bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Novel Strategies to Enforce an Epithelial Phenotype in Mesenchymal Cells
Authors: Dragoi Ana-Maria , Swiss Rachel , Gao Beile , Agaisse Hervé
Published: March 2024

Abstract
E-cadherin downregulation in cancer cells is associated with epithelial-to-mesenchymal transition (EMT) and metastatic prowess, but the underlying mechanisms are incompletely characterized. In this study, we probed E-cadherin expression at the plasma membrane as a functional assay to identify genes involved in E-cadherin downregulation. The assay was based on the E-cadherin–dependent invasion properties of the intracellular pathogen Listeria monocytogenes. On the basis of a functional readout, automated microscopy and computer-assisted image analysis were used to screen siRNAs targeting 7,000 human genes. The validity of the screen was supported by its definition of several known regulators of E-cadherin expression, including ZEB1, HDAC1, and MMP14. We identified three new regulators (FLASH, CASP7, and PCGF1), the silencing of which was sufficient to restore high levels of E-cadherin transcription. In addition, we identified two new regulators (FBXL5 and CAV2), the silencing of which was sufficient to increase E-cadherin expression at a posttranscriptional level. FLASH silencing regulated the expression of E-cadherin and other ZEB1-dependent genes, through posttranscriptional regulation of ZEB1, but it also regulated the expression of numerous ZEB1-independent genes with functions predicted to contribute to a restoration of the epithelial phenotype. Finally, we also report the identification of siRNA duplexes that potently restored the epithelial phenotype by mimicking the activity of known and putative microRNAs. Our findings suggest new ways to enforce epithelial phenotypes as a general strategy to treat cancer by blocking invasive and metastatic phenotypes associated with EMT. Cancer Res; 74(14); 3659–72. ©2014 AACR.
Data from Curcumin Targets Breast Cancer Stem–like Cells with Microtentacles That Persist in Mammospheres and Promote Reattachment
Authors: Charpentier Monica S. , Whipple Rebecca A. , Vitolo Michele I. , Boggs Amanda E. , Slovic Jana , Thompson Keyata N. , Bhandary Lekhana , Martin Stuart S.
Published: March 2024

Abstract
Cancer stem–like cells (CSC) and circulating tumor cells (CTC) have related properties associated with distant metastasis, but the mechanisms through which CSCs promote metastasis are unclear. In this study, we report that breast cancer cell lines with more stem-like properties display higher levels of microtentacles (McTN), a type of tubulin-based protrusion of the plasma cell membrane that forms on detached or suspended cells and aid in cell reattachment. We hypothesized that CSCs with large numbers of McTNs would more efficiently attach to distant tissues, promoting metastatic efficiency. The naturally occurring stem-like subpopulation of the human mammary epithelial (HMLE) cell line presents increased McTNs compared with its isogenic non–stem-like subpopulation. This increase was supported by elevated α-tubulin detyrosination and vimentin protein levels and organization. Increased McTNs in stem-like HMLEs promoted a faster initial reattachment of suspended cells that was inhibited by the tubulin-directed drug, colchicine, confirming a functional role for McTNs in stem cell reattachment. Moreover, live-cell confocal microscopy showed that McTNs persist in breast stem cell mammospheres as flexible, motile protrusions on the surface of the mammosphere. Although exposed to the environment, they also function as extensions between adjacent cells along cell–cell junctions. We found that treatment with the breast CSC-targeting compound curcumin rapidly extinguished McTN in breast CSC, preventing reattachment from suspension. Together, our results support a model in which breast CSCs with cytoskeletal alterations that promote McTNs can mediate attachment and metastasis but might be targeted by curcumin as an antimetastatic strategy. Cancer Res; 74(4); 1250–60. ©2013 AACR.
Data from MDR1 Synonymous Polymorphisms Alter Transporter Specificity and Protein Stability in a Stable Epithelial Monolayer
Authors: Fung King Leung , Pan James , Ohnuma Shinobu , Lund Paul E. , Pixley Jessica N. , Kimchi-Sarfaty Chava , Ambudkar Suresh V. , Gottesman Michael M.
Published: March 2024

Abstract
The drug efflux function of P-glycoprotein (P-gp) encoded by MDR1 can be influenced by genetic polymorphisms, including two synonymous changes in the coding region of MDR1. Here we report that the conformation of P-gp and its drug efflux activity can be altered by synonymous polymorphisms in stable epithelial monolayers expressing P-gp. Several cell lines with similar MDR1 DNA copy number were developed and termed LLC-MDR1-WT (expresses wild-type P-gp), LLC-MDR1-3H (expresses common haplotype P-gp), and LLC-MDR1-3HA (a mutant that carries a different valine codon in position 3435). These cell lines express similar levels of recombinant mRNA and protein. P-gp in each case is localized on the apical surface of polarized cells. However, the haplotype and its mutant P-gps fold differently from the wild-type, as determined by UIC2 antibody shift assays and limited proteolysis assays. Surface biotinylation experiments suggest that the non-wild-type P-gps have longer recycling times. Drug transport assays show that wild-type and haplotype P-gp respond differently to P-gp inhibitors that block efflux of rhodamine 123 or mitoxantrone. In addition, cytotoxicity assays show that the LLC-MDR1-3H cells are more resistant to mitoxantrone than the LLC-MDR1-WT cells after being treated with a P-gp inhibitor. Expression of polymorphic P-gp, however, does not affect the host cell's morphology, growth rate, or monolayer formation. Also, ATPase activity assays indicate that neither basal nor drug-stimulated ATPase activities are affected in the variant P-gps. Taken together, our findings indicate that “silent” polymorphisms significantly change P-gp function, which would be expected to affect interindividual drug disposition and response. Cancer Res; 74(2); 598–608. ©2013 AACR.
Data from Cancer Cells Exploit eIF4E2-Directed Synthesis of Hypoxia Response Proteins to Drive Tumor Progression
Authors: Uniacke James , Kishan Perera J. , Lachance Gabriel , Francisco Camille B. , Lee Stephen
Published: March 2024

