bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Covalent JNK Inhibitor, JNK-IN-8, Suppresses Tumor Growth in Triple-Negative Breast Cancer by Activating TFEB- and TFE3-Mediated Lysosome Biogenesis and Autophagy
Authors: Soleimani Milad , Somma Alexander , Kaoud Tamer , Goyal Ria , Bustamante Jorge , Wylie Dennis C. , Holay Nisha , Looney Agnieszka , Giri Uma , Triplett Todd , Dalby Kevin , Kowalski Jeanne , Eckhardt S. Gail , Van Den Berg Carla
Published: March 2024

Abstract
The heterogeneity and aggressiveness of triple-negative breast cancer (TNBC) contribute to its early recurrence and metastasis. Despite substantial research to identify effective therapeutic targets, TNBC remains elusive in terms of improving patient outcomes. Here, we report that a covalent JNK inhibitor, JNK-IN-8, suppresses TNBC growth both in vitro and in vivo. JNK-IN-8 reduced colony formation, cell viability, and organoid growth in vitro and slowed patient-derived xenograft and syngeneic tumor growth in vivo. Cells treated with JNK-IN-8 exhibited large, cytoplasmic vacuoles with lysosomal markers. To examine the molecular mechanism of this phenotype, we looked at the master regulators of lysosome biogenesis and autophagy transcription factor EB (TFEB) and TFE3. JNK-IN-8 inhibited TFEB phosphorylation and induced nuclear translocation of unphosphorylated TFEB and TFE3. This was accompanied by an upregulation of TFEB/TFE3 target genes associated with lysosome biogenesis and autophagy. Depletion of both TFEB and TFE3 diminished the JNK-IN-8–driven upregulation of lysosome biogenesis and/or autophagy markers. TFEB and TFE3 are phosphorylated by a number of kinases, including mTOR. JNK-IN-8 reduced phosphorylation of mTOR targets in a concentration-dependent manner. Knockout of JNK1 and/or JNK2 had no impact on TFEB/TFE3 activation or mTOR inhibition by JNK-IN-8 but inhibited colony formation. Similarly, reexpression of either wildtype or drug-nonbinding JNK (C116S) in JNK knockout cells did not reverse JNK-IN-8–induced TFEB dephosphorylation. In summary, JNK-IN-8 induced lysosome biogenesis and autophagy by activating TFEB/TFE3 via mTOR inhibition independently of JNK. Together, these findings demonstrate the efficacy of JNK-IN-8 as a targeted therapy for TNBC and reveal its novel lysosome- and autophagy-mediated mechanism of action.
Data from Hypoxia-Independent Gene Expression Mediated by SOX9 Promotes Aggressive Pancreatic Tumor Biology
Authors: Camaj Peter , Jäckel Carsten , Krebs Stefan , DeToni Enrico N. , Blum Helmut , Jauch Karl-Walter , Nelson Peter J. , Bruns Christiane J.
Published: March 2024

Abstract
Pancreatic cancer aggressiveness is characterized by its high capacity for local invasion, ability to promote angiogenesis, and potential to metastasize. Hypoxia is known to represent a crucial step in the development of aggressive malignant features of many human cancers. However, micrometastatic tumors are not typically subjected to hypoxic events during early stages of dissemination; therefore, it is unclear how these tumors are able to maintain their aggressive phenotype. Thus, the identification of regulators of hypoxia-related genes in aggressive/metastatic tumors represents a fundamental step for the design of future therapies to treat pancreatic cancer. To this end, transcriptomic profiles were compared between the nonmetastatic pancreatic cancer cell line FG (LMET) and its angiogenic/metastatic derivate L3.6pl (HMET) under normoxic or hypoxic conditions. Cluster analysis revealed a number of transcripts that were induced by hypoxia in nonmetastatic cancer cells. Strikingly, this cluster was determined to be constitutively activated under normoxia in the metastatic cancer cells and could not be further induced by hypoxia. A subset of these transcripts were regulated by the transcription factor SOX9 in the aggressive-metastatic cells, but driven by hypoxia-inducible factor-1α (HIF-1α) in the parental nonmetastatic cell line. Moreover, these transcripts were enriched in cancer-related networks including: WNT, CXCR4, retinoic acid, and (FAK) focal adhesion kinase, gene PTK2 signaling pathways. In functional assays, inhibition of SOX9 expression in HMET cells led to increased apoptosis and reduced migration in vitro and a significant reduction in primary tumor growth, angiogenesis, and metastasis following orthotopic tumor cell injection. At the molecular level, the control of SOX9 expression was associated with changes in the methylation status of the SOX9 promoter. Finally, SOX9 upregulation was verified in a series of tumor specimens of patients with pancreatic carcinoma.

Implications: SOX9 represents a novel target for pancreatic cancer therapy. Mol Cancer Res; 12(3); 421–32. ©2013 AACR.
Data from Breast Cancer Metastasis Suppressor-1 Promoter Methylation in Primary Breast Tumors and Corresponding Circulating Tumor Cells
Authors: Chimonidou Maria , Kallergi Galatea , Georgoulias Vassilis , Welch Danny R. , Lianidou Evi S.
Published: March 2024

Abstract
Breast cancer metastasis suppressor-1 (BRMS1) differentially regulates the expression of multiple genes, leading to metastasis suppression without affecting orthotopic tumor growth. For the first time, BRMS1 promoter methylation was evaluated as a prognostic biomarker in primary breast tumors and a subset of corresponding circulating tumor cells (CTC). Formalin-fixed paraffin embedded samples were analyzed for BRMS1 methylation status using methylation-specific PCR in a human specimen cohort consisting of noncancerous tissues, benign fibroadenomas, and primary breast tumors, including some with adjacent noncancerous tissues. Peripheral blood mononuclear cells from a large subset of these patients were fixed in cytospins and analyzed. In addition, BRMS1 expression in cytospins was examined by double-immunofluorescence using anti-BRMS1 and pan-cytokeratin antibodies. BRMS1 promoter methylation was not detected in noncancerous breast tissues or benign fibroadenomas; however, methylation was observed in more than a third of primary breast tumors. Critically, BRMS1 promoter methylation in primary tumors was significantly associated with reduced disease-free survival with a trend toward reduced overall survival. Similarly, a third of cytospin samples were positive for the presence of CTCs, and the total number of detected CTCs was 41. Although a large fraction of CTCs were negative or maintained low expression of BRMS1, promoter methylation was observed in a small fraction of samples, implying that BRMS1 expression in CTCs was either downregulated or heterogeneous. In summary, these data define BRMS1 promoter methylation in primary breast tumors and associated CTCs.

