RESUMO
Cell-cell communication (CCC) is essential to how life forms and functions. However, accurate, high-throughput mapping of how expression of all genes in one cell affects expression of all genes in another cell is made possible only recently through the introduction of spatially resolved transcriptomics (SRT) technologies, especially those that achieve single-cell resolution. Nevertheless, substantial challenges remain to analyze such highly complex data properly. Here, we introduce a multiple-instance learning framework, Spacia, to detect CCCs from data generated by SRTs, by uniquely exploiting their spatial modality. We highlight Spacia's power to overcome fundamental limitations of popular analytical tools for inference of CCCs, including losing single-cell resolution, limited to ligand-receptor relationships and prior interaction databases, high false positive rates and, most importantly, the lack of consideration of the multiple-sender-to-one-receiver paradigm. We evaluated the fitness of Spacia for three commercialized single-cell resolution SRT technologies: MERSCOPE/Vizgen, CosMx/NanoString and Xenium/10x. Overall, Spacia represents a notable step in advancing quantitative theories of cellular communications.
Assuntos
Comunicação Celular , Perfilação da Expressão Gênica , Análise de Célula Única , Transcriptoma , Análise de Célula Única/métodos , Humanos , Comunicação Celular/genética , Perfilação da Expressão Gênica/métodos , AnimaisRESUMO
BACKGROUND: Immune-positron emission tomography (PET) imaging with tracers that target CD8 and granzyme B has shown promise in predicting the therapeutic response following immune checkpoint blockade (ICB) in immunologically "hot" tumors. However, immune dynamics in the low T-cell infiltrating "cold" tumor immune microenvironment during ICB remain poorly understood. This study uses molecular imaging to evaluate changes in CD4 + T cells and CD8 + T cells during ICB in breast cancer models and examines biomarkers of response. METHODS: [89Zr]Zr-DFO-CD4 and [89Zr]Zr-DFO-CD8 radiotracers were used to quantify changes in intratumoral and splenic CD4 T cells and CD8 T cells in response to ICB treatment in 4T1 and MMTV-HER2 mouse models, which represent immunologically "cold" tumors. A correlation between PET quantification metrics and long-term anti-tumor response was observed. Further biological validation was obtained by autoradiography and immunofluorescence. RESULTS: Following ICB treatment, an increase in the CD8-specific PET signal was observed within 6 days, and an increase in the CD4-specific PET signal was observed within 2 days in tumors that eventually responded to immunotherapy, while no significant differences in CD4 or CD8 were found at the baseline of treatment that differentiated responders from nonresponders. Furthermore, mice whose tumors responded to ICB had a lower CD8 PET signal in the spleen and a higher CD4 PET signal in the spleen compared to non-responders. Intratumoral spatial heterogeneity of the CD8 and CD4-specific PET signals was lower in responders compared to non-responders. Finally, PET imaging, autoradiography, and immunofluorescence signals were correlated when comparing in vivo imaging to ex vivo validations. CONCLUSIONS: CD4- and CD8-specific immuno-PET imaging can be used to characterize the in vivo distribution of CD4 + and CD8 + T cells in response to immune checkpoint blockade. Imaging metrics that describe the overall levels and distribution of CD8 + T cells and CD4 + T cells can provide insight into immunological alterations, predict biomarkers of response to immunotherapy, and guide clinical decision-making in those tumors where the kinetics of the response differ.
Assuntos
Neoplasias da Mama , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Modelos Animais de Doenças , Inibidores de Checkpoint Imunológico , Tomografia por Emissão de Pósitrons , Microambiente Tumoral , Animais , Microambiente Tumoral/imunologia , Feminino , Camundongos , Linfócitos T CD8-Positivos/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/imunologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linhagem Celular Tumoral , Zircônio , Compostos Radiofarmacêuticos , RadioisótoposRESUMO
BACKGROUND: Although trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms. METHODS: Immunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4 expression. Three-dimensional co-culture (tumor cells/breast cancer-associated fibroblasts/human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effects of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms. RESULTS: Using a panel of cell lines and patient breast cancer samples, we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using a panel of trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel. CONCLUSIONS: Our findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.
