RESUMO
Compound VBT-5445 was identified as an inhibitor to block the association of Pim and the protein Enhancer of Decapping 3 (EDC3), a Pim substrate, which normally functions to enhance the decapping of messenger RNA (mRNA). It was also shown to inhibit both the Pim and mTORC protein kinases. The activity of this compound class can be fine-tuned by structural modification. A series of VBT analogs were designed, synthesized, and evaluated. These compounds decrease the growth of multiple cancer types, including pancreas, prostate, breast, lung, and leukemia. Notably, 6-methyl (GRG-1-31, 6d), 4-Bromo (GRG-1-34, 6e), 4-Chloro (GRG-1-35, 6f), and phenylthio substituted (GRG-1-104, 6n) derivatives are highly potent at inhibiting tumor growth. The ability of these compounds to block cancer growth in vitro is highly correlated with their activity as mTORC inhibitors. The toxicity of GRG 1-34 is low in mice treated with twice-daily gavage for 30 days and did not induce weight loss. Pharmacokinetics of a single oral dose demonstrated a peak concentration at 0.5 hours after gavage. In summary, further development of this compound class has the potential to inhibit important signaling pathways and impact cancer treatment.
RESUMO
The primary objective was to determine if multi-omic molecular profiling (MMP) informed selection of approved cancer treatments could change the clinical course of disease for patients with previously treated metastatic breast cancer (MBC) (i.e., produce a growth modulation index (GMI) ≥1.3). GMI was calculated as the ratio of progression free survival on MMP-selected therapy/time to progression on last prior treatment. To meet the primary objective at least 35 % of the subjects should demonstrate a GMI ≥1.3. Secondary endpoints included determining the response rate (according to RECIST 1.1), the percent of patients with non-progression at 4 months, and overall survival in patients whose therapy is selected by molecular profiling and proteomic analysis. Eligible patients had MBC, with ≥3 prior lines of therapy. A multi-omic based approach was performed incorporating multiplexed immunohistochemistry, c-DNA microarray, and phosphoprotein pathway activation mapping by reverse phase protein array. MMP was performed on fresh core biopsies; results were generated and sent to a Treatment Selection Committee (TSC) for review and treatment selection. Three sites enrolled 28 patients, of which 25 were evaluable. The median range of prior treatment was 7 (range 3-12). The MMP analysis and treatment recommendation were delivered within a median of 15.5 days from biopsy (range 12-23). The TSC selected MMP-rationalized treatment in 100 % (25/25) of cases. None of the MMP-based therapies were the same as what the clinician would have selected if the MMP had not been performed. GMI ≥1.3 was reported in 11/25 (44 %) patients. Partial responses were noted in 5/25 (20 %), stable disease in 8/25 (32 %) and 9/25 (36 %) had no progression at 4 months. This pilot study demonstrates the feasibility of finding possible treatments for patients with previously treated MBC using a multiplexed MMP-rationalized treatment recommendation. This MMP approach merits further investigation.
Assuntos
Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Medicina de Precisão/métodos , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biópsia , Neoplasias da Mama/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Projetos Piloto , Resultado do TratamentoRESUMO
Human tumors often contain slowly proliferating cancer cells that resist treatment, but we do not know precisely how these cells arise. We show that rapidly proliferating cancer cells can divide asymmetrically to produce slowly proliferating "G0-like" progeny that are enriched following chemotherapy in breast cancer patients. Asymmetric cancer cell division results from asymmetric suppression of AKT/PKB kinase signaling in one daughter cell during telophase of mitosis. Moreover, inhibition of AKT signaling with small-molecule drugs can induce asymmetric cancer cell division and the production of slow proliferators. Cancer cells therefore appear to continuously flux between symmetric and asymmetric division depending on the precise state of their AKT signaling network. This model may have significant implications for understanding how tumors grow, evade treatment, and recur.
