bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from HO-3867, a Safe STAT3 Inhibitor, Is Selectively Cytotoxic to Ovarian Cancer
Authors: Rath Kellie S. , Naidu Shan K. , Lata Pushpa , Bid Hemant K. , Rivera Brian K. , McCann Georgia A. , Tierney Brent J. , ElNaggar Adam C. , Bravo Veronica , Leone Gustavo , Houghton Peter , Hideg Kálmán , Kuppusamy Periannan , Cohn David E. , Selvendiran Karuppaiyah
Published: March 2024

Abstract
STAT3 is well corroborated preclinically as a cancer therapeutic target, but tractable translational strategies for its blockade by small molecule inhibitors have remained elusive. In this study, we report the development of a novel class of bifunctional STAT3 inhibitors, based on conjugation of a diarylidenyl-piperidone (DAP) backbone to an N-hydroxypyrroline (–NOH) group, which exhibits minimal toxicity against normal cells and good oral bioavailability. Molecular modeling studies of this class suggested direct interaction with the STAT3 DNA binding domain. In particular, the DAP compound HO-3867 selectively inhibited STAT3 phosphorylation, transcription, and DNA binding without affecting the expression of other active STATs. HO-3867 exhibited minimal toxicity toward noncancerous cells and tissues but induced apoptosis in ovarian cancer cells. Pharmacologic analysis revealed greater bioabsorption and bioavailability of the active (cytotoxic) metabolites in cancer cells compared with normal cells. The selective cytotoxicity of HO-3867 seemed to be multifaceted, eliciting differential activation of the Akt pathway in normal versus cancer cells. RNAi attenuation experiments confirmed the requirement of STAT3 for HO-3867–mediated apoptosis in ovarian cancer cells. In vivo testing showed that HO-3867 could block xenograft tumor growth without toxic side effects. Furthermore, in primary human ovarian cancer cells isolated from patient ascites, HO-3867 inhibited cell migration/invasion and survival. Our results offer preclinical proof-of-concept for HO-3867 as a selective STAT3 inhibitor to treat ovarian cancer and other solid tumors where STAT3 is widely upregulated. Cancer Res; 74(8); 2316–27. ©2014 AACR.
Data from Activation of the FGFR–STAT3 Pathway in Breast Cancer Cells Induces a Hyaluronan-Rich Microenvironment That Licenses Tumor Formation
Authors: Bohrer Laura R. , Chuntova Pavlina , Bade Lindsey K. , Beadnell Thomas C. , Leon Ronald P. , Brady Nicholas J. , Ryu Yungil , Goldberg Jodi E. , Schmechel Stephen C. , Koopmeiners Joseph S. , McCarthy James B. , Schwertfeger Kathryn L.
Published: March 2024

Abstract
Aberrant activation of fibroblast growth factor receptors (FGFR) contributes to breast cancer growth, progression, and therapeutic resistance. Because of the complex nature of the FGF/FGFR axis, and the numerous effects of FGFR activation on tumor cells and the surrounding microenvironment, the specific mechanisms through which aberrant FGFR activity contributes to breast cancer are not completely understood. We show here that FGFR activation induces accumulation of hyaluronan within the extracellular matrix and that blocking hyaluronan synthesis decreases proliferation, migration, and therapeutic resistance. Furthermore, FGFR-mediated hyaluronan accumulation requires activation of the STAT3 pathway, which regulates expression of hyaluronan synthase 2 (HAS2) and subsequent hyaluronan synthesis. Using a novel in vivo model of FGFR-dependent tumor growth, we demonstrate that STAT3 inhibition decreases both FGFR-driven tumor growth and hyaluronan levels within the tumor. Finally, our results suggest that combinatorial therapies inhibiting both FGFR activity and hyaluronan synthesis is more effective than targeting either pathway alone and may be a relevant therapeutic approach for breast cancers associated with high levels of FGFR activity. In conclusion, these studies indicate a novel targetable mechanism through which FGFR activation in breast cancer cells induces a protumorigenic microenvironment. Cancer Res; 74(1); 374–86. ©2013 AACR.
Data from Metastatic Growth Progression Caused by PSGL-1–Mediated Recruitment of Monocytes to Metastatic Sites
Authors: Hoos Alexandra , Protsyuk Darya , Borsig Lubor
Published: March 2024

Abstract
Tumor cell–derived selectin ligands mediate contact to the endothelium, platelets, and leukocytes through binding to selectins that facilitates metastasis. Here, we describe the mechanism of how endogenous (non–tumor derived) selectin ligands contribute to metastasis using α(1,3)fucosyltransferase 7 (Fuc-TVII−/−)–deficient mice. Experimental metastasis of MC-38GFP and Lewis lung (3LL) carcinoma cells was attenuated in Fuc-TVII−/− mice, which express minimal amount of selectin ligands. We show that metastasis is dependent on selectin ligands carried on hematopoietic cells. P-selectin glycoprotein ligand-1 (PSGL-1) was identified as the major ligand facilitating monocyte accumulation at metastatic sites. Reduced recruitment of monocytes to metastasizing tumor cells in Fuc-TVII−/− mice correlated with attenuated metastasis. Adoptive transfer of Fuc-T7+ monocytes rescued metastasis in Fuc-TVII−/− mice, indicating that selectin ligand–dependent recruitment of monocytes is required for cancer progression. Cytokine analysis in metastatic lungs revealed high expression of CCL2 in C57BL/6 mice that was significantly lower in Fuc-TVII−/− mice. The absence of monocyte recruitment in Fuc-TVII−/− mice correlated with increased apoptosis of tumor cells. Thus, the recruitment of monocytes to metastasizing tumor cells is facilitated by endogenous selectin ligands on monocytes that enable efficient tumor cell survival, extravasation, and metastasis. Cancer Res; 74(3); 695–704. ©2013 AACR.
Data from miR149 Functions as a Tumor Suppressor by Controlling Breast Epithelial Cell Migration and Invasion
Authors: Bischoff Annabell , Huck Bettina , Keller Bettina , Strotbek Michaela , Schmid Simone , Boerries Melanie , Busch Hauke , Müller Dafne , Olayioye Monilola A.
Published: March 2024

