bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from CXADR-Mediated Formation of an AKT Inhibitory Signalosome at Tight Junctions Controls Epithelial–Mesenchymal Plasticity in Breast Cancer
Authors: Nilchian Azadeh , Johansson Joel , Ghalali Aram , Asanin Sandra T. , Santiago Ana , Rosencrantz Oskar , Sollerbrant Kerstin , Vincent C. Theresa , Sund Malin , Stenius Ulla , Fuxe Jonas
Published: March 2023

Abstract
Tight junctions (TJ) act as hubs for intracellular signaling pathways controlling epithelial cell fate and function. Deregulation of TJ is a hallmark of epithelial–mesenchymal transition (EMT), which contributes to carcinoma progression and metastasis. However, the signaling mechanisms linking TJ to the induction of EMT are not understood. Here, we identify a TJ-based signalosome, which controls AKT signaling and EMT in breast cancer. The coxsackie and adenovirus receptor (CXADR), a TJ protein with an essential yet uncharacterized role in organogenesis and tissue homeostasis, was identified as a key component of the signalosome. CXADR regulated the stability and function of the phosphatases and AKT inhibitors PTEN and PHLPP2. Loss of CXADR led to hyperactivation of AKT and sensitized cells to TGFβ1–induced EMT. Conversely, restoration of CXADR stabilized PHLPP2 and PTEN, inhibited AKT, and promoted epithelial differentiation. Loss of CXADR in luminal A breast cancer correlated with loss of PHLPP2 and PTEN and poor prognosis. These results show that CXADR promotes the formation of an AKT-inhibitory signalosome at TJ and regulates epithelial–mesenchymal plasticity in breast cancer cells. Moreover, loss of CXADR might be used as a prognostic marker in luminal breast cancer.
Significance:
The tight junction protein CXADR controls epithelial-mesenchymal plasticity in breast cancer by stabilizing the AKT regulators PTEN and PHLPP2.
Data from Infiltrating Mast Cell–Mediated Stimulation of Estrogen Receptor Activity in Breast Cancer Cells Promotes the Luminal Phenotype
Authors: Majorini Maria Teresa , Cancila Valeria , Rigoni Alice , Botti Laura , Dugo Matteo , Triulzi Tiziana , De Cecco Loris , Fontanella Enrico , Jachetti Elena , Tagliabue Elda , Chiodoni Claudia , Tripodo Claudio , Colombo Mario P. , Lecis Daniele
Published: March 2023

Abstract
Tumor growth and development is determined by both cancer cell–autonomous and microenvironmental mechanisms, including the contribution of infiltrating immune cells. Because the role of mast cells (MC) in this process is poorly characterized and even controversial, we investigated their part in breast cancer. Crossing C57BL/6 MMTV-PyMT mice, which spontaneously develop mammary carcinomas, with MC-deficient C57BL/6-KitW-sh/W-sh (Wsh) mice, showed that MCs promote tumor growth and prevent the development of basal CK5-positive areas in favor of a luminal gene program. When cocultured with breast cancer cells in vitro, MCs hindered activation of cMET, a master regulator of the basal program, and simultaneously promoted expression and activation of estrogen receptor (ESR1/ER) and its target genes (PGR, KRT8/CK8, BCL2), which are all luminal markers. Moreover, MCs reduced ERBB2/HER2 levels, whose inhibition further increased ESR1 expression. In vivo and in silico analysis of patients with breast cancer revealed a direct correlation between MC density and ESR1 expression. In mice engrafted with HER2-positive breast cancer tumors, coinjection of MCs increased tumor engraftment and outgrowth, supporting the link between MCs and increased risk of relapse in patients with breast cancer. Together, our findings support the notion that MCs influence the phenotype of breast cancer cells by stimulating a luminal phenotype and ultimately modifying the outcome of the disease.
Significance:
Mast cells impact breast cancer outcome by directly affecting the phenotype of tumor cells through stimulation of the estrogen receptor pathway.
Data from Inhibition of NF-κB–Dependent Signaling Enhances Sensitivity and Overcomes Resistance to BET Inhibition in Uveal Melanoma
Authors: Ambrosini Grazia , Do Catherine , Tycko Benjamin , Realubit Ronald B. , Karan Charles , Musi Elgilda , Carvajal Richard D. , Chua Vivian , Aplin Andrew E. , Schwartz Gary K.
Published: March 2023

Abstract
Bromodomain and extraterminal protein inhibitors (BETi) are epigenetic therapies aimed to target dysregulated gene expression in cancer cells. Despite early successes of BETi in a range of malignancies, the development of drug resistance may limit their clinical application. Here, we evaluated the mechanisms of BETi resistance in uveal melanoma, a disease with little treatment options, using two approaches: a high-throughput combinatorial drug screen with the clinical BET inhibitor PLX51107 and RNA sequencing of BETi-resistant cells. NF-κB inhibitors synergistically sensitized uveal melanoma cells to PLX51107 treatment. Furthermore, genes involved in NF-κB signaling were upregulated in BETi-resistant cells, and the transcription factor CEBPD contributed to the mechanism of resistance. These findings suggest that inhibitors of NF-κB signaling may improve the efficacy of BET inhibition in patients with advanced uveal melanoma.
Significance:
These findings provide evidence that inhibitors of NF-κB signaling synergize with BET inhibition in in vitro and in vivo models, suggesting a clinical utility of these targeted therapies in patients with uveal melanoma.
Data from esiCancer: Evolutionary In Silico Cancer Simulator
Authors: Minussi Darlan Conterno , Henz Bernardo , dos Santos Oliveira Mariana , Filippi-Chiela Eduardo C. , Oliveira Manuel M. , Lenz Guido
Published: March 2023