Implications: This study indicates that BRMS1 promoter methylation status has biomarker potential in breast cancer. Mol Cancer Res; 11(10); 1248–57. ©2013 AACR.
Data from Energy Management by Enhanced Glycolysis in G1-phase in Human Colon Cancer Cells In Vitro and In Vivo
Authors: Bao Yan , Mukai Kuniaki , Hishiki Takako , Kubo Akiko , Ohmura Mitsuyo , Sugiura Yuki , Matsuura Tomomi , Nagahata Yoshiko , Hayakawa Noriyo , Yamamoto Takehiro , Fukuda Ryo , Saya Hideyuki , Suematsu Makoto , Minamishima Yoji Andrew
Published: March 2024

Abstract
Activation of aerobic glycolysis in cancer cells is well known as the Warburg effect, although its relation to cell- cycle progression remains unknown. In this study, human colon cancer cells were labeled with a cell-cycle phase-dependent fluorescent marker Fucci to distinguish cells in G1-phase and those in S + G2/M phases. Fucci-labeled cells served as splenic xenograft transplants in super-immunodeficient NOG mice and exhibited multiple metastases in the livers, frozen sections of which were analyzed by semiquantitative microscopic imaging mass spectrometry. Results showed that cells in G1-phase exhibited higher concentrations of ATP, NADH, and UDP-N-acetylglucosamine than those in S and G2–M phases, suggesting accelerated glycolysis in G1-phase cells in vivo. Quantitative determination of metabolites in cells synchronized in S, G2–M, and G1 phases suggested that efflux of lactate was elevated significantly in G1-phase. By contrast, ATP production in G2–M was highly dependent on mitochondrial respiration, whereas cells in S-phase mostly exhibited an intermediary energy metabolism between G1 and G2–M phases. Isogenic cells carrying a p53-null mutation appeared more active in glycolysis throughout the cell cycle than wild-type cells. Thus, as the cell cycle progressed from G2–M to G1 phases, the dependency of energy production on glycolysis was increased while the mitochondrial energy production was reciprocally decreased.

Implications: These results shed light on distinct features of the phase-specific phenotypes of metabolic systems in cancer cells. Mol Cancer Res; 11(9); 973–85. ©2013 AACR.
Data from Fer Protein-Tyrosine Kinase Promotes Lung Adenocarcinoma Cell Invasion and Tumor Metastasis
Authors: Ahn Joseph , Truesdell Peter , Meens Jalna , Kadish Carli , Yang Xiaolong , Boag Alexander H. , Craig Andrew W.B.
Published: March 2024

Abstract
Epidermal growth factor receptor (EGFR) is frequently amplified or mutated in non–small cell lung cancer (NSCLC). Although Fer protein-tyrosine kinase signals downstream of EGFR, its role in NSCLC tumor progression has not been reported. Here, Fer kinase was elevated in NSCLC tumors compared to normal lung epithelium. EGFR signaling in NSCLC cells fosters rapid Fer activation and increased localization to lamellipodia. Stable silencing of Fer in H1299 lung adenocarcinoma cells (Fer KD) caused impaired EGFR-induced lamellipodia formation compared to control cells. Fer KD NSCLC cells showed reduced Vav2 tyrosine phosphorylation that was correlated with direct Fer-mediated phosphorylation of Vav2 on tyrosine-172, which was previously reported to increase the guanine nucleotide exchange factor activity of Vav2. Indeed, Fer KD cells displayed defects in Rac-GTP localization to lamellipodia, cell migration, and cell invasion in vitro. To test the role of Fer in NSCLC progression and metastasis, control and Fer KD cells were grown as subcutaneous tumors in mice. Although Fer was not required for tumor growth, Fer KD tumor-bearing mice had significantly fewer numbers of spontaneous metastases. Combined, these data demonstrate that Fer kinase is elevated in NSCLC tumors and is important for cellular invasion and metastasis.

Implications: Fer protein-tyrosine kinase is a potential therapeutic target in metastatic lung cancer. Mol Cancer Res; 11(8); 952–63. ©2013 AACR.
Data from ARF Regulates the Stability of p16 Protein Via REGγ-Dependent Proteasome Degradation
Authors: Kobayashi Takashi , Wang Jingqiang , Al-Ahmadie Hikmat , Abate-Shen Cory
Published: March 2024

Abstract
The cell-cycle regulatory gene INK4A-ARF (CDKN2A) has two alternative transcripts that produce entirely different proteins, namely p14ARF and p16, which have complementary functions as regulators of p53 and pRB tumor suppressor pathways, respectively. The unusual organization of INK4A-ARF has long led to speculation of a need for coordinated regulation of p14ARF and p16. We now show that p14ARF (ARF) regulates the stability of p16 protein in human cancer cell lines, as well as in mouse embryonic fibroblasts (MEFs). In particular, ARF promotes rapid degradation of p16 protein, which is mediated by the proteasome and, more specifically, by interaction of ARF with one of its subunits, REGγ. Furthermore, this ARF-dependent destabilization of p16 can be abrogated by knockdown of REGγ or by pharmacologic blockade of its nuclear export. Thus, our findings have uncovered a novel crosstalk of 2 key tumor suppressors mediated by a REGγ-dependent mechanism. The ability of ARF to control p16 stability may influence cell-cycle function.