Assuntos
Neoplasias da Mama , Humanos , Animais , Camundongos , Feminino , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Docetaxel/farmacologia , Apoptose , Leucócitos Mononucleares/metabolismo , Receptor ErbB-2/metabolismo , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular , Mitose , Resistencia a Medicamentos Antineoplásicos , Microambiente Tumoral , Receptores CXCR4/genéticaRESUMO
Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.
Assuntos
Neoplasias da Mama/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mutação/genética , Comunicação Parácrina , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais , Anfirregulina/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida , Classe I de Fosfatidilinositol 3-Quinases , Intervalo Livre de Doença , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Humanos , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteômica , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em Tandem , Regulação para Cima/efeitos dos fármacosRESUMO
PURPOSE: Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in human breast cancer. Furthermore, PIK3CA mutations are commonly associated with resistance to anti-epidermal growth factor receptor 2 (HER2) or anti-estrogen receptor (ER) agents in HER2 or ER positive (HER2+/ER+) breast cancer. Hence, deciphering the underlying mechanisms of PIK3CA mutations in HER2+/ER+ breast cancer would provide novel insights into elucidating resistance to anti-HER2/ER therapies. METHODS: In this study, we systematically investigated the biological consequences of PIK3CA H1047R in HER2+/ER+ breast cancer by uniquely incorporating mRNA transcriptomic data from The Cancer Genome Atlas and proteomic data from reverse-phase protein arrays. RESULTS: Our integrative bioinformatics analyses revealed that several important pathways such as STAT3 and VEGF/hypoxia were selectively altered by PIK3CA H1047R in HER2+/ER+ breast cancer. Protein differential expression analysis indicated that an elevated eIF4G might promote tumor angiogenesis and growth via regulation of the hypoxia-activated switch in HER2+ PIK3CA H1047R breast cancer. We observed hypo-phosphorylation of EGFR in HER2+ PIK3CA H1047R breast cancer versus HER2+PIK3CAwild-type (PIK3CA WT). In addition, ER and PIK3CA H1047R might cooperate to activate STAT3, MAPK, AKT, and Hippo pathways in ER+ PIK3CA H1047R breast cancer. A higher YAPpS127 level was observed in ER+ PIK3CA H1047R patients than that in an ER+ PIK3CA WT subgroup. By examining breast cancer cell lines having both microarray gene expression and drug treatment data from the Genomics of Drug Sensitivity in Cancer and the Stand Up to Cancer datasets, we found that the elevated YAP1 mRNA expression was associated with the resistance of BCL-2 family inhibitors, but with the sensitivity to MEK/MAPK inhibitors in breast cancer cells. CONCLUSIONS: In summary, these findings shed light on the functional consequences of PIK3CA H1047R-driven breast tumorigenesis and resistance to the existing therapeutic agents in HER2+/ER+ breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor ErbB-2/genética , Receptores de Estrogênio/genética , Fator de Transcrição STAT3/metabolismo , Biomarcadores , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Análise por Conglomerados , Biologia Computacional/métodos , Bases de Dados Genéticas , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Hippo , Humanos , Terapia de Alvo Molecular , Mutação , Fosforilação , Proteômica/métodos , Transdução de SinaisRESUMO
To employ in vivo imaging and histological techniques to identify and quantify vascular changes early in the course of treatment with trastuzumab in a murine model of HER2+ breast cancer. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to quantitatively characterize vessel perfusion/permeability (via the parameter K (trans) ) and the extravascular extracellular volume fraction (v e ) in the BT474 mouse model of HER2+ breast cancer (N = 20) at baseline, day one, and day four following trastuzumab treatment (10 mg/kg). Additional cohorts of mice were used to quantify proliferation (Ki67), microvessel density (CD31), pericyte coverage (α-SMA) by immunohistochemistry (N = 44), and to quantify human VEGF-A expression (N = 29) throughout the course of therapy. Longitudinal assessment of combination doxorubicin ± trastuzumab (N = 42) tested the hypothesis that prior treatment with trastuzumab will increase the efficacy of subsequent doxorubicin therapy. Compared to control tumors, trastuzumab-treated tumors exhibited a significant increase in K (trans) (P = 0.035) on day four, indicating increased perfusion and/or vessel permeability and a simultaneous