bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from BACH1 Promotes Pancreatic Cancer Metastasis by Repressing Epithelial Genes and Enhancing Epithelial–Mesenchymal Transition
Authors: Sato Masaki , Matsumoto Mitsuyo , Saiki Yuriko , Alam Mahabub , Nishizawa Hironari , Rokugo Masahiro , Brydun Andrey , Yamada Shinji , Kaneko Mika K. , Funayama Ryo , Ito Mamoru , Kato Yukinari , Nakayama Keiko , Unno Michiaki , Igarashi Kazuhiko
Published: March 2023

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is among the cancers with the poorest prognoses due to its highly malignant features. BTB and CNC homology 1 (BACH1) has been implicated in RAS-driven tumor formation. We focused on the role of BACH1 in PDAC, more than 90% of which have KRAS mutation. Knockdown of BACH1 in PDAC cell lines reduced cell migration and invasion, in part, by increasing E-cadherin expression, whereas its overexpression showed opposite effects. BACH1 directly repressed the expression of FOXA1 that is known to activate the expression of CDH1 encoding E-cadherin and to inhibit epithelial-to-mesenchymal transition. BACH1 also directly repressed the expression of genes important for epithelial cell adhesion including CLDN3 and CLDN4. In a mouse orthotopic implantation model, BACH1 was required for the high metastatic ability of AsPC-1 cells. IHC analysis of clinical specimens with a newly developed anti-BACH1 mAb revealed that high expression of BACH1 is a poor prognostic factor. These results suggest that the gene regulatory network of BACH1 and downstream genes including CDH1 contribute to the malignant features of PDAC by regulating epithelial-to-mesenchymal transition.
Significance:
Greater understanding of the gene regulatory network involved in epithelial-to-mesenchymal transition of pancreatic cancer cells will provide novel therapeutic targets and diagnostic markers.
Data from Sustained Coevolution in a Stochastic Model of Cancer–Immune Interaction
Authors: George Jason T. , Levine Herbert
Published: March 2023

Abstract
The dynamic interactions between an evolving malignancy and the adaptive immune system generate diverse evolutionary trajectories that ultimately result in tumor clearance or immune escape. Here, we create a simple mathematical model coupling T-cell recognition with an evolving cancer population that may randomly produce evasive subclones, imparting transient protection against the effector T cells. T-cell turnover declines and evasion rates together explained differences in early incidence data across almost all cancer types. Fitting the model to TRACERx evolutionary data argued in favor of substantial and sustained immune pressure exerted upon a developing tumor, suggesting that clinically observed incidence is a small proportion of all cancer initiation events. This dynamical model promises to increase our quantitative understanding of many immune escape contexts, including cancer progression and intracellular pathogenic infections.
Significance:
The early cancer–immune interaction sculpts intratumor heterogeneity through the selection of immune-evasive clones. This study provides a mathematical framework for investigating the coevolution between an immune-evasive cancer population and the adaptive immune system.
Data from Increased Tumor Penetration of Single-Domain Antibody–Drug Conjugates Improves In Vivo Efficacy in Prostate Cancer Models
Authors: Nessler Ian , Khera Eshita , Vance Steven , Kopp Anna , Qiu Qifeng , Keating Thomas A. , Abu-Yousif Adnan O. , Sandal Thomas , Legg James , Thompson Lorraine , Goodwin Normann , Thurber Greg M.
Published: March 2023

Abstract
Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrating drugs at the site-of-action, a method embodied by the seven current FDA-approved antibody–drug conjugates (ADC). However, a variety of pharmacokinetic challenges result in relatively narrow therapeutic windows for these agents, hampering the development of new drugs. Here, we use a series of prostate-specific membrane antigen–binding single-domain (Humabody) ADC constructs to demonstrate that tissue penetration of protein–drug conjugates plays a major role in therapeutic efficacy. Counterintuitively, a construct with lower in vitro potency resulted in higher in vivo efficacy than other protein–drug conjugates. Biodistribution data, tumor histology images, spheroid experiments, in vivo single-cell measurements, and computational results demonstrate that a smaller size and slower internalization rate enabled higher tissue penetration and more cell killing. The results also illustrate the benefits of linking an albumin-binding domain to the single-domain ADCs. A construct lacking an albumin-binding domain was rapidly cleared, leading to lower tumor uptake (%ID/g) and decreased in vivo efficacy. In conclusion, these results provide evidence that reaching the maximum number of cells with a lethal payload dose correlates more strongly with in vivo efficacy than total tumor uptake or in vitro potency alone for these protein–drug conjugates. Computational modeling and protein engineering can be used to custom design an optimal framework for controlling internalization, clearance, and tissue penetration to maximize cell killing.
Significance:
A mechanistic study of protein–drug conjugates demonstrates that a lower potency compound is more effective in vivo than other agents with equal tumor uptake due to improved tissue penetration and cellular distribution.
Data from Beclin 1 Promotes Endosome Recruitment of Hepatocyte Growth Factor Tyrosine Kinase Substrate to Suppress Tumor Proliferation
Authors: Matthew-Onabanjo Asia N. , Janusis Jenny , Mercado-Matos Jose , Carlisle Anne E. , Kim Dohoon , Levine Fayola , Cruz-Gordillo Peter , Richards Ryan , Lee Michael J. , Shaw Leslie M.
Published: March 2023

