The PP2A-Integrator-CDK9 axis fine-tunes transcription and can be targeted therapeutically in cancer.

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.

Piecing Together a Broken Tumor Suppressor Phosphatase for Cancer Therapy.

Members of the PP2A family of serine/threonine phosphatases are important human tumor suppressor genes. Unlike most tumor suppressors, they are rarely mutated/deleted, but rather are impaired by "inhibitor proteins." Two papers in this issue of Cell show how some phenothiazine derivatives reactivate specific PP2A isozymes with potential benefit in cancer and other diseases.

SHARPIN S146 phosphorylation mediates ARP2/3 interaction, cancer cell invasion and metastasis.

SHARPIN is involved in several cellular processes and promotes cancer progression. However, how the choice between different functions of SHARPIN is post-translationally regulated is unclear. Here, we characterized SHARPIN phosphorylation by mass spectrometry and in vitro kinase assay. Focusing on S131 and S146, we demonstrate that they have a role in SHARPIN-ARP2/3 complex interaction, but play no role in integrin inhibition or LUBAC activation. Consistent with its novel role in ARP2/3 regulation, S146 phosphorylation of SHARPIN promoted lamellipodia formation. We also demonstrate that SHARPIN S146 phosphorylation-mediated ARP2/3 interaction is sensitive to inhibition of ERK1/2 or reactivation of protein phosphatase 2A (PP2A). Notably, CRISPR/Cas9-mediated knockout of SHARPIN abrogated three-dimensional (3D) invasion of several cancer cell lines. The 3D invasion of cancer cells was rescued by overexpression of the wild-type SHARPIN, but not by SHARPIN S146A mutant. Finally, we demonstrate that inhibition of phosphorylation at S146 significantly reduces in vivo metastasis in a zebrafish model. Collectively, these results map SHARPIN phosphorylation sites and identify S146 as a novel phosphorylation switch defining ARP2/3 interaction and cancer cell invasion. This article has an associated First Person interview with the first author of the paper.

CIP2A deficiency promotes depression-like behaviors in mice through inhibition of dendritic arborization.

Major depressive disorder (MDD) is a severe mental illness. Decreased brain plasticity and dendritic fields have been consistently found in MDD patients and animal models; however, the underlying molecular mechanisms remain to be clarified. Here, we demonstrate that the deletion of cancerous inhibitor of PP2A (CIP2A), an endogenous inhibitor of protein phosphatase 2A (PP2A), leads to depression-like behaviors in mice. Hippocampal RNA sequencing analysis of CIP2A knockout mice shows alterations in the PI3K-AKT pathway and central nervous system development. In primary neurons, CIP2A stimulates AKT activity and promotes dendritic development. Further analysis reveals that the effect of CIP2A in promoting dendritic development is dependent on PP2A-AKT signaling. In vivo, CIP2A deficiency-induced depression-like behaviors and impaired dendritic arborization are rescued by AKT activation. Decreased CIP2A expression and impaired dendrite branching are observed in a mouse model of chronic unpredictable mild stress (CUMS). Indicative of clinical relevance to humans, CIP2A expression is found decreased in transcriptomes from MDD patients. In conclusion, we discover a novel mechanism that CIP2A deficiency promotes depression through the regulation of PP2A-AKT signaling and dendritic arborization.

Cancer stem cell phosphatases.

Cancer stem cells (CSCs) are involved in the initiation and progression of human malignancies by enabling cancer tissue self-renewal capacity and constituting the therapy-resistant population of tumor cells. However, despite the exhausting characterization of CSC genetics, epigenetics, and kinase signaling, eradication of CSCs remains an unattainable goal in most human malignancies. While phosphatases contribute equally with kinases to cellular phosphoregulation, our understanding of phosphatases in CSCs lags severely behind our knowledge about other CSC signaling mechanisms. Many cancer-relevant phosphatases have recently become druggable, indicating that further understanding of the CSC phosphatases might provide novel therapeutic opportunities. This review summarizes the current knowledge about fundamental, but yet poorly understood involvement of phosphatases in the regulation of major CSC signaling pathways. We also review the functional roles of phosphatases in CSC self-renewal, cancer progression, and therapy resistance; focusing particularly on hematological cancers and glioblastoma. We further discuss the small molecule targeting of CSC phosphatases and their therapeutic potential in cancer combination therapies.

Phosphoproteome and drug-response effects mediated by the three protein phosphatase 2A inhibitor proteins CIP2A, SET, and PME-1.