significant increase in v e (P = 0.01), indicating increased cell death. Immunohistochemical and ELISA analyses revealed that by day four the trastuzumab-treated tumors had a significant increase in vessel maturation index (i.e., the ratio of α-SMA to CD31 staining) compared to controls (P < 0.001) and a significant decrease in VEGF-A (P = 0.03). Additionally, trastuzumab dosing prior to doxorubicin improved the overall effectiveness of the therapies (P < 0.001). This study identifies and validates improved perfusion characteristics following trastuzumab therapy, resulting in an improvement in trastuzumab-doxorubicin combination therapy in a murine model of HER2+ breast cancer. This data suggests properties of vessel maturation. In particular, the use of DCE-MRI, a clinically available imaging method, following treatment with trastuzumab may provide an opportunity to optimize the scheduling and improve delivery of subsequent cytotoxic therapy.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Doxorrubicina/farmacologia , Quimioterapia Combinada/métodos , Feminino , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Human epidermal growth factor receptor 2 (HER2; ERBB2) amplification and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations often co-occur in breast cancer. Aberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway has been shown to correlate with a diminished response to HER2-directed therapies. We generated a mouse model of HER2-overexpressing (HER2(+)), PIK3CA(H1047R)-mutant breast cancer. Mice expressing both human HER2 and mutant PIK3CA in the mammary epithelium developed tumors with shorter latencies compared with mice expressing either oncogene alone. HER2 and mutant PIK3CA also cooperated to promote lung metastases. By microarray analysis, HER2-driven tumors clustered with luminal breast cancers, whereas mutant PIK3CA tumors were associated with claudin-low breast cancers. PIK3CA and HER2(+)/PIK3CA tumors expressed elevated transcripts encoding markers of epithelial-to-mesenchymal transition and stem cells. Cells from HER2(+)/PIK3CA tumors more efficiently formed mammospheres and lung metastases. Finally, HER2(+)/PIK3CA tumors were resistant to trastuzumab alone and in combination with lapatinib or pertuzumab. Both drug resistance and enhanced mammosphere formation were reversed by treatment with a PI3K inhibitor. In sum, PIK3CA(H1047R) accelerates HER2-mediated breast epithelial transformation and metastatic progression, alters the intrinsic phenotype of HER2-overexpressing cancers, and generates resistance to approved combinations of anti-HER2 therapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Genes erbB-2 , Neoplasias Mamárias Experimentais/patologia , Mutação , Fosfatidilinositol 3-Quinases/genética , Animais , Classe I de Fosfatidilinositol 3-Quinases , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/enzimologia , Camundongos , Camundongos TransgênicosRESUMO
Luminal Androgen Receptor (LAR) triple-negative breast cancers (TNBC) express androgen receptors (AR), exhibit high frequency of PIK3CA mutations and intact RB. Herein, we investigated combined blockade of the CDK4/6 and PI3K signaling with palbociclib, alpelisib, and capivasertib, which inhibit CDK4/6, PI3Kα, and AKT1-3, respectively. The combination of palbociclib/capivasertib, but not palbociclib/alpelisib, synergistically inhibited proliferation of MDA-MB-453 and MFM-223 LAR cells [synergy score 7.34 (p = 5.81x10-11) and 4.78 (p = 0.012), respectively]. The AR antagonist enzalutamide was inactive against MDA-MB-453, MFM-223, and CAL148 cells and did not enhance the efficacy of either combination. Palbociclib/capivasertib inhibited growth of LAR patient-derived xenografts more potently than palbociclib/alpelisib. Treatment of LAR cells with palbociclib suppressed phosphorylated-RB and resulted in adaptive phosphorylation/activation of S473 pAKT and AKT substrates GSK3ß, PRAS40, and FoxO3a. Capivasertib blocked palbociclib-induced phosphorylation of AKT substrates more potently than alpelisib. Treatment with PI3Kß inhibitors did not block phosphorylation of AKT substrates, suggesting that PI3Kß did not mediate the adaptive response to CDK4/6 inhibition. Phosphokinase arrays of MDA-MB-453 cells treated with palbociclib showed time-dependent upregulation of PDGFRß, GSK3ß, STAT3, and STAT6. RNA silencing of PDGFRß in palbociclib-treated MDA-MB-453 and MFM-223 cells blocked the upregulation of S473 pAKT, suggesting that the adaptive response to CDK4/6 blockade involves PDGFRß signaling. Finally, treatment with palbociclib and the PDGFR inhibitor CP637451 arrested growth of MDA-MB-453 and MFM-223 cells to the same degree as palbociclib/capivasertib. These findings support testing the combination of CDK4/6 and AKT inhibitors in patients with LAR TNBC, and further investigation of PDGFR antagonists in this breast cancer subtype.