Assuntos
Divisão Celular , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Modelos Biológicos , Estrutura Molecular , Neoplasias/genética , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Fase de Repouso do Ciclo Celular , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Malignant peripheral nerve sheath tumors (MPNSTs) are chemotherapy resistant sarcomas that are a leading cause of death in neurofibromatosis type 1 (NF1). Although NF1-related MPNSTs derive from neural crest cell origin, they also exhibit intratumoral heterogeneity. TP53 mutations are associated with significantly decreased survival in MPNSTs, however the mechanisms underlying TP53-mediated therapy responses are unclear in the context of NF1-deficiency. We evaluated the role of two commonly altered genes, MET and TP53, in kinome reprograming and cellular differentiation in preclinical MPNST mouse models. We previously showed that MET amplification occurs early in human MPNST progression and that Trp53 loss abrogated MET-addiction resulting in MET inhibitor resistance. Here we demonstrate a novel mechanism of therapy resistance whereby p53 alters MET stability, localization, and downstream signaling leading to kinome reprogramming and lineage plasticity. Trp53 loss also resulted in a shift from RAS/ERK to AKT signaling and enhanced sensitivity to MEK and mTOR inhibition. In response to MET, MEK and mTOR inhibition, we observed broad and heterogeneous activation of key differentiation genes in Trp53-deficient lines suggesting Trp53 loss also impacts lineage plasticity in MPNSTs. These results demonstrate the mechanisms by which p53 loss alters MET dependency and therapy resistance in MPNSTS through kinome reprogramming and phenotypic flexibility.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neurofibromatose 1 , Inibidores de Proteínas Quinases , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Camundongos , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Neurofibromina 1/genética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Neoplasias de Bainha Neural/genética , Neoplasias de Bainha Neural/patologia , Neoplasias de Bainha Neural/tratamento farmacológico , Linhagem Celular Tumoral , Transdução de Sinais , Linhagem da Célula/genética , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Neurofibrossarcoma/genética , Neurofibrossarcoma/patologia , Neurofibrossarcoma/tratamento farmacológico , Plasticidade Celular/efeitos dos fármacos , Plasticidade Celular/genéticaRESUMO
Combinations of immune checkpoint inhibitors (ICI, including anti-PD-1/PD-L1) and chemotherapy have been FDA approved for metastatic and early-stage triple-negative breast cancer (TNBC), but most patients do not benefit. B7-H4 is a B7 family ligand with proposed immunosuppressive functions being explored as a cancer immunotherapy target and may be associated with anti-PD-L1 resistance. However, little is known about its regulation and effect on immune cell function in breast cancers. We assessed murine and human breast cancer cells to identify regulation mechanisms of B7-H4 in vitro. We used an immunocompetent anti-PD-L1-sensitive orthotopic mammary cancer model and induced ectopic expression of B7-H4. We assessed therapy response and transcriptional changes at baseline and under treatment with anti-PD-L1. We observed B7-H4 was highly associated with epithelial cell status and transcription factors and found to be regulated by PI3K activity. EMT6 tumors with cell-surface B7-H4 expression were more resistant to immunotherapy. In addition, tumor-infiltrating immune cells had reduced immune activation signaling based on transcriptomic analysis. Paradoxically, in human breast cancer, B7-H4 expression was associated with survival benefit for patients with metastatic TNBC treated with carboplatin plus anti-PD-L1 and was associated with no change in response or survival for patients with early breast cancer receiving chemotherapy plus anti-PD-1. While B7-H4 induces tumor resistance to anti-PD-L1 in murine models, there are alternative mechanisms of signaling and function in human cancers. In addition, the strong correlation of B7-H4 to epithelial cell markers suggests a potential regulatory mechanism of B7-H4 independent of PD-L1. SIGNIFICANCE: This translational study confirms the association of B7-H4 expression with a cold immune microenvironment in breast cancer and offers preclinical studies demonstrating a potential role for B7-H4 in suppressing response to checkpoint therapy. However, analysis of two clinical trials with checkpoint inhibitors in the early and metastatic settings argue against B7-H4 as being a mechanism of clinical resistance to checkpoints, with clear implications for its candidacy as a therapeutic target.