Abstract
Deregulated molecular signaling pathways are responsible for the altered adhesive, migratory, and invasive properties of cancer cells. The different breast cancer subtypes are characterized by the expression of distinct miRNAs, short non-coding RNAs that posttranscriptionally modulate the expression of entire gene networks. Profiling studies have revealed downregulation of miR149 in basal breast cancer. Here, we show that miR149 expression severely impairs cell spreading, migration, and invasion of basal-like breast cancer cells. We identify signaling molecules, including the small GTPases Rap1a and Rap1b, downstream of integrin receptors as miR149 targets, providing an explanation for the defective Src and Rac activation during cell adhesion and spreading upon miR149 expression. Suppression of cell spreading by miR149 could be rescued, at least in part, by expression of constitutively active Rac. Finally, we demonstrate that increased miR149 levels block lung colonization in vivo. On the basis of our findings, we propose that miR149 downregulation in basal breast cancer facilitates the metastatic dissemination of tumor cells by supporting aberrant Rac activation. Cancer Res; 74(18); 5256–65. ©2014 AACR.
Data from Hypoxia-Induced SUMOylation of E3 Ligase HAF Determines Specific Activation of HIF2 in Clear-Cell Renal Cell Carcinoma
Authors: Koh Mei Yee , Nguyen Vuvi , Lemos Robert , Darnay Bryant G. , Kiriakova Galina , Abdelmelek Mena , Ho Thai H. , Karam Jose , Monzon Federico A. , Jonasch Eric , Powis Garth
Published: March 2024

Abstract
Clear-cell renal cell cancer (CRCC) is initiated typically by loss of the tumor-suppressor VHL, driving constitutive activation of hypoxia-inducible factor-1 (HIF1) and HIF2. However, whereas HIF1 has a tumor-suppressor role, HIF2 plays a distinct role in driving CRCC. In this study, we show that the HIF1α E3 ligase hypoxia-associated factor (HAF) complexes with HIF2α at DNA to promote HIF2-dependent transcription through a mechanism relying upon HAF SUMOylation. HAF SUMOylation was induced by hypoxia, whereas HAF-mediated HIF1α degradation was SUMOylation independent. HAF overexpression in mice increased CRCC growth and metastasis. Clinically, HAF overexpression was associated with poor prognosis. Taken together, our results show that HAF is a specific mediator of HIF2 activation that is critical for CRCC development and morbidity. Cancer Res; 75(2); 316–29. ©2014 AACR.
Data from High Numbers of Differentiated Effector CD4 T Cells Are Found in Patients with Cancer and Correlate with Clinical Response after Neoadjuvant Therapy of Breast Cancer
Authors: Péguillet Isabelle , Milder Maud , Louis Delphine , Vincent-Salomon Anne , Dorval Thierry , Piperno-Neumann Sophie , Scholl Suzy M. , Lantz Olivier
Published: March 2024

Abstract
CD4+ T cells influence tumor immunity in complex ways that are not fully understood. In this study, we characterized a population of human differentiated effector CD4+ T cells that is defined by low levels of the interleukin (IL)-2 and IL-7 receptors (CD25−CD127−). We found that this cell population expands in patients with various types of cancer, including breast cancer, to represent 2% to 20% of total CD4+ blood T lymphocytes as compared with only 0.2% to 2% in healthy individuals. Notably, these CD25−CD127−CD4 T cells expressed effector markers such as CD244 and CD11b with low levels of CD27, contrasting with the memory phenotype dominating this population in healthy individuals. These cells did not cycle in patients, nor did they secrete IL-10 or IL-17, but instead displayed cytotoxic features. Moreover, they encompassed oligoclonal expansions paralleling an expansion of effector CD8+ T cells that included tumor antigen–specific T cells. During neoadjuvant chemotherapy in patients with breast cancer, we found that the increase in CD25−CD127− CD4+ T cells correlated with tumor regression. This observation suggested that CD4+ T cells included tumor antigen–specific cells, which may be generated by or participate in tumor regressions during chemotherapy. In summary, our results lend support to the hypothesis that CD4+ T cells are involved in human antitumor responses. Cancer Res; 74(8); 2204–16. ©2014 AACR.
Data from Universal Marker and Detection Tool for Human Sarcoma Circulating Tumor Cells
Authors: Satelli Arun , Mitra Abhisek , Cutrera Jeffry J. , Devarie Marcos , Xia Xueqing , Ingram Davis R. , Dibra Denada , Somaiah Neeta , Torres Keila E. , Ravi Vinod , Ludwig Joseph A. , Kleinerman Eugenie S. , Li Shulin
Published: March 2024

Abstract
To date, no specific marker exists for the detection of circulating tumor cells (CTC) from different types of sarcomas, though tools are available for detection of CTCs in peripheral blood of patients with cancer for epithelial cancers. Here, we report cell-surface vimentin (CSV) as an exclusive marker on sarcoma CTC regardless of the tissue origin of the sarcoma as detected by a novel monoclonal antibody. Utilizing CSV as a probe, we isolated and enumerated sarcoma CTCs with high sensitivity and specificity from the blood of patients bearing different types of sarcoma, validating their phenotype by single cell genomic amplification, mutation detection, and FISH. Our results establish the first universal and specific CTC marker described for enumerating CTCs from different types of sarcoma, thereby providing a key prognosis tool to monitor cancer metastasis and relapse. Cancer Res; 74(6); 1645–50. ©2014 AACR.
Data from Extracellular RNA Liberates Tumor Necrosis Factor-α to Promote Tumor Cell Trafficking and Progression
Authors: Fischer Silvia , Gesierich Sabine , Griemert Barbara , Schänzer Anne , Acker Till , Augustin Hellmut G. , Olsson Anna-Karin , Preissner Klaus T.
Published: March 2024