Abstract
Beclin 1 has nonautophagic functions that include its ability to regulate endocytic receptor trafficking. However, the contribution of this function to tumor suppression is poorly understood. Here, we provide in vivo evidence that Beclin 1 suppresses tumor proliferation by regulating the endocytic trafficking and degradation of the EGFR and transferrin (TFR1) receptors. Beclin 1 promoted endosomal recruitment of hepatocyte growth factor tyrosine kinase substrate (HRS), which was necessary for sorting surface receptors to intraluminal vesicles for signal silencing and lysosomal degradation. In tumors with low Beclin 1 expression, endosomal HRS recruitment was diminished and receptor function was sustained. Collectively, our results demonstrate a novel role for Beclin 1 in impeding tumor growth by coordinating the regulation of key growth factor and nutrient receptors. These data provide an explanation for how low levels of Beclin 1 facilitate tumor proliferation and contribute to poor cancer outcomes.
Significance:
Beclin 1 controls the trafficking fate of growth regulatory receptors to suppress tumor proliferation.
Data from β-Galactosylceramidase Promotes Melanoma Growth via Modulation of Ceramide Metabolism
Authors: Belleri Mirella , Paganini Giuseppe , Coltrini Daniela , Ronca Roberto , Zizioli Daniela , Corsini Michela , Barbieri Andrea , Grillo Elisabetta , Calza Stefano , Bresciani Roberto , Maiorano Eugenio , Mastropasqua Mauro G. , Annese Tiziana , Giacomini Arianna , Ribatti Domenico , Casas Josefina , Levade Thierry , Fabrias Gemma , Presta Marco
Published: March 2023

Abstract
Disturbance of sphingolipid metabolism may represent a novel therapeutic target in metastatic melanoma, the most lethal form of skin cancer. β-Galactosylceramidase (GALC) removes β-galactose from galactosylceramide and other sphingolipids. In this study, we show that downregulation of galcb, a zebrafish ortholog of human GALC, affects melanoblast and melanocyte differentiation in zebrafish embryos, suggesting a possible role for GALC in melanoma. On this basis, the impact of GALC expression in murine B16-F10 and human A2058 melanoma cells was investigated following its silencing or upregulation. Galc knockdown hampered growth, motility, and invasive capacity of B16-F10 cells and their tumorigenic and metastatic activity when grafted in syngeneic mice or zebrafish embryos. Galc-silenced cells displayed altered sphingolipid metabolism and increased intracellular levels of ceramide, paralleled by a nonredundant upregulation of Smpd3, which encodes for the ceramide-generating enzyme neutral sphingomyelinase 2. Accordingly, GALC downregulation caused SMPD3 upregulation, increased ceramide levels, and inhibited the tumorigenic activity of human melanoma A2058 cells, whereas GALC upregulation exerted opposite effects. In concordance with information from melanoma database mining, RNAscope analysis demonstrated a progressive increase of GALC expression from common nevi to stage IV human melanoma samples that was paralleled by increases in microphthalmia transcription factor and tyrosinase immunoreactivity inversely related to SMPD3 and ceramide levels. Overall, these findings indicate that GALC may play an oncogenic role in melanoma by modulating the levels of intracellular ceramide, thus providing novel opportunities for melanoma therapy.
Significance:
Data from zebrafish embryos, murine and human cell melanoma lines, and patient-derived tumor specimens indicate that β-galactosylceramidase plays an oncogenic role in melanoma and may serve as a therapeutic target.
Data from Adipocyte-Induced FABP4 Expression in Ovarian Cancer Cells Promotes Metastasis and Mediates Carboplatin Resistance
Authors: Mukherjee Abir , Chiang Chun-Yi , Daifotis Helen A. , Nieman Kristin M. , Fahrmann Johannes F. , Lastra Ricardo R. , Romero Iris L. , Fiehn Oliver , Lengyel Ernst
Published: March 2023

Abstract
Adipocytes are critical for ovarian cancer cells to home to the omentum, but the metabolic changes initiated by this interaction are unknown. To this end, we carried out unbiased mass spectrometry–based metabolomic and proteomic profiling of cancer cells cocultured with primary human omental adipocytes. Cancer cells underwent significant proteo-metabolomic alteration(s), typified by changes in the lipidome with corresponding upregulation of lipid metabolism proteins. FABP4, a lipid chaperone protein, was identified as the critical regulator of lipid responses in ovarian cancer cells cocultured with adipocytes. Subsequently, knockdown of FABP4 resulted in increased 5-hydroxymethylcytosine levels in the DNA, downregulation of gene signatures associated with ovarian cancer metastasis, and reduced clonogenic cancer cell survival. In addition, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated knockout of FABP4 in high-grade serous ovarian cancer cells reduced metastatic tumor burden in mice. Consequently, a small-molecule inhibitor of FABP4 (BMS309403) not only significantly reduced tumor burden in a syngeneic orthotopic mouse model but also increased the sensitivity of cancer cells toward carboplatin both in vitro and in vivo. Taken together, these results show that targeting FABP4 in ovarian cancer cells can inhibit their ability to adapt and colonize lipid-rich tumor microenvironments, providing an opportunity for specific metabolic targeting of ovarian cancer metastasis.
Significance:
Ovarian cancer metastatic progression can be restricted by targeting a critical regulator of lipid responses, FABP4.
Data from USP22 Functions as an Oncogenic Driver in Prostate Cancer by Regulating Cell Proliferation and DNA Repair
Authors: McCann Jennifer J. , Vasilevskaya Irina A. , Poudel Neupane Neermala , Shafi Ayesha A. , McNair Christopher , Dylgjeri Emanuela , Mandigo Amy C. , Schiewer Matthew J. , Schrecengost Randy S. , Gallagher Peter , Stanek Timothy J. , McMahon Steven B. , Berman-Booty Lisa D. , Ostrander William F. , Knudsen Karen E.
Published: March 2023

