Mutant KRAS drives metabolic reprogramming and autophagic flux in premalignant pancreatic cells

AbstractMutational activation of the KRAS gene occurs in almost all pancreatic ductal adenocarcinoma (PDAC) and is the earliest molecular event in their carcinogenesis. Evidence has accumulated of the metabolic reprogramming in PDAC, such as amino acid homeostasis and autophagic flux. However, the biological effects of KRAS mutation on metabolic reprogramming at the earlier stages of PDAC carcinogenesis are unclear. Here we report dynamic metabolic reprogramming in immortalized human non-cancerous pancreatic ductal epithelial cells, in which a KRAS mutation was induced by gene-editing, which may mimic early pancreatic carcinogenesis. Similar to the cases of PDAC, KRAS gene mutation increased the dependency on glucose and glutamine for maintaining the intracellular redox balance. In addition, the intracellular levels of amino acids were significantly decreased because of active protein synthesis, and the cells required greater autophagic flux to maintain their viability. The lysosomal inhibitor chloroquine significantly inhibited cell proliferation. Therefore, metabolic reprogramming is an early event in carcinogenesis initiated by KRAS gene mutation, suggesting a rationale for the development of nutritional interventions that suppress or delay the development of PDAC.

Download Full-text

KRAS Mutation Dictates the Cancer Immune Environment in Pancreatic Ductal Adenocarcinoma and Other Adenocarcinomas

Cancers ◽

10.3390/cancers13102429 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2429

Author(s):

Meichen Gu ◽

Yanli Gao ◽

Pengyu Chang

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Kras Mutation ◽

Checkpoint Inhibitors ◽

Metabolic Reprogramming ◽

Ductal Adenocarcinoma ◽

Genomic Alteration ◽

Pancreatic Carcinogenesis ◽

Novel Method ◽

Almost All ◽

Lung Adenocarcinomas

Generally, patients with pancreatic ductal adenocarcinoma, especially those with wide metastatic lesions, have a poor prognosis. Recently, a breakthrough in improving their survival has been achieved by using first-line chemotherapy, such as gemcitabine plus nab-paclitaxel or oxaliplatin plus irinotecan plus 5-fluorouracil plus calcium folinate. Unfortunately, regimens with high effectiveness are still absent in second- or later-line settings. In addition, although immunotherapy using checkpoint inhibitors definitively represents a novel method for metastatic cancers, monotherapy using checkpoint inhibitors is almost completely ineffective for pancreatic ductal adenocarcinomas largely due to the suppressive immune milieu in such tumors. Critically, the genomic alteration pattern is believed to impact cancer immune environment. Surprisingly, KRAS gene mutation is found in almost all pancreatic ductal adenocarcinomas. Moreover, KRAS mutation is indispensable for pancreatic carcinogenesis. On these bases, a relationship likely exists between this oncogene and immunosuppression in this cancer. During pancreatic carcinogenesis, KRAS mutation-driven events, such as metabolic reprogramming, cell autophagy, and persistent activation of the yes-associated protein pathway, converge to cause immune evasion. However, intriguingly, KRAS mutation can dictate a different immune environment in other types of adenocarcinoma, such as colorectal adenocarcinoma and lung adenocarcinoma. Overall, the KRAS mutation can drive an immunosuppression in pancreatic ductal adenocarcinomas or in colorectal carcinomas, but this mechanism is not true in KRAS-mutant lung adenocarcinomas, especially in the presence of TP53 inactivation. As a result, the response of these adenocarcinomas to checkpoint inhibitors will vary.

Download Full-text

4-Hydroxyphenylpyruvate Dioxygenase-Like Protein Promotes Pancreatic Cancer Cell Progression and Is Associated With Glutamine-Mediated Redox Balance

Frontiers in Oncology ◽

10.3389/fonc.2020.617190 ◽

2021 ◽

Vol 10 ◽

Author(s):

Xianglai Ye ◽

Xiujuan Wei ◽

Jing Liao ◽

Peipei Chen ◽

Xueyun Li ◽

...