Abstract
Extracellular RNA (eRNA) released from injured cells promotes tissue permeability, thrombosis, and inflammation in vitro and in vivo, and RNase1 pretreatment can reduce all these effects. In this study, we investigated the role of the eRNA/RNase1 system in tumor progression and metastasis. Under quiescent and stimulatory conditions, tumor cells released much higher levels of endogenous extracellular RNA (eRNA) than nontumor cells. In glioblastomas, eRNA was detected at higher levels in tumors than nontumor tissue. eRNA induced tumor cells to adhere to and migrate through human cerebral microvascular endothelial cells (HCMEC/D3), in a manner requiring activation of VEGF signaling. In addition, eRNA liberated TNF-α from macrophages in a manner requiring activation of the TNF-α–converting enzyme TACE. Accordingly, supernatants derived from eRNA-treated macrophages enhanced tumor cell adhesion to HCMEC/D3. TNF-α release evoked by eRNA relied upon signaling activation of mitogen-activated protein kinases and the NF-κB pathway. In subcutaneous xenograft models of human cancer, administration of RNase1 but not DNase decreased tumor volume and weight. Taken together, these results suggest that eRNA released from tumor cells has the capacity to promote tumor cell invasion through endothelial barriers by both direct and indirect mechanisms, including through a mechanism involving TNF-α release from tumor-infiltrating monocytes/macrophages. Our findings establish a crucial role for eRNA in driving tumor progression, and they suggest applications for extracellular RNase1 as an antiinvasive regimen for cancer treatment. Cancer Res; 73(16); 5080–9. ©2013 AACR.
Data from COX-2 Drives Metastatic Breast Cells from Brain Lesions into the Cerebrospinal Fluid and Systemic Circulation
Authors: Allen Joshua E. , Patel Akshal S. , Prabhu Varun V. , Dicker David T. , Sheehan Jonas M. , Glantz Michael J. , El-Deiry Wafik S.
Published: March 2024

Abstract
Breast cancer is among the most common malignancies that metastasize to the brain, with 15% to 20% of patients with metastatic breast cancer eventually developing brain metastases. We previously reported a method to enumerate tumor cells in the cerebrospinal fluid (CSF) of patients with breast cancer with central nervous system (CNS) metastases, a setting that lacks sufficiently informative biomarkers. Here, we show that breast cancer cells can spontaneously disseminate into the CSF from brain lesions in mice in a COX-2–dependent manner and can escape from the CNS to systemic circulation. Enumeration of tumor cells in the peripheral blood (circulating tumor cells, CTC) and CSF (cerebrospinal fluid tumor cells, CSFTC) of nine breast cancer patients with brain metastases revealed dynamic changes in tumor cell burden in both the peripheral blood and CSF compartments that correlated with clinical disease progression. Interestingly, four of the enrolled patients exhibited rapid intercompartmental transitioning of the disease reflected in the CTC and CSFTC counts that preceded corresponding evidence by clinical imaging or neurologic symptoms. Two of these patients had systemic disease recurrence involving the primary malignant site. Intercompartmental cycling of tumor cells may represent an important mechanism for disease persistence and recurrence that may involve tumor self-seeding. Our findings demonstrate the involvement of COX-2 in the genesis of CSFTCs and suggest that COX-2 inhibitors should be investigated in patients with breast cancer with brain metastases for their ability to reduce CSFTC counts and prevent systemic recurrence. Cancer Res; 74(9); 2385–90. ©2014 AACR.
Data from Glioblastoma Cell Enrichment Is Critical for Analysis of Phosphorylated Drug Targets and Proteomic–Genomic Correlations
Authors: Mueller Claudius , deCarvalho Ana C. , Mikkelsen Tom , Lehman Norman L. , Calvert Valerie , Espina Virginia , Liotta Lance A. , Petricoin Emanuel F.
Published: March 2024

Abstract
The quality of cancer genomic and proteomic data relies upon the quality of the clinical specimens examined. Here, we show that data derived from non-microdissected glioblastoma multiforme tumor tissue is either masked or not accurate, producing correlations between genomic and proteomic data that lead to false classifications for therapeutic stratification. We analyzed the level of 133 key signaling proteins and phosphoproteins in laser capture microdissected (LCM) primary tumors from a study set of tissues used for the Cancer Genome Atlas (TCGA) profiling efforts, comparing the results to tissue-matched, nontumor cell–enriched lysates from adjacent sections. Among the analytes, 44%, including targets for clinically important inhibitors, such as phosphorylated mTOR, AKT, STAT1, VEGFR2, or BCL2, differed between matched tumor cell–enriched and nonenriched specimens (even in tumor sections with 90% tumor cell content). While total EGFR protein levels were higher in tumors with EGFR mutations, regardless of tumor cell enrichment, EGFR phosphorylation was increased only in LCM-enriched tumor specimens carrying EGFR mutations. Phosphorylated and total PTEN, which is highly expressed in normal brain, was reduced only in LCM-enriched tumor specimens with either PTEN mutation or loss in PTEN copy number, with no differences observed in non-microdissected samples. These results were confirmed in an independent, non-microdissected, publicly available protein data set from the TCGA database. Our findings highlight the necessity for careful upfront cellular enrichment in biospecimens that form the basis for targeted therapy selection and for molecular characterization efforts such as TCGA. Cancer Res; 74(3); 818–28. ©2013 AACR.
Data from High-Throughput Time-Resolved FRET Reveals Akt/PKB Activation as a Poor Prognostic Marker in Breast Cancer
Authors: Veeriah Selvaraju , Leboucher Pierre , de Naurois Julien , Jethwa Nirmal , Nye Emma , Bunting Tamara , Stone Richard , Stamp Gordon , Calleja Véronique , Jeffrey Stefanie S. , Parker Peter J. , Larijani Banafshé
Published: March 2024