Abstract
Emerging evidence indicates the deubiquitinase USP22 regulates transcriptional activation and modification of target substrates to promote pro-oncogenic phenotypes. Here, in vivo characterization of tumor-associated USP22 upregulation and unbiased interrogation of USP22-regulated functions in vitro demonstrated critical roles for USP22 in prostate cancer. Specifically, clinical datasets validated that USP22 expression is elevated in prostate cancer, and a novel murine model demonstrated a hyperproliferative phenotype with prostate-specific USP22 overexpression. Accordingly, upon overexpression or depletion of USP22, enrichment of cell-cycle and DNA repair pathways was observed in the USP22-sensitive transcriptome and ubiquitylome using prostate cancer models of clinical relevance. Depletion of USP22 sensitized cells to genotoxic insult, and the role of USP22 in response to genotoxic insult was further confirmed using mouse adult fibroblasts from the novel murine model of USP22 expression. As it was hypothesized that USP22 deubiquitylates target substrates to promote protumorigenic phenotypes, analysis of the USP22-sensitive ubiquitylome identified the nucleotide excision repair protein, XPC, as a critical mediator of the USP22-mediated response to genotoxic insult. Thus, XPC undergoes deubiquitylation as a result of USP22 function and promotes USP22-mediated survival to DNA damage. Combined, these findings reveal unexpected functions of USP22 as a driver of protumorigenic phenotypes and have significant implications for the role of USP22 in therapeutic outcomes.
Significance:
The studies herein present a novel mouse model of tumor-associated USP22 overexpression and implicate USP22 in modulation of cellular survival and DNA repair, in part through regulation of XPC.
Data from The Deubiquitinase USP38 Promotes NHEJ Repair through Regulation of HDAC1 Activity and Regulates Cancer Cell Response to Genotoxic Insults
Authors: Yang Yongfeng , Yang Chuanzhen , Li Tingting , Yu Shuyu , Gan Tingting , Hu Jiazhi , Cui Jun , Zheng Xiaofeng
Published: March 2023

Abstract
The DNA damage response (DDR) is essential for maintaining genome integrity. Mounting evidence reveals that protein modifications play vital roles in the DDR. Here, we show that USP38 is involved in the DDR by regulating the activity of HDAC1. In response to DNA damage, USP38 interacted with HDAC1 and specifically removed the K63-linked ubiquitin chain promoting the deacetylase activity of HDAC1. As a result, HDAC1 was able to deacetylate H3K56. USP38 deletion resulted in persistent focal accumulation of nonhomologous end joining (NHEJ) factors at DNA damage sites and impaired NHEJ efficiency, causing genome instability and sensitizing cancer cells to genotoxic insults. Knockout of USP38 rendered mice hypersensitive to irradiation and shortened survival. In addition, USP38 was expressed at low levels in certain types of cancers including renal cell carcinoma, indicating dysregulation of USP38 expression contributes to genomic instability and may lead to tumorigenesis. In summary, this study identifies a critical role of USP38 in modulating genome integrity and cancer cell resistance to genotoxic insults by deubiquitinating HDAC1 and regulating its deacetylation activity.
Significance:
This study demonstrates that USP38 regulates genome stability and mediates cancer cell resistance to DNA-damaging therapy, providing insight into tumorigenesis and implicating USP38 as a potential target for cancer diagnosis.
Data from EpCAM Signaling Promotes Tumor Progression and Protein Stability of PD-L1 through the EGFR Pathway
Authors: Chen Hao-Nien , Liang Kang-Hao , Lai Jun-Kai , Lan Chun-Hsin , Liao Mei-Ying , Hung Shao-Hsi , Chuang Yi-Ting , Chen Kai-Chi , Tsuei William Wei-Fu , Wu Han-Chung
Published: March 2023

Abstract
Although epithelial cell adhesion molecule (EpCAM) has previously been shown to promote tumor progression, the underlying mechanisms remain largely unknown. Here, we report that the EGF-like domain I within the extracellular domain of EpCAM (EpEX) binds EGFR, activating both AKT and MAPK signaling to inhibit forkhead transcription factor O3a (FOXO3a) function and stabilize PD-L1 protein, respectively. Treatment with the EpCAM neutralizing antibody, EpAb2-6, inhibited AKT and FOXO3a phosphorylation, increased FOXO3a nuclear translocation, and upregulated high temperature requirement A2 (HtrA2) expression to promote apoptosis while decreasing PD-L1 protein levels to enhance the cytotoxic activity of CD8+ T cells. In vivo, EpAb2-6 markedly extended survival in mouse metastasis and orthotopic models of human colorectal cancer. The combination of EpAb2-6 with atezolizumab, an anti-PD-L1 antibody, almost completely eliminated tumors. Moreover, the number of CD8+ T cells in combination-treated tumors was increased compared with atezolizumab alone. Our findings suggest a new combination strategy for cancer immunotherapy in patients with EpCAM-expressing tumors.
Significance:
This study shows that treatment with an EpCAM neutralizing antibody promotes apoptosis while decreasing PD-L1 protein to enhance cytotoxic activity of CD8+ T cells.
Data from Therapeutic Targeting of the Premetastatic Stage in Human Lung-to-Brain Metastasis
Authors: Singh Mohini , Venugopal Chitra , Tokar Tomas , McFarlane Nicole , Subapanditha Minomi K. , Qazi Maleeha , Bakhshinyan David , Vora Parvez , Murty Naresh K. , Jurisica Igor , Singh Sheila K.
Published: March 2023