Protein phosphatase 2A (PP2A) critically regulates cell signaling and is a human tumor suppressor. PP2A complexes are modulated by proteins such as cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) that often are deregulated in cancers. However, how they impact cellular phosphorylation and how redundant they are in cellular regulation is poorly understood. Here, we conducted a systematic phosphoproteomics screen for phosphotargets modulated by siRNA-mediated depletion of CIP2A, PME-1, and SET (to reactivate PP2A) or the scaffolding A-subunit of PP2A (PPP2R1A) (to inhibit PP2A) in HeLa cells. We identified PP2A-modulated targets in diverse cellular pathways, including kinase signaling, cytoskeleton, RNA splicing, DNA repair, and nuclear lamina. The results indicate nonredundancy among CIP2A, PME-1, and SET in phosphotarget regulation. Notably, PP2A inhibition or reactivation affected largely distinct phosphopeptides, introducing a concept of nonoverlapping phosphatase inhibition- and activation-responsive sites (PIRS and PARS, respectively). This phenomenon is explained by the PPP2R1A inhibition impacting primarily dephosphorylated threonines, whereas PP2A reactivation results in dephosphorylation of clustered and acidophilic sites. Using comprehensive drug-sensitivity screening in PP2A-modulated cells to evaluate the functional impact of PP2A across diverse cellular pathways targeted by these drugs, we found that consistent with global phosphoproteome effects, PP2A modulations broadly affect responses to more than 200 drugs inhibiting a broad spectrum of cancer-relevant targets. These findings advance our understanding of the phosphoproteins, pharmacological responses, and cellular processes regulated by PP2A modulation and may enable the development of combination therapies.

Cancer cell line microarray as a novel screening method for identification of radioresistance biomarkers in head and neck squamous cell carcinoma.

BACKGROUND: Currently, no clinically useful biomarkers for radioresistance are available in head and neck squamous cell carcinoma (HNSCC). This study assesses the usefulness of Cell Line Microarray (CMA) method to enhance immunohistochemical screening of potential immunohistochemical biomarkers for radioresistance in HNSCC cell lines. METHODS: Twenty-nine HNSCC cell lines were cultured, cell pellets formalin-fixed, paraffin-embedded, and arrayed. Radioresistance features of the cell lines were combined to immunohistochemical stains for p53, NDFIP1, EGFR, stem cell marker Oct4, and PP2A inhibitor CIP2A. RESULTS: Expression of p53, EGFR or CIP2A did not indicate intrinsic radioresistance in vitro. Stem cell marker Oct4 nuclear positivity and NDFIP1 nuclear positivity was correlated with increased intrinsic radioresistance. CONCLUSION: The usefulness of CMA in analysis of HNSCC cell lines and discovery of biomarkers is demonstrated. CMA is very well adapted to both testing of antibodies in a large panel of cell lines as well as correlating staining results with other cell line characteristics. In addition, CMA-based antibody screening proved an efficient and relatively simple method to identify potential radioresistance biomarkers in HNSCC.

Enhanced expression of MycN/CIP2A drives neural crest toward a neural stem cell-like fate: Implications for priming of neuroblastoma.

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 MycN is expressed together with phosphorylation-stabilizing factor CIP2A 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 MycN/CIP2A 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 MycN, its paralogue cMyc 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.

Thioridazine inhibits autophagy and sensitizes glioblastoma cells to temozolomide.

Glioblastoma multiforme (GBM) has a poor prognosis with an overall survival of 14-15 months after surgery, radiation and chemotherapy using temozolomide (TMZ). A major problem is that the tumors acquire resistance to therapy. In an effort to improve the therapeutic efficacy of TMZ, we performed a genome-wide RNA interference (RNAi) synthetic lethality screen to establish a functional gene signature for TMZ sensitivity in human GBM cells. We then queried the Connectivity Map database to search for drugs that would induce corresponding changes in gene expression. By this approach we identified several potential pharmacological sensitizers to TMZ, where the most potent drug was the established antipsychotic agent Thioridazine, which significantly improved TMZ sensitivity while not demonstrating any significant toxicity alone. Mechanistically, we show that the specific chemosensitizing effect of Thioridazine is mediated by impairing autophagy, thereby preventing adaptive metabolic alterations associated with TMZ resistance. Moreover, we demonstrate that Thioridazine inhibits late-stage autophagy by impairing fusion between autophagosomes and lysosomes. Finally, Thioridazine in combination with TMZ significantly inhibits brain tumor growth in vivo, demonstrating the potential clinical benefits of compounds targeting the autophagy-lysosome pathway. Our study emphasizes the feasibility of exploiting drug repurposing for the design of novel therapeutic strategies for GBM.

