31 results on '"Sizemore ST"'
Search Results
2. The RAL Small G Proteins Are Clinically Relevant Targets in Triple Negative Breast Cancer.
- Author
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Han D, Spehar JM, Richardson DS, Leelananda S, Chakravarthy P, Grecco S, Reardon J, Stover DG, Bennett C, Sizemore GM, Li Z, Lindert S, and Sizemore ST
- Abstract
Breast cancer (BC) is the most frequent cancer and second-leading cause of cancer deaths in women in the United States. While RAS mutations are infrequent in BC, triple-negative (TN) and HER2-positive (HER2+) BC both exhibit increased RAS activity. Here, we tested the RAS effectors RALA and RALB, which are overexpressed in BC, as tractable molecular targets in these subtypes. While analysis of the breast cancer patient sample data suggests that the RALs are associated with poor outcome in both TNBC and HER2+ BC, our in vivo and in vitro experimental findings revealed the RALs to be essential in only the TNBC cell lines. While testing the response of the BC cell lines to the RAL inhibitors RBC8 and BQU57, we observed no correlation between drug efficacy and cell line dependency on RAL expression for survival, suggesting that these compounds kill via off-target effects. Finally, we report the discovery of a new small molecule inhibitor, OSURALi, which exhibits strong RAL binding, effectively inhibits RAL activation, and is significantly more toxic to RAL-dependent TNBC cells than RAL-independent HER2+ and normal cell lines. These results support the RALs as viable molecular targets in TNBC and the further investigation of OSURALi as a therapeutic agent.
- Published
- 2024
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3. PP2A B55α inhibits epithelial-mesenchymal transition via regulation of Slug expression in non-small cell lung cancer.
- Author
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Singh D, Qiu Z, Jonathan SM, Fa P, Thomas H, Prasad CB, Cai S, Wang JJ, Yan C, Zhang X, Venere M, Li Z, Sizemore ST, Wang QE, and Zhang J
- Subjects
- Humans, Animals, Cell Movement, Cell Line, Tumor, Glycogen Synthase Kinase 3 beta metabolism, Glycogen Synthase Kinase 3 beta genetics, A549 Cells, Mice, Neoplasm Invasiveness, Epithelial-Mesenchymal Transition genetics, Snail Family Transcription Factors genetics, Snail Family Transcription Factors metabolism, Carcinoma, Non-Small-Cell Lung genetics, Carcinoma, Non-Small-Cell Lung pathology, Carcinoma, Non-Small-Cell Lung metabolism, Protein Phosphatase 2 genetics, Protein Phosphatase 2 metabolism, Lung Neoplasms genetics, Lung Neoplasms pathology, Lung Neoplasms metabolism, Gene Expression Regulation, Neoplastic
- Abstract
PP2A B55α, encoded by PPP2R2A, acts as a regulatory subunit of the serine/threonine phosphatase PP2A. Despite a frequent loss of heterozygosity of PPP2R2A in cases of non-small cell lung cancer (NSCLC), research on PP2A B55α's functions remains limited and controversial. To investigate the biological roles of PP2A B55α, we conducted bulk RNA-sequencing to assess the impact of PPP2R2A knockdown using two shRNAs in a NSCLC cell line. Gene set enrichment analysis (GSEA) of the RNA-sequencing data revealed significant enrichment of the epithelial-mesenchymal transition (EMT) pathway, with SNAI2 (the gene encoding Slug) emerging as one of the top candidates. Our findings demonstrate that PP2A B55α suppresses EMT, as PPP2R2A deficiency through knockdown or homozygous or hemizygous depletion promotes EMT and metastatic behavior in NSCLC cells, as evidenced by changes in EMT biomarkers, invasion and migration abilities, as well as metastasis in a tail vein assay. Mechanistically, PP2A B55α inhibits EMT by downregulating SNAI2 expression via the GSK3β-β-catenin pathway. Importantly, PPP2R2A deficiency also slows cell proliferation by disrupting DNA replication, particularly in PPP2R2A
-/- cells. Furthermore, PPP2R2A deficiency, especially PPP2R2A-/- cells, leads to an increase in the cancer stem cell population, which correlates with enhanced resistance to chemotherapy. Overall, the decrease in PP2A B55α levels due to hemizygous/homozygous depletion heightens EMT and the metastatic or stemness/drug resistance potential of NSCLC cells despite their proliferation disadvantage. Our study highlights the significance of PP2A B55α in EMT and metastasis and suggests that targeting EMT/stemness could be a potential therapeutic strategy for treating PPP2R2A-deficient NSCLC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2024
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4. TLE3 Sustains Luminal Breast Cancer Lineage Fidelity to Suppress Metastasis.
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Anstine LJ, Majmudar PR, Aponte A, Singh S, Zhao R, Weber-Bonk KL, Abdul-Karim FW, Valentine M, Seachrist DD, Grennel-Nickelson KE, Cuellar-Vite L, Sizemore GM, Sizemore ST, Webb BM, Thompson CL, and Keri RA
- Subjects
- Cell Differentiation, Co-Repressor Proteins genetics, Receptors, Estrogen metabolism, Transforming Growth Factor beta, Breast Neoplasms metabolism, Humans, Neoplasms, Transcription Factors
- Abstract
Breast cancer subtypes and their phenotypes parallel different stages of the mammary epithelial cell developmental hierarchy. Discovering mechanisms that control lineage identity could provide novel avenues for mitigating disease progression. Here we report that the transcriptional corepressor TLE3 is a guardian of luminal cell fate in breast cancer and operates independently of the estrogen receptor. In luminal breast cancer, TLE3 actively repressed the gene-expression signature associated with highly aggressive basal-like breast cancers (BLBC). Moreover, maintenance of the luminal lineage depended on the appropriate localization of TLE3 to its transcriptional targets, a process mediated by interactions with FOXA1. By repressing genes that drive BLBC phenotypes, including SOX9 and TGFβ2, TLE3 prevented the acquisition of a hybrid epithelial-mesenchymal state and reduced metastatic capacity and aggressive cellular behaviors. These results establish TLE3 as an essential transcriptional repressor that sustains the more differentiated and less metastatic nature of luminal breast cancers. Approaches to induce TLE3 expression could promote the acquisition of less aggressive, more treatable disease states to extend patient survival., Significance: Transcriptional corepressor TLE3 actively suppresses SOX9 and TGFβ transcriptional programs to sustain the luminal lineage identity of breast cancer cells and to inhibit metastatic progression., (©2023 American Association for Cancer Research.)
- Published
- 2023
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5. A miniaturized screening platform to identify novel regulators of extracellular matrix alignment.
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Jones CE, Sharick JT, Sizemore ST, Cukierman E, Strohecker AM, and Leight JL
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- Humans, Cell Movement, Cell Line, Tumor, Fibroblasts, Cytoskeletal Proteins metabolism, Extracellular Matrix genetics, Signal Transduction
- Abstract
Extracellular matrix alignment contributes to metastasis in a number of cancers and is a known prognostic stromal factor; however, the mechanisms controlling matrix organization remain unclear. Cancer-associated fibroblasts (CAF) play a critical role in this process, particularly via matrix production and modulation of key signaling pathways controlling cell adhesion and contractility. Stroma normalization, as opposed to elimination, is a highly sought strategy, and screening for drugs that effectively alter extracellular matrix (ECM) alignment is a practical way to identify novel CAF-normalizing targets that modulate ECM organization. To meet this need, we developed a novel high-throughput screening platform in which fibroblast-derived matrices were produced in 384-well plates, imaged with automated confocal microscopy, and analyzed using a customized MATLAB script. This platform is a technical advance because it miniaturizes the assay, eliminates costly and time-consuming experimental steps, and streamlines data acquisition and analysis to enable high-throughput screening applications. As a proof of concept, this platform was used to screen a kinase inhibitor library to identify modulators of matrix alignment. A number of novel potential regulators were identified, including several receptor tyrosine kinases (c-MET, tropomyosin receptor kinase 1 (NTRK1), HER2/ERBB2) and the serine/threonine kinases protein kinase A, C, and G (PKA, PKC, and PKG). The expression of these regulators was analyzed in publicly available patient datasets to examine the association between stromal gene expression and patient outcomes., Competing Interests: Conflict of interest: EC is a scientific consultant for Phenomic AI. All other authors declare no potential conflicts of interest.
