RESUMEN
Chimeric antigen receptor (CAR) T cell therapy effectively treats human cancer, but the loss of the antigen recognized by the CAR poses a major obstacle. We found that in vivo vaccine boosting of CAR T cells triggers the engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR T promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited the priming of endogenous anti-tumor T cells. This process was accompanied by shifts in CAR T metabolism toward oxidative phosphorylation (OXPHOS) and was critically dependent on CAR-T-derived IFN-γ. Antigen spreading (AS) induced by vaccine-boosted CAR T enabled a proportion of complete responses even when the initial tumor was 50% CAR antigen negative, and heterogeneous tumor control was further enhanced by the genetic amplification of CAR T IFN-γ expression. Thus, CAR-T-cell-derived IFN-γ plays a critical role in promoting AS, and vaccine boosting provides a clinically translatable strategy to drive such responses against solid tumors.
Asunto(s)
Vacunas contra el Cáncer , Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Neoplasias/terapia , Linfocitos T , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
For over a century, fasting regimens have improved health, lifespan and tissue regeneration in diverse organisms, including humans1-6. However, how fasting and post-fast refeeding affect adult stem cells and tumour formation has yet to be explored in depth. Here we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation; post-fast refeeding augments the regenerative capacity of Lgr5+ ISCs, and loss of the tumour suppressor gene Apc in post-fast-refed ISCs leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum-fed states, demonstrating that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust mTORC1 induction in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production or protein synthesis abrogates the regenerative or tumorigenic effects of post-fast refeeding. Given our findings, fast-refeeding cycles must be carefully considered and tested when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst in stem-cell-driven regeneration and tumorigenicity.
RESUMEN
Aneuploidy, defined as whole-chromosome gain or loss, causes cellular stress but, paradoxically, is a frequent occurrence in cancers. Here, we investigate why â¼50% of Ewing sarcomas, driven by the EWS-FLI1 fusion oncogene, harbor chromosome 8 gains. Expression of the EWS-FLI1 fusion in primary cells causes replication stress that can result in cellular senescence. Using an evolution approach, we show that trisomy 8 mitigates EWS-FLI1-induced replication stress through gain of a copy of RAD21. Low-level ectopic expression of RAD21 is sufficient to dampen replication stress and improve proliferation in EWS-FLI1-expressing cells. Conversely, deleting one copy in trisomy 8 cells largely neutralizes the fitness benefit of chromosome 8 gain and reduces tumorgenicity of a Ewing sarcoma cancer cell line in soft agar assays. We propose that RAD21 promotes tumorigenesis through single gene copy gain. Such genes may explain some recurrent aneuploidies in cancer.
Asunto(s)
Carcinogénesis/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Sarcoma de Ewing/genética , Estrés Fisiológico/genética , Trisomía/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Cromosomas Humanos Par 8/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Duplicación de Gen/genética , Regulación Neoplásica de la Expresión Génica , HumanosRESUMEN
Aneuploidy causes a proliferative disadvantage in all normal cells analyzed to date, yet this condition is associated with a disease characterized by unabated proliferative potential, cancer. The mechanisms that allow cancer cells to tolerate the adverse effects of aneuploidy are not known. To probe this question, we identified aneuploid yeast strains with improved proliferative abilities. Their molecular characterization revealed strain-specific genetic alterations as well as mutations shared between different aneuploid strains. Among the latter, a loss-of-function mutation in the gene encoding the deubiquitinating enzyme Ubp6 improves growth rates in four different aneuploid yeast strains by attenuating the changes in intracellular protein composition caused by aneuploidy. Our results demonstrate the existence of aneuploidy-tolerating mutations that improve the fitness of multiple different aneuploidies and highlight the importance of ubiquitin-proteasomal degradation in suppressing the adverse effects of aneuploidy.