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Proliferação de Células , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Piperazinas , Proteínas Proto-Oncogênicas c-akt , Piridinas , Receptores Androgênicos , Neoplasias de Mama Triplo Negativas , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Piperazinas/farmacologia , Animais , Feminino , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/metabolismo , Piridinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proliferação de Células/efeitos dos fármacos , Camundongos , Pirimidinas/farmacologia , Sinergismo Farmacológico , Transdução de Sinais/efeitos dos fármacos , Feniltioidantoína/farmacologia , Feniltioidantoína/análogos & derivados , Benzamidas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Nitrilas/farmacologia , Pirróis/farmacologia , TiazóisRESUMO
Cell-cell communication (CCC) is essential to how life forms and functions. However, accurate, high-throughput mapping of how expression of all genes in one cell affects expression of all genes in another cell is made possible only recently, through the introduction of spatially resolved transcriptomics technologies (SRTs), especially those that achieve single cell resolution. However, significant challenges remain to analyze such highly complex data properly. Here, we introduce a Bayesian multi-instance learning framework, spacia, to detect CCCs from data generated by SRTs, by uniquely exploiting their spatial modality. We highlight spacia's power to overcome fundamental limitations of popular analytical tools for inference of CCCs, including losing single-cell resolution, limited to ligand-receptor relationships and prior interaction databases, high false positive rates, and most importantly the lack of consideration of the multiple-sender-to-one-receiver paradigm. We evaluated the fitness of spacia for all three commercialized single cell resolution ST technologies: MERSCOPE/Vizgen, CosMx/Nanostring, and Xenium/10X. Spacia unveiled how endothelial cells, fibroblasts and B cells in the tumor microenvironment contribute to Epithelial-Mesenchymal Transition and lineage plasticity in prostate cancer cells. We deployed spacia in a set of pan-cancer datasets and showed that B cells also participate in PDL1/PD1 signaling in tumors. We demonstrated that a CD8+ T cell/PDL1 effectiveness signature derived from spacia analyses is associated with patient survival and response to immune checkpoint inhibitor treatments in 3,354 patients. We revealed differential spatial interaction patterns between γδ T cells and liver hepatocytes in healthy and cancerous contexts. Overall, spacia represents a notable step in advancing quantitative theories of cellular communications.
RESUMO
Therapy resistance and metastatic progression are primary causes of cancer-related mortality. Disseminated tumor cells possess adaptive traits that enable them to reprogram their metabolism, maintain stemness, and resist cell death, facilitating their persistence to drive recurrence. The survival of disseminated tumor cells also depends on their ability to modulate replication stress in response to therapy while colonizing inhospitable microenvironments. In this study, we discovered that the nuclear translocation of AXL, a TAM receptor tyrosine kinase, and its interaction with WRNIP1, a DNA replication stress response factor, promotes the survival of HER2+ breast cancer cells that are resistant to HER2-targeted therapy and metastasize to the brain. In preclinical models, knocking down or pharmacologically inhibiting AXL or WRNIP1 attenuated protection of stalled replication forks. Furthermore, deficiency or inhibition of AXL and WRNIP1 also prolonged metastatic latency and delayed relapse. Together, these findings suggest that targeting the replication stress response, which is a shared adaptive mechanism in therapy-resistant and metastasis-initiating cells, could reduce metachronous metastasis and enhance the response to standard-of-care therapies. SIGNIFICANCE: Nuclear AXL and WRNIP1 interact and mediate replication stress response, promote therapy resistance, and support metastatic progression, indicating that targeting the AXL/WRNIP1 axis is a potentially viable therapeutic strategy for breast cancer.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Receptor Tirosina Quinase Axl , Proteínas Proto-Oncogênicas/metabolismo , Recidiva Local de Neoplasia , Receptores Proteína Tirosina Quinases/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Microambiente Tumoral , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação a DNA/metabolismoRESUMO
CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Through a genome-wide CRISPR screen, we identify protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout breast cancer cells. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle independent of RB, leading to growth arrest in RB1-knockout cells. Proteomics analysis uncovers fused in sarcoma (FUS) as a downstream effector of PRMT5. Inhibition of PRMT5 results in dissociation of FUS from RNA polymerase II, leading to hyperphosphorylation of serine 2 in RNA polymerase II, intron retention, and subsequent downregulation of proteins involved in DNA synthesis. Furthermore, treatment with the PRMT5 inhibitor pemrametostat and a selective ER degrader fulvestrant synergistically inhibits growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings highlight dual ER and PRMT5 blockade as a potential therapeutic strategy to overcome resistance to CDK4/6i in ER+/RB-deficient breast cancer.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , RNA Polimerase II , Quinase 4 Dependente de Ciclina/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Cancer cells exhibit phenotypical plasticity and epigenetic reprogramming that allows them to evade lineage-dependent targeted treatments by adopting lineage plasticity. The underlying mechanisms by which cancer cells exploit the epigenetic regulatory machinery to acquire lineage plasticity and therapy resistance remain poorly understood. We identified zinc finger protein 397 (ZNF397) as a bona fide coactivator of the androgen receptor (AR), essential for the transcriptional program governing AR-driven luminal lineage. ZNF397 deficiency facilitates the transition of cancer cell from an AR-driven luminal lineage to a ten-eleven translocation 2 (TET2)-driven lineage plastic state, ultimately promoting resistance to therapies inhibiting AR signaling. Intriguingly, our findings indicate that a TET2 inhibitor can eliminate the resistance to AR-targeted therapies in ZNF397-deficient tumors. These insights uncover a novel mechanism through which prostate cancer acquires lineage plasticity via epigenetic rewiring and offer promising implications for clinical interventions designed to overcome therapy resistance dictated by lineage plasticity. Significance: This study reveals a bifurcated role of ZNF397, and a TET2-driven epigenetic mechanism regulating tumor lineage plasticity and therapy response in prostate cancer, enhances the understanding of drug resistance, and unveils a new therapeutic strategy for overcoming androgen receptor-targeted therapy resistance.
Assuntos
Proteínas de Ligação a DNA , Dioxigenases , Resistencia a Medicamentos Antineoplásicos , Neoplasias da Próstata , Receptores Androgênicos , Masculino , Humanos , Proteínas de Ligação a DNA/genética , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Camundongos , Animais , Linhagem Celular Tumoral , Epigênese Genética , Linhagem da CélulaRESUMO
HER2 mutations drive the growth of a subset of breast cancers and are targeted with HER2 tyrosine kinase inhibitors (TKI) such as neratinib. However, acquired resistance is common and limits the durability of clinical responses. Most HER2-mutant breast cancers progressing on neratinib-based therapy acquire secondary mutations in HER2. It is unknown whether these secondary HER2 mutations, other than the HER2T798I gatekeeper mutation, are causal to neratinib resistance. Herein, we show that secondary acquired HER2T862A and HER2L755S mutations promote resistance to HER2 TKIs via enhanced HER2 activation and impaired neratinib binding. While cells expressing each acquired HER2 mutation alone were sensitive to neratinib, expression of acquired double mutations enhanced HER2 signaling and reduced neratinib sensitivity. Computational structural modeling suggested that secondary HER2 mutations stabilize the HER2 active state and reduce neratinib binding affinity. Cells expressing double HER2 mutations exhibited resistance to most HER2 TKIs but retained sensitivity to mobocertinib and poziotinib. Double-mutant cells showed enhanced MEK/ERK signaling, which was blocked by combined inhibition of HER2 and MEK. Together, these findings reveal the driver function of secondary HER2 mutations in resistance to HER2 inhibition and provide a potential treatment strategy to overcome acquired resistance to HER2 TKIs in HER2-mutant breast cancer. SIGNIFICANCE: HER2-mutant breast cancers acquire secondary HER2 mutations that drive resistance to HER2 tyrosine kinase inhibitors, which can be overcome by combined inhibition of HER2 and MEK.