- Published
- 2022
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6. The RAL Enigma: Distinct Roles of RALA and RALB in Cancer.
- Author
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Richardson DS, Spehar JM, Han DT, Chakravarthy PA, and Sizemore ST
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- Cell Survival, GTPase-Activating Proteins metabolism, Humans, ral GTP-Binding Proteins genetics, ral GTP-Binding Proteins metabolism, Monomeric GTP-Binding Proteins metabolism, Neoplasms
- Abstract
RALA and RALB are highly homologous small G proteins belonging to the RAS superfamily. Like other small GTPases, the RALs are molecular switches that can be toggled between inactive GDP-bound and active GTP-bound states to regulate diverse and critical cellular functions such as vesicle trafficking, filopodia formation, mitochondrial fission, and cytokinesis. The RAL paralogs are activated and inactivated by a shared set of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) and utilize similar sets of downstream effectors. In addition to their important roles in normal cell biology, the RALs are known to be critical mediators of cancer cell survival, invasion, migration, and metastasis. However, despite their substantial similarities, the RALs often display striking functional disparities in cancer. RALA and RALB can have redundant, unique, or even antagonistic functions depending on cancer type. The molecular basis for these discrepancies remains an important unanswered question in the field of cancer biology. In this review we examine the functions of the RAL paralogs in normal cellular physiology and cancer biology with special consideration provided to situations where the roles of RALA and RALB are non-redundant.
- Published
- 2022
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7. The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer.
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Thies KA, Cole MW, Schafer RE, Spehar JM, Richardson DS, Steck SA, Das M, Lian AW, Ray A, Shakya R, Knoblaugh SE, Timmers CD, Ostrowski MC, Chakravarti A, Sizemore GM, and Sizemore ST
- Subjects
- Animals, Cell Line, Tumor, Cell Movement, Cell Proliferation, Cell Survival drug effects, Enzyme Inhibitors therapeutic use, Female, Humans, Mice, Neoplasm Metastasis, Paclitaxel therapeutic use, Prognosis, Triple Negative Breast Neoplasms drug therapy, Xenograft Model Antitumor Assays, ral GTP-Binding Proteins antagonists & inhibitors, ral GTP-Binding Proteins genetics, Triple Negative Breast Neoplasms metabolism, Triple Negative Breast Neoplasms pathology, ral GTP-Binding Proteins metabolism
- Abstract
Background: Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC., Methods: The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models., Results: Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo., Conclusions: Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC.
- Published
- 2021
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8. Genomic features of rapid versus late relapse in triple negative breast cancer.
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Zhang Y, Asad S, Weber Z, Tallman D, Nock W, Wyse M, Bey JF, Dean KL, Adams EJ, Stockard S, Singh J, Winer EP, Lin NU, Jiang YZ, Ma D, Wang P, Shi L, Huang W, Shao ZM, Cherian M, Lustberg MB, Ramaswamy B, Sardesai S, VanDeusen J, Williams N, Wesolowski R, Obeng-Gyasi S, Sizemore GM, Sizemore ST, Verschraegen C, and Stover DG
- Subjects
- Adult, Chemotherapy, Adjuvant statistics & numerical data, DNA Copy Number Variations, Datasets as Topic, Disease-Free Survival, Female, Follow-Up Studies, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, Humans, Logistic Models, Middle Aged, Models, Genetic, Mutation, Neoplasm Recurrence, Local epidemiology, Neoplasm Recurrence, Local prevention & control, Prognosis, Risk Assessment methods, Risk Assessment statistics & numerical data, Time Factors, Triple Negative Breast Neoplasms genetics, Triple Negative Breast Neoplasms mortality, Biomarkers, Tumor genetics, Mastectomy, Neoadjuvant Therapy statistics & numerical data, Neoplasm Recurrence, Local genetics, Triple Negative Breast Neoplasms therapy
- Abstract
Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease and we have previously shown that rapid relapse of TNBC is associated with distinct sociodemographic features. We hypothesized that rapid versus late relapse in TNBC is also defined by distinct clinical and genomic features of primary tumors., Methods: Using three publicly-available datasets, we identified 453 patients diagnosed with primary TNBC with adequate follow-up to be characterized as 'rapid relapse' (rrTNBC; distant relapse or death ≤2 years of diagnosis), 'late relapse' (lrTNBC; > 2 years) or 'no relapse' (nrTNBC: > 5 years no relapse/death). We explored basic clinical and primary tumor multi-omic data, including whole transcriptome (n = 453), and whole genome copy number and mutation data for 171 cancer-related genes (n = 317). Association of rapid relapse with clinical and genomic features were assessed using Pearson chi-squared tests, t-tests, ANOVA, and Fisher exact tests. We evaluated logistic regression models of clinical features with subtype versus two models that integrated significant genomic features., Results: Relative to nrTNBC, both rrTNBC and lrTNBC had significantly lower immune signatures and immune signatures were highly correlated to anti-tumor CD8 T-cell, M1 macrophage, and gamma-delta T-cell CIBERSORT inferred immune subsets. Intriguingly, lrTNBCs were enriched for luminal signatures. There was no difference in tumor mutation burden or percent genome altered across groups. Logistic regression mModels that incorporate genomic features significantly outperformed standard clinical/subtype models in training (n = 63 patients), testing (n = 63) and independent validation (n = 34) cohorts, although performance of all models were overall modest., Conclusions: We identify clinical and genomic features associated with rapid relapse TNBC for further study of this aggressive TNBC subset.
- Published
- 2021
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9. Pten regulates collagen fibrillogenesis by fibroblasts through SPARC.
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Jones CE, Sharick JT, Colbert SE, Shukla VC, Zent JM, Ostrowski MC, Ghadiali SN, Sizemore ST, and Leight JL
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- Animals, Cell Line, Extracellular Matrix metabolism, Fibroblasts, Humans, Mammary Glands, Human cytology, Mammary Glands, Human pathology, Mice, Mice, Knockout, Collagen metabolism, Mammary Glands, Human metabolism, Osteonectin metabolism, PTEN Phosphohydrolase physiology
- Abstract
Collagen deposition contributes to both high mammographic density and breast cancer progression. Low stromal PTEN expression has been observed in as many as half of breast tumors and is associated with increases in collagen deposition, however the mechanism connecting PTEN loss to increased collagen deposition remains unclear. Here, we demonstrate that Pten knockout in fibroblasts using an Fsp-Cre;PtenloxP/loxP mouse model increases collagen fiber number and fiber size within the mammary gland. Pten knockout additionally upregulated Sparc transcription in fibroblasts and promoted collagen shuttling out of the cell. Interestingly, SPARC mRNA expression was observed to be significantly elevated in the tumor stroma as compared to the normal breast in several patient cohorts. While SPARC knockdown via shRNA did not affect collagen shuttling, it notably decreased assembly of exogenous collagen. In addition, SPARC knockdown decreased fibronectin assembly and alignment of the extracellular matrix in an in vitro fibroblast-derived matrix model. Overall, these data indicate upregulation of SPARC is a mechanism by which PTEN regulates collagen deposition in the mammary gland stroma., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
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10. Stromal Platelet-Derived Growth Factor Receptor-β Signaling Promotes Breast Cancer Metastasis in the Brain.
