Stem Cell Theory of Cancer: Origin of Metastasis and Sub-clonality.

Metastasis may be the secret weapon cancer uses to dominate and subjugate, to persist and prevail. However, it is no longer a secret when we realize that a stem cell has the same ways and means to fulfill its own omnipotence and accomplish its own omnipresence… and when we realize that a cancer cell has its own version of stem-ness origin and stem-like nature. In this perspective, we discuss whether stem-ness enables metastasis or mutations drive metastasis. We ponder about low-grade versus high-grade tumors and about primary versus metastatic tumors. We wonder about stochasticity and hierarchy in the genesis and evolution of cancer and of metastasis. We postulate that metastasis may hold the elusive code that makes or breaks a stem-cell versus a genetic theory of cancer. We speculate that the vaunted model of multistep carcinogenesis may be in error and needs some belated remodeling and a major overhaul. We propose that subsequent malignant neoplasms from germ cell tumors and donor-derived malignancies in organ transplants are quintessential experiments of nature and by man that may eventually empower us to elucidate a stem-cell origin of cancer and metastasis. Unfortunately, even the best experiments of cancer and of metastasis will be left unfinished, overlooked, or forgotten, when we do not formulate a proper cancer theory derived from pertinent and illuminating clinical observations. Ultimately, there should be no consternations when we realize that metastasis has a stem-cell rather than a genetic origin, and no reservations when we recognize that metastasis has been providing us some of the most enduring tests and endearing proofs to demonstrate that cancer is indeed a stem-cell rather than a genetic disease after all.

PET/MR Imaging of a Lung Metastasis Model of Clear Cell Renal Cell Carcinoma with (2S,4R)-4-[18F]Fluoroglutamine.

PURPOSE: Metabolic reprogramming plays an important role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Currently, positron emission tomography (PET) reporters are not used clinically to visualize altered glutamine metabolism in ccRCC, which greatly hinders detection, staging, and real-time therapeutic assessment. We sought to determine if (2S,4R)-4-[18F]fluoroglutamine ([18F]FGln) could be used to interrogate altered glutamine metabolism in ccRCC lesions in the lung. PROCEDURES: We generated a novel ccRCC lung lesion model using the ccRCC cell line UMRC3 stably transfected with GFP and luciferase constructs. This cell line was used for characterization of [18F]FGln uptake and retention by transport analysis in cell culture and by PET/MRI (magnetic resonance imaging) in animal models. Tumor growth in animal models was monitored using bioluminescence (BLI) and MRI. After necropsy, UMRC3 tumor growth in lung tissue was verified by fluorescence imaging and histology. RESULTS: In UMRC3 cells, [18F]FGln cell uptake was twofold higher than cell uptake in normal kidney HEK293 cells. Tracer cell uptake was reduced by 60-90% in the presence of excess glutamine in the media and by 20-50% upon treatment with V-9302, an inhibitor of the major glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2). Furthermore, in UMRC3 cells, [18F]FGln cell uptake was reduced by siRNA knockdown of ASCT2 to levels obtained by the addition of excess exogenous glutamine. Conversely, [18F]FGln cellular uptake was increased in the presence of the glutaminase inhibitor CB-839. Using simultaneous PET/MRI for visualization, retention of [18F]FGln in vivo in ccRCC lung tumors was 1.5-fold greater than normal lung tissue and twofold greater than muscle. In ccRCC lung tumors, [18F]FGln retention did not change significantly upon treatment with CB-839. CONCLUSIONS: We report one of the first direct orthotopic mouse models of ccRCC lung lesions. Using PET/MR imaging, lung tumors were easily discerned from normal tissue. Higher uptake of [18F]FGln was observed in a ccRCC cell line and lung lesions compared to HEK293 cells and normal lung tissue, respectively. [18F]FGln cell uptake was modulated by exogenous glutamine, V-9302, siRNA knockdown of ASCT2, and CB-839. Interestingly, in a pilot therapeutic study with CB-839, we observed no difference in treated tumors relative to untreated controls. This was in contrast with cellular studies, where CB-839 increased glutamine uptake.

Cysteine Modification by Ebselen Reduces the Stability and Cellular Levels of 14-3-3 Proteins.

