RESUMO
Loss of the tumor suppressive activity of the protein phosphatase 2A (PP2A) is associated with cancer, but the underlying molecular mechanisms are unclear. PP2A holoenzyme comprises a heterodimeric core, a scaffolding A subunit and a catalytic C subunit, and one of over 20 distinct substrate-directing regulatory B subunits. Methylation of the C subunit regulates PP2A heterotrimerization, affecting B subunit binding and substrate specificity. Here, we report that the leucine carboxy methyltransferase (LCMT1), which methylates the L309 residue of the C subunit, acts as a suppressor of androgen receptor (AR) addicted prostate cancer (PCa). Decreased methyl-PP2A-C levels in prostate tumors is associated with biochemical recurrence and metastasis. Silencing LCMT1 increases AR activity and promotes castration-resistant prostate cancer growth. LCMT1-dependent methyl-sensitive AB56αCme heterotrimers target AR and its critical coactivator MED1 for dephosphorylation, resulting in the eviction of the AR-MED1 complex from chromatin and loss of target gene expression. Mechanistically, LCMT1 is regulated by S6K1-mediated phosphorylation-induced degradation requiring the ß-TRCP, leading to acquired resistance to anti-androgens. Finally, feedforward stabilization of LCMT1 by small molecule activator of phosphatase (SMAP) results in attenuation of AR-signaling and tumor growth inhibition in anti-androgen refractory PCa. These findings highlight methyl-PP2A-C as a prognostic marker and that the loss of LCMT1 is a major determinant in AR-addicted PCa, suggesting therapeutic potential for AR degraders or PP2A modulators in prostate cancer treatment.
Assuntos
Neoplasias da Próstata , Proteína Fosfatase 2 , Humanos , Masculino , Antagonistas de Androgênios , Leucina , Metiltransferases , Próstata , Neoplasias da Próstata/genética , Proteína Fosfatase 2/genéticaRESUMO
Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, which encodes the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30% to 40% of USC cases, the clinical actionability of these mutations has not been studied. Using a high-throughput screening approach, we showed that mutations in Aα results in synthetic lethality following treatment with inhibitors of ribonucleotide reductase (RNR). In vivo, multiple models of Aα mutant uterine serous tumors were sensitive to clofarabine, an RNR inhibitor (RNRi). Aα-mutant cells displayed impaired checkpoint signaling upon RNRi treatment and subsequently accumulated more DNA damage than wild-type (WT) cells. Consistently, inhibition of PP2A activity using LB-100, a catalytic inhibitor, sensitized WT USC cells to RNRi. Analysis of The Cancer Genome Atlas data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of patients with USC harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNRi are not routinely used for uterine cancers, a retrospective analysis of patients treated with gemcitabine as a second- or later-line therapy revealed a trend for improved outcomes in patients with USC treated with RNRi gemcitabine compared with patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC. SIGNIFICANCE: A drug repurposing screen identifies synthetic lethal interactions in PP2A-deficient uterine serous carcinoma, providing potential therapeutic avenues for treating this deadly endometrial cancer.
Assuntos
Cistadenocarcinoma Seroso/genética , Proteína Fosfatase 2/genética , Ribonucleotídeo Redutases/genética , Mutações Sintéticas Letais/genética , Neoplasias Uterinas/genética , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Clofarabina/farmacologia , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/metabolismo , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteína Fosfatase 2/metabolismo , Ratos Sprague-Dawley , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/metabolismo , Mutações Sintéticas Letais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Neoplasias Uterinas/tratamento farmacológico , Neoplasias Uterinas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Fms-like tyrosine-like kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients and is associated with short disease-free survival. FLT3 inhibitor efficacy is limited and transient but may be enhanced by multitargeting of FLT3-ITD signaling pathways. FLT3-ITD drives both STAT5-dependent transcription of oncogenic Pim-1 kinase and inactivation of the tumor-suppressor protein phosphatase 2A (PP2A), and FLT3-ITD, Pim-1, and PP2A all regulate the c-Myc oncogene. We studied mechanisms of action of cotreatment of FLT3-ITD-expressing cells with FLT3 inhibitors and PP2A-activating drugs (PADs), which are in development. PADs, including FTY720 and DT-061, enhanced FLT3 inhibitor growth suppression and apoptosis induction in FLT3-ITD-expressing cell lines and primary AML cells in vitro and MV4-11 growth suppression in vivo PAD and FLT3 inhibitor cotreatment independently downregulated c-Myc and Pim-1 protein through enhanced proteasomal degradation. c-Myc and Pim-1 downregulation was preceded by AKT inactivation, did not occur in cells expressing myristoylated (constitutively active) AKT1, and could be induced by AKT inhibition. AKT inactivation resulted in activation of GSK-3ß, and GSK-3ß inhibition blocked downregulation of both c-Myc and Pim-1 by PAD and FLT3 inhibitor cotreatment. GSK-3ß activation increased c-Myc proteasomal degradation through c-Myc phosphorylation on T58; infection with c-Myc with T58A substitution, preventing phosphorylation, blocked downregulation of c-Myc by PAD and FLT3 inhibitor cotreatment. GSK-3ß also phosphorylated Pim-1L/Pim-1S on S95/S4. Thus, PADs enhance efficacy of FLT3 inhibitors in FLT3-ITD-expressing cells through a novel mechanism involving AKT inhibition-dependent GSK-3ß-mediated increased c-Myc and Pim-1 proteasomal degradation.