Asunto(s)
Aneuploidia , Saccharomyces cerevisiae/genética , Proliferación Celular , Aberraciones Cromosómicas , Endopeptidasas/genética , Eliminación de Gen , Humanos , Neoplasias/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismoRESUMEN
BACKGROUND: The yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins by deleting unneeded endogenous proteins. In non-model microorganisms such as K. phaffii, however, genetic engineering is limited by lack gene annotation and knowledge of gene essentiality. RESULTS: We identified a set of endogenous secreted proteins in K. phaffii by mass spectrometry and signal peptide prediction. Our efforts to disrupt these genes were hindered by limited annotation of essential genes. To predict essential genes, therefore, we designed, transformed, and sequenced a pooled library of guide RNAs for CRISPR-Cas9-mediated knockout of all endogenous secreted proteins. We then used predicted gene essentiality to guide iterative disruptions of up to 11 non-essential genes. Engineered strains exhibited a ~20× increase in the production of human serum albumin and a twofold increase in the production of a monoclonal antibody. CONCLUSIONS: We demonstrated that disruption of as few as six genes can increase production of recombinant proteins. Further reduction of the endogenous proteome of K. phaffii may further improve strain performance. The pooled library of secretome-targeted guides for CRISPR-Cas9 and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant proteins and enzymes.
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Sistemas CRISPR-Cas , Proteínas Recombinantes , Saccharomycetales , Saccharomycetales/genética , Saccharomycetales/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Humanos , Técnicas de Inactivación de Genes/métodos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteoma/metabolismo , Anticuerpos Monoclonales/biosíntesis , Albúmina Sérica Humana/genética , Albúmina Sérica Humana/metabolismoRESUMEN
Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.
Asunto(s)
Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , Ingeniería de Proteínas/métodos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales , Sitios de Unión , COVID-19/virología , Vacunas contra la COVID-19/economía , Humanos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Unión Proteica , Conformación Proteica , Saccharomycetales/metabolismo , Vacunas de SubunidadRESUMEN
Aneuploidy, a condition characterized by whole chromosome gains and losses, is often associated with significant cellular stress and decreased fitness. However, how cells respond to the aneuploid state has remained controversial. In aneuploid budding yeast, two opposing gene-expression patterns have been reported: the "environmental stress response" (ESR) and the "common aneuploidy gene-expression" (CAGE) signature, in which many ESR genes are oppositely regulated. Here, we investigate this controversy. We show that the CAGE signature is not an aneuploidy-specific gene-expression signature but the result of normalizing the gene-expression profile of actively proliferating aneuploid cells to that of euploid cells grown into stationary phase. Because growth into stationary phase is among the strongest inducers of the ESR, the ESR in aneuploid cells was masked when stationary phase euploid cells were used for normalization in transcriptomic studies. When exponentially growing euploid cells are used in gene-expression comparisons with aneuploid cells, the CAGE signature is no longer evident in aneuploid cells. Instead, aneuploid cells exhibit the ESR. We further show that the ESR causes selective ribosome loss in aneuploid cells, providing an explanation for the decreased cellular density of aneuploid cells. We conclude that aneuploid budding yeast cells mount the ESR, rather than the CAGE signature, in response to aneuploidy-induced cellular stresses, resulting in selective ribosome loss. We propose that the ESR serves two purposes in aneuploid cells: protecting cells from aneuploidy-induced cellular stresses and preventing excessive cellular enlargement during slowed cell cycles by down-regulating translation capacity.
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Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico/genética , Aneuploidia , Ambiente , Regulación Fúngica de la Expresión Génica , ARN de Hongos/genética , ARN de Hongos/metabolismo , Análisis de Secuencia de ARN , Transcriptoma/genéticaRESUMEN
Despite the fact that the majority of lung cancer deaths are due to metastasis, the molecular mechanisms driving metastatic progression are poorly understood. Here, we present evidence that loss of Foxa2 and Cdx2 synergizes with loss of Nkx2-1 to fully activate the metastatic program. These three lineage-specific transcription factors are consistently down-regulated in metastatic cells compared with nonmetastatic cells. Knockdown of these three factors acts synergistically and is sufficient to promote the metastatic potential of nonmetastatic cells to that of naturally arising metastatic cells in vivo. Furthermore, silencing of these three transcription factors is sufficient to account for a significant fraction of the gene expression differences between the nonmetastatic and metastatic states in lung adenocarcinoma, including up-regulated expression of the invadopodia component Tks5long, the embryonal proto-oncogene Hmga2, and the epithelial-to-mesenchymal mediator Snail. Finally, analyses of tumors from a genetically engineered mouse model and patients show that low expression of Nkx2-1, Foxa2, and Cdx2 strongly correlates with more advanced tumors and worse survival. Our findings reveal that a large part of the complex transcriptional network in metastasis can be controlled by a small number of regulatory nodes that function redundantly, and loss of multiple nodes is required to fully activate the metastatic program.