The 14-3-3 proteins constitute a family of adaptor proteins with many binding partners and biological functions, and they are considered promising drug targets in cancer and neuropsychiatry. By screening 1280 small-molecule drugs using differential scanning fluorimetry (DSF), we found 15 compounds that decreased the thermal stability of 14-3-3ζ Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ζ, ε, γ, and η Ebselen bonding decreased 14-3-3ζ binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3ζ (C25, C94, and C189) by DSF and mass spectroscopy revealed covalent modification by ebselen of all cysteines through a selenylsulfide bond. C25 appeared to be the preferential site of ebselen interaction in vitro, whereas modification of C94 was the main determinant for protein destabilization. At therapeutically relevant concentrations, ebselen and ebselen oxide caused decreased 14-3-3 levels in SH-SY5Y cells, accompanied with an increased degradation, most probably by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed decreased brain 14-3-3 content, a freezing phenotype, and reduced mobility, resembling the effects of lithium, consistent with its proposed action as a safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, such as cancer, neuropsychiatric disorders, and infectious diseases, including coronavirus disease 2019. Its pleiotropic actions are attributed to antioxidant effects and formation of selenosulfides with critical cysteine residues in proteins. Our work indicates that a destabilization of 14-3-3 may affect the protein interaction networks of this protein family, contributing to the therapeutic potential of ebselen. SIGNIFICANCE STATEMENT: There is currently great interest in the repurposing of established drugs for new indications and therapeutic targets. This study shows that ebselen, which is a promising drug candidate against cancer, bipolar disorder, and the viral infection coronavirus disease 2019, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering destabilization and increased degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral, anti-inflammatory, and antineoplastic effects of this drug.

Endobronchial ultrasound-guided injection of NBTXR3 radio-enhancing nanoparticles into mediastinal and hilar lymph nodes: a swine model to evaluate feasibility, injection technique, safety, nanoparticle retention and dispersion.

BACKGROUND: Loco-regionally advanced lung cancer is typically treated with a combination of chemotherapy and radiation therapy, but overall survival and local control remain poor. Radio-enhancing nanoparticles such as NBTXR3 activated by radiotherapy results in increased cell death and potentially an anti-tumor immune response. The goal of this study was to assess the feasibility and safety of endobronchial ultrasound (EBUS)-guided injection of NBTXR3 into mediastinal and hilar lymph nodes (LN), as well as assess nanoparticle retention in the LN post-injection. METHODS: Animals underwent bronchoscopy under general anesthesia with EBUS-guided injection of NBTXR3 into hilar and mediastinal LN. LN and injection volumes were calculated based on pre-injection computed tomography (CT) scans. CT scans were repeated at 5 min, 30 min, and 8 days post-injection. Blood-draws were also obtained at baseline and post-injection. Animals were then housed, monitored, and sacrificed 8 days post-injection. Necropsy was then performed with gross and histologic analysis of LN. RESULTS: A total of 20 LN were injected in 5 pigs (4 LN per animal). Nanoparticles were retained in 100% of LN at 30 min, and 90% of LN at 8 days. Extravasation of nanoparticles was seen in 4 out of the 20 LN. There were no cases of nanoparticle embolization visible by CT in distant organs. Small air-bubbles were introduced in the targets and surrounding tissue in 3 out of 20 LN. Of note, at 8 days, none of these air-bubbles were present on CT scan. There were no intra-procedural or post-procedural complications in either CT scans or necropsy findings. Pigs remained clinically stable and neither laboratory values nor necropsy showed evidence of inflammation. CONCLUSIONS: EBUS-guided injection of NBTXR3 radio-enhancing nanoparticles can be safely performed achieving a high rate of nanoparticle retention, low extravasation, and no visible nanoparticle embolization.

Current practice of anticoagulant in the treatment of splanchnic vein thrombosis secondary to acute pancreatitis.

BACKGROUND: Severe acute pancreatitis is a common diagnosis in emergency general surgery and can be a cause of significant morbidity and mortality. A consequence of severe acute pancreatitis is thrombus in the splanchnic veins. These thrombi can potentially lead to bowel ischemia or hepatic failure. However, another complication of severe acute pancreatitis is retroperitoneal bleeding. At this time, it is unclear if treating patients for splanchnic vein thrombosis in the context of severe acute pancreatitis is associated with any outcome benefit. A systematic review might clarify this question. DATA SOURCES: A two-fold search strategy (one broad and one precise) looked at all published literature. The review was registered on PROSPERO (ID: CRD42018102705). MEDLINE, EMBASE, PubMed, Cochrane and Web of Science databases were searched and potentially relevant papers were reviewed independently by two researchers. Any disagreement was reviewed by a third independent researcher. Primary outcome was reestablishment of flow in the thrombosed vein versus bleeding complications. RESULTS: Of 1462 papers assessed, a total of 16 papers were eligible for inclusion. There were no randomized controlled trials, 2 were case series, 5 retrospective single-center studies and 9 case reports. There were a total of 198 patients in these studies of whom 92 (46.5%) received anticoagulation therapy. The rates of recanalization of veins in the treated and non-treated groups was 14% and 11% and bleeding complications were 16% and 5%, respectively. However, the included studies were too heterogeneous to undertake a meta-analysis. CONCLUSIONS: The systematic review highlights the lack evidence addressing this clinical question. Therefore a randomized controlled trial would be appropriate to undertake.

A novel patient-derived orthotopic xenograft model of esophageal adenocarcinoma provides a platform for translational discoveries.

Mouse models of gastroesophageal junction (GEJ) cancer strive to recapitulate the intratumoral heterogeneity and cellular crosstalk within patient tumors to improve clinical translation. GEJ cancers remain a therapeutic challenge due to the lack of a reliable mouse model for preclinical drug testing. In this study, a novel patient-derived orthotopic xenograft (PDOX) was established from GEJ cancer via transabdominal surgical implantation. Patient tumor was compared to subcutaneously implanted patient-derived tumor xenograft (PDX) and PDOX by Hematoxylin and Eosin staining, immunohistochemistry and next-generation sequencing. Treatment efficacy studies of radiotherapy were performed. We observed that mechanical abrasion of mouse GEJ prior to surgical implantation of a patient-derived tumor in situ promotes tumor engraftment (100%, n=6). Complete PDOX engraftment was observed with rapid intra- and extraluminal tumor growth, as evidenced by magnetic resonance imaging. PDOXs contain fibroblasts, tumor-associated macrophages, immune and inflammatory cells, vascular and lymphatic vessels. Stromal hallmarks of aggressive GEJ cancers are recapitulated in a GEJ PDOX mouse model. PDOXs demonstrate tumor invasion into vasculature and perineural space. Next-generation sequencing revealed loss of heterozygosity with very high allelic frequency in NOTCH3, TGFB1, EZH2 and KMT2C in the patient tumor, the subcutaneous PDX and the PDOX. Immunohistochemical analysis of Her2/neu (also known as ERBB2), p53 (also known as TP53) and p16 (also known as CDKN2A) in PDX and PDOX revealed maintenance of expression of proteins found in patient tumors, but membranous EGFR overexpression in patient tumor cells was absent in both xenografts. Targeted radiotherapy in this model suggested a decrease in size by 61% according to Response Evaluation Criteria in Solid Tumors (RECIST), indicating a partial response to radiation therapy. Our GEJ PDOX model exhibits remarkable fidelity to human disease and captures the precise tissue microenvironment present within the local GEJ architecture, providing a novel tool for translating findings from studies on human GEJ cancer. This model can be applied to study metastatic progression and to develop novel therapeutic approaches for the treatment of GEJ cancer.This article has an associated First Person interview with the first author of the paper.

Metabolic Differences in Glutamine Utilization Lead to Metabolic Vulnerabilities in Prostate Cancer.

