bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Bicyclic Peptides as a New Modality for Imaging and Targeting of Proteins Overexpressed by Tumors
Authors: Eder Matthias , Pavan Silvia , Bauder-Wüst Ulrike , van Rietschoten Katerine , Baranski Ann-Christin , Harrison Helen , Campbell Spencer , Stace Catherine L. , Walker Edward H. , Chen Liuhong , Bennett Gavin , Mudd Gemma , Schierbaum Ursula , Leotta Karin , Haberkorn Uwe , Kopka Klaus , Teufel Daniel P.
Published: March 2023

Abstract
Molecular imaging of cancers using probes specific for tumor-associated target proteins offers a powerful solution for providing information regarding selection of targeted therapy, patient stratification, and response to therapy. Here we demonstrate the power of bicyclic peptides as targeting probes, exemplified with the tumor-overexpressed matrix metalloproteinase MT1-MMP as a target. A bicyclic peptide with subnanomolar affinity towards MT1-MMP was identified, and its radioconjugate showed selective tumor uptake in an HT1080 xenograft mouse model. Proteolytic stabilization of the peptide by chemical modification significantly enhanced the in vivo tumor signal [from 2.5%ID/g to 12%ID/g at 1 hour post injection (p.i.)]. Studies using mouse xenograft models with different cell lines show a robust correlation between tumor signals and in vivo MT1-MMP expression levels. Fatty acid modification of the bicyclic peptide extended its circulating half-life, resulting in increased tumor signals (36%ID/g at 6 hours p.i.). Comparative work with an equipotent radiolabeled MT1-MMP targeting antibody demonstrated starkly differential biodistribution and tumor accumulation properties, with the tumor signal slowly increasing to 6.2%ID/g within 48 hours. The rapid tumor penetration characteristics of bicyclic peptides, coupled with high potency and chemical versatility, thus offer high-contrast imaging probes for clinical diagnostics with compelling additional potential in targeted therapy.

Significance: This work demonstrates the potential of bicyclic peptides as a platform for the development of high-contrast imaging probes for potential use in clinical cancer diagnostics and molecularly targeted therapeutics.
Data from Deoxycytidine Release from Pancreatic Stellate Cells Promotes Gemcitabine Resistance
Authors: Dalin Simona , Sullivan Mark R. , Lau Allison N. , Grauman-Boss Beatrice , Mueller Helen S. , Kreidl Emanuel , Fenoglio Silvia , Luengo Alba , Lees Jacqueline A. , Vander Heiden Matthew G. , Lauffenburger Douglas A. , Hemann Michael T.
Published: March 2023

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths in the United States. The deoxynucleoside analogue gemcitabine is among the most effective therapies to treat PDAC, however, nearly all patients treated with gemcitabine either fail to respond or rapidly develop resistance. One hallmark of PDAC is a striking accumulation of stromal tissue surrounding the tumor, and this accumulation of stroma can contribute to therapy resistance. To better understand how stroma limits response to therapy, we investigated cell-extrinsic mechanisms of resistance to gemcitabine. Conditioned media from pancreatic stellate cells (PSC), as well as from other fibroblasts, protected PDAC cells from gemcitabine toxicity. The protective effect of PSC-conditioned media was mediated by secretion of deoxycytidine, but not other deoxynucleosides, through equilibrative nucleoside transporters. Deoxycytidine inhibited the processing of gemcitabine in PDAC cells, thus reducing the effect of gemcitabine and other nucleoside analogues on cancer cells. These results suggest that reducing deoxycytidine production in PSCs may increase the efficacy of nucleoside analog therapies.
Significance:
This study provides important new insight into mechanisms that contribute to gemcitabine resistance in PDAC and suggests new avenues for improving gemcitabine efficacy.
Data from Novel Third-Generation EGFR Tyrosine Kinase Inhibitors and Strategies to Overcome Therapeutic Resistance in Lung Cancer
Authors: Murtuza Ayesha , Bulbul Ajaz , Shen John Paul , Keshavarzian Parissa , Woodward Brian D. , Lopez-Diaz Fernando J. , Lippman Scott M. , Husain Hatim
Published: March 2023

Abstract
EGFR-activating mutations are observed in approximately 15% to 20% of patients with non–small cell lung cancer. Tyrosine kinase inhibitors have provided an illustrative example of the successes in targeting oncogene addiction in cancer and the role of tumor-specific adaptations conferring therapeutic resistance. The compound osimertinib is a third-generation tyrosine kinase inhibitor, which was granted full FDA approval in March 2017 based on targeting EGFR T790M resistance. The compound has received additional FDA approval as first-line therapy with improvement in progression-free survival by suppressing the activating mutation and preventing the rise of the dominant resistance clone. Drug development has been breathtaking in this space with other third-generation compounds at various stages of development: rociletinib (CO-1686), olmutinib (HM61713), nazartinib (EGF816), naquotinib (ASP8273), mavelertinib (PF-0647775), and AC0010. However, therapeutic resistance after the administration of third-generation inhibitors is complex and not fully understood, with significant intertumoral and intratumoral heterogeneity. Repeat tissue and plasma analyses on therapy have revealed insights into multiple mechanisms of resistance, including novel second site EGFR mutations, activated bypass pathways such as MET amplification, HER2 amplification, RAS mutations, BRAF mutations, PIK3CA mutations, and novel fusion events. Strategies to understand and predict patterns of mutagenesis are still in their infancy; however, technologies to understand synthetically lethal dependencies and track cancer evolution through therapy are being explored. The expansion of combinatorial therapies is a direction forward targeting minimal residual disease and bypass pathways early based on projected resistance.
Data from The Pro-Oncogenic Adaptor CIN85 Acts as an Inhibitory Binding Partner of Hypoxia-Inducible Factor Prolyl Hydroxylase 2
Authors: Kozlova Nina , Mennerich Daniela , Samoylenko Anatoly , Dimova Elitsa Y. , Koivunen Peppi , Biterova Ekaterina , Richter Kati , Hassinen Antti , Kellokumpu Sakari , Manninen Aki , Miinalainen Ilkka , Glumoff Virpi , Ruddock Lloyd , Drobot Lyudmyla Borysivna , Kietzmann Thomas
Published: March 2023