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Thies KA, Hammer AM, Hildreth BE 3rd, Steck SA, Spehar JM, Kladney RD, Geisler JA, Das M, Russell LO, Bey JF 4th, Bolyard CM, Pilarski R, Trimboli AJ, Cuitiño MC, Koivisto CS, Stover DG, Schoenfield L, Otero J, Godbout JP, Chakravarti A, Ringel MD, Ramaswamy B, Li Z, Kaur B, Leone G, Ostrowski MC, Sizemore ST, and Sizemore GM
- Subjects
- Animals, Brain metabolism, Endothelial Cells metabolism, Humans, Mice, Receptor, Platelet-Derived Growth Factor beta, Breast Neoplasms genetics, MicroRNAs
- Abstract
Platelet-derived growth factor receptor-beta (PDGFRβ) is a receptor tyrosine kinase found in cells of mesenchymal origin such as fibroblasts and pericytes. Activation of this receptor is dependent on paracrine ligand induction, and its preferred ligand PDGFB is released by neighboring epithelial and endothelial cells. While expression of both PDGFRβ and PDGFB has been noted in patient breast tumors for decades, how PDGFB-to-PDGFRβ tumor-stroma signaling mediates breast cancer initiation, progression, and metastasis remains unclear. Here we demonstrate this paracrine signaling pathway that mediates both primary tumor growth and metastasis, specifically, metastasis to the brain. Elevated levels of PDGFB accelerated orthotopic tumor growth and intracranial growth of mammary tumor cells, while mesenchymal-specific expression of an activating mutant PDGFRβ (PDGFRβ
D849V ) exerted proproliferative signals on adjacent mammary tumor cells. Stromal expression of PDGFRβD849V also promoted brain metastases of mammary tumor cells expressing high PDGFB when injected intravenously. In the brain, expression of PDGFRβD849V was observed within a subset of astrocytes, and aged mice expressing PDGFRβD849V exhibited reactive gliosis. Importantly, the PDGFR-specific inhibitor crenolanib significantly reduced intracranial growth of mammary tumor cells. In a tissue microarray comprised of 363 primary human breast tumors, high PDGFB protein expression was prognostic for brain metastases, but not metastases to other sites. Our results advocate the use of mice expressing PDGFRβD849V in their stromal cells as a preclinical model of breast cancer-associated brain metastases and support continued investigation into the clinical prognostic and therapeutic use of PDGFB-to-PDGFRβ signaling in women with breast cancer. SIGNIFICANCE: These studies reveal a previously unknown role for PDGFB-to-PDGFRβ paracrine signaling in the promotion of breast cancer brain metastases and support the prognostic and therapeutic clinical utility of this pathway for patients. See related article by Wyss and colleagues, p. 594 ., (©2020 American Association for Cancer Research.)- Published
- 2021
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11. A Genome-Wide Pooled shRNA Screen Identifies PPP2R2A as a Predictive Biomarker for the Response to ATR and CHK1 Inhibitors.
- Author
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Qiu Z, Fa P, Liu T, Prasad CB, Ma S, Hong Z, Chan ER, Wang H, Li Z, He K, Wang QE, Williams TM, Yan C, Sizemore ST, Narla G, and Zhang J
- Subjects
- Animals, Biomarkers, Tumor genetics, Biomarkers, Tumor metabolism, Carcinoma, Non-Small-Cell Lung drug therapy, Carcinoma, Non-Small-Cell Lung genetics, Carcinoma, Non-Small-Cell Lung metabolism, Cell Line, Tumor, DNA Damage, DNA Replication, Drug Resistance, Neoplasm, Female, Gene Knockdown Techniques, Genes, p53, Genome-Wide Association Study, Heterografts, Humans, Lung Neoplasms drug therapy, Lung Neoplasms genetics, Lung Neoplasms metabolism, Male, Mice, Mice, Nude, Protein Phosphatase 2 genetics, Protein Phosphatase 2 metabolism, Proto-Oncogene Proteins c-myc metabolism, RNA, Small Interfering, Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors, Biomarkers, Tumor deficiency, Carcinoma, Non-Small-Cell Lung chemistry, Checkpoint Kinase 1 antagonists & inhibitors, Lung Neoplasms chemistry, Protein Phosphatase 2 deficiency
- Abstract
There is currently a lack of precise predictive biomarkers for patient selection in clinical trials of inhibitors targeting replication stress (RS) response proteins ATR and CHK1. The objective of this study was to identify novel predictive biomarkers for the response to these agents in treating non-small cell lung cancer (NSCLC). A genome-wide loss-of-function screen revealed that tumor suppressor PPP2R2A, a B regulatory subunit of protein phosphatase 2 (PP2A), determines sensitivity to CHK1 inhibition. A synthetic lethal interaction between PPP2R2A deficiency and ATR or CHK1 inhibition was observed in NSCLC in vitro and in vivo and was independent of p53 status. ATR and CHK1 inhibition resulted in significantly increased levels of RS and altered replication dynamics, particularly in PPP2R2A-deficient NSCLC cells. Mechanistically, PPP2R2A negatively regulated translation of oncogene c-Myc protein. c-Myc activity was required for PPP2R2A deficiency-induced alterations of replication initiation/RS and sensitivity to ATR/CHK1 inhibitors. We conclude that PPP2R2A deficiency elevates RS by upregulating c-Myc activity, rendering cells reliant on the ATR/CHK1 axis for survival. Our studies show a novel synthetic lethal interaction and identify PPP2R2A as a potential new predictive biomarker for patient stratification in the clinical use of ATR and CHK1 inhibitors. SIGNIFICANCE: This study reveals new approaches to specifically target PPP2R2A-deficient lung cancer cells and provides a novel biomarker that will significantly improve treatment outcome with ATR and CHK1 inhibitors., (©2020 American Association for Cancer Research.)
- Published
- 2020
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12. The transcriptional repressor BCL11A promotes breast cancer metastasis.
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Seachrist DD, Hannigan MM, Ingles NN, Webb BM, Weber-Bonk KL, Yu P, Bebek G, Singh S, Sizemore ST, Varadan V, Licatalosi DD, and Keri RA
- Subjects
- Cell Line, Tumor, Disease Progression, Female, Humans, Neoplasm Invasiveness pathology, Neoplasm Metastasis genetics, Neoplasm Metastasis pathology, Triple Negative Breast Neoplasms pathology, Gene Expression Regulation, Neoplastic, Neoplasm Invasiveness genetics, Repressor Proteins genetics, Triple Negative Breast Neoplasms genetics, Up-Regulation
- Abstract
The phenotypes of each breast cancer subtype are defined by their transcriptomes. However, the transcription factors that regulate differential patterns of gene expression that contribute to specific disease outcomes are not well understood. Here, using gene silencing and overexpression approaches, RNA-Seq, and splicing analysis, we report that the transcription factor B-cell leukemia/lymphoma 11A (BCL11A) is highly expressed in triple-negative breast cancer (TNBC) and drives metastatic disease. Moreover, BCL11A promotes cancer cell invasion by suppressing the expression of muscleblind-like splicing regulator 1 ( MBNL1 ), a splicing regulator that suppresses metastasis. This ultimately increases the levels of an alternatively spliced isoform of integrin-α6 ( ITGA6 ), which is associated with worse patient outcomes. These results suggest that BCL11A sustains TNBC cell invasion and metastatic growth by repressing MBNL1-directed splicing of ITGA6 Our findings also indicate that BCL11A lies at the interface of transcription and splicing and promotes aggressive TNBC phenotypes., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Seachrist et al.)
- Published
- 2020
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13. Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells.
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Thies KA, Steck S, Knoblaugh SE, and Sizemore ST
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- Animals, Cell Count, Cell Line, Tumor, Cell Transformation, Neoplastic, Humans, Image Processing, Computer-Assisted, Injections, Intravenous, Mice, Neoplasm Metastasis, Lung Neoplasms pathology, Pathology methods, Tail
- Abstract
Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.
- Published
- 2020
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14. LIN9 and NEK2 Are Core Regulators of Mitotic Fidelity That Can Be Therapeutically Targeted to Overcome Taxane Resistance.
