bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from FOXD3 Modulates Migration through Direct Transcriptional Repression of TWIST1 in Melanoma
Authors: Weiss Michele B. , Abel Ethan V. , Dadpey Neda , Aplin Andrew E.
Published: March 2024

Abstract
The neural crest is a multipotent, highly migratory cell population that gives rise to diverse cell types, including melanocytes. Factors regulating the development of the neural crest and emigration of its cells are likely to influence melanoma metastasis. The transcription factor FOXD3 plays an essential role in premigratory neural crest development and has been implicated in melanoma cell dormancy and response to therapeutics. FOXD3 is downregulated during the migration of the melanocyte lineage from the neural crest, and our previous work supports a role for FOXD3 in suppressing melanoma cell migration and invasion. Alternatively, TWIST1 is known to have promigratory and proinvasive roles in a number of cancers, including melanoma. Using ChIP-seq analysis, TWIST1 was identified as a potential transcriptional target of FOXD3. Mechanistically, FOXD3 directly binds to regions of the TWIST1 gene locus, leading to transcriptional repression of TWIST1 in human mutant BRAF melanoma cells. In addition, depletion of endogenous FOXD3 promotes upregulation of TWIST1 transcripts and protein. Finally, FOXD3 expression leads to a significant decrease in cell migration that can be efficiently reversed by the overexpression of TWIST1. These findings uncover the novel interplay between FOXD3 and TWIST1, which is likely to be important in the melanoma metastatic cascade.

Implications: FOXD3 and TWIST1 define distinct subgroups of cells within a heterogeneous tumor. Mol Cancer Res; 12(9); 1314–23. ©2014 AACR.
Data from Notch3 Overexpression Promotes Anoikis Resistance in Epithelial Ovarian Cancer via Upregulation of COL4A2
Authors: Brown Caitlin W. , Brodsky Alexander S. , Freiman Richard N.
Published: March 2024

Abstract
Ovarian cancer is a lethal disease with the majority of diagnosed women having distant metastases. Interestingly, although Notch3 overexpression has been correlated with poor survival in epithelial ovarian cancer (EOC), little is known about its mechanism of action. Data show that Notch3 specifically promotes anoikis resistance. In addition, data indicate a positive role for focal adhesion kinase (FAK) as well as downstream signaling kinases such as Akt and Erk 1/2 in promoting anchorage-independent growth. Mechanistically, both mRNA transcript and protein levels of type IV collagen (COL4A2) are reduced when Notch3 levels are decreased and exogenous collagen IV supplementation reverses the anoikis sensitivity. Reduction of COL4A2 expression by RNAI-mediated knockdown induces cell death. Finally, elevated Notch3 expression levels correlate with higher COL4A2 expression in human ovarian tumor specimens.

Implications: These data highlight type IV collagen as a novel therapeutic target for metastatic EOC.

Visual Overview: http://mcr.aacrjournals.org/content/early/2014/11/25/1541-7786.MCR-14-0334/F1.large.jpg

Mol Cancer Res; 13(1); 78–85. ©2014 AACR.
Data from Novel Potent and Selective Inhibitors of p90 Ribosomal S6 Kinase Reveal the Heterogeneity of RSK Function in MAPK-Driven Cancers
Authors: Aronchik Ida , Appleton Brent A. , Basham Stephen E. , Crawford Kenneth , Del Rosario Mercedita , Doyle Laura V. , Estacio William F. , Lan Jiong , Lindvall Mika K. , Luu Catherine A. , Ornelas Elizabeth , Venetsanakos Eleni , Shafer Cynthia M. , Jefferson Anne B.
Published: March 2024

Abstract
The p90 ribosomal S6 kinase (RSK) family of serine/threonine kinases is expressed in a variety of cancers and its substrate phosphorylation has been implicated in direct regulation of cell survival, proliferation, and cell polarity. This study characterizes and presents the most selective and potent RSK inhibitors known to date, LJH685 and LJI308. Structural analysis confirms binding of LJH685 to the RSK2 N-terminal kinase ATP-binding site and reveals that the inhibitor adopts an unusual nonplanar conformation that explains its excellent selectivity for RSK family kinases. LJH685 and LJI308 efficiently inhibit RSK activity in vitro and in cells. Furthermore, cellular inhibition of RSK and its phosphorylation of YB1 on Ser102 correlate closely with inhibition of cell growth, but only in an anchorage-independent growth setting, and in a subset of examined cell lines. Thus, RSK inhibition reveals dynamic functional responses among the inhibitor-sensitive cell lines, underscoring the heterogeneous nature of RSK dependence in cancer.

Implications: Two novel potent and selective RSK inhibitors will now allow a full assessment of the potential of RSK as a therapeutic target for oncology. Mol Cancer Res; 12(5); 803–12. ©2014 AACR.
Data from PKCϵ Is an Essential Mediator of Prostate Cancer Bone Metastasis
Authors: Gutierrez-Uzquiza Alvaro , Lopez-Haber Cynthia , Jernigan Danielle L. , Fatatis Alessandro , Kazanietz Marcelo G.
Published: March 2024

Abstract
The bone is a preferred site for metastatic homing of prostate cancer cells. Once prostate cancer patients develop skeletal metastases, they eventually succumb to the disease; therefore, it is imperative to identify key molecular drivers of this process. This study examines the involvement of protein kinase C epsilon (PKCϵ), an oncogenic protein that is abnormally overexpressed in human tumor specimens and cell lines, on prostate cancer cell bone metastasis. PC3-ML cells, a highly invasive prostate cancer PC3 derivative with bone metastatic colonization properties, failed to induce skeletal metastatic foci upon inoculation into nude mice when PKCϵ expression was silenced using shRNA. Interestingly, while PKCϵ depletion had only marginal effects on the proliferative, adhesive, and migratory capacities of PC3-ML cells in vitro or in the growth of xenografts upon s.c. inoculation, it caused a significant reduction in cell invasiveness. Notably, PKCϵ was required for transendothelial cell migration (TEM) as well as for the growth of PC3-ML cells in a bone biomimetic environment. At a mechanistic level, PKCϵ depletion abrogates the expression of IL1β, a cytokine implicated in skeletal metastasis. Taken together, PKCϵ is a key factor for driving the formation of bone metastasis by prostate cancer cells and is a potential therapeutic target for advanced stages of the disease.