Abstract
The EGFR adaptor protein, CIN85, has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill defined. Here we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase, PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SRC homology 3 domains of CIN85 interacted with the proline-arginine–rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF degradation. This activity is essential in vivo, as specific loss of the CIN85–PHD2 interaction in CRISPR/Cas9-edited cells affected growth and migration properties, as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2 and uncovered an essential survival function in tumor cells by linking growth factor adaptors with hypoxia signaling.
Significance:
This study provides unprecedented evidence for an oxygen-independent mechanism of PHD2 regulation that has important implications in cancer cell survival.
Data from Targeting Hippo-Dependent and Hippo-Independent YAP1 Signaling for the Treatment of Childhood Rhabdomyosarcoma
Authors: Slemmons Katherine K. , Yeung Choh , Baumgart Joshua T. , Juarez Jhazeel O. Martinez , McCalla Amy , Helman Lee J.
Published: March 2023

Abstract
Rhabdomyosarcoma is the most common childhood soft-tissue sarcoma, yet patients with metastatic or recurrent disease continue to do poorly, indicating a need for new treatments. The SRC family tyrosine kinase YES1 is upregulated in rhabdomyosarcoma and is necessary for growth, but clinical trials using single agent dasatinib, a SRC family kinase inhibitor, have failed in sarcomas. YAP1 (YES-associated protein) is highly expressed in rhabdomyosarcoma, driving growth and survival when the upstream Hippo tumor suppressor pathway is silenced, but efforts to pharmacologically inhibit YAP1 have been unsuccessful. Here we demonstrate that treatment of rhabdomyosarcoma with DNA methyltransferase inhibitor (DNMTi) upregulates Hippo activators RASSF1 and RASSF5 by promoter demethylation, activating canonical Hippo signaling and increasing inactivation of YAP1 by phosphorylation. Treatment with DNMTi decreased rhabdomyosarcoma cell growth and increased apoptosis and differentiation, an effect partially rescued by expression of constitutively active YAP (S127A), suggesting the effects of DNMTi treatment are, in part, due to Hippo-dependent inhibition of YAP1. In addition, YES1 and YAP1 interacted in the nucleus of rhabdomyosarcoma cells, and genetic or pharmacologic suppression of YES1 resulted in cytoplasmic retention of YAP1 and decreased YAP1 target gene expression, suggesting YES1 regulates YAP1 in a Hippo-independent manner. Combined treatment with DNMTi and dasatinib targeted both Hippo-dependent and Hippo-independent regulation of YAP1, ablating rhabdomyosarcoma cell growth in vitro and trending toward decreased tumor growth in vivo. These results show that the mechanisms regulating YAP1 in rhabdomyosarcoma can be inhibited by combinatorial therapy of DNMTi and dasatinib, laying the groundwork for future clinical investigations.
Significance:
This study elucidates the signaling pathways that regulate the oncogenic protein YAP1 and identifies a combination therapy to target these pathways in the childhood tumor rhabdomyosarcoma.
Data from NRAS Status Determines Sensitivity to SHP2 Inhibitor Combination Therapies Targeting the RAS–MAPK Pathway in Neuroblastoma
Authors: Valencia-Sama Ivette , Ladumor Yagnesh , Kee Lynn , Adderley Teresa , Christopher Gabriella , Robinson Claire M. , Kano Yoshihito , Ohh Michael , Irwin Meredith S.
Published: March 2023