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Roberts MS, Sahni JM, Schrock MS, Piemonte KM, Weber-Bonk KL, Seachrist DD, Avril S, Anstine LJ, Singh S, Sizemore ST, Varadan V, Summers MK, and Keri RA
- Subjects
- Antineoplastic Agents administration & dosage, Antineoplastic Agents pharmacology, Apoptosis, Cell Line, Tumor, Cellular Senescence, Centrosome enzymology, Female, Gene Expression Regulation, Neoplastic, Gene Silencing, Heterografts, Humans, Mitosis genetics, NIMA-Related Kinases metabolism, Neoplasm Proteins antagonists & inhibitors, Neoplasm Proteins metabolism, Nuclear Proteins metabolism, Paclitaxel administration & dosage, Survival Rate, Taxoids administration & dosage, Triple Negative Breast Neoplasms metabolism, Triple Negative Breast Neoplasms mortality, Tumor Stem Cell Assay, Tumor Suppressor Proteins metabolism, Up-Regulation, Drug Resistance, Neoplasm drug effects, Mitosis drug effects, NIMA-Related Kinases antagonists & inhibitors, Nuclear Proteins antagonists & inhibitors, Paclitaxel pharmacology, Taxoids pharmacology, Triple Negative Breast Neoplasms drug therapy, Tumor Suppressor Proteins antagonists & inhibitors
- Abstract
A significant therapeutic challenge for patients with cancer is resistance to chemotherapies such as taxanes. Overexpression of LIN9, a transcriptional regulator of cell-cycle progression, occurs in 65% of patients with triple-negative breast cancer (TNBC), a disease commonly treated with these drugs. Here, we report that LIN9 is further elevated with acquisition of taxane resistance. Inhibiting LIN9 genetically or by suppressing its expression with a global BET inhibitor restored taxane sensitivity by inducing mitotic progression errors and apoptosis. While sustained LIN9 is necessary to maintain taxane resistance, there are no inhibitors that directly repress its function. Hence, we sought to discover a druggable downstream transcriptional target of LIN9. Using a computational approach, we identified NIMA-related kinase 2 (NEK2), a regulator of centrosome separation that is also elevated in taxane-resistant cells. High expression of NEK2 was predictive of low survival rates in patients who had residual disease following treatment with taxanes plus an anthracycline, suggesting a role for this kinase in modulating taxane sensitivity. Like LIN9, genetic or pharmacologic blockade of NEK2 activity in the presence of paclitaxel synergistically induced mitotic abnormalities in nearly 100% of cells and completely restored sensitivity to paclitaxel, in vitro . In addition, suppressing NEK2 activity with two distinct small molecules potentiated taxane response in multiple in vivo models of TNBC, including a patient-derived xenograft, without inducing toxicity. These data demonstrate that the LIN9/NEK2 pathway is a therapeutically targetable mediator of taxane resistance that can be leveraged to improve response to this core chemotherapy. SIGNIFICANCE: Resistance to chemotherapy is a major hurdle for treating patients with cancer. Combining NEK2 inhibitors with taxanes may be a viable approach for improving patient outcomes by enhancing mitotic defects induced by taxanes alone., (©2020 American Association for Cancer Research.)
- Published
- 2020
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15. NEDD4 expression is associated with breast cancer progression and is predictive of a poor prognosis.
- Author
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Wan L, Liu T, Hong Z, Pan Y, Sizemore ST, Zhang J, and Ma Z
- Subjects
- Adult, Biomarkers, Tumor, Breast Neoplasms pathology, Cell Line, Tumor, Disease Progression, Female, Humans, Immunohistochemistry, Kaplan-Meier Estimate, Middle Aged, Nedd4 Ubiquitin Protein Ligases metabolism, Neoplasm Grading, Neoplasm Staging, Prognosis, Proportional Hazards Models, Proto-Oncogene Proteins c-akt metabolism, RNA Interference, Receptor, IGF Type 1 metabolism, Young Adult, Breast Neoplasms genetics, Breast Neoplasms mortality, Gene Expression, Nedd4 Ubiquitin Protein Ligases genetics
- Abstract
Background: A role for neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) in tumorigenesis has been suggested. However, information is lacking on its role in breast tumor biology. The purpose of this study was to determine the role of NEDD4 in the promotion of the growth and progression of breast cancer (BC) and to evaluate the clinicopathologic and prognostic significance of NEDD4., Methods: The impact of NEDD4 expression in BC cell growth was determined by Cell Counting Kit-8 and colony formation assays. Formalin-fixed paraffin-embedded specimens were collected from 133 adjacent normal tissues (ANTs), 445 BC cases composed of pre-invasive ductal carcinoma in situ (DCIS, n = 37), invasive ductal carcinomas (IDC, n = 408, 226 without and 182 with lymph node metastasis), and 116 invaded lymph nodes. The expression of NEDD4 was analyzed by immunohistochemistry. The association between NEDD4 expression and clinicopathological characteristics was analyzed by chi-square test. Survival was evaluated using the Kaplan-Meier method, and curves were compared using a log-rank test. Univariate and multivariate analyses were performed using the Cox regression method., Results: NEDD4 promoted BC growth in vitro. In clinical retrospective studies, 16.5% of ANTs (22/133) demonstrated positive NEDD4 staining. Strikingly, the proportion of cases showing NEDD4-positive staining increased to 51.4% (19/37) in DCIS, 58.4% (132/226) in IDC without lymph node metastasis, and 73.1% (133/182) in BC with lymph node metastasis (BCLNM). In addition, NEDD4-positive staining was associated with clinical parameters, including tumor size (P = 0.030), nodal status (P = 0.001), estrogen receptor status (P = 0.035), and progesterone receptor status (P = 0.023). Moreover, subset analysis in BCLNM revealed that high NEDD4 expression correlated with an elevated risk of relapse (P = 0.0276). Further, NEDD4 expression was an independent prognostic predictor. Lastly, the rates for 10-year overall survival and disease-free survival were significantly lower in patients with positive NEDD4 staining than those in BC patients with negative NEDD4 staining BC (P = 0.0024 and P = 0.0011, respectively)., Conclusions: NEDD4 expression is elevated in BC and is associated with BC growth. NEDD4 correlated with clinicopathological parameters and predicts a poor prognosis. Thus, NEDD4 is a potential biomarker of poor prognosis and a potential therapeutic target for BC treatment.
- Published
- 2019
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16. Pyruvate kinase M2 regulates homologous recombination-mediated DNA double-strand break repair.
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Sizemore ST, Zhang M, Cho JH, Sizemore GM, Hurwitz B, Kaur B, Lehman NL, Ostrowski MC, Robe PA, Miao W, Wang Y, Chakravarti A, and Xia F
- Subjects
- Animals, Cell Line, Tumor, Cell Survival, Female, Humans, Mice, Mice, Nude, Thyroid Hormone-Binding Proteins, Carrier Proteins metabolism, DNA Breaks, Double-Stranded, DNA Repair, Membrane Proteins metabolism, Thyroid Hormones metabolism
- Abstract
Resistance to genotoxic therapies is a primary cause of treatment failure and tumor recurrence. The underlying mechanisms that activate the DNA damage response (DDR) and allow cancer cells to escape the lethal effects of genotoxic therapies remain unclear. Here, we uncover an unexpected mechanism through which pyruvate kinase M2 (PKM2), the highly expressed PK isoform in cancer cells and a master regulator of cancer metabolic reprogramming, integrates with the DDR to directly promote DNA double-strand break (DSB) repair. In response to ionizing radiation and oxidative stress, ATM phosphorylates PKM2 at T328 resulting in its nuclear accumulation. pT328-PKM2 is required and sufficient to promote homologous recombination (HR)-mediated DNA DSB repair through phosphorylation of CtBP-interacting protein (CtIP) on T126 to increase CtIP's recruitment at DSBs and resection of DNA ends. Disruption of the ATM-PKM2-CtIP axis sensitizes cancer cells to a variety of DNA-damaging agents and PARP1 inhibition. Furthermore, increased nuclear pT328-PKM2 level is associated with significantly worse survival in glioblastoma patients. Combined, these data advocate the use of PKM2-targeting strategies as a means to not only disrupt cancer metabolism but also inhibit an important mechanism of resistance to genotoxic therapies.
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- 2018
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17. Stromal PTEN determines mammary epithelial response to radiotherapy.