Implications: The ability of ARF to control p16 stability may influence cell cycle function.

Visual Overview: http://mcr.aacrjournals.org/content/11/8/828/F1.large.jpg. Mol Cancer Res; 11(8); 828–33. ©2013 AACR.
Data from Interleukin-6 and Oncostatin-M Synergize with the PI3K/AKT Pathway to Promote Aggressive Prostate Malignancy in Mouse and Human Tissues
Authors: Smith Daniel A. , Kiba Atsushi , Zong Yang , Witte Owen N.
Published: March 2024

Abstract
Chronic inflammation has been proposed as an etiological and progression factor in prostate cancer. In this study, we used a dissociated prostate tissue recombination system to interrogate the role of interleukin 6 (IL6) and the related cytokine oncostatin-M (OSM) in the initiation and progression of prostate cancer. We identified that prostatic intraepithelial neoplasia (PIN) lesions induced by PTEN loss of function (PTENLOF) progress to invasive adenocarcinoma following paracrine expression of either cytokine. Increased expression of OSM was also able to drive progression of benign human epithelium when combined with constitutively activated AKT. Malignant progression in the mouse was associated with invasion into the surrounding mesenchyme and increased activation of STAT3 in PTENLOF grafts expressing IL6 or OSM. Collectively, our work indicates that pro-inflammatory cytokines such as IL6 or OSM could activate pathways associated with prostate cancer progression and synergize with cell-autonomous oncogenic events to promote aggressive malignancy.

Implications: Increased expression of IL6 or OSM synergizes with loss of PTEN to promote invasive prostate cancer.

Visual Overview: http://mcr.aacrjournals.org/content/early/2013/09/02/1541-7786.MCR-13-0238/F1.large.jpg. Mol Cancer Res; 11(10); 1159–65. ©2013 AACR.
Data from AKR1B10, a Transcriptional Target of p53, Is Downregulated in Colorectal Cancers Associated with Poor Prognosis
Authors: Ohashi Tomoko , Idogawa Masashi , Sasaki Yasushi , Suzuki Hiromu , Tokino Takashi
Published: March 2024

Abstract
p53 is one of the most important tumor suppressor genes, and it is frequently inactivated in various cancers. p53 modulates various cellular functions, such as apoptosis and cell-cycle arrest via transcriptional regulation. Recently, p53 has been reported to be involved in a wide range of cellular metabolic pathways, including glycolysis, oxidative phosphorylation, glutaminolysis, and the antioxidant response. To understand the functional mechanism of p53, it is important to find out the direct transcriptional targets of p53. In this study, aldo-keto reductase family 1, member B10 (AKR1B10) was identified as a direct target of the p53 family by cDNA microarray analysis after comparing the mRNA expression of control and H1299 cells that overexpressed with p53 family members. In addition, we found that the expression of AKR1B10 was significantly decreased in colorectal cancers and adenomas as compared with normal colon tissues. Knockdown of AKR1B10 significantly inhibited p53-induced apoptosis in colorectal cancer cells, whereas the overexpression of AKR1B10 enhanced p53-induced apoptosis and inhibited tumor proliferation in vivo. Furthermore, low expression of AKR1B10 in colon cancer patients was correlated with decreased survival and poor prognosis. These results suggest that decreased expression of AKR1B10 could disrupt the tumor suppressive function of p53, which result in decreased survival in colorectal cancer patients. In summary, AKR1B10 may be a novel prognostic predictor and a novel therapeutic target for colorectal cancer.

Implications: AKR1B10, a transcriptional target of p53, is also a novel prognostic and therapeutic molecule in colorectal cancer. Mol Cancer Res; 11(12); 1554–63. ©2013 AACR.
Data from FGFR3 Translocations in Bladder Cancer: Differential Sensitivity to HSP90 Inhibition Based on Drug Metabolism
Authors: Acquaviva Jaime , He Suqin , Zhang Chaohua , Jimenez John-Paul , Nagai Masazumi , Sang Jim , Sequeira Manuel , Smith Donald L. , Ogawa Luisa Shin , Inoue Takayo , Tatsuta Noriaki , Knowles Margaret A. , Bates Richard C. , Proia David A.
Published: March 2024

Abstract
Activating mutations and/or overexpression of FGFR3 are common in bladder cancer, making FGFR3 an attractive therapeutic target in this disease. In addition, FGFR3 gene rearrangements have recently been described that define a unique subset of bladder tumors. Here, a selective HSP90 inhibitor, ganetespib, induced loss of FGFR3-TACC3 fusion protein expression and depletion of multiple oncogenic signaling proteins in RT112 bladder cells, resulting in potent cytotoxicity comparable with the pan-FGFR tyrosine kinase inhibitor BGJ398. However, in contrast to BGJ398, ganetespib exerted pleiotropic effects on additional mitogenic and survival pathways and could overcome the FGFR inhibitor–resistant phenotype of FGFR3 mutant–expressing 97-7 and MHG-U3 cells. Combinatorial benefit was observed when ganetespib was used with BGJ398 both in vitro and in vivo. Interestingly, two additional FGFR3 fusion-positive lines (RT4 and SW480) retained sensitivity to HSP90 inhibitor treatment by the ansamycins 17-AAG and 17-DMAG yet displayed intrinsic resistance to ganetespib or AUY922, both second-generation resorcinol-based compounds. Both cell lines, compared with RT112, expressed considerably higher levels of endogenous UGT1A enzyme; this phenotype resulted in a rapid glucuronidation-dependent metabolism and subsequent efflux of ganetespib from SW780 cells, thus providing a mechanism to account for the lack of bioactivity.