Implications: This study uncovers an important new function of PKCϵ in the dissemination of cancer cells to the bone; thus, highlighting the promising potential of this oncogenic kinase as a therapeutic target for skeletal metastasis. Mol Cancer Res; 13(9); 1336–46. ©2015 AACR.
Data from AKT-Induced Tamoxifen Resistance Is Overturned by RRM2 Inhibition
Authors: Shah Khyati N. , Mehta Kshama R. , Peterson David , Evangelista Marie , Livesey John C. , Faridi Jesika S.
Published: March 2024

Abstract
Acquired tamoxifen resistance develops in the majority of hormone-responsive breast cancers and frequently involves overexpression of the PI3K/AKT axis. Here, breast cancer cells with elevated endogenous AKT or overexpression of activated AKT exhibited tamoxifen-stimulated cell proliferation and enhanced cell motility. To gain mechanistic insight on AKT-induced endocrine resistance, gene expression profiling was performed to determine the transcripts that are differentially expressed post-tamoxifen therapy under conditions of AKT overexpression. Consistent with the biologic outcome, many of these transcripts function in cell proliferation and cell motility networks and were quantitatively validated in a larger panel of breast cancer cells. Moreover, ribonucleotide reductase M2 (RRM2) was revealed as a key contributor to AKT-induced tamoxifen resistance. Inhibition of RRM2 by RNA interference (RNAi)–mediated approaches significantly reversed the tamoxifen-resistant cell growth, inhibited cell motility, and activated DNA damage and proapoptotic pathways. In addition, treatment of tamoxifen-resistant breast cancer cells with the small molecule RRM inhibitor didox significantly reduced in vitro and in vivo growth. Thus, AKT-expressing breast cancer cells upregulate RRM2 expression, leading to increased DNA repair and protection from tamoxifen-induced apoptosis.

Implications: These findings identify RRM2 as an AKT-regulated gene, which plays a role in tamoxifen resistance and may prove to be a novel target for effective diagnostic and preventative strategies. Mol Cancer Res; 12(3); 394–407. ©2013 AACR.
Data from HIC1 Tumor Suppressor Loss Potentiates TLR2/NF-κB Signaling and Promotes Tissue Damage–Associated Tumorigenesis
Authors: Janeckova Lucie , Pospichalova Vendula , Fafilek Bohumil , Vojtechova Martina , Tureckova Jolana , Dobes Jan , Dubuissez Marion , Leprince Dominique , Baloghova Nikol , Horazna Monika , Hlavata Adela , Stancikova Jitka , Sloncova Eva , Galuskova Katerina , Strnad Hynek , Korinek Vladimir
Published: March 2024

Abstract
Hypermethylated in cancer 1 (HIC1) represents a prototypic tumor suppressor gene frequently inactivated by DNA methylation in many types of solid tumors. The gene encodes a sequence-specific transcriptional repressor controlling expression of several genes involved in cell cycle or stress control. In this study, a Hic1 allele was conditionally deleted, using a Cre/loxP system, to identify genes influenced by the loss of Hic1. One of the transcripts upregulated upon Hic1 ablation is the toll-like receptor 2 (TLR2). Tlr2 expression levels increased in Hic1-deficient mouse embryonic fibroblasts (MEF) and cultured intestinal organoids or in human cells upon HIC1 knockdown. In addition, HIC1 associated with the TLR2 gene regulatory elements, as detected by chromatin immunoprecipitation, indicating that Tlr2 indeed represents a direct Hic1 target. The Tlr2 receptor senses “danger” signals of microbial or endogenous origin to trigger multiple signaling pathways, including NF-κB signaling. Interestingly, Hic1 deficiency promoted NF-κB pathway activity not only in cells stimulated with Tlr2 ligand, but also in cells treated with NF-κB activators that stimulate different surface receptors. In the intestine, Hic1 is mainly expressed in differentiated epithelial cells and its ablation leads to increased Tlr2 production. Finally, in a chemical-induced mouse model of carcinogenesis, Hic1 absence resulted in larger Tlr2-positive colonic tumors that showed increased proportion of proliferating cells.

Implications: The tumor-suppressive function of Hic1 in colon is related to its inhibitory action on proproliferative signaling mediated by the Tlr2 receptor present on tumor cells. Mol Cancer Res; 13(7); 1139–48. ©2015 AACR.
Data from AMPK Causes Cell Cycle Arrest in LKB1-Deficient Cells via Activation of CAMKK2
Authors: Fogarty Sarah , Ross Fiona A. , Vara Ciruelos Diana , Gray Alexander , Gowans Graeme J. , Hardie D. Grahame
Published: March 2024

Abstract
The AMP-activated protein kinase (AMPK) is activated by phosphorylation at Thr172, either by the tumor suppressor kinase LKB1 or by an alternate pathway involving the Ca2+/calmodulin-dependent kinase, CAMKK2. Increases in AMP:ATP and ADP:ATP ratios, signifying energy deficit, promote allosteric activation and net Thr172 phosphorylation mediated by LKB1, so that the LKB1–AMPK pathway acts as an energy sensor. Many tumor cells carry loss-of-function mutations in the STK11 gene encoding LKB1, but LKB1 reexpression in these cells causes cell-cycle arrest. Therefore, it was investigated as to whether arrest by LKB1 is caused by activation of AMPK or of one of the AMPK-related kinases, which are also dependent on LKB1 but are not activated by CAMKK2. In three LKB1-null tumor cell lines, treatment with the Ca2+ ionophore A23187 caused a G1 arrest that correlated with AMPK activation and Thr172 phosphorylation. In G361 cells, expression of a truncated, Ca2+/calmodulin-independent CAMKK2 mutant also caused G1 arrest similar to that caused by expression of LKB1, while expression of a dominant-negative AMPK mutant, or a double knockout of both AMPK-α subunits, also prevented the cell-cycle arrest caused by A23187. These mechanistic findings confirm that AMPK activation triggers cell-cycle arrest, and also suggest that the rapid proliferation of LKB1-null tumor cells is due to lack of the restraining influence of AMPK. However, cell-cycle arrest can be restored by reexpressing LKB1 or a constitutively active CAMKK2, or by pharmacologic agents that increase intracellular Ca2+ and thus activate endogenous CAMKK2.