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Sizemore GM, Balakrishnan S, Thies KA, Hammer AM, Sizemore ST, Trimboli AJ, Cuitiño MC, Steck SA, Tozbikian G, Kladney RD, Shinde N, Das M, Park D, Majumder S, Krishnan S, Yu L, Fernandez SA, Chakravarti A, Shields PG, White JR, Yee LD, Rosol TJ, Ludwig T, Park M, Leone G, and Ostrowski MC
- Subjects
- Animals, Antineoplastic Agents pharmacology, Breast Neoplasms metabolism, Breast Neoplasms pathology, Breast Neoplasms radiotherapy, Cell Proliferation drug effects, Cell Proliferation radiation effects, Cell Transformation, Neoplastic, Epithelial Cells drug effects, Epithelial Cells metabolism, Epithelial Cells radiation effects, ErbB Receptors antagonists & inhibitors, ErbB Receptors genetics, ErbB Receptors metabolism, Female, Gamma Rays adverse effects, Genomic Instability drug effects, Genomic Instability radiation effects, Humans, Mammary Glands, Animal drug effects, Mammary Glands, Animal metabolism, Mammary Glands, Animal radiation effects, Mammary Glands, Human drug effects, Mammary Glands, Human metabolism, Mammary Glands, Human radiation effects, Mammary Neoplasms, Experimental metabolism, Mammary Neoplasms, Experimental pathology, Mammary Neoplasms, Experimental radiotherapy, Mice, PTEN Phosphohydrolase deficiency, Protein Kinase Inhibitors pharmacology, Signal Transduction, Stromal Cells drug effects, Stromal Cells metabolism, Stromal Cells radiation effects, Breast Neoplasms genetics, Gene Expression Regulation, Neoplastic, Mammary Neoplasms, Experimental genetics, PTEN Phosphohydrolase genetics, Radiation Tolerance genetics
- Abstract
The importance of the tumor-associated stroma in cancer progression is clear. However, it remains uncertain whether early events in the stroma are capable of initiating breast tumorigenesis. Here, we show that in the mammary glands of non-tumor bearing mice, stromal-specific phosphatase and tensin homolog (Pten) deletion invokes radiation-induced genomic instability in neighboring epithelium. In these animals, a single dose of whole-body radiation causes focal mammary lobuloalveolar hyperplasia through paracrine epidermal growth factor receptor (EGFR) activation, and EGFR inhibition abrogates these cellular changes. By analyzing human tissue, we discover that stromal PTEN is lost in a subset of normal breast samples obtained from reduction mammoplasty, and is predictive of recurrence in breast cancer patients. Combined, these data indicate that diagnostic or therapeutic chest radiation may predispose patients with decreased stromal PTEN expression to secondary breast cancer, and that prophylactic EGFR inhibition may reduce this risk.
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- 2018
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18. Synthetic Lethality of PARP Inhibition and Ionizing Radiation is p53-dependent.
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Sizemore ST, Mohammad R, Sizemore GM, Nowsheen S, Yu H, Ostrowski MC, Chakravarti A, and Xia F
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- Breast Neoplasms genetics, Breast Neoplasms pathology, Cell Line, Tumor, Cell Proliferation genetics, Cell Proliferation radiation effects, Female, Glioma genetics, Glioma pathology, Humans, Radiation, Ionizing, Recombinational DNA Repair genetics, Recombinational DNA Repair radiation effects, Synthetic Lethal Mutations genetics, BRCA1 Protein genetics, Breast Neoplasms drug therapy, Glioma drug therapy, Poly(ADP-ribose) Polymerase Inhibitors pharmacology, Tumor Suppressor Protein p53 genetics
- Abstract
PARP inhibitors (PARPi) are potentially effective therapeutic agents capable of inducing synthetic lethality in tumors with deficiencies in homologous recombination (HR)-mediated DNA repair such as those carrying BRCA1 mutations. However, BRCA mutations are rare, the majority of tumors are proficient in HR repair, and thus most tumors are resistant to PARPi. Previously, we observed that ionizing radiation (IR) initiates cytoplasmic translocation of BRCA1 leading to suppression of HR-mediated DNA repair and induction of synthetic PARPi lethality in wild-type BRCA1 and HR-proficient tumor cells. The tumor suppressor p53 was identified as a key factor that regulates DNA damage-induced BRCA1 cytoplasmic sequestration following IR. However, the role of p53 in IR-induced PARPi sensitization remains unclear. This study elucidates the role of p53 in IR-induced PARPi cytotoxicity in HR-proficient cancer cells and suggests p53 status may help define a patient population that might benefit from this treatment strategy. Sensitization to PARPi following IR was determined in vitro and in vivo utilizing human breast and glioma tumor cells carrying wild-type BRCA1 and p53, and in associated cells in which p53 function was modified by knockdown or mutation. In breast and glioma cells with proficient HR repair, IR-induced BRCA1 cytoplasmic sequestration, HR repair inhibition, and subsequent PARPi sensitization in vitro and in vivo was dependent upon functional p53. Implications: Implications: p53 status determines PARP inhibitor sensitization by ionizing radiation in multiple BRCA1 and HR-proficient tumor types and may predict which patients are most likely to benefit from combination therapy. Mol Cancer Res; 16(7); 1092-102. ©2018 AACR ., (©2018 American Association for Cancer Research.)
- Published
- 2018
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19. A hedgehog pathway-dependent gene signature is associated with poor clinical outcomes in Luminal A breast cancer.
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Rudolph M, Sizemore ST, Lu Y, Teng KY, Basree MM, Reinbolt R, Timmers CD, Leone G, Ostrowski MC, Majumder S, and Ramaswamy B
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- Adult, Biomarkers, Tumor, Breast Neoplasms mortality, Breast Neoplasms pathology, Cell Line, Tumor, Cell Survival, Drug Resistance, Neoplasm, Ectopic Gene Expression, Female, Gene Expression Profiling, Humans, Middle Aged, Neoplasm Grading, Neoplasm Staging, Prognosis, Receptor, ErbB-2 metabolism, Receptors, Estrogen metabolism, Survival Analysis, Transcriptome, Zinc Finger Protein GLI1 genetics, Zinc Finger Protein GLI1 metabolism, Breast Neoplasms genetics, Breast Neoplasms metabolism, Gene Expression Regulation, Neoplastic, Hedgehog Proteins metabolism, Signal Transduction
- Abstract
Purpose: High expression of glioma-associated oncogene homolog-1 (GLI1) is associated with poor prognosis in estrogen receptor (ER) positive breast cancers. We sought to define a GLI1-dependent gene signature in ER-positive tumors that could further stratify patients at higher risk for disease recurrence and potentially lead to novel combination therapies., Methods: We identified an inverse correlation between GLI1 expression and distant disease-free survival (DFS) using a dataset developed at MD Anderson Cancer Center (Hatzis dataset) containing clinical data from 508 breast cancer patients. Using a qPCR-based microarray platform, we identified genes differentially regulated by GLI1 in MCF7 cells and then determined if expression of these genes correlated with GLI1 expression in patient tumor samples. Statistical comparison between the groups was performed by ANOVA. Direct comparison of two groups was done by a two-tailed t test. Correlations between variables were done by Pearson's method., Results: Expression of GLI1 and its target genes correlated significantly with worse distant DFS in breast cancer patients with Luminal A molecular subtype. Particularly, co-expression of GLI1 with EGFR and/or SNAI1, two of the identified GLI1 targets, was predictive of worse distant DFS in this subtype. Furthermore, patients with Luminal A tumors with a high GLI1 signature had a shorter distant DFS compared to the Luminal B subtype and the outcome for this group was comparable to patients with HER2-positive or basal-like tumors., Conclusion: We have identified a novel GLI1 gene signature that is associated with worse clinical outcomes among the patients with Luminal A subtype of breast cancer.
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- 2018
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20. Stromal PTEN inhibits the expansion of mammary epithelial stem cells through Jagged-1.
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Sizemore GM, Balakrishnan S, Hammer AM, Thies KA, Trimboli AJ, Wallace JA, Sizemore ST, Kladney RD, Woelke SA, Yu L, Fernandez SA, Chakravarti A, Leone G, and Ostrowski MC
- Abstract
This corrects the article DOI: 10.1038/onc.2016.383.
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- 2017
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21. Mitotic Vulnerability in Triple-Negative Breast Cancer Associated with LIN9 Is Targetable with BET Inhibitors.