Abstract
Human tumors display considerable diversity in their genetic makeup but share common physiologic attributes such as a hypoxic microenvironment that contribute to the malignant phenotype. Hypoxic cells switch from eukaryotic initiation factor 4E (eIF4E) to eIF4E2 cap-dependent translation to synthesize a portion of their proteins. Here, we show that genetically distinct human cancer cells exploit eIF4E2-directed protein synthesis to form cellular masses larger than approximately 0.15 mm, the diffusion limit of oxygen. Cancer cells depleted of eIF4E2 are indistinguishable from control cells under normoxic conditions, but are unable to survive and proliferate in low oxygen conditions. Activation of eIF4E2-directed translation is essential for cancer cells to form a hypoxic tumor core in in vitro spheroids and to form detectable tumors in in vivo xenograft assays. In contrast, the eIF4E-directed protein synthesis pathway alone cannot sustain cellular adaptation to hypoxia in vitro or confer tumorigenic potential in xenograft assays. These data demonstrate that the phenotypic expression of the cancer genome requires translation by the eIF4E2-directed hypoxic protein synthesis machinery. Cancer Res; 74(5); 1379–89. ©2014 AACR.
Data from ERK1/2 Blockade Prevents Epithelial–Mesenchymal Transition in Lung Cancer Cells and Promotes Their Sensitivity to EGFR Inhibition
Authors: Buonato Janine M. , Lazzara Matthew J.
Published: March 2024

Abstract
Overcoming cellular mechanisms of de novo and acquired resistance to drug therapy remains a central challenge in the clinical management of many cancers, including non–small cell lung cancer (NSCLC). Although much work has linked the epithelial–mesenchymal transition (EMT) in cancer cells to the emergence of drug resistance, it is less clear where tractable routes may exist to reverse or inhibit EMT as a strategy for drug sensitization. Here, we demonstrate that extracellular signal-regulated kinase (ERK) 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) signaling plays a key role in directing the mesenchymal character of NSCLC cells and that blocking ERK signaling is sufficient to heighten therapeutic responses to EGF receptor (EGFR) inhibitors. MEK1/2 (MAPKK1/2) inhibition promoted an epithelial phenotype in NSCLC cells, preventing induction of EMT by exogenous TGF-β. Moreover, in cells exhibiting de novo or acquired resistance to the EGFR inhibitor gefitinib, MEK inhibition enhanced the sensitivity to gefitinib and slowed cell migration. These effects only occurred, however, if MEK was inhibited for a period sufficient to trigger changes in EMT marker expression. Consistent with these findings, changes in EMT phenotypes and markers were also induced by the expression of mutant KRAS in a MEK-dependent manner. Our results suggest that prolonged exposure to MEK or ERK inhibitors may not only restrain EMT but also overcome naïve or acquired resistance of NSCLC to EGFR-targeted therapy in the clinic. Cancer Res; 74(1); 309–19. ©2013 AACR.
Data from Chromosome 10, Frequently Lost in Human Melanoma, Encodes Multiple Tumor-Suppressive Functions
Authors: Kwong Lawrence N. , Chin Lynda
Published: March 2024

Abstract
Although many DNA aberrations in melanoma have been well characterized, including focal amplification and deletions of oncogenes and tumor suppressors, broad regions of chromosomal gain and loss are less well understood. One possibility is that these broad events are a consequence of collateral damage from targeting single loci. Another possibility is that the loss of large regions permits the simultaneous repression of multiple tumor suppressors by broadly decreasing the resident gene dosage and expression. Here, we test this hypothesis in a targeted fashion using RNA interference to suppress multiple candidate residents in broad regions of loss. We find that loss of chromosome regions 6q, 10, and 11q21-ter is correlated with broadly decreased expression of most resident genes and that multiple resident genes impacted by broad regional loss of chromosome 10 are tumor suppressors capable of affecting tumor growth and/or invasion. We also provide additional functional support for Ablim1 as a novel tumor suppressor. Our results support the hypothesis that multiple cancer genes are targeted by regional chromosome copy number aberrations. Cancer Res; 74(6); 1814–21. ©2014 AACR.
Data from Dendritic Cells in Tumor-Associated Tertiary Lymphoid Structures Signal a Th1 Cytotoxic Immune Contexture and License the Positive Prognostic Value of Infiltrating CD8+ T Cells
Authors: Goc Jérémy , Germain Claire , Vo-Bourgais Thi Kim Duy , Lupo Audrey , Klein Christophe , Knockaert Samantha , de Chaisemartin Luc , Ouakrim Hanane , Becht Etienne , Alifano Marco , Validire Pierre , Remark Romain , Hammond Scott A. , Cremer Isabelle , Damotte Diane , Fridman Wolf-Herman , Sautès-Fridman Catherine , Dieu-Nosjean Marie-Caroline
Published: March 2024

