RESUMO
Cancer stem cells (CSCs) are thought to drive tumor growth, metastasis and chemoresistance. Although surface markers such as CD133 and CD44 have been successfully used to isolate CSCs, their expression is not exclusively linked to the CSC phenotype and is prone to environmental alteration. We identified cells with an autofluorescent subcellular compartment that exclusively showed CSC features across different human tumor types. Primary tumor-derived autofluorescent cells did not overlap with side-population (SP) cells, were enriched in sphere culture and during chemotherapy, strongly expressed pluripotency-associated genes, were highly metastatic and showed long-term in vivo tumorigenicity, even at the single-cell level. Autofluorescence was due to riboflavin accumulation in membrane-bounded cytoplasmic structures bearing ATP-dependent ABCG2 transporters. In summary, we identified and characterized an intrinsic autofluorescent phenotype in CSCs of diverse epithelial cancers and used this marker to isolate and characterize these cells.
Assuntos
Biomarcadores Tumorais/metabolismo , Separação Celular/métodos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Imagem Óptica/métodos , Riboflavina/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Autofagia , Proteína 12 Relacionada à Autofagia , Carcinoma Hepatocelular/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Ductal Pancreático/patologia , Neoplasias Colorretais/patologia , Feminino , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/biossíntese , Células Tumorais CultivadasRESUMO
OBJECTIVES: The tumour stroma/microenvironment not only provides structural support for tumour development, but more importantly it provides cues to cancer stem cells (CSCs) that regulate their self-renewal and metastatic potential. This is certainly true for pancreatic ductal adenocarcinomas (PDAC), where tumour-associated fibroblasts, pancreatic stellate cells and immune cells create an abundant paracrine niche for CSCs via microenvironment-secreted factors. Thus understanding the role that tumour stroma cells play in PDAC development and CSC biology is of utmost importance. DESIGN: Microarray analyses, tumour microarray immunohistochemical assays, in vitro co-culture experiments, recombinant protein treatment approaches and in vivo intervention studies were performed to understand the role that the immunomodulatory cationic antimicrobial peptide 18/LL-37 (hCAP-18/LL-37) plays in PDAC biology. RESULTS: We found that hCAP-18/LL-37 was strongly expressed in the stroma of advanced primary and secondary PDAC tumours and is secreted by immune cells of the stroma (eg, tumour-associated macrophages) in response to tumour growth factor-ß1 and particularly CSC-secreted Nodal/ActivinA. Treatment of pancreatic CSCs with recombinant LL-37 increased pluripotency-associated gene expression, self-renewal, invasion and tumourigenicity via formyl peptide receptor 2 (FPR2)- and P2X purinoceptor 7 receptor (P2X7R)-dependent mechanisms, which could be reversed by inhibiting these receptors. Importantly, in a genetically engineered mouse model of K-Ras-driven pancreatic tumourigenesis, we also showed that tumour formation was inhibited by either reconstituting these mice with bone marrow from cathelicidin-related antimicrobial peptide (ie, murine homologue of hCAP-18/LL-37) knockout mice or by pharmacologically inhibiting FPR2 and P2X7R. CONCLUSIONS: Thus, hCAP-18/LL-37 represents a previously unrecognised PDAC microenvironment factor that plays a critical role in pancreatic CSC-mediated tumourigenesis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Ativinas/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinoma Ductal Pancreático/genética , Autorrenovação Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Pancreáticas/genética , Análise Serial de Proteínas , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores de Formil Peptídeo/antagonistas & inibidores , Receptores de Formil Peptídeo/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise Serial de Tecidos , Fator de Crescimento Transformador beta1/farmacologia , CatelicidinasRESUMO
OBJECTIVE: Cancer stem cells (CSCs) represent the root of many solid cancers including pancreatic ductal adenocarcinoma, are highly chemoresistant and represent the cellular source for disease relapse. However the mechanisms involved in these processes still need to be fully elucidated. Understanding the mechanisms implicated in chemoresistance and metastasis of pancreatic cancer is critical to improving patient outcomes. DESIGN: Micro-RNA (miRNA) expression analyses were performed to identify functionally defining epigenetic signatures in pancreatic CSC-enriched sphere-derived cells and gemcitabine-resistant pancreatic CSCs. RESULTS: We found the miR-17-92 cluster to be downregulated in chemoresistant CSCs versus non-CSCs and demonstrate its crucial relevance for CSC biology. In particular, overexpression of miR-17-92 reduced CSC self-renewal capacity, in vivo tumourigenicity and chemoresistance by targeting multiple NODAL/ACTIVIN/TGF-ß1 signalling cascade members as well as directly inhibiting the downstream targets p21, p57 and TBX3. Overexpression of miR-17-92 translated into increased CSC proliferation and their eventual exhaustion via downregulation of p21 and p57. Finally, the translational impact of our findings could be confirmed in preclinical models for pancreatic cancer. CONCLUSIONS: Our findings therefore identify the miR-17-92 cluster as a functionally determining family of miRNAs in CSCs, and highlight the putative potential of developing modulators of this cluster to overcome drug resistance in pancreatic CSCs.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/metabolismo , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Ativinas/metabolismo , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Autorrenovação Celular , Transformação Celular Neoplásica , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Regulação para Baixo , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteína Nodal/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , RNA Longo não Codificante , Transdução de Sinais , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Transcriptoma , Fator de Crescimento Transformador beta1/metabolismo , GencitabinaRESUMO