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Sahni JM, Gayle SS, Webb BM, Weber-Bonk KL, Seachrist DD, Singh S, Sizemore ST, Restrepo NA, Bebek G, Scacheri PC, Varadan V, Summers MK, and Keri RA
- Subjects
- Animals, Apoptosis drug effects, Cell Proliferation drug effects, Female, Humans, Mice, Mice, Inbred NOD, Mice, SCID, Triple Negative Breast Neoplasms metabolism, Triple Negative Breast Neoplasms pathology, Tumor Cells, Cultured, Xenograft Model Antitumor Assays, Mitosis drug effects, Nuclear Proteins antagonists & inhibitors, Protein Kinase Inhibitors pharmacology, Protein Serine-Threonine Kinases antagonists & inhibitors, Triple Negative Breast Neoplasms drug therapy, Tumor Suppressor Proteins antagonists & inhibitors
- Abstract
Triple-negative breast cancers (TNBC) are highly aggressive, lack FDA-approved targeted therapies, and frequently recur, making the discovery of novel therapeutic targets for this disease imperative. Our previous analysis of the molecular mechanisms of action of bromodomain and extraterminal protein inhibitors (BETi) in TNBC revealed these drugs cause multinucleation, indicating BET proteins are essential for efficient mitosis and cytokinesis. Here, using live cell imaging, we show that BET inhibition prolonged mitotic progression and induced mitotic cell death, both of which are indicative of mitotic catastrophe. Mechanistically, the mitosis regulator LIN9 was a direct target of BET proteins that mediated the effects of BET proteins on mitosis in TNBC. Although BETi have been proposed to function by dismantling super-enhancers (SE), the LIN9 gene lacks an SE but was amplified or overexpressed in the majority of TNBCs. In addition, its mRNA expression predicted poor outcome across breast cancer subtypes. Together, these results provide a mechanism for cancer selectivity of BETi that extends beyond modulation of SE-associated genes and suggest that cancers dependent upon LIN9 overexpression may be particularly vulnerable to BETi. Cancer Res; 77(19); 5395-408. ©2017 AACR ., (©2017 American Association for Cancer Research.)
- Published
- 2017
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22. Follistatin is a metastasis suppressor in a mouse model of HER2-positive breast cancer.
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Seachrist DD, Sizemore ST, Johnson E, Abdul-Karim FW, Weber Bonk KL, and Keri RA
- Subjects
- Animals, Breast Neoplasms metabolism, Breast Neoplasms mortality, Cell Line, Tumor, Cell Movement genetics, Cell Proliferation, Cell Survival genetics, Databases, Genetic, Female, Follistatin metabolism, Humans, Kaplan-Meier Estimate, Mice, Mice, Transgenic, Neoplasm Metastasis, Neoplasm Staging, Prognosis, Receptor, ErbB-2 metabolism, Tumor Suppressor Proteins metabolism, Breast Neoplasms genetics, Breast Neoplasms pathology, Follistatin genetics, Receptor, ErbB-2 genetics, Tumor Suppressor Proteins genetics
- Abstract
Background: Follistatin (FST) is an intrinsic inhibitor of activin, a member of the transforming growth factor-β superfamily of ligands. The prognostic value of FST and its family members, the follistatin-like (FSTL) proteins, have been studied in various cancers. However, these studies, as well as limited functional analyses of the FSTL proteins, have yielded conflicting results on the role of these proteins in disease progression. Furthermore, very few have been focused on FST itself. We assessed whether FST may be a suppressor of tumorigenesis and/or metastatic progression in breast cancer., Methods: Using publicly available gene expression data, we examined the expression patterns of FST and INHBA, a subunit of activin, in normal and cancerous breast tissue and the prognostic value of FST in breast cancer metastases, recurrence-free survival, and overall survival. The functional effects of activin and FST on in vitro proliferation, migration, and invasion of breast cancer cells were also examined. FST overexpression in an autochthonous mouse model of breast cancer was then used to assess the in vivo impact of FST on metastatic progression., Results: Examination of multiple breast cancer datasets revealed that FST expression is reduced in breast cancers compared with normal tissue and that low FST expression predicts increased metastasis and reduced overall survival. FST expression was also reduced in a mouse model of HER2/Neu-induced metastatic breast cancer. We found that FST blocks activin-induced breast epithelial cell migration in vitro, suggesting that its loss may promote breast cancer aggressiveness. To directly determine if FST restoration could inhibit metastatic progression, we transgenically expressed FST in the HER2/Neu model. Although FST had no impact on tumor initiation or growth, it completely blocked the formation of lung metastases., Conclusions: These data indicate that FST is a bona fide metastasis suppressor in this mouse model and support future efforts to develop an FST mimetic to suppress metastatic progression.
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- 2017
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23. Stromal PDGFR-α Activation Enhances Matrix Stiffness, Impedes Mammary Ductal Development, and Accelerates Tumor Growth.
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Hammer AM, Sizemore GM, Shukla VC, Avendano A, Sizemore ST, Chang JJ, Kladney RD, Cuitiño MC, Thies KA, Verfurth Q, Chakravarti A, Yee LD, Leone G, Song JW, Ghadiali SN, and Ostrowski MC
- Subjects
- Animals, Cell Differentiation genetics, Extracellular Matrix genetics, Female, Gene Expression Regulation, Developmental genetics, Gene Expression Regulation, Neoplastic genetics, Humans, Hyaluronic Acid administration & dosage, Mammary Glands, Animal pathology, Mammary Glands, Human pathology, Mammary Neoplasms, Animal pathology, Mice, Morphogenesis genetics, Signal Transduction, Stromal Cells pathology, Mammary Glands, Animal growth & development, Mammary Glands, Human growth & development, Mammary Neoplasms, Animal genetics, Receptor, Platelet-Derived Growth Factor alpha genetics
- Abstract
The extracellular matrix (ECM) is critical for mammary ductal development and differentiation, but how mammary fibroblasts regulate ECM remodeling remains to be elucidated. Herein, we used a mouse genetic model to activate platelet derived growth factor receptor-alpha (PDGFRα) specifically in the stroma. Hyperactivation of PDGFRα in the mammary stroma severely hindered pubertal mammary ductal morphogenesis, but did not interrupt the lobuloalveolar differentiation program. Increased stromal PDGFRα signaling induced mammary fat pad fibrosis with a corresponding increase in interstitial hyaluronic acid (HA) and collagen deposition. Mammary fibroblasts with PDGFRα hyperactivation also decreased hydraulic permeability of a collagen substrate in an in vitro microfluidic device assay, which was mitigated by inhibition of either PDGFRα or HA. Fibrosis seen in this model significantly increased the overall stiffness of the mammary gland as measured by atomic force microscopy. Further, mammary tumor cells injected orthotopically in the fat pads of mice with stromal activation of PDGFRα grew larger tumors compared to controls. Taken together, our data establish that aberrant stromal PDGFRα signaling disrupts ECM homeostasis during mammary gland development, resulting in increased mammary stiffness and increased potential for tumor growth., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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24. Changes in BAI1 and nestin expression are prognostic indicators for survival and metastases in breast cancer and provide opportunities for dual targeted therapies.
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Meisen WH, Dubin S, Sizemore ST, Mathsyaraja H, Thies K, Lehman NL, Boyer P, Jaime-Ramirez AC, Elder JB, Powell K, Chakravarti A, Ostrowski MC, and Kaur B
- Subjects
- Animals, Breast Neoplasms pathology, Cell Line, Tumor, Chlorocebus aethiops, Female, Gene Expression Regulation, Neoplastic drug effects, Humans, MCF-7 Cells, Mice, Neoplasm Metastasis, Oncolytic Virotherapy, Prognosis, Receptors, G-Protein-Coupled, Vero Cells, Angiogenic Proteins metabolism, Biomarkers, Tumor metabolism, Breast Neoplasms metabolism, Breast Neoplasms therapy, Nestin metabolism, Oncolytic Viruses genetics
- Abstract
The 2-year survival rate of patients with breast cancer brain metastases is less than 2%. Treatment options for breast cancer brain metastases are limited, and there is an unmet need to identify novel therapies for this disease. Brain angiogenesis inhibitor 1 (BAI1) is a GPCR involved in tumor angiogenesis, invasion, phagocytosis, and synaptogenesis. For the first time, we identify that BAI1 expression is significantly reduced in breast cancer and higher expression is associated with better patient survival. Nestin is an intermediate filament whose expression is upregulated in several cancers. We found that higher Nestin expression significantly correlated with breast cancer lung and brain metastases, suggesting both BAI1 and Nestin can be therapeutic targets for this disease. Here, we demonstrate the ability of an oncolytic virus, 34.5ENVE, to target and kill high Nestin-expressing cells and deliver Vstat120 (extracellular fragment of BAI1). Finally, we created two orthotopic immune-competent murine models of breast cancer brain metastases and demonstrated 34.5ENVE extended the survival of immune-competent mice bearing intracranial breast cancer tumors., (©2014 American Association for Cancer Research.)