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Adenocarcinoma/fisiopatología , Factor Nuclear 3-beta del Hepatocito/metabolismo , Proteínas de Homeodominio/metabolismo , Neoplasias Pulmonares/fisiopatología , Metástasis de la Neoplasia/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/mortalidad , Adenocarcinoma del Pulmón , Animales , Animales Modificados Genéticamente , Factor de Transcripción CDX2 , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Factor Nuclear 3-beta del Hepatocito/genética , Proteínas de Homeodominio/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Ratones , Ratones Desnudos , Proteínas Nucleares/genética , Proto-Oncogenes Mas , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genéticaRESUMEN
Prevention of COVID-19 on a global scale will require the continued development of high-volume, low-cost platforms for the manufacturing of vaccines to supply ongoing demand. Vaccine candidates based on recombinant protein subunits remain important because they can be manufactured at low costs in existing large-scale production facilities that use microbial hosts like Komagataella phaffii (Pichia pastoris). Here, we report an improved and scalable manufacturing approach for the SARS-CoV-2 spike protein receptor-binding domain (RBD); this protein is a key antigen for several reported vaccine candidates. We genetically engineered a manufacturing strain of K. phaffii to obviate the requirement for methanol induction of the recombinant gene. Methanol-free production improved the secreted titer of the RBD protein by >5X by alleviating protein folding stress. Removal of methanol from the production process enabled to scale up to a 1200 L pre-existing production facility. This engineered strain is now used to produce an RBD-based vaccine antigen that is currently in clinical trials and could be used to produce other variants of RBD as needed for future vaccines.
RESUMEN
Aneuploidy, defined as chromosome gains and losses, is a hallmark of cancer. However, compared with other tumor types, extensive aneuploidy is relatively rare in prostate cancer. Thus, whether numerical chromosome aberrations dictate disease progression in prostate cancer patients is not known. Here, we report the development of a method based on whole-transcriptome profiling that allowed us to identify chromosome-arm gains and losses in 333 primary prostate tumors. In two independent cohorts (n = 404) followed prospectively for metastases and prostate cancer-specific death for a median of 15 years, increasing extent of tumor aneuploidy as predicted from the tumor transcriptome was strongly associated with higher risk of lethal disease. The 23% of patients whose tumors had five or more predicted chromosome-arm alterations had 5.3 times higher odds of lethal cancer (95% confidence interval, 2.2 to 13.1) than those with the same Gleason score and no predicted aneuploidy. Aneuploidy was associated with lethality even among men with high-risk Gleason score 8-to-10 tumors. These results point to a key role of aneuploidy in driving aggressive disease in primary prostate cancer.
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Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Adulto , Anciano , Aneuploidia , Aberraciones Cromosómicas , Progresión de la Enfermedad , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor/métodos , Estadificación de Neoplasias/métodos , Próstata/patología , Transcriptoma/genéticaRESUMEN
The threat of viral pandemics demands a comprehensive understanding of evolution at the host-pathogen interface. Here, we show that the accessibility of adaptive mutations in influenza nucleoprotein at fever-like temperatures is mediated by host chaperones. Particularly noteworthy, we observe that the Pro283 nucleoprotein variant, which (1) is conserved across human influenza strains, (2) confers resistance to the Myxovirus resistance protein A (MxA) restriction factor, and (3) critically contributed to adaptation to humans in the 1918 pandemic influenza strain, is rendered unfit by heat shock factor 1 inhibition-mediated host chaperone depletion at febrile temperatures. This fitness loss is due to biophysical defects that chaperones are unavailable to address when heat shock factor 1 is inhibited. Thus, influenza subverts host chaperones to uncouple the biophysically deleterious consequences of viral protein variants from the benefits of immune escape. In summary, host proteostasis plays a central role in shaping influenza adaptation, with implications for the evolution of other viruses, for viral host switching, and for antiviral drug development.