Abstract
The evolution of cancer is inferred mainly from samples taken at discrete points that represent glimpses of the complete process. In this study, we present esiCancer as a cancer-evolution simulator. It uses a branching process, randomly applying events to a diploid oncogenome, altering probabilities of proliferation and death of the affected cells. Multiple events that occur over hundreds of generations may lead to a gradual change in cell fitness and the establishment of a fast-growing population. esiCancer provides a platform to study the impact of several factors on tumor evolution, including dominance, fitness, event rate, and interactions among genes as well as factors affecting the tumor microenvironment. The output of esiCancer can be used to reconstruct clonal composition and Kaplan-Meier–like survival curves of multiple evolutionary stories. esiCancer is an open-source, standalone software to model evolutionary aspects of cancer biology.
Significance:
This study provides a customizable and hands-on simulation tool to model the effect of diverse types of genomic alterations on the fate of tumor cells.
Data from CD38-Driven Mitochondrial Trafficking Promotes Bioenergetic Plasticity in Multiple Myeloma
Authors: Marlein Christopher R. , Piddock Rachel E. , Mistry Jayna J. , Zaitseva Lyubov , Hellmich Charlotte , Horton Rebecca H. , Zhou Zhigang , Auger Martin J. , Bowles Kristian M. , Rushworth Stuart A.
Published: March 2023

Abstract
Metabolic adjustments are necessary for the initiation, proliferation, and spread of cancer cells. Although mitochondria have been shown to move to cancer cells from their microenvironment, the metabolic consequences of this phenomenon have yet to be fully elucidated. Here, we report that multiple myeloma cells use mitochondrial-based metabolism as well as glycolysis when located within the bone marrow microenvironment. The reliance of multiple myeloma cells on oxidative phosphorylation was caused by intercellular mitochondrial transfer to multiple myeloma cells from neighboring nonmalignant bone marrow stromal cells. This mitochondrial transfer occurred through tumor-derived tunneling nanotubes (TNT). Moreover, shRNA-mediated knockdown of CD38 inhibits mitochondrial transfer and TNT formation in vitro and blocks mitochondrial transfer and improves animal survival in vivo. This study describes a potential treatment strategy to inhibit mitochondrial transfer for clinical benefit and scientifically expands the understanding of the functional effects of mitochondrial transfer on tumor metabolism.
Significance:
Multiple myeloma relies on both oxidative phosphorylation and glycolysis following acquisition of mitochondria from its bone marrow microenvironment.

See related commentary by Boise and Shanmugam, p. 2102
Data from A Pygopus 2-Histone Interaction Is Critical for Cancer Cell Dedifferentiation and Progression in Malignant Breast Cancer
Authors: Saxena Meera , Kalathur Ravi K.R. , Rubinstein Natalia , Vettiger Andrea , Sugiyama Nami , Neutzner Melanie , Coto-Llerena Mairene , Kancherla Venkatesh , Ercan Caner , Piscuoglio Salvatore , Fischer Jonas , Fagiani Ernesta , Cantù Claudio , Basler Konrad , Christofori Gerhard
Published: March 2023

Abstract
Pygopus 2 (Pygo2) is a coactivator of Wnt/β-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing. It remains unknown whether the Pygo2–H3K4me2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer, we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective. Loss of Pygo2–histone interaction resulted in smaller, differentiated, and less metastatic tumors, due, in part, to decreased canonical Wnt/β-catenin signaling. RNA- and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGFβ signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically, the Pygo2–histone interaction potentiated Wnt/β-catenin signaling, in part, by repressing the expression of Wnt signaling antagonists. Furthermore, Pygo2 and β-catenin regulated the expression of miR-29 family members, which, in turn, repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively, these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors, and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2–H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer.
Significance:
Pygo2 represents a potential therapeutic target in metastatic breast cancer, as its histone-binding capability promotes β-catenin–mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation, EMT, and metastasis.
Data from Modeling Acquired Resistance to the Second-Generation Androgen Receptor Antagonist Enzalutamide in the TRAMP Model of Prostate Cancer
Authors: Cerasuolo Marianna , Maccarinelli Federica , Coltrini Daniela , Mahmoud Ali Mokhtar , Marolda Viviana , Ghedini Gaia Cristina , Rezzola Sara , Giacomini Arianna , Triggiani Luca , Kostrzewa Magdalena , Verde Roberta , Paris Debora , Melck Dominique , Presta Marco , Ligresti Alessia , Ronca Roberto
Published: March 2023

Abstract
Enzalutamide (MDV3100) is a potent second-generation androgen receptor antagonist approved for the treatment of castration-resistant prostate cancer (CRPC) in chemotherapy-naïve as well as in patients previously exposed to chemotherapy. However, resistance to enzalutamide and enzalutamide withdrawal syndrome have been reported. Thus, reliable and integrated preclinical models are required to elucidate the mechanisms of resistance and to assess therapeutic settings that may delay or prevent the onset of resistance. In this study, the prostate cancer multistage murine model TRAMP and TRAMP-derived cells have been used to extensively characterize in vitro and in vivo the response and resistance to enzalutamide. The therapeutic profile as well as the resistance onset were characterized and a multiscale stochastic mathematical model was proposed to link the in vitro and in vivo evolution of prostate cancer. The model showed that all therapeutic strategies that use enzalutamide result in the onset of resistance. The model also showed that combination therapies can delay the onset of resistance to enzalutamide, and in the best scenario, can eliminate the disease. These results set the basis for the exploitation of this “TRAMP-based platform” to test novel therapeutic approaches and build further mathematical models of combination therapies to treat prostate cancer and CRPC.