Abstract
Dysregulation of the Akt/PKB pathway has been associated with poor prognosis in several human carcinomas. Current approaches to assess Akt activation rely on intensity-based methods, which are limited by the subjectivity of manual scoring and poor specificity. Here, we report the development of a novel assay using amplified, time-resolved Förster resonance energy transfer (FRET), which is highly specific and sensitive and can be adapted to any protein. Using this approach to analyze primary breast tissue microarrays, we quantified levels of activated pAkt at a spatial resolution that revealed molecular heterogeneity within tumors. High pAkt status assessed by amplified FRET correlated with worse disease-free survival. Our findings support the use of amplified FRET to determine pAkt status in cancer tissues as candidate biomarker for the identification of high-risk patients. Cancer Res; 74(18); 4983–95. ©2014 AACR.
Data from Collagen Signaling Enhances Tumor Progression after Anti-VEGF Therapy in a Murine Model of Pancreatic Ductal Adenocarcinoma
Authors: Aguilera Kristina Y. , Rivera Lee B. , Hur Hoon , Carbon Juliet G. , Toombs Jason E. , Goldstein Courtney D. , Dellinger Michael T. , Castrillon Diego H. , Brekken Rolf A.
Published: March 2024

Abstract
There is growing evidence that antiangiogenic therapy stimulates cancer cell invasion and metastasis. However, the underlying molecular mechanisms responsible for these changes have not been fully defined. Here, we report that anti-VEGF therapy promotes local invasion and metastasis by inducing collagen signaling in cancer cells. We show that chronic VEGF inhibition in a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDA) induces hypoxia, a less differentiated mesenchymal-like tumor cell phenotype, TGF-β expression, and collagen deposition and signaling. In addition, we show that collagen signaling is critical for protumorigenic activity of TGF-β in vitro. To further model the impact of collagen signaling in tumors, we evaluated PDA in mice lacking Sparc, a protein that reduces collagen binding to cell surface receptors. Importantly, we show that loss of Sparc increases collagen signaling and tumor progression. Together, these findings suggest that collagen actively promotes PDA spread and that enhanced disease progression associated with anti-VEGF therapy can arise from elevated extracellular matrix–mediated signaling. Cancer Res; 74(4); 1032–44. ©2013 AACR.
Data from The DREAM Complex Mediates GIST Cell Quiescence and Is a Novel Therapeutic Target to Enhance Imatinib-Induced Apoptosis
Authors: Boichuk Sergei , Parry Joshua A. , Makielski Kathleen R. , Litovchick Larisa , Baron Julianne L. , Zewe James P. , Wozniak Agnieszka , Mehalek Keith R. , Korzeniewski Nina , Seneviratne Danushka S. , Schöffski Patrick , Debiec-Rychter Maria , DeCaprio James A. , Duensing Anette
Published: March 2024

Abstract
Gastrointestinal stromal tumors (GIST) can be successfully treated with imatinib mesylate (Gleevec); however, complete remissions are rare and patients frequently achieve disease stabilization in the presence of residual tumor masses. The clinical observation that discontinuation of treatment can lead to tumor progression suggests that residual tumor cells are, in fact, quiescent and, therefore, able to re-enter the cell-division cycle. In line with this notion, we have previously shown that imatinib induces GIST cell quiescence in vitro through the APCCDH1–SKP2–p27Kip1 signaling axis. Here, we provide evidence that imatinib induces GIST cell quiescence in vivo and that this process also involves the DREAM complex, a multisubunit complex that has recently been identified as an additional key regulator of quiescence. Importantly, inhibition of DREAM complex formation by depletion of the DREAM regulatory kinase DYRK1A or its target LIN52 was found to enhance imatinib-induced cell death. Our results show that imatinib induces apoptosis in a fraction of GIST cells while, at the same time, a subset of cells undergoes quiescence involving the DREAM complex. Inhibition of this process enhances imatinib-induced apoptosis, which opens the opportunity for future therapeutic interventions to target the DREAM complex for more efficient imatinib responses. Cancer Res; 73(16); 5120–9. ©2013 AACR.
Data from Genetic and Phenotypic Diversity in Breast Tumor Metastases
Authors: Almendro Vanessa , Kim Hee Jung , Cheng Yu-Kang , Gönen Mithat , Itzkovitz Shalev , Argani Pedram , van Oudenaarden Alexander , Sukumar Saraswati , Michor Franziska , Polyak Kornelia
Published: March 2024

Abstract
Metastatic disease is the main cause of cancer-related mortality due to almost universal therapeutic resistance. Despite its high clinical relevance, our knowledge of how cancer cell populations change during metastatic progression is limited. Here, we investigated intratumor genetic and phenotypic heterogeneity during metastatic progression of breast cancer. We analyzed cellular genotypes and phenotypes at the single cell level by performing immunoFISH in intact tissue sections of distant metastatic tumors from rapid autopsy cases and from primary tumors and matched lymph node metastases collected before systemic therapy. We calculated the Shannon index of intratumor diversity in all cancer cells and within phenotypically distinct cell populations. We found that the extent of intratumor genetic diversity was similar regardless of the chromosomal region analyzed, implying that it may reflect an inherent property of the tumors. We observed that genetic diversity was highest in distant metastases and was generally concordant across lesions within the same patient, whereas treatment-naïve primary tumors and matched lymph node metastases were frequently genetically more divergent. In contrast, cellular phenotypes were more discordant between distant metastases than primary tumors and matched lymph node metastases. Diversity for 8q24 was consistently higher in HER2+ tumors compared with other subtypes and in metastases of triple-negative tumors relative to primary sites. We conclude that our integrative method that couples ecologic models with experimental data in human tissue samples could be used for the improved prognostication of patients with cancer and for the design of more effective therapies for progressive disease.Cancer Res; 74(5); 1338–48. ©2014 AACR.
Data from Identification of ATR–Chk1 Pathway Inhibitors That Selectively Target p53-Deficient Cells without Directly Suppressing ATR Catalytic Activity
Authors: Kawasumi Masaoki , Bradner James E. , Tolliday Nicola , Thibodeau Renee , Sloan Heather , Brummond Kay M. , Nghiem Paul
Published: March 2024