Assuntos
Genes myc/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Animais , Proliferação de Células , Humanos , Camundongos , Camundongos Endogâmicos NOD , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , TransfecçãoRESUMO
We have created the immunodeficient SRG rat, a Sprague-Dawley Rag2/Il2rg double knockout that lacks mature B cells, T cells, and circulating NK cells. This model has been tested and validated for use in oncology (SRG OncoRat®). The SRG rat demonstrates efficient tumor take rates and growth kinetics with different human cancer cell lines and PDXs. Although multiple immunodeficient rodent strains are available, some important human cancer cell lines exhibit poor tumor growth and high variability in those models. The VCaP prostate cancer model is one such cell line that engrafts unreliably and grows irregularly in existing models but displays over 90% engraftment rate in the SRG rat with uniform growth kinetics. Since rats can support much larger tumors than mice, the SRG rat is an attractive host for PDX establishment. Surgically resected NSCLC tissue from nine patients were implanted in SRG rats, seven of which engrafted and grew for an overall success rate of 78%. These developed into a large tumor volume, over 20,000 mm3 in the first passage, which would provide an ample source of tissue for characterization and/or subsequent passage into NSG mice for drug efficacy studies. Molecular characterization and histological analyses were performed for three PDX lines and showed high concordance between passages 1, 2 and 3 (P1, P2, P3), and the original patient sample. Our data suggest the SRG OncoRat is a valuable tool for establishing PDX banks and thus serves as an alternative to current PDX mouse models hindered by low engraftment rates, slow tumor growth kinetics, and multiple passages to develop adequate tissue banks.
Assuntos
Subunidade gama Comum de Receptores de Interleucina/genética , Neoplasias Experimentais/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Deleção de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Experimentais/genética , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de Xenoenxerto/normasRESUMO
Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.
Assuntos
Cromograninas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Neoplasias Pulmonares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteína Fosfatase 2/metabolismo , Proteômica/métodos , Carcinoma de Pequenas Células do Pulmão/metabolismo , Células A549 , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cromograninas/genética , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteína Fosfatase 2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The tumor suppressor protein phosphatase 2A (PP2A) is a serine/threonine phosphatase whose activity is inhibited in most human cancers. One of the best-characterized PP2A substrates is MYC proto-oncogene basic helix-loop-helix transcription factor (MYC), whose overexpression is commonly associated with aggressive forms of this disease. PP2A directly dephosphorylates MYC, resulting in its degradation. To explore the therapeutic potential of direct PP2A activation in a diverse set of MYC-driven cancers, here we used biochemical assays, recombinant cell lines, gene expression analyses, and immunohistochemistry to evaluate a series of first-in-class small-molecule activators of PP2A (SMAPs) in Burkitt lymphoma, KRAS-driven non-small cell lung cancer, and triple-negative breast cancer. In all tested models of MYC-driven cancer, the SMAP treatment rapidly and persistently inhibited MYC expression through proteasome-mediated degradation, inhibition of MYC transcriptional activity, decreased cancer cell proliferation, and tumor growth inhibition. Importantly, we generated a series of cell lines expressing PP2A-dependent phosphodegron variants of MYC and demonstrated that the antitumorigenic activity of SMAPs depends on MYC degradation. Collectively, the findings presented here indicate a pharmacologically tractable approach to drive MYC degradation by using SMAPs for the management of a broad range of MYC-driven cancers.