Abstract
Survival for high-risk neuroblastoma remains poor and treatment for relapsed disease rarely leads to long-term cures. Large sequencing studies of neuroblastoma tumors from diagnosis have not identified common targetable driver mutations other than the 10% of tumors that harbor mutations in the anaplastic lymphoma kinase (ALK) gene. However, at neuroblastoma recurrence, more frequent mutations in genes in the RAS–MAPK pathway have been detected. The PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and we and others have shown that pharmacologic inhibition of SHP2 suppresses the growth of various tumor types harboring KRAS mutations such as pancreatic and lung cancers. Here we report inhibition of growth and downstream RAS–MAPK signaling in neuroblastoma cells in response to treatment with the SHP2 inhibitors SHP099, II-B08, and RMC-4550. However, neuroblastoma cell lines harboring endogenous NRASQ61K mutation (which is commonly detected at relapse) or isogenic neuroblastoma cells engineered to overexpress NRASQ61K were distinctly resistant to SHP2 inhibitors. Combinations of SHP2 inhibitors with other RAS pathway inhibitors such as trametinib, vemurafenib, and ulixertinib were synergistic and reversed resistance to SHP2 inhibition in neuroblastoma in vitro and in vivo. These results suggest for the first time that combination therapies targeting SHP2 and other components of the RAS–MAPK pathway may be effective against conventional therapy-resistant relapsed neuroblastoma, including those that have acquired NRAS mutations.
Significance:
These findings suggest that conventional therapy–resistant, relapsed neuroblastoma may be effectively treated via combined inhibition of SHP2 and MEK or ERK of the RAS–MAPK pathway.
Data from IL33 Is a Key Driver of Treatment Resistance of Cancer
Authors: Kudo-Saito Chie , Miyamoto Takahiro , Imazeki Hiroshi , Shoji Hirokazu , Aoki Kazunori , Boku Narikazu
Published: March 2023

Abstract
Recurrence and treatment resistance are major causes of cancer-associated death. There has been a growing interest in better understanding epithelial–mesenchymal transition, stemness of cancer cells, and exhaustion and dysfunction of the immune system for which numerous genomic, proteomic, microenvironmental, and immunologic mechanisms have been demonstrated. However, practical treatments for such patients have not yet been established. Here we identified IL33 as a key driver of polyploidy, followed by rapid proliferation after treatment. IL33 induction transformed tumor cells into polyploid giant cells, showing abnormal cell cycle without cell division accompanied by Snail deregulation and p53 inactivation; small progeny cells were generated in response to treatment stress. Simultaneously, soluble IL33 was released from tumor cells, leading to expansion of receptor ST2-expressing cells including IL17RB+GATA3+ cells, which promoted tumor progression and metastasis directly and indirectly via induction of immune exhaustion and dysfunction. Blocking IL33 with a specific mAb in murine IL33+ metastatic tumor models abrogated negative consequences and successfully elicited antitumor efficacy induced by other combined treatments. Ex vivo assays using tumor tissues and peripheral blood mononuclear cells of patients with cancer validated the clinical relevancy of these findings. Together, these data suggest that targeting the IL33-ST2 axis is a promising strategy for diagnosis and treatment of patients likely to be resistant to treatments in the clinical settings.
Significance:
These findings indicate that the functional role of IL33 in cancer polyploidy contributes to intrinsic and extrinsic mechanisms underlying treatment failure.
Data from Activation of Estrogen Receptor Alpha by Decitabine Inhibits Osteosarcoma Growth and Metastasis
Authors: Lillo Osuna Maria Angeles , Garcia-Lopez Jesus , El Ayachi Ikbale , Fatima Iram , Khalid Aysha B. , Kumpati Jerusha , Slayden Alexandria V. , Seagroves Tiffany N. , Miranda-Carboni Gustavo A. , Krum Susan A.
Published: March 2023

Abstract
Osteosarcoma is a malignant tumor in the bone, which originates from normal osteoblasts or osteoblast precursors. Normal osteoblasts express estrogen receptor alpha (ERα); however, osteosarcomas do not express ERα due to promoter DNA methylation. Here we show that treatment of 143B osteosarcoma cells with decitabine (DAC, 5-Aza-2′-deoxycytidine) induces expression of ERα and leads to decreased proliferation and concurrent induction of osteoblast differentiation. DAC exposure reduced protein expression of metastasis-associated markers VIMENTIN, SLUG, ZEB1, and MMP9, with a concurrent decrease in mRNA expression of known stem cell markers SOX2, OCT4, and NANOG. Treatment with 17β-estradiol (E2) synergized with DAC to reduce proliferation. Overexpression of ERα inhibited proliferation and induced osteoblast differentiation, whereas knockout of ERα by CRISPR/Cas9 prevented the effects of DAC. In an orthotopic model of osteosarcoma, DAC inhibited tumor growth and metastasis of 143B cells injected into the tibia of NOD SCID gamma mice. Furthermore, ERα overexpression reduced tumor growth and metastasis, and ERα knockout prevented the effects of DAC in vivo. Together, these experiments provide preclinical evidence that the FDA-approved DNA methylation inhibitor DAC may be repurposed to treat patients with osteosarcoma based on its efficacy to decrease proliferation, to induce osteoblast differentiation, and to reduce metastasis to visceral organs.

Significance: These findings describe the effects of DNA methyltransferase inhibition on ERα and its potential role as a tumor suppressor in osteosarcoma.

