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Oncogenic mutations in KRAS are present in approximately 95% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) and are considered the initiating event of pancreatic intraepithelial neoplasia (PanIN) precursor lesions. While it is well established that KRAS mutations drive the activation of oncogenic kinase cascades during pancreatic oncogenesis, the effects of oncogenic KRAS signaling on regulation of phosphatases during this process is not fully appreciated. Protein Phosphatase 2A (PP2A) has been implicated in suppressing KRAS-driven cellular transformation. However, low PP2A activity is observed in PDAC cells compared to non-transformed cells, suggesting that suppression of PP2A activity is an important step in the overall development of PDAC. In the current study, we demonstrate that KRASG12D induces the expression of both an endogenous inhibitor of PP2A activity, Cancerous Inhibitor of PP2A (CIP2A), and the PP2A substrate, c-MYC. Consistent with these findings, KRASG12D sequestered the specific PP2A subunit responsible for c-MYC degradation, B56α, away from the active PP2A holoenzyme in a CIP2A-dependent manner. During PDAC initiation in vivo, knockout of B56α promoted KRASG12D tumorigenesis by accelerating acinar-to-ductal metaplasia (ADM) and the formation of PanIN lesions. The process of ADM was attenuated ex vivo in response to pharmacological re-activation of PP2A utilizing direct small molecule activators of PP2A (SMAPs). Together, our results suggest that suppression of PP2A-B56α through KRAS signaling can promote the MYC-driven initiation of pancreatic tumorigenesis.
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Tumor progression is accompanied by fibrosis, a condition of excessive extracellular matrix accumulation, which is associated with diminished antitumor immune infiltration. Here we demonstrate that tumor-associated macrophages (TAMs) respond to the stiffened fibrotic tumor microenvironment (TME) by initiating a collagen biosynthesis program directed by transforming growth factor-ß. A collateral effect of this programming is an untenable metabolic milieu for productive CD8+ T cell antitumor responses, as collagen-synthesizing macrophages consume environmental arginine, synthesize proline and secrete ornithine that compromises CD8+ T cell function in female breast cancer. Thus, a stiff and fibrotic TME may impede antitumor immunity not only by direct physical exclusion of CD8+ T cells but also through secondary effects of a mechano-metabolic programming of TAMs, which creates an inhospitable metabolic milieu for CD8+ T cells to respond to anticancer immunotherapies.
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Neoplasias de la Mama , Linfocitos T CD8-positivos , Colágeno , Microambiente Tumoral , Macrófagos Asociados a Tumores , Microambiente Tumoral/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Femenino , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Colágeno/metabolismo , Animales , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismo , Humanos , Ratones , Factor de Crecimiento Transformador beta/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Reprogramación MetabólicaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) subsists in a nutrient-deregulated microenvironment, making it particularly susceptible to treatments that interfere with cancer metabolism12. For example, PDAC utilizes and is dependent on high levels of autophagy and other lysosomal processes3-5. Although targeting these pathways has shown potential in preclinical studies, progress has been hampered by the challenge of identifying and characterizing favorable targets for drug development6. Here, we characterize PIKfyve, a lipid kinase integral to lysosomal functioning7, as a novel and targetable vulnerability in PDAC. In human patient and murine PDAC samples, we discovered that PIKFYVE is overexpressed in PDAC cells compared to adjacent normal cells. Employing a genetically engineered mouse model, we established the essential role of PIKfyve in PDAC progression. Further, through comprehensive metabolic analyses, we found that PIKfyve inhibition obligated PDAC to upregulate de novo lipid synthesis, a relationship previously undescribed. PIKfyve inhibition triggered a distinct lipogenic gene expression and metabolic program, creating a dependency on de novo lipid metabolism pathways, by upregulating genes such as FASN and ACACA. In PDAC, the KRAS-MAPK signaling pathway is a primary driver of de novo lipid synthesis, specifically enhancing FASN and ACACA levels. Accordingly, the simultaneous targeting of PIKfyve and KRAS-MAPK resulted in the elimination of tumor burden in a syngeneic orthotopic model and tumor regression in a xenograft model of PDAC. Taken together, these studies suggest that disrupting lipid metabolism through PIKfyve inhibition induces synthetic lethality in conjunction with KRAS-MAPK-directed therapies for PDAC.