Abstract
Resistance to DNA-damaging chemotherapy is a barrier to effective treatment that appears to be augmented by p53 functional deficiency in many cancers. In p53-deficient cells in which the G1–S checkpoint is compromised, cell viability after DNA damage relies upon intact intra-S and G2–M checkpoints mediated by the ATR (ataxia telangiectasia and Rad3 related) and Chk1 kinases. Thus, a logical rationale to sensitize p53-deficient cancers to DNA-damaging chemotherapy is through the use of ATP-competitive inhibitors of ATR or Chk1. To discover small molecules that may act on uncharacterized components of the ATR pathway, we performed a phenotype-based screen of 9,195 compounds for their ability to inhibit hydroxyurea-induced phosphorylation of Ser345 on Chk1, known to be a critical ATR substrate. This effort led to the identification of four small-molecule compounds, three of which were derived from known bioactive library (anthothecol, dihydrocelastryl, and erysolin) and one of which was a novel synthetic compound termed MARPIN. These compounds all inhibited ATR-selective phosphorylation and sensitized p53-deficient cancer cells to DNA-damaging agents in vitro and in vivo. Notably, these compounds did not inhibit ATR catalytic activity in vitro, unlike typical ATP-competitive inhibitors, but acted in a mechanistically distinct manner to disable ATR–Chk1 function. Our results highlight a set of novel molecular probes to further elucidate druggable mechanisms to improve cancer therapeutic responses produced by DNA-damaging drugs. Cancer Res; 74(24); 7534–45. ©2014 AACR.
Data from Oncogenic Properties of a Spermatogenic Meiotic Variant of Fer Kinase Expressed in Somatic Cells
Authors: Yaffe Etai , Hikri Elad , Elkis Yoav , Cohen Ortal , Segal Ariela , Makovski Adar , Varvak Alexander , Shpungin Sally , Nir Uri
Published: March 2024

Abstract
The kinase Fer and its spermatogenic meiotic variant, FerT, are coexpressed in normal testes and cancerous tumors, but whether they exert related roles in spermatogenic or malignant cells has not been known. Here, we show that Fer and FerT reside in the mitochondria of spermatogenic cells and are harnessed to the reprogrammed mitochondria of colon carcinoma cells. Both kinases bound complex I of the mitochondrial electron transport chain (ETC) in spermatogenic and in colon carcinoma cells, and silencing of either Fer or FerT was sufficient to impair the activity of this complex. Directed mitochondrial accumulation of FerT in nonmalignant NIH3T3 cells increased their ETC complex I activity, ATP production, and survival, contingent upon stress conditions caused by nutrient and oxygen deprivation. Strikingly, directed mitochondrial accumulation of FerT endowed nonmalignant cells with tumor-forming ability. Thus, recruitment of a meiotic mitochondrial component to cancer cell mitochondria highlights a pivotal role for reprogrammed mitochondria in tumorigenesis. Cancer Res; 74(22); 6474–85. ©2014 AACR.
Data from Complex Formation and Function of Estrogen Receptor α in Transcription Requires RIP140
Authors: Rosell Meritxell , Nevedomskaya Ekaterina , Stelloo Suzan , Nautiyal Jaya , Poliandri Ariel , Steel Jennifer H. , Wessels Lodewyk F.A. , Carroll Jason S. , Parker Malcolm G. , Zwart Wilbert
Published: March 2024

Abstract
RIP140 is a transcriptional coregulator involved in energy homeostasis, ovulation, and mammary gland development. Although conclusive evidence is lacking, reports have implicated a role for RIP140 in breast cancer. Here, we explored the mechanistic role of RIP140 in breast cancer and its involvement in estrogen receptor α (ERα) transcriptional regulation of gene expression. Using ChIP-seq analysis, we demonstrate that RIP140 shares more than 80% of its binding sites with ERα, colocalizing with its interaction partners FOXA1, GATA3, p300, CBP, and p160 family members at H3K4me1-demarcated enhancer regions. RIP140 is required for ERα-complex formation, ERα-mediated gene expression, and ERα-dependent breast cancer cell proliferation. Genes affected following RIP140 silencing could be used to stratify tamoxifen-treated breast cancer cohorts, based on clinical outcome. Importantly, this gene signature was only effective in endocrine-treated conditions. Cumulatively, our data suggest that RIP140 plays an important role in ERα-mediated transcriptional regulation in breast cancer and response to tamoxifen treatment. Cancer Res; 74(19); 5469–79. ©2014 AACR.
Data from Distinct Subpopulations of Head and Neck Cancer Cells with Different Levels of Intracellular Reactive Oxygen Species Exhibit Diverse Stemness, Proliferation, and Chemosensitivity
Authors: Chang Ching-Wen , Chen Yu-Syuan , Chou Shiu-Huey , Han Chia-Li , Chen Yu-Ju , Yang Cheng-Chieh , Huang Chih-Yang , Lo Jeng-Fan
Published: March 2024

Abstract
Head and neck squamous cell carcinoma (HNSCC) is driven by cancer-initiating cells (CIC), but their maintenance mechanisms are obscure. For hematopoietic stem cells, low levels of intracellular reactive oxygen species (ROSLow) is known to help sustain stemness properties. In this report, we evaluated the hypothesis that ROSLow character conferred CIC properties in HNSCC. Sphere cultures define CIC in HNSCC cell populations (HN-CIC). We found that ROSLow cells in HN-CIC defined in this manner were more numerous than in parental HNSCC cells. Further, ROSLow cells frequently coexpressed CIC surface markers such as memGrp78 and Glut3. Exploiting flow cytometry to sort cells on the basis of their ROS level, we found that isolated ROSLow cells displayed relatively more CIC properties, including quiescence, chemoresistance, in vitro malignant properties, and tumorigenicity. Pharmacological depletion of ROS modulators in cisplatin-treated HN-CIC reduced CIC properties, enhancing cell differentiation and enhancing cisplatin-induced cell death. Overall, our work defined cell subpopulations in HNSCC on the basis of differential intracellular ROS levels, which associated with stemness and chemoresistance properties. On the basis of our findings, we suggest that strategies to promote intracellular ROS levels may heighten the efficacy of conventional chemotherapy used for HNSCC treatment. Cancer Res; 74(21); 6291–305. ©2014 AACR.
Data from Cell Surface Lactate Receptor GPR81 Is Crucial for Cancer Cell Survival
Authors: Roland Christina L. , Arumugam Thiruvengadam , Deng Defeng , Liu Shi He , Philip Bincy , Gomez Sobeyda , Burns William R. , Ramachandran Vijaya , Wang Huamin , Cruz-Monserrate Zobeida , Logsdon Craig D.
Published: March 2024