Assuntos
Neoplasias da Mama , Quinolinas , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Receptor ErbB-2/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Mutação , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Background: Although trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms. Methods: Immunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4expression. Three-dimensional co-culture (tumor cells/ breast cancer-associated fibroblasts / human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effect of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms. Results: Using multiple cell lines and patient breast cancer samples we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated that the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using multiple trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel. Conclusions: Our findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.
RESUMO
NF1 is a key tumor suppressor that represses both RAS and estrogen receptor-α (ER) signaling in breast cancer. Blocking both pathways by fulvestrant (F), a selective ER degrader, together with binimetinib (B), a MEK inhibitor, promotes tumor regression in NF1-depleted ER+ models. We aimed to establish approaches to determine how NF1 protein levels impact B+F treatment response to improve our ability to identify B+F sensitive tumors. We examined a panel of ER+ patient-derived xenograft (PDX) models by DNA and mRNA sequencing and found that more than half of these models carried an NF1 shallow deletion and generally have low mRNA levels. Consistent with RAS and ER activation, RET and MEK levels in NF1-depleted tumors were elevated when profiled by mass spectrometry (MS) after kinase inhibitor bead pulldown. MS showed that NF1 can also directly and selectively bind to palbociclib-conjugated beads, aiding quantification. An IHC assay was also established to measure NF1, but the MS-based approach was more quantitative. Combined IHC and MS analysis defined a threshold of NF1 protein loss in ER+ breast PDX, below which tumors regressed upon treatment with B+F. These results suggest that we now have a MS-verified NF1 IHC assay that can be used for patient selection as a complement to somatic genomic analysis. Significance: A major challenge for targeting the consequence of tumor suppressor disruption is the accurate assessment of protein functional inactivation. NF1 can repress both RAS and ER signaling, and a ComboMATCH trial is underway to treat the patients with binimetinib and fulvestrant. Herein we report a MS-verified NF1 IHC assay that can determine a threshold for NF1 loss to predict treatment response. These approaches may be used to identify and expand the eligible patient population.
Assuntos
Neoplasias da Mama , Proteogenômica , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neurofibromina 1/genética , Fulvestranto/farmacologia , Receptores de Estrogênio/genética , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição NFI , RNA Mensageiro , Quinases de Proteína Quinase Ativadas por MitógenoRESUMO
CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer acquired resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Using a genome-wide CRISPR screen, we identified protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout (RBKO) breast cancer cells. PRMT5 inhibition blocked cell cycle G1-to-S transition independent of RB, thus arresting growth of RBKO cells. Proteomics analysis uncovered fused in sarcoma (FUS) as a downstream effector of PRMT5. Pharmacological inhibition of PRMT5 resulted in dissociation of FUS from RNA polymerase II (Pol II), Ser2 Pol II hyperphosphorylation, and intron retention in genes that promote DNA synthesis. Treatment with the PRMT5i inhibitor pemrametostat and fulvestrant synergistically inhibited growth of ER+/RB-deficient patient-derived xenografts, suggesting dual ER and PRMT5 blockade as a novel therapeutic strategy to treat ER+/RB-deficient breast cancer.
RESUMO
Disseminated tumor cells with metabolic flexibility to utilize available nutrients in distal organs persist, but the precise mechanisms that facilitate metabolic adaptations remain unclear. Here we show fragmented mitochondrial puncta in latent brain metastatic (Lat) cells enable fatty acid oxidation (FAO) to sustain cellular bioenergetics and maintain redox homeostasis. Depleting the enriched dynamin-related protein 1 (DRP1) and limiting mitochondrial plasticity in Lat cells results in increased lipid droplet accumulation, impaired FAO and attenuated metastasis. Likewise, pharmacological inhibition of DRP1 using a small-molecule brain-permeable inhibitor attenuated metastatic burden in preclinical models. In agreement with these findings, increased phospho-DRP1 expression was observed in metachronous brain metastasis compared with patient-matched primary tumors. Overall, our findings reveal the pivotal role of mitochondrial plasticity in supporting the survival of Lat cells and highlight the therapeutic potential of targeting cellular plasticity programs in combination with tumor-specific alterations to prevent metastatic recurrences.