Abstract
Tumor-infiltrating T cells, particularly CD45RO+CD8+ memory T cells, confer a positive prognostic value in human cancers. However, the mechanisms that promote a protective T-cell response in the tumor microenvironment remain unclear. In chronic inflammatory settings such as the tumor microenvironment, lymphoid neogenesis can occur to create local lymph node–like structures known as tertiary lymphoid structures (TLS). These structures can exacerbate a local immune response, such that TLS formation in tumors may help promote an efficacious immune contexture. However, the role of TLS in tumors has yet to be investigated carefully. In lung tumors, mature dendritic cells (DC) present in tumor-associated TLS can provide a specific marker of these structures. In this study, we evaluated the influence of TLS on the characteristics of the immune infiltrate in cohorts of prospective and retrospective human primary lung tumors (n = 458). We found that a high density of mature DC correlated closely to a strong infiltration of T cells that are predominantly of the effector–memory phenotype. Moreover, mature DC density correlated with expression of genes related to T-cell activation, T-helper 1 (Th1) phenotype, and cytotoxic orientation. Lastly, a high density of TLS-associated DC correlated with long-term survival, which also allowed a distinction of patients with high CD8+ T-cell infiltration but a high risk of death. Taken together, our results show how tumors infiltrated by TLS-associated mature DC generate a specific immune contexture characterized by a strong Th1 and cytotoxic orientation that confers the lowest risk of death. Furthermore, our findings highlight the pivotal function of TLS in shaping the immune character of the tumor microenvironment, in promoting a protective immune response mediated by T cells against cancer. Cancer Res; 74(3); 705–15. ©2013 AACR.
Data from Differential Regulation of Estrogen Receptor α Expression in Breast Cancer Cells by Metastasis-Associated Protein 1
Authors: Kang Hyun-Jin , Lee Min-Ho , Kang Hae-Lim , Kim Sung-Hye , Ahn Jung-Ranh , Na Hyelin , Na Tae-Young , Kim Yo Na , Seong Je Kyung , Lee Mi-Ock
Published: March 2024

Abstract
Metastasis-associated protein 1 (MTA1) is a component of the nucleosome remodeling and histone deacetylase (HDAC) complex, which plays an important role in progression of breast cancer. Although MTA1 is known as a repressor of the transactivation function of estrogen receptor α (ERα), its involvement in the epigenetic control of transcription of the ERα gene ESR1 has not been studied. Here, we show that silencing of MTA1 reduced the level of expression of ERα in ERα-positive cells but increased it in ERα-negative cells. In both MCF7 and MDA-MB-231, MTA1 was recruited to the region +146 to +461 bp downstream of the transcription start site of ESR1 (ERpro315). Proteomics analysis of the MTA1 complex that was pulled down by an oligonucleotide encoding ERpro315 revealed that the transcription factor AP-2γ (TFAP2C) and the IFN-γ–inducible protein 16 (IFI16) were components of the complex. Interestingly, in MCF7, TFAP2C activated the reporter encoding ERpro315 and the level of ERα mRNA. By contrast, in MDA-MB-231, IFI16 repressed the promoter activity and silencing of MTA1 increased expression of ERα. Importantly, class II HDACs are involved in the MTA1-mediated differential regulation of ERα. Finally, an MDA-MB-231-derived cell line that stably expressed shIFI16 or shMTA1 was more susceptible to tamoxifen-induced growth inhibition in in vitro and in vivo experiments. Taken together, our findings suggest that the MTA1–TFAP2C or the MTA1–IFI16 complex may contribute to the epigenetic regulation of ESR1 expression in breast cancer and may determine the chemosensitivity of tumors to tamoxifen therapy in patients with breast cancer. Cancer Res; 74(5); 1484–94. ©2014 AACR.
Data from HuR Posttranscriptionally Regulates WEE1: Implications for the DNA Damage Response in Pancreatic Cancer Cells
Authors: Lal Shruti , Burkhart Richard A. , Beeharry Neil , Bhattacharjee Vikram , Londin Eric R. , Cozzitorto Joseph A. , Romeo Carmella , Jimbo Masaya , Norris Zoë A. , Yeo Charles J. , Sawicki Janet A. , Winter Jordan M. , Rigoutsos Isidore , Yen Timothy J. , Brody Jonathan R.
Published: March 2024

Abstract
HuR (ELAV1), an RNA-binding protein abundant in cancer cells, primarily resides in the nucleus, but under specific stress (e.g., gemcitabine), HuR translocates to the cytoplasm in which it tightly modulates the expression of mRNA survival cargo. Here, we demonstrate for the first time that stressing pancreatic ductal adenocarcinoma (PDA) cells by treatment with DNA-damaging anticancer agents (mitomycin C, oxaliplatin, cisplatin, carboplatin, and a PARP inhibitor) results in HuR's translocation from the nucleus to the cytoplasm. Importantly, silencing HuR in PDA cells sensitized the cells to these agents, whereas overexpressing HuR caused resistance. HuR's role in the efficacy of DNA-damaging agents in PDA cells was, in part, attributed to the acute upregulation of WEE1 by HuR. WEE1, a mitotic inhibitor kinase, regulates the DNA damage repair pathway, and therapeutic inhibition of WEE1 in combination with chemotherapy is currently in early phase trials for the treatment of cancer. We validate WEE1 as a HuR target in vitro and in vivo by demonstrating (i) direct binding of HuR to WEE1′s mRNA (a discrete 56-bp region residing in the 3′ untranslated region) and (ii) HuR siRNA silencing and overexpression directly affects the protein levels of WEE1, especially after DNA damage. HuR's positive regulation of WEE1 increases γ-H2AX levels, induces Cdk1 phosphorylation, and promotes cell-cycle arrest at the G2–M transition. We describe a novel mechanism that PDA cells use to protect against DNA damage in which HuR posttranscriptionally regulates the expression and downstream function of WEE1 upon exposure to DNA-damaging agents. Cancer Res; 74(4); 1128–40. ©2014 AACR.
Data from Comparative Functional Analysis of DPYD Variants of Potential Clinical Relevance to Dihydropyrimidine Dehydrogenase Activity
Authors: Offer Steven M. , Fossum Croix C. , Wegner Natalie J. , Stuflesser Alexander J. , Butterfield Gabriel L. , Diasio Robert B.
Published: March 2024