Abstract
Brain metastases (BM) result from the spread of primary tumors to the brain and are a leading cause of cancer mortality in adults. Secondary tissue colonization remains the main bottleneck in metastatic development, yet this “premetastatic” stage of the metastatic cascade, when primary tumor cells cross the blood–brain barrier and seed the brain before initiating a secondary tumor, remains poorly characterized. Current studies rely on specimens from fully developed macrometastases to identify therapeutic options in cancer treatment, overlooking the potentially more treatable “premetastatic” phase when colonizing cancer cells could be targeted before they initiate the secondary brain tumor. Here we use our established brain metastasis initiating cell (BMIC) models and gene expression analyses to characterize premetastasis in human lung-to-BM. Premetastatic BMIC engaged invasive and epithelial developmental mechanisms while simultaneously impeding proliferation and apoptosis. We identified the dopamine agonist apomorphine to be a potential premetastasis-targeting drug. In vivo treatment with apomorphine prevented BM formation, potentially by targeting premetastasis-associated genes KIF16B, SEPW1, and TESK2. Low expression of these genes was associated with poor survival of patients with lung adenocarcinoma. These results illuminate the cellular and molecular dynamics of premetastasis, which is subclinical and currently impossible to identify or interrogate in human patients with BM. These data present several novel therapeutic targets and associated pathways to prevent BM initiation.

Significance: These findings unveil molecular features of the premetastatic stage of lung-to-brain metastases and offer a potential therapeutic strategy to prevent brain metastases. Cancer Res; 78(17); 5124–34. ©2018 AACR.
Data from Structural Optimization and Enhanced Prodrug-Mediated Delivery Overcomes Camptothecin Resistance in High-Risk Solid Tumors
Authors: Nguyen Ferro , Guan Peng , Guerrero David T. , Kolla Venkatadri , Naraparaju Koumudi , Perry Lauren M. , Soberman Danielle , Pressly Benjamin B. , Alferiev Ivan S. , Chorny Michael , Brodeur Garrett M.
Published: March 2023

Abstract
Camptothecins are potent topoisomerase I inhibitors used to treat high-risk pediatric solid tumors, but they often show poor efficacy due to intrinsic or acquired chemoresistance. Here, we developed a multivalent, polymer-based prodrug of a structurally optimized camptothecin (SN22) designed to overcome key chemoresistance mechanisms. The ability of SN22 vs. SN38 (the active form of irinotecan/CPT-11) to overcome efflux pump-driven drug resistance was tested. Tumor uptake and biodistribution of SN22 as a polymer-based prodrug (PEG-[SN22]4) compared with SN38 was determined. The therapeutic efficacy of PEG-[SN22]4 to CPT-11 was compared in: (i) spontaneous neuroblastomas (NB) in transgenic TH-MYCN mice; (ii) orthotopic xenografts of a drug-resistant NB line SK-N-BE(2)C (mutated TP53); (iii) flank xenografts of a drug-resistant NB-PDX; and (iv) xenografts of Ewing sarcoma and rhabdomyosarcoma. Unlike SN38, SN22 inhibited NB cell growth regardless of ABCG2 expression levels. SN22 prodrug delivery resulted in sustained intratumoral drug concentrations, dramatically higher than those of SN38 at all time points. CPT-11/SN38 treatment had only marginal effects on tumors in transgenic mice, but PEG-[SN22]4 treatment caused complete tumor regression lasting over 6 months (tumor free at necropsy). PEG-[SN22]4 also markedly extended survival of mice with drug-resistant, orthotopic NB and it caused long-term (6+ months) remissions in 80% to 100% of NB and sarcoma xenografts. SN22 administered as a multivalent polymeric prodrug resulted in increased and protracted tumor drug exposure compared with CPT-11, leading to long-term “cures” in NB models of intrinsic or acquired drug resistance, and models of high-risk sarcomas, warranting its further development for clinical trials.
Significance:
SN22 is an effective and curative multivalent macromolecular agent in multiple solid tumor mouse models, overcoming common mechanisms of drug resistance with the potential to elicit fewer toxicities than most cancer therapeutics.
Data from Cancer Cell CD44 Mediates Macrophage/Monocyte-Driven Regulation of Head and Neck Cancer Stem Cells
Authors: Gomez Karina E. , Wu FangLong , Keysar Stephen B. , Morton J. Jason , Miller Bettina , Chimed Tugs-Saikhan , Le Phuong N. , Nieto Cera , Chowdhury Farshad N. , Tyagi Anit , Lyons Traci R. , Young Christian D. , Zhou Hongmei , Somerset Hilary L. , Wang Xiao-Jing , Jimeno Antonio
Published: March 2023

Abstract
Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K–4EBP1–SOX2 activation and signaling regulate CSC properties, yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME, we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K–4EBP1–SOX2 pathway, possibly by modulating availability of hyaluronic acid (HA), the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships, and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC–derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions, respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K–4EBP1–SOX2 signaling and the CSC fraction, whereas CD44–VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo, targeting CD44 decreased PI3K–4EBP1–SOX2 signaling, tumor growth, and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally, a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes, respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals.
Significance:
These findings establish a mechanistic link between tumor cell CD44, TAM, and CSC properties at the tumor–stroma interface that can serve as a vital area of focus for target and drug discovery.
Data from Extracellular Vesicle–Derived miR-124 Resolves Radiation-Induced Brain Injury
Authors: Leavitt Ron J. , Acharya Munjal M. , Baulch Janet E. , Limoli Charles L.
Published: March 2023