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Adaptación Fisiológica , Interacciones Huésped-Patógeno , Evasión Inmune , Sistema Inmunológico/virología , Inmunidad Innata , Chaperonas Moleculares/metabolismo , Orthomyxoviridae/inmunología , Secuencia de Aminoácidos , Animales , Fenómenos Biofísicos , Análisis Mutacional de ADN , Perros , Humanos , Células de Riñón Canino Madin Darby , Modelos Biológicos , Proteínas de Resistencia a Mixovirus/metabolismo , Nucleoproteínas/química , Estructura Secundaria de Proteína , Temperatura , Proteínas Virales/químicaRESUMEN
BACKGROUND: Vaccines comprising recombinant subunit proteins are well-suited to low-cost and high-volume production for global use. The design of manufacturing processes to produce subunit vaccines depends, however, on the inherent biophysical traits presented by an individual antigen of interest. New candidate antigens typically require developing custom processes for each one and may require unique steps to ensure sufficient yields without product-related variants. RESULTS: We describe a holistic approach for the molecular design of recombinant protein antigens-considering both their manufacturability and antigenicity-informed by bioinformatic analyses such as RNA-seq, ribosome profiling, and sequence-based prediction tools. We demonstrate this approach by engineering the product sequences of a trivalent non-replicating rotavirus vaccine (NRRV) candidate to improve titers and mitigate product variants caused by N-terminal truncation, hypermannosylation, and aggregation. The three engineered NRRV antigens retained their original antigenicity and immunogenicity, while their improved manufacturability enabled concomitant production and purification of all three serotypes in a single, end-to-end perfusion-based process using the biotechnical yeast Komagataella phaffii. CONCLUSIONS: This study demonstrates that molecular engineering of subunit antigens using advanced genomic methods can facilitate their manufacturing in continuous production. Such capabilities have potential to lower the cost and volumetric requirements in manufacturing vaccines based on recombinant protein subunits.
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Antígenos Virales/genética , Ingeniería Genética/métodos , Vacunas contra Rotavirus/genética , Rotavirus/inmunología , Saccharomycetales/genética , Antígenos Virales/inmunología , Biología Computacional , Genómica/métodos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Rotavirus/genética , Vacunas contra Rotavirus/inmunología , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunologíaRESUMEN
Tissue-specific differentiation programs become dysregulated during cancer evolution. The transcription factor Nkx2-1 is a master regulator of pulmonary differentiation that is downregulated in poorly differentiated lung adenocarcinoma. Here we use conditional murine genetics to determine how the identity of lung epithelial cells changes upon loss of their master cell-fate regulator. Nkx2-1 deletion in normal and neoplastic lungs causes not only loss of pulmonary identity but also conversion to a gastric lineage. Nkx2-1 is likely to maintain pulmonary identity by recruiting transcription factors Foxa1 and Foxa2 to lung-specific loci, thus preventing them from binding gastrointestinal targets. Nkx2-1-negative murine lung tumors mimic mucinous human lung adenocarcinomas, which express gastric markers. Loss of the gastrointestinal transcription factor Hnf4α leads to derepression of the embryonal proto-oncogene Hmga2 in Nkx2-1-negative tumors. These observations suggest that loss of both active and latent differentiation programs is required for tumors to reach a primitive, poorly differentiated state.
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Adenocarcinoma/metabolismo , Diferenciación Celular , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Adenocarcinoma/patología , Animales , Sitios de Unión , Proliferación Celular , Transformación Celular Neoplásica , Mucosa Gástrica/metabolismo , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Hiperplasia/metabolismo , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/patología , Ratones , Ratones Transgénicos , Mutación Missense , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Especificidad de Órganos , Unión Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Estómago/patología , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Activación Transcripcional , Transcriptoma , Carga TumoralRESUMEN
Classically, the unfolded protein response (UPR) safeguards secretory pathway proteostasis. The most ancient arm of the UPR, the IRE1-activated spliced X-box binding protein 1 (XBP1s)-mediated response, has roles in secretory pathway maturation beyond resolving proteostatic stress. Understanding the consequences of XBP1s activation for cellular processes is critical for elucidating mechanistic connections between XBP1s and development, immunity, and disease. Here, we show that a key functional output of XBP1s activation is a cell type-dependent shift in the distribution of N-glycan structures on endogenous membrane and secreted proteomes. For example, XBP1s activity decreased levels of sialylation and bisecting GlcNAc in the HEK293 membrane proteome and secretome, while substantially increasing the population of oligomannose N-glycans only in the secretome. In HeLa cell membranes, stress-independent XBP1s activation increased the population of high-mannose and tetraantennary N-glycans, and also enhanced core fucosylation. mRNA profiling experiments suggest that XBP1s-mediated remodeling of the N-glycome is, at least in part, a consequence of coordinated transcriptional resculpting of N-glycan maturation pathways by XBP1s. The discovery of XBP1s-induced N-glycan structural remodeling on a glycome-wide scale suggests that XBP1s can act as a master regulator of N-glycan maturation. Moreover, because the sugars on cell-surface proteins or on proteins secreted from an XBP1s-activated cell can be molecularly distinct from those of an unactivated cell, these findings reveal a potential new mechanism for translating intracellular stress signaling into altered interactions with the extracellular environment.