Implications: Pharmacologic blockade of the molecular chaperone HSP90 represents a promising approach for treating bladder tumors driven by oncogenic gene rearrangements of FGFR3. Furthermore, UDP-glucuronosyltransferase enzyme expression may serve as a predictive factor for clinical response to resorcinol-based HSP90 inhibitors. Mol Cancer Res; 12(7); 1042–54. ©2014 AACR.
Data from Comprehensive Molecular Profiling of Olfactory Neuroblastoma Identifies Potentially Targetable FGFR3 Amplifications
Authors: Lazo de la Vega Lorena , McHugh Jonathan B. , Cani Andi K. , Kunder Komal , Walocko Frances M. , Liu Chia-Jen , Hovelson Daniel H. , Robinson Dan , Chinnaiyan Arul M. , Tomlins Scott A. , Harms Paul W.
Published: March 2024

Abstract
Olfactory neuroblastomas (ONBs), also known as esthesioneuroblastomas, are malignant round-cell tumors that represent up to 5% of sinonasal malignancies. Despite their aggressive course, molecular studies of ONBs have been limited, and targeted therapies are lacking. To identify potential oncogenic drivers and targetable pathways in ONBs, we characterized 20 ONBs, including archived ONBs profiled by targeted, multiplexed PCR (mxPCR)–based DNA next-generation sequencing (NGS) of the coding sequence of over 400 cancer-relevant genes (n = 16), mxPCR-based RNA NGS of 108 target genes (n = 15), and 2 ONBs profiled by comprehensive hybrid-capture–based clinical grade NGS of >1,500 genes. Somatic mutations were infrequent in our cohort, with 7 prioritized nonsynonymous mutations in 5 of 18 (28%) ONBs, and no genes were recurrently mutated. We detected arm/chromosome-level copy-number alterations in all tumors, most frequently gains involving all or part of chromosome 20, chromosome 5, and chromosome 11. Recurrent focal amplifications, often but not exclusively in the context of arm-level gains, included CCND1 [n = 4/18 (22%) tumors] and the targetable receptor tyrosine kinase FGFR3 [n = 5/18 (28%) tumors]. Targeted RNA NGS confirmed high expression of FGFR3 in ONB (at levels equivalent to bladder cancer), with the highest expression observed in FGFR3-amplified ONB cases. Importantly, our findings suggest that FGFR3 may be a therapeutic target in a subset of these aggressive tumors.

Implications: ONBs harbor recurrent chromosomal copy-number changes, including FGFR3 amplification associated with overexpression. Hence, FGFR3 may represent a novel therapeutic target in these tumors. Mol Cancer Res; 15(11); 1551–7. ©2017 AACR.
Data from Fhit Regulates EMT Targets through an EGFR/Src/ERK/Slug Signaling Axis in Human Bronchial Cells
Authors: Joannes Audrey , Grelet Simon , Duca Laurent , Gilles Christine , Kileztky Claire , Dalstein Véronique , Birembaut Philippe , Polette Myriam , Nawrocki-Raby Béatrice
Published: March 2024

Abstract
In many cancers, including lung carcinomas, Fragile histidine triad (Fhit) is frequently decreased or lost. Fhit status has recently been shown to be associated with elevated in vitro and in vivo invasiveness in lung cancer. Tumor cell invasion is facilitated by epithelial–mesenchymal transition (EMT), a process by which tumor cells lose their epithelial features to acquire a mesenchymal cell-like phenotype. In this study, the mechanism underlying Fhit-regulated EMT was deciphered. Using Slug knockdown, pharmacologic inhibitors PD98059, PP1, and gefitinib as well as an anti-EGFR antibody, it was demonstrated that Fhit silencing in bronchial cells induced overexpression of two primary EMT-associated targets, MMP-9 and vimentin, to regulate cell invasion dependent on an EGFR/Src/ERK/Slug signaling pathway. Moreover, ectopic expression of Fhit in Fhit-deficient lung cancer cells downregulated this pathway. Finally, an inverse correlation was observed between Fhit and phospho-EGFR levels in a cohort of human squamous cell lung carcinoma specimens. These results demonstrate a Fhit-dependent mechanism in the control of EMT-regulated EGFR signaling.

Implications: This study adds new insight into the regulatory mechanism of EMT, a process known to increase resistance to conventional and targeted therapies in lung cancer. Mol Cancer Res; 12(5); 775–83. ©2014 AACR.
Data from Tumor Cell-Derived Periostin Regulates Cytokines That Maintain Breast Cancer Stem Cells
Authors: Lambert Arthur W. , Wong Chen Khuan , Ozturk Sait , Papageorgis Panagiotis , Raghunathan Rekha , Alekseyev Yuriy , Gower Adam C. , Reinhard Björn M. , Abdolmaleky Hamid M. , Thiagalingam Sam
Published: March 2024

Abstract
Basal-like breast cancer (BLBC) is an aggressive subtype of breast cancer which is often enriched with cancer stem cells (CSC), but the underlying molecular basis for this connection remains elusive. We hypothesized that BLBC cells are able to establish a niche permissive to the maintenance of CSCs and found that tumor cell-derived periostin (POSTN), a component of the extracellular matrix, as well as a corresponding cognate receptor, integrin αvβ3, are highly expressed in a subset of BLBC cell lines as well as in CSC-enriched populations. Furthermore, we demonstrated that an intact periostin–integrin β3 signaling axis is required for the maintenance of breast CSCs. POSTN activates the ERK signaling pathway and regulates NF-κB–mediated transcription of key cytokines, namely IL6 and IL8, which in turn control downstream activation of STAT3. In summary, these findings suggest that BLBC cells have an innate ability to establish a microenvironmental niche supportive of CSCs.