Implications: Evidence here reveals that the rapid growth and proliferation of cancer cells lacking the tumor suppressor LKB1 is due to reduced activity of AMPK, and suggests a therapeutic approach by which this block might be circumvented. Mol Cancer Res; 14(8); 683–95. ©2016 AACR.
Data from Differential Effects of Tyrosine Kinase Inhibitors on Normal and Oncogenic EGFR Signaling and Downstream Effectors
Authors: Kim Youngjoo , Apetri Mihaela , Luo BeiBei , Settleman Jeffrey E. , Anderson Karen S.
Published: March 2024

Abstract
Constitutive activation of EGFR due to overexpression or mutation in tumor cells leads to dysregulated downstream cellular signaling pathways. Therefore, EGFR as well as its downstream effectors have been identified as important therapeutic targets. The FDA-approved small-molecule inhibitors of EGFR, gefitinib (Iressa) and erlotinib (Tarceva), are clinically effective in a subset of patients with non–small cell lung cancer (NSCLC) whose tumors harbor activating mutations within the kinase domain of EGFR. The current study examined effects of these drugs in 32D cells expressing native (WT) or oncogenic (L858R) EGFR as well as in cancer cell lines A431 and H3255. Distinct patterns for gefitinib and erlotinib inhibition of EGFR autophosphorylation at individual tyrosines were revealed for wild-type (WT) and L858R EGFR. Phosphorylation of Y845 has been shown to be important in cancer cells and Y1045 phosphorylation is linked to Cbl-mediated ubiquitination and degradation. Dramatic differences were observed by greater potency of these drugs for inhibiting downstream effectors for L858R EGFR including Cbl and STAT5. Selective targeting of Cbl may play a role in oncogene addiction and effects on STAT5 identify features of signaling circuitry for L858R EGFR that contribute to drug sensitivity and clinical efficacy. These data provide new understanding of the EGFR signaling environment and suggest useful paradigms for predicting patient response to EGFR-targeted therapy as well as combination treatments.

Implications: This study offers fundamental insights for understanding molecular mechanisms of drug sensitivity on oncogenic forms of EGFR and downstream signaling components as well as considerations for further drug optimization and design of combination therapy. Mol Cancer Res; 13(4); 765–74. ©2015 AACR.
Data from Exposure of Patient-Derived Mesenchymal Stromal Cells to TGFB1 Supports Fibrosis Induction in a Pediatric Acute Megakaryoblastic Leukemia Model
Authors: Hack Theresa , Bertram Stefanie , Blair Helen , Börger Verena , Büsche Guntram , Denson Lora , Fruth Enrico , Giebel Bernd , Heidenreich Olaf , Klein–Hitpass Ludger , Kollipara Laxmikanth , Sendker Stephanie , Sickmann Albert , Walter Christiane , von Neuhoff Nils , Hanenberg Helmut , Reinhardt Dirk , Schneider Markus , Rasche Mareike
Published: March 2024

Abstract
Bone marrow fibrosis (BMF) is a rare complication in acute leukemia. In pediatrics, it predominantly occurs in acute megakaryoblastic leukemia (AMKL) and especially in patients with trisomy 21, called myeloid leukemia in Down syndrome (ML-DS). Defects in mesenchymal stromal cells (MSC) and cytokines specifically released by the myeloid blasts are thought to be the main drivers of fibrosis in the bone marrow niche (BMN). To model the BMN of pediatric patients with AMKL in mice, we first established MSCs from pediatric patients with AMKL (n = 5) and ML-DS (n = 9). Healthy donor control MSCs (n = 6) were generated from unaffected children and adolescents ≤18 years of age. Steady-state analyses of the MSCs revealed that patient-derived MSCs exhibited decreased adipogenic differentiation potential and enrichment of proliferation-associated genes. Importantly, TGFB1 exposure in vitro promoted early profibrotic changes in all three MSC entities. To study BMF induction for longer periods of time, we created an in vivo humanized artificial BMN subcutaneously in immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, using a mixture of MSCs, human umbilical vein endothelial cell, and Matrigel. Injection of AMKL blasts as producers of TGFB1 into this BMN after 8 weeks induced fibrosis grade I/II in a dose-dependent fashion over a time period of 4 weeks. Thus, our study developed a humanized mouse model that will be instrumental to specifically examine leukemogenesis and therapeutic targets for AMKL blasts in future.
Implications:
TGFB1 supports fibrosis induction in a pediatric AMKL model generated with patient-derived MSCs.
Visual Overview:
http://mcr.aacrjournals.org/content/molcanres/18/10/1603/F1.large.jpg.
Data from Amplification of TLK2 Induces Genomic Instability via Impairing the G2–M Checkpoint
Authors: Kim Jin-Ah , Anurag Meenakshi , Veeraraghavan Jamunarani , Schiff Rachel , Li Kaiyi , Wang Xiao-Song
Published: March 2024

Abstract
Managing aggressive breast cancers with enhanced chromosomal instability (CIN) is a significant challenge in clinics. Previously, we described that a cell cycle–associated kinase called Tousled-like kinase 2 (TLK2) is frequently deregulated by genomic amplifications in aggressive estrogen receptor–positive (ER+) breast cancers. In this study, it was discovered that TLK2 amplification and overexpression mechanistically impair Chk1/2-induced DNA damage checkpoint signaling, leading to a G2–M checkpoint defect, delayed DNA repair process, and increased CIN. In addition, TLK2 overexpression modestly sensitizes breast cancer cells to DNA-damaging agents, such as irradiation or doxorubicin. To our knowledge, this is the first report linking TLK2 function to CIN, in contrast to the function of its paralog TLK1 as a guardian of genome stability. This finding yields new insight into the deregulated DNA damage pathway and increased genomic instability in aggressive ER+ breast cancers.