Abstract
Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme of the uracil catabolic pathway, being critically important for inactivation of the commonly prescribed anti-cancer drug 5-fluorouracil (5-FU). DPD impairment leads to increased exposure to 5-FU and, in turn, increased anabolism of 5-FU to cytotoxic nucleotides, resulting in more severe clinical adverse effects. Numerous variants within the gene coding for DPD, DPYD, have been described, although only a few have been demonstrated to reduce DPD enzyme activity. To identify DPYD variants that alter enzyme function, we expressed 80 protein-coding variants in an isogenic mammalian system and measured their capacities to convert 5-FU to dihydro-fluorouracil, the product of DPD catabolism. The M166V, E828K, K861R, and P1023T variants exhibited significantly higher enzyme activity than wild-type DPD (120%, P = 0.025; 116%, P = 0.049; 130%, P = 0.0077; 138%, P = 0.048, respectively). Consistent with clinical association studies of 5-FU toxicity, the D949V substitution reduced enzyme activity by 41% (P = 0.0031). Enzyme activity was also significantly reduced for 30 additional variants, 19 of which had <25% activity. None of those 30 variants have been previously reported to associate with 5-FU toxicity in clinical association studies, which have been conducted primarily in populations of European ancestry. Using publicly available genotype databases, we confirmed the rarity of these variants in European populations but showed that they are detected at appreciable frequencies in other populations. These data strongly suggest that testing for the reported deficient DPYD variations could dramatically improve predictive genetic tests for 5-FU sensitivity, especially in individuals of non-European descent. Cancer Res; 74(9); 2545–54. ©2014 AACR.
Data from ATR Inhibitors VE-821 and VX-970 Sensitize Cancer Cells to Topoisomerase I Inhibitors by Disabling DNA Replication Initiation and Fork Elongation Responses
Authors: Jossé Rozenn , Martin Scott E. , Guha Rajarshi , Ormanoglu Pinar , Pfister Thomas D. , Reaper Philip M. , Barnes Christopher S. , Jones Julie , Charlton Peter , Pollard John R. , Morris Joel , Doroshow James H. , Pommier Yves
Published: March 2024

Abstract
Camptothecin and its derivatives, topotecan and irinotecan, are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress, came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821 confirmed marked antiproliferative synergy with camptothecin and even greater synergy with LMP-400. Single-cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin–firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970, enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof of principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials. Cancer Res; 74(23); 6968–79. ©2014 AACR.
Data from shRNA Kinome Screen Identifies TBK1 as a Therapeutic Target for HER2+ Breast Cancer
Authors: Deng Tao , Liu Jeff C. , Chung Philip E.D. , Uehling David , Aman Ahmed , Joseph Babu , Ketela Troy , Jiang Zhe , Schachter Nathan F. , Rottapel Robert , Egan Sean E. , Al-awar Rima , Moffat Jason , Zacksenhaus Eldad
Published: March 2024

Abstract
HER2+ breast cancer is currently treated with chemotherapy plus anti-HER2 inhibitors. Many patients do not respond or relapse with aggressive metastatic disease. Therefore, there is an urgent need for new therapeutics that can target HER2+ breast cancer and potentiate the effect of anti-HER2 inhibitors, in particular those that can target tumor-initiating cells (TIC). Here, we show that MMTV-Her2/Neu mammary tumor cells cultured as nonadherent spheres or as adherent monolayer cells select for stabilizing mutations in p53 that “immortalize” the cultures and that, after serial passages, sphere conditions maintain TICs, whereas monolayer cells gradually lose these tumorigenic cells. Using tumorsphere formation as surrogate for TICs, we screened p53-mutant Her2/Neu+ tumorsphere versus monolayer cells with a lentivirus short hairpin RNA kinome library. We identified kinases such as the mitogen-activated protein kinase and the TGFβR protein family, previously implicated in HER2+ breast cancer, as well as autophagy factor ATG1/ULK1 and the noncanonical IκB kinase (IKK), TANK-binding kinase 1 (TBK1), which have not been previously linked to HER2+ breast cancer. Knockdown of TBK1 or pharmacologic inhibition of TBK1 and the related protein, IKKϵ, suppressed growth of both mouse and human HER2+ breast cancer cells. TBK1/IKKϵ inhibition promoted cellular senescence by suppressing p65–NF-κB and inducing p16Ink4a. In addition, TBK1/IKKϵ inhibition cooperated with lapatinib, a HER2/EGFR1–targeted drug, to accelerate apoptosis and kill HER2+ breast cancer cells both in culture and in xenografts. Our results suggest that patients with HER2+ breast cancer may benefit from anti-TBK1/IKKϵ plus anti-HER2 combination therapies and establish conditions that can be used to screen for additional TIC-specific inhibitors of HER2+ breast cancer. Cancer Res; 74(7); 2119–30. ©2014 AACR.
Data from Phosphorylation at Ser-181 of Oncogenic KRAS Is Required for Tumor Growth
Authors: Barceló Carles , Paco Noelia , Morell Mireia , Alvarez-Moya Blanca , Bota-Rabassedas Neus , Jaumot Montserrat , Vilardell Felip , Capella Gabriel , Agell Neus
Published: March 2024