Implications: The findings reported here indicate that POSTN produced by CSCs acts to reinforce the stem cell state through the activation of integrin receptors and the production of key cytokines. Mol Cancer Res; 14(1); 103–13. ©2015 AACR.
Data from FOXC1 Regulates FGFR1 Isoform Switching to Promote Invasion Following TGFβ-Induced EMT
Authors: Hopkins Alex , Coatham Mackenzie L. , Berry Fred B.
Published: March 2024

Abstract
Epithelial-to-mesenchymal transition (EMT) is an important physiologic process that drives tissue formation during development, but also contributes to disease pathogenesis, including fibrosis and cancer metastasis. Elevated expression of the FOXC1 transcription factor has been detected in several metastatic cancers that have undergone EMT. Therefore, mechanistic insight into the role of FOXC1 in the initiation of the EMT process was sought. It was determined that although Foxc1 transcript expression was elevated following TGFβ1-induced EMT of NMuMG cells, FOXC1 was not required for this induction. RNA sequencing revealed that the mRNA levels of FGF receptor 1-isoform IIIc (Fgfr1-IIIc), normally activated upon TGFβ1 treatment, were reduced in Foxc1 knockdown cells, and overexpression of Foxc1 was sufficient to induce Fgfr1-IIIc expression, but not EMT. Chromatin immunoprecipitation experiments demonstrated that FOXC1 binds to an Fgfr1 upstream regulatory region and that FOXC1 activates an Fgfr1 promoter element. Furthermore, elevated expression of Foxc1 led to increased Fgfr1-IIIc transcript. Foxc1 knockdown impaired the FGF2-mediated three-dimensional migratory ability of NMuMG cells, which was rescued by expression of FGFR1. In addition, elevated expression of FOXC1 and FGFR1 was also observed in migratory mesenchymal MDA-MB-231 breast cancer cells. Together, these results define a role for FOXC1 in specifying an invasive mesenchymal cell type by promoting FGFR1 isoform switching following induction of TGFβ1-mediated EMT. Mol Cancer Res; 15(10); 1341–53. ©2017 AACR.
Data from Constitutively Active ErbB2 Regulates Cisplatin-Induced Cell Death in Breast Cancer Cells via Pro- and Antiapoptotic Mechanisms
Authors: Sigurðsson Haraldur H. , Olesen Christina W. , Dybboe Rie , Lauritzen Gitte , Pedersen Stine F.
Published: March 2024

Abstract
Despite the frequent expression of N-terminally truncated ErbB2 (ΔNErbB2/p95HER2) in breast cancer and its association with Herceptin resistance and poor prognosis, it remains poorly understood how ΔNErbB2 affects chemotherapy-induced cell death. Previously it was shown that ΔNErbB2 upregulates acid extrusion from MCF-7 breast cancer cells and that inhibition of the Na+/H+ exchanger (SLC9A1/NHE1) strongly sensitizes ΔNErbB2-expressing MCF-7 cells to cisplatin chemotherapy. The aim of this study was to identify the mechanism through which ΔNErbB2 regulates cisplatin-induced breast cancer cell death, and determine how NHE1 regulates this process. Cisplatin treatment elicited apoptosis, ATM phosphorylation, upregulation of p53, Noxa (PMAIP1), and PUMA (BBC3), and cleavage of caspase-9, -7, fodrin, and PARP-1 in MCF-7 cells. Inducible ΔNErbB2 expression strongly reduced cisplatin-induced ATM- and p53-phosphorylation, augmented Noxa upregulation and caspase-9 and -7 cleavage, doubled p21WAF1/Cip1 (CDKN1A) expression, and nearly abolished Bcl-2 expression. LC3-GFP analysis demonstrated that autophagic flux was reduced by cisplatin in a manner augmented by ΔNErbB2, yet did not contribute to cisplatin-induced death. Using knockdown approaches, it was shown that cisplatin-induced caspase-7 cleavage in ΔNErbB2-MCF-7 cells was Noxa- and caspase-9 dependent. This pathway was augmented by NHE1 inhibition, while the Na+/HCO3− cotransporter (SLC4A7/NBCn1) was internalized following cisplatin exposure.

Implications: This work reveals that ΔNErbB2 strongly affects several major pro- and antiapoptotic pathways and provides mechanistic insight into the role of NHE1 in chemotherapy resistance. These findings have relevance for defining therapy regimens in breast cancers with ΔNErbB2 and/or NHE1 overexpression. Mol Cancer Res; 13(1); 63–77. ©2014 AACR.
Data from Metabolic Reprogramming by Folate Restriction Leads to a Less Aggressive Cancer Phenotype
Authors: Ashkavand Zahra , O'Flanagan Ciara , Hennig Mirko , Du Xiuxia , Hursting Stephen D. , Krupenko Sergey A.
Published: March 2024

Abstract
Folate coenzymes are involved in biochemical reactions of one-carbon transfer, and deficiency of this vitamin impairs cellular proliferation, migration, and survival in many cell types. Here, the effect of folate restriction on mammary cancer was evaluated using three distinct breast cancer subtypes differing in their aggressiveness and metastatic potential: noninvasive basal-like (E-Wnt), invasive but minimally metastatic claudin-low (M-Wnt), and highly metastatic claudin-low (metM-Wntliver) cell lines, each derived from the same pool of MMTV-Wnt-1 transgenic mouse mammary tumors. NMR-based metabolomics was used to quantitate 41 major metabolites in cells grown in folate-free medium versus standard medium. Each cell line demonstrated metabolic reprogramming when grown in folate-free medium. In E-Wnt, M-Wnt, and metM-Wntliver cells, 12, 29, and 25 metabolites, respectively, were significantly different (P < 0.05 and at least 1.5-fold change). The levels of eight metabolites (aspartate, ATP, creatine, creatine phosphate, formate, serine, taurine and β-alanine) were changed in each folate-restricted cell line. Increased glucose, decreased lactate, and inhibition of glycolysis, cellular proliferation, migration, and invasion occurred in M-Wnt and metM-Wntliver cells (but not E-Wnt cells) grown in folate-free versus standard medium. These effects were accompanied by altered levels of several folate-metabolizing enzymes, indicating that the observed metabolic reprogramming may result from both decreased folate availability and altered folate metabolism. These findings reveal that folate restriction results in metabolic and bioenergetic changes and a less aggressive cancer cell phenotype.