Implications: Targeting TLK2 presents an attractive therapeutic strategy for the TLK2-amplified breast cancers that possess enhanced genomic instability and aggressiveness. Mol Cancer Res; 14(10); 920–7. ©2016 AACR.
Data from Evolution of Dermatofibrosarcoma Protuberans to DFSP-Derived Fibrosarcoma: An Event Marked by Epithelial–Mesenchymal Transition–like Process and 22q Loss
Authors: Stacchiotti Silvia , Astolfi Annalisa , Gronchi Alessandro , Fontana Andrea , Pantaleo Maria A. , Negri Tiziana , Brenca Monica , Tazzari Marcella , Urbini Milena , Indio Valentina , Colombo Chiara , Radaelli Stefano , Brich Silvia , Dei Tos Angelo P. , Casali Paolo G. , Castelli Chiara , Dagrada Gian Paolo , Pilotti Silvana , Maestro Roberta
Published: March 2024

Abstract
Dermatofibrosarcoma protuberans (DFSP) is a rare and indolent cutaneous sarcoma. At times, a fibrosarcomatous transformation marked by a more aggressive clinical behavior may be present. We investigated the natural history and the molecular bases of progression from classic DFSP to the fibrosarcomatous form (FS-DFSP), looking, retrospectively, at the outcome of all patients affected by primary DFSP treated at our institution from 1993 to 2012 and analyzing the molecular profile of 5 DFSPs and 5 FS-DFSPs by an integrated genomics approach (whole transcriptome sequencing, copy number analysis, FISH, qRT-PCR, IHC). The presence of fibrosarcomatous features was identified in 20 (7.6%) patients out of 263 DFSP. All cases were treated with macroscopic complete surgery. A local relapse occurred in 4 of 23 patients who received a microscopic marginal surgery (2 classic DFSP, 2 FS-DFSP), while metastasis affected 2 patients, both FS-DFSP (10% of FS-DFSP), being the first event. DFSP evolution to FS-DFSP was paralleled by a transcriptional reprogramming. The recurrent loss of chromosome 22q appeared to contribute to this phenomenon by promoting the expression of epigenetic regulators, such as EZH2. Loss of the p16/CDKN2A/INK4A locus at 9p was also observed in two FS-DFSP metastatic cases.

Implications: FS-DFSP is a rare subgroup among DFSP, with a 10% metastatic risk, that was independent from local recurrence and that was not observed in DFSP, that were all cured by wide surgery. Chromosome 22q deletion might play a role in FS-DFSP, and p16 loss may convey a poor outcome. EZH2 dysregulation was also found and represents a druggable target. Mol Cancer Res; 14(9); 820–9. ©2016 AACR.
Data from Hepatitis B Virus X Protein Induces RHAMM-Dependent Motility in Hepatocellular Carcinoma Cells via PI3K–Akt–Oct-1 Signaling
Authors: Liu Yu-Chin , Lu Li-Feng , Li Chia-Jung , Sun Nian-Kang , Guo Jing-You , Huang Ya-Hui , Yeh Chau-Ting , Chao Chuck C.-K.
Published: March 2024

Abstract
Chronic hepatitis B virus (HBV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC), which represents one of the most common cancers worldwide. Recent studies suggest that HBV's protein X (HBx) plays a crucial role in HCC development and progression. Earlier, genome-wide analysis identified that the receptor for hyaluronan-mediated motility (RHAMM) represents a putative oncogene and is overexpressed in many human cancers, including HCC. However, the mechanism underlying RHAMM upregulation and its role in tumorigenesis remain unclear. Here, we show that ectopic expression of HBx activates the PI3K/Akt/Oct-1 pathway and upregulates RHAMM expression in HCC cells. HBx overexpression leads to dissociation of C/EBPβ from the RHAMM gene promoter, thereby inducing RHAMM upregulation. RHAMM knockdown attenuates HBx-induced cell migration and invasion in vitro. In mice, HBx promotes cancer cell colonization via RHAMM upregulation, resulting in enhanced metastasis. Analysis of gene expression datasets reveals that RHAMM mRNA level is upregulated in patients with HCC with poor prognosis.
Implications:
These results indicate that RHAMM expression is upregulated by HBx, a process that depends on the inhibition of C/EBPβ activity and activation of the PI3K/Akt/Oct-1 pathway. These results have several implications for the treatment of HBV-positive HCC involving upregulation of RHAMM and cancer metastasis.
Visual Overview:
http://mcr.aacrjournals.org/content/molcanres/18/3/375/F1.large.jpg.
Data from Combination Lenalidomide/Bortezomib Treatment Synergistically Induces Calpain-Dependent Ikaros Cleavage and Apoptosis in Myeloma Cells
Authors: Ganesan Saravanan , Palani Hamenth Kumar , Balasundaram Nithya , David Sachin , Devasia Anup J. , George Biju , Mathews Vikram
Published: March 2024

Abstract
Multiple myeloma had been successfully treated by combining lenalidomide and bortezomib with reports suggesting benefits of such a combination even in relapsed/refractory cases. Recently, it was demonstrated that Ikaros degradation by lenalidomide happens via proteasome-dependent pathway and this process is critical for the eradication of myeloma cells. On the basis of this, an antagonistic effect should be observed if a combination of both these agents were used, which however is not the observation seen in the clinical setting. Our study demonstrates that when these agents are combined they exhibit a synergistic activity against myeloma cells and degradation of Ikaros happens by a proteasome-independent calcium-induced calpain pathway. Our study identifies the crucial role of calcium-induced calpain pathway in inducing apoptosis of myeloma cells when this combination or lenalidomide and bortezomib is used. We also report that this combination enhanced the expression of CD38 compared with lenalidomide alone. Thus, data from our study would establish the rationale for the addition of daratumumab along with this combination to further enhance therapeutic activity against multiple myeloma.
Implications:
Lenalidomide and bortezomib combination degrades IKZF1 in multiple myeloma through a calcium-dependent calpain and caspase pathway.
Visual Overview:
http://mcr.aacrjournals.org/content/molcanres/18/4/529/F1.large.jpg.
Data from A New Pipeline to Predict and Confirm Tumor Neoantigens Predict Better Response to Immune Checkpoint Blockade
Authors: Lazdun Yelena , Si Han , Creasy Todd , Ranade Koustubh , Higgs Brandon W. , Streicher Katie , Durham Nicholas M.
Published: March 2024