Oncoprotein CIP2A is stabilized via interaction with tumor suppressor PP2A/B56.

Protein phosphatase 2A (PP2A) is a critical human tumor suppressor. Cancerous inhibitor of PP2A (CIP2A) supports the activity of several critical cancer drivers (Akt, MYC, E2F1) and promotes malignancy in most cancer types via PP2A inhibition. However, the 3D structure of CIP2A has not been solved, and it remains enigmatic how it interacts with PP2A. Here, we show by yeast two-hybrid assays, and subsequent validation experiments, that CIP2A forms homodimers. The homodimerization of CIP2A is confirmed by solving the crystal structure of an N-terminal CIP2A fragment (amino acids 1-560) at 3.0 Å resolution, and by subsequent structure-based mutational analyses of the dimerization interface. We further describe that the CIP2A dimer interacts with the PP2A subunits B56α and B56γ. CIP2A binds to the B56 proteins via a conserved N-terminal region, and dimerization promotes B56 binding. Intriguingly, inhibition of either CIP2A dimerization or B56α/γ expression destabilizes CIP2A, indicating opportunities for controlled degradation. These results provide the first structure-function analysis of the interaction of CIP2A with PP2A/B56 and have direct implications for its targeting in cancer therapy.

Relevance Rank Platform (RRP) for Functional Filtering of High Content Protein-Protein Interaction Data.

High content protein interaction screens have revolutionized our understanding of protein complex assembly. However, one of the major challenges in translation of high content protein interaction data is identification of those interactions that are functionally relevant for a particular biological question. To address this challenge, we developed a relevance ranking platform (RRP), which consist of modular functional and bioinformatic filters to provide relevance rank among the interactome proteins. We demonstrate the versatility of RRP to enable a systematic prioritization of the most relevant interaction partners from high content data, highlighted by the analysis of cancer relevant protein interactions for oncoproteins Pin1 and PME-1. We validated the importance of selected interactions by demonstration of PTOV1 and CSKN2B as novel regulators of Pin1 target c-Jun phosphorylation and reveal previously unknown interacting proteins that may mediate PME-1 effects via PP2A-inhibition. The RRP framework is modular and can be modified to answer versatile research problems depending on the nature of the biological question under study. Based on comparison of RRP to other existing filtering tools, the presented data indicate that RRP offers added value especially for the analysis of interacting proteins for which there is no sufficient prior knowledge available. Finally, we encourage the use of RRP in combination with either SAINT or CRAPome computational tools for selecting the candidate interactors that fulfill the both important requirements, functional relevance, and high confidence interaction detection.

Potential role for inhibition of protein phosphatase 2A tumor suppressor in salivary gland malignancies.

The aetiology and pathogenesis of salivary gland malignancies remain unknown. To reveal novel molecular factors behind the development of salivary gland cancer, we performed gene expression analyses from Smgb-Tag mouse salivary gland samples. The overall purpose was to apply these results for clinical use to find new approaches for both possible therapeutic targets and more accurate diagnostic tools. Smgb-Tag mouse strain, in which salivary neoplasms arise through a dysplastic phase in submandibular glands, was investigated using genome-wide microarray expression analysis, ingenuity pathway analysis, RT-PCR, and immunohistochemistry. Thirty-eight human salivary gland adenoid cystic carcinoma samples were investigated using immunohistochemistry for validation purposes. Our genome-wide study showed that Ppp2r1b, a PP2A subunit encoding tumor suppressor gene, is underexpressed in submandibular gland tumors of Smgb-Tag mice. mTOR signaling pathway was significantly enriched and mTOR linked PP2A subunit gene B55 gamma was significantly underexpressed in the analyses. Furthermore, parallel immunohistochemical analysis of three PP2A inhibitors demonstrated that two PP2A inhibitors, CIP2A and SET, are highly expressed in both dysplastic and adenocarcinomatous tumors of the Smgb-Tag mice. In addition, all 38 investigated human salivary adenoid cystic carcinoma samples stained positively for CIP2A and most for SET. Finally, p-S6 staining showed activation of mTOR pathway in human adenoid cystic carcinoma samples. Our results suggest that PP2A inhibition either via PP2A subunit underexpression or PP2A inhibitor overexpression play an important role in the formation of salivary gland malignancy, potentially due to mTOR signaling activation.