Abstract
KRAS phosphorylation has been reported recently to modulate the activity of mutant KRAS protein in vitro. In this study, we defined S181 as a specific phosphorylation site required to license the oncogenic function of mutant KRAS in vivo. The phosphomutant S181A failed to induce tumors in mice, whereas the phosphomimetic mutant S181D exhibited an enhanced tumor formation capacity, compared with the wild-type KRAS protein. Reduced growth of tumors composed of cells expressing the nonphosphorylatable KRAS S181A mutant was correlated with increased apoptosis. Conversely, increased growth of tumors composed of cells expressing the phosphomimetic KRAS S181D mutant was correlated with increased activation of AKT and ERK, two major downstream effectors of KRAS. Pharmacologic treatment with PKC inhibitors impaired tumor growth associated with reduced levels of phosphorylated KRAS and reduced effector activation. In a panel of human tumor cell lines expressing various KRAS isoforms, we showed that KRAS phosphorylation was essential for survival and tumorigenic activity. Furthermore, we identified phosphorylated KRAS in a panel of primary human pancreatic tumors. Taken together, our findings establish that KRAS requires S181 phosphorylation to manifest its oncogenic properties, implying that its inhibition represents a relevant target to attack KRAS-driven tumors. Cancer Res; 74(4); 1190–9. ©2013 AACR.
Data from Sequential Gene Targeting to Make Chimeric Tumor Models with De Novo Chromosomal Abnormalities
Authors: Chambers Jennifer S. , Tanaka Tomoyuki , Brend Tim , Ali Hanif , Geisler Nicola J. , Khazin Leah , Cigudosa Juan C. , Neil Dear T. , MacLennan Kenneth , Rabbitts Terence H.
Published: March 2024

Abstract
The discovery of chromosomal translocations in leukemia/lymphoma and sarcomas presaged a widespread discovery in epithelial tumors. With the advent of new-generation whole-genome sequencing, many consistent chromosomal abnormalities have been described together with putative driver and passenger mutations. The multiple genetic changes required in mouse models to assess the interrelationship of abnormalities and other mutations are severe limitations. Here, we show that sequential gene targeting of embryonic stem cells can be used to yield progenitor cells to generate chimeric offspring carrying all the genetic changes needed for cell-specific cancer. Illustrating the technology, we show that MLL–ENL fusion is sufficient for lethal leukocytosis and proof of genome integrity comes from germline transmission of the sequentially targeted alleles. This accelerated technology leads to a reduction in mouse numbers (contributing significantly to the 3Rs), allows fluorescence tagging of cancer-initiating cells, and provides a flexible platform for interrogating the interaction of chromosomal abnormalities with mutations. Cancer Res; 74(5); 1588–97. ©2014 AACR.
Data from Bcl2 Induces DNA Replication Stress by Inhibiting Ribonucleotide Reductase
Authors: Xie Maohua , Yen Yun , Owonikoko Taofeek K. , Ramalingam Suresh S. , Khuri Fadlo R. , Curran Walter J. , Doetsch Paul W. , Deng Xingming
Published: March 2024

Abstract
DNA replication stress is an inefficient DNA synthesis process that leads replication forks to progress slowly or stall. Two main factors that cause replication stress are alterations in pools of deoxyribonucleotide (dNTP) precursors required for DNA synthesis and changes in the activity of proteins required for synthesis of dNTPs. Ribonucleotide reductase (RNR), containing regulatory hRRM1 and catalytic hRRM2 subunits, is the enzyme that catalyzes the conversion of ribonucleoside diphosphates (NDP) to deoxyribonucleoside diphosphates (dNDP) and thereby provides dNTP precursors needed for the synthesis of DNA. Here, we demonstrate that either endogenous or exogenous expression of Bcl2 results in decreases in RNR activity and intracellular dNTP, retardation of DNA replication fork progression, and increased rate of fork asymmetry leading to DNA replication stress. Bcl2 colocalizes with hRRM1 and hRRM2 in the cytoplasm and directly interacts via its BH4 domain with hRRM2 but not hRRM1. Removal of the BH4 domain of Bcl2 abrogates its inhibitory effects on RNR activity, dNTP pool level, and DNA replication. Intriguingly, Bcl2 directly inhibits RNR activity by disrupting the functional hRRM1/hRRM2 complex via its BH4 domain. Our findings argue that Bcl2 reduces intracellular dNTPs by inhibiting ribonucleotide reductase activity, thereby providing insight into how Bcl2 triggers DNA replication stress. Cancer Res; 74(1); 212–23. ©2013 AACR.
Data from Critical Tumor Suppressor Function Mediated by Epithelial Mig-6 in Endometrial Cancer
Authors: Kim Tae Hoon , Lee Dong-Kee , Cho Sung-Nam , Orvis Grant D. , Behringer Richard R. , Lydon John P. , Ku Bon Jeong , McCampbell Adrienne S. , Broaddus Russell R. , Jeong Jae-Wook
Published: March 2024