Abstract
Mutations that drive oncogenesis in cancer can generate neoantigens that may be recognized by the immune system. Identification of these neoantigens remains challenging due to the complexity of the MHC antigen and T-cell receptor interaction. Here, we describe the development of a systematic approach to efficiently identify and validate immunogenic neoantigens. Whole-exome sequencing of tissue from a patient with melanoma was used to identify nonsynonymous mutations, followed by MHC binding prediction and identification of tumor clonal architecture. The top 18 putative class I neoantigens were selected for immunogenicity testing via a novel in vitro pipeline in HLA-A201 healthy donor blood. Naïve CD8 T cells from donors were stimulated with allogeneic dendritic cells pulsed with peptide pools and then with individual peptides. The presence of antigen-specific T cells was determined via functional assays. We identified one putative neoantigen that expanded T cells specific to the mutant form of the peptide and validated this pipeline in a subset of patients with bladder tumors treated with durvalumab (n = 5). Within this cohort, the top predicted neoantigens from all patients were immunogenic in vitro. Finally, we looked at overall survival in the whole durvalumab-treated bladder cohort (N = 37) by stratifying patients by tertile measure of tumor mutation burden (TMB) or neoantigen load. Patients with higher neoantigen and TMB load tended to show better overall survival.
Implications:
This pipeline can enable accurate and rapid identification of personalized neoantigens that may help to identify patients who will survive longer on durvalumab.
Data from Decreased eIF3e Expression Can Mediate Epithelial-to-Mesenchymal Transition through Activation of the TGFβ Signaling Pathway
Authors: Desnoyers Guillaume , Frost Laura D. , Courteau Lynn , Wall Michael L. , Lewis Stephen M.
Published: March 2024

Abstract
The eIF3e protein is a component of the multisubunit eIF3 complex, which is essential for cap-dependent translation initiation. Decreased eIF3e expression is often observed in breast and lung cancer and has been shown to induce epithelial-to-mesenchymal transition (EMT) in breast epithelial cells by an unknown mechanism. Here, we study the effect of decreased eIF3e expression in lung epithelial cells by creating stable clones of lung epithelial cells (A549) that express an eIF3e-targeting shRNA. Our data indicate that decreased eIF3e expression in lung epithelial cells leads to EMT, as it does in breast epithelial cells. Importantly, we show that decreased eIF3e expression in both lung and breast epithelial cells leads to the overproduction of the TGFβ cytokine and that inhibition of TGFβ signaling can reverse eIF3e-regulated EMT in lung epithelial cells. In addition, we discovered that several mRNAs that encode important EMT regulators are translated by a cap-independent mechanism when eIF3e levels are reduced. These findings indicate that EMT mediated by a decrease in eIF3e expression may be a general phenomenon in epithelial cells and that it requires activation and maintenance of the TGFβ signaling pathway.

Implications: These results indicate that inhibition of TGFβ signaling could be an efficient way to prevent metastasis in patients with NSCLC that display reduced eIF3e expression. Mol Cancer Res; 13(10); 1421–30. ©2015 AACR.
Data from Stromal-MDM2 Promotes Lung Cancer Cell Invasion through Tumor–Host Feedback Signaling
Authors: Kamer Iris , Daniel-Meshulam Inbal , Zadok Oranit , Bab-Dinitz Elizabeta , Perry Gili , Feniger-Barish Rotem , Perelman Marina , Barshack Iris , Ben-Nun Alon , Onn Amir , Bar Jair
Published: March 2024

Abstract
Tumor–host interactions play a major role in malignancies' initiation and progression. We have reported in the past that tumor cells attenuate genotoxic stress–induced p53 activation in neighboring stromal cells. Herein, we aim to further elucidate cancer cells' impact on signaling within lung cancer stroma. Primary cancer-associated fibroblasts were grown from resected human lung tumors. Lung cancer lines as well as fresh cultures of resected human lung cancers were used to produce conditioned medium (CM) or cocultured with stromal cells. Invasiveness of cancer cells was evaluated by transwell assays, and in vivo tumor growth was tested in Athymic nude mice. We found CM of a large variety of cancer cell lines as well as ex vivo–cultured lung cancers to rapidly induce protein levels of stromal-MDM2. CM of nontransformed cells had no such effect. Mdm2 induction occurred through enhanced translation, was mTORC1-dependent, and correlated with activation of AKT and p70 S6 Kinase. AKT or MDM2 knockdown in fibroblasts reduced the invasion of neighboring cancer cells, independently of stromal-p53. MDM2 overexpression in fibroblasts enhanced cancer cells' invasion and growth of inoculated tumors in mice. Our results indicate that stromal-MDM2 participates in a p53-independent cancer–host feedback mechanism. Soluble cancer-originated signals induce enhanced translation of stromal-MDM2 through AKT/mTORC1 signaling, which in turn enhances the neighboring cancer cells' invasion ability. The role of these tumor–host interactions needs to be further explored.
Implications:
We uncovered a novel tumor–stroma signaling loop, which is a potentially new therapeutic target in lung cancer and possibly in additional types of cancer.
Data from Systematic Screening of Promoter Regions Pinpoints Functional Cis-Regulatory Mutations in a Cutaneous Melanoma Genome
Authors: Poulos Rebecca C. , Thoms Julie A.I. , Shah Anushi , Beck Dominik , Pimanda John E. , Wong Jason W.H.
Published: March 2024