Diaphyseal medullary stenosis with malignant fibrous histiocytoma (DMS-MFH) is an autosomal-dominant syndrome characterized by bone dysplasia, myopathy, and bone cancer. We previously mapped the DMS-MFH tumor-suppressing-gene locus to chromosomal region 9p21-22 but failed to identify mutations in known genes in this region. We now demonstrate that DMS-MFH results from mutations in the most proximal of three previously uncharacterized terminal exons of the gene encoding methylthioadenosine phosphorylase, MTAP. Intriguingly, two of these MTAP exons arose from early and independent retroviral-integration events in primate genomes at least 40 million years ago, and since then, their genomic integration has gained a functional role. MTAP is a ubiquitously expressed homotrimeric-subunit enzyme critical to polyamine metabolism and adenine and methionine salvage pathways and was believed to be encoded as a single transcript from the eight previously described exons. Six distinct retroviral-sequence-containing MTAP isoforms, each of which can physically interact with archetype MTAP, have been identified. The disease-causing mutations occur within one of these retroviral-derived exons and result in exon skipping and dysregulated alternative splicing of all MTAP isoforms. Our results identify a gene involved in the development of bone sarcoma, provide evidence of the primate-specific evolution of certain parts of an existing gene, and demonstrate that mutations in parts of this gene can result in human disease despite its relatively recent origin.
Assuntos
Doenças do Desenvolvimento Ósseo/genética , Neoplasias Ósseas/genética , Genoma , Histiocitoma Fibroso Benigno/genética , Síndromes Neoplásicas Hereditárias/genética , Purina-Núcleosídeo Fosforilase/genética , Retroviridae/genética , Processamento Alternativo/genética , Animais , Sequência de Bases , Evolução Biológica , Cromossomos Humanos Par 9/genética , Éxons , Humanos , Isoenzimas/genética , Dados de Sequência Molecular , Distrofias Musculares/genética , Mutação , Primatas/genética , Sarcoma/genéticaRESUMO
Alternative splicing represents a unique post-transcriptional mechanism that increases the complexity of the eukaryotic proteome-generating protein isoforms whose functions can be novel, diverse, and/or even antagonistic when compared to its full-length transcript. The KLF family of genes consists of ≥17 members, which are involved in the regulation of numerous critical cellular processes, including differentiation, cell proliferation, growth-related signal transduction, angiogenesis, and apoptosis. Using a strategy based on RT-PCR, selective cloning, and promoter-based assays of cancer-relevant genes, we identify and characterize the existence of multiple biologically active KLF splice forms across the entire family of proteins. We demonstrate biological function for a number of these isoforms. Furthermore, we highlight a possible functional interaction between full-length KLF4 and one of its splice variants in up-regulating cellular proliferation. Taken together, this report identifies for the first time a more complete view of the genomic and proteomic breadth and complexity of the KLF transcription factor family, revealing the existence of highly expressed and biologically active isoforms previously uncharacterized. In essence, knowing that these KLF isoforms exist provides the first step toward understanding the roles of these genes in human health and disease.
Assuntos
Processamento Alternativo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sequência de Bases , Feminino , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Células MCF-7 , Masculino , Modelos Biológicos , Dados de Sequência Molecular , Família Multigênica , Gravidez , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual , TransfecçãoRESUMO
The identification of cancer stem cells (CSCs), which are implicated in tumor initiation, progression, therapy resistance, and relapse, is of great biological and clinical relevance. In glioblastoma (GBM), this is still a challenge, as no single marker is able to universally identify populations of GBM cancer stem cells (GSCs). Indeed, there is still controversy on whether biomarker-expressing cells fulfill the functional criteria of bona fide GSCs, despite being widely used. Here, we describe a novel subpopulation of autofluorescent (Fluo+) cells in GBM that bear all the functional characteristics of GSCs, including higher capacity to grow as neurospheres, long-term self-renewal ability, increased expression of stem cell markers, and enhanced in vivo tumorigenicity. Mechanistically, the autofluorescent phenotype is largely due to the intracellular accumulation of riboflavin, mediated by the ABC transporter ABCG2. In summary, our work identifies an intrinsic cellular autofluorescent phenotype enriched in GBM cells with functional stem cells features that can be used as a novel, simple and reliable biomarker to target these highly malignant tumors, with implications for GBM biological and clinical research.