See related commentary by Roberts, p. 1034

See related article by El Ayachi and colleagues; Cancer Res 79(5);982–93.
Data from Functional Analysis and Fine Mapping of the 9p22.2 Ovarian Cancer Susceptibility Locus
Authors: Buckley Melissa A. , Woods Nicholas T. , Tyrer Jonathan P. , Mendoza-Fandiño Gustavo , Lawrenson Kate , Hazelett Dennis J. , Najafabadi Hamed S. , Gjyshi Anxhela , Carvalho Renato S. , Lyra Paulo C. , Coetzee Simon G. , Shen Howard C. , Yang Ally W. , Earp Madalene A. , Yoder Sean J. , Risch Harvey , Chenevix-Trench Georgia , Ramus Susan J. , Phelan Catherine M. , Coetzee Gerhard A. , Noushmehr Houtan , Hughes Timothy R. , Sellers Thomas A. , Goode Ellen L. , Pharoah Paul D. , Gayther Simon A. , Monteiro Alvaro N.A.
Published: March 2023

Abstract
Genome-wide association studies have identified 40 ovarian cancer risk loci. However, the mechanisms underlying these associations remain elusive. In this study, we conducted a two-pronged approach to identify candidate causal SNPs and assess underlying biological mechanisms at chromosome 9p22.2, the first and most statistically significant associated locus for ovarian cancer susceptibility. Three transcriptional regulatory elements with allele-specific effects and a scaffold/matrix attachment region were characterized and, through physical DNA interactions, BNC2 was established as the most likely target gene. We determined the consensus binding sequence for BNC2 in vitro, verified its enrichment in BNC2 ChIP-seq regions, and validated a set of its downstream target genes. Fine-mapping by dense regional genotyping in over 15,000 ovarian cancer cases and 30,000 controls identified SNPs in the scaffold/matrix attachment region as among the most likely causal variants. This study reveals a comprehensive regulatory landscape at 9p22.2 and proposes a likely mechanism of susceptibility to ovarian cancer.
Significance:
Mapping the 9p22.2 ovarian cancer risk locus identifies BNC2 as an ovarian cancer risk gene.

See related commentary by Choi and Brown, p. 439
Data from Phosphorylation of RAB7 by TBK1/IKKϵ Regulates Innate Immune Signaling in Triple-Negative Breast Cancer
Authors: Ritter Jessica L. , Zhu Zehua , Thai Tran C. , Mahadevan Navin R. , Mertins Philipp , Knelson Erik H. , Piel Brandon P. , Han Saemi , Jaffe Jacob D. , Carr Steven A. , Barbie David A. , Barbie Thanh U.
Published: March 2023

Abstract
Triple-negative breast cancer (TNBC) is a heterogeneous disease enriched for mutations in PTEN and dysregulation of innate immune signaling. Here, we demonstrate that Rab7, a recently identified substrate of PTEN phosphatase activity, is also a substrate of the innate immune signaling kinases TANK-binding kinase 1 (TBK1)/IκB kinase ϵ (IKKϵ) on the same serine-72 (S72) site. An unbiased search for novel TBK1/IKKϵ substrates using stable isotope labeling with amino acids in cell culture phosphoproteomic analysis identified Rab7-S72 as a top hit. PTEN-null TNBC cells expressing a phosphomimetic version of Rab7-S72 exhibited diffuse cytosolic Rab7 localization and enhanced innate immune signaling, in contrast to a kinase-resistant version, which localized to active puncta that promote lysosomal-mediated stimulator of interferon genes (STING) degradation. Thus, convergence of PTEN loss and TBK1/IKKϵ activation on Rab7-S72 phosphorylation limited STING turnover and increased downstream production of IRF3 targets including CXCL10, CCL5, and IFNβ. Consistent with this data, PTEN-null TNBC tumors expressed higher levels of STING, and PTEN-null TNBC cell lines were hyperresponsive to STING agonists. Together, these findings begin to uncover how innate immune signaling is dysregulated downstream of TBK1/IKKϵ in a subset of TNBCs and reveals previously unrecognized cross-talk with STING recycling that may have implications for STING agonism in the clinic.
Significance:
These findings identify Rab7 as a substrate for TBK1 for regulation of innate immune signaling, thereby providing important insight for strategies aimed at manipulating the immune response to enhance therapeutic efficacy in TNBC.
Data from SIRT1-Mediated Expression of CD24 and Epigenetic Suppression of Novel Tumor Suppressor miR-1185-1 Increases Colorectal Cancer Stemness
Authors: Wang Teh-Wei , Chern Edward , Hsu Chao-Wei , Tseng Kuo-Chang , Chao Hsiao-Mei
Published: March 2023

Abstract
NAD-dependent deacetylase sirtuin-1 (SIRT1) is a class III histone deacetylase that positively regulates cancer-related pathways such as proliferation and stress resistance. SIRT1 has been shown to promote progression of colorectal cancer and is associated with cancer stemness, yet the precise mechanism between colorectal cancer stemness and SIRT1 remains to be further clarified. Here we report that SIRT1 signaling regulates colorectal cancer stemness by enhancing expression of CD24, a colorectal cancer stemness promoter. A novel miRNA, miR-1185-1, suppressed the expression of CD24 by targeting its 3′UTR (untranslated region) and could be inhibited by SIRT1 via histone deacetylation. Targeting SIRT1 by RNAi led to elevated H3 lysine 9 acetylation on the promoter region of miR-1185-1, which increased expression of miR-1185-1 and further repressed CD24 translation and colorectal cancer stemness. In a mouse xenograft model, overexpression of miR-1185-1 in colorectal cancer cells substantially reduced tumor growth. In addition, expression of miR-1185-1 was downregulated in human colorectal cancer tissues, whereas expression of CD24 was increased. In conclusion, this study not only demonstrates the essential roles of a SIRT1–miR-1185-1–CD24 axis in both colorectal cancer stemness properties and tumorigenesis but provides a potential therapeutic target for colorectal cancer treatment.
Significance:
A novel tumor suppressor miR-1185-1 is involved in molecular regulation of CD24- and SIRT1-related cancer stemness networks, marking it a potential therapeutic target in colorectal cancer.
Data from Raman Spectroscopy Reveals That Biochemical Composition of Breast Microcalcifications Correlates with Histopathologic Features
Authors: Vanna Renzo , Morasso Carlo , Marcinnò Beatrice , Piccotti Francesca , Torti Emanuele , Altamura Davide , Albasini Sara , Agozzino Manuela , Villani Laura , Sorrentino Luca , Bunk Oliver , Leporati Francesco , Giannini Cinzia , Corsi Fabio
Published: March 2023