Abstract
Radiation-induced cognitive dysfunction (RICD) is a progressive and debilitating health issue facing patients following cranial radiotherapy to control central nervous system cancers. There has been some success treating RICD in rodents using human neural stem cell (hNSC) transplantation, but the procedure is invasive, requires immunosuppression, and could cause other complications such as teratoma formation. Extracellular vesicles (EV) are nanoscale membrane-bound structures that contain biological contents including mRNA, miRNA, proteins, and lipids that can be readily isolated from conditioned culture media. It has been previously shown that hNSC-derived EV resolves RICD following cranial irradiation using an immunocompromised rodent model. Here, we use immunocompetent wild-type mice to show that hNSC-derived EV treatment administered either intravenously via retro-orbital vein injection or via intracranial transplantation can ameliorate cognitive deficits following 9 Gy head-only irradiation. Cognitive function assessed on the novel place recognition, novel object recognition, and temporal order tasks was not only improved at early (5 weeks) but also at delayed (6 months) postirradiation times with just a single EV treatment. Improved behavioral outcomes were also associated with reduced neuroinflammation as measured by a reduction in activated microglia. To identify the mechanism of action, analysis of EV cargo implicated miRNA (miR-124) as a potential candidate in the mitigation of RICD. Furthermore, viral vector–mediated overexpression of miR-124 in the irradiated brain ameliorated RICD and reduced microglial activation. Our findings demonstrate for the first time that systemic administration of hNSC-derived EV abrogates RICD and neuroinflammation in cranially irradiated wild-type rodents through a mechanism involving miR-124.
Significance:
Radiation-induced neurocognitive decrements in immunocompetent mice can be resolved by systemic delivery of hNSC-derived EVs involving a mechanism dependent on expression of miR-124.
Data from Cotargeting Ephrin Receptor Tyrosine Kinases A2 and A3 in Cancer Stem Cells Reduces Growth of Recurrent Glioblastoma
Authors: Qazi Maleeha A. , Vora Parvez , Venugopal Chitra , Adams Jarrett , Singh Mohini , Hu Amy , Gorelik Maryna , Subapanditha Minomi K. , Savage Neil , Yang Jiahe , Chokshi Chirayu , London Max , Gont Alexander , Bobrowski David , Grinshtein Natalie , Brown Kevin R. , Murty Naresh K. , Nilvebrant Johan , Kaplan David , Moffat Jason , Sidhu Sachdev , Singh Sheila K.
Published: March 2023

Abstract
Glioblastoma (GBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many members of the Eph receptor tyrosine kinase (EphR) family are expressed by GBM stem cells (GSC), which have been implicated in resistance to GBM therapy. In this study, we identify several EphRs that mark a therapeutically targetable GSC population in treatment-refractory, recurrent GBM (rGBM). Using a highly specific EphR antibody panel and CyTOF (cytometry by time-of-flight), we characterized the expression of all 14 EphR in primary and recurrent patient-derived GSCs to identify putative rGBM-specific EphR. EPHA2 and EPHA3 coexpression marked a highly tumorigenic cell population in rGBM that was enriched in GSC marker expression. Knockdown of EPHA2 and EPHA3 together led to increased expression of differentiation marker GFAP and blocked clonogenic and tumorigenic potential, promoting significantly higher survival in vivo. Treatment of rGBM with a bispecific antibody against EPHA2/A3 reduced clonogenicity in vitro and tumorigenic potential of xenografted recurrent GBM in vivo via downregulation of AKT and ERK and increased cellular differentiation. In conclusion, we show that EPHA2 and EPHA3 together mark a GSC population in rGBM and that strategic cotargeting of EPHA2 and EPHA3 presents a novel and rational therapeutic approach for rGBM.

Significance: Treatment of rGBM with a novel bispecific antibody against EPHA2 and EPHA3 reduces tumor burden, paving the way for the development of therapeutic approaches against biologically relevant targets in rGBM. Cancer Res; 78(17); 5023–37. ©2018 AACR.
Data from The MDM2/MDMX-p53 Antagonist PM2 Radiosensitizes Wild-Type p53 Tumors
Authors: Spiegelberg Diana , Mortensen Anja C. , Lundsten Sara , Brown Christopher J. , Lane David P. , Nestor Marika
Published: March 2023

Abstract
Radiotherapy amplifies p53 expression in cancer cells with wild-type (wt) p53. Blocking the negative regulators MDM2 and MDMX stabilizes p53 and may therefore potentiate radiotherapy outcomes. In this study, we investigate the efficacy of the novel anti-MDM2/X stapled peptide PM2 alone and in combination with external gamma radiation in vitro and in vivo. PM2 therapy combined with radiotherapy elicited synergistic therapeutic effects compared with monotherapy in cells with wt p53 in both in vitro and in vivo assays, whereas these effects did not manifest in p53 −/− cells. Biodistribution and autoradiography of 125I-PM2 revealed high and retained uptake homogenously distributed throughout the tumor. In mice carrying wt p53 tumors, PM2 combined with radiotherapy significantly prolonged the median survival by 50%, whereas effects of PM2 therapy on mutant and p53 −/− tumors were negligible. PM2-dependent stabilization of p53 was confirmed with ex vivo immunohistochemistry. These data demonstrate the potential of the stapled peptide PM2 as a radiotherapy potentiator in vivo and suggest that clinical application of PM2 with radiotherapy in wt p53 cancers might improve tumor control.