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We find that NUPR1, a stress-associated intrinsically disordered protein, induced droplet formation via liquid-liquid phase separation (LLPS). NUPR1-driven LLPS was crucial for the creation of NUPR1-dependent stress granules (SGs) in pancreatic cancer cells since genetic or pharmacological inhibition by ZZW-115 of NUPR1 activity impeded SGs formation. The KrasG12D mutation induced oncogenic stress, NUPR1 overexpression, and promoted SGs development. Notably, enforced NUPR1 expression induced SGs formation independently of mutated KrasG12D. Mechanistically, KrasG12D expression strengthened sensitivity to NUPR1 inactivation, inducing cell death, activating caspase 3 and releasing LDH. Remarkably, ZZW-115-mediated SG-formation inhibition hampered the development of pancreatic intraepithelial neoplasia (PanINs) in Pdx1-cre;LSL-KrasG12D (KC) mice. ZZW-115-treatment of KC mice triggered caspase 3 activation, DNA fragmentation, and formation of the apoptotic bodies, leading to cell death, specifically in KrasG12D-expressing cells. We further demonstrated that, in developed PanINs, short-term ZZW-115 treatment prevented NUPR1-associated SGs presence. Lastly, a four-week ZZW-115 treatment significantly reduced the number and size of PanINs in KC mice. This study proposes that targeting NUPR1-dependent SGs formation could be a therapeutic approach to induce cell death in KrasG12D-dependent tumors.
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Carcinoma in Situ , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Piperazinas , Tiazinas , Animales , Ratones , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Carcinoma Ductal Pancreático/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Gránulos de Estrés , Mutaciones Letales SintéticasRESUMEN
This manuscript has been withdrawn by the authors due to a dispute over co-first authorship that is currently being arbitrated by the medical school at our institution. Therefore, the authors do not wish this work to be cited as reference for the project. Upon completion of the arbitration process, we will take steps to revert the current withdrawn status. If you have any questions, please contact the corresponding author.
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Although KMT2D, also known as MLL2, is known to play an essential role in development, differentiation, and tumor suppression, its role in pancreatic cancer development is not well understood. Here, we discovered a novel signaling axis mediated by KMT2D, which links TGF-ß to the activin A pathway. We found that TGF-ß upregulates a microRNA, miR-147b, which in turn leads to post-transcriptional silencing of KMT2D. Loss of KMT2D induces the expression and secretion of activin A, which activates a noncanonical p38 MAPK-mediated pathway to modulate cancer cell plasticity, promote a mesenchymal phenotype, and enhance tumor invasion and metastasis in mice. We observed a decreased KMT2D expression in human primary and metastatic pancreatic cancer. Furthermore, inhibition or knockdown of activin A reversed the protumoral role of KMT2D loss. These findings support a tumor-suppressive role of KMT2D in pancreatic cancer and identify miR-147b and activin A as novel therapeutic targets.
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MicroARNs , Neoplasias Pancreáticas , Humanos , Animales , Ratones , Plasticidad de la Célula , Línea Celular Tumoral , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias Pancreáticas/patología , Factor de Crecimiento Transformador beta/metabolismo , Activinas/genética , Neoplasias PancreáticasRESUMEN
Cancer cells rely on certain extracellular nutrients to sustain their metabolism and growth. Solute carrier (SLC) transporters enable cells to acquire extracellular nutrients or shuttle intracellular nutrients across organelles. However, the function of many SLC transporters in cancer is unknown. Determining the key SLC transporters promoting cancer growth could reveal important therapeutic opportunities. Here we summarize recent findings and knowledge gaps on SLC transporters in cancer. We highlight existing inhibitors for studying these transporters, clinical trials on treating cancer by blocking transporters, and compensatory transporters used by cancer cells to evade treatment. We propose targeting transporters simultaneously or in combination with targeted therapy or immunotherapy as alternative strategies for effective cancer therapy.