Assuntos
Imunoterapia , Neoplasias de Mama Triplo Negativas , Inibidor 1 da Ativação de Células T com Domínio V-Set , Inibidor 1 da Ativação de Células T com Domínio V-Set/genética , Inibidor 1 da Ativação de Células T com Domínio V-Set/metabolismo , Animais , Humanos , Camundongos , Feminino , Linhagem Celular Tumoral , Imunoterapia/métodos , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/terapia , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias da Mama/imunologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Células Epiteliais/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
PURPOSE: The neutralizing peptibody trebananib prevents angiopoietin-1 and angiopoietin-2 from binding with Tie2 receptors, inhibiting angiogenesis and proliferation. Trebananib was combined with paclitaxel±trastuzumab in the I-SPY2 breast cancer trial. PATIENTS AND METHODS: I-SPY2, a phase II neoadjuvant trial, adaptively randomizes patients with high-risk, early-stage breast cancer to one of several experimental therapies or control based on receptor subtypes as defined by hormone receptor (HR) and HER2 status and MammaPrint risk (MP1, MP2). The primary endpoint is pathologic complete response (pCR). A therapy "graduates" if/when it achieves 85% Bayesian probability of success in a phase III trial within a given subtype. Patients received weekly paclitaxel (plus trastuzumab if HER2-positive) without (control) or with weekly intravenous trebananib, followed by doxorubicin/cyclophosphamide and surgery. Pathway-specific biomarkers were assessed for response prediction. RESULTS: There were 134 participants randomized to trebananib and 133 to control. Although trebananib did not graduate in any signature [phase III probabilities: Hazard ratio (HR)-negative (78%), HR-negative/HER2-positive (74%), HR-negative/HER2-negative (77%), and MP2 (79%)], it demonstrated high probability of superior pCR rates over control (92%-99%) among these subtypes. Trebananib improved 3-year event-free survival (HR 0.67), with no significant increase in adverse events. Activation levels of the Tie2 receptor and downstream signaling partners predicted trebananib response in HER2-positive disease; high expression of a CD8 T-cell gene signature predicted response in HR-negative/HER2-negative disease. CONCLUSIONS: The angiopoietin (Ang)/Tie2 axis inhibitor trebananib combined with standard neoadjuvant therapy increased estimated pCR rates across HR-negative and MP2 subtypes, with probabilities of superiority >90%. Further study of Ang/Tie2 receptor axis inhibitors in validated, biomarker-predicted sensitive subtypes is warranted.
Assuntos
Neoplasias da Mama , Proteínas Recombinantes de Fusão , Feminino , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Teorema de Bayes , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Terapia Neoadjuvante , Paclitaxel/efeitos adversos , Receptor ErbB-2/metabolismo , Receptor TIE-2 , Trastuzumab/efeitos adversosRESUMO
INTRODUCTION: The human epidermal growth factor receptor 2 (HER2) receptor tyrosine kinase (RTK) oncogene is an attractive therapeutic target for the treatment of HER2-addicted tumors. Although lapatinib, an FDA-approved small-molecule HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), represents a significant therapeutic advancement in the treatment of HER2+ breast cancers, responses to lapatinib have not been durable. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER-directed therapies is of critical importance. METHODS: Using a functional protein-pathway activation mapping strategy, along with targeted genomic knockdowns applied to a series of isogenic-matched pairs of lapatinib-sensitive and resistant cell lines, we now report an unexpected mechanism of acquired resistance to lapatinib and similar TKIs. RESULTS: The signaling analysis revealed that whereas HER2 was appropriately inhibited in lapatinib-resistant cells, EGFR tyrosine phosphorylation was incompletely inhibited. Using a targeted molecular knockdown approach to interrogate the causal molecular underpinnings of EGFR-persistent activation, we found that lapatinib-resistant cells were no longer oncogene addicted to HER2-HER3-PI3K signaling, as seen in the parental lapatinib-sensitive cell lines, but instead were dependent on a heregulin (HRG)-driven HER3-EGFR-PI3K-PDK1 signaling axis. Two FDA-approved EGFR TKIs could not overcome HRG-HER3-mediated activation of EGFR, or reverse lapatinib resistance. The ability to overcome EGFR-mediated acquired therapeutic resistance to lapatinib was demonstrated through molecular knockdown of EGFR and treatment with the irreversible pan-HER TKI neratinib, which blocked HRG-dependent phosphorylation of HER3 and EGFR, resulting in apoptosis of resistant cells. In addition, whereas HRG reversed lapatinib-mediated antitumor effects in parental HER2+ breast cancer cells, neratinib was comparatively resistant to the effects of HRG in parental cells. Finally, we showed that HRG expression is an independent negative predictor of clinical outcome in HER2+ breast cancers, providing potential clinical relevance to our findings. CONCLUSIONS: Molecular analysis of acquired therapeutic resistance to lapatinib identified a new resistance mechanism based on incomplete and "leaky" inhibition of EGFR by lapatinib. The selective pressure applied by incomplete inhibition of the EGFR drug target resulted in selection of ligand-driven feedback that sustained EGFR activation in the face of constant exposure to the drug. Inadequate target inhibition driven by a ligand-mediated autocrine feedback loop may represent a broader mechanism of therapeutic resistance to HER TKIs and suggests adopting a different strategy for selecting more effective TKIs to advance into the clinic.
