Regulation and role of the PP2A-B56 holoenzyme family in cancer

Protein phosphatase 2A (PP2A) inactivation is common in cancer, leading to sustained activation of pro-survival and growth-promoting pathways. PP2A consists of a scaffolding A-subunit, a catalytic C-subunit, and a regulatory B-subunit. The functional complexity of PP2A holoenzymes arises mainly through the vast repertoire of regulatory B-subunits, which determine both their substrate specificity and their subcellular localization. Therefore, a major challenge for developing more effective therapeutic strategies for cancer is to identify the specific PP2A complexes to be targeted. Of note, the development of small molecules specifically directed at PP2A-B56α has opened new therapeutic avenues in both solid and hematological tumors. Here, we focus on the B56/PR61 family of PP2A regulatory subunits, which have a central role in directing PP2A tumor suppressor activity. We provide an overview of the mechanisms controlling the formation and regulation of these complexes, the pathways they control, and the mechanisms underlying their deregulation in cancer

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 interacts with TopBP1 and drives basal-like breast cancer tumorigenesis

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The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination

Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination

Regulation and role of the PP2A-B56 holoenzyme family in cancer

Protein phosphatase 2A (PP2A) inactivation is common in cancer, leading to sustained activation of pro-survival and growth-promoting pathways. PP2A consists of a scaffolding A-subunit, a catalytic C-subunit, and a regulatory B-subunit. The functional complexity of PP2A holoenzymes arises mainly through the vast repertoire of regulatory B-subunits, which determine both their substrate specificity and their subcellular localization. Therefore, a major challenge for developing more effective therapeutic strategies for cancer is to identify the specific PP2A complexes to be targeted. Of note, the development of small molecules specifically directed at PP2A-B56α has opened new therapeutic avenues in both solid and hematological tumors. Here, we focus on the B56/PR61 family of PP2A regulatory subunits, which have a central role in directing PP2A tumor suppressor activity. We provide an overview of the mechanisms controlling the formation and regulation of these complexes, the pathways they control, and the mechanisms underlying their deregulation in cancer

The Sustained Induction of c-MYC Drives Nab-Paclitaxel Resistance in Primary Pancreatic Ductal Carcinoma Cells

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited and, very often, ineffective medical and surgical therapeutic options. The treatment of patients with advanced unresectable PDAC is restricted to systemic chemotherapy, a therapeutic intervention to which most eventually develop resistance. Recently, nab-paclitaxel (n-PTX) has been added to the arsenal of first-line therapies, and the combination of gemcitabine and n-PTX has modestly prolonged median overall survival. However, patients almost invariably succumb to the disease, and little is known about the mechanisms underlying n-PTX resistance. Using the conditionally reprogrammed (CR) cell approach, we established and verified continuously growing cell cultures from treatment-naïve patients with PDAC. To study the mechanisms of primary drug resistance, nab-paclitaxel-resistant (n-PTX-R) cells were generated from primary cultures and drug resistance was verified in vivo, both in zebrafish and in athymic nude mouse xenograft models. Molecular analyses identified the sustained induction of c-MYC in the n-PTX-R cells. Depletion of c-MYC restored n-PTX sensitivity, as did treatment with either the MEK inhibitor, trametinib, or a small-molecule activator of protein phosphatase 2a. IMPLICATIONS: The strategies we have devised, including the patient-derived primary cells and the unique, drug-resistant isogenic cells, are rapid and easily applied in vitro and in vivo platforms to better understand the mechanisms of drug resistance and for defining effective therapeutic options on a patient by patient basis

Conditional <i>Klf6</i> inactivation in vivo causes selective loss of differentiating oligodendrocytes.