Keyword(s):

Redox Balance ◽

Metabolic Reprogramming ◽

Glutamine Metabolism ◽

Ductal Adenocarcinoma ◽

Intermembrane Space ◽

Dependent Manner ◽

Uncharacterized Protein ◽

Cell Progression ◽

Mitochondrial Intermembrane Space

Tumor cells develop a series of metabolic reprogramming mechanisms to meet the metabolic needs for tumor progression. As metabolic hubs in cells, mitochondria play a significant role in this process, including energy production, biosynthesis, and redox hemostasis. In this study, we show that 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL), a previously uncharacterized protein, is positively associated with the development of pancreatic ductal adenocarcinoma (PDAC) and disease prognosis. We found that overexpression of HPDL in PDAC cells promotes tumorigenesis in vitro, whereas knockdown of HPDL inhibits cell proliferation and colony formation. Mechanistically, we found that HPDL is a mitochondrial intermembrane space localized protein that positively regulates mitochondrial bioenergetic processes and adenosine triphosphate (ATP) generation in a glutamine dependent manner. Our results further reveal that HPDL protects cells from oxidative stress by reprogramming the metabolic profile of PDAC cells toward glutamine metabolism. In short, we conclude that HPDL promotes PDAC likely through its effects on glutamine metabolism and redox balance.

Download Full-text

The Use of Genotoxicity Endpoints as Biomarkers of Low Dose Radiation Exposure in Interventional Cardiology

Frontiers in Public Health ◽

10.3389/fpubh.2021.701878 ◽

2021 ◽

Vol 9 ◽

Author(s):

Martha Habibi ◽

Panagiotis K. Karyofyllis ◽

Aggeliki Nikolakopoulou ◽

Panagiotis Papagiannis ◽

Pantelis Karaiskos ◽

...

Keyword(s):

Risk Estimation ◽

Biological Effects ◽

Interventional Cardiology ◽

Mean Values ◽

Individual Level ◽

Cardiac Procedures ◽

Before And After ◽

Radiation Induced ◽

The Individual ◽

Almost All

The effect of the reportedly low ionizing radiation doses, such as those very often delivered to patients in interventional cardiology, remains ambiguous. As interventional cardiac procedures may have a significant impact on total collective effective dose, there are radiation protection concerns for patients and physicians regarding potential late health effects. Given that very low doses (<100 mSv) are expected to be delivered during these procedures, the purpose of this study was to assess the potency and suitability of current genotoxicity biomarkers to detect and quantitate biological effects essential for risk estimation in interventional cardiology. Specifically, the biomarkers γ-H2AX foci, dicentric chromosomes, and micronuclei, which underpin radiation-induced DNA damage, were studied in blood lymphocytes of 25 adult patients before and after interventional cardiac procedures. Even though the mean values of all patients as a group for all three endpoints tested show increased yields relative to baseline following medical exposure, our results demonstrate that only the γ-H2AX biomarker enables detection of statistically significant differences at the individual level (p < 0.001) for almost all patients (91%). Furthermore, 24 h after exposure, residual γ-H2AX foci were still detectable in irradiated lymphocytes. Their decline was found to vary significantly among the individuals and the repair kinetics of γ-H2AX foci was found to range from 25 to 95.6% of their maximum values obtained.

Download Full-text

Positive Crosstalk Between Hedgehog and NF-κB Pathways Is Dependent on KRAS Mutation in Pancreatic Ductal Adenocarcinoma

Frontiers in Oncology ◽

10.3389/fonc.2021.652283 ◽

2021 ◽

Vol 11 ◽

Author(s):

Yuqiong Wang ◽

Dan Wang ◽

Yanmiao Dai ◽

Xiangyu Kong ◽

Xian Zhu ◽

...