Assuntos
Comunicação Autócrina , Neoplasias da Mama/metabolismo , Receptores ErbB/metabolismo , Neuregulina-1/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Transdução de Sinais , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Lapatinib , Neuregulina-1/genética , Fosfatidilinositol 3-Quinases , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Prognóstico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Piruvato Desidrogenase Quinase de Transferência de Acetil , Quinazolinas/farmacologia , Receptor ErbB-3/genética , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Quinases da Família src/antagonistas & inibidoresAssuntos
Antineoplásicos/farmacologia , Terapia de Alvo Molecular/métodos , Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Fosfotransferases/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Mapeamento de Peptídeos , Fosforilação , Fator de Transcrição STAT1/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The expression level of proteins governing the electrical excitability of and conduction within ventricular myocardium are known to vary as a function of distance through the heart wall. The expression patterns of a subset of these proteins are altered in disease. Precise measurement of such patterns is therefore essential to understanding structure-function relationships within the heart in health and disease. Here, we report a new experimental approach using reverse-phase protein microarrays to map the left ventricular transmural proteome. This approach can yield submillimeter spatial resolution, and when coupled with the method of array microenvironment normalization, reduces nonbiological components of variability to â¼10% of overall study variability. In addition, the experimental design provides sufficient statistical power to detect small, yet potentially biologically significant expression changes on the order of 1.1-fold. The usefulness of this technique is demonstrated by mapping the transmural expression of Serca2a in the left ventricle of 12 canine hearts, each in one of three states: normal, dyssynchronous heart failure, and dyssynchronous heart failure followed by cardiac resynchronization therapy. We confirm the existence of a 40% transmural gradient (epi>endo) of Serca2a, and demonstrate the ability of this technique to yield highly significant transmural expression differences within each individual heart.
Assuntos
Ventrículos do Coração/metabolismo , Miocárdio/metabolismo , Mapeamento de Peptídeos/métodos , Análise Serial de Proteínas/métodos , Proteoma/metabolismo , Animais , Terapia de Ressincronização Cardíaca , Cães , Regulação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/terapia , Especificidade de Órgãos , Proteoma/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
The ability of physicians to effectively treat and cure cancer is directly dependent on their ability to detect cancers at their earliest stages. Proteomic analyses of early-stage cancers have provided new insights into the changes that occur in the early phases of tumorigenesis and represent a new resource of candidate biomarkers for early-stage disease. Studies that profile proteomic patterns in body fluids also present new opportunities for the development of novel, highly sensitive diagnostic tools for the early detection of cancer.