Assuntos
Proteína Fosfatase 2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Supressoras de Tumor/genética , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteólise/efeitos dos fármacos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/química , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The serine/threonine Protein Phosphatase 2A (PP2A) functions as a tumor suppressor by negatively regulating multiple oncogenic signaling pathways. The canonical PP2A holoenzyme comprises a scaffolding subunit (PP2A Aα/ß), which serves as the platform for binding of both the catalytic C subunit and one regulatory B subunit. Somatic heterozygous missense mutations in PPP2R1A, the gene encoding the PP2A Aα scaffolding subunit, have been identified across multiple cancer types, but the effects of the most commonly mutated residue, Arg-183, on PP2A function have yet to be fully elucidated. In this study, we used a series of cellular and in vivo models and discovered that the most frequent Aα R183W mutation formed alternative holoenzymes by binding of different PP2A regulatory subunits compared with wild-type Aα, suggesting a rededication of PP2A functions. Unlike wild-type Aα, which suppressed tumorigenesis, the R183W mutant failed to suppress tumor growth in vivo through activation of the MAPK pathway in RAS-mutant transformed cells. Furthermore, cells expressing R183W were less sensitive to MEK inhibitors. Taken together, our results demonstrate that the R183W mutation in PP2A Aα scaffold abrogates the tumor suppressive actions of PP2A, thereby potentiating oncogenic signaling and reducing drug sensitivity of RAS-mutant cells.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2/genética , Proteínas Recombinantes/genética , Substituição de Aminoácidos , Arginina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Proteínas de Membrana/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Neoplasias/genética , Proteínas do Tecido Nervoso/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteína Fosfatase 2/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Transfecção , Tirosina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Somatic mutation of the protein phosphatase 2A (PP2A) Aα-subunit gene PPP2R1A is highly prevalent in high-grade endometrial carcinoma. The structural, molecular, and biological basis by which the most recurrent endometrial carcinoma-specific mutation site P179 facilitates features of endometrial carcinoma malignancy has yet to be fully determined. Here, we used a series of structural, biochemical, and biological approaches to investigate the impact of the P179R missense mutation on PP2A function. Enhanced sampling molecular dynamics simulations showed that arginine-to-proline substitution at the P179 residue changes the protein's stable conformation profile. A crystal structure of the tumor-derived PP2A mutant revealed marked changes in A-subunit conformation. Binding to the PP2A catalytic subunit was significantly impaired, disrupting holoenzyme formation and enzymatic activity. Cancer cells were dependent on PP2A disruption for sustained tumorigenic potential, and restoration of wild-type Aα in a patient-derived P179R-mutant cell line restored enzyme function and significantly attenuated tumorigenesis and metastasis in vivo. Furthermore, small molecule-mediated therapeutic reactivation of PP2A significantly inhibited tumorigenicity in vivo. These outcomes implicate PP2A functional inactivation as a critical component of high-grade endometrial carcinoma disease pathogenesis. Moreover, they highlight PP2A reactivation as a potential therapeutic strategy for patients who harbor P179R PPP2R1A mutations. SIGNIFICANCE: This study characterizes a highly recurrent, disease-specific PP2A PPP2R1A mutation as a driver of endometrial carcinoma and a target for novel therapeutic development.See related commentary by Haines and Huang, p. 4009.
Assuntos
Neoplasias do Endométrio , Proteína Fosfatase 2/genética , Carcinogênese , Feminino , Humanos , Mutação , Recidiva Local de NeoplasiaRESUMO
Although tyrosine kinase inhibitors (TKIs) have demonstrated significant efficacy in advanced lung adenocarcinoma (LUAD) patients with pathogenic alterations in EGFR, most patients develop acquired resistance to these agents via mechanisms enabling the sustained activation of the PI3K and MAPK oncogenic pathways downstream of EGFR. The tumor suppressor protein phosphatase 2A (PP2A) acts as a negative regulator of these pathways. We hypothesize that activation of PP2A simultaneously inhibits the PI3K and MAPK pathways and represents a promising therapeutic strategy for the treatment of TKI-resistant LUAD. After establishing the efficacy of small molecule activators of PP2A (SMAPs) in a transgenic EGFRL858R model and TKI-sensitive cell lines, we evaluated their therapeutic potential in vitro and in vivo in TKI-resistant models. PP2A activation resulted in apoptosis, significant tumor growth inhibition, and downregulation of PI3K and MAPK pathways. Combination of SMAPs and TKI afatinib resulted in an enhanced effect on the downregulation of the PI3K pathway via degradation of the PP2A endogenous inhibitor CIP2A. An improved effect on tumor growth inhibition was observed in a TKI-resistant xenograft mouse model treated with a combination of both agents. These collective data support the development of PP2A activators for the treatment of TKI-resistant LUAD.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Fosfoproteínas Fosfatases/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Ativadores de Enzimas/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The serine/threonine phosphatase PP2A regulates a vast portion of the phosphoproteome including pathways involved in apoptosis, proliferation and DNA damage response and PP2A inactivation is a vital step in malignant transformation. Many groups have explored the therapeutic venue of combining PP2A reactivation with kinase inhibition to counteract the very changes in tumor suppressors and oncogenes that lead to cancer development. Conversely, inhibition of PP2A to complement chemotherapy and radiation-induced cancer cell death is also an area of active investigation. Here we review the studies that utilize PP2A targeted agents as combination therapy in cancer. A potential role for PP2A in tumor immunity is also highlighted.