Keyword(s):

Tumor Growth ◽

Pancreatic Ductal Adenocarcinoma ◽

Signaling Pathways ◽

Kras Mutation ◽

Biological Effects ◽

Ductal Adenocarcinoma ◽

Luciferase Reporter ◽

Hh Signaling

It has been shown that aberrant activation of the Hedgehog (Hh) and nuclear factor-kappa B (NF-κB) signaling pathways plays an important role in the pancreatic carcinogenesis, and KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDAC). Until now, the role of KRAS mutation in the context of crosstalk between Hh and NF-κB signaling pathways in PDAC has not been investigated. This study was to determine whether the crosstalk between the Hh and NF-κB pathways is dependent on KRAS mutation in PDAC. The correlation between Gli1, Shh, NF-κB p65 expression and KRAS mutation in PDAC tissues was firstly examined by immunohistochemistry. Next, Western blotting, qPCR, and immunofluorescence were conducted to examine the biological effects of interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α) as NF-κB signaling agonists, Shh as an Hh ligand alone or in combination with KRAS small interfering RNA (si-KRAS) in KRAS-mutant PDAC cells (MT-KRAS; SW1990 and Panc-1), wild-type KRAS PDAC cells (WT-KRAS; BxPC-3) and mutant KRAS knock-in BxPC-3 cells in vitro as well as tumor growth in vivo. KRAS mutation-dependent crosstalk between Hh and NF-κB in PDAC cells was further assessed by Ras activity and luciferase reporter assays. The aberrant Hh and NF-κB pathway activation was found in PDAC tissues with KRAS mutation. The same findings were confirmed in MT-KRAS PDAC cells and MT-KRAS knock-in BxPC-3 cells, whereas this activation was not observed in WT-KRAS PDAC cells. However, the activation was significantly down-regulated by KRAS silencing in MT-KRAS PDAC cells. Furthermore, MT-KRAS cancer cell proliferation and survival in vitro and tumor growth after inoculation with MT-KRAS cells in vivo were promoted by NF-κB and Hh signaling activation. The pivotal factor for co-activation of NF-κB and Hh signaling is MT-KRAS protein upregulation, showing that positive crosstalk between Hh and NF-κB pathways is dependent upon KRAS mutation in PDAC.

Download Full-text

Patient-specific iPSCs carrying an SFTPC mutation reveal the intrinsic alveolar epithelial dysfunction at the inception of interstitial lung disease

10.1101/2020.11.13.382390 ◽

2020 ◽

Author(s):

Konstantinos-Dionysios Alysandratos ◽

Scott J. Russo ◽

Anton Petcherski ◽

Evan P. Taddeo ◽

Rebeca Acín-Pérez ◽

...

Keyword(s):

Interstitial Lung Disease ◽

Pulmonary Fibrosis ◽

Lung Disease ◽

Disease Onset ◽

Metabolic Reprogramming ◽

Patient Specific ◽

Autophagic Flux ◽

Alveolar Epithelial ◽

Epithelial Dysfunction

SummaryThe incompletely understood pathogenesis of pulmonary fibrosis (PF) and lack of reliable preclinical disease models have limited development of effective therapies. An emerging literature now implicates alveolar epithelial type 2 cell (AEC2) dysfunction as an initiating pathogenic event in the onset of a variety of PF syndromes, including adult idiopathic pulmonary fibrosis (IPF) and childhood interstitial lung disease (chILD). However, inability to access primary AEC2s from patients, particularly at early disease stages, has impeded identification of disease-initiating mechanisms. Here we present an in vitro reductionist model system that permits investigation of epithelial-intrinsic events that lead to AEC2 dysfunction over time using patient-derived cells that carry a disease-associated variant, SFTPCI73T, known to be expressed solely in AEC2s. After generating patient-specific induced pluripotent stem cells (iPSCs) and engineering their gene-edited (corrected) counterparts, we employ directed differentiation to produce pure populations of syngeneic corrected and mutant AEC2s, which we expand >1015 fold in vitro, providing a renewable source of cells for modeling disease onset. We find that mutant iPSC-derived AEC2s (iAEC2s) accumulate large amounts of misprocessed pro-SFTPC protein which mistrafficks to the plasma membrane, similar to changes observed in vivo in the donor patient’s AEC2s. These changes result in marked reduction in AEC2 progenitor capacity and several downstream perturbations in AEC2 proteostatic and bioenergetic programs, including a late block in autophagic flux, accumulation of dysfunctional mitochondria with consequent time-dependent metabolic reprograming from oxidative phosphorylation to glycolysis, and activation of an NF-κB dependent inflammatory response. Treatment of SFTPCI73T expressing iAEC2s with hydroxychloroquine, a medication commonly prescribed to these patients, results in aggravation of autophagy perturbations and metabolic reprogramming. Thus, iAEC2s provide a patientspecific preclinical platform for modeling the intrinsic epithelial dysfunction associated with the inception of interstitial lung disease.