Significance: These findings contribute advances to cancer radiotherapy by using novel p53-reactivating stapled peptides as radiosensitizers in wild-type p53 cancers. Cancer Res; 78(17); 5084–93. ©2018 AACR.
Data from Resolution of Spatial and Temporal Heterogeneity in Bevacizumab-Treated Breast Tumors by Eigenspectra Multispectral Optoacoustic Tomography
Authors: Liapis Evangelos , Klemm Uwe , Karlas Angelos , Reber Josefine , Ntziachristos Vasilis
Published: March 2023

Abstract
Understanding temporal and spatial hemodynamic heterogeneity as a function of tumor growth or therapy affects the development of novel therapeutic strategies. In this study, we employed eigenspectra multispectral optoacoustic tomography (eMSOT) as a next-generation optoacoustic method to impart high accuracy in resolving tumor hemodynamics during bevacizumab therapy in two types of breast cancer xenografts (KPL-4 and MDA-MB-468). Patterns of tumor total hemoglobin concentration (THb) and oxygen saturation (sO2) were imaged in control and bevacizumab-treated tumors over the course of 58 days (KPL-4) and 16 days (MDA-MB-468), and the evolution of functional vasculature “normalization” was resolved macroscopically. An initial sharp drop in tumor sO2 and THb content shortly after the initiation of bevacizumab treatment was followed by a recovery in oxygenation levels. Rim–core subregion analysis revealed steep spatial oxygenation gradients in growing tumors that were reduced after bevacizumab treatment. Critically, eMSOT imaging findings were validated directly by histopathologic assessment of hypoxia (pimonidazole) and vascularity (CD31). These data demonstrate how eMSOT brings new abilities for accurate observation of entire tumor responses to challenges at spatial and temporal dimensions not available by other techniques today.
Significance:
Accurate assessment of hypoxia and vascularization over space and time is critical for understanding tumor development and the role of spatial heterogeneity in tumor aggressiveness, metastasis, and response to treatment.
Data from Downregulation of miR-223 Expression Is an Early Event during Mammary Transformation and Confers Resistance to CDK4/6 Inhibitors in Luminal Breast Cancer
Authors: Citron Francesca , Segatto Ilenia , Vinciguerra Gian Luca Rampioni , Musco Lorena , Russo Francesca , Mungo Giorgia , D'Andrea Sara , Mattevi Maria Chiara , Perin Tiziana , Schiappacassi Monica , Massarut Samuele , Marchini Cristina , Amici Augusto , Vecchione Andrea , Baldassarre Gustavo , Belletti Barbara
Published: March 2023

Abstract
miR-223 is an anti-inflammatory miRNA that in cancer acts either as an oncosuppressor or oncopromoter, in a context-dependent manner. In breast cancer, we demonstrated that it dampens the activation of the EGF pathway. However, little is known on the role of miR-223 during breast cancer onset and progression. miR-223 expression was decreased in breast cancer of luminal and HER2 subtypes and inversely correlated with patients' prognosis. In normal luminal mammary epithelial cells, miR-223 acted cell autonomously in the control of their growth and morphology in three-dimensional context. In the MMTV-Δ16HER2 transgenic mouse model, oncogene transformation resulted in a timely abrogation of miR-223 expression, likely due to activation of E2F1, a known repressor of miR-223 transcription. Accordingly, treatment with CDK4/6 inhibitors, which eventually results in restraining E2F1 activity, restored miR-223 expression and miR-223 ablation induced luminal breast cancer resistance to CDK4/6 inhibition, both in vitro and in vivo. Notably, miR-223 expression was lost in microdissected ductal carcinoma in situ (DCIS) from patients with luminal and HER2-positive breast cancer. Altogether, these results identify downmodulation of miR-223 as an early step in luminal breast cancer onset and suggest that it could be used to identify aggressive DCIS and predict the response to targeted therapy.
Significance:
miR-223 may represent a predictive biomarker of response to CDK4/6 inhibitors and its loss could identify DCIS lesions that are likely to progress into invasive breast cancer.
Data from YAP Enhances Tumor Cell Dissemination by Promoting Intravascular Motility and Reentry into Systemic Circulation
Authors: Benjamin David C. , Kang Joon Ho , Hamza Bashar , King Emily M. , Lamar John M. , Manalis Scott R. , Hynes Richard O.
Published: March 2023

Abstract
The oncogene YAP has been shown previously to promote tumor growth and metastasis. However, how YAP influences the behavior of tumor cells traveling within the circulatory system has not been as well explored. Given that rate-limiting steps of metastasis are known to occur while tumor cells enter, travel through, or exit circulation, we sought to study how YAP influences tumor cell behavior within the circulatory system. Intravital imaging in live zebrafish embryos revealed that YAP influenced the distribution of tumor cells within the animal following intravenous injection. Control cells became lodged in the first capillary bed encountered in the tail, whereas cells overexpressing constitutively active YAP were able to travel through this capillary plexus, reenter systemic circulation, and seed in the brain. YAP controlled transit through these capillaries by promoting active migration within the vasculature. These results were corroborated in a mouse model following intravenous injection, where active YAP increased the number of circulating tumor cells over time. Our results suggest a possible mechanism whereby tumor cells can spread to organs beyond the first capillary bed downstream from the primary tumor. These results also show that a specific gene can affect the distribution of tumor cells within an animal, thereby influencing the global pattern of metastasis in that animal.
Significance:
These findings demonstrate that YAP endows tumor cells with the ability to move through capillaries, allowing them to return to and persist in circulation, thereby increasing their metastatic spread.