Regulation of protein phosphatase 2A (PP2A) tumor suppressor function by PME-1.

Protein phosphatase 2A (PP2A) plays a major role in maintaining cellular signaling homeostasis by dephosphorylation of a variety of signaling proteins and acts as a tumor suppressor. Protein phosphatase methylesterase-1 (PME-1) negatively regulates PP2A activity by highly complex mechanisms that are reviewed here. Importantly, recent studies have shown that PME-1 promotes oncogenic MAPK/ERK and AKT pathway activities in various cancer types. In human glioma, high PME-1 expression correlates with tumor progression and kinase inhibitor resistance. We discuss the emerging cancer-associated function of PME-1 and its potential clinical relevance.

Copy number increase of oncoprotein CIP2A is associated with poor patient survival in human head and neck squamous cell carcinoma.

BACKGROUND: CIP2A, an inhibitor of PP2A tumour suppressor function, is a widely overexpressed biomarker of aggressive disease and poor therapy response in multiple human cancer types. METHODS: CIP2A and DPPA4 copy number alterations and expression were analysed by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) in different cell lines and a tissue microarray of 52 HNSCC patients. Results were correlated with patient survival and other clinicopathological data. RESULTS: CIP2A and DPPA4 copy number increase occurred at a relatively high frequency in human HNSCC patient samples. CIP2A but not DPPA4 FISH status was significantly associated with patient survival. CIP2A detection by combining IHC with FISH yielded superior resolution in the prognostication of HNSCC. CONCLUSIONS: CIP2A copy number increase is associated with poor patient survival in human HNSCC. We suggest that the reliability and prognostic value of CIP2A detection can be improved by performing FISH analysis to CIP2A IHC positive tumours.

Identification of protein interactions involved in cellular signaling.

Protein-protein interactions drive biological processes. They are critical for all intra- and extracellular functions, and the technologies to analyze them are widely applied throughout the various fields of biological sciences. This study takes an in-depth view of some common principles of cellular regulation and provides a detailed account of approaches required to comprehensively map signaling protein-protein interactions in any particular cellular system or condition. We provide a critical review of the benefits and disadvantages of the yeast two-hybrid method and affinity purification coupled with mass spectrometric procedures for identification of signaling protein-protein interactions. In particular, we emphasize the quantitative and qualitative differences between tandem affinity and one-step purification (such as FLAG and Strep tag) methods. Although applicable to all types of interaction studies, a special section is devoted in this review to aspects that should be considered when attempting to identify signaling protein interactions that often are transient and weak by nature. Finally, we discuss shotgun and quantitative information that can be gleaned by MS-coupled methods for analysis of multiprotein complexes.

CIP2A coordinates phosphosignaling, mitosis, and the DNA damage response.

Human cancers share requirements for phosphorylation-dependent signaling, mitotic hyperactivity, and survival after DNA damage. The oncoprotein CIP2A (cancerous inhibitor of PP2A) can coordinate all these cancer cell characteristics. In addition to controlling cancer cell phosphoproteomes via inhibition of protein phosphatase PP2A, CIP2A directly interacts with the DNA damage protein TopBP1 (topoisomerase II-binding protein 1). Consequently, CIP2A allows DNA-damaged cells to enter mitosis and is essential for mitotic cells that are defective in homologous recombination (HR)-mediated DNA repair (e.g., BRCA mutants). The CIP2A-TopBP1 complex is also important for clustering fragmented chromosomes at mitosis. Clinically, CIP2A is a disease driver for basal-like triple-negative breast cancer (BL-TNBC) and a promising cancer therapy target across many cancer types.

DUSP6 inhibition overcomes neuregulin/HER3-driven therapy tolerance in HER2+ breast cancer.