- Published
- 2015
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25. GABA(A) receptor pi (GABRP) stimulates basal-like breast cancer cell migration through activation of extracellular-regulated kinase 1/2 (ERK1/2).
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Sizemore GM, Sizemore ST, Seachrist DD, and Keri RA
- Subjects
- Breast Neoplasms enzymology, Cell Line, Enzyme Activation, Gene Expression Profiling, Gene Silencing, Humans, Real-Time Polymerase Chain Reaction, Receptors, GABA-A genetics, Survival Analysis, Breast Neoplasms pathology, MAP Kinase Signaling System, Receptors, GABA-A physiology
- Abstract
Breast cancer is a heterogeneous disease comprised of distinct subtypes predictive of patient outcome. Tumors of the basal-like subtype have a poor prognosis due to inherent aggressiveness and the lack of targeted therapeutics. Basal-like tumors typically lack estrogen receptor-α, progesterone receptor and HER2/ERBB2, or in other words they are triple negative (TN). Continued evaluation of basal-like breast cancer (BLBC) biology is essential to identify novel therapeutic targets. Expression of the pi subunit of the GABA(A) receptor (GABRP) is associated with the BLBC/TN subtype, and herein, we reveal its expression also correlates with metastases to the brain and poorer patient outcome. GABRP expression in breast cancer cell lines also demonstrates a significant correlation with the basal-like subtype suggesting that GABRP functions in the initiation and/or progression of basal-like tumors. To address this postulate, we stably silenced GABRP in two BLBC cell lines, HCC1187 and HCC70 cells. Decreased GABRP reduces in vitro tumorigenic potential and migration concurrent with alterations in the cytoskeleton, specifically diminished cellular protrusions and expression of the BLBC-associated cytokeratins, KRT5, KRT6B, KRT14, and KRT17. Silencing GABRP also decreases phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) in both cell lines and selective inhibition of ERK1/2 similarly decreases the basal-like cytokeratins as well as migration. Combined, these data reveal a GABRP-ERK1/2-cytokeratin axis that maintains the migratory phenotype of basal-like breast cancer. GABRP is a component of a cell surface receptor, thus, these findings suggest that targeting this new signaling axis may have therapeutic potential in BLBC., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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26. Hypomethylation of the MMP7 promoter and increased expression of MMP7 distinguishes the basal-like breast cancer subtype from other triple-negative tumors.
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Sizemore ST, Sizemore GM, Booth CN, Thompson CL, Silverman P, Bebek G, Abdul-Karim FW, Avril S, and Keri RA
- Subjects
- Adult, Aged, Breast Neoplasms diagnosis, Breast Neoplasms mortality, Cell Line, Tumor, CpG Islands, Datasets as Topic, Female, Follow-Up Studies, Gene Expression, Humans, Middle Aged, Neoplasm Grading, Neoplasm Staging, Neoplasms, Basal Cell diagnosis, Neoplasms, Basal Cell mortality, Triple Negative Breast Neoplasms diagnosis, Triple Negative Breast Neoplasms mortality, Tumor Burden, Breast Neoplasms genetics, DNA Methylation, Matrix Metalloproteinase 7 genetics, Neoplasms, Basal Cell genetics, Promoter Regions, Genetic, Triple Negative Breast Neoplasms genetics
- Abstract
Identification of novel targets for the treatment of basal-like breast cancer is essential for improved outcomes in patients with this disease. This study investigates the association of MMP7 expression and MMP7 promoter methylation with subtype and outcome in breast cancer patient cohorts. Immunohistochemical analysis was performed on a breast cancer tissue microarray and validated in independent histological samples. MMP7 expression significantly correlated with patient age, tumor size, triple-negative (TN) status, and recurrence. Analysis of publically available datasets confirmed MMP7 gene expression as a prognostic marker of breast cancer metastasis, particularly metastasis to the brain and lungs. Methylation of the MMP7 promoter was assessed by methylation-specific PCR in a panel of breast cancer cell lines and patient tumor samples. Hypomethylation of the MMP7 promoter significantly correlated with TN status in DNA from patient tumor samples, and this association was confirmed using The Cancer Genome Atlas (TCGA) dataset. Evaluation of a panel of breast cancer cell lines and data from the Curtis and TCGA breast carcinoma datasets revealed that elevated MMP7 expression and MMP7 promoter hypomethylation are specific biomarkers of the basal-like molecular subtype which shares considerable, but not complete, overlap with the clinical TN subtype. Importantly, MMP7 expression was identified as an independent predictor of pathological complete response in a large breast cancer patient cohort. Combined, these data suggest that MMP7 expression and MMP7 promoter methylation may be useful as prognostic biomarkers. Furthermore, MMP7 expression and promoter methylation analysis may be effective mechanisms to distinguish basal-like breast cancers from other triple-negative subtypes. Finally, these data implicate MMP7 as a potential therapeutic target for the treatment of basal-like breast cancers.
- Published
- 2014
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27. Protein kinase C Beta in the tumor microenvironment promotes mammary tumorigenesis.
- Author
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Wallace JA, Pitarresi JR, Sharma N, Palettas M, Cuitiño MC, Sizemore ST, Yu L, Sanderlin A, Rosol TJ, Mehta KD, Sizemore GM, and Ostrowski MC
- Abstract
Protein kinase C beta (PKCβ) expression in breast cancer is associated with a more aggressive tumor phenotype, yet the mechanism for how PKCβ is pro-tumorigenic in this disease is still unclear. Interestingly, while it is known that PKCβ mediates angiogenesis, immunity, fibroblast function and adipogenesis, all components of the mammary tumor microenvironment (TME), no study to date has investigated whether stromal PKCβ is functionally relevant in breast cancer. Herein, we evaluate mouse mammary tumor virus-polyoma middle T-antigen (MMTV-PyMT) induced mammary tumorigenesis in the presence and absence of PKCβ. We utilize two model systems: one where PKCβ is deleted in both the epithelial and stromal compartments to test the global requirement for PKCβ on tumor formation, and second, where PKCβ is deleted only in the stromal compartment to test its role in the TME. MMTV-PyMT mice globally lacking PKCβ live longer and develop smaller tumors with decreased proliferation and decreased macrophage infiltration. Similarly, when PKCβ is null exclusively in the stroma, PyMT-driven B6 cells form smaller tumors with diminished collagen deposition. These experiments reveal for the first time a tumor promoting role for stromal PKCβ in MMTV-PyMT tumorigenesis. In corroboration with these results, PKCβ mRNA (Prkcb) is increased in fibroblasts isolated from MMTV-PyMT tumors. These data were confirmed in a breast cancer patient cohort. Combined these data suggest the continued investigation of PKCβ in the mammary TME is necessary to elucidate how to effectively target this signaling pathway in breast cancer.
- Published
- 2014
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28. FOXC1 is enriched in the mammary luminal progenitor population, but is not necessary for mouse mammary ductal morphogenesis.