Abstract
Breast microcalcifications are a common mammographic finding. Microcalcifications are considered suspicious signs of breast cancer and a breast biopsy is required, however, cancer is diagnosed in only a few patients. Reducing unnecessary biopsies and rapid characterization of breast microcalcifications are unmet clinical needs. In this study, 473 microcalcifications detected on breast biopsy specimens from 56 patients were characterized entirely by Raman mapping and confirmed by X-ray scattering. Microcalcifications from malignant samples were generally more homogeneous, more crystalline, and characterized by a less substituted crystal lattice compared with benign samples. There were significant differences in Raman features corresponding to the phosphate and carbonate bands between the benign and malignant groups. In addition to the heterogeneous composition, the presence of whitlockite specifically emerged as marker of benignity in benign microcalcifications. The whole Raman signature of each microcalcification was then used to build a classification model that distinguishes microcalcifications according to their overall biochemical composition. After validation, microcalcifications found in benign and malignant samples were correctly recognized with 93.5% sensitivity and 80.6% specificity. Finally, microcalcifications identified in malignant biopsies, but located outside the lesion, reported malignant features in 65% of in situ and 98% of invasive cancer cases, respectively, suggesting that the local microenvironment influences microcalcification features. This study confirms that the composition and structural features of microcalcifications correlate with breast pathology and indicates new diagnostic potentialities based on microcalcifications assessment.
Significance:
Raman spectroscopy could be a quick and accurate diagnostic tool to precisely characterize and distinguish benign from malignant breast microcalcifications detected on mammography.
Data from ETV4 Is Necessary for Estrogen Signaling and Growth in Endometrial Cancer Cells
Authors: Rodriguez Adriana C. , Vahrenkamp Jeffery M. , Berrett Kristofer C. , Clark Kathleen A. , Guillen Katrin P. , Scherer Sandra D. , Yang Chieh-Hsiang , Welm Bryan E. , Janát-Amsbury Margit M. , Graves Barbara J. , Gertz Jason
Published: March 2023

Abstract
Estrogen signaling through estrogen receptor alpha (ER) plays a major role in endometrial cancer risk and progression, however, the molecular mechanisms underlying ER's regulatory role in endometrial cancer are poorly understood. In breast cancer cells, ER genomic binding is enabled by FOXA1 and GATA3, but the transcription factors that control ER genomic binding in endometrial cancer cells remain unknown. We previously identified ETV4 as a candidate factor controlling ER genomic binding in endometrial cancer cells, and here we explore the functional importance of ETV4. Homozygous deletion of ETV4, using CRISPR/Cas9, led to greatly reduced ER binding at the majority of loci normally bound by ER. Consistent with the dramatic loss of ER binding, the gene expression response to estradiol was dampened for most genes. ETV4 contributes to estrogen signaling in two distinct ways. ETV4 loss affects chromatin accessibility at some ER bound loci and impairs ER nuclear translocation. The diminished estrogen signaling upon ETV4 deletion led to decreased growth, particularly in 3D culture, where hollow organoids were formed and in vivo in the context of estrogen-dependent growth. These results show that ETV4 plays an important role in estrogen signaling in endometrial cancer cells.
Significance:
Estrogen receptor alpha (ER) is a key oncogene in endometrial cancer. This study uncovers ETV4 as an important factor in controlling the activity of ER and the growth of endometrial cancer cells.
Data from Topological Tumor Graphs: A Graph-Based Spatial Model to Infer Stromal Recruitment for Immunosuppression in Melanoma Histology
Authors: Failmezger Henrik , Muralidhar Sathya , Rullan Antonio , de Andrea Carlos E. , Sahai Erik , Yuan Yinyin
Published: March 2023