Assuntos
Biomarcadores Tumorais/sangue , Proteínas de Neoplasias/sangue , Neoplasias/diagnóstico , Proteoma/análise , Proteômica/métodos , Biomarcadores Tumorais/genética , Humanos , Proteínas de Neoplasias/genética , Neoplasias/sangue , Neoplasias/genéticaRESUMO
Targeted monoclonal antibody therapy has emerged as a powerful therapeutic strategy for cancer. However, only a minority of patients have durable responses and the development of resistance remains a major clinical obstacle. Antibody-dependent cell-mediated cytotoxicity (ADCC) represents a crucial therapeutic mechanism of action; however, few studies have explored ADCC resistance. Using multiple in vitro models of ADCC selection pressure, we have uncovered both shared and distinct resistance mechanisms. Persistent ADCC selection pressure yielded ADCC-resistant cells that are characterized by a loss of NK cell conjugation and this shared resistance phenotype is associated with cell-line dependent modulation of cell surface proteins that contribute to immune synapse formation and NK cell function. We employed single-cell RNA sequencing and proteomic screens to interrogate molecular mechanisms of resistance. We demonstrate that ADCC resistance involves upregulation of interferon/STAT1 and DNA damage response signaling as well as activation of the immunoproteasome. Here, we identify pathways that modulate ADCC sensitivity and report strategies to enhance ADCC-mediated elimination of cancer cells. ADCC resistance could not be reversed with combinatorial treatment approaches. Hence, our findings indicate that tumor cells utilize multiple strategies to inhibit NK cell mediated-ADCC. Future research and development of NK cell-based immunotherapies must incorporate plans to address or potentially prevent the induction of resistance.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Proteômica , Humanos , Linhagem Celular Tumoral , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Células Matadoras NaturaisRESUMO
Pleomorphic xanthoastrocytoma (PXA) is a rare subset of primary pediatric glioma with 70% 5-year disease free survival. However, up to 20% of cases present with local recurrence and malignant transformation into more aggressive type anaplastic PXA (AXPA) or glioblastoma. The understanding of disease etiology and mechanisms driving PXA and APXA are limited, and there is no standard of care. Therefore, development of relevant preclinical models to investigate molecular underpinnings of disease and to guide novel therapeutic approaches are of interest. Here, for the first time we established, and characterized a patient-derived xenograft (PDX) from a leptomeningeal spread of a patient with recurrent APXA bearing a novel CDC42SE2-BRAF fusion. An integrated -omics analysis was conducted to assess model fidelity of the genomic, transcriptomic, and proteomic/phosphoproteomic landscapes. A stable xenoline was derived directly from the patient recurrent tumor and maintained in 2D and 3D culture systems. Conserved histology features between the PDX and matched APXA specimen were maintained through serial passages. Whole exome sequencing (WES) demonstrated a high degree of conservation in the genomic landscape between PDX and matched human tumor, including small variants (Pearson's r = 0.794-0.839) and tumor mutational burden (~ 3 mutations/MB). Large chromosomal variations including chromosomal gains and losses were preserved in PDX. Notably, chromosomal gain in chromosomes 4-9, 17 and 18 and loss in the short arm of chromosome 9 associated with homozygous 9p21.3 deletion involving CDKN2A/B locus were identified in both patient tumor and PDX sample. Moreover, chromosomal rearrangement involving 7q34 fusion; CDC42SE-BRAF t (5;7) (q31.1, q34) (5:130,721,239, 7:140,482,820) was identified in the PDX tumor, xenoline and matched human tumor. Transcriptomic profile of the patient's tumor was retained in PDX (Pearson r = 0.88) and in xenoline (Pearson r = 0.63) as well as preservation of enriched signaling pathways (FDR Adjusted P < 0.05) including MAPK, EGFR and PI3K/AKT pathways. The multi-omics data of (WES, transcriptome, and reverse phase protein array (RPPA) was integrated to deduce potential actionable pathways for treatment (FDR < 0.05) including KEGG01521, KEGG05202, and KEGG05200. Both xenoline and PDX were resistant to the MEK inhibitors trametinib or mirdametinib at clinically relevant doses, recapitulating the patient's resistance to such treatment in the clinic. This set of APXA models will serve as a preclinical resource for developing novel therapeutic regimens for rare anaplastic PXAs and pediatric high-grade gliomas bearing BRAF fusions.