Abstract
Endometrial cancer is preceded by endometrial hyperplasia, unopposed estrogen exposure, and genetic alterations, but the precise causes of endometrial cancer remain uncertain. Mig-6, mainly known as a negative regulator of the EGF receptor, is an important mediator of progesterone signaling in the uterus, where it mediates tumor suppression by modulating endometrial stromal–epithelial communications. In this study, we investigated the function of Mig-6 in the uterine epithelium using a tissue-specific gene knockout strategy, in which floxed Mig-6 (Mig-6f/f) mice were crossed to Wnt7a-Cre mice (Wnt7acre+Mig-6f/f). Wnt7acre+Mig-6f/f mice developed endometrial hyperplasia and estrogen-dependent endometrial cancer, exhibiting increased proliferation in epithelial cells as well as apoptosis in subepithelial stromal cells. We documented increased expression of NOTCH1 and BIRC3 in epithelial cells of Wnt7acre+Mig-6f/f mice and decreased expression of the progesterone receptor (PR) in stromal cells. Progesterone therapy controls endometrial growth and prevents endometrial cancer, but the effectiveness of progesterone as a treatment for women with endometrial cancer is less clear. We noted that the hyperplasic phenotype of Wnt7acre+Mig-6f/f mice was prevented by progesterone treatment, whereas this treatment had no effect in PRcre/+Mig-6f/f mice where Mig-6 was deleted in both the epithelial and stromal compartments of the uterus. In contrast, activation of progesterone signaling in the stroma regulated proliferation and apoptosis in the epithelium via suppression of ERα signaling. In summary, our results establish that epithelial Mig-6 functions as a critical tumor suppressor that mediates the ability of progesterone to prevent the development of endometrial cancer. Cancer Res; 73(16); 5090–9. ©2013 AACR.
Data from Vemurafenib Cooperates with HPV to Promote Initiation of Cutaneous Tumors
Authors: Holderfield Matthew , Lorenzana Edward , Weisburd Ben , Lomovasky Lisa , Boussemart Lise , Lacroix Ludovic , Tomasic Gorana , Favre Michel , Vagner Stephan , Robert Caroline , Ghoddusi Majid , Daniel Dylan , Pryer Nancy , McCormick Frank , Stuart Darrin
Published: March 2024

Abstract
Treatment with RAF inhibitors such as vemurafenib causes the development of cutaneous squamous cell carcinomas (cSCC) or keratoacanthomas as a side effect in 18% to 30% of patients. It is known that RAF inhibitors activate the mitogen—activated protein kinase (MAPK) pathway and stimulate growth of RAS-mutated cells, possibly accounting for up to 60% of cSCC or keratoacanthoma lesions with RAS mutations, but other contributing events are obscure. To identify such events, we evaluated tumors from patients treated with vemurafenib for the presence of human papilloma virus (HPV) DNA and identified 13% to be positive. Using a transgenic murine model of HPV-driven cSCC (K14-HPV16 mice), we conducted a functional test to determine whether administration of RAF inhibitors could promote cSCC in HPV-infected tissues. Vemurafenib treatment elevated MAPK markers and increased cSCC incidence from 22% to 70% in this model. Furthermore, 55% of the cSCCs arising in vemurafenib-treated mice exhibited a wild-type Ras genotype, consistent with the frequency observed in human patients. Our results argue that HPV cooperates with vemurafenib to promote tumorigenesis, in either the presence or absence of RAS mutations. Cancer Res; 74(8); 2238–45. ©2014 AACR.
Data from Sequential Application of a Cytotoxic Nanoparticle and a PI3K Inhibitor Enhances Antitumor Efficacy
Authors: Pandey Ambarish , Kulkarni Ashish , Roy Bhaskar , Goldman Aaron , Sarangi Sasmit , Sengupta Poulomi , Phipps Colin , Kopparam Jawahar , Oh Michael , Basu Sudipta , Kohandel Mohammad , Sengupta Shiladitya
Published: March 2024

Abstract
Nanomedicines that preferentially deploy cytotoxic agents to tumors and molecular targeted therapeutics that inhibit specific aberrant oncogenic drivers are emerging as the new paradigm for the management of cancer. While combination therapies are a mainstay of cancer chemotherapy, few studies have addressed the combination of nanomedicines and molecular targeted therapeutics. Furthermore, limited knowledge exists on the impact of sequencing of such therapeutics and nanomedicines on the antitumor outcome. Here, we engineered a supramolecular cis-platinum nanoparticle, which induced apoptosis in breast cancer cells but also elicited prosurvival signaling via an EGF receptor/phosphoinositide 3-kinase (PI3K) pathway. A combination of mathematical modeling and in vitro and in vivo validation using a pharmacologic inhibitor of PI3K, PI828, demonstrate that administration of PI828 following treatment with the supramolecular cis-platinum nanoparticle results in enhanced antitumor efficacy in breast cancer as compared with when the sequence is reversed or when the two treatments are administered simultaneously. This study addresses, for the first time, the impact of drug sequencing in the case of a combination of a nanomedicine and a targeted therapeutic. Furthermore, our results indicate that a rational combination of cis-platinum nanoparticles and a PI3K-targeted therapeutic can emerge as a potential therapy for breast cancer. Cancer Res; 74(3); 675–85. ©2013 AACR.
Data from An Antibody–Drug Conjugate That Targets Tissue Factor Exhibits Potent Therapeutic Activity against a Broad Range of Solid Tumors
Authors: Breij Esther C.W. , de Goeij Bart E.C.G. , Verploegen Sandra , Schuurhuis Danita H. , Amirkhosravi Ali , Francis John , Miller Vibeke Breinholt , Houtkamp Mischa , Bleeker Wim K. , Satijn David , Parren Paul W.H.I.
Published: March 2024