See related commentary by Davidson, p. 3797
Data from Decreased Mitochondrial DNA Content Drives OXPHOS Dysregulation in Chromophobe Renal Cell Carcinoma
Authors: Xiao Yi , Clima Rosanna , Busch Jonas , Rabien Anja , Kilic Ergin , Villegas Sonia L. , Timmermann Bernd , Attimonelli Marcella , Jung Klaus , Meierhofer David
Published: March 2023

Abstract
Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are closely related, rare kidney tumors. Mutations in complex I (CI)-encoding genes play an important role in dysfunction of the oxidative phosphorylation (OXPHOS) system in renal oncocytoma, but are less frequently observed in chRCC. As such, the relevance of OXPHOS status and role of CI mutations in chRCC remain unknown. To address this issue, we performed proteome and metabolome profiling as well as mitochondrial whole-exome sequencing to detect mitochondrial alterations in chRCC tissue specimens. Multiomic analysis revealed downregulation of electron transport chain (ETC) components in chRCC that differed from the expression profile in renal oncocytoma. A decrease in mitochondrial (mt)DNA content, rather than CI mutations, was the main cause for reduced OXPHOS in chRCC. There was a negative correlation between protein and transcript levels of nuclear DNA- but not mtDNA-encoded ETC complex subunits in chRCC. In addition, the reactive oxygen species scavenger glutathione (GSH) was upregulated in chRCC due to decreased expression of proteins involved in GSH degradation. These results demonstrate that distinct mechanisms of OXPHOS exist in chRCC and renal oncocytoma and that expression levels of ETC complex subunits can serve as a diagnostic marker for this rare malignancy.
Significance:
These findings establish potential diagnostic markers to distinguish malignant chRCC from its highly similar but benign counterpart, renal oncocytoma.
Data from IL2RA Promotes Aggressiveness and Stem Cell–Related Properties of Acute Myeloid Leukemia
Authors: Nguyen Chi Huu , Schlerka Angela , Grandits Alexander M. , Koller Elisabeth , van der Kouwe Emiel , Vassiliou George S. , Staber Philipp B. , Heller Gerwin , Wieser Rotraud
Published: March 2023

Abstract
Overexpression of IL2RA, which encodes the alpha chain of the IL2 receptor, is associated with chemotherapy resistance and poor outcome in acute myeloid leukemia (AML). The clinical potential of anti-IL2RA therapy is, therefore, being explored in early-stage clinical trials. Notwithstanding, only very limited information regarding the biological function of IL2RA in AML is available. Using genetic manipulation of IL2RA expression as well as antibody-mediated inhibition of IL2RA in human cell lines, mouse models, and primary patient samples, we investigated the effects of IL2RA on AML cell proliferation and apoptosis, and on pertinent signaling pathways. The impact of IL2RA on the properties of leukemic stem cells (LSC) and on leukemogenesis were queried. IL2RA promoted proliferation and cell-cycle activity and inhibited apoptosis in human AML cell lines and primary cells. These phenotypes were accompanied by corresponding alterations in cell-cycle machinery and in pathways associated with cell survival and apoptosis. The biological roles of IL2RA were confirmed in two genetically distinct AML mouse models, revealing that IL2RA inhibits differentiation, promotes stem cell–related properties, and is required for leukemogenesis. IL2RA antibodies inhibited leukemic, but not normal, hematopoietic cells and synergized with other antileukemic agents in this regard. Collectively, these data show for the first time that IL2RA plays key biological roles in AML and underscore its value as a potential therapeutic target in this disease.
Significance:
This study identifies IL2RA as a potential therapeutic target in AML, where it is shown to regulate proliferation, differentiation, apoptosis, stem cell–related properties, and leukemogenesis.
Data from A Rare TP53 Mutation Predominant in Ashkenazi Jews Confers Risk of Multiple Cancers
Authors: Powers Jacquelyn , Pinto Emilia M. , Barnoud Thibaut , Leung Jessica C. , Martynyuk Tetyana , Kossenkov Andrew V. , Philips Aaron H. , Desai Heena , Hausler Ryan , Kelly Gregory , Le Anh N. , Li Marilyn M. , MacFarland Suzanne P. , Pyle Louise C. , Zelley Kristin , Nathanson Katherine L. , Domchek Susan M. , Slavin Thomas P. , Weitzel Jeffrey N. , Stopfer Jill E. , Garber Judy E. , Joseph Vijai , Offit Kenneth , Dolinsky Jill S. , Gutierrez Stephanie , McGoldrick Kelly , Couch Fergus J. , Levin Brooke , Edelman Morris C. , Levy Carolyn Fein , Spunt Sheri L. , Kriwacki Richard W. , Zambetti Gerard P. , Ribeiro Raul C. , Murphy Maureen E. , Maxwell Kara N.
Published: March 2023

Abstract
Germline mutations in TP53 cause a rare high penetrance cancer syndrome, Li–Fraumeni syndrome (LFS). Here, we identified a rare TP53 tetramerization domain missense mutation, c.1000G>C;p.G334R, in a family with multiple late-onset LFS-spectrum cancers. Twenty additional c.1000G>C probands and one c.1000G>A proband were identified, and available tumors showed biallelic somatic inactivation of TP53. The majority of families were of Ashkenazi Jewish descent, and the TP53 c.1000G>C allele was found on a commonly inherited chromosome 17p13.1 haplotype. Transient transfection of the p.G334R allele conferred a mild defect in colony suppression assays. Lymphoblastoid cell lines from the index family in comparison with TP53 normal lines showed that although classical p53 target gene activation was maintained, a subset of p53 target genes (including PCLO, PLTP, PLXNB3, and LCN15) showed defective transactivation when treated with Nutlin-3a. Structural analysis demonstrated thermal instability of the G334R-mutant tetramer, and the G334R-mutant protein showed increased preponderance of mutant conformation. Clinical case review in comparison with classic LFS cohorts demonstrated similar rates of pediatric adrenocortical tumors and other LFS component cancers, but the latter at significantly later ages of onset. Our data show that TP53 c.1000G>C;p.G334R is found predominantly in Ashkenazi Jewish individuals, causes a mild defect in p53 function, and leads to low penetrance LFS.
Significance:
TP53 c.1000C>G;p.G334R is a pathogenic, Ashkenazi Jewish–predominant mutation associated with a familial multiple cancer syndrome in which carriers should undergo screening and preventive measures to reduce cancer risk.
Data from LDtrait: An Online Tool for Identifying Published Phenotype Associations in Linkage Disequilibrium
Authors: Lin Shu-Hong , Brown Derek W. , Machiela Mitchell J.
Published: March 2023