Assuntos
Astrocitoma , Neoplasias Encefálicas , Glioma , Humanos , Criança , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Xenoenxertos , Fosfatidilinositol 3-Quinases/genética , Proteômica , Recidiva Local de Neoplasia/patologia , Astrocitoma/patologia , Glioma/patologia , Mutação , Aberrações Cromossômicas , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas de Membrana/genética , Peptídeos e Proteínas de Sinalização Intracelular/genéticaRESUMO
This clinical trial combined fulvestrant with the anti-androgen enzalutamide in women with metastatic ER+/HER2- breast cancer (BC). Eligible patients were women with ECOG 0-2, ER+/HER2- measurable or evaluable metastatic BC. Prior fulvestrant was allowed. Fulvestrant was administered at 500 mg IM on days 1, 15, 29, and every 4 weeks thereafter. Enzalutamide was given at 160 mg po daily. Fresh tumor biopsies were required at study entry and after 4 weeks of treatment. The primary efficacy endpoint of the trial was the clinical benefit rate at 24 weeks (CBR24). The median age was 61 years (46-87); PS 1 (0-1); median of 4 prior non-hormonal and 3 prior hormonal therapies for metastatic disease. Twelve had prior fulvestrant, and 91% had visceral disease. CBR24 was 25% (7/28 evaluable). Median progression-free survival (PFS) was 8 weeks (95% CI: 2-52). Adverse events were as expected for hormonal therapy. Significant (p < 0.1) univariate relationships existed between PFS and ER%, AR%, and PIK3CA and/or PTEN mutations. Baseline levels of phospho-proteins in the mTOR pathway were more highly expressed in biopsies of patients with shorter PFS. Fulvestrant plus enzalutamide had manageable side effects. The primary endpoint of CBR24 was 25% in heavily pretreated metastatic ER+/HER2- BC. Short PFS was associated with activation of the mTOR pathway, and PIK3CA and/or PTEN mutations were associated with an increased hazard of progression. Thus, a combination of fulvestrant or other SERD plus AKT/PI3K/mTOR inhibitor with or without AR inhibition warrants investigation in second-line endocrine therapy of metastatic ER+ BC.
RESUMO
Molecular subtyping of breast cancer is based mostly on HR/HER2 and gene expression-based immune, DNA repair deficiency, and luminal signatures. We extend this description via functional protein pathway activation mapping using pre-treatment, quantitative expression data from 139 proteins/phosphoproteins from 736 patients across 8 treatment arms of the I-SPY 2 Trial (ClinicalTrials.gov: NCT01042379). We identify predictive fit-for-purpose, mechanism-of-action-based signatures and individual predictive protein biomarker candidates by evaluating associations with pathologic complete response. Elevated levels of cyclin D1, estrogen receptor alpha, and androgen receptor S650 associate with non-response and are biomarkers for global resistance. We uncover protein/phosphoprotein-based signatures that can be utilized both for molecularly rationalized therapeutic selection and for response prediction. We introduce a dichotomous HER2 activation response predictive signature for stratifying triple-negative breast cancer patients to either HER2 or immune checkpoint therapy response as a model for how protein activation signatures provide a different lens to view the molecular landscape of breast cancer and synergize with transcriptomic-defined signatures.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias de Mama Triplo Negativas , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Terapia Neoadjuvante , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Biomarcadores , Perfilação da Expressão GênicaRESUMO
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC-MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC-MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately 2-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC-MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers.