Significance: Merging mathematical modeling with experimental data, this study presents the “TRAMP-based platform” as a novel experimental tool to study the in vitro and in vivo evolution of prostate cancer resistance to enzalutamide.
Data from A Tissue-Engineered 3D Microvessel Model Reveals the Dynamics of Mosaic Vessel Formation in Breast Cancer
Authors: Silvestri Vanesa L. , Henriet Elodie , Linville Raleigh M. , Wong Andrew D. , Searson Peter C. , Ewald Andrew J.
Published: March 2023

Abstract
In solid tumors, vascular structure and function varies from the core to the periphery. This structural heterogeneity has been proposed to influence the mechanisms by which tumor cells enter the circulation. Blood vessels exhibit regional defects in endothelial coverage, which can result in cancer cells directly exposed to flow and potentially promoting intravasation. Consistent with prior reports, we observed in human breast tumors and in a mouse model of breast cancer that approximately 6% of vessels consisted of both endothelial cells and tumor cells, so-called mosaic vessels. Due, in part, to the challenges associated with observing tumor–vessel interactions deep within tumors in real-time, the mechanisms by which mosaic vessels form remain incompletely understood. We developed a tissue-engineered model containing a physiologically realistic microvessel in coculture with mammary tumor organoids. This approach allows real-time and quantitative assessment of tumor–vessel interactions under conditions that recapitulate many in vivo features. Imaging revealed that tumor organoids integrate into the endothelial cell lining, resulting in mosaic vessels with gaps in the basement membrane. While mosaic vessel formation was the most frequently observed interaction, tumor organoids also actively constricted and displaced vessels. Furthermore, intravasation of cancer cell clusters was observed following the formation of a mosaic vessel. Taken together, our data reveal that cancer cells can rapidly reshape, destroy, or integrate into existing blood vessels, thereby affecting oxygenation, perfusion, and systemic dissemination. Our novel assay also enables future studies to identify targetable mechanisms of vascular recruitment and intravasation.
Significance:
A tissue-engineered microdevice that recapitulates the tumor–vascular microenvironment enables real-time imaging of the cellular mechanisms of mosaic vessel formation and vascular defect generation.
Data from Netrin-1 and Its Receptor DCC Are Causally Implicated in Melanoma Progression
Authors: Boussouar Amina , Tortereau Antonin , Manceau Ambroise , Paradisi Andrea , Gadot Nicolas , Vial Jonathan , Neves David , Larue Lionel , Battistella Maxime , Leboeuf Christophe , Lebbé Celeste , Janin Anne , Mehlen Patrick
Published: March 2023

Abstract
Deleted in colorectal cancer (DCC), the receptor for the multifunctional cue netrin-1, acts as a tumor suppressor in intestinal cancer and lung metastasis by triggering cancer cell death when netrin-1 is lowly expressed. Recent genomic data highlighted that DCC is the third most frequently mutated gene in melanoma; we therefore investigated whether DCC could act as a melanoma tumor suppressor. Reexpressing DCC in human melanoma cell lines promoted tumor cell death and tumor growth inhibition in xenograft mouse models. Genetic silencing of DCC prodeath activity in a BRAFV600E mouse model increased the proportion of mice with melanoma, further supporting that DCC is a melanoma tumor suppressor. Netrin-1 expression was elevated in melanoma compared with benign melanocytic lesions. Upregulation of netrin-1 in the skin cells of a BRAFV600E-mutated murine model reduced cancer cell death and promoted melanoma progression. Therapeutic antibody blockade of netrin-1 combined with dacarbazine increased overall survival in several mouse melanoma models. Together, these data support that interfering with netrin-1 could be a viable therapeutic approach in patients with netrin-1–expressing melanoma.
Significance:
Netrin-1 and its receptor DCC regulate melanoma progression, suggesting therapeutic targeting of this signaling axis as a viable option for melanoma treatment.
Data from Combined MEK and PI3K/p110β Inhibition as a Novel Targeted Therapy for Malignant Mesothelioma Displaying Sarcomatoid Features
Authors: Marqués Miriam , Tranchant Robin , Risa-Ebrí Blanca , Suárez-Solís María L. , Fernández Luis C. , Carrillo-de-Santa-Pau Enrique , del Pozo Natalia , Martínez de Villarreal Jaime , Meiller Clément , Allory Yves , Blum Yuna , Pirker Christine , Hegedus Balazs , Barry Simon T. , Carnero Amancio , Berger Walter , Jean Didier , Real Francisco X.
Published: March 2023