Abstract
The mechanisms that allow cancer cells to adapt to the typical tumor microenvironment of low oxygen and glucose and high lactate are not well understood. GPR81 is a lactate receptor recently identified in adipose and muscle cells that has not been investigated in cancer. In the current study, we examined GPR81 expression and function in cancer cells. We found that GPR81 was present in colon, breast, lung, hepatocellular, salivary gland, cervical, and pancreatic carcinoma cell lines. Examination of tumors resected from patients with pancreatic cancer indicated that 94% (148 of 158) expressed high levels of GPR81. Functionally, we observed that the reduction of GPR81 levels using shRNA-mediated silencing had little effect on pancreatic cancer cells cultured in high glucose, but led to the rapid death of cancer cells cultured in conditions of low glucose supplemented with lactate. We also observed that lactate addition to culture media induced the expression of genes involved in lactate metabolism, including monocarboxylase transporters in control, but not in GPR81-silenced cells. In vivo, GPR81 expression levels correlated with the rate of pancreatic cancer tumor growth and metastasis. Cells in which GPR81 was silenced showed a dramatic decrease in growth and metastasis. Implantation of cancer cells in vivo was also observed to lead to greatly elevated levels of GPR81. These data support that GPR81 is important for cancer cell regulation of lactate transport mechanisms. Furthermore, lactate transport is important for the survival of cancer cells in the tumor microenvironment. Cancer Res; 74(18); 5301–10. ©2014 AACR.
Data from Deubiquitination of γ-Tubulin by BAP1 Prevents Chromosome Instability in Breast Cancer Cells
Authors: Zarrizi Reihaneh , Menard Julien Albert , Belting Mattias , Massoumi Ramin
Published: March 2024

Abstract
Microtubule nucleation requires the γ-tubulin ring complex, and during the M-phase (mitosis) this complex accumulates at the centrosome to support mitotic spindle formation. The posttranslational modification of γ-tubulin through ubiquitination is vital for regulating microtubule nucleation and centrosome duplication. Blocking the BRCA1/BARD1-dependent ubiquitination of γ-tubulin causes centrosome amplification. In the current study, we identified BRCA1-associated protein-1 (BAP1) as a deubiquitination enzyme for γ-tubulin. BAP1 was downregulated in metastatic adenocarcinoma breast cell lines compared with noncancerous human breast epithelial cells. Furthermore, low expression of BAP1 was associated with reduced overall survival of patients with breast cancer. Reduced expression of BAP1 in breast cancer cell lines was associated with mitotic abnormalities. Importantly, rescue experiments including expression of full length but not the catalytic mutant of BAP1 reduced ubiquitination of γ-tubulin and prevented mitotic defects. Our study uncovers a new mechanism for BAP1 involved in deubiquitination of γ-tubulin, which is required to prevent abnormal mitotic spindle formation and genome instability. Cancer Res; 74(22); 6499–508. ©2014 AACR.
Data from Natural Killer Cells Eradicate Galectin-1–Deficient Glioma in the Absence of Adaptive Immunity
Authors: Baker Gregory J. , Chockley Peter , Yadav Viveka Nand , Doherty Robert , Ritt Michael , Sivaramakrishnan Sivaraj , Castro Maria G. , Lowenstein Pedro R.
Published: March 2024

Abstract
Natural killer (NK) cells safeguard against early tumor formation by destroying transformed target cells in a process referred to as NK immune surveillance. However, the immune escape mechanisms used by malignant brain tumors to subvert this innate type of immune surveillance remain unclear. Here we show that malignant glioma cells suppress NK immune surveillance by overexpressing the β-galactoside–binding lectin galectin-1. Conversely, galectin-1–deficient glioma cells could be eradicated by host NK cells before the initiation of an antitumor T-cell response. In vitro experiments demonstrated that galectin-1–deficient GL26-Cit glioma cells are ∼3-fold more sensitive to NK-mediated tumor lysis than galectin-1–expressing cells. Our findings suggest that galectin-1 suppression in human glioma could improve patient survival by restoring NK immune surveillance that can eradicate glioma cells. Cancer Res; 74(18); 5079–90. ©2014 AACR.
Data from Validation and Structural Characterization of the LEDGF/p75–MLL Interface as a New Target for the Treatment of MLL-Dependent Leukemia
Authors: Čermáková Kateřina , Tesina Petr , Demeulemeester Jonas , El Ashkar Sara , Méreau Hélène , Schwaller Juerg , Řezáčová Pavlína , Veverka Vaclav , De Rijck Jan
Published: March 2024

