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BACKGROUND: Anti-HER2 therapies, including the HER2 antibody-drug conjugates (ADCs) trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd), have led to improved survival outcomes in patients with HER2-overexpressing (HER2+) metastatic breast cancer. However, intrinsic or acquired resistance to anti-HER2-based therapies remains a clinical challenge in these patients, as there is no standard of care following disease progression. The purpose of this study was to elucidate the mechanisms of resistance to T-DM1 and T-DXd in HER2+ BC patients and preclinical models and identify targets whose inhibition enhances the antitumor activity of T-DXd in HER2-directed ADC-resistant HER2+ breast cancer in vitro and in vivo. METHODS: Targeted DNA and whole transcriptome sequencing were performed in breast cancer patient tissue samples to investigate genetic aberrations that arose after anti-HER2 therapy. We generated T-DM1 and T-DXd-resistant HER2+ breast cancer cell lines. To elucidate their resistance mechanisms and to identify potential synergistic kinase targets for enhancing the efficacy of T-DXd, we used fluorescence in situ hybridization, droplet digital PCR, Western blotting, whole-genome sequencing, cDNA microarray, and synthetic lethal kinome RNA interference screening. In addition, cell viability, colony formation, and xenograft assays were used to determine the synergistic antitumor effect of T-DXd combinations. RESULTS: We found reduced HER2 expression in patients and amplified DNA repair-related genes in patients after anti-HER2 therapy. Reduced ERBB2 gene amplification in HER2-directed ADC-resistant HER2+ breast cancer cell lines was through DNA damage and epigenetic mechanisms. In HER2-directed ADC-resistant HER2+ breast cancer cell lines, our non-biased RNA interference screening identified the DNA repair pathway as a potential target within the canonical pathways to enhance the efficacy of T-DXd. We validated that the combination of T-DXd with ataxia telangiectasia and Rad3-related inhibitor, elimusertib, led to significant breast cancer cell death in vitro (P < 0.01) and in vivo (P < 0.01) compared to single agents. CONCLUSIONS: The DNA repair pathways contribute to HER2-directed ADC resistance. Our data justify exploring the combination treatment of T-DXd with DNA repair-targeting drugs to treat HER2-directed ADC-resistant HER2+ breast cancer in clinical trials.
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Neoplasias de la Mama , Reparación del ADN , Resistencia a Antineoplásicos , Inmunoconjugados , Receptor ErbB-2 , Trastuzumab , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Trastuzumab/farmacología , Trastuzumab/uso terapéutico , Animales , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Ratones , Receptor ErbB-2/metabolismo , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Camptotecina/análogos & derivados , Camptotecina/farmacología , Camptotecina/uso terapéutico , Sinergismo FarmacológicoRESUMEN
Cyclin E is a regulatory subunit of CDK2 that mediates S phase entry and progression. Cleavage of full-length cyclin E (FL-cycE) to low molecular weight isoforms (LMW-E) dramatically alters the substrate specificity, promoting G1/S cell cycle transition and accelerating mitotic exit. Approximately 70% of triple-negative breast cancers (TNBC) express LMW-E, which correlates with poor prognosis. PKMYT1 also plays an important role in mitosis by inhibiting CDK1 to block premature mitotic entry, suggesting it could be a therapeutic target in TNBC expressing LMW-E. Here, analysis of TNBC patient tumor samples revealed that co-expression of LMW-E and PKMYT1-catalyzed CDK1 phosphorylation predicted poor response to neoadjuvant chemotherapy. Compared to FL-cycE, LMW-E specifically upregulated PKMYT1 expression and protein stability, elevating CDK1 phosphorylation. Inhibiting PKMYT1 with the selective inhibitor RP-6306 (lunresertib) elicited LMW-E dependent antitumor effects, accelerating premature mitotic entry, inhibiting replication fork restart, and enhancing DNA damage, chromosomal breaks, apoptosis, and replication stress. Importantly, TNBC cell line xenografts expressing LMW-E showed greater sensitivity to RP-6306 than tumors with empty vector or FL-cycE. Furthermore, RP-6306 exerted tumor suppressive effects in LMW-E transgenic murine mammary tumors and LMW-E-high TNBC patient-derived xenografts but not in the LMW-E null models examined in parallel. Lastly, transcriptomic and immune profiling demonstrated that RP-6306 treatment induced interferon responses and T-cell infiltration in the LMW-E-high tumor microenvironment, enhancing the antitumor immune response. These findings highlight the LMW-E/PKMYT1/CDK1 regulatory axis as a promising therapeutic target in TNBC, providing the rationale for further clinical development of PKMYT1 inhibitors in this aggressive breast cancer subtype.