Abstract
Tissue factor (TF) is aberrantly expressed in solid cancers and is thought to contribute to disease progression through its procoagulant activity and its capacity to induce intracellular signaling in complex with factor VIIa (FVIIa). To explore the possibility of using tissue factor as a target for an antibody-drug conjugate (ADC), a panel of human tissue factor–specific antibodies (TF HuMab) was generated. Three tissue factor HuMab, that induced efficient inhibition of TF:FVIIa-dependent intracellular signaling, antibody-dependent cell-mediated cytotoxicity, and rapid target internalization, but had minimal impact on tissue factor procoagulant activity in vitro, were conjugated with the cytotoxic agents monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). Tissue factor–specific ADCs showed potent cytotoxicity in vitro and in vivo, which was dependent on tissue factor expression. TF-011-MMAE (HuMax-TF-ADC) was the most potent ADC, and the dominant mechanism of action in vivo was auristatin-mediated tumor cell killing. Importantly, TF-011-MMAE showed excellent antitumor activity in patient-derived xenograft (PDX) models with variable levels of tissue factor expression, derived from seven different solid cancers. Complete tumor regression was observed in all PDX models, including models that showed tissue factor expression in only 25% to 50% of the tumor cells. In conclusion, TF-011-MMAE is a promising novel antitumor agent with potent activity in xenograft models that represent the heterogeneity of human tumors, including heterogeneous target expression. Cancer Res; 74(4); 1214–26. ©2013 AACR.
Data from NDY1/KDM2B Functions as a Master Regulator of Polycomb Complexes and Controls Self-Renewal of Breast Cancer Stem Cells
Authors: Kottakis Filippos , Foltopoulou Parthena , Sanidas Ioannis , Keller Patricia , Wronski Ania , Dake Benjamin T. , Ezell Scott A. , Shen Zhu , Naber Stephen P. , Hinds Philip W. , McNiel Elizabeth , Kuperwasser Charlotte , Tsichlis Philip N.
Published: March 2024

Abstract
The JmjC domain histone H3K36me2/me1 demethylase NDY1/KDM2B is overexpressed in various types of cancer. Here we show that knocking down NDY1 in a set of 10 cell lines derived from a broad range of human tumors inhibited their anchorage-dependent and anchorage-independent growth by inducing senescence and/or apoptosis in some and by inhibiting G1 progression in all. We further show that the knockdown of NDY1 in mammary adenocarcinoma cell lines decreased the number, size, and replating efficiency of mammospheres and downregulated the stem cell markers ALDH and CD44, while upregulating CD24. Together, these findings suggest that NDY1 is required for the self-renewal of cancer stem cells and are in agreement with additional findings showing that tumor cells in which NDY1 was knocked down undergo differentiation and a higher number of them is required to induce mammary adenocarcinomas, upon orthotopic injection in animals. Mechanistically, NDY1 functions as a master regulator of a set of miRNAs that target several members of the polycomb complexes PRC1 and PRC2, and its knockdown results in the de-repression of these miRNAs and the downregulation of their polycomb targets. Consistent with these observations, NDY1/KDM2B is expressed at higher levels in basal-like triple-negative breast cancers, and its overexpression is associated with higher rates of relapse after treatment. In addition, NDY1-regulated miRNAs are downregulated in both normal and cancer mammary stem cells. Finally, in primary human breast cancer, NDY1/KDM2B expression correlates negatively with the expression of the NDY1-regulated miRNAs and positively with the expression of their PRC targets. Cancer Res; 74(14); 3935–46. ©2014 AACR.
Data from The NADH Oxidase ENOX1, a Critical Mediator of Endothelial Cell Radiosensitization, Is Crucial for Vascular Development
Authors: Venkateswaran Amudhan , Sekhar Konjeti R. , Levic Daniel S. , Melville David B. , Clark Travis A. , Rybski Witold M. , Walsh Alexandra J. , Skala Melissa C. , Crooks Peter A. , Knapik Ela W. , Freeman Michael L.
Published: March 2024

Abstract
ENOX1 is a highly conserved NADH oxidase that helps to regulate intracellular nicotinamide adenine dinucleotide levels in many cell types, including endothelial cells. Pharmacologic and RNA interference (RNAi)–mediated suppression of ENOX1 impairs surrogate markers of tumor angiogenesis/vasculogenesis, providing support for the concept that ENOX1 represents an antiangiogenic druggable target. However, direct genetic evidence that demonstrates a role for ENOX1 in vascular development is lacking. In this study, we exploited a zebrafish embryonic model of development to address this question. Whole-mount in situ hybridization coupled with immunofluorescence performed on zebrafish embryos demonstrate that enox1 message and translated protein are expressed in most tissues, and its expression is enriched in blood vessels and heart. Morpholino-mediated suppression of Enox1 in Tg(fli1-eGFP) and Tg(flk1-eGFP) zebrafish embryos significantly impairs the development of vasculature and blood circulation. Using in vivo multiphoton microscopy, we show that morpholino-mediated knockdown of enox1 increases NADH levels, consistent with loss of enzyme. VJ115 is a small-molecule inhibitor of Enox1′s oxidase activity shown to increase intracellular NADH in endothelial cells; we used VJ115 to determine if the oxidase activity was crucial for vascular development. We found that VJ115 suppressed vasculogenesis in Tg(fli1-eGFP) embryos and impaired circulation. Previously, it was shown that suppression of ENOX1 radiosensitizes proliferating tumor vasculature, a consequence of enhanced endothelial cell apoptosis. Thus, our current findings, coupled with previous research, support the hypothesis that ENOX1 represents a potential cancer therapy target, one that combines molecular targeting with cytotoxic sensitization. Cancer Res; 74(1); 38–43. ©2013 AACR.
Data from Acquired Expression of NFATc1 Downregulates E-Cadherin and Promotes Cancer Cell Invasion
Authors: Oikawa Tsukasa , Nakamura Atsuko , Onishi Nobuyuki , Yamada Taketo , Matsuo Koichi , Saya Hideyuki
Published: March 2024