Assuntos
Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/fisiologia , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Proliferação de Células/fisiologia , Reparo do DNA/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Terapia de Alvo Molecular/métodos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Neoplasias/terapia , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Subunidades Proteicas/fisiologia , Transdução de Sinais/fisiologiaRESUMO
The rat is the preferred model for toxicology studies, and it offers distinctive advantages over the mouse as a preclinical research model including larger sample size collection, lower rates of drug clearance, and relative ease of surgical manipulation. An immunodeficient rat would allow for larger tumor size development, prolonged dosing and drug efficacy studies, and preliminary toxicologic testing and pharmacokinetic/pharmacodynamic studies in the same model animal. Here, we created an immunodeficient rat with a functional deletion of the Recombination Activating Gene 2 (Rag2) gene, using genetically modified spermatogonial stem cells (SSC). We targeted the Rag2 gene in rat SSCs with TALENs and transplanted these Rag2-deficient SSCs into sterile recipients. Offspring were genotyped, and a founder with a 27 bp deletion mutation was identified and bred to homozygosity to produce the Sprague-Dawley Rag2 - Rag2 tm1Hera (SDR) knockout rat. We demonstrated that SDR rat lacks mature B and T cells. Furthermore, the SDR rat model was permissive to growth of human glioblastoma cell line subcutaneously resulting in successful growth of tumors. In addition, a human KRAS-mutant non-small cell lung cancer cell line (H358), a patient-derived high-grade serous ovarian cancer cell line (OV81), and a patient-derived recurrent endometrial cancer cell line (OV185) were transplanted subcutaneously to test the ability of the SDR rat to accommodate human xenografts from multiple tissue types. All human cancer cell lines showed efficient tumor uptake and growth kinetics indicating that the SDR rat is a viable host for a range of xenograft studies. Mol Cancer Ther; 17(11); 2481-9. ©2018 AACR.
Assuntos
Proteínas de Ligação a DNA/deficiência , Espermatogônias/citologia , Células-Tronco/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linfócitos B/citologia , Sequência de Bases , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Técnicas de Inativação de Genes , Genoma , Humanos , Masculino , Ratos Sprague-Dawley , Tela Subcutânea/patologia , Linfócitos T/citologiaRESUMO
Kinase inhibitor resistance constitutes a major unresolved clinical challenge in cancer. Furthermore, the role of serine/threonine phosphatase deregulation as a potential cause for resistance to kinase inhibitors has not been thoroughly addressed. We characterize protein phosphatase 2A (PP2A) activity as a global determinant of KRAS-mutant lung cancer cell resistance across a library of >200 kinase inhibitors. The results show that PP2A activity modulation alters cancer cell sensitivities to a large number of kinase inhibitors. Specifically, PP2A inhibition ablated mitogen-activated protein kinase kinase (MEK) inhibitor response through the collateral activation of AKT/mammalian target of rapamycin (mTOR) signaling. Combination of mTOR and MEK inhibitors induced cytotoxicity in PP2A-inhibited cells, but even this drug combination could not abrogate MYC up-regulation in PP2A-inhibited cells. Treatment with an orally bioavailable small-molecule activator of PP2A DT-061, in combination with the MEK inhibitor AZD6244, resulted in suppression of both p-AKT and MYC, as well as tumor regression in two KRAS-driven lung cancer mouse models. DT-061 therapy also abrogated MYC-driven tumorigenesis. These data demonstrate that PP2A deregulation drives MEK inhibitor resistance in KRAS-mutant cells. These results emphasize the need for better understanding of phosphatases as key modulators of cancer therapy responses.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteína Fosfatase 2/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
The dynamic and stringent coordination of kinase and phosphatase activity controls a myriad of physiologic processes. Aberrations that disrupt the balance of this interplay represent the basis of numerous diseases. For a variety of reasons, early work in this area portrayed kinases as the dominant actors in these signaling events with phosphatases playing a secondary role. In oncology, these efforts led to breakthroughs that have dramatically altered the course of certain diseases and directed vast resources toward the development of additional kinase-targeted therapies. Yet, more recent scientific efforts have demonstrated a prominent and sometimes driving role for phosphatases across numerous malignancies. This maturation of the phosphatase field has brought with it the promise of further therapeutic advances in the field of oncology. In this review, we discuss the role of phosphatases in the regulation of cellular proliferation and survival signaling using the examples of the MAPK and PI3K/AKT pathways, c-Myc and the apoptosis machinery. Emphasis is placed on instances where these signaling networks are perturbed by dysregulation of specific phosphatases to favor growth and persistence of human cancer.