Assuntos
Glândulas Écrinas/metabolismo , Proteômica/métodos , Esquizofrenia/metabolismo , Suor/química , Adolescente , Adulto , Sequência de Aminoácidos , Biomarcadores/análise , Cromatografia Líquida/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteoma/análise , Espectrometria de Massas em Tandem/métodosRESUMO
Previously, we have identified a panel of breast cancer antiestrogen resistance (BCAR) genes. Several of these genes have clinical relevance because mRNA or protein levels associate with tamoxifen resistance or tumor aggressiveness. We postulated that changes in activation status of protein signaling networks induced by BCAR genes may provide better insight into the mechanisms underlying antiestrogen resistance. Key signal transduction pathways were analyzed for changes in activation or expression using reverse-phase protein microarrays probed with 78 antibodies against signaling proteins with known roles in tumorigenesis. We used ZR-75-1-derived cell lines transduced with AKT1, AKT2, BCAR1, BCAR3, BCAR4, EGFR, GRB7, HRAS, HRAS(v12) or HEF1 and MCF7-derived cell lines transduced with BCAR3, BCAR4 or EGFR. In the antiestrogen-resistant cell lines, we observed increased phosphorylation of several pathways involved in cell proliferation and survival. All tamoxifen-resistant cell lines contained high levels of phosphorylated AKT and its biochemically linked substrates Forkhead box O1/3. The activation of ERBB2, ERBB3 and the downstream modulators focal adhesion kinase and SHC were activated in cells with overexpression of BCAR4. Remarkable differences were observed for the levels of activated AMPK alpha1, cyclins, STAT5, STAT6, ERK1/2 and BCL2. The comparison of the cell signaling networks in estrogen-dependent and -independent cell lines revealed biochemically linked kinase-substrate markers that comprised systemically activated signaling pathways involved in tamoxifen resistance. Our results show that this model provides insights into the molecular and cellular mechanisms of breast cancer progression and antiestrogen resistance. This knowledge may help the development of novel targeted treatments.
Assuntos
Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Moduladores de Receptor Estrogênico/farmacologia , Mapeamento de Interação de Proteínas , Transdução de Sinais , Tamoxifeno/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Proteína Substrato Associada a Crk/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras da Sinalização Shc/biossínteseRESUMO
Normal mammary gland homeostasis requires the coordinated regulation of protein signaling networks. However, we have little prospective information on whether activation of protein signaling occurs in premalignant mammary epithelial cells, as represented by cells with cytological atypia from women who are at high risk for breast cancer. This information is critical for understanding the role of deregulated signaling pathways in the initiation of breast cancer and for developing targeted prevention and/or treatment strategies for breast cancer in the future. In this pilot and feasibility study, we examined the expression of 52 phosphorylated, total, and cleaved proteins in 31 microdissected Random Periareolar Fine Needle Aspiration (RPFNA) samples by high-throughput Reverse Phase Protein Microarray. Unsupervised hierarchical clustering analysis indicated the presence of four clusters of proteins that represent the following signaling pathways: (1) receptor tyrosine kinase/Akt/mammalian target of rapamycin (RTK/Akt/mTOR), (2) RTK/Akt/extracellular signal-regulated kinase (RTK/Akt/ERK), (3) mitochondrial apoptosis, and (4) indeterminate. Clusters 1 through 3 comprised moderately to highly expressed proteins, while Cluster 4 comprised proteins that are lowly expressed in a majority of RPFNA samples. Our exploratory study showed that the interlinked components of mitochondrial apoptosis pathway are highly expressed in all mammary epithelial cells obtained from high-risk women. In particular, the expression levels of anti-apoptotic Bcl-xL and pro-apoptotic Bad are positively correlated in both non-atypical and atypical samples (unadjusted P < 0.0001), suggesting a delicate balance between the pro-apoptotic and anti-apoptotic regulation of cell proliferation during the early steps of mammary carcinogenesis. Our feasibility study suggests that the activation of key proteins along the RTK/Akt pathway may tip this balance to cell survival. Taken together, our results demonstrate the feasibility of mapping proteomic signaling networks in limited RPFNA samples obtained from high-risk women and the promise of developing rational drug targets or preventative strategies for breast cancer in future proteomic studies with a larger cohort of high-risk women.
Assuntos
Proteínas Reguladoras de Apoptose/análise , Neoplasias da Mama/química , Proteínas de Ciclo Celular/análise , Glândulas Mamárias Humanas/química , Proteômica , Transdução de Sinais , Adulto , Idoso , Apoptose , Biópsia por Agulha Fina , Neoplasias da Mama/patologia , Sobrevivência Celular , Análise por Conglomerados , Estudos de Viabilidade , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Modelos Logísticos , Glândulas Mamárias Humanas/patologia , Microdissecção , Pessoa de Meia-Idade , North Carolina , Projetos Piloto , Estudos Prospectivos , Análise Serial de Proteínas , Proteômica/métodos , Medição de Risco , Fatores de RiscoRESUMO
Protein microarrays, one emerging class of proteomic technologies, have broad applications for discovery and quantitative analysis. A rapidly expanding use of this technology is the acquisition of information about the posttranslational modifications of proteins reflecting the activity state of signal pathways and networks, and is now employed for the analysis of biopsy samples in clinical trial research.