Abstract
With the recent discovery of recurrent mutations in the TERT promoter in melanoma, identification of other somatic causal promoter mutations is of considerable interest. Yet, the impact of sequence variation on the regulatory potential of gene promoters has not been systematically evaluated. This study assesses the impact of promoter mutations on promoter activity in the whole-genome sequenced malignant melanoma cell line COLO-829. Combining somatic mutation calls from COLO-829 with genome-wide chromatin accessibility and histone modification data revealed mutations within promoter elements. Interestingly, a high number of potential promoter mutations (n = 23) were found, a result mirrored in subsequent analysis of TCGA whole-melanoma genomes. The impact of wild-type and mutant promoter sequences were evaluated by subcloning into luciferase reporter vectors and testing their transcriptional activity in COLO-829 cells. Of the 23 promoter regions tested, four mutations significantly altered reporter activity relative to wild-type sequences. These data were then subjected to multiple computational algorithms that score the cis-regulatory altering potential of mutations. These analyses identified one mutation, located within the promoter region of NDUFB9, which encodes the mitochondrial NADH dehydrogenase (ubiquinone) 1 beta subcomplex 9, to be recurrent in 4.4% (19 of 432) of TCGA whole-melanoma exomes. The mutation is predicted to disrupt a highly conserved SP1/KLF transcription factor binding motif and its frequent co-occurrence with mutations in the coding sequence of NF1 supports a pathologic role for this mutation in melanoma. Taken together, these data show the relatively high prevalence of promoter mutations in the COLO-829 melanoma genome, and indicate that a proportion of these significantly alter the regulatory potential of gene promoters.

Implications: Genomic-based screening within gene promoter regions suggests that functional cis-regulatory mutations may be common in melanoma genomes, highlighting the need to examine their role in tumorigenesis. Mol Cancer Res; 13(8); 1218–26. ©2015 AACR.
Data from Autophagy Supports Breast Cancer Stem Cell Maintenance by Regulating IL6 Secretion
Authors: Maycotte Paola , Jones Kenneth L. , Goodall Megan L. , Thorburn Jacqueline , Thorburn Andrew
Published: March 2024

Abstract
Autophagy is a mechanism by which cells degrade cellular material to provide nutrients and energy for survival during stress. The autophagy is thought to be a critical process for cancer stem cell (CSC) or tumor-initiating cell maintenance but the mechanisms by which autophagy supports survival of CSCs remain poorly understood. In this study, inhibition of autophagy by knockdown of ATG7 or BECN1 modified the CD44+/CD24low/− population in breast cancer cells by regulating CD24 and IL6 secretion. In a breast cancer cell line that is independent of autophagy for survival, autophagy inhibition increased IL6 secretion to the media. On the other hand, in an autophagy-dependent cell line, autophagy inhibition decreased IL6 secretion, cell survival, and mammosphere formation. In these cells, IL6 treatment or conditioned media from autophagy-competent cells rescued the deficiency in mammosphere formation induced by autophagy inhibition. These results reveal that autophagy regulates breast CSC maintenance in autophagy-dependent breast cancer cells by modulating IL6 secretion implicating autophagy as a potential therapeutic target in breast cancer.

Implications: Modulation of autophagy in breast cancer has different and even opposite effects, indicating the need for a selection strategy when trying to manipulate autophagy in the context of cancer therapy. Mol Cancer Res; 13(4); 651–8. ©2015 AACR.
Data from Convergent and Divergent Cellular Responses by ErbB4 Isoforms in Mammary Epithelial Cells
Authors: Wali Vikram B. , Haskins Jonathan W. , Gilmore-Hebert Maureen , Platt James T. , Liu Zongzhi , Stern David F.
Published: March 2024

Abstract
Associations of ErbB4 (ERBB4/HER4), the fourth member of the EGFR family, with cancer are variable, possibly as a result of structural diversity of this receptor. There are multiple structural isoforms of ERBB4 arising by alternative mRNA splicing, and a subset undergo proteolysis that releases membrane-anchored and soluble isoforms that associate with transcription factors and coregulators to modulate transcription. To compare the differential and common signaling activities of full-length (FL) and soluble intracellular isoforms of ERBB4, four JM-a isoforms (FL and soluble intracellular domain (ICD) CYT-1 and CYT-2) were expressed in isogenic MCF10A cells and their biologic activities were analyzed. Both FL and ICD CYT-2 promoted cell proliferation and invasion, and CYT-1 suppressed cell growth. Transcriptional profiling revealed several new and underexplored ERBB4-regulated transcripts, including: proteases/protease inhibitors (MMP3 and SERPINE2), the YAP/Hippo pathway (CTGF, CYR61, and SPARC), the mevalonate/cholesterol pathway (HMGCR, HMGCS1, LDLR, and DHCR7), and cytokines (IL8, CCL20, and CXCL1). Many of these transcripts were subsequently validated in a luminal breast cancer cell line that normally expresses ERBB4. Furthermore, ChIP-seq experiments identified ADAP1, APOE, SPARC, STMN1, and MXD1 as novel molecular targets of ERBB4. These findings clarify the diverse biologic activities of ERBB4 isoforms, and reveal new and divergent functions.

Implications: ErbB4 as a regulator of Hippo and mevalonate pathways provides new insight into milk production and anabolic processes in normal mammary epithelia and cancer. Mol Cancer Res; 12(8); 1140–55. ©2014 AACR.
Data from Prognostic Potential of DNA Methylation and Transcript Levels of HIF1A and EPAS1 in Colorectal Cancer
Authors: Rawłuszko-Wieczorek Agnieszka Anna , Horbacka Karolina , Krokowicz Piotr , Misztal Matthew , Jagodziński Paweł Piotr
Published: March 2024