Abstract
Among malignant mesotheliomas (MM), the sarcomatoid subtype is associated with higher chemoresistance and worst survival. Due to its low incidence, there has been little progress in the knowledge of the molecular mechanisms associated with sarcomatoid MM, which might help to define novel therapeutic targets. In this work, we show that loss of PTEN expression is frequent in human sarcomatoid MM and PTEN expression levels are lower in sarcomatoid MM than in the biphasic and epithelioid subtypes. Combined Pten and Trp53 deletion in mouse mesothelium led to nonepithelioid MM development. In Pten;Trp53-null mice developing MM, the Gαi2-coupled receptor subunit activated MEK/ERK and PI3K, resulting in aggressive, immune-suppressed tumors. Combined inhibition of MEK and p110β/PI3K reduced mouse tumor cell growth in vitro. Therapeutic inhibition of MEK and p110β/PI3K using selumetinib (AZD6244, ARRY-142886) and AZD8186, two drugs that are currently in clinical trials, increased the survival of Pten;Trp53-null mice without major toxicity. This drug combination effectively reduced the proliferation of primary cultures of human pleural (Pl) MM, implicating nonepithelioid histology and high vimentin, AKT1/2, and Gαi2 expression levels as predictive markers of response to combined MEK and p110β/PI3K inhibition. Our findings provide a rationale for the use of selumetinib and AZD8186 in patients with MM with sarcomatoid features. This constitutes a novel targeted therapy for a poor prognosis and frequently chemoresistant group of patients with MM, for whom therapeutic options are currently lacking.
Significance:
Mesothelioma is highly aggressive; its sarcomatoid variants have worse prognosis. Building on a genetic mouse model, a novel combination therapy is uncovered that is relevant to human tumors.
Data from Cellular Senescence Promotes Skin Carcinogenesis through p38MAPK and p44/42MAPK Signaling
Authors: Alimirah Fatouma , Pulido Tanya , Valdovinos Alexis , Alptekin Sena , Chang Emily , Jones Elijah , Diaz Diego A. , Flores Jose , Velarde Michael C. , Demaria Marco , Davalos Albert R. , Wiley Christopher D. , Limbad Chandani , Desprez Pierre-Yves , Campisi Judith
Published: March 2023

Abstract
Cellular senescence entails an irreversible growth arrest that evolved in part to prevent cancer. Paradoxically, senescent cells secrete proinflammatory and growth-stimulatory molecules, termed the senescence-associated secretory phenotype (SASP), which is correlated with cancer cell proliferation in culture and xenograft models. However, at what tumor stage and how senescence and the SASP act on endogenous tumor growth in vivo is unknown. To understand the role of senescence in cancer etiology, we subjected p16-3MR transgenic mice, which permit the identification and selective elimination of senescent cells in vivo, to the well-established two-step protocol of squamous cell skin carcinoma, in which tumorigenesis is initiated by a carcinogen 7,12-dimethylbenz[α]anthracene, and then promoted by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We show that TPA promotes skin carcinogenesis by inducing senescence and a SASP. Systemic induction of senescence in nontumor-bearing p16-3MR mice using a chemotherapy followed by the two-step carcinogenesis protocol potentiated the conversion of benign papillomas to carcinomas by elevating p38MAPK and MAPK/ERK signaling. Ablation of senescent cells reduced p38MAPK and MAPK/ERK signaling, thereby preventing the progression of benign papillomas to carcinomas. Thus, we show for the first time that senescent cells are tumor promoters, not tumor initiators, and that they stimulate skin carcinogenesis by elevating p38MAPK and MAPK/ERK signaling. These findings pave the way for developing novel therapeutics against senescence-fueled cancers.
Significance:
These findings identify chemotherapy-induced senescence as a culprit behind tumor promotion, suggesting that elimination of senescent cells after chemotherapy may reduce occurrence of second cancers decades later.
Data from A High-Content Screen Identifies Drugs That Restrict Tumor Cell Extravasation across the Endothelial Barrier
Authors: Hilfenhaus Georg , Mompeón Ana , Freshman Jonathan , Prajapati Divya P. , Hernandez Gloria , Freitas Vanessa M. , Ma Feiyang , Langenbacher Adam D. , Mirkov Snezana , Song Dana , Cho Byoung-Kyu , Goo Young Ah , Pellegrini Matteo , Chen Jau-Nian , Damoiseaux Robert , Iruela-Arispe M. Luisa
Published: March 2023

Abstract
Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell–matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor–endothelial cell interactions.
Significance:
A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor–endothelial cell interactions.
Data from Single-Cell Analyses Reveal Diverse Mechanisms of Resistance to EGFR Tyrosine Kinase Inhibitors in Lung Cancer
Authors: Kashima Yukie , Shibahara Daisuke , Suzuki Ayako , Muto Kyoko , Kobayashi Ikei S. , Plotnick David , Udagawa Hibiki , Izumi Hiroki , Shibata Yuji , Tanaka Kosuke , Fujii Masanori , Ohashi Akihiro , Seki Masahide , Goto Koichi , Tsuchihara Katsuya , Suzuki Yutaka , Kobayashi Susumu S.
Published: March 2023