- Author
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Sizemore GM, Sizemore ST, Pal B, Booth CN, Seachrist DD, Abdul-Karim FW, Kume T, and Keri RA
- Subjects
- Adult Stem Cells cytology, Adult Stem Cells physiology, Animals, Epithelial Cells cytology, Female, Forkhead Transcription Factors physiology, Male, Mammary Glands, Animal growth & development, Mice, Mice, Inbred C57BL, Morphogenesis, Epithelial Cells physiology, Forkhead Transcription Factors metabolism, Mammary Glands, Animal cytology, Mammary Glands, Animal metabolism
- Abstract
Expression of FOXC1, a forkhead box transcription factor, correlates with the human basal-like breast cancer (BLBC) subtype, and functional analyses have revealed its importance for in vitro invasiveness of BLBC cells. Women diagnosed with this breast tumor subtype have a poorer outcome because of the lack of targeted therapies; thus, continued investigation of factors driving these tumors is critical to uncover novel therapeutic targets. Several processes that dictate normal mammary morphogenesis parallel cancer progression, and enforced expression of FOXC1 can induce a progenitor state in more-differentiated mammary epithelial cells. Consequently, evaluating how FOXC1 functions in the normal gland is critical to further understand BLBC biology. Although FOXC1 is well known to control normal development of a number of tissues, its role in the mammary gland has not yet been investigated. Herein, we describe FOXC1 expression patterning in the normal breast, where it is localized to the basal/myoepithelium, suggesting that FOXC1 would be required for normal development. However, mammary glands lacking Foxc1 have no overt defect in ductal outgrowth, alveologenesis, or lineage specification. Of significant interest, we found that expression of FOXC1 is enriched in the normal luminal progenitor population, which is the postulated cell of origin of BLBC. These results indicate that FOXC1 is unnecessary for mammary morphogenesis and that its role in BLBC likely involves processes that are unrelated to cell lineage specification.
- Published
- 2013
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29. FOXA1 represses the molecular phenotype of basal breast cancer cells.
- Author
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Bernardo GM, Bebek G, Ginther CL, Sizemore ST, Lozada KL, Miedler JD, Anderson LA, Godwin AK, Abdul-Karim FW, Slamon DJ, and Keri RA
- Subjects
- Breast Neoplasms pathology, Cell Line, Tumor, Cell Movement, Female, Gene Expression Regulation, Neoplastic, Hepatocyte Nuclear Factor 3-alpha genetics, Humans, Phenotype, Prognosis, Receptors, Estrogen metabolism, Breast Neoplasms metabolism, Hepatocyte Nuclear Factor 3-alpha metabolism, Neoplasms, Basal Cell metabolism
- Abstract
Breast cancer is a heterogeneous disease that comprises multiple subtypes. Luminal subtype tumors confer a more favorable patient prognosis, which is, in part, attributed to estrogen receptor (ER)-α positivity and antihormone responsiveness. Expression of the forkhead box transcription factor, FOXA1, similarly correlates with the luminal subtype and patient survival, but is also present in a subset of ER-negative tumors. FOXA1 is also consistently expressed in luminal breast cancer cell lines even in the absence of ER. In contrast, breast cancer cell lines representing the basal subtype do not express FOXA1. To delineate an ER-independent role for FOXA1 in maintaining the luminal phenotype, and hence a more favorable prognosis, we performed expression microarray analyses on FOXA1-positive and ER-positive (MCF7, T47D), or FOXA1-positive and ER-negative (MDA-MB-453, SKBR3) luminal cell lines in the presence or absence of transient FOXA1 silencing. This resulted in three FOXA1 transcriptomes: (1) a luminal signature (consistent across cell lines), (2) an ER-positive signature (restricted to MCF7 and T47D) and (3) an ER-negative signature (restricted to MDA-MB-453 and SKBR3). Gene set enrichment analyses revealed FOXA1 silencing causes a partial transcriptome shift from luminal to basal gene expression signatures. FOXA1 binds to a subset of both luminal and basal genes within luminal breast cancer cells, and loss of FOXA1 increases enhancer RNA transcription for a representative basal gene (CD58). These data suggest FOXA1 directly represses a subset of basal signature genes. Functionally, FOXA1 silencing increases migration and invasion of luminal cancer cells, both of which are characteristics of basal subtype cells. We conclude FOXA1 controls plasticity between basal and luminal breast cancer cells, not only by inducing luminal genes but also by repressing the basal phenotype, and thus aggressiveness. Although it has been proposed that FOXA1-targeting agents may be useful for treating luminal tumors, these data suggest that this approach may promote transitions toward more aggressive cancers.
- Published
- 2013
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30. The forkhead box transcription factor FOXC1 promotes breast cancer invasion by inducing matrix metalloprotease 7 (MMP7) expression.
- Author
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Sizemore ST and Keri RA
- Subjects
- Blotting, Western, Cell Line, Tumor, Cell Movement genetics, Cell Movement physiology, Forkhead Transcription Factors genetics, Gene Expression Regulation, Neoplastic genetics, Gene Expression Regulation, Neoplastic physiology, Gene Silencing physiology, Humans, Matrix Metalloproteinase 7 genetics, Real-Time Polymerase Chain Reaction, Breast Neoplasms enzymology, Breast Neoplasms metabolism, Forkhead Transcription Factors metabolism, Matrix Metalloproteinase 7 metabolism
- Abstract
Therapeutic options for treatment of basal-like breast cancers are limited and identification of molecular targets for novel therapies to treat this aggressive cancer is urgently needed. Recently, FOXC1, a forkhead box transcription factor, was identified as a functionally important biomarker of breast cancer aggressiveness and the basal-like breast cancer subtype. However, the mechanism through which FOXC1 controls aggressiveness of basal-like breast cancer remains to be elucidated. Here, we identify matrix metalloprotease 7 (MMP7) as a key downstream effector of FOXC1-mediated invasiveness. Expression of FOXC1 and MMP7 is significantly correlated in breast cancer samples and cell lines at both the mRNA and protein levels. Transient expression of FOXC1 in nontransformed mammary epithelial cell lines resulted in significantly increased expression of MMP7 and an MMP7-dependent increase in invasiveness. In reciprocal experiments, silencing endogenous FOXC1 in basal-like breast cancer cell lines resulted in decreased expression of MMP7 without decreased expression of other matrix metalloproteinases. We also demonstrate that elevated co-expression of FOXC1 and MMP7 is an independent predictor of patient outcome in multivariate analyses of two breast cancer patient cohorts. Together, our findings identify MMP7 as a novel mechanism through which FOXC1 may regulate the basal-like breast cancer invasive phenotype and the propensity of these cancers to metastasize. Furthermore, our findings demonstrate for the first time a correlation between MMP7 expression and basal-like breast cancers, suggesting that MMP7 may be a useful therapeutic target for treatment of this disease.
- Published
- 2012
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31. Cloning and characterization of scon-3+, a new member of the Neurospora crassa sulfur regulatory system.
- Author
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Sizemore ST and Paietta JV
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Arylsulfatases metabolism, Base Sequence, Blotting, Northern, Cloning, Molecular, Ligases, Molecular Sequence Data, Mutation, Phenotype, Plasmids metabolism, Point Mutation, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, RNA, Messenger metabolism, Recombinant Fusion Proteins metabolism, Sequence Homology, Amino Acid, Transcription, Genetic, Two-Hybrid System Techniques, Ubiquitin-Protein Ligases, Fungal Proteins chemistry, Fungal Proteins genetics, Neurospora crassa metabolism, Sulfur metabolism, Transcription Factors chemistry, Transcription Factors genetics
- Abstract
The sulfur regulatory system of Neurospora crassa consists of a group of sulfur-regulated structural genes (e.g., arylsulfatase) that are under coordinate control of the CYS3 positive regulator and sulfur controller (SCON) negative regulators. Here we report on the cloning of scon-3(+), which encodes a polypeptide of 171 amino acids and is a Skp1 family homolog. Repeat-induced point mutation of scon-3(+) resulted in a phenotype of constitutive expression of arylsulfatase, a phenotype consistent with other sulfur controller mutants. Northern analysis indicated that, unlike other members of the sulfur regulatory system, expression of scon-3(+) is not under the direct control of the CYS3 transcriptional activator. In particular, scon-3(+) mRNA was detectable under sulfur repressing or derepressing conditions in a Deltacys-3 mutant. In yeast, Skp1p and an F-box protein binding partner are core constituents of a class of E3 ubiquitin ligases known as SCF complexes. The N. crassa negative regulator SCON2 contains an F-box motif essential for the operation of the sulfur regulatory system and suggests a role for an SCF complex in the N. crassa sulfur regulatory system. A crucial set of experiments, by using a yeast two-hybrid approach with confirming coimmunoprecipitation assays, demonstrated that SCON3 interacts with SCON2 in a manner dependent upon the F-box motif of SCON2. The protein-protein interaction detected between SCON2 and SCON3 represents the initial demonstration in a filamentous fungus of functional interaction between putative core components of a SCF complex.
- Published
- 2002
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