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Inactivating TP53 mutations leads to a loss of function of p53, but can also often result in oncogenic gain-of-function (GOF) of mutant p53 (mutp53) proteins which promotes tumor development and progression. The GOF activities of TP53 mutations are well documented, but the mechanisms involved remain poorly understood. Here, we study the mutp53 interactome and find that by targeting minichromosome maintenance complex components (MCMs), GOF mutp53 predisposes cells to replication stress and chromosomal instability (CIN), leading to a tumor cell-autonomous and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent cytosolic DNA response that activates downstream non-canonical nuclear factor kappa light chain enhancer of activated B cell (NC-NF-κB) signaling. Consequently, GOF mutp53-MCMs-CIN-cytosolic DNA-cGAS-STING-NC-NF-κB signaling promotes tumor cell metastasis and an immunosuppressive tumor microenvironment through antagonizing interferon signaling and regulating genes associated with pro-tumorigenic inflammation. Our findings have important implications for understanding not only the GOF activities of TP53 mutations but also the genome-guardian role of p53 and its inactivation during tumor development and progression.
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Neoplasias , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Neoplasias/genética , ADN , Inestabilidad Cromosómica/genética , Nucleotidiltransferasas/metabolismo , Interferones/metabolismo , Microambiente TumoralRESUMEN
Low-molecular-weight cyclin E (LMW-E) is an N-terminus deleted (40 amino acid) form of cyclin E detected in breast cancer, but not in normal cells or tissues. LMW-E overexpression predicts poor survival in breast cancer patients independent of tumor proliferation rate, but the oncogenic mechanism of LMW-E and its unique function(s) independent of full-length cyclin E (FL-cycE) remain unclear. In the current study, we found LMW-E was associated with genomic instability in early-stage breast tumors (n = 725) and promoted genomic instability in human mammary epithelial cells (hMECs). Mechanistically, FL-cycE overexpression inhibited the proliferation of hMECs by replication stress and DNA damage accumulation, but LMW-E facilitated replication stress tolerance by upregulating DNA replication and damage repair. Specifically, LMW-E interacted with chromatin and upregulated the loading of minichromosome maintenance complex proteins (MCMs) in a CDC6 dependent manner and promoted DNA repair in a RAD51- and C17orf53-dependent manner. Targeting the ATR-CHK1-RAD51 pathway with ATR inhibitor (ceralasertib), CHK1 inhibitor (rabusertib), or RAD51 inhibitor (B02) significantly decreased the viability of LMW-E-overexpressing hMECs and breast cancer cells. Collectively, our findings delineate a novel role for LMW-E in tumorigenesis mediated by replication stress tolerance and genomic instability, providing novel therapeutic strategies for LMW-E-overexpressing breast cancers.
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Neoplasias de la Mama , Ciclina E , Humanos , Femenino , Ciclina E/genética , Ciclina E/metabolismo , Neoplasias de la Mama/patología , Quinasa 2 Dependiente de la Ciclina/genética , Biomarcadores de Tumor/metabolismo , Inestabilidad Genómica , Inhibidores de Proteínas Quinasas/farmacología , Replicación del ADN/genética , Daño del ADN/genética , Reparación del ADN/genéticaRESUMEN
The structure of the genome shapes the distribution of genetic diversity and sequence divergence. To investigate how the relationship between chromosome size and recombination rate affects sequence divergence between species, we combined empirical analyses and evolutionary simulations. We estimated pairwise sequence divergence among 15 species from three different mammalian clades-Peromyscus rodents, Mus mice, and great apes-from chromosome-level genome assemblies. We found a strong significant negative correlation between chromosome size and sequence divergence in all species comparisons within the Peromyscus and great apes clades but not the Mus clade, suggesting that the dramatic chromosomal rearrangements among Mus species may have masked the ancestral genomic landscape of divergence in many comparisons. Our evolutionary simulations showed that the main factor determining differences in divergence among chromosomes of different sizes is the interplay of recombination rate and selection, with greater variation in larger populations than in smaller ones. In ancestral populations, shorter chromosomes harbor greater nucleotide diversity. As ancestral populations diverge, diversity present at the onset of the split contributes to greater sequence divergence in shorter chromosomes among daughter species. The combination of empirical data and evolutionary simulations revealed that chromosomal rearrangements, demography, and divergence times may also affect the relationship between chromosome size and divergence, thus deepening our understanding of the role of genome structure in the evolution of species divergence.