Abstract
Tumor heterogeneity underlies resistance to tyrosine kinase inhibitors (TKI) in lung cancers harboring EGFR mutations. Previous evidence suggested that subsets of preexisting resistant cells are selected by EGFR-TKI treatment, or alternatively, that diverse acquired resistance mechanisms emerge from drug-tolerant persister (DTP) cells. Many studies have used bulk tumor specimens or subcloned resistant cell lines to identify resistance mechanism. However, intratumoral heterogeneity can result in divergent responses to therapies, requiring additional approaches to reveal the complete spectrum of resistance mechanisms. Using EGFR-TKI-resistant cell models and clinical specimens, we performed single-cell RNA-seq and single-cell ATAC-seq analyses to define the transcriptional and epigenetic landscape of parental cells, DTPs, and tumor cells in a fully resistant state. In addition to AURKA, VIM, and AXL, which are all known to induce EGFR-TKI resistance, CD74 was identified as a novel gene that plays a critical role in the drug-tolerant state. In vitro and in vivo experiments demonstrated that CD74 upregulation confers resistance to the EGFR-TKI osimertinib and blocks apoptosis, enabling tumor regrowth. Overall, this study provides new insight into the mechanisms underlying resistance to EGFR-TKIs.
Significance:
Single-cell analyses identify diverse mechanisms of resistance as well as the state of tolerant cells that give rise to resistance to EGFR tyrosine kinase inhibitors.
Data from E2A Modulates Stemness, Metastasis, and Therapeutic Resistance of Breast Cancer
Authors: López-Menéndez Celia , Vázquez-Naharro Alberto , Santos Vanesa , Dubus Pierre , Santamaría Patricia G. , Martínez-Ramírez Ángel , Portillo Francisco , Moreno-Bueno Gema , Faraldo Marisa M. , Cano Amparo
Published: March 2023

Abstract
Cancer stem cells (CSC) are considered responsible for tumor initiation, therapeutic resistance, and metastasis. A comprehensive knowledge of the mechanisms governing the acquisition and maintenance of cancer stemness is crucial for the development of new therapeutic approaches in oncology. E2A basic helix–loop–helix (bHLH) transcription factors are associated with epithelial–mesenchymal transition (EMT) and tumor progression, but knowledge of their functional contributions to cancer biology is still limited. Using a combination of in vivo and in vitro analyses in a novel PyMT-E2A conditional knockout mouse model and derived primary tumor cell lines, we report here an essential role of E2A in stemness, metastasis, and therapeutic resistance in breast cancer. Targeted deletion of E2A in the mammary gland impaired tumor-initiating ability and dedifferentiation potential and severely compromised metastatic competence of PyMT-driven mammary tumors. Mechanistic studies in PyMT-derived cell lines indicated that E2A actions are mediated by the upregulation of Snai1 transcription. Importantly, high E2A and SNAIL1 expression occurred in aggressive human basal-like breast carcinomas, highlighting the relevance of the E2A–Snail1 axis in metastatic breast cancer. In addition, E2A factors contributed to the maintenance of genomic integrity and resistance to PARP inhibitors in PyMT and human triple-negative breast cancer cells. Collectively, these results support the potential for E2A transcription factors as novel targets worthy of translational consideration in breast cancer.
Significance:
These findings identify key functions of E2A factors in breast cancer cell stemness, metastasis, and drug resistance, supporting a therapeutic vulnerability to targeting E2A proteins in breast cancer.
Data from A Conformation Selective Mode of Inhibiting SRC Improves Drug Efficacy and Tolerability
Authors: Temps Carolin , Lietha Daniel , Webb Emily R. , Li Xue-Feng , Dawson John C. , Muir Morwenna , Macleod Kenneth G. , Valero Teresa , Munro Alison F. , Contreras-Montoya Rafael , Luque-Ortega Juan R. , Fraser Craig , Beetham Henry , Schoenherr Christina , Lopalco Maria , Arends Mark J. , Frame Margaret C. , Qian Bin-Zhi , Brunton Valerie G. , Carragher Neil O. , Unciti-Broceta Asier
Published: March 2023

Abstract
Despite the approval of several multikinase inhibitors that target SRC and the overwhelming evidence of the role of SRC in the progression and resistance mechanisms of many solid malignancies, inhibition of its kinase activity has thus far failed to improve patient outcomes. Here we report the small molecule eCF506 locks SRC in its native inactive conformation, thereby inhibiting both enzymatic and scaffolding functions that prevent phosphorylation and complex formation with its partner FAK. This mechanism of action resulted in highly potent and selective pathway inhibition in culture and in vivo. Treatment with eCF506 resulted in increased antitumor efficacy and tolerability in syngeneic murine cancer models, demonstrating significant therapeutic advantages over existing SRC/ABL inhibitors. Therefore, this mode of inhibiting SRC could lead to improved treatment of SRC-associated disorders.
Significance:
Small molecule–mediated inhibition of SRC impairing both catalytic and scaffolding functions confers increased anticancer properties and tolerability compared with other SRC/ABL inhibitors.
Data from Fragmentation of Small-Cell Lung Cancer Regulatory States in Heterotypic Microenvironments
Authors: Schaff Dylan L. , Singh Shambhavi , Kim Kee-Beom , Sutcliffe Matthew D. , Park Kwon-Sik , Janes Kevin A.
Published: March 2023