Abstract
Hypoxic conditions during the formation of colorectal cancer may support the development of more aggressive tumors. Hypoxia-inducible factor (HIF) is a heterodimeric complex, composed of oxygen-induced HIFα and constitutively expressed HIFβ subunits, which mediates the primary transcriptional response to hypoxic stress. Among HIFα isoforms, HIF1α (HIF1A) and endothelial PAS domain–containing protein 1 (EPAS1) are able to robustly activate hypoxia-responsive gene signatures. Although posttranslational regulation of HIFα subunits is well described, less is known about their transcriptional regulation. Here, molecular analysis determined that EPAS1 mRNA was significantly reduced in primary colonic adenocarcinoma specimens compared with histopathologically nonneoplastic tissue from 120 patients. In contrast, no difference in HIF1A mRNA levels was observed between cancerous and noncancerous tissue. Bisulfite DNA sequencing and high-resolution melting analysis identified significant DNA hypermethylation in the EPAS1 regulatory region from cancerous tissue compared with nonneoplastic tissue. Importantly, multivariate Cox regression analysis revealed a high HR for patients with cancer with low EPAS1 transcript levels (HR, 4.91; 95% confidence interval, CI, 0.42–56.15; P = 0.047) and hypermethylated EPAS1 DNA (HR, 33.94; 95% CI, 2.84–405.95; P = 0.0054). Treatment with a DNA methyltransferase inhibitor, 5-Aza-2′-deoxycytidine (5-aza-dC/Decitabine), upregulated EPAS1 expression in hypoxic colorectal cancer cells that were associated with DNA demethylation of the EPAS1 regulatory region. In summary, EPAS1 is transcriptionally regulated by DNA methylation in colorectal cancer.

Implications: DNA methylation and mRNA status of EPAS1 have novel prognostic potential for colorectal cancer. Mol Cancer Res; 12(8); 1112–27. ©2014 AACR.
Data from A Macrophage-Dominant PI3K Isoform Controls Hypoxia-Induced HIF1α and HIF2α Stability and Tumor Growth, Angiogenesis, and Metastasis
Authors: Joshi Shweta , Singh Alok R. , Zulcic Muamera , Durden Donald L.
Published: March 2024

Abstract
Tumor growth, progression, and response to the hypoxic tumor microenvironment involve the action of hypoxia-inducible transcription factors, HIF1 and HIF2. HIF is a heterodimeric transcription factor containing an inducible HIFα subunit and a constitutively expressed HIFβ subunit. The signaling pathways operational in macrophages regulating hypoxia-induced HIFα stabilization remain the subject of intense investigation. Here, it was discovered that the PTEN/PI3K/AKT signaling axis controls hypoxia-induced HIF1α (HIF1A) and HIF2α (EPAS1) stability in macrophages. Using genetic mouse models and pan-PI3K as well as isoform-specific inhibitors, inhibition of the PI3K/AKT pathway blocked the accumulation of HIFα protein and its primary transcriptional target VEGF in response to hypoxia. Moreover, blocking the PI3K/AKT signaling axis promoted the hypoxic degradation of HIFα via the 26S proteasome. Mechanistically, a macrophage-dominant PI3K isoform (p110γ) directed tumor growth, angiogenesis, metastasis, and the HIFα/VEGF axis. Moreover, a pan-PI3K inhibitor (SF1126) blocked tumor-induced angiogenesis and inhibited VEGF and other proangiogenic factors secreted by macrophages. These data define a novel molecular mechanism by which PTEN/PI3K/AKT regulates the proteasome-dependent stability of HIFα under hypoxic conditions, a signaling pathway in macrophages that controls tumor-induced angiogenesis and metastasis.

Implications: This study indicates that PI3K inhibitors are excellent candidates for the treatment of cancers where macrophages promote tumor progression. Mol Cancer Res; 12(10); 1520–31. ©2014 AACR.
Data from Novel Bispecific Domain Antibody to LRP6 Inhibits Wnt and R-spondin Ligand-Induced Wnt Signaling and Tumor Growth
Authors: Jackson Heather , Granger David , Jones Gavin , Anderson Louisa , Friel Sarah , Rycroft Daniel , Fieles William , Tunstead James , Steward Michael , Wattam Trevor , Walker Adam , Griggs Jeremy , Al-Hajj Muhammad , Shelton Christopher
Published: March 2024

Abstract
Aberrant WNT signaling is associated with the formation and growth of numerous human cancer types. The low-density lipoprotein receptor-related protein 6 (LRP6) is the least redundant component of the WNT receptor complex with two independent WNT ligand-binding sites. Using domain antibody (dAb) technology, a bispecific antibody (GSK3178022) to LRP6 was identified that is capable of blocking stimulation in the presence of a range of WNT and R-spondin (RSPO) ligands in vitro. GSK3178022 was also efficacious in reducing WNT target gene expression in vivo, in both cancer cell line and patient-derived xenograft models, and delays tumor growth in a patient-derived RSPO fusion model of colorectal cancer.

Implications: This article demonstrates the inhibition of a key oncogenic receptor, intractable to mAb inhibition due to multiple independent ligand interaction sites, using an innovative dAb approach. Mol Cancer Res; 14(9); 859–68. ©2016 AACR.
Data from EMT Transition Alters Interstitial Fluid Flow–Induced Signaling in ERBB2-Positive Breast Cancer Cells
Authors: Tchafa Alimatou M. , Ta Mi , Reginato Mauricio J. , Shieh Adrian C.
Published: March 2024

Abstract
A variety of biophysical forces are altered in the tumor microenvironment (TME) and these forces can influence cancer progression. One such force is interstitial fluid flow (IFF)—the movement of fluid through the tissue matrix. IFF was previously shown to induce invasion of cancer cells, but the activated signaling cascades remain poorly understood. Here, it is demonstrated that IFF induces invasion of ERBB2/HER2-expressing breast cancer cells via activation of phosphoinositide-3-kinase (PI3K). In constitutively activate ERBB2-expressing cells that have undergone epithelial-to-mesenchymal transition (EMT), IFF-mediated invasion requires the chemokine receptor CXCR4, a gradient of its ligand CXCL12, and activity of the PI3K catalytic subunits p110α and β. In wild-type ERBB2-expressing cells, IFF-mediated invasion is chemokine receptor–independent and requires only p110α activation. To test whether cells undergoing EMT alter their signaling response to IFF, TGFβ1 was used to induce EMT in wild-type ERBB2-expressing cells, resulting in IFF-induced invasion dependent on CXCR4 and p110β.