Download Full-text

Upregulated MicroRNA-483-3p is an Early Event in Pancreatic Ductal Adenocarcinoma (PDAC) and as a Powerful Liquid Biopsy Biomarker in PDAC

OncoTargets and Therapy ◽

10.2147/ott.s288936 ◽

2021 ◽

Vol Volume 14 ◽

pp. 2163-2175

Author(s):

Huilin Shao ◽

Yue Zhang ◽

Jie Yan ◽

Xinchao Ban ◽

Xiaojie Fan ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Liquid Biopsy ◽

Ductal Adenocarcinoma ◽

Early Event

Download Full-text

Organotypic Culture of Acinar Cells for the Study of Pancreatic Cancer Initiation

Cancers ◽

10.3390/cancers12092606 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2606

Author(s):

Carlotta Paoli ◽

Alessandro Carrer

Keyword(s):

Pancreatic Cancer ◽

Ex Vivo ◽

Acinar Cells ◽

Lineage Tracing ◽

Metabolic Reprogramming ◽

Pancreatic Acinar Cells ◽

Ductal Adenocarcinoma ◽

Molecular Features ◽

The Impact

The carcinogenesis of pancreatic ductal adenocarcinoma (PDA) progresses according to multi-step evolution, whereby the disease acquires increasingly aggressive pathological features. On the other hand, disease inception is poorly investigated. Decoding the cascade of events that leads to oncogenic transformation is crucial to design strategies for early diagnosis as well as to tackle tumor onset. Lineage-tracing experiments demonstrated that pancreatic cancerous lesions originate from acinar cells, a highly specialized cell type in the pancreatic epithelium. Primary acinar cells can survive in vitro as organoid-like 3D spheroids, which can transdifferentiate into cells with a clear ductal morphology in response to different cell- and non-cell-autonomous stimuli. This event, termed acinar-to-ductal metaplasia, recapitulates the histological and molecular features of disease initiation. Here, we will discuss the isolation and culture of primary pancreatic acinar cells, providing a historical and technical perspective. The impact of pancreatic cancer research will also be debated. In particular, we will dissect the roles of transcriptional, epigenetic, and metabolic reprogramming for tumor initiation and we will show how that can be modeled using ex vivo acinar cell cultures. Finally, mechanisms of PDA initiation described using organotypical cultures will be reviewed.

Download Full-text

Reciprocal interactions between mtDNA and lifespan control in budding yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e18-06-0356 ◽

2019 ◽

Vol 30 (24) ◽

pp. 2943-2952 ◽

Cited By ~ 2

Author(s):

Enrique J. Garcia ◽

Janeska J. de Jonge ◽

Pin-Chao Liao ◽

Elizabeth Stivison ◽

Cierra N. Sing ◽

...