Abstract
Despite the advent of immunotherapy, metastatic melanoma represents an aggressive tumor type with a poor survival outcome. The successful application of immunotherapy requires in-depth understanding of the biological basis and immunosuppressive mechanisms within the tumor microenvironment. In this study, we conducted spatially explicit analyses of the stromal-immune interface across 400 melanoma hematoxylin and eosin (H&E) specimens from The Cancer Genome Atlas. A computational pathology pipeline (CRImage) was used to classify cells in the H&E specimen into stromal, immune, or cancer cells. The estimated proportions of these cell types were validated by independent measures of tumor purity, pathologists' estimate of lymphocyte density, imputed immune cell subtypes, and pathway analyses. Spatial interactions between these cell types were computed using a graph-based algorithm (topological tumor graphs, TTG). This approach identified two stromal features, namely stromal clustering and stromal barrier, which represented the melanoma stromal microenvironment. Tumors with increased stromal clustering and barrier were associated with reduced intratumoral lymphocyte distribution and poor overall survival independent of existing prognostic factors. To explore the genomic basis of these TTG-derived stromal phenotypes, we used a deep learning approach integrating genomic (copy number) and transcriptomic data, thereby inferring a compressed representation of copy number-driven alterations in gene expression. This integrative analysis revealed that tumors with high stromal clustering and barrier had reduced expression of pathways involved in naïve CD4 signaling, MAPK, and PI3K signaling. Taken together, our findings support the immunosuppressive role of stromal cells and T-cell exclusion within the vicinity of melanoma cells.
Significance:
Computational histology-based stromal phenotypes within the tumor microenvironment are significantly associated with prognosis and immune exclusion in melanoma.
Data from ERK-Mediated Loss of miR-199a-3p and Induction of EGR1 Act as a “Toggle Switch” of GBM Cell Dedifferentiation into NANOG- and OCT4-Positive Cells
Authors: Almairac Fabien , Turchi Laurent , Sakakini Nathalie , Debruyne David Nicolas , Elkeurti Sarah , Gjernes Elisabet , Polo Beatrice , Bianchini Laurence , Fontaine Denys , Paquis Philippe , Chneiweiss Herve , Junier Marie-Pierre , Verrando Patrick , Burel-Vandenbos Fanny , Virolle Thierry
Published: March 2023

Abstract
There is great interest in understanding how the cancer stem cell population may be maintained in solid tumors. Here, we show that tumor cells exhibiting stem-like properties and expression of pluripotency markers NANOG and OCT4 can arise from original differentiated tumor cells freshly isolated from human glioblastomas (GBM) and that have never known any serum culture conditions. Induction of EGR1 by EGFR/ERK signaling promoted cell conversion from a less aggressive, more differentiated cellular state to a self-renewing and strongly tumorigenic state, expressing NANOG and OCT4. Expression of these pluripotency markers occurred before the cells re-entered the cell cycle, demonstrating their capacity to change and dedifferentiate without any cell divisions. In differentiated GBM cells, ERK-mediated repression of miR-199a-3p induced EGR1 protein expression and triggered dedifferentiation. Overall, this signaling pathway constitutes an ERK-mediated "toggle switch" that promotes pluripotency marker expression and stem-like features in GBM cells.
Significance:
This study defines an ERK-mediated molecular mechanism of dedifferentiation of GBM cells into a stem-like state, expressing markers of pluripotency.

See related commentary by Koncar and Agnihotri, p. 3195
Data from A Novel Bioengineered miR-127 Prodrug Suppresses the Growth and Metastatic Potential of Triple-Negative Breast Cancer Cells
Authors: Umeh-Garcia Maxine , Simion Catalina , Ho Pui-Yan , Batra Neelu , Berg Anastasia L. , Carraway Kermit L. , Yu Aiming , Sweeney Colleen
Published: March 2023

Abstract
miR-127 is downregulated in breast cancer, where it has been shown to suppress the proliferation, migration, and invasion of breast cancer cells. In triple-negative breast cancer (TNBC), miR-127 downregulation correlates with decreased disease-free and overall patient survival. Tumor suppressor miRNAs may hold therapeutic promise but progress has been limited by several factors, including the lability and high cost of miRNA mimics. Here, we take a novel approach to produce a miR-127 prodrug (miR-127PD), which we demonstrate is processed to mature, functional miR-127-3p in TNBC tumor cells. miR-127PD decreased the viability and motility of TNBC cells, sensitized TNBC cells to chemotherapy, and restricted the TNBC stem cell population. Furthermore, systemic delivery of miR-127PD suppressed tumor growth of MDA-MB-231 and MDA-MB-468 TNBC cells and spontaneous metastasis of MDA-MB-231 cells. In addition, CERK, NANOS1, FOXO6, SOX11, SOX12, FASN, and SUSD2 were identified as novel, functionally important targets of miR-127. In conclusion, our study demonstrates that miR-127 functions as a tumor and metastasis suppressor in TNBC and that delivery of miR-127 may hold promise as a novel therapy.
Significance:
Exogenous administration of miR-127, which is functionally activated in target cells, inhibits growth and spontaneous metastasis of triple-negative breast cancer.
Data from Cancer-Associated Point Mutations in the DLC1 Tumor Suppressor and Other Rho-GAPs Occur Frequently and Are Associated with Decreased Function
Authors: Wang Dunrui , Qian Xiaolan , Sanchez-Solana Beatriz , Tripathi Brajendra K. , Durkin Marian E. , Lowy Douglas R.
Published: March 2023