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Evolución Molecular , Hominidae , Animales , Cromosomas/genética , Genoma , Hominidae/genética , Mamíferos/genética , Recombinación GenéticaRESUMEN
Focal amplifications (FA) can mediate targeted therapy resistance in cancer. Understanding the structure and dynamics of FAs is critical for designing treatments that overcome plasticity-mediated resistance. We developed a melanoma model of dual MAPK inhibitor (MAPKi) resistance that bears BRAFV600 amplifications through either extrachromosomal DNA (ecDNA)/double minutes (DM) or intrachromosomal homogenously staining regions (HSR). Cells harboring BRAFV600E FAs displayed mode switching between DMs and HSRs, from both de novo genetic changes and selection of preexisting subpopulations. Plasticity is not exclusive to ecDNAs, as cells harboring HSRs exhibit drug addiction-driven structural loss of BRAF amplicons upon dose reduction. FA mechanisms can couple with kinase domain duplications and alternative splicing to enhance resistance. Drug-responsive amplicon plasticity is observed in the clinic and can involve other MAPK pathway genes, such as RAF1 and NRAS. BRAF FA-mediated dual MAPKi-resistant cells are more sensitive to proferroptotic drugs, extending the spectrum of ferroptosis sensitivity in MAPKi resistance beyond cases of dedifferentiation. SIGNIFICANCE: Understanding the structure and dynamics of oncogene amplifications is critical for overcoming tumor relapse. BRAF amplifications are highly plastic under MAPKi dosage challenges in melanoma, through involvement of de novo genomic alterations, even in the HSR mode. Moreover, BRAF FA-driven, dual MAPKi-resistant cells extend the spectrum of resistance-linked ferroptosis sensitivity. This article is highlighted in the In This Issue feature, p. 873.
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Melanoma , Proteínas Proto-Oncogénicas B-raf , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Mutación , Recurrencia Local de Neoplasia/tratamiento farmacológico , Oncogenes , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.
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Corteza Suprarrenal , Adenoma Corticosuprarrenal , Humanos , Zona Reticular/metabolismo , Adenoma Corticosuprarrenal/genética , Adenoma Corticosuprarrenal/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/metabolismo , Corticoesteroides/metabolismo , Línea CelularRESUMEN
BRAF-activating mutations are the most frequent driver mutations in papillary thyroid cancer (PTC). Targeted inhibitors such as dabrafenib have been used in advanced BRAF-mutated PTC; however, acquired resistance to the drug is common and little is known about other effectors that may play integral roles in this resistance. In addition, the induction of PTC dedifferentiation into highly aggressive KRAS-driven anaplastic thyroid cancer (ATC) has been reported. We detected a novel RAC1 (P34R) mutation acquired during dabrafenib treatment in a progressive metastatic lesion with ATC phenotype. To identify a potential functional link between this novel mutation and tumor dedifferentiation, we developed a cell line derived from the metastatic lesion and compared its behavior to isogenic cell lines and primary tumor samples. Our data demonstrated that RAC1 mutations induce changes in cell morphology, reorganization of F-actin almost exclusively at the cell cortex, and changes in cell adhesion properties. We also established that RAC1 amplification, with or without mutation, is sufficient to drive cell proliferation and resistance to BRAF inhibition. Further, we identified polyploidy of chromosome 7, which harbors RAC1, in both the metastatic lesion and its derived cell line. Copy number amplification and overexpression of other genes located on this chromosome, such as TWIST1, EGFR, and MET were also detected, which might also lead to dabrafenib resistance. Our study suggests that polyploidy leading to increased expression of specific genes, particularly those located on chromosome 7, should be considered when analyzing aggressive thyroid tumor samples and in further treatments.
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Inflammatory bowel disease (IBD) is a chronic inflammatory condition driven by diverse genetic and nongenetic programs that converge to disrupt immune homeostasis in the intestine. We have reported that, in murine intestinal epithelium with telomere dysfunction, DNA damage-induced activation of ataxia-telangiectasia mutated (ATM) results in ATM-mediated phosphorylation and activation of the YAP1 transcriptional coactivator, which in turn up-regulates pro-IL-18, a pivotal immune regulator in IBD pathogenesis. Moreover, individuals with germline defects in telomere maintenance genes experience increased occurrence of intestinal inflammation and show activation of the ATM/YAP1/pro-IL-18 pathway in the intestinal epithelium. Here, we sought to determine the relevance of the ATM/YAP1/pro-IL-18 pathway as a potential driver of IBD, particularly older-onset IBD. Analysis of intestinal biopsy specimens and organoids from older-onset IBD patients documented the presence of telomere dysfunction and activation of the ATM/YAP1/precursor of interleukin 18 (pro-IL-18) pathway in the intestinal epithelium. Employing intestinal organoids from healthy individuals, we demonstrated that experimental induction of telomere dysfunction activates this inflammatory pathway. In organoid models from ulcerative colitis and Crohn's disease patients, pharmacological interventions of telomerase reactivation, suppression of DNA damage signaling, or YAP1 inhibition reduced pro-IL-18 production. Together, these findings support a model wherein telomere dysfunction in the intestinal epithelium can initiate the inflammatory process in IBD, pointing to therapeutic interventions for this disease.
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Enfermedades Inflamatorias del Intestino/inmunología , Telómero/inmunología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/inmunología , Humanos , Enfermedades Inflamatorias del Intestino/genética , Interleucina-18/genética , Interleucina-18/inmunología , Mucosa Intestinal/inmunología , Ratones , Telomerasa/genética , Telomerasa/inmunología , Telómero/genética , Proteínas Señalizadoras YAP/genética , Proteínas Señalizadoras YAP/inmunologíaRESUMEN
Our knowledge of copy number evolution during the expansion of primary breast tumours is limited1,2. Here, to investigate this process, we developed a single-cell, single-molecule DNA-sequencing method and performed copy number analysis of 16,178 single cells from 8 human triple-negative breast cancers and 4 cell lines. The results show that breast tumours and cell lines comprise a large milieu of subclones (7-22) that are organized into a few (3-5) major superclones. Evolutionary analysis suggests that after clonal TP53 mutations, multiple loss-of-heterozygosity events and genome doubling, there was a period of transient genomic instability followed by ongoing copy number evolution during the primary tumour expansion. By subcloning single daughter cells in culture, we show that tumour cells rediversify their genomes and do not retain isogenic properties. These data show that triple-negative breast cancers continue to evolve chromosome aberrations and maintain a reservoir of subclonal diversity during primary tumour growth.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular , Células Clonales/metabolismo , Células Clonales/patología , Evolución Molecular , Secuencia de Bases , Línea Celular Tumoral , Linaje de la Célula , Aberraciones Cromosómicas , Variaciones en el Número de Copia de ADN/genética , Análisis Mutacional de ADN , Inestabilidad Genómica/genética , Humanos , Pérdida de Heterocigocidad/genética , Modelos Genéticos , Tasa de Mutación , Imagen Individual de Molécula , Análisis de la Célula Individual , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Despite efforts in understanding its underlying mechanisms, the etiology of chromosomal instability (CIN) remains unclear for many tumor types. Here, we identify CIN initiation as a previously undescribed function for APOBEC3A (A3A), a cytidine deaminase upregulated across cancer types. Using genetic mouse models of pancreatic ductal adenocarcinoma (PDA) and genomics analyses in human tumor cells we show that A3A-induced CIN leads to aggressive tumors characterized by enhanced early dissemination and metastasis in a STING-dependent manner and independently of the canonical deaminase functions of A3A. We show that A3A upregulation recapitulates numerous copy number alterations commonly observed in patients with PDA, including co-deletions in DNA repair pathway genes, which in turn render these tumors susceptible to poly (ADP-ribose) polymerase inhibition. Overall, our results demonstrate that A3A plays an unexpected role in PDA as a specific driver of CIN, with significant effects on disease progression and treatment.
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Citidina Desaminasa , Neoplasias Pancreáticas , Animales , Inestabilidad Cromosómica/genética , Citidina Desaminasa/genética , Humanos , Ratones , Neoplasias Pancreáticas/genética , Proteínas/genética , Neoplasias PancreáticasRESUMEN
BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic. METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, γ-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients. RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients. CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.
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Inestabilidad Cromosómica , Neoplasias Colorrectales/genética , Neoplasias Experimentales/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Aurora Quinasa B/metabolismo , Azoximetano/toxicidad , Carcinogénesis/genética , Línea Celular Tumoral , Colon/citología , Colon/patología , Neoplasias Colorrectales/inducido químicamente , Neoplasias Colorrectales/patología , Análisis Citogenético , Dextranos/toxicidad , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Transgénicos , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/patología , Organoides , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genéticaRESUMEN
Renal medullary carcinoma (RMC) is a highly lethal malignancy that mainly afflicts young individuals of African descent and is resistant to all targeted agents used to treat other renal cell carcinomas. Comprehensive genomic and transcriptomic profiling of untreated primary RMC tissues was performed to elucidate the molecular landscape of these tumors. We found that RMC was characterized by high replication stress and an abundance of focal copy-number alterations associated with activation of the stimulator of the cyclic GMP-AMP synthase interferon genes (cGAS-STING) innate immune pathway. Replication stress conferred a therapeutic vulnerability to drugs targeting DNA-damage repair pathways. Elucidation of these previously unknown RMC hallmarks paves the way to new clinical trials for this rare but highly lethal malignancy.
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Biomarcadores de Tumor/metabolismo , Carcinoma Medular/patología , Carcinoma de Células Renales/patología , Aberraciones Cromosómicas , Replicación del ADN , Neoplasias Renales/patología , Proteína SMARCB1/metabolismo , Adulto , Animales , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma Medular/genética , Carcinoma Medular/inmunología , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/inmunología , Proliferación Celular , Estudios de Cohortes , Variaciones en el Número de Copia de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Renales/genética , Neoplasias Renales/inmunología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Desnudos , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteína SMARCB1/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Exploration of novel strategies to extend the benefit of PARP inhibitors beyond BRCA-mutant cancers is of great interest in personalized medicine. Here, we identified EGFR amplification as a potential biomarker to predict sensitivity to PARP inhibition, providing selection for the glioblastoma (GBM) patient population who will benefit from PARP inhibition therapy. EXPERIMENTAL DESIGN: Selective sensitivity to the PARP inhibitor talazoparib was screened and validated in two sets [test set (n = 14) and validation set (n = 13)] of well-characterized patient-derived glioma sphere-forming cells (GSC). FISH was used to detect EGFR copy number. DNA damage response following talazoparib treatment was evaluated by γH2AX and 53BP1 staining and neutral comet assay. PARP-DNA trapping was analyzed by subcellular fractionation. The selective monotherapy of talazoparib was confirmed using in vivo glioma models. RESULTS: EGFR-amplified GSCs showed remarkable sensitivity to talazoparib treatment. EGFR amplification was associated with increased reactive oxygen species (ROS) and subsequent increased basal expression of DNA-repair pathways to counterelevated oxidative stress, and thus rendered vulnerability to PARP inhibition. Following talazoparib treatment, EGFR-amplified GSCs showed enhanced DNA damage and increased PARP-DNA trapping, which augmented the cytotoxicity. EGFR amplification-associated selective sensitivity was further supported by the in vivo experimental results showing that talazoparib significantly suppressed tumor growth in EGFR-amplified subcutaneous models but not in nonamplified models. CONCLUSIONS: EGFR-amplified cells are highly sensitive to talazoparib. Our data provide insight into the potential of using EGFR amplification as a selection biomarker for the development of personalized therapy.
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Neoplasias Encefálicas/tratamiento farmacológico , Daño del ADN , Amplificación de Genes , Glioblastoma/tratamiento farmacológico , Estrés Oxidativo , Ftalazinas/farmacología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Receptores ErbB/genética , Glioblastoma/genética , Glioblastoma/patología , Humanos , Masculino , Ratones , Ratones Desnudos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Esferoides Celulares , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Though human prostate cancer (PCa) heterogeneity can best be studied using multiple cell types isolated from clinical specimens, the difficulty of establishing cell lines from clinical tumors has hampered this approach. In this proof-of-concept study, we established a human PCa cell line from a prostatectomy surgical specimen without the need for retroviral transduction. In a previous report, we characterized the stromal cells derived from PCa specimens. Here, we characterized the epithelial cells isolated from the same tumors. Compared to the ease of establishing prostate stromal cell lines, prostatic epithelial cell lines are challenging. From three matched pairs of normal and tumor tissues, we established one new PCa cell line, HPE-15. We confirmed the origin of HPE-15 cells by short tandem repeat microsatellite polymorphism analysis. HPE-15 cells are androgen-insensitive and express marginal androgen receptor, prostate-specific antigen and prostate-specific membrane antigen proteins. HPE-15 expresses luminal epithelial markers of E-cadherin and cytokeratin 18, basal cell markers of cytokeratin 5 and p63 and neuroendocrine marker of chromogranin A. Interestingly, HPE-15 Cells exhibited no tumorigenicity in different strains of immune-deficient mice but can become tumorigenic through interaction with aggressive cancer cell types. HPE-15 cells can thus serve as an experimental model for the study of PCa progression, metastasis and tumor cell dormancy.
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Células Epiteliales/citología , Mesodermo/citología , Próstata/citología , Neoplasias de la Próstata/patología , Células del Estroma/citología , Animales , Carcinogénesis , Comunicación Celular , Línea Celular , Línea Celular Transformada , Línea Celular Tumoral , Células Epiteliales/metabolismo , Humanos , Calicreínas/metabolismo , Masculino , Mesodermo/metabolismo , Ratones , Próstata/metabolismo , Antígeno Prostático Específico/metabolismo , Prostatectomía , Neoplasias de la Próstata/metabolismo , Células del Estroma/metabolismo , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Bone marrow (BM) sclerosis is commonly found in patients with late-stage myelofibrosis (MF). Because osteoclasts (OCs) and osteoblasts play a key role in bone remodeling, and MF monocytes, the OC precursors, are derived from the neoplastic clone, we wondered whether decreased OC numbers or impairment in their osteolytic function affects the development of osteosclerosis. Analysis of BM biopsies from 50 MF patients showed increased numbers of multinucleated tartrate-resistant acid phosphatase (TRAP)/cathepsin K+ OCs expressing phosphorylated Janus kinase 2 (JAK2). Randomly microdissected TRAP+ OCs from 16 MF patients harbored JAK2 or calreticulin (CALR) mutations, confirming MF OCs are clonal. To study OC function, CD14+ monocytes from MF patients and healthy individuals were cultured and differentiated into OCs. Unlike normal OCs, MF OCs appeared small and round, with few protrusions, and carried the mutations and chromosomal abnormalities of neoplastic clones. In addition, MF OCs lacked F-actin-rich ring-like structures and had fewer nuclei and reduced colocalization signals, compatible with decreased fusion events, and their mineral resorption capacity was significantly reduced, indicating impaired osteolytic function. Taken together, our data suggest that, although the numbers of MF OCs are increased, their impaired osteolytic activity distorts bone remodeling and contributes to the induction of osteosclerosis.
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Remodelación Ósea , Osteoclastos , Osteólisis , Mielofibrosis Primaria , Calreticulina/metabolismo , Catepsina K/genética , Catepsina K/metabolismo , Femenino , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Masculino , Mutación , Osteoclastos/metabolismo , Osteoclastos/patología , Osteólisis/genética , Osteólisis/metabolismo , Osteólisis/patología , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/metabolismo , Mielofibrosis Primaria/patología , Fosfatasa Ácida Tartratorresistente/genética , Fosfatasa Ácida Tartratorresistente/metabolismoRESUMEN
HIV-1 integration into the host genome favors actively transcribed genes. Prior work indicated that the nuclear periphery provides the architectural basis for integration site selection, with viral capsid-binding host cofactor CPSF6 and viral integrase-binding cofactor LEDGF/p75 contributing to selection of individual sites. Here, by investigating the early phase of infection, we determine that HIV-1 traffics throughout the nucleus for integration. CPSF6-capsid interactions allow the virus to bypass peripheral heterochromatin and penetrate the nuclear structure for integration. Loss of interaction with CPSF6 dramatically alters virus localization toward the nuclear periphery and integration into transcriptionally repressed lamina-associated heterochromatin, while loss of LEDGF/p75 does not significantly affect intranuclear HIV-1 localization. Thus, CPSF6 serves as a master regulator of HIV-1 intranuclear localization by trafficking viral preintegration complexes away from heterochromatin at the periphery toward gene-dense chromosomal regions within the nuclear interior.
Asunto(s)
Cápside/metabolismo , Núcleo Celular/virología , ADN Viral/genética , Infecciones por VIH/metabolismo , VIH-1/fisiología , Integración Viral , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , ADN Viral/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Interacciones Huésped-Patógeno , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Unión Proteica , Replicación Viral , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
PURPOSE: To evaluate the incidence of cMET proto-oncogene aberration in a cohort of triple negative breast cancers using immunohistochemistry and fluorescence in situ hybridization (FISH) methods and correlated with patient outcome. PATIENTS AND METHODS: One hundred and six female patients with diagnosis of triple negative invasive breast carcinoma at The University of Texas-M D Anderson Cancer Center from 1983 to 2009 were included in the study. Expression of cMET was assessed by IHC using rabbit monoclonal anti-total cMET antibody (SP44 from Ventana). Staining intensity was scored on a scale of 0, 1+, 2+ and 3+. cMET overexpression was defined as at least moderate membranous/cytoplasmic staining in ≥50% of tumor cells (scoreâ¯≥â¯2+). FISH analysis was performed using MET (7q31) specific probe (BAC clone RP11-95i20, Abbott Molecular Inc.) and the centromere probe (CEP7/D7Z1, Abbott Molecular Inc.) as internal control. cMET amplification was defined as gene copy numbers ≥4 per cell or cMET/CEP7 ratioâ¯≥â¯2. cMET status was tested for correlation using Fisher's exact test with other clinicopathological parameters. The Kaplan-Meier product limit method was used to estimate the survival outcomes. Cox proportional hazards models were fit to determine the association of cMET status by IHC, or by FISH, or by copy number with survival outcomes after adjustment for other patient and disease characteristics. RESULTS: Medium follow up is 69.4â¯months (range 9-317â¯months). cMET was successfully evaluated by both IHC and FISH methods in ninety-six patients. There were 13 patients whose tumors overexpressed cMET was by IHC. Two patients had cMET amplification by FISH using definitive of cMET/CEP7 ratio of ≥2 and four patients had cMET copy number >4. Only one patient showed cMET/CEP7 ratio of 2.53 and one was positive for cMET overexpression by IHC. No significant association between cMET overexpression by IHC and by FISH using cut-off of with either cMET/CEP7 ratio of ≥2 or cMET copy number of >4 (Pâ¯=â¯1.0). There was no significant correlation between the cMET overexpression and other clinicopathological characteristics, such as patient demographics, tumor grade, stage, or chemotherapy treatment history. cMET overexpression and gene amplification did not correlate with the prognosis of TNBC regarding OS or DFS. CONCLUSION: MET amplification is a rare incidence in TNBCs. cMET overexpression is infrequent in TNBCs and may not be driven by gene amplification. Neither have significant prognostic value nor do they correlate with other clinicopathological characteristics in this TNBC cohort.
Asunto(s)
Carcinoma Ductal de Mama/genética , Amplificación de Genes , Proteínas Proto-Oncogénicas c-met/genética , Neoplasias de la Mama Triple Negativas/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Persona de Mediana Edad , Proto-Oncogenes Mas , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
To understand how genomic heterogeneity of glioblastoma (GBM) contributes to poor therapy response, we performed DNA and RNA sequencing on GBM samples and the neurospheres and orthotopic xenograft models derived from them. We used the resulting dataset to show that somatic driver alterations including single-nucleotide variants, focal DNA alterations and oncogene amplification on extrachromosomal DNA (ecDNA) elements were in majority propagated from tumor to model systems. In several instances, ecDNAs and chromosomal alterations demonstrated divergent inheritance patterns and clonal selection dynamics during cell culture and xenografting. We infer that ecDNA was unevenly inherited by offspring cells, a characteristic that affects the oncogenic potential of cells with more or fewer ecDNAs. Longitudinal patient tumor profiling found that oncogenic ecDNAs are frequently retained throughout the course of disease. Our analysis shows that extrachromosomal elements allow rapid increase of genomic heterogeneity during GBM evolution, independently of chromosomal DNA alterations.