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Proteínas de Transporte de Membrana , Neoplasias , Humanos , Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico , Neoplasias/metabolismo , InmunoterapiaRESUMEN
Pancreatic ductal adenocarcinoma (PDA) continues to have a dismal prognosis. The poor survival of patients with PDA has been attributed to a high rate of early metastasis and low efficacy of current therapies, which partly result from its complex immunosuppressive tumor microenvironment. Previous studies from our group and others have shown that tumor-associated macrophages (TAMs) are instrumental in maintaining immunosuppression in PDA. Here, we explored the role of Notch signaling, a key regulator of immune response, within the PDA microenvironment. We identified Notch pathway components in multiple immune cell types within human and mouse pancreatic cancer. TAMs, the most abundant immune cell population in the tumor microenvironment, express high levels of Notch receptors with cognate ligands such as JAG1 expressed on tumor epithelial cells, endothelial cells and fibroblasts. TAMs with activated Notch signaling expressed higher levels of immunosuppressive mediators including arginase 1 (Arg1) suggesting that Notch signaling plays a role in macrophage polarization within the PDA microenvironment. Combination of Notch inhibition with PD-1 blockade resulted in increased cytotoxic T cell infiltration, tumor cell apoptosis, and smaller tumor size. Our work implicates macrophage Notch signaling in the establishment of immunosuppression and indicates that targeting the Notch pathway may improve the efficacy of immune-based therapies in PDA patients.
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The adult healthy human pancreas has been poorly studied given lack of indication to obtain tissue from the pancreas in the absence of disease and rapid postmortem degradation. We obtained pancreata from brain dead donors thus avoiding any warm ischemia time. The 30 donors were diverse in age and race and had no known pancreas disease. Histopathological analysis of the samples revealed PanIN lesions in most individuals irrespective of age. Using a combination of multiplex immunohistochemistry, single cell RNA sequencing, and spatial transcriptomics, we provide the first ever characterization of the unique microenvironment of the adult human pancreas and of sporadic PanIN lesions. We compared healthy pancreata to pancreatic cancer and peritumoral tissue and observed distinct transcriptomic signatures in fibroblasts, and, to a lesser extent, macrophages. PanIN epithelial cells from healthy pancreata were remarkably transcriptionally similar to cancer cells, suggesting that neoplastic pathways are initiated early in tumorigenesis. Statement of significance: The causes underlying the onset of pancreatic cancer remain largely unknown, hampering early detection and prevention strategies. Here, we show that PanIN are abundant in healthy individuals and present at a much higher rate than the incidence of pancreatic cancer, setting the stage for efforts to elucidate the microenvironmental and cell intrinsic factors that restrain, or, conversely, promote, malignant progression.
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Despite advances in therapy over the past decades, metastatic colorectal cancer (mCRC) remains a highly morbid disease. While the impact of MHC-I on immune infiltration in mCRC has been well studied, data on the consequences of MHC-II loss are lacking. Multiplex fluorescent immunohistochemistry (mfIHC) was performed on 149 patients undergoing curative intent resection for mCRC and stratified into high and low human leukocyte antigen isotype DR (HLA-DR) expressing tumors. Intratumoral HLA-DR expression was found in stromal bands, and its expression level was associated with different infiltrating immune cell makeup and distribution. Low HLA-DR expression was associated with increased intercellular distances and decreased population mixing of T helper cells and antigen-presenting cells (APC), suggestive of decreased interactions. This was associated with less co-localization of tumor cells and cytotoxic T lymphocytes (CTLs), which tended to be in a less activated state as determined by Ki67 and granzyme B expression. These findings suggest that low HLA-DR in the tumor microenvironment of mCRC may reflect a state of poor helper T-cell interactions with APCs and CTL-mediated anti-tumor activity. Efforts to restore/enhance MHC-II presentation may be a useful strategy to enhance checkpoint inhibition therapy in the future.
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BACKGROUND: Pancreatic ductal adenocarcinoma initiation is most frequently caused by Kras mutations. RESULTS: Here, we apply biological, biochemical, and network biology methods to validate GEMM-derived cell models using inducible KrasG12D expression. We describe the time-dependent, chromatin remodeling program that impacts function during early oncogenic signaling. We find that the KrasG12D-induced transcriptional response is dominated by downregulated expression concordant with layers of epigenetic events. More open chromatin characterizes the ATAC-seq profile associated with a smaller group of upregulated genes and epigenetic marks. RRBS demonstrates that promoter hypermethylation does not account for the silencing of the extensive gene promoter network. Moreover, ChIP-Seq reveals that heterochromatin reorganization plays little role in this early transcriptional program. Notably, both gene activation and silencing primarily depend on the marking of genes with a combination of H3K27ac, H3K4me3, and H3K36me3. Indeed, integrated modeling of all these datasets shows that KrasG12D regulates its transcriptional program primarily through unique super-enhancers and enhancers, and marking specific gene promoters and bodies. We also report chromatin remodeling across genomic areas that, although not contributing directly to cis-gene transcription, are likely important for KrasG12D functions. CONCLUSIONS: In summary, we report a comprehensive, time-dependent, and coordinated early epigenomic program for KrasG12D in pancreatic cells, which is mechanistically relevant to understanding chromatin remodeling events underlying transcriptional outcomes needed for the function of this oncogene.
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Reprogramación Celular/genética , Cromatina/metabolismo , Epigénesis Genética , Genes ras , Neoplasias Pancreáticas/genética , Animales , Línea Celular , Núcleo Celular/genética , Metilación de ADN , Genoma , Código de Histonas , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción GenéticaRESUMEN
Cancer metabolism is rewired to support cell survival in response to intrinsic and environmental stressors. Identification of strategies to target these adaptions is an area of active research. We previously described a cytosolic aspartate aminotransaminase (GOT1)-driven pathway in pancreatic cancer used to maintain redox balance. Here, we sought to identify metabolic dependencies following GOT1 inhibition to exploit this feature of pancreatic cancer and to provide additional insight into regulation of redox metabolism. Using pharmacological methods, we identify cysteine, glutathione, and lipid antioxidant function as metabolic vulnerabilities following GOT1 withdrawal. We demonstrate that targeting any of these pathways triggers ferroptosis, an oxidative, iron-dependent form of cell death, in GOT1 knockdown cells. Mechanistically, we reveal that GOT1 inhibition represses mitochondrial metabolism and promotes a catabolic state. Consequently, we find that this enhances labile iron availability through autophagy, which potentiates the activity of ferroptotic stimuli. Overall, our study identifies a biochemical connection between GOT1, iron regulation, and ferroptosis.
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Aspartato Aminotransferasa Citoplasmática/antagonistas & inhibidores , Ferroptosis , Neoplasias Pancreáticas/metabolismo , Animales , Antioxidantes/farmacología , Aspartato Aminotransferasa Citoplasmática/genética , Aspartato Aminotransferasa Citoplasmática/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Cistina/metabolismo , Ferroptosis/efectos de los fármacos , Glutatión/biosíntesis , Humanos , Hierro/metabolismo , Ratones , Mitocondrias/metabolismo , Neoplasias Pancreáticas/patologíaRESUMEN
The unfolded protein response (UPR) is activated in pancreatic pathologies and suggested as a target for therapeutic intervention. In this study, we examined activating transcription factor 3 (ATF3), a mediator of the UPR that promotes acinar-to-ductal metaplasia (ADM) in response to pancreatic injury. Since ADM is an initial step in the progression to pancreatic ductal adenocarcinoma (PDAC), we hypothesized that ATF3 is required for initiation and progression of PDAC. We generated mice carrying a germline mutation of Atf3 (Atf3-/-) combined with acinar-specific induction of oncogenic KRAS (Ptf1acreERT/+KrasG12D/+). Atf3-/- mice with (termed APK) and without KRASG12D were exposed to cerulein-induced pancreatitis. In response to recurrent pancreatitis, Atf3-/- mice showed decreased ADM and enhanced regeneration based on morphological and biochemical analysis. Similarly, an absence of ATF3 reduced spontaneous pancreatic intraepithelial neoplasia (PanIN) formation and PDAC in Ptf1acreERT/+KrasG12D/+ mice. In response to injury, KRASG12D bypassed the requirement for ATF3 with a dramatic loss in acinar tissue and PanIN formation observed regardless of ATF3 status. Compared to Ptf1acreERT/+KrasG12D/+ mice, APK mice exhibited a significant decrease in pancreatic and total body weight, did not progress through to PDAC, and showed altered pancreatic fibrosis and immune cell infiltration. These findings suggest a complex, multifaceted role for ATF3 in pancreatic cancer pathology.
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Factor de Transcripción Activador 3 , Células Acinares , Animales , Ceruletida , Humanos , Ratones , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Neoplasias PancreáticasRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) remains a lethal cancer with an urgent need for better medical therapies. Efforts have been made to investigate the efficacy of immunotherapy, particularly given the hallmarks of immune suppression and exhaustion in PDAC tumors. Here, we review the molecular components responsible for the immune-privileged state in PDAC and provide an overview of the immunotherapeutic strategies for PDAC including vaccine therapy, checkpoint blockade, myeloid-targeted therapy, and immune agonist therapy.
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Carcinoma Ductal Pancreático/terapia , Inmunoterapia/métodos , Neoplasias Pancreáticas/terapia , Animales , Vacunas contra el Cáncer/uso terapéutico , Carcinoma Ductal Pancreático/inmunología , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Pancreáticas/inmunología , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
The Center for Basic and Translational Science was formed to address the unique challenges faced by surgeon-scientists. Shortly after its inception, COVID-19 upended research workflows at our institution. We discuss how the collaborative Center for Basic and Translational Science framework was adapted to support laboratories during the pandemic by assisting with ramp-down, promoting mentorship and community building, and maintaining research productivity.
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COVID-19/prevención & control , Colaboración Intersectorial , Investigadores/organización & administración , Cirujanos/organización & administración , Investigación Biomédica Traslacional/organización & administración , COVID-19/epidemiología , Eficiencia , Humanos , Mentores , Michigan/epidemiología , PandemiasRESUMEN
BACKGROUND: The extracellular signal-regulated kinase (ERK) pathway regulates cell growth, and is hyper-activated and associated with drug resistance in hepatocellular carcinoma (HCC). Metabolic pathways are profoundly dysregulated in HCC. Whether an altered metabolic state is linked to activated ERK pathway and drug response in HCC is unaddressed. METHODS: We deprived HCC cells of glutamine to induce metabolic alterations and performed various assays, including metabolomics (with 13C-glucose isotope tracing), microarray analysis, and cell proliferation assays. Glutamine-deprived cells were also treated with kinase inhibitors (e.g. Sorafenib, Erlotinib, U0126 amongst other MEK inhibitors). We performed bioinformatics analysis and stratification of HCC tumour microarrays to determine upregulated ERK gene signatures in patients. FINDINGS: In a subset of HCC cells, the withdrawal of glutamine triggers a severe metabolic alteration and ERK phosphorylation (pERK). This is accompanied by resistance to the anti-proliferative effect of kinase inhibitors, despite pERK inhibition. High intracellular serine is a consistent feature of an altered metabolic state and contributes to pERK induction and the kinase inhibitor resistance. Blocking the ERK pathway facilitates cell proliferation by reprogramming metabolism, notably enhancing aerobic glycolysis. We have identified 24 highly expressed ERK gene signatures that their combined expression strongly indicates a dysregulated metabolic gene network in human HCC tissues. INTERPRETATION: A severely compromised metabolism lead to ERK pathway induction, and primes some HCC cells to pro-survival phenotypes upon ERK pathway blockade. Our findings offer novel insights for understanding, predicting and overcoming drug resistance in liver cancer patients. FUND: DFG, BMBF and Sino-German Cooperation Project.
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Carcinoma Hepatocelular/metabolismo , Resistencia a Antineoplásicos , Neoplasias Hepáticas/metabolismo , Sistema de Señalización de MAP Quinasas , Antineoplásicos/toxicidad , Carcinoma Hepatocelular/genética , Proliferación Celular , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Metaboloma , Inhibidores de Proteínas Quinasas/toxicidad , TranscriptomaRESUMEN
BACKGROUND: Although immune-based therapy has proven efficacious for some patients with microsatellite instability (MSI) colon cancers, a majority of patients receive limited benefit. Conversely, select patients with microsatellite stable (MSS) tumors respond to checkpoint blockade, necessitating novel ways to study the immune tumor microenvironment (TME). We used phenotypic and spatial data from infiltrating immune and tumor cells to model cellular mixing to predict disease specific outcomes in patients with colorectal liver metastases. METHODS: Formalin fixed paraffin embedded metastatic colon cancer tissue from 195 patients were subjected to multiplex immunohistochemistry (mfIHC). After phenotyping, the G-function was calculated for each patient and cell type. Data was correlated with clinical outcomes and survival. RESULTS: High tumor cell to cytotoxic T lymphocyte (TC-CTL) mixing was associated with both a pro-inflammatory and immunosuppressive TME characterized by increased CTL infiltration and PD-L1+ expression, respectively. Presence and engagement of antigen presenting cells (APC) and helper T cells (Th) were associated with greater TC-CTL mixing and improved 5-year disease specific survival compared to patients with a low degree of mixing (42% vs. 16%, p = 0.0275). Comparison of measured mixing to a calculated theoretical random mixing revealed that PD-L1 expression on APCs resulted in an environment where CTLs were non-randomly less associated with TCs, highlighting their biologic significance. CONCLUSION: Evaluation of immune interactions within the TME of metastatic colon cancer using mfIHC in combination with mathematical modeling characterized cellular mixing of TCs and CTLs, providing a novel strategy to better predict clinical outcomes while identifying potential candidates for immune based therapies.
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Células Presentadoras de Antígenos/inmunología , Antígeno B7-H1/metabolismo , Neoplasias Colorrectales/inmunología , Neoplasias Hepáticas/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Modelos Teóricos , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral/inmunología , Antígeno B7-H1/inmunología , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , Pronóstico , Tasa de SupervivenciaRESUMEN
Pancreatic adenocarcinoma (PDA) is an aggressive disease driven by oncogenic KRAS and characterized by late diagnosis and therapeutic resistance. Here we show that deletion of the ataxia-telangiectasia group D-complementing (Atdc) gene, whose human homolog is up-regulated in the majority of pancreatic adenocarcinoma, completely prevents PDA development in the context of oncogenic KRAS. ATDC is required for KRAS-driven acinar-ductal metaplasia (ADM) and its progression to pancreatic intraepithelial neoplasia (PanIN). As a result, mice lacking ATDC are protected from developing PDA. Mechanistically, we show ATDC promotes ADM progression to PanIN through activation of ß-catenin signaling and subsequent SOX9 up-regulation. These results provide new insight into PDA initiation and reveal ATDC as a potential target for preventing early tumor-initiating events.
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Carcinogénesis , Carcinoma Ductal Pancreático/fisiopatología , Neoplasias Pancreáticas/fisiopatología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Factores de Transcripción/fisiología , Células Acinares/metabolismo , Células Acinares/patología , Animales , Carcinoma in Situ/patología , Carcinoma in Situ/fisiopatología , Carcinoma Ductal Pancreático/patología , Transdiferenciación Celular , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Humanos , Metaplasia , Ratones , Ratones Transgénicos , Conductos Pancreáticos/metabolismo , Conductos Pancreáticos/patología , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta Catenina/metabolismoRESUMEN
BACKGROUND & AIMS: Pancreatitis is a major cause of morbidity and mortality and is a risk factor for pancreatic tumorigenesis. Upon tissue damage, an inflammatory response, made up largely of macrophages, provides multiple growth factors that promote repair. Here, we examine the molecular pathways initiated by macrophages to promote pancreas recovery from pancreatitis. METHODS: To induce organ damage, mice were subjected to cerulein-induced experimental pancreatitis and analyzed at various times of recovery. CD11b-DTR mice were used to deplete myeloid cells. Hbegff/f;LysM-Cre mice were used to ablate myeloid cell-derived heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). To ablate EGFR specifically during recovery, pancreatitis was induced in Egfrf/f;Ptf1aFlpO/+;FSF-Rosa26CAG-CreERT2 mice followed by tamoxifen treatment. RESULTS: Macrophages infiltrating the pancreas in experimental pancreatitis make high levels of HB-EGF. Both depletion of myeloid cells and ablation of myeloid cell HB-EGF delayed recovery from experimental pancreatitis, resulting from a decrease in cell proliferation and an increase in apoptosis. Mechanistically, ablation of myeloid cell HB-EGF impaired epithelial cell DNA repair, ultimately leading to cell death. Soluble HB-EGF induced EGFR nuclear translocation and methylation of histone H4, facilitating resolution of DNA damage in pancreatic acinar cells in vitro. Consistent with its role as the primary receptor of HB-EGF, in vivo ablation of EGFR from pancreatic epithelium during recovery from pancreatitis resulted in accumulation of DNA damage. CONCLUSIONS: By using novel conditional knockout mouse models, we determined that HB-EGF derived exclusively from myeloid cells induces epithelial cell proliferation and EGFR-dependent DNA repair, facilitating pancreas healing after injury.
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Reparación del ADN , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Células Mieloides/metabolismo , Páncreas/fisiología , Pancreatitis/fisiopatología , Regeneración , Animales , ADN/metabolismo , Factor de Crecimiento Similar a EGF de Unión a Heparina/farmacología , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND: The tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) contains abundant immunosuppressive tumor-associated macrophages. High level of infiltration is associated with poor outcome and is thought to represent a major roadblock to lymphocyte-based immunotherapy. Efforts to block macrophage infiltration have been met with some success, but noninvasive means to track tumor-associated macrophagess in PDAC are lacking. Translocator protein (TSPO) is a mitochondrial membrane receptor which is upregulated in activated macrophages. We sought to identify if a radiotracer-labeled cognate ligand could track macrophages in PDAC. MATERIALS AND METHODS: A murine PDAC cell line was established from a transgenic mouse with pancreas-specific mutations in KRAS and p53. After confirming lack of endogenous TSPO expression, tumors were established in syngeneic mice. A radiolabeled TSPO-specific ligand ([11C] peripheral benzodiazepine receptor [PBR]28) was delivered intravenously, and tumor uptake was assessed by autoradiography, ex vivo, or micro-positron emission tomography imaging. RESULTS: Resected tumors contained abundant macrophages as determined by immunohistochemistry and flow cytometry. Immunoblotting revealed murine macrophages expressed TSPO with increasing concentration on activation and polarization. Autoradiography of resected tumors confirmed [11C]PBR28 uptake, and whole mount sections demonstrated the ability to localize tumors. To confirm the findings were macrophage specific, experiments were repeated in CD11b-deficient mice, and the radiotracer uptake was diminished. Micro-positron emission tomography imaging validated radiotracer uptake and tumor localization in a clinically applicable manner. CONCLUSIONS: As new immunotherapeutics reshape the PDAC microenvironment, tools are needed to better measure and track immune cell subsets. We have demonstrated the potential to measure changes in macrophage infiltration in PDAC using [11C]PBR28.