CD3 and anti-CD28 for 24h. The mean + S.E.M. of three independent experiments is shown. Student's t test.</p

Characterization of CIP2A^HOZ lymphocytes.

<p>Characterization of CIP2A^HOZ lymphocytes.</p

Impaired T-cell activation in CIP2A^HOZ mice <i>in vivo</i>.

<p>(A-H) Flow cytometry analysis of splenocytes from 3 WT and 4 CIP2A^HOZ mice five days after recall infection with high-dose <i>L</i>.<i>m</i>.-OVA. (A) Representative flow cytometry analysis for CD4⁺ and CD8⁺ T cells. (B) Percentage of CD4⁺ and CD8⁺ splenocytes from analysis described in (A). * p < 0.05, Two-tailed T-test. (C) Representative dot plots of antigen-specific CD8⁺ T cells identified by H-2K^b/SIINFEKL multimer staining. Dot plots are gated on living CD45⁺ CD3⁺ CD8⁺ cells. (D) Bar chart numbers indicate H2-Kb/SIINFEKL multimer+ cells as percentages of CD8+ T cells. T-test. (E-H) Analysis of OVA-specific CD8⁺ T lymphocytes from control and mutant mice 5 days after recall infection with high-dose <i>L</i>.<i>m</i>.-OVA and unchallenged control mice. (E) CD62L and CD127 surface expression on OVA-specific (H-2K^b/SIINFEKL multimer⁺) allows the characterization of secondary T-cell subsets: central memory phenotype (T_CM, CD127⁺ CD62L⁺) and effector memory phenotype (T_EM, CD127⁺ CD62L^-) can be observed in immunized, unchallenged mice; whereas immunized challenged mice present two main populations of effector T cells differentiated by their CD127 expression. (F) Bar chart numbers indicate percentages of effector phenotype CD8⁺ T-cells in regards of total splenocytes or OVA-specific CD8⁺ T lymphocytes (right). * p < 0.05, Two-tailed T-test. (G) Cytokine production by antigen-specific T-cells from CIP2A^HOZ and WT mice on day 5 after recall infection with high-dose <i>L</i>.<i>m</i>.-OVA. (H) Proportion of T_EM and T_CM from total splenocytes. * p < 0.05, Two-tailed T-test.</p