Implications: Metabolic reprogramming driven by folate restriction represents a therapeutic target for reducing the burden of breast cancer. Mol Cancer Res; 15(2); 189–200. ©2016 AACR.
Data from Beneficial Effects of RAF Inhibitor in Mutant BRAF Splice Variant–Expressing Melanoma
Authors: Hartsough Edward J. , Basile Kevin J. , Aplin Andrew E.
Published: March 2024

Abstract
Resistance to RAF inhibitors such as vemurafenib and dabrafenib is a major clinical problem in the treatment of melanoma. Patients with mutant BRAF melanoma that progress on RAF inhibitors have limited treatment options, and drug removal from resistant tumors may elicit multiple effects. A frequent mechanism of resistance to RAF inhibitors is caused by expression of mutant BRAF splice variants. RAF inhibitor–resistant cell lines, generated in vivo, were tested as to whether or not mutant BRAF splice variants confer a fitness advantage in the presence of RAF inhibitor. Critically, cells expressing distinct mutant BRAF splice variants grow more efficiently in vitro and in vivo in the presence of the vemurafenib analog, PLX4720, compared with in the absence of inhibitor. PLX4720-treated BRAF splice variant–expressing cells exhibited levels of phospho-extracellular signal–regulated kinase (ERK)1/2 comparable to untreated parental cells. In addition, a reduction in phospho-ERK1/2 levels following treatment with the MEK inhibitor, trametinib (GSK1120212) phenocopied the fitness benefit provided by PLX4720. These data indicate that mutant BRAF splice variant–expressing melanoma cells are benefited by defined concentrations of RAF inhibitors.

Implications: This study provides evidence that RAF inhibitor–resistant melanoma cells benefit from continued therapy. Mol Cancer Res; 12(5); 795–802. ©2014 AACR.
Data from β-Arrestin-2 Counters CXCR7-Mediated EGFR Transactivation and Proliferation
Authors: Kallifatidis Georgios , Munoz Daniel , Singh Rajendra Kumar , Salazar Nicole , Hoy James J. , Lokeshwar Bal L.
Published: March 2024

Abstract
The atypical 7-transmembrane chemokine receptor, CXCR7, transactivates the EGFR leading to increased tumor growth in several tumor types. However, the molecular mechanism of CXCR7 ligand–independent EGFR transactivation is unknown. We used cDNA knock-in, RNAi and analysis of mitogenic signaling components in both normal prostate epithelial cells and prostate cancer cells to decipher the proliferation-inducing mechanism of the CXCR7–EGFR interaction. The data demonstrate that CXCR7-induced EGFR transactivation is independent of both the release of cryptic EGFR ligands (e.g., AREG/amphiregulin) and G-protein–coupled receptor signaling. An alternate signaling mechanism involving β-arrestin-2 (ARRB2/β-AR2) was examined by manipulating the levels of β-AR2 and analyzing changes in LNCaP cell growth and phosphorylation of EGFR, ERK1/2, Src, and Akt. Depletion of β-AR2 in LNCaP cells increased proliferation/colony formation and significantly increased activation of Src, phosphorylation of EGFR at Tyr-1110, and phosphorylation/activation of ERK1/2 compared with that with control shRNA. Moreover, β-AR2 depletion downregulated the proliferation suppressor p21. Stimulation of β-AR2–expressing cells with EGF resulted in rapid nuclear translocation of phosphorylated/activated EGFR. Downregulation of β-AR2 enhanced this nuclear translocation. These results demonstrate that β-AR2 is a negative regulator of CXCR7/Src/EGFR–mediated mitogenic signaling.

Implications: This study reveals that β-AR2 functions as a tumor suppressor, underscoring its clinical importance in regulating CXCR7/EGFR–mediated tumor cell proliferation. Mol Cancer Res; 14(5); 493–503. ©2016 AACR.
Data from Immunophenotyping and Transcriptomic Outcomes in PDX-Derived TNBC Tissue
Authors: Snowden Eileen , Porter Warren , Hahn Friedrich , Ferguson Mitchell , Tong Frances , Parker Joel S. , Middlebrook Aaron , Ghanekar Smita , Dillmore W. Shannon , Blaesius Rainer
Published: March 2024

Abstract
Cancer tissue functions as an ecosystem of a diverse set of cells that interact in a complex tumor microenvironment. Genomic tools applied to biopsies in bulk fail to account for this tumor heterogeneity, whereas single-cell imaging methods limit the number of cells which can be assessed or are very resource intensive. The current study presents methods based on flow cytometric analysis and cell sorting using known cell surface markers (CXCR4/CD184, CD24, THY1/CD90) to identify and interrogate distinct groups of cells in triple-negative breast cancer clinical biopsy specimens from patient-derived xenograft (PDX) models. The results demonstrate that flow cytometric analysis allows a relevant subgrouping of cancer tissue and that sorting of these subgroups provides insights into cancer cell populations with unique, reproducible, and functionally divergent gene expression profiles. The discovery of a drug resistance signature implies that uncovering the functional interaction between these populations will lead to deeper understanding of cancer progression and drug response.