Abstract
Mixed lineage leukemia (MLL) fusion–driven acute leukemias represent a genetically distinct subset of leukemias with poor prognosis. MLL forms a ternary complex with the lens epithelium–derived growth factor (LEDGF/p75) and MENIN. LEDGF/p75, a chromatin reader recognizing H3K36me3 marks, contributes to the association of the MLL multiprotein complex to chromatin. Formation of this complex is critical for the development of MLL leukemia. Available X-ray data represent only a partial structure of the LEDGF/p75–MLL–MENIN complex. Using nuclear magnetic resonance spectroscopy, we identified an additional LEDGF/p75–MLL interface, which overlaps with the binding site of known LEDGF/p75 interactors—HIV-1 integrase, PogZ, and JPO2. Binding of these proteins or MLL to LEDGF/p75 is mutually exclusive. The resolved structure, as well as mutational analysis, shows that the interaction is primarily sustained via two aromatic residues of MLL (F148 and F151). Colony-forming assays in MLL–AF9+ leukemic cells expressing MLL interaction-defective LEDGF/p75 mutants revealed that this interaction is essential for transformation. Finally, we show that the clonogenic growth of primary murine MLL-AF9–expressing leukemic blasts is selectively impaired upon overexpression of a LEDGF/p75-binding cyclic peptide CP65, originally developed to inhibit the LEDGF/p75–HIV-1 integrase interaction. The newly defined protein–protein interface therefore represents a new target for the development of therapeutics against LEDGF/p75–dependent MLL fusion–driven leukemic disorders. Cancer Res; 74(18); 5139–51. ©2014 AACR.
Data from Neuronal Pentraxin 2 Supports Clear Cell Renal Cell Carcinoma by Activating the AMPA-Selective Glutamate Receptor-4
Authors: von Roemeling Christina A. , Radisky Derek C. , Marlow Laura A. , Cooper Simon J. , Grebe Stefan K. , Anastasiadis Panagiotis Z. , Tun Han W. , Copland John A.
Published: March 2024

Abstract
Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer and has the highest propensity to manifest as metastatic disease. Recent characterizations of the genetic signature of ccRCC have revealed several factors correlated with tumor cell migration and invasion; however, the specific events driving malignancy are not well defined. Furthermore, there remains a lack of targeted therapies that result in long-term, sustainable response in patients with metastatic disease. We show here that neuronal pentraxin 2 (NPTX2) is overexpressed specifically in ccRCC primary tumors and metastases, and that it contributes to tumor cell viability and promotes cell migration through its interaction with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluR4. We propose NPTX2 as a novel molecular target for therapy for patients with ccRCC diagnosed with or at risk of developing metastatic disease. Cancer Res; 74(17); 4796–810. ©2014 AACR.
Data from Discrepancies in Cancer Genomic Sequencing Highlight Opportunities for Driver Mutation Discovery
Authors: Hudson Andrew M. , Yates Tim , Li Yaoyong , Trotter Eleanor W. , Fawdar Shameem , Chapman Phil , Lorigan Paul , Biankin Andrew , Miller Crispin J. , Brognard John
Published: March 2024

Abstract
Cancer genome sequencing is being used at an increasing rate to identify actionable driver mutations that can inform therapeutic intervention strategies. A comparison of two of the most prominent cancer genome sequencing databases from different institutes (Cancer Cell Line Encyclopedia and Catalogue of Somatic Mutations in Cancer) revealed marked discrepancies in the detection of missense mutations in identical cell lines (57.38% conformity). The main reason for this discrepancy is inadequate sequencing of GC-rich areas of the exome. We have therefore mapped over 400 regions of consistent inadequate sequencing (cold-spots) in known cancer-causing genes and kinases, in 368 of which neither institute finds mutations. We demonstrate, using a newly identified PAK4 mutation as proof of principle, that specific targeting and sequencing of these GC-rich cold-spot regions can lead to the identification of novel driver mutations in known tumor suppressors and oncogenes. We highlight that cross-referencing between genomic databases is required to comprehensively assess genomic alterations in commonly used cell lines and that there are still significant opportunities to identify novel drivers of tumorigenesis in poorly sequenced areas of the exome. Finally, we assess other reasons for the observed discrepancy, such as variations in dbSNP filtering and the acquisition/loss of mutations, to give explanations as to why there is a discrepancy in pharmacogenomic studies, given recent concerns with poor reproducibility of data. Cancer Res; 74(22); 6390–6. ©2014 AACR.
Data from Harnessing the Fcμ Receptor for Potent and Selective Cytotoxic Therapy of Chronic Lymphocytic Leukemia
Authors: Vire Bérengère , Skarzynski Martin , Thomas Joshua D. , Nelson Christopher G. , David Alexandre , Aue Georg , Burke Terrence R. , Rader Christoph , Wiestner Adrian
Published: March 2024

Abstract
Chronic lymphocytic leukemia (CLL) is a B-cell malignancy in need of new, effective, and safe therapies. The recently identified IgM receptor FcμR is overexpressed on malignant B cells in CLL and mediates the rapid internalization and lysosomal shuttling of IgM via its Fc fragment (Fcμ). To exploit this internalization and trafficking pathway for targeted drug delivery, we engineered an IgM-derived protein scaffold (Fcμ) and linked it with the cytotoxic agent monomethylauristatin F. This Fcμ–drug conjugate was selectively toxic for FcμR-expressing cell lines in vitro and for CLL cells but not autologous normal T cells ex vivo. Notably, the cytotoxic activity of the Fcμ–drug conjugate was maintained in CLL cells carrying a 17p deletion, which predicts resistance to standard chemotherapy. Next, we tested the possible therapeutic application of the Fcμ–drug conjugate in immunodeficient NOD/SCID/IL-2Rγnull (NSG) mice engrafted with peripheral blood cells from patients with leukemia. Three intravenous injections of the Fcμ–drug conjugate over a 10-day period were well tolerated and selectively killed the human CLL cells but not the coengrafted autologous human T cells. In summary, we developed a novel strategy for targeted cytotoxic therapy of CLL based on the unique properties of FcμR. FcμR-targeted drug delivery showed potent and specific therapeutic activity in CLL, thus providing proof of concept for FcμR as a valuable therapeutic target in CLL and for IgM-based antibody–drug conjugates as a new targeting platform. Cancer Res; 74(24); 7510–20. ©2014 AACR.
Data from Hippo Coactivator YAP1 Upregulates SOX9 and Endows Esophageal Cancer Cells with Stem-like Properties
Authors: Song Shumei , Ajani Jaffer A. , Honjo Soichiro , Maru Dipen M. , Chen Qiongrong , Scott Ailing W. , Heallen Todd R. , Xiao Lianchun , Hofstetter Wayne L. , Weston Brian , Lee Jeffrey H. , Wadhwa Roopma , Sudo Kazuki , Stroehlein John R. , Martin James F. , Hung Mien-Chie , Johnson Randy L.
Published: March 2024