Assuntos
Neoplasias Encefálicas , Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Linhagem Celular Tumoral , Neoplasias Encefálicas/tratamento farmacológicoRESUMO
HER2+ breast cancer patients are presented with either synchronous (S-BM), latent (Lat), or metachronous (M-BM) brain metastases. However, the basis for disparate metastatic fitness among disseminated tumor cells of similar oncotype within a distal organ remains unknown. Here, employing brain metastatic models, we show that metabolic diversity and plasticity within brain-tropic cells determine metastatic fitness. Lactate secreted by aggressive metastatic cells or lactate supplementation to mice bearing Lat cells limits innate immunosurveillance and triggers overt metastasis. Attenuating lactate metabolism in S-BM impedes metastasis, while M-BM adapt and survive as residual disease. In contrast to S-BM, Lat and M-BM survive in equilibrium with innate immunosurveillance, oxidize glutamine, and maintain cellular redox homeostasis through the anionic amino acid transporter xCT. Moreover, xCT expression is significantly higher in matched M-BM brain metastatic samples compared to primary tumors from HER2+ breast cancer patients. Inhibiting xCT function attenuates residual disease and recurrence in these preclinical models.
Assuntos
Neoplasias Encefálicas , Neoplasias da Mama , Animais , Encéfalo/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Feminino , Humanos , CamundongosRESUMO
The MYC oncogene is frequently amplified in triple-negative breast cancer (TNBC). Here, we show that MYC suppression induces immune-related hallmark gene set expression and tumor-infiltrating T cells in MYC-hyperactivated TNBCs. Mechanistically, MYC repressed stimulator of interferon genes (STING) expression via direct binding to the STING1 enhancer region, resulting in downregulation of the T-cell chemokines CCL5, CXCL10, and CXCL11. In primary and metastatic TNBC cohorts, tumors with high MYC expression or activity exhibited low STING expression. Using a CRISPR-mediated enhancer perturbation approach, we demonstrated that MYC-driven immune evasion is mediated by STING repression. STING repression induced resistance to PD-L1 blockade in mouse models of TNBC. Finally, a small-molecule inhibitor of MYC combined with PD-L1 blockade elicited a durable response in immune-cold TNBC with high MYC expression, suggesting a strategy to restore PD-L1 inhibitor sensitivity in MYC-overexpressing TNBC.
Assuntos
Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Neoplasias de Mama Triplo Negativas , Animais , Antígeno B7-H1 , Linhagem Celular Tumoral , Repressão Epigenética , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Evasão da Resposta Imune , Camundongos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
PURPOSE: FGFR1 overexpression has been associated with endocrine resistance in ER+ breast cancer. We found FGFR1 localized in the nucleus of breast cancer cells in primary tumors resistant to estrogen suppression. We investigated a role of nuclear FGFR1 on gene transcription and antiestrogen resistance. EXPERIMENTAL DESIGN: Tumors from patients treated with letrozole were subjected to Ki67 and FGFR1 IHC. MCF7 cells were transduced with FGFR1(SP-)(NLS) to promote nuclear FGFR1 overexpression. FGFR1 genomic activity in ER+/FGFR1-amplified breast cancer cells ± FOXA1 siRNA or ± the FGFR tyrosine kinase inhibitor (TKI) erdafitinib was examined by chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). The nuclear and chromatin-bound FGFR1 interactome was investigated by mass spectrometry (MS). RESULTS: High nuclear FGFR1 expression in ER+ primary tumors positively correlated with post-letrozole Ki67 values. Nuclear FGFR1 overexpression influenced gene transcription and promoted resistance to estrogen suppression and to fulvestrant in vivo. A gene expression signature induced by nuclear FGFR1 correlated with shorter survival in the METABRIC cohort of patients treated with antiestrogens. ChIP-Seq revealed FGFR1 occupancy at transcription start sites, overlapping with active transcription histone marks. MS analysis of the nuclear FGFR1 interactome identified phosphorylated RNA-Polymerase II and FOXA1, with FOXA1 RNAi impairing FGFR1 recruitment to chromatin. Treatment with erdafitinib did not impair nuclear FGFR1 translocation and genomic activity. CONCLUSIONS: These data suggest nuclear FGFR1 contributes to endocrine resistance by modulating gene transcription in ER+ breast cancer. Nuclear FGFR1 activity was unaffected by FGFR TKIs, thus supporting the development of treatment strategies to inhibit nuclear FGFR1 in ER+/FGFR1 overexpressing breast cancer.