Assuntos
Proliferação de Células , Neoplasias/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Apoptose , Sobrevivência Celular , Humanos , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment.Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065-80. ©2018 AACR.
Assuntos
Ativadores de Enzimas/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/enzimologia , Proteína Fosfatase 2C/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Linhagem Celular Tumoral , Ativadores de Enzimas/farmacologia , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos SCID , Fosfoproteínas/metabolismo , Proteína Fosfatase 2C/metabolismo , Proteômica , RNA Mensageiro/genética , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase that regulates many cellular processes. Given the central role of PP2A in regulating diverse biological functions and its dysregulation in many diseases, including cancer, PP2A directed therapeutics have become of great interest. The main approaches leveraged thus far can be categorized as follows: 1) inhibiting endogenous inhibitors of PP2A, 2) targeted disruption of post translational modifications on PP2A subunits, or 3) direct targeting of PP2A. Additional insight into the structural, molecular, and biological framework driving the efficacy of these therapeutic strategies will provide a foundation for the refinement and development of novel and clinically tractable PP2A targeted therapies.
Assuntos
Antineoplásicos , Sistemas de Liberação de Medicamentos/métodos , Inibidores Enzimáticos , Proteínas de Neoplasias , Neoplasias , Proteína Fosfatase 2 , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacosRESUMO
Activation of protein phosphatase 2A (PP2A) is a promising anticancer therapeutic strategy, as this tumor suppressor has the ability to coordinately downregulate multiple pathways involved in the regulation of cellular growth and proliferation. In order to understand the systems-level perturbations mediated by PP2A activation, we carried out mass spectrometry-based phosphoproteomic analysis of two KRAS mutated non-small cell lung cancer (NSCLC) cell lines (A549 and H358) treated with a novel small molecule activator of PP2A (SMAP). Overall, this permitted quantification of differential signaling across over 1600 phosphoproteins and 3000 phosphosites. Kinase activity assessment and pathway enrichment implicate collective downregulation of RAS and cell cycle kinases in the case of both cell lines upon PP2A activation. However, the effects on RAS-related signaling are attenuated for A549 compared to H358, while the effects on cell cycle-related kinases are noticeably more prominent in A549. Network-based analyses and validation experiments confirm these detailed differences in signaling. These studies reveal the power of phosphoproteomics studies, coupled to computational systems biology, to elucidate global patterns of phosphatase activation and understand the variations in response to PP2A activation across genetically similar NSCLC cell lines.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatase 2/metabolismo , Proteômica/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Espectrometria de Massas , Fosforilação , Transdução de SinaisRESUMO
Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.
Assuntos
Antineoplásicos/farmacologia , Ativadores de Enzimas/farmacologia , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Ativadores de Enzimas/química , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Ligação Proteica , Proteína Fosfatase 2/química , Transdução de Sinais , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase involved in the regulation of many cellular processes. A confirmed tumor suppressor protein, PP2A is genetically altered or functionally inactivated in many cancers highlighting a need for its therapeutic reactivation. In this review we discuss recent literature on PP2A: the elucidation of its structure and the functions of its subunits, and the identification of molecular lesions and post-translational modifications leading to its dysregulation in cancer. A final section will discuss the proteins and small molecules that modulate PP2A and how these might be used to target dysregulated forms of PP2A to treat cancers and other diseases.
Assuntos
Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Domínio Catalítico , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Moleculares , Mutação , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/terapia , Fosforilação , Proteína Fosfatase 2/genética , Processamento de Proteína Pós-Traducional , Subunidades Proteicas , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The phenothiazine and dibenzazepine tricyclics are potent neurotropic drugs with a documented but underutilized anti-cancer side effect. Reengineering these agents (TFP, CPZ, CIP) by replacing the basic amine with a neutral polar functional group (e.g., RTC-1, RTC-2) abrogated their CNS effects as demonstrated by in vitro pharmacological assays and in vivo behavioral models. Further optimization generated several phenothiazines and dibenzazepines with improved anti-cancer potency, exemplified by RTC-5. This new lead demonstrated efficacy against a xenograft model of an EGFR driven cancer without the neurotropic effects exhibited by the parent molecules. Its effects were attributed to concomitant negative regulation of PI3K-AKT and RAS-ERK signaling.