<p><b>(A–F)</b> Confocal and morphometric analysis of oligodendrocyte numbers in developing <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and control <i>Klf6</i>^<i>fl/fl</i> spinal cords. Panel <b>(A)</b> shows samples from E12.5, immunostained for Olig2 and either Mnr (upper panels) or NeuN (lower panels). At E12.5, Olig2⁺ numbers in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and control samples are identical, and no differences are seen in neuronal markers. In controls, Olig2⁺ cell numbers then increase from E16.5 through P14, but no increase is seen in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> samples <b>(B–D)</b>. Areas outlined in panel <b>(B)</b> are shown at higher magnification, inset. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s006" target="_blank">S4B Fig</a>. At P1, myelin proteins are expressed in controls, but not in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> spinal cords <b>(C)</b>. <b>(E,F)</b> Analysis of stage-specific markers. Early events in differentiation, such as Nkx2.2 co-expression at E14.5, occur normally in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> mice, but differentiating cells are selectively lost at subsequent timepoints <b>(E)</b>. In contrast, numbers of Olig2⁺Sox9⁺ OLP remain identical to controls <b>(F)</b>. There are no changes in Mnr⁺ motor neurons, which share the same origin as ventral OLP in the pMN domain (<b>A</b> upper panels, <b>B, G</b>). <b>(H,I)</b> Selective loss of differentiating cells is associated with increased apoptosis <b>(H)</b>, but OLP proliferation is unaffected <b>(I)</b>. <b>(J–L)</b> Postnatal stage-specific analysis confirms absence of differentiating (Apc⁺) oligodendrocytes from <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> mice <b>(J,L)</b>, whereas OLP (Olig2⁺Ng2⁺) numbers are comparable to controls <b>(K,L)</b>. Representative cells are arrowed. <b>(M)</b> Analysis of <i>NG2creER</i>^<i>–</i>:<i>Klf6</i>^<i>fl/fl</i> mice, in which <i>Klf6</i> inactivation is inducibly targeted to OLP. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s006" target="_blank">S4C and S4D Fig</a>. Similar to <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> embryos, these mice also display selective loss of differentiating oligodendrocytes. Data are mean ± SEM. Statistics, <b>(D–I,L)</b> ANOVA plus Bonferroni post test, <b>(M)</b> Student’s <i>t</i> test, <i>***p <</i> 0.001. Scale: <b>(A,B)</b> 100 μm, inset 20 μm, <b>(C)</b> 250 μm, <b>(J,K)</b> 10 μm. Data shown are from lumbar sections of two to six mice per genotype per timepoint. Thoracic sections showed compatible findings. Individual values are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s002" target="_blank">S1 Data</a>.</p

Klf6 is induced by gp130-Stat3 signals and is required for CNS myelination.

<p><b>(A)</b> Stage-specific markers in differentiation. Following specification to the Olig2⁺ oligodendrocyte lineage, oligodendrocyte progenitors (OLP) express Ng2, Sox9, and Pdgfrα. Pro-myelinating signals induce differentiation to immature oligodendrocytes (iOL), marked by co-expression of Nkx2.2 and acquisition of Apc and O4. These undergo terminal differentiation to mature OL, which express myelin proteins. <b>(B)</b> Klf/Sp family responses to the pro-myelinating factors Cntf (100ng/ml) and T3 (40ng/ml), as determined by quantitative PCR (qPCR). <b>(C)</b> Immunoblotting for Klf6 in Oli-neu cultures pretreated with 0–1,000 nM Jak Inhibitor I 2 h, then exposed to 100 ng/ml Cntf 1 h. <b>(D)</b> Confocal imaging for Klf6 and the gp130 effector Stat3 in mouse OLP exposed to 100 ng/ml Cntf or vehicle 1 h. Cntf upregulation of Klf6 is associated with translocation to the nucleus, where it colocalizes with Stat3. <b>(E,F)</b> Klf6 expression visualized via confocal imaging in vivo. <b>(E)</b> In the postnatal CNS (spinal cord shown), immunoreactivity is heterogeneous in Olig2⁺ cells and more homogeneous in astrocytes (Gfap⁺) and neurons (NeuN⁺). Boxes and arrows highlight representative cells. <b>(F)</b> Klf6 is highly expressed (arrowheads) in OLP (left panel). In contrast, expression is lower (arrowhead) or undetectable (asterisks) in more mature Apc⁺ iOL (center panel), and Klf6 is not seen in mature Mag⁺ cells (right panel). <b>(G)</b> Still from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s015" target="_blank">S1 Video</a> comparing a P11 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> mutant (cko, right) with sex-matched <i>Klf6</i>^<i>fl/fl</i> littermate (ctrl, left). The mutant is ataxic. <b>(H)</b> CNS white matter tracts in P14 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> mice display hypomyelination (arrowed), whereas <i>mGfapCre</i>:<i>Klf6</i>^<i>fl/fl</i> mice and <i>Klf6</i>^<i>fl/fl</i> littermate controls show no gross abnormalities. Brains are shown at the same magnification (scale bar, upper right). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s003" target="_blank">S1 Fig</a>. <b>(I,J)</b> Confocal analysis of lumbar spinal cords of P14 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and <i>mGfapCre</i>:<i>Klf6</i>^<i>fl/fl</i> mice and <i>Klf6</i>^<i>fl/fl</i> controls. Myelin proteins are almost absent from <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> spinal cord, whereas the peripheral nervous system (PNS) appears normal (arrowed). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s004" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s005" target="_blank">S3 Figs</a>. <b>(K,L)</b> Electron micrographs of P14 <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> and <i>Klf6</i>^<i>fl/fl</i> spinal cords and optic nerves. Almost no myelin sheaths are present in <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> CNS samples. <b>(M)</b> Gene ontology of BeadArray profiling of P1 CNS from <i>Olig1Cre</i>:<i>Klf6</i>^<i>fl/fl</i> pups and sex-matched <i>Klf6</i>^<i>fl/fl</i> littermates. The five most significant results are shown for physiologic and disease relevance. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s006" target="_blank">S4 Fig</a>. Data are mean ± SEM. Statistics: <b>(B,J)</b> ANOVA plus Bonferroni test, *<i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001. Data are representative of two to four mice per genotype (for confocal imaging data) or three mice per genotype (for electron microscopy data). Scale, <b>(A)</b> 5 μm, <b>(E)</b> 20 μm, <b>(F)</b> 10 μm, <b>(H)</b> 3 mm <b>(I)</b> 150 μm, inset 15 μm. Magnifications, <b>(K)</b> x5,000, inset x20,000, <b>(L)</b> x3,000. Individual values are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s002" target="_blank">S1 Data</a>.</p