Implications: PDX-derived human breast cancer tissue was investigated at the single-cell level, and cell subpopulations defined by surface markers were identified which suggest specific roles for distinct cellular compartments within a solid tumor. Mol Cancer Res; 15(4); 429–38. ©2016 AACR.
Data from GR and ER Coactivation Alters the Expression of Differentiation Genes and Associates with Improved ER+ Breast Cancer Outcome
Authors: West Diana C. , Pan Deng , Tonsing-Carter Eva Y. , Hernandez Kyle M. , Pierce Charles F. , Styke Sarah C. , Bowie Kathleen R. , Garcia Tzintzuni I. , Kocherginsky Masha , Conzen Suzanne D.
Published: March 2024

Abstract
In estrogen receptor (ER)–negative breast cancer, high tumor glucocorticoid receptor (GR) expression has been associated with a relatively poor outcome. In contrast, using a meta-analysis of several genomic datasets, here we find that tumor GR mRNA expression is associated with improved ER+ relapse-free survival (RFS; independently of progesterone receptor expression). To understand the mechanism by which GR expression is associated with a better ER+ breast cancer outcome, the global effect of GR-mediated transcriptional activation in ER+ breast cancer cells was studied. Analysis of GR chromatin immunoprecipitation followed by high-throughput sequencing in ER+/GR+ MCF-7 cells revealed that upon coactivation of GR and ER, GR chromatin association became enriched at proximal promoter regions. Furthermore, following ER activation, increased GR chromatin association was observed at ER, FOXO, and AP1 response elements. In addition, ER associated with GR response elements, suggesting that ER and GR interact in a complex. Coactivation of GR and ER resulted in increased expression (relative to ER activation alone) of transcripts that encode proteins promoting cellular differentiation (e.g., KDM4B, VDR) and inhibiting the Wnt signaling pathway (IGFBP4). Finally, expression of these individual prodifferentiation genes was associated with significantly improved RFS in ER+ breast cancer patients. Together, these data suggest that the coexpression and subsequent activity of tumor cell GR and ER contribute to the less aggressive natural history of early-stage breast cancer by coordinating the altered expression of genes favoring differentiation.

Implications: The interaction between ER and GR activity highlights the importance of context-dependent nuclear receptor function in cancer. Mol Cancer Res; 14(8); 707–19. ©2016 AACR.
Data from Identification of mTORC2 as a Necessary Component of HRG/ErbB2-Dependent Cellular Transformation
Authors: Lin Miao-chong J. , Rojas Katherine S. , Cerione Richard A. , Wilson Kristin F.
Published: March 2024

Abstract
Overexpression of the receptor tyrosine kinase HER2/ErbB2 (ERBB2) has been linked to a poor prognosis for patients with breast cancer; thus, its activity is a central target for cancer therapy. Likewise, overexpression of heregulin (HRG/NRG1), a growth factor responsible for ErbB2 activation, has also been shown to be a driver of breast cancer progression. Although ErbB2 inhibitors offer a major advancement in the treatment of ErbB2-dependent breast cancers, patients are highly susceptible to developing clinical resistance to these drugs. Therefore, a detailed understanding of the molecular mechanism that underlies HRG/ErbB2-induced tumorigenesis is essential for the development of effective therapeutic strategies for this subset of patients with breast cancer. Here, it was demonstrated that HRG promoted anchorage-independent breast cancer cell growth more potently than EGF, and that the HRG-dependent activation of phosphoinositide 3-kinase and mTORC1 are necessary events for cell transformation. Functional evaluation of two distinct mTOR (MTOR) inhibitors, rapamycin and INK-128, on HRG-dependent signaling activities, uncovered a necessary role for mTORC2 in the regulation of the AKT/TSC2/mTORC1 axis by affecting the phosphorylation of AKT at the PDK1(PDPK1)-dependent site (T308) as well as at the mTORC2-dependent site (S473). The elimination of Rictor (RICTOR), a critical component of mTORC2, is detrimental to both the activation of mTORC1 and HRG-mediated cellular transformation. Similar results were obtained in multiple breast cancer model systems, highlighting an important role for mTORC2 in HRG/ErbB2-dependent breast cancer.

Implications: These findings suggest the potential benefits of targeting mTORC2 in HRG/ErbB2-induced breast cancer. Mol Cancer Res; 12(6); 940–52. ©2014 AACR.
Data from PCBP1/HNRNP E1 Protects Chromosomal Integrity by Translational Regulation of CDC27
Authors: Link Laura A. , Howley Breege V. , Hussey George S. , Howe Philip H.
Published: March 2024

Abstract
CDC27 is a core component of the anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase, whose oscillatory activity is responsible for the metaphase-to-anaphase transition and mitotic exit. Here, in normal murine mammary gland epithelial cells (NMuMG), CDC27 expression is controlled posttranscriptionally through the RNA binding protein poly(rC) binding protein 1 (PCBP1)/heterogeneous nuclear ribonucleoprotein E1 (HNRNP E1). shRNA-mediated knockdown of HNRNP E1 abrogates translational silencing of the Cdc27 transcript, resulting in constitutive expression of CDC27. Dysregulated expression of CDC27 leads to premature activation of the G2–M–APC/C–CDC20 complex, resulting in the aberrant degradation of FZR1/CDH1, a cofactor of the G1 and late G2–M–APC/C and a substrate normally reserved for the SCF-βTRCP ligase. Loss of CDH1 expression and of APC/C-CDH1 activity, upon constitutive expression of CDC27, results in mitotic aberrations and aneuploidy in NMuMG cells. Furthermore, tissue microarray of breast cancer patient tumor samples reveals high CDC27 levels compared with nonneoplastic breast tissue and a significant correlation between disease recurrence and CDC27 expression. These results suggest that dysregulation of HNRNP E1-mediated translational regulation of Cdc27 leads to chromosomal instability and aneuploidy and that CDC27 expression represents a significant predictor of breast cancer recurrence.