Abstract
Small-cell lung cancers derive from pulmonary neuroendocrine cells, which have stem-like properties to reprogram into other cell types upon lung injury. It is difficult to uncouple transcriptional plasticity of these transformed cells from genetic changes that evolve in primary tumors or secondary metastases. Profiling of single cells is also problematic if the required sample dissociation activates injury-like signaling and reprogramming. Here we defined cell-state heterogeneities in situ through laser capture microdissection–based 10-cell transcriptomics coupled with stochastic-profiling fluctuation analysis. In labeled cells from a small-cell lung cancer mouse model initiated by neuroendocrine deletion of Rb1-Trp53, variations in transcript abundance revealed cell-to-cell differences in regulatory state in vitro and in vivo. Fluctuating transcripts in spheroid culture were partly shared among Rb1-Trp53–null models, and heterogeneities increased considerably when cells were delivered intravenously to colonize the liver. Colonization of immunocompromised animals drove a fractional appearance of alveolar type II–like markers and poised cells for paracrine stimulation from immune cells and hepatocytes. Immunocompetency further exaggerated the fragmentation of tumor states in the liver, yielding mixed stromal signatures evident in bulk sequencing from autochthonous tumors and metastases. Dozens of transcript heterogeneities recurred irrespective of biological context; their mapped orthologs brought together observations of murine and human small-cell lung cancer. Candidate heterogeneities recurrent in the liver also stratified primary human tumors into discrete groups not readily explained by molecular subtype but with prognostic relevance. These data suggest that heterotypic interactions in the liver and lung are an accelerant for intratumor heterogeneity in small-cell lung cancer.
Significance:
These findings demonstrate that the single-cell regulatory heterogeneity of small-cell lung cancer becomes increasingly elaborate in the liver, a common metastatic site for the disease.

See related articles by Singh and colleagues, p. 1840 and Sutcliffe and colleagues, p. 1868
Data from Protein Signatures of NK Cell–Mediated Melanoma Killing Predict Response to Immunotherapies
Authors: Cappello Sabrina , Sung Hsu-Min , Ickes Christian , Gibhardt Christine S. , Vultur Adina , Bhat Hilal , Hu Zhongwen , Brafford Patricia , Denger Andreas , Stejerean-Todoran Ioana , Köhn Rixa-Mareike , Lorenz Verena , Künzel Nicolas , Salinas Gabriela , Stanisz Hedwig , Legler Tobias , Rehling Peter , Schön Michael P. , Lang Karl S. , Helms Volkhard , Herlyn Meenhard , Hoth Markus , Kummerow Carsten , Bogeski Ivan
Published: March 2023

Abstract
Despite impressive advances in melanoma-directed immunotherapies, resistance is common and many patients still succumb to metastatic disease. In this context, harnessing natural killer (NK) cells, which have thus far been sidelined in the development of melanoma immunotherapy, could provide therapeutic benefits for cancer treatment. To identify molecular determinants of NK cell–mediated melanoma killing (NKmK), we quantified NK-cell cytotoxicity against a panel of genetically diverse melanoma cell lines and observed highly heterogeneous susceptibility. Melanoma protein microarrays revealed a correlation between NKmK and the abundance and activity of a subset of proteins, including several metabolic factors. Oxidative phoshorylation, measured by oxygen consumption rate, negatively correlated with melanoma cell sensitivity toward NKmK, and proteins involved in mitochondrial metabolism and epithelial–mesenchymal transition were confirmed to regulate NKmK. Two- and three-dimensional killing assays and melanoma xenografts established that the PI3K/AKT/mTOR signaling axis controls NKmK via regulation of NK cell–relevant surface proteins. A “protein-killing-signature” based on the protein analysis predicted NKmK of additional melanoma cell lines and the response of patients with melanoma to anti-PD-1 checkpoint therapy. Collectively, these findings identify novel NK cell–related prognostic biomarkers and may contribute to improved and personalized melanoma-directed immunotherapies.
Significance:
NK-cell cytotoxicity assays and protein microarrays reveal novel biomarkers of NK cell–mediated melanoma killing and enable development of signatures to predict melanoma patient responsiveness to immunotherapies.
Data from Intravital Imaging Identifies the VEGF–TXA2 Axis as a Critical Promoter of PGE2 Secretion from Tumor Cells and Immune Evasion
Authors: Konishi Yoshinobu , Ichise Hiroshi , Watabe Tetsuya , Oki Choji , Tsukiji Shinya , Hamazaki Yoko , Murakawa Yasuhiro , Takaori-Kondo Akifumi , Terai Kenta , Matsuda Michiyuki
Published: March 2023

Abstract
Prostaglandin E2 (PGE2) promotes tumor progression through evasion of antitumor immunity. In stark contrast to cyclooxygenase-dependent production of PGE2, little is known whether PGE2 secretion is regulated within tumor tissues. Here, we show that VEGF-dependent release of thromboxane A2 (TXA2) triggers Ca2+ transients in tumor cells, culminating in PGE2 secretion and subsequent immune evasion in the early stages of tumorigenesis. Ca2+ transients caused cPLA2 activation and triggered the arachidonic acid cascade. Ca2+ transients were monitored as the surrogate marker of PGE2 secretion. Intravital imaging of BrafV600E mouse melanoma cells revealed that the proportion of cells exhibiting Ca2+ transients is markedly higher in vivo than in vitro. The TXA2 receptor was indispensable for the Ca2+ transients in vivo, high intratumoral PGE2 concentration, and evasion of antitumor immunity. Notably, treatment with a VEGF receptor antagonist and an anti-VEGF antibody rapidly suppressed Ca2+ transients and reduced TXA2 and PGE2 concentrations in tumor tissues. These results identify the VEGF–TXA2 axis as a critical promoter of PGE2-dependent tumor immune evasion, providing a molecular basis underlying the immunomodulatory effect of anti-VEGF therapies.
Significance:
This study identifies the VEGF–TXA2 axis as a potentially targetable regulator of PGE2 secretion, which provides novel strategies for prevention and treatment of multiple types of malignancies.
Data from Multimodal Molecular Imaging Detects Early Responses to Immune Checkpoint Blockade
Authors: Saida Yu , Brender Jeffrey R. , Yamamoto Kazutoshi , Mitchell James B. , Krishna Murali C. , Kishimoto Shun
Published: March 2023