Assuntos
Neoplasias/genética , Análise Serial de Proteínas/métodos , Transdução de Sinais , Animais , Anticorpos , Biotecnologia , Humanos , Neoplasias/terapia , Proteoma , Sensibilidade e EspecificidadeRESUMO
PURPOSE: Despite extensive genomic and transcriptomic profiling, it remains unknown how signaling pathways are differentially activated and how tumors are differentially sensitized to certain perturbations. Here, we aim to characterize AKT signaling activity and its association with other genomic or IHC-based PI3K/AKT pathway biomarkers as well as the clinical activity of ipatasertib (AKT inhibitor) in the FAIRLANE trial. EXPERIMENTAL DESIGN: In FAIRLANE, 151 patients with early triple-negative breast cancer (TNBC) were randomized 1:1 to receive paclitaxel with ipatasertib or placebo for 12 weeks prior to surgery. Adding ipatasertib did not increase pathologic complete response rate and numerically improved overall response rate by MRI. We used reverse-phase protein microarrays (RPPA) to examine the total level and/or phosphorylation states of over 100 proteins in various signaling or cell processes including PI3K/AKT and mTOR signaling. One hundred and twenty-five baseline and 127 on-treatment samples were evaluable by RPPA, with 110 paired samples at both time points. RESULTS: Tumors with genomic/protein alterations in PIK3CA/AKT1/PTEN were associated with higher levels of AKT phosphorylation. In addition, phosphorylated AKT (pAKT) levels exhibited a significant association with enriched clinical benefit of ipatasertib, and identified patients who received benefit in the absence of PIK3CA/AKT1/PTEN alterations. Ipatasertib treatment led to a downregulation of AKT/mTORC1 signaling, which was more pronounced among the tumors with PIK3CA/AKT1/PTEN alterations or among the responders to the treatment. CONCLUSIONS: We showed that the high baseline pAKT levels are associated with the alterations of PI3K/AKT pathway components and enriched benefit of ipatasertib in TNBC.
Assuntos
Neoplasias de Mama Triplo Negativas , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores , Classe I de Fosfatidilinositol 3-Quinases/genética , Humanos , Terapia Neoadjuvante , Paclitaxel , Fosfatidilinositol 3-Quinases/genética , Piperazinas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Epigenetic variation plays a significant role in normal development and human diseases including cancer, in part through post-translational modifications (PTMs) of histones. Identification and profiling of changes in histone PTMs, and in proteins regulating PTMs, are crucial to understanding diseases, and for discovery of epigenetic therapeutic agents. In this study, we have adapted and validated an antibody-based reverse phase protein array (RPPA) platform for profiling 20 histone PTMs and expression of 40 proteins that modify histones and other epigenomic regulators. The specificity of the RPPA assay for histone PTMs was validated with synthetic peptides corresponding to histone PTMs and by detection of histone PTM changes in response to inhibitors of histone modifier proteins in cell cultures. The useful application of the RPPA platform was demonstrated with two models: induction of pluripotent stem cells and a mouse mammary tumor progression model. Described here is a robust platform that includes a rapid microscale method for histone isolation and partially automated workflows for analysis of histone PTMs and histone modifiers that can be performed in a high-throughput manner with hundreds of samples. This RPPA platform has potential for translational applications through the discovery and validation of epigenetic states as therapeutic targets and biomarkers. SIGNIFICANCE: Our study has established an antibody-based reverse phase protein array platform for global profiling of a wide range of post-translational modifications of histones and histone modifier proteins. The high-throughput platform provides comprehensive analyses of epigenetics for biological research and disease studies and may serve as screening assay for diagnostic purpose or therapy development.