Abstract
Cancer stem cells (CSC) are purported to initiate and maintain tumor growth. Deregulation of normal stem cell signaling may lead to the generation of CSCs; however, the molecular determinants of this process remain poorly understood. Here we show that the transcriptional coactivator YAP1 is a major determinant of CSC properties in nontransformed cells and in esophageal cancer cells by direct upregulation of SOX9. YAP1 regulates the transcription of SOX9 through a conserved TEAD binding site in the SOX9 promoter. Expression of exogenous YAP1 in vitro or inhibition of its upstream negative regulators in vivo results in elevated SOX9 expression accompanied by the acquisition of CSC properties. Conversely, shRNA-mediated knockdown of YAP1 or SOX9 in transformed cells attenuates CSC phenotypes in vitro and tumorigenicity in vivo. The small-molecule inhibitor of YAP1, verteporfin, significantly blocks CSC properties in cells with high YAP1 and a high proportion of ALDH1+. Our findings identify YAP1-driven SOX9 expression as a critical event in the acquisition of CSC properties, suggesting that YAP1 inhibition may offer an effective means of therapeutically targeting the CSC population. Cancer Res; 74(15); 4170–82. ©2014 AACR.
Data from MYC Activates Stem-like Cell Potential in Hepatocarcinoma by a p53-Dependent Mechanism
Authors: Akita Hirofumi , Marquardt Jens U. , Durkin Marian E. , Kitade Mitsuteru , Seo Daekwan , Conner Elizabeth A. , Andersen Jesper B. , Factor Valentina M. , Thorgeirsson Snorri S.
Published: March 2024

Abstract
Activation of c-MYC is an oncogenic hallmark of many cancers, including liver cancer, and is associated with a variety of adverse prognostic characteristics. Despite a causative role during malignant transformation and progression in hepatocarcinogenesis, consequences of c-MYC activation for the biology of hepatic cancer stem cells (CSC) are undefined. Here, distinct levels of c-MYC overexpression were established by using two dose-dependent tetracycline-inducible systems in four hepatoma cell lines with different p53 mutational status. The CSCs were evaluated using side population (SP) approach as well as standard in vitro and in vivo assays. Functional repression of p53 was achieved by lentiviral shRNA transduction. The results show that c-MYC expression levels have a differential impact on liver CSC characteristics. At low levels, c-MYC activation led to increased proliferation and enhanced CSC properties including activation of reprogramming transcription factors and CSC marker expression (e.g., NANOG, OCT4, and EpCAM), expansion of SP, and acceleration of tumor growth upon subcutaneous transplantation into immunocompromised mice. However, when exceeding a threshold level, c-MYC induced a proapoptotic program and loss of CSC potential both in vitro and in vivo. Mechanistically, c-MYC–induced self-renewal capacity of liver cancer cells was exerted in a p53-dependent manner. Low c-MYC activation increased spheroid formation in p53-deficient tumor cells, whereas p53-dependent effects were blunted in the absence of c-MYC overexpression. Together, our results confirm the role of c-MYC as a master regulator during hepatocarcinogenesis and establish a new gatekeeper role for p53 in repressing c-MYC–induced CSC phenotype in liver cancer cells. Cancer Res; 74(20); 5903–13. ©2014 AACR.
Data from Development of Novel ADCs: Conjugation of Tubulysin Analogues to Trastuzumab Monitored by Dual Radiolabeling
Authors: Cohen Ruth , Vugts Danielle J. , Visser Gerard W.M. , Stigter-van Walsum Marijke , Bolijn Marije , Spiga Marco , Lazzari Paolo , Shankar Sreejith , Sani Monica , Zanda Matteo , van Dongen Guus A.M.S.
Published: March 2024

Abstract
Tubulysins are highly toxic tubulin-targeting agents with a narrow therapeutic window that are interesting for application in antibody–drug conjugates (ADC). For full control over drug–antibody ratio (DAR) and the effect thereof on pharmacokinetics and tumor targeting, a dual-labeling approach was developed, wherein the drug, tubulysin variants, and the antibody, the anti-HER2 monoclonal antibody (mAb) trastuzumab, are radiolabeled. 131I-radioiodination of two synthetic tubulysin A analogues, the less potent TUB-OH (IC50 > 100 nmol/L) and the potent TUB-OMOM (IC50, ∼1 nmol/L), and their direct covalent conjugation to 89Zr-trastuzumab were established. Radioiodination of tubulysins was 92% to 98% efficient and conversion to N-hydroxysuccinimide (NHS) esters more than 99%; esters were isolated in an overall yield of 68% ± 5% with radiochemical purity of more than 99.5%. Conjugation of 131I-tubulysin–NHS esters to 89Zr-trastuzumab was 45% to 55% efficient, resulting in ADCs with 96% to 98% radiochemical purity after size-exclusion chromatography. ADCs were evaluated for their tumor-targeting potential and antitumor effects in nude mice with tumors that were sensitive or resistant to trastuzumab, using ado-trastuzumab emtansine as a reference. ADCs appeared stable in vivo. An average DAR of 2 and 4 conferred pharmacokinetics and tumor-targeting behavior similar to parental trastuzumab. Efficacy studies using single-dose TUB-OMOM–trastuzumab (DAR 4) showed dose-dependent antitumor effects, including complete tumor eradications in trastuzumab-sensitive tumors in vivo. TUB-OMOM–trastuzumab (60 mg/kg) displayed efficacy similar to ado-trastuzumab emtansine (15 mg/kg) yet more effective than trastuzumab. Our findings illustrate the potential of synthetic tubulysins in ADCs for cancer treatment. Cancer Res; 74(20); 5700–10. ©2014 AACR.