Keyword(s):

Mitochondrial Function ◽

Cell Cycle Progression ◽

Genome Instability ◽

Nuclear Genome ◽

Metabolic Reprogramming ◽

Yeast Cells ◽

Early Event ◽

Cycle Progression ◽

Genome Stabilization ◽

Mitochondrial Respiratory Activity

Loss of mitochondrial DNA (mtDNA) results in loss of mitochondrial respiratory activity, checkpoint-regulated inhibition of cell cycle progression, defects in growth, and nuclear genome instability. However, after several generations, yeast cells can adapt to the loss of mtDNA. During this adaptation, rho0 cells, which have no mtDNA, exhibit increased growth rates and nuclear genome stabilization. Here, we report that an immediate response to loss of mtDNA is a decrease in replicative lifespan (RLS). Moreover, we find that adapted rho0 cells bypass the mtDNA inheritance checkpoint, exhibit increased mitochondrial function, and undergo an increase in RLS as they adapt to the loss of mtDNA. Transcriptome analysis reveals that metabolic reprogramming to compensate for defects in mitochondrial function is an early event during adaptation and that up-regulation of stress response genes occurs later in the adaptation process. We also find that specific subtelomeric genes are silenced during adaptation to loss of mtDNA. Moreover, we find that deletion of SIR3, a subtelomeric gene silencing protein, inhibits silencing of subtelomeric genes associated with adaptation to loss of mtDNA, as well as adaptation-associated increases in mitochondrial function and RLS extension.

Download Full-text

Mo1395 – Metabolic Reprogramming and Subsequent Autophagy Triggered by Kras Gene Mutation is a Novel Preventive Target Against Pancreatic Carcinogenesis

Gastroenterology ◽

10.1016/s0016-5085(19)38847-x ◽

2019 ◽

Vol 156 (6) ◽

pp. S-762

Author(s):

Tatsunori Suzuki ◽

Motoyuki Otsuka ◽

Takahiro Kishikawa ◽

Motoko Ohno ◽

Kazuma Sekiba ◽

...

Keyword(s):

Gene Mutation ◽

Metabolic Reprogramming ◽

Kras Gene ◽

Pancreatic Carcinogenesis

Download Full-text

RAS in pancreatic cancer

Biochemical Society Transactions ◽

10.1042/bst20170521 ◽

2019 ◽

Vol 47 (4) ◽

pp. 961-972 ◽

Cited By ~ 7

Author(s):

Simone Lanfredini ◽

Asmita Thapa ◽

Eric O'Neill

Keyword(s):

Pancreatic Cancer ◽

Neuroendocrine Tumour ◽

Critical Role ◽

Acinar Cell Carcinoma ◽

Genetically Engineered ◽

Ductal Adenocarcinoma ◽

Kras Mutations ◽

Activating Mutations ◽

Downstream Effectors ◽

Almost All

Abstract The pancreas is a gland composed mainly by endocrine and exocrine cells, giving rise to three main tumour types. Pancreatic neuroendocrine tumour or PNET arise from the endocrine portion of the pancreas. On the contrary, pancreatic exocrine neoplasms include pancreatic ductal adenocarcinoma (PDAC) and acinar cell carcinoma. PDAC is the most common type of pancreatic cancer and one of the leading causes of cancer-related death. It has been shown that less than 3% of PDAC patients have an overall survival of up to 5 years in the U.K. This mainly arises since the majority of patients diagnosed with PDAC present with advanced unresectable disease, which is highly resistant to all forms of chemotherapy and radiotherapy. Activating mutations of an isoform of the RAS protein, KRAS, are found in almost all PDAC cases and occur during early stages of malignant transformation. KRAS mutations play a critical role as they are involved in both initiating and maintaining PDAC development. The interaction of RAS with GDP/GTP along with its recruitment to the membrane affects transduction of its activating signals to downstream effectors. In this review, we aim to summarise different mutations of RAS and their prevalence in pancreatic cancer along with other RAS-induced tumours. In addition, we briefly discuss the genetically engineered mouse models that have been developed to study KRAS-mutated adenocarcinomas in the pancreas. These provide an opportunity to also address the importance of targeting RAS for better treatment response in PDAC patients along with the challenges incurred herein.

Download Full-text