Abstract
In advanced cancer, the RHOA GTPase is often active together with reduced expression of genes encoding Rho-specific GTPase-accelerating proteins (Rho-GAP), which negatively regulate RHOA and related GTPases. Here we used the The Cancer Genome Atlas dataset to examine 12 tumor types (including colon, breast, prostate, pancreas, lung adenocarcinoma, and squamous cell carcinoma) for the frequency of codon mutations of 10 Rho-GAP and experimentally tested biochemical and biological consequences for cancer-associated mutants that arose in the DLC1 tumor suppressor gene. DLC1 was the Rho-GAP gene mutated most frequently, with 5%–8% of tumors in five of the tumor types evaluated having DLC1 missense mutations. Furthermore, 20%–26% of the tumors in four of these five tumor types harbored missense mutations in at least one of the 10 Rho-GAPs. Experimental analysis of the DLC1 mutants indicated 7 of 9 mutants whose lesions were located in the Rho-GAP domain were deficient for Rho-GAP activity and for suppressing cell migration and anchorage-independent growth. Analysis of a DLC1 linker region mutant and a START domain mutant showed each was deficient for suppressing migration and growth in agar, but their Rho-GAP activity was similar to that of wild-type DLC1. Compared with the wild-type, the linker region mutant bound 14-3-3 proteins less efficiently, while the START domain mutant displayed reduced binding to Caveolin-1. Thus, mutation of Rho-GAP genes occurs frequently in some cancer types and the majority of cancer-associated DLC1 mutants evaluated were deficient biologically, with various mechanisms contributing to their reduced activity.
Significance:
These findings indicate that point mutation of Rho-GAP genes is unexpectedly frequent in several cancer types, with DLC1 mutants exhibiting reduced function by various mechanisms.
Data from Soft Microenvironments Induce Chemoresistance by Increasing Autophagy Downstream of Integrin-Linked Kinase
Authors: Anlaş Alişya A. , Nelson Celeste M.
Published: March 2023

Abstract
Breast cancer relapse can develop over the course of years as a result of dormant cancer cells that disseminate to secondary sites. These dormant cells are often resistant to conventional hormone and chemotherapy. Although recurrence is the main cause of death from cancer, microenvironmental factors that may influence resistance to therapy and duration of dormancy are largely unknown. Breast cancer relapse is often detected in tissues that are softer than the normal mammary gland or the primary breast tumor, such as bone marrow, brain, and lung. We therefore explored how stiffness of the microenvironment at secondary sites regulates tumor dormancy and the response of breast cancer cells to hormone and chemotherapy. In soft microenvironments reminiscent of metastatic sites, breast cancer cells were more resistant to the estrogen receptor modulator tamoxifen as a result of increased autophagy and decreased expression of estrogen receptor-α. Consistently, pharmacologic inhibition or genetic downregulation of autophagy increased the response of breast cancer cells to tamoxifen on soft substrata. In addition, autophagy was decreased downstream of integrin-linked kinase on stiff substrata. Altogether, our data show that tissue mechanics regulates therapeutic outcome and long-term survival of breast cancer cells by influencing autophagy.
Significance:
These findings characterize the persistence of dormant cells at metastatic sites, where soft microenvironments downregulate estrogen receptor expression and upregulate autophagy, thereby promoting therapy resistance in breast cancer cells.
Data from Fulvestrant-Mediated Attenuation of the Innate Immune Response Decreases ER+ Breast Cancer Growth In Vivo More Effectively than Tamoxifen
Authors: Abrahamsson Annelie , Rodriguez Gabriela Vazquez , Dabrosin Charlotta
Published: March 2023

Abstract
Although blocking estrogen-dependent signaling is a cornerstone of adjuvant treatment for breast cancer, 25% of patients experience recurrent disease. Stroma events including innate immune responses are key in cancer progression. How different estrogen receptor (ER)–targeting therapies, including the partial agonist tamoxifen and the pure antagonist fulvestrant, affect the tumor stroma has not yet been elucidated. Fulvestrant is used in only postmenopausal patients, and its effects in the presence of estradiol remain undetermined. Here we observe that fulvestrant decreases ER+ breast cancer growth compared with tamoxifen in the presence of physiologic levels of estradiol in human breast cancer in nude mice and in murine breast cancer in immune-competent mice. Fulvestrant significantly inhibited macrophage and neutrophil infiltration in both models. These effects were corroborated in a zebrafish model where fulvestrant inhibited neutrophil- and macrophage-dependent cancer cell dissemination more effectively than tamoxifen. A comprehensive analysis of 234 human proteins released into the cancer microenvironment by the cancer cells sampled via microdialysis in vivo revealed that 38 proteins were altered following both treatments; 25 of these proteins were associated with immune response and were altered by fulvestrant only. Compared with tamoxifen, fulvestrant significantly affected inflammatory proteins released by murine stroma cells. Importantly, in vivo microdialysis of human ER+ breast cancer revealed that the majority of affected proteins in murine models were upregulated in patients. Together, these results suggest that fulvestrant targets ER+ breast cancer more effectively than tamoxifen even in the presence of estradiol, mainly by attenuation of the innate immune response.
Significance:
These findings demonstrate novel effects of the pure antiestrogen fulvestrant in ER+ breast cancer and evaluate its effects under physiologic levels of estradiol, representative of premenopausal patients.
Data from Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System
Authors: Tubeuf Hélène , Caputo Sandrine M. , Sullivan Teresa , Rondeaux Julie , Krieger Sophie , Caux-Moncoutier Virginie , Hauchard Julie , Castelain Gaia , Fiévet Alice , Meulemans Laëtitia , Révillion Françoise , Léoné Mélanie , Boutry-Kryza Nadia , Delnatte Capucine , Guillaud-Bataille Marine , Cleveland Linda , Reid Susan , Southon Eileen , Soukarieh Omar , Drouet Aurélie , Di Giacomo Daniela , Vezain Myriam , Bonnet-Dorion Françoise , Bourdon Violaine , Larbre Hélène , Muller Danièle , Pujol Pascal , Vaz Fátima , Audebert-Bellanger Séverine , Colas Chrystelle , Venat-Bouvet Laurence , Solano Angela R. , Stoppa-Lyonnet Dominique , Houdayer Claude , Frebourg Thierry , Gaildrat Pascaline , Sharan Shyam K. , Martins Alexandra
Published: March 2023