Implications: This study identifies a novel signaling mechanism for interstitial flow–induced invasion of ERBB2-expressing breast cancer cells, one that depends on EMT and acts through a CXCR4–PI3K pathway. These findings suggest that the response of cancer cells to interstitial flow depends on EMT status and malignancy. Mol Cancer Res; 13(4); 755–64. ©2015 AACR.
Data from c-MYC Generates Repair Errors via Increased Transcription of Alternative-NHEJ Factors, LIG3 and PARP1, in Tyrosine Kinase–Activated Leukemias
Authors: Muvarak Nidal , Kelley Shannon , Robert Carine , Baer Maria R. , Perrotti Danilo , Gambacorti-Passerini Carlo , Civin Curt , Scheibner Kara , Rassool Feyruz V.
Published: March 2024

Abstract
Leukemias expressing the constitutively activated tyrosine kinases (TK) BCR-ABL1 and FLT3/ITD activate signaling pathways that increase genomic instability through generation of reactive oxygen species (ROS), DNA double-strand breaks (DSB), and error-prone repair. The nonhomologous end-joining (NHEJ) pathway is a major pathway for DSB repair and is highly aberrant in TK-activated leukemias; an alternative form of NHEJ (ALT-NHEJ) predominates, evidenced by increased expression of DNA ligase IIIα (LIG3) and PARP1, increased frequency of large genomic deletions, and repair using DNA sequence microhomologies. This study, for the first time, demonstrates that the TK target c-MYC plays a role in transcriptional activation and subsequent expression of LIG3 and PARP1 and contributes to the increased error-prone repair observed in TK-activated leukemias. c-MYC negatively regulates microRNAs miR-150 and miR-22, which demonstrate an inverse correlation with LIG3 and PARP1 expression in primary and cultured leukemia cells and chronic myelogenous leukemia human patient samples. Notably, inhibition of c-MYC and overexpression of miR-150 and -22 decreases ALT-NHEJ activity. Thus, BCR-ABL1 or FLT3/ITD induces c-MYC expression, leading to genomic instability via augmented expression of ALT-NHEJ repair factors that generate repair errors.

Implications: In the context of TK-activated leukemias, c-MYC contributes to aberrant DNA repair through downstream targets LIG3 and PARP1, which represent viable and attractive therapeutic targets. Mol Cancer Res; 13(4); 699–712. ©2015 AACR.
Data from BET Bromodomain Inhibitors Enhance Efficacy and Disrupt Resistance to AR Antagonists in the Treatment of Prostate Cancer
Authors: Asangani Irfan A. , Wilder-Romans Kari , Dommeti Vijaya L. , Krishnamurthy Pranathi M. , Apel Ingrid J. , Escara-Wilke June , Plymate Stephen R. , Navone Nora M. , Wang Shaomeng , Feng Felix Y. , Chinnaiyan Arul M.
Published: March 2024

Abstract
Next-generation antiandrogen therapies, such as enzalutamide and abiraterone, have had a profound impact on the management of metastatic castration-resistant prostate cancer (mCRPC). However, mCRPC patients invariably develop resistance to these agents. Here, a series of clonal cell lines were developed from enzalutamide-resistant prostate tumor xenografts to study the molecular mechanism of resistance and test their oncogenic potential under various treatment conditions. Androgen receptor (AR) signaling was maintained in these cell lines, which acquired potential resistance mechanisms, including expression of AR-variant 7 (AR-v7) and glucocorticoid receptor. BET bromodomain inhibitors were shown previously to attenuate AR signaling in mCRPC; here, we demonstrate the efficacy of bromodomain and extraterminal (BET) inhibitors in enzalutamide-resistant prostate cancer models. AR antagonists, enzalutamide, and ARN509 exhibit enhanced prostate tumor growth inhibition when combined with BET inhibitors, JQ1 and OTX015, respectively. Taken together, these data provide a compelling preclinical rationale to combine BET inhibitors with AR antagonists to subvert resistance mechanisms.

Implications: Therapeutic combinations of BET inhibitors and AR antagonists may enhance the clinical efficacy in the treatment of mCRPC.

Visual Overview: http://mcr.aacrjournals.org/content/14/4/324/F1.large.jpg. Mol Cancer Res; 14(4); 324–31. ©2016 AACR.
Data from AMPK-Independent LKB1 Activity Is Required for Efficient Epithelial Ovarian Cancer Metastasis
Authors: Buensuceso Adrian , Ramos-Valdes Yudith , DiMattia Gabriel E. , Shepherd Trevor G.
Published: March 2024

Abstract
Epithelial ovarian cancer (EOC) spreads by direct dissemination of malignant cells and multicellular clusters, known as spheroids, into the peritoneum followed by implantation and growth on abdominal surfaces. Using a spheroid model system of EOC metastasis, we discovered that Liver kinase B1 (LKB1), encoded by the STK11 gene, and its canonical substrate AMP-activated protein kinase (AMPK) are activated in EOC spheroids, yet only LKB1 is required for cell survival. We have now generated STK11-knockout cell lines using normal human FT190 cells and three EOC cell lines, OVCAR8, HeyA8, and iOvCa147. STK11KO did not affect growth and viability in adherent culture, but it decreased anchorage-independent growth of EOC cells. EOC spheroids lacking LKB1 had markedly impaired growth and viability, whereas there was no difference in normal FT190 spheroids. To test whether LKB1 loss affects EOC metastasis, we performed intraperitoneal injections of OVCAR8-, HeyA8-, and iOvCa147-STK11KO cells, and respective controls. LKB1 loss exhibited a dramatic reduction on tumor burden and metastatic potential; in particular, OVCAR8-STK11KO tumors had evidence of extensive necrosis, apoptosis, and hypoxia. Interestingly, LKB1 loss did not affect AMPKα phosphorylation in EOC spheroids and tumor xenografts, indicating that LKB1 signaling to support EOC cell survival in spheroids and metastatic tumor growth occurs via other downstream mediators. We identified the dual-specificity phosphatase DUSP4 as a commonly upregulated protein due to LKB1 loss; indeed, DUSP4 knockdown in HeyA8-STK11KO cells partially restored spheroid formation and viability.
Implications:
LKB1 possesses key tumor-promoting activity independent of downstream AMPK signaling during EOC metastasis.