The new oncologic paradigm of precision medicine is focused on identifying metabolic, proteomic, transcriptomic and genomic variabilities in tumors that can be exploited to tailor treatments and improve patient outcomes. Metabolic changes are a hallmark of cancer, and inhibition of metabolic pathways is now a major strategy in medicinal chemistry for targeting cancers. However, non-invasive biomarkers to categorize metabolic subtypes are in short supply. The purpose of this study was to characterize the intracellular and extracellular metabolic profiles of four prostate cancer cell lines with varying degrees of aggressiveness. We observed metabolic differences between the aggressive prostate cancer cell line PC3 and the even more aggressive, metastatic subline PC3M assessed by hyperpolarized in vivo pyruvate studies, nuclear magnetic resonance spectroscopy, and carbon-13 feeding studies. On further examination of the differences between these two cell lines, we found increased glutamine utilization in the metastatic PC3M subline that led directly to sensitivity to glutaminase inhibitor CB-839. Our study supports the theory that metastatic progression increases glutamine utilization and the inhibition of glutaminolysis could have clinical implications.

ΔNp63/DGCR8-Dependent MicroRNAs Mediate Therapeutic Efficacy of HDAC Inhibitors in Cancer.

ΔNp63 is an oncogenic member of the p53 family and acts to inhibit the tumor-suppressive activities of the p53 family. By performing a chemical library screen, we identified histone deacetylase inhibitors (HDACi) as agents reducing ΔNp63 protein stability through the E3 ubiquitin ligase, Fbw7. ΔNp63 inhibition decreases the levels of its transcriptional target, DGCR8, and the maturation of let-7d and miR-128, which we found to be critical for HDACi function in vitro and in vivo. Our work identified Fbw7 as a predictive marker for HDACi response in squamous cell carcinomas and lymphomas, and unveiled let-7d and miR-128 as specific targets to bypass tumor resistance to HDACi treatment.

Refinements for embryo implantation surgery in the mouse: comparison of injectable and inhalant anesthesias - tribromoethanol, ketamine and isoflurane - on pregnancy and pup survival.

An essential aspect of genetically-engineered mice (GEM) is the ability to produce live animals after the appropriate injection procedure. Animals are produced by implantation of manipulated embryos into pseudopregnant females for gestation, parturition, and growth to the weaning stage. This study was carried out to test whether the anesthesia used during surgery could affect the number of pups produced. Anesthetics commonly used for implant surgery include tribromoethanol (Avertin) delivered by intraperitoneal (IP) injection, IP-injected ketamine:xylazine or ketamine:medetomidine mix, and inhaled isoflurane. To determine if the anesthesia used might affect the number of animals produced, each anesthetic agent was tested in implant surgeries and the numbers of pups produced using both wild-type and GEM embryos were assessed. Parallel studies were conducted in institutions in the EU and in the USA. Based on a direct comparison of pregnancy status, number of pups born, and number of pups weaned for each agent, we found no statistical differences among the three anesthetics. We conclude that all three anesthetic agents tested are equally useful for implantation surgery.

Novel therapeutic interventions for p53-altered tumors through manipulation of its family members, p63 and p73.

TP53 is highly mutated in human cancers, thus targeting this tumor suppressor pathway is highly desirable and will impact many cancer patients. (1,2) Therapeutic strategies to reactivate the p53-pathway have been challenging, (3,4) and no effective treatment exists. (5) We utilized the p53-family members, p63 and p73, which are not frequently mutated in cancer, to treat p53-defective cancers. The N-terminal splice variants of p63 and p73 are denoted as the TA and ΔN isoforms. We recently demonstrated that deletion of either ΔNp63 or ΔNp73 in p53-deficient mouse tumors results in tumor regression mediated by metabolic programming. Using this strategy, we identified pramlintide, a synthetic analog of amylin, as an effective treatment for p53 deficient and mutant tumors. Here, we show the utility of using pramlintide, as a potential cancer preventive option for p53-deficient tumors in mouse models. Additionally, we found that in vivo inhibition of both ΔNp63 and ΔNp73 in combination accelerates tumor regression and increases survival of p53-deficient mice. We report that inhibition of both ΔNp63 and ΔNp73 in combination results in upregulation of 3 key metabolic regulators, IAPP, GLS2, and TIGAR resulting in an increase in apoptosis and tumor regression in ΔNp63/ΔNp73/p53 deficient thymic lymphomas. These data highlight the value of generating inhibitors that will simultaneously target ΔNp63 and ΔNp73 to treat cancer patients with alterations in p53.

IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo.

TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas the ΔN isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes that promote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ΔN isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the ß cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.