Abstract
Immune checkpoint blockade (ICB) has become a standard therapy for several cancers, however, the response to ICB is inconsistent and a method for noninvasive assessment has not been established to date. To investigate the capability of multimodal imaging to evaluate treatment response to ICB therapy, hyperpolarized 13C MRI using [1–13C] pyruvate and [1,4–13C2] fumarate and dynamic contrast enhanced (DCE) MRI was evaluated to detect early changes in tumor glycolysis, necrosis, and intratumor perfusion/permeability, respectively. Mouse tumor models served as platforms for high (MC38 colon adenocarcinoma) and low (B16-F10 melanoma) sensitivity to dual ICB of PD-L1 and CTLA4. Glycolytic flux significantly decreased following treatment only in the less sensitive B16-F10 tumors. Imaging [1,4–13C2] fumarate conversion to [1,4–13C2] malate showed a significant increase in necrotic cell death following treatment in the ICB-sensitive MC38 tumors, with essentially no change in B16-F10 tumors. DCE-MRI showed significantly increased perfusion/permeability in MC38-treated tumors, whereas a similar, but statistically nonsignificant, trend was observed in B16-F10 tumors. When tumor volume was also taken into consideration, each imaging biomarker was linearly correlated with future survival in both models. These results suggest that hyperpolarized 13C MRI and DCE MRI may serve as useful noninvasive imaging markers to detect early response to ICB therapy.
Significance:
Hyperpolarized 13C MRI and dynamic contrast enhanced MRI in murine tumor models provide useful insight into evaluating early response to immune checkpoint blockade therapy.

See related commentary by Cullen and Keshari, p. 3444
Data from Treatment with HIV-Protease Inhibitor Nelfinavir Identifies Membrane Lipid Composition and Fluidity as a Therapeutic Target in Advanced Multiple Myeloma
Authors: Besse Lenka , Besse Andrej , Stolze Sara C. , Sobh Amin , Zaal Esther A. , van der Ham Alwin J. , Ruiz Mario , Phuyal Santosh , Büchler Lorina , Sathianathan Marc , Florea Bogdan I. , Borén Jan , Ståhlman Marcus , Huber Julia , Bolomsky Arnold , Ludwig Heinz , Hannich J. Thomas , Loguinov Alex , Everts Bart , Berkers Celia R. , Pilon Marc , Farhan Hesso , Vulpe Christopher D. , Overkleeft Herman S. , Driessen Christoph
Published: March 2023

Abstract
The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)–refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9–based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI–refractory multiple myeloma.
Significance:
Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.
Data from Pan-Cancer Drivers Are Recurrent Transcriptional Regulatory Heterogeneities in Early-Stage Luminal Breast Cancer
Authors: Singh Shambhavi , Sutcliffe Matthew D. , Repich Kathy , Atkins Kristen A. , Harvey Jennifer A. , Janes Kevin A.
Published: March 2023

Abstract
The heterogeneous composition of solid tumors is known to impact disease progression and response to therapy. Malignant cells coexist in different regulatory states that can be accessed transcriptomically by single-cell RNA sequencing, but these methods have many caveats related to sensitivity, noise, and sample handling. We revised a statistical fluctuation analysis called stochastic profiling to combine with 10-cell RNA sequencing, which was designed for laser-capture microdissection (LCM) and extended here for immuno-LCM. When applied to a cohort of late-onset, early-stage luminal breast cancers, the integrated approach identified thousands of candidate regulatory heterogeneities. Intersecting the candidates from different tumors yielded a relatively stable set of 710 recurrent heterogeneously expressed genes (RHEG), which were significantly variable in >50% of patients. RHEGs were not strongly confounded by dissociation artifacts, cell-cycle oscillations, or driving mutations for breast cancer. Rather, RHEGs were enriched for epithelial-to-mesenchymal transition genes and, unexpectedly, the latest pan-cancer assembly of driver genes across cancer types other than breast. These findings indicate that heterogeneous transcriptional regulation conceivably provides a faster, reversible mechanism for malignant cells to evaluate the effects of potential oncogenes or tumor suppressors on cancer hallmarks.
Significance:
Profiling intratumor heterogeneity of luminal breast carcinoma cells identifies a recurrent set of genes, suggesting sporadic activation of pathways known to drive other types of cancer.