Despite clinical benefits of tyrosine kinase inhibitors (TKIs) in cancer, most tumors can reactivate proliferation under TKI therapy. Here we present transcriptional profiling of HER2+ breast cancer cells transitioning from dormant drug tolerant cells to re-proliferating cells under continuous HER2 inhibitor (HER2i) therapy. Focusing on phosphatases, expression of dual-specificity phosphatase DUSP6 was found inhibited in dormant cells, but strongly induced upon regrowth. DUSP6 expression also selectively associated with poor patient survival in HER2+ breast cancers. DUSP6 overexpression conferred apoptosis resistance, whereas its pharmacological blockade prevented therapy tolerance development under HER2i therapy. DUSP6 targeting also synergized with clinically used HER2i combination therapies. Mechanistically DUSP6 is a positive regulator of HER3 expression, and its impact on HER2i tolerance was mediated by neuregulin-HER3 axis. In vivo, genetic targeting of DUSP6 reduced tumor growth in brain metastasis model, whereas its pharmacological targeting induced synthetic lethal therapeutic effect in combination with HER2i. Collectively this work demonstrates that DUSP6 drives escape from HER2i-induced dormancy, and that DUSP6 is a druggable target to overcome HER3-driven TKI resistance.

Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

Cancer cells undergo major epigenetic alterations and transcriptomic changes, including ectopic expression of tissue- and cell-type-specific genes. Here, we show that the germline-specific RNA helicase DDX4 forms germ-granule-like cytoplasmic ribonucleoprotein granules in various human tumors, but not in cultured cancer cells. These cancerous DDX4 complexes contain RNA-binding proteins and splicing regulators, including many known germ granule components. The deletion of DDX4 in cancer cells induces transcriptomic changes and affects the alternative splicing landscape of a number of genes involved in cancer growth and invasiveness, leading to compromised capability of DDX4-null cancer cells to form xenograft tumors in immunocompromised mice. Importantly, the occurrence of DDX4 granules is associated with poor survival in patients with head and neck squamous cell carcinoma and higher histological grade of prostate cancer. Taken together, these results show that the germ-granule-resembling cancerous DDX4 granules control gene expression and promote malignant and invasive properties of cancer cells.

Identification and regulation of a stage-specific stem cell niche enriched by Nanog-positive spermatogonial stem cells in the mouse testis.

The ability of spermatogonial stem cells to acquire embryonic stem cell (ESC) properties in vitro has recently been of great interest. However, studies focused on the in vivo regulation of testicular stem cells have been hampered because the exact anatomical location of these cells is unknown. Moreover, no specialized stem cell niche substructure has been identified in the mammalian testis thus far. It has also been unclear whether the adult mammalian testis houses pluripotent stem cells or whether pluripotency can be induced only in vitro. Here, we demonstrate, for the first time, the existence of a Nanog-positive spermatogonial stem cell subpopulation located in stage XII of the mouse seminiferous epithelial cycle. The efficiency of the cells from seminiferous tubules with respect to prolonged pluripotent gene expression was correlated directly with stage-specific expression levels of Nanog and Oct4, demonstrating the previously unknown stage-specific regulation of undifferentiated spermatogonia (SPG). Testicular Nanog expression marked a radioresistant spermatogonial subpopulation, supporting its stem cell nature. Furthermore, we demonstrated that p21 acts as an upstream regulator of Nanog in SPG and mouse ESCs, and our results demonstrate that promyelocytic leukemia zinc finger is a specific marker of progenitor SPG. Additionally, we describe a novel method to cultivate Nanog-positive SPG in vitro. This study demonstrates the existence and location of a previously unknown stage-specific spermatogonial stem cell niche and reports the regulation of radioresistant spermatogonial stem cells.

Inhibition of adaptive therapy tolerance in cancer: is triplet mitochondrial targeting the key?

Targeted therapies have become a mainstay in the treatment of cancer, but their long-term efficacy is compromised by acquired drug resistance. Acquired therapy resistance develops via two phases-first through adaptive development of nongenetic drug tolerance, which is followed by stable resistance through the acquisition of genetic mutations. Drug tolerance has been described in practically all clinical cancer treatment contexts, and detectable drug-tolerant tumors are highly associated with treatment relapse and poor survival. Thereby, novel therapeutic strategies are needed to overcome cancer therapy tolerance. Recent studies have identified a critical role of mitochondrial mechanisms in defining cancer cell sensitivity to targeted therapies and the surprising effects of established cancer therapies on mitochondria. Here, these recent studies are reviewed emphasizing an emerging concept of triplet therapies including three compounds targeting different cancer cell vulnerabilities but including at least one compound that targets the mitochondria. These mitochondria-targeting triplet therapies have very promising preclinical effects in overcoming cancer therapy tolerance. Potential strategies of how to overcome challenges in the clinical translation of mitochondria-targeting triplet therapies are also discussed.