Abstract
Genome-wide association studies (GWAS) have identified thousands of germline susceptibility loci associated with risk for cancer as well as a wide range of other traits and diseases. An interest of many investigators is identifying traits or diseases that share common susceptibility loci. We developed LDtrait (https://ldlink.nci.nih.gov/?tab=ldtrait) as an open access web tool for finding germline variation associated with multiple traits. LDtrait searches the NHGRI-EBI GWAS Catalog to identify susceptibility loci in linkage disequilibrium (LD) with a user-provided list of query variants. Options allow for modifying LD thresholds, calculating LD from a diverse set of reference populations, and downloading annotated variant lists. Results from example query searches highlight the utility of LDtrait in uncovering cross-trait associations for cancer risk and other traits. LDtrait accelerates etiologic understanding of cancer genetics by rapidly identifying genetic similarities with other traits or diseases.
Significance:
The new GWAS search tool LDtrait will expedite discovery of shared genetic components underlying seemingly unrelated diseases and may offer novel insights into cancer research.
Data from Targeting Pan-ETS Factors Inhibits Melanoma Progression
Authors: Huang Lee , Zhai Yougang , La Jennifer , Lui Jason W. , Moore Stephen P.G. , Little Elizabeth C. , Xiao Sixia , Haresi Adil J. , Brem Candice , Bhawan Jag , Lang Deborah
Published: March 2023

Abstract
The failure of once promising target-specific therapeutic strategies often arises from redundancies in gene expression pathways. Even with new melanoma treatments, many patients are not responsive or develop resistance, leading to disease progression in terms of growth and metastasis. We previously discovered that the transcription factors ETS1 and PAX3 drive melanoma growth and metastasis by promoting the expression of the MET receptor. Here, we find that there are multiple ETS family members expressed in melanoma and that these factors have redundant functions. The small molecule YK-4-279, initially developed to target the ETS gene–containing translocation product EWS-FLI1, significantly inhibited cellular growth, invasion, and ETS factor function in melanoma cell lines and a clinically relevant transgenic mouse model, BrafCA;Tyr-CreERT2;Ptenf/f. One of the antitumor effects of YK-4-279 in melanoma is achieved via interference of multiple ETS family members with PAX3 and the expression of the PAX3-ETS downstream gene MET. Expression of exogenous MET provided partial rescue of the effects of YK-4-279, further supporting that MET loss is a significant contributor to the antitumor effects of the drug. This is the first study identifying multiple overlapping functions of the ETS family promoting melanoma. In addition, targeting all factors, rather than individual members, demonstrated impactful deleterious consequences in melanoma progression. Given that multiple ETS factors are known to have oncogenic functions in other malignancies, these findings have a high therapeutic impact.
Significance:
These findings identify YK-4-279 as a promising therapeutic agent against melanoma by targeting multiple ETS family members and blocking their ability to act as transcription factors.
Data from UBR5 Is Coamplified with MYC in Breast Tumors and Encodes an Ubiquitin Ligase That Limits MYC-Dependent Apoptosis
Authors: Qiao Xi , Liu Ying , Prada Maria Llamazares , Mohan Aravind K. , Gupta Abhishekh , Jaiswal Alok , Sharma Mukund , Merisaari Joni , Haikala Heidi M. , Talvinen Kati , Yetukuri Laxman , Pylvänäinen Joanna W. , Klefström Juha , Kronqvist Pauliina , Meinander Annika , Aittokallio Tero , Hietakangas Ville , Eilers Martin , Westermarck Jukka
Published: March 2023

Abstract
For maximal oncogenic activity, cellular MYC protein levels need to be tightly controlled so that they do not induce apoptosis. Here, we show how ubiquitin ligase UBR5 functions as a molecular rheostat to prevent excess accumulation of MYC protein. UBR5 ubiquitinates MYC and its effects on MYC protein stability are independent of FBXW7. Silencing of endogenous UBR5 induced MYC protein expression and regulated MYC target genes. Consistent with the tumor suppressor function of UBR5 (HYD) in Drosophila, HYD suppressed dMYC-dependent overgrowth of wing imaginal discs. In contrast, in cancer cells, UBR5 suppressed MYC-dependent priming to therapy-induced apoptosis. Of direct cancer relevance, MYC and UBR5 genes were coamplified in MYC-driven human cancers. Functionally, UBR5 suppressed MYC-mediated apoptosis in p53-mutant breast cancer cells with UBR5/MYC coamplification. Furthermore, single-cell immunofluorescence analysis demonstrated reciprocal expression of UBR5 and MYC in human basal-type breast cancer tissues. In summary, UBR5 is a novel MYC ubiquitin ligase and an endogenous rheostat for MYC activity. In MYC-amplified, and p53-mutant breast cancer cells, UBR5 has an important role in suppressing MYC-mediated apoptosis priming and in protection from drug-induced apoptosis.
Significance:
These findings identify UBR5 as a novel MYC regulator, the inactivation of which could be very important for understanding of MYC dysregulation on cancer cells.