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Polisacáridos/metabolismo , Proteína 1 de Unión a la X-Box/metabolismo , Línea Celular , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Manosa/metabolismo , Proteoma/metabolismo , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
BACKGROUND: Individuals with peanut allergy range in clinical sensitivity: some can consume grams of peanut before experiencing any symptoms, whereas others suffer systemic reactions to 10 mg or less. Current diagnostic testing only partially predicts this clinical heterogeneity. OBJECTIVE: We sought to identify characteristics of the peanut-specific CD4+ T-cell response in peanut-allergic patients that correlate with high clinical sensitivity. METHODS: We studied the T-cell receptor ß-chain (TCRß) usage and phenotypes of peanut-activated, CD154+ CD4+ memory T cells using fluorescence-activated cell sorting, TCRß sequencing, and RNA-Seq, in reactive and hyporeactive patients who were stratified by clinical sensitivity. RESULTS: TCRß analysis of the CD154+ and CD154- fractions revealed more than 6000 complementarity determining region 3 sequences and motifs that were significantly enriched in the activated cells and 17% of the sequences were shared between peanut-allergic individuals, suggesting strong convergent selection of peanut-specific clones. These clones were more numerous among the reactive patients, and this expansion was identified within effector, but not regulatory T-cell populations. The transcriptional profile of CD154+ T cells in the reactive group skewed toward a polarized TH2 effector phenotype, and expression of TH2 cytokines strongly correlated with peanut-specific IgE levels. There were, however, also non-TH2-related differences in phenotype. Furthermore, the ratio of peanut-specific clones in the effector versus regulatory T-cell compartment, which distinguished the clinical groups, was independent of specific IgE concentration. CONCLUSIONS: Expansion of the peanut-specific effector T-cell repertoire is correlated with clinical sensitivity, and this observation may be useful to inform our assessment of disease phenotype and to monitor disease longitudinally.
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Citocinas/inmunología , Memoria Inmunológica , Hipersensibilidad al Cacahuete/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Células Th2/inmunología , Adulto , Femenino , Humanos , Masculino , Hipersensibilidad al Cacahuete/patología , Células Th2/patologíaRESUMEN
MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function.