Abstract
NFATc1 is a transcription factor that regulates T-cell development, osteoclastogenesis, and macrophage function. Given that T cells, osteoclasts, and macrophages in the tumor microenvironment are thought to modulate tumor progression, tumor cells may acquire NFATc1 expression through fusion with these NFATc1-expressing normal cells. We here revealed that a small proportion of tumor cells in human carcinoma specimens expressed NFATc1. To investigate the consequences of NFATc1 acquisition by tumor cells, we established A549 and MCF7 cell lines expressing a constitutively active form of NFATc1 (NFATc1CA) in an inducible manner. The expression of NFATc1CA promoted cancer cell invasion in association with changes in cell morphology. Analysis of gene expression and RNA interference experiments revealed that NFATc1CA suppressed E-cadherin expression by upregulating the transcriptional repressors Snail and Zeb1 in a manner independent of TGF-β signaling. Induced expression of NFATc1CA also downregulated E-cadherin expression and increased invasive activity in tumor xenografts in vivo. Our results thus suggest that the acquisition of NFATc1 expression contributes to tumor progression. Cancer Res; 73(16); 5100–9. ©2013 AACR.
Data from Osteopontin Shapes Immunosuppression in the Metastatic Niche
Authors: Sangaletti Sabina , Tripodo Claudio , Sandri Sara , Torselli Ilaria , Vitali Caterina , Ratti Chiara , Botti Laura , Burocchi Alessia , Porcasi Rossana , Tomirotti Andrea , Colombo Mario P. , Chiodoni Claudia
Published: March 2024

Abstract
The matricellular protein osteopontin (OPN, Spp-1) is widely associated with cancer aggressiveness when produced by tumor cells, but its impact is uncertain when produced by leukocytes in the context of the tumor stroma. In a broad study using Spp1−/− mice along with gene silencing in tumor cells, we obtained evidence of distinct and common activities of OPN when produced by tumor or host cells in a spontaneously metastatic model of breast cancer. Different cellular localization of OPN is associated with its distinct activities, being mainly secreted in tumor cells while intracellular in myeloid cells. OPN produced by tumor cells supported their survival in the blood stream, whereas both tumor- and host-derived OPN, particularly from myeloid cells, rendered the metastatic site more immunosuppressive. Myeloid-derived suppressor cells (MDSC) expanded with tumor progression at both primary and lung metastatic sites. Of the expanded monocytic and granulocytic cell populations of MDSCs, the monocytic subset was the predominant source of OPN. In Spp1−/− mice, the inhibition of lung metastases correlated with the expansion of granulocyte-oriented MDSCs. Notably, monocytic MDSCs in Spp1−/− mice were less suppressive than their wild-type counterparts due to lower expression of arginase-1, IL6, and phospho-Stat3. Moreover, fewer regulatory T cells accumulated at the metastatic site in Spp1−/− mice. Our data find correlation with lung metastases of human mammary carcinomas that are associated with myeloid cells expressing OPN. Overall, our results unveiled novel functions for OPN in shaping local immunosuppression in the lung metastatic niche. Cancer Res; 74(17); 4706–19. ©2014 AACR.
Data from RAE1 Ligands for the NKG2D Receptor Are Regulated by STING-Dependent DNA Sensor Pathways in Lymphoma
Authors: Lam Adeline R. , Le Bert Nina , Ho Samantha S.W. , Shen Yu J. , Tang Melissa L.F. , Xiong Gordon M. , Croxford J. Ludovic , Koo Christine X. , Ishii Ken J. , Akira Shizuo , Raulet David H. , Gasser Stephan
Published: March 2024

Abstract
The immunoreceptor NKG2D originally identified in natural killer (NK) cells recognizes ligands that are upregulated on tumor cells. Expression of NKG2D ligands (NKG2DL) is induced by the DNA damage response (DDR), which is often activated constitutively in cancer cells, revealing them to NK cells as a mechanism of immunosurveillance. Here, we report that the induction of retinoic acid early transcript 1 (RAE1) ligands for NKG2D by the DDR relies on a STING-dependent DNA sensor pathway involving the effector molecules TBK1 and IRF3. Cytosolic DNA was detected in lymphoma cell lines that express RAE1 and its occurrence required activation of the DDR. Transfection of DNA into ligand-negative cells was sufficient to induce RAE1 expression. Irf3+/−;Eμ-Myc mice expressed lower levels of RAE1 on tumor cells and showed a reduced survival rate compared with Irf3+/+;Eμ-Myc mice. Taken together, our results suggest that genomic damage in tumor cells leads to activation of STING-dependent DNA sensor pathways, thereby activating RAE1 and enabling tumor immunosurveillance. Cancer Res; 74(8); 2193–203. ©2014 AACR.
Data from The Transcriptional Regulatory Network of Proneural Glioma Determines the Genetic Alterations Selected during Tumor Progression
Authors: Sonabend Adam M. , Bansal Mukesh , Guarnieri Paolo , Lei Liang , Amendolara Benjamin , Soderquist Craig , Leung Richard , Yun Jonathan , Kennedy Benjamin , Sisti Julia , Bruce Samuel , Bruce Rachel , Shakya Reena , Ludwig Thomas , Rosenfeld Steven , Sims Peter A. , Bruce Jeffrey N. , Califano Andrea , Canoll Peter
Published: March 2024

Abstract
Proneural glioblastoma is defined by an expression pattern resembling that of oligodendrocyte progenitor cells and carries a distinctive set of genetic alterations. Whether there is a functional relationship between the proneural phenotype and the associated genetic alterations is unknown. To evaluate this possible relationship, we performed a longitudinal molecular characterization of tumor progression in a mouse model of proneural glioma. In this setting, the tumors acquired remarkably consistent genetic deletions at late stages of progression, similar to those deleted in human proneural glioblastoma. Further investigations revealed that p53 is a master regulator of the transcriptional network underlying the proneural phenotype. This p53-centric transcriptional network and its associated phenotype were observed at both the early and late stages of progression, and preceded the proneural-specific deletions. Remarkably, deletion of p53 at the time of tumor initiation obviated the acquisition of later deletions, establishing a link between the proneural transcriptional network and the subtype-specific deletions selected during glioma progression. Cancer Res; 74(5); 1440–51. ©2014 AACR.