RNA sequencing identifies Klf6-dependence of gp130-driven transcriptional patterns.

<p><b>(A)</b> Overview of approach used to define key Klf6 effectors. Initial RNA-seq analysis of primary mouse cultures identifies 212 unique Klf6-regulated transcripts in OLP and 91 in iOL, of which 40 are shared. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s009" target="_blank">S1</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s011" target="_blank">S3</a> Tables and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s007" target="_blank">S5A and S5B Fig</a>. <b>(B,C)</b> Results of functional inference <b>(B)</b> and GO analysis <b>(C)</b> of RNA-seq data, from primary OLP (red), iOL (blue), or both (purple). In <b>(B)</b>, numbers of Klf6-regulated genes are indicated for each function. Implicated signaling pathways in OLP are presented as a smaller Venn diagram, inset. <b>(D)</b> Examples of qPCR validation of RNA-seq data for select OLP, iOL, and shared genes. A larger cohort of validation data is presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s007" target="_blank">S5C Fig</a>. <b>(E)</b> Gp130 sensitivity of select validated differentially expressed transcripts. Results are shown from Oli-neu cells treated with Cntf (100 ng/ml) for up to 24 h. Colored areas indicate the time period before peak response of Klf6 to Cntf. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s007" target="_blank">S5D Fig</a>. <b>(F)</b> Klf6-dependence of Cntf-induced responses. qPCR analysis of <i>Klf6-</i>silenced and control Oli-neu cells treated with Cntf for up to 24 h. Cntf sensitivity of differentially expressed targets is blunted in <i>Klf6</i>-silenced samples. Note also that some genes are Cntf-independent but Klf6-dependent during differentiation. Data are mean ± SEM. Statistics, <b>(D)</b> Student’s <i>t</i> test, <b>(F)</b> Two-way ANOVA plus Bonferroni post-test, <i>*p <</i> 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. Data are representative of two to three independent studies. RNA-seq data are presented in full in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s009" target="_blank">S1</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s011" target="_blank">S3</a> Tables and are available on the GEO website (<a href="http://www.ncbi.nlm.nih.gov/geo/" target="_blank">http://www.ncbi.nlm.nih.gov/geo/</a>) (Accession number GSE79245). Individual values for all other quantifications are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002467#pbio.1002467.s002" target="_blank">S1 Data</a>.</p