RESUMO
Pancreatic ductal adenocarcinoma is one of the deadliest carcinomas and is characterized by highly tumorigenic and metastatic cancer stem cells (CSC). CSCs evade available therapies, which preferentially target highly proliferative and more differentiated progenies, leaving behind CSCs as a putative source for disease relapse. Thus, to identify potentially more effective treatment regimens, we screened established and new compounds for their ability to eliminate CSCs in primary pancreatic cancer (stem) cells in vitro and corresponding patient-derived pancreatic cancer tissue xenografts in vivo. Intriguingly, we found that in vitro treatment with the antimalarial agent chloroquine significantly decreased CSCs, translating into diminished in vivo tumorigenicity and invasiveness in a large panel of pancreatic cancers. In vivo treatment in combination with gemcitabine was capable of more effectively eliminating established tumors and improved overall survival. The inhibitory effect of chloroquine was not related to inhibition of autophagy, but was due to inhibition of CXCL12/CXCR4 signaling, resulting in reduced phosphorylation of ERK and STAT3. Furthermore, chloroquine showed potent inhibition of hedgehog signaling by decreasing the production of Smoothened, translating into a significant reduction in sonic hedgehog-induced chemotaxis and downregulation of downstream targets in CSCs and the surrounding stroma. Our study demonstrates that via to date unreported effects, chloroquine is an effective adjuvant therapy to chemotherapy, offering more efficient tumor elimination and improved cure rates. Chloroquine should be further explored in the clinical setting as its success may help to more rapidly improve the poor prognosis of patients with pancreatic cancer.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Cloroquina/farmacologia , Proteínas Hedgehog/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Receptores CXCR4/antagonistas & inibidores , Animais , Autofagia/efeitos dos fármacos , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Distribuição Aleatória , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Since the identification of self-renewing cells in the hematopoietic system several decades ago, stem cells have changed the way we study biology and medicine. Solid tumors contain a distinct subpopulation of cells that have stem cell characteristics and are exclusively responsible for tumorigenicity. This discovery has led to the development of the stem cell concept of cancer, which proposes that a subpopulation of self-renewing tumor cells, also termed cancer stem cells, is responsible for tumorigenesis and metastasis. This contrasts with the stochastic model of tumor development, which holds that all tumor cells are capable of tumor initiation. Different subpopulations of cancer stem cells have been identified in pancreatic ductal adenocarcinoma, based on the use of combinations of surface markers that allow their isolation, propagation, and further characterization. Importantly, cancer stem cells are not only capable of self-renewal and differentiation, but may also confer virulence via immune system evasion and multidrug resistance, and potentially via vasculogenic mimicry and transition to migratory and metastasizing derivatives. Therapeutic targeting of this subset of cells and the pathways defining their virulence holds great promise for the development of more effective strategies for the amelioration and eradication of this most lethal form of cancer.
Assuntos
Carcinoma Ductal Pancreático/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/terapia , Humanos , Modelos Biológicos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/terapiaRESUMO
Nodal and Activin belong to the TGF-ß superfamily and are important regulators of embryonic stem cell fate. Here we investigated whether Nodal and Activin regulate self-renewal of pancreatic cancer stem cells. Nodal and Activin were hardly detectable in more differentiated pancreatic cancer cells, while cancer stem cells and stroma-derived pancreatic stellate cells markedly overexpressed Nodal and Activin, but not TGF-ß. Knockdown or pharmacological inhibition of the Nodal/Activin receptor Alk4/7 in cancer stem cells virtually abrogated their self-renewal capacity and in vivo tumorigenicity, and reversed the resistance of orthotopically engrafted cancer stem cells to gemcitabine. However, engrafted primary human pancreatic cancer tissue with a substantial stroma showed no response due to limited drug delivery. The addition of a stroma-targeting hedgehog pathway inhibitor enhanced delivery of the Nodal/Activin inhibitor and translated into long-term, progression-free survival. Therefore, inhibition of the Alk4/7 pathway, if combined with hedgehog pathway inhibition and gemcitabine, provides a therapeutic strategy for targeting cancer stem cells.