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Fibroblastos/citología , Regulación de la Expresión Génica , MicroARNs/genética , MicroARNs/metabolismo , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares/metabolismo , Animales , Antígenos de Neoplasias/metabolismo , Línea Celular , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Fibroblastos/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Unión Proteica , Ribonucleasa III/genética , Ribonucleasa III/metabolismoRESUMEN
Metastasis accounts for the vast majority of cancer-related deaths, yet the molecular mechanisms that drive metastatic spread remain poorly understood. Here we report that Tks5, which has been linked to the formation of proteolytic cellular protrusions known as invadopodia, undergoes an isoform switch during metastatic progression in a genetically engineered mouse model of lung adenocarcinoma. Nonmetastatic primary tumor-derived cells predominantly expressed a short isoform, Tks5short, while metastatic primary tumor- and metastasis-derived cells acquired increased expression of the full-length isoform Tks5long. This elevation of Tks5long to Tks5short ratio correlated with a commensurate increase in invadopodia activity in metastatic cells compared with nonmetastatic cells. Further characterization of these isoforms by knockdown and overexpression experiments demonstrated that Tks5long promoted invadopodia in vitro and increased metastasis in transplant models and an autochthonous model of lung adenocarcinoma. Conversely, Tks5short decreased invadopodia stability and proteolysis, acting as a natural dominant-negative inhibitor to Tks5long. Importantly, high Tks5long and low Tks5short expressions in human lung adenocarcinomas correlated with metastatic disease and predicted worse survival of early stage patients. These data indicate that tipping the Tks5 isoform balance to a high Tks5long to Tks5short ratio promotes invadopodia-mediated invasion and metastasis.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Fosfoproteínas/genética , Adenocarcinoma/mortalidad , Adenocarcinoma del Pulmón , Animales , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/mortalidad , Ratones , Ratones Desnudos , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Proteínas de Unión a Fosfato , Isoformas de Proteínas , Análisis de SupervivenciaRESUMEN
Komagataella phaffii, also known as Pichia pastoris, is a common host for the production of biologics and enzymes, due to fast growth, high productivity, and advancements in host engineering. Several K. phaffii variants are commonly used as interchangeable base strains, which confounds efforts to improve this host. In this study, genomic and transcriptomic analyses of Y-11430 (CBS7435), GS115, X-33, and eight other variants enabled a comparative assessment of the relative fitness of these hosts for recombinant protein expression. Cell wall integrity explained the majority of the variation among strains, impacting transformation efficiency, growth, methanol metabolism, and secretion of heterologous proteins. Y-11430 exhibited the highest activity of genes involved in methanol utilization, up to two-fold higher transcription of heterologous genes, and robust growth. With a more permeable cell wall, X-33 displayed a six-fold higher transformation efficiency and up to 1.2-fold higher titers than Y-11430. X-33 also shared nearly all mutations, and a defective variant of HIS4, with GS115, precluding robust growth. Transferring two beneficial mutations identified in X-33 into Y-11430 resulted in an optimized base strain that provided up to four-fold higher transformation efficiency and three-fold higher protein titers, while retaining robust growth. The approach employed here to assess unique banked variants in a species and then transfer key beneficial variants into a base strain should also facilitate rational assessment of a broad set of other recombinant hosts.
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Proteínas Fúngicas/genética , Genoma Fúngico/genética , Pichia/genética , Proteínas Recombinantes/genética , Transcriptoma/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Genómica , Pichia/metabolismo , ARN de Hongos/análisis , ARN de Hongos/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Análisis de Secuencia de ARNRESUMEN
Small cell lung cancer (SCLC) is an aggressive cancer often diagnosed after it has metastasized. Despite the need to better understand this disease, SCLC remains poorly characterized at the molecular and genomic levels. Using a genetically engineered mouse model of SCLC driven by conditional deletion of Trp53 and Rb1 in the lung, we identified several frequent, high-magnitude focal DNA copy number alterations in SCLC. We uncovered amplification of a novel, oncogenic transcription factor, Nuclear factor I/B (Nfib), in the mouse SCLC model and in human SCLC. Functional studies indicate that NFIB regulates cell viability and proliferation during transformation.
Asunto(s)
Factores de Transcripción NFI/genética , Factores de Transcripción NFI/metabolismo , Oncogenes/fisiología , Carcinoma Pulmonar de Células Pequeñas/genética , Animales , Animales Modificados Genéticamente , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Oncogenes/genéticaRESUMEN
Chemotherapy resistance is a major obstacle in cancer treatment, yet the mechanisms of response to specific therapies have been largely unexplored in vivo. Employing genetic, genomic, and imaging approaches, we examined the dynamics of response to a mainstay chemotherapeutic, cisplatin, in multiple mouse models of human non-small-cell lung cancer (NSCLC). We show that lung tumors initially respond to cisplatin by sensing DNA damage, undergoing cell cycle arrest, and inducing apoptosis-leading to a significant reduction in tumor burden. Importantly, we demonstrate that this response does not depend on the tumor suppressor p53 or its transcriptional target, p21. Prolonged cisplatin treatment promotes the emergence of resistant tumors with enhanced repair capacity that are cross-resistant to platinum analogs, exhibit advanced histopathology, and possess an increased frequency of genomic alterations. Cisplatin-resistant tumors express elevated levels of multiple DNA damage repair and cell cycle arrest-related genes, including p53-inducible protein with a death domain (Pidd). We demonstrate a novel role for PIDD as a regulator of chemotherapy response in human lung tumor cells.