Implications: The RNA-binding protein HNRNP E1 mediates translational regulation of the cell-cycle regulator CDC27 and that dysregulation of CDC27 leads to aneuploidy. In addition, high CDC27 expression in breast cancer patient tumor specimens significantly predicts disease recurrence, suggesting a novel role for CDC27 as a predictor of relapse. Mol Cancer Res; 14(7); 634–46. ©2016 AACR.
Data from Functional Genomics Approach Identifies Novel Signaling Regulators of TGFα Ectodomain Shedding
Authors: Wilson Jennifer L. , Kefaloyianni Eirini , Stopfer Lauren , Harrison Christina , Sabbisetti Venkata S. , Fraenkel Ernest , Lauffenburger Douglas A. , Herrlich Andreas
Published: March 2024

Abstract
Ectodomain shedding of cell-surface precursor proteins by metalloproteases generates important cellular signaling molecules. Of importance for disease is the release of ligands that activate the EGFR, such as TGFα, which is mostly carried out by ADAM17 [a member of the A-disintegrin and metalloprotease (ADAM) domain family]. EGFR ligand shedding has been linked to many diseases, in particular cancer development, growth and metastasis, as well as resistance to cancer therapeutics. Excessive EGFR ligand release can outcompete therapeutic EGFR inhibition or the inhibition of other growth factor pathways by providing bypass signaling via EGFR activation. Drugging metalloproteases directly have failed clinically because it indiscriminately affected shedding of numerous substrates. It is therefore essential to identify regulators for EGFR ligand cleavage. Here, integration of a functional shRNA genomic screen, computational network analysis, and dedicated validation tests succeeded in identifying several key signaling pathways as novel regulators of TGFα shedding in cancer cells. Most notably, a cluster of genes with NFκB pathway regulatory functions was found to strongly influence TGFα release, albeit independent of their NFκB regulatory functions. Inflammatory regulators thus also govern cancer cell growth–promoting ectodomain cleavage, lending mechanistic understanding to the well-known connection between inflammation and cancer.

Implications: Using genomic screens and network analysis, this study defines targets that regulate ectodomain shedding and suggests new treatment opportunities for EGFR-driven cancers. Mol Cancer Res; 16(1); 147–61. ©2017 AACR.
Data from Prognostic Relevance of Tumor Purity and Interaction with MGMT Methylation in Glioblastoma
Authors: Schulze Heuling Eva , Knab Felix , Radke Josefine , Eskilsson Eskil , Martinez-Ledesma Emmanuel , Koch Arend , Czabanka Marcus , Dieterich Christoph , Verhaak Roel G. , Harms Christoph , Euskirchen Philipp
Published: March 2024

Abstract
Promoter methylation status of O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, is a critical biomarker in glioblastoma (GBM), as treatment decisions and clinical trial inclusion rely on its accurate assessment. However, interpretation of results is complicated by poor interassay reproducibility as well as a weak correlation between methylation status and expression levels of MGMT. This study systematically investigates the influence of tumor purity on tissue subjected to MGMT analysis. A quantitative, allele-specific real-time PCR (qAS-PCR) assay was developed to determine genotype and mutant allele frequency of telomerase promoter (pTERT) mutations as a direct measure of tumor purity. We studied tumor purity, pTERT mutation by Sanger sequencing, MGMT methylation by pyrosequencing, IDH1 mutation status, and clinical parameters in a cohort of high-grade gliomas (n = 97). The qAS-PCR reliably predicted pTERT genotype and tumor purity compared with independent methods. Tumor purity positively and significantly correlated with the extent of methylation in MGMT methylated GBMs. Extent of MGMT methylation differed significantly with respect to pTERT mutation hotspot (C228T vs. C250T). Interestingly, frontal lobe tumors showed greater tumor purity than those in other locations. Above all, tumor purity was identified as an independent prognostic factor in GBM. In conclusion, we determined mutual associations of tumor purity with MGMT methylation and pTERT mutations and found that the extent of MGMT methylation reflects tumor purity. In turn, tumor purity is prognostic in IDH1 wild-type GBM.

Implications: Tumor purity is an independent prognostic marker in glioblastoma and is associated with the extent of MGMT methylation. Mol Cancer Res; 15(5); 532–40. ©2017 AACR.
Data from Key Survival Factor, Mcl-1, Correlates with Sensitivity to Combined Bcl-2/Bcl-xL Blockade
Authors: Williams Michelle M. , Lee Linus , Hicks Donna J. , Joly Meghan M. , Elion David , Rahman Bushra , McKernan Courtney , Sanchez Violeta , Balko Justin M. , Stricker Thomas , Estrada Monica Valeria , Cook Rebecca S.
Published: March 2024

Abstract
An estimated 40,000 deaths will be attributed to breast cancer in 2016, underscoring the need for improved therapies. Evading cell death is a major hallmark of cancer, driving tumor progression and therapeutic resistance. To evade apoptosis, cancers use antiapoptotic Bcl-2 proteins to bind to and neutralize apoptotic activators, such as Bim. Investigation of antiapoptotic Bcl-2 family members in clinical breast cancer datasets revealed greater expression and more frequent gene amplification of MCL1 as compared with BCL2 or BCL2L1 (Bcl-xL) across three major molecular breast cancer subtypes, Luminal (A and B), HER2-enriched, and Basal-like. While Mcl-1 protein expression was elevated in estrogen receptor α (ERα)-positive and ERα-negative tumors as compared with normal breast, Mcl-1 staining was higher in ERα+ tumors. Targeted Mcl-1 blockade using RNAi increased caspase-mediated cell death in ERα+ breast cancer cells, resulting in sustained growth inhibition. In contrast, combined blockade of Bcl-2 and Bcl-xL only transiently induced apoptosis, as cells rapidly acclimated through Mcl-1 upregulation and enhanced Mcl-1 activity, as measured in situ using Mcl-1/Bim proximity ligation assays. Importantly, MCL1 gene expression levels correlated inversely with sensitivity to pharmacologic Bcl-2/Bcl-xL inhibition in luminal breast cancer cells, whereas no relationship was seen between the gene expression of BCL2 or BCL2L1 and sensitivity to Bcl-2/Bcl-xL inhibition. These results demonstrate that breast cancers rapidly deploy Mcl-1 to promote cell survival, particularly when challenged with blockade of other Bcl-2 family members, warranting the continued development of Mcl-1–selective inhibitors for targeted tumor cell killing.

Implications: Mcl-1 levels predict breast cancer response to inhibitors targeting other Bcl-2 family members, and demonstrate the key role played by Mcl-1 in resistance to this drug class. Mol Cancer Res; 15(3); 259–68. ©2016 AACR.