See related articles by Schaff and colleagues, p. 1853 and Sutcliffe and colleagues, p. 1868
Data from STINGing Viral Tumors: What We Know from Head and Neck Cancers
Authors: Saulters Emma , Woolley John F. , Varadarajan Shankar , Jones Terence M. , Dahal Lekh N.
Published: March 2023

Abstract
It has now become increasingly clear that viruses, which may not be directly oncogenic, can affect the biology of tumors as well as immune behavior against tumors. This has led to a fundamental question: Should tumors associated with viral infection be considered distinct from those without? Typically, viruses activate the host innate immune responses by stimulating pathogen recognition receptors and DNA-sensing pathways, including the stimulator of interferon genes (STING) pathway. However, regulation of the STING pathway in a virus-associated tumor microenvironment is poorly understood. Human papillomavirus (HPV) infection within a subset of head and neck squamous cell carcinomas (HNSCC) promotes a unique etiology and clinical outcome. For reasons currently not well understood, patients with HPV+ tumors have a better outcome in terms of both overall survival and reduced risk of recurrence compared with HPV− HNSCC. This observation may reflect a greater intrinsic immunogenicity associated with HPV infection, pertaining to innate immune system pathways activated following recognition of viral nucleotides. Here we discuss how HNSCC provides a unique model to study the STING pathway in the context of viral-induced tumor type as well as recent advances in our understanding of this pathway in HSNCC.
Data from SMARCA2 Is a Novel Interactor of NSD2 and Regulates Prometastatic PTP4A3 through Chromatin Remodeling in t(4;14) Multiple Myeloma
Authors: Chong Phyllis S.Y. , Chooi Jing Yuan , Lim Julia S.L. , Toh Sabrina Hui Min , Tan Tuan Zea , Chng Wee-Joo
Published: March 2023

Abstract
NSD2 is the primary oncogenic driver in t(4;14) multiple myeloma. Using SILAC-based mass spectrometry, we demonstrate a novel role of NSD2 in chromatin remodeling through its interaction with the SWI/SNF ATPase subunit SMARCA2. SMARCA2 was primarily expressed in t(4;14) myeloma cells, and its interaction with NSD2 was noncanonical and independent of the SWI/SNF complex. RNA sequencing identified PTP4A3 as a downstream target of NSD2 and mapped NSD2–SMARCA2 complex on PTP4A3 promoter. This led to a focal increase in the permissive H3K36me2 mark and transcriptional activation of PTP4A3. High levels of PTP4A3 maintained MYC expression and correlated with a 54-gene MYC signature in t(4;14) multiple myeloma. Importantly, this mechanism was druggable by targeting the bromodomain of SMARCA2 using the specific BET inhibitor PFI-3, leading to the displacement of NSD2 from PTP4A3 promoter and inhibiting t(4;14) myeloma cell viability. In vivo, treatment with PFI-3 reduced the growth of t(4;14) xenograft tumors. Together, our study reveals an interplay between histone-modifying enzymes and chromatin remodelers in the regulation of myeloma-specific genes that can be clinically intervened.
Significance:
This study uncovers a novel, SWI/SNF–independent interaction between SMARCA2 and NSD2 that facilitates chromatin remodeling and transcriptional regulation of oncogenes in t(4;14) multiple myeloma, revealing a therapeutic vulnerability targetable by BET inhibition.
Data from Suppression of Endothelial Cell FAK Expression Reduces Pancreatic Ductal Adenocarcinoma Metastasis after Gemcitabine Treatment
Authors: Roy-Luzarraga Marina , Reynolds Louise E. , de Luxán-Delgado Beatriz , Maiques Oscar , Wisniewski Laura , Newport Emma , Rajeeve Vinothini , Drake Rebecca J.G. , Gómez-Escudero Jesús , Richards Frances M. , Weller Céline , Dormann Christof , Meng Ya-Ming , Vermeulen Peter B. , Saur Dieter , Sanz-Moreno Victoria , Wong Ping-Pui , Géraud Cyrill , Cutillas Pedro R. , Hodivala-Dilke Kairbaan
Published: March 2023

Abstract
Despite substantial advances in the treatment of solid cancers, resistance to therapy remains a major obstacle to prolonged progression-free survival. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, with a high level of liver metastasis. Primary PDAC is highly hypoxic, and metastases are resistant to first-line treatment, including gemcitabine. Recent studies have indicated that endothelial cell (EC) focal adhesion kinase (FAK) regulates DNA-damaging therapy–induced angiocrine factors and chemosensitivity in primary tumor models. Here, we show that inducible loss of EC-FAK in both orthotopic and spontaneous mouse models of PDAC is not sufficient to affect primary tumor growth but reduces liver and lung metastasis load and improves survival rates in gemcitabine-treated, but not untreated, mice. EC-FAK loss did not affect primary tumor angiogenesis, tumor blood vessel leakage, or early events in metastasis, including the numbers of circulating tumor cells, tumor cell homing, or metastatic seeding. Phosphoproteomics analysis showed a downregulation of the MAPK, RAF, and PAK signaling pathways in gemcitabine-treated FAK-depleted ECs compared with gemcitabine-treated wild-type ECs. Moreover, low levels of EC-FAK correlated with increased survival and reduced relapse in gemcitabine-treated patients with PDAC, supporting the clinical relevance of these findings. Altogether, we have identified a new role of EC-FAK in regulating PDAC metastasis upon gemcitabine treatment that impacts outcome.
Significance:
These findings establish the potential utility of combinatorial endothelial cell FAK targeting together with gemcitabine in future clinical applications to control metastasis in patients with pancreatic ductal adenocarcinoma.