Abstract
BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons.
Significance:
These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.
Data from ATF3 Coordinates Antitumor Synergy between Epigenetic Drugs and Protein Disulfide Isomerase Inhibitors
Authors: Duncan Ravyn M. , Reyes Leticia , Moats Katelyn , Robinson Reeder M. , Murphy Sara A. , Kaur Balveen , Stessman Holly A.F. , Dolloff Nathan G.
Published: March 2023

Abstract
Histone deacetylase inhibitors (HDACi) are largely ineffective in the treatment of solid tumors. In this study, we describe a new class of protein disulfide isomerase (PDI) inhibitors that significantly and synergistically enhance the antitumor activity of HDACi in glioblastoma and pancreatic cancer preclinical models. RNA-sequencing screening coupled with gene silencing studies identified ATF3 as the driver of this antitumor synergy. ATF3 was highly induced by combined PDI and HDACi treatment as a result of increased acetylation of key histone lysine residues (acetylated histone 3 lysine 27 and histone 3 lysine 18) flanking the ATF3 promoter region. These chromatin marks were associated with increased RNA polymerase II recruitment to the ATF3 promoter, a synergistic upregulation of ATF3, and a subsequent apoptotic response in cancer cells. The HSP40/HSP70 family genes DNAJB1 and HSPA6 were found to be critical ATF3-dependent genes that elicited the antitumor response after PDI and HDAC inhibition. In summary, this study presents a synergistic antitumor combination of PDI and HDAC inhibitors and demonstrates a mechanistic and tumor suppressive role of ATF3. Combined treatment with PDI and HDACi offers a dual therapeutic strategy in solid tumors and the opportunity to achieve previously unrealized activity of HDACi in oncology.
Significance:
This study uses a first-in-class PDI inhibitor entering clinical development to enhance the effects of epigenetic drugs in some of the deadliest forms of cancer.
Data from Reactive Oxygen Species Drive Proliferation in Acute Myeloid Leukemia via the Glycolytic Regulator PFKFB3
Authors: Robinson Andrew J. , Hopkins Goitseone L. , Rastogi Namrata , Hodges Marie , Doyle Michelle , Davies Sara , Hole Paul S. , Omidvar Nader , Darley Richard L. , Tonks Alex
Published: March 2023

Abstract
Acute myeloid leukemia (AML) is a heterogeneous clonal disorder with a poor clinical outcome. Previously, we showed that overproduction of reactive oxygen species (ROS), arising from constitutive activation of NOX2 oxidase, occurs in >60% of patients with AML and that ROS production promotes proliferation of AML cells. We show here that the process most significantly affected by ROS overproduction is glycolysis. Whole metabolome analysis of 20 human primary AML showed that blasts generating high levels of ROS have increased glucose uptake and correspondingly increased glucose metabolism. In support of this, exogenous ROS increased glucose consumption while inhibition of NOX2 oxidase decreased glucose consumption. Mechanistically, ROS promoted uncoupling protein 2 (UCP2) protein expression and phosphorylation of AMPK, upregulating the expression of a key regulatory glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Overexpression of PFKFB3 promoted glucose uptake and cell proliferation, whereas downregulation of PFKFB3 strongly suppressed leukemia growth both in vitro and in vivo in the NSG model. These experiments provide direct evidence that oxidase-derived ROS promotes the growth of leukemia cells via the glycolytic regulator PFKFB3. Targeting PFKFB3 may therefore present a new mode of therapy for this disease with a poor outcome.
Significance:
These findings show that ROS generated by NOX2 in AML cells promotes glycolysis by activating PFKFB3 and suggest PFKFB3 as a novel therapeutic target in AML.
Data from 3D Functional Genomics Screens Identify CREBBP as a Targetable Driver in Aggressive Triple-Negative Breast Cancer
Authors: Peck Barrie , Bland Philip , Mavrommati Ioanna , Muirhead Gareth , Cottom Hannah , Wai Patty T. , Maguire Sarah L. , Barker Holly E. , Morrison Eamonn , Kriplani Divya , Yu Lu , Gibson Amy , Falgari Giulia , Brennan Keith , Farnie Gillian , Buus Richard , Marlow Rebecca , Novo Daniela , Knight Eleanor , Guppy Naomi , Kolarevic Daniela , Susnjar Snezana , Milijic Natasa Medic , Naidoo Kalnisha , Gazinska Patrycja , Roxanis Ioannis , Pancholi Sunil , Martin Lesley-Ann , Holgersen Erle M. , Cheang Maggie C.U. , Noor Farzana , Postel-Vinay Sophie , Quinn Gerard , McDade Simon , Krasny Lukas , Huang Paul , Daley Frances , Wallberg Fredrik , Choudhary Jyoti S. , Haider Syed , Tutt Andrew N. , Natrajan Rachael
Published: March 2023

Abstract
Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo–like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population.
Significance:
This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors.