RESUMO
The cellular changes during an epithelial-mesenchymal transition (EMT) largely rely on global changes in gene expression orchestrated by transcription factors. Tead transcription factors and their transcriptional co-activators Yap and Taz have been previously implicated in promoting an EMT; however, their direct transcriptional target genes and their functional role during EMT have remained elusive. We have uncovered a previously unanticipated role of the transcription factor Tead2 during EMT. During EMT in mammary gland epithelial cells and breast cancer cells, levels of Tead2 increase in the nucleus of cells, thereby directing a predominant nuclear localization of its co-factors Yap and Taz via the formation of Tead2-Yap-Taz complexes. Genome-wide chromatin immunoprecipitation and next generation sequencing in combination with gene expression profiling revealed the transcriptional targets of Tead2 during EMT. Among these, zyxin contributes to the migratory and invasive phenotype evoked by Tead2. The results demonstrate that Tead transcription factors are crucial regulators of the cellular distribution of Yap and Taz, and together they control the expression of genes critical for EMT and metastasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/biossíntese , Transição Epitelial-Mesenquimal/fisiologia , Fosfoproteínas/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Zixina/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Feminino , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Fosfoproteínas/genética , Transdução de Sinais , Fatores de Transcrição de Domínio TEA , Transativadores , Fatores de Transcrição/genética , Ativação Transcricional , Proteínas de Sinalização YAP , Zixina/genéticaRESUMO
Acquiring resistance against transforming growth factor ß (TGFß)-induced growth inhibition at early stages of carcinogenesis and shifting to TGFß's tumour-promoting functions at later stages is a pre-requisite for malignant tumour progression and metastasis. We have identified the transcription factor distal-less homeobox 2 (Dlx2) to exert critical functions during this switch. Dlx2 counteracts TGFß-induced cell-cycle arrest and apoptosis in mammary epithelial cells by at least two molecular mechanisms: Dlx2 acts as a direct transcriptional repressor of TGFß receptor II (TGFßRII) gene expression and reduces canonical, Smad-dependent TGFß signalling and expression of the cell-cycle inhibitor p21(CIP1) and increases expression of the mitogenic transcription factor c-Myc. On the other hand, Dlx2 directly induces the expression of the epidermal growth factor (EGF) family member betacellulin, which promotes cell survival by stimulating EGF receptor signalling. Finally, Dlx2 expression supports experimental tumour growth and metastasis of B16 melanoma cells and correlates with tumour malignancy in a variety of human cancer types. These results establish Dlx2 as one critical player in shifting TGFß from its tumour suppressive to its tumour-promoting functions.
Assuntos
Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Genes Supressores de Tumor/fisiologia , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The Wnt receptor ROR1 has generated increased interest as a cancer therapeutic target. Research on several therapeutic approaches involving this receptor is ongoing; however, ROR1 tissue expression remains understudied. We performed an immunohistochemistry analysis of ROR1 protein expression in a large cohort of multiple tumor and histologic types. We analyzed 12 anonymized multi-tumor tissue microarrays (TMAs), including mesothelioma, esophageal and upper gastrointestinal carcinomas, and uterine endometrioid carcinoma, among other tumor types. Additionally, we studied 5 different sarcoma types of TMAs and 6 patient-derived xenografts (PDX) TMAs developed from 19 different anatomic sites and tumor histologic types. A total of 1142 patient cases from different histologic types and 140 PDXs placed in TMAs were evaluated. Pathologists assessed the percentage of tumor cells in each case that were positive for ROR1 and the intensity of staining. For determining the prevalence of staining for each tumor type, a case was considered positive if >1% of its tumor cells showed ROR1 staining. Our immunohistochemistry assays revealed a heterogeneous ROR1 expression profile. A high prevalence of ROR1 expression was found in mesothelioma (84.6%), liposarcoma (36.1%), gastrointestinal stromal tumors (33.3%), and uterine endometrioid carcinoma (28.9%). Other histologic types such as breast, lung, renal cell, hepatocellular, urothelial carcinoma, and colon carcinomas; glioblastoma; cholangiocarcinoma; and leiomyosarcoma showed less ROR1 overall expression, ranging between 0.9 and 13%. No ROR1 expression was seen in mesenchymal chondrosarcoma, rhabdomyosarcoma, or gastric adenocarcinoma cases. Overall, ROR1 expression was relatively infrequent and low in most tumor types investigated; however, ROR1 expression was infrequent but high in selected tumor types, such as gastroesophageal GIST, suggesting that ROR1 prescreening may be preferable for those indications. Further, mesothelioma exhibited frequent and high levels of ROR1 expression, which represents a previously unrecognized therapeutic opportunity. These findings can contribute to the development of ROR1-targeted therapies.
RESUMO
The mammalian Golgi apparatus consists of individual cisternae that are stacked in a polarized manner to form the compact zones of the Golgi. Several stacks are linked to form a ribbon via dynamic lateral bridges. The determinants required for maintaining the characteristic Golgi structure are incompletely understood. Here, we have characterized p28, a new gamma-subfamily member of p24 membrane proteins. p28 localized to endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and cis Golgi and accumulated in the ERGIC upon Brefeldin A treatment, typical for a protein cycling in the early secretory pathway. p28 interacted with a subset of p24 proteins. Its depletion by small interfering RNA (siRNA) led to fragmentation of the Golgi without affecting the overall organization of microtubules but considerably reducing the amount of acetylated tubulin. The distribution of COPI and tethers, including GM130, was not affected. At the ultrastructural level, the Golgi fragments appeared as mini-stacks with apparently unchanged cis-trans topology. Golgi fragmentation did not impair anterograde or retrograde traffic. Fluorescence recovery after photobleaching (FRAP) experiments revealed that silencing p28 prevents protein exchange between Golgi stacks during reassembly after Brefeldin A-induced Golgi breakdown. These results show that the formation of a Golgi ribbon requires the structural membrane protein p28 in addition to previously identified SNAREs, coat proteins and tethers.
Assuntos
Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Retículo Endoplasmático/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Complexo de Golgi/metabolismo , Células HeLa , Células Hep G2 , Humanos , Immunoblotting , Imunoprecipitação , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Dados de Sequência Molecular , Subunidades Proteicas , Transporte Proteico , Alinhamento de SequênciaRESUMO
Antibody-drug conjugates directed against tumor-specific targets have allowed targeted delivery of highly potent chemotherapy to malignant cells while sparing normal cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein with limited expression on normal adult tissues and is overexpressed on the surface of malignant cells in mantle cell lymphoma, acute lymphocytic leukemia with t(1;19)(q23;p13) translocation, and chronic lymphocytic leukemia. This differential expression makes ROR1 an attractive target for antibody-drug conjugate therapy, especially in malignancies such as mantle cell lymphoma and acute lymphocytic leukemia, in which systemic chemotherapy remains the gold standard. Several preclinical and phase 1 clinical studies have established the safety and effectiveness of anti-ROR1 monoclonal antibody-based therapies. Herein we describe a humanized, first-in-class anti-ROR1 antibody-drug conjugate, huXBR1-402-G5-PNU, which links a novel anti-ROR1 antibody (huXBR1-402) to a highly potent anthracycline derivative (PNU). We found that huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ malignant cells in vitro and suppressed leukemia proliferation and extended survival in multiple models of mice engrafted with human ROR1+ leukemia. Lastly, we show that the B-cell lymphoma 2 (BCL2)-dependent cytotoxicity of huXBR1-402-G5-PNU can be leveraged by combined treatment strategies with the BCL2 inhibitor venetoclax. Together, our data present compelling preclinical evidence for the efficacy of huXBR1-402-G5-PNU in treating ROR1+ hematologic malignancies.
Assuntos
Neoplasias Hematológicas , Imunoconjugados , Leucemia Linfocítica Crônica de Células B , Linfoma de Célula do Manto , Animais , Anticorpos Monoclonais , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , CamundongosRESUMO
Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the de novo discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B lymphocytes ("Transpo-mAb Display") for ROR2 binding. Individual cellular "Transpo-mAb" clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs.
Assuntos
Anticorpos Monoclonais Humanizados/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Imunoconjugados/isolamento & purificação , Neoplasias/terapia , Células Precursoras de Linfócitos B/fisiologia , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunização , Imunoconjugados/farmacologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Imunotoxinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/imunologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/imunologia , Análise de Célula Única , Éxons VDJ/genéticaRESUMO
Antibody-drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the specific targeting of mAbs with the potency of small-molecule toxic payloads. ADCs generated by conventional chemical conjugation yield heterogeneous mixtures with variable pharmacokinetics, stability, safety, and efficacy profiles. To address these issues, numerous site-specific conjugation technologies are currently being developed allowing the manufacturing of homogeneous ADCs with predetermined drug-to-antibody ratios. Here, we used sortase-mediated antibody conjugation (SMAC) technology to generate homogeneous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a noncleavable peptide linker, using the anti-HER2 antibody trastuzumab (part of Kadcyla) and the anti-CD30 antibody cAC10 (part of Adcetris). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conventional tubulin-targeting payloads, such as Kadcyla and Adcetris. The data presented here suggest that such novel and highly potent ADC formats may help to increase the number of targets available to ADC approaches, by reducing the threshold levels of target expression required. Mol Cancer Ther; 16(5); 879-92. ©2017 AACR.
Assuntos
Antraciclinas/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Imunoconjugados/administração & dosagem , Neoplasias/tratamento farmacológico , Ado-Trastuzumab Emtansina , Aminoaciltransferases/química , Animais , Antraciclinas/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/química , Brentuximab Vedotin , Linhagem Celular Tumoral , Cisteína Endopeptidases/química , Doxorrubicina/administração & dosagem , Doxorrubicina/análogos & derivados , Doxorrubicina/química , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Antígeno Ki-1/química , Antígeno Ki-1/imunologia , Maitansina/análogos & derivados , Maitansina/química , Maitansina/imunologia , Camundongos , Neoplasias/imunologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Trastuzumab/administração & dosagem , Trastuzumab/química , Trastuzumab/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates.
Assuntos
Anticorpos Monoclonais/análise , Técnicas de Visualização da Superfície Celular/métodos , Ensaios de Triagem em Larga Escala/métodos , Imunoglobulina G/análise , Animais , Elementos de DNA Transponíveis , Avaliação Pré-Clínica de Medicamentos/métodos , Vetores Genéticos , HumanosRESUMO
Gene expression profiling has uncovered the transcription factor Sox4 with upregulated activity during TGF-ß-induced epithelial-mesenchymal transition (EMT) in normal and cancerous breast epithelial cells. Sox4 is indispensable for EMT and cell survival in vitro and for primary tumor growth and metastasis in vivo. Among several EMT-relevant genes, Sox4 directly regulates the expression of Ezh2, encoding the Polycomb group histone methyltransferase that trimethylates histone 3 lysine 27 (H3K27me3) for gene repression. Ablation of Ezh2 expression prevents EMT, whereas forced expression of Ezh2 restores EMT in Sox4-deficient cells. Ezh2-mediated H3K27me3 marks associate with key EMT genes, representing an epigenetic EMT signature that predicts patient survival. Our results identify Sox4 as a master regulator of EMT by governing the expression of the epigenetic modifier Ezh2.
Assuntos
Epigênese Genética , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/genética , Complexo Repressor Polycomb 2/genética , Fatores de Transcrição SOXC/fisiologia , Animais , Linhagem Celular , Movimento Celular/genética , Sobrevivência Celular/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Histonas/metabolismo , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Metilação , Camundongos , Metástase Neoplásica/genética , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/fisiologia , Regiões Promotoras Genéticas , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Transcrição GênicaRESUMO
INTRODUCTION: Increasing evidence supports a role of an epithelial to mesenchymal transition (EMT) process in endowing subsets of tumor cells with properties driving malignant tumor progression and resistance to cancer therapy. To advance our understanding of the underlying mechanisms, we sought to generate a transplantable cellular model system that allows defined experimental manipulation and analysis of EMT in vitro and at the same time recapitulates oncogenic EMT in vivo. METHODOLOGY/RESULTS: We have established a stable murine breast cancer cell line (Py2T) from a breast tumor of an MMTV-PyMT transgenic mouse. Py2T cells display a metastable epithelial phenotype characterized by concomitant expression of luminal and basal cytokeratins and sheet migration. Exposure of Py2T cells to transforming growth factor ß (TGFß) in vitro induces reversible EMT accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers, including EMT transcription factors, and a gain in single cell motility and invasiveness. Py2T cells give rise to tumors after orthotopic injection into syngeneic FVB/N mice. Notably, transplantation of epithelial Py2T cells results in the formation of invasive primary tumors with low to absent E-cadherin expression, indicating that the cells undergo EMT-like changes in vivo. This process appears to at least in part depend on TGFß signaling, since tumors formed by Py2T cells expressing a dominant-negative version of TGFß receptor widely maintain their epithelial differentiation status. CONCLUSIONS/SIGNIFICANCE: Together, the data demonstrate that the Py2T cell line represents a versatile model system to study the EMT process in vitro and in vivo. The observation that Py2T cells give rise to tumors and collectively undergo EMT-like changes in vivo highlights the suitability of the Py2T model system as a tool to study tumor-related EMT. In particular, Py2T cells may serve to corroborate recent findings relating EMT to cancer cell stemness, to therapy resistance and to tumor recurrence.