Publications by authors named "Petra den Hollander"

25 Publications

  • Page 1 of 1

Vimentin and cytokeratin: Good alone, bad together.

Semin Cancer Biol 2021 Dec 22. Epub 2021 Dec 22.

Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, United States. Electronic address:

The cytoskeleton plays an integral role in maintaining the integrity of epithelial cells. Epithelial cells primarily employ cytokeratin in their cytoskeleton, whereas mesenchymal cells use vimentin. During the epithelial-mesenchymal transition (EMT), cytokeratin-positive epithelial cells begin to express vimentin. EMT induces stem cell properties and drives metastasis, chemoresistance, and tumor relapse. Most studies of the functions of cytokeratin and vimentin have relied on the use of either epithelial or mesenchymal cell types. However, it is important to understand how these two cytoskeleton intermediate filaments function when co-expressed in cells undergoing EMT. Here, we discuss the individual and shared functions of cytokeratin and vimentin that coalesce during EMT and how alterations in intermediate filament expression influence carcinoma progression.
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http://dx.doi.org/10.1016/j.semcancer.2021.12.006DOI Listing
December 2021

Breast cancer dormancy: need for clinically relevant models to address current gaps in knowledge.

NPJ Breast Cancer 2021 May 28;7(1):66. Epub 2021 May 28.

Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.

Breast cancer is the most commonly diagnosed cancer in the USA. Although advances in treatment over the past several decades have significantly improved the outlook for this disease, most women who are diagnosed with estrogen receptor positive disease remain at risk of metastatic relapse for the remainder of their life. The cellular source of late relapse in these patients is thought to be disseminated tumor cells that reactivate after a long period of dormancy. The biology of these dormant cells and their natural history over a patient's lifetime is largely unclear. We posit that research on tumor dormancy has been significantly limited by the lack of clinically relevant models. This review will discuss existing dormancy models, gaps in biological understanding, and propose criteria for future models to enhance their clinical relevance.
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http://dx.doi.org/10.1038/s41523-021-00269-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163741PMC
May 2021

Identification of EMT signaling cross-talk and gene regulatory networks by single-cell RNA sequencing.

Proc Natl Acad Sci U S A 2021 05;118(19)

Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX 77030;

The epithelial-to-mesenchymal transition (EMT) plays a critical role during normal development and in cancer progression. EMT is induced by various signaling pathways, including TGF-β, BMP, Wnt-β-catenin, NOTCH, Shh, and receptor tyrosine kinases. In this study, we performed single-cell RNA sequencing on MCF10A cells undergoing EMT by TGF-β1 stimulation. Our comprehensive analysis revealed that cells progress through EMT at different paces. Using pseudotime clustering reconstruction of gene-expression profiles during EMT, we found sequential and parallel activation of EMT signaling pathways. We also observed various transitional cellular states during EMT. We identified regulatory signaling nodes that drive EMT with the expression of important microRNAs and transcription factors. Using a random circuit perturbation methodology, we demonstrate that the NOTCH signaling pathway acts as a key driver of TGF-β-induced EMT. Furthermore, we demonstrate that the gene signatures of pseudotime clusters corresponding to the intermediate hybrid EMT state are associated with poor patient outcome. Overall, this study provides insight into context-specific drivers of cancer progression and highlights the complexities of the EMT process.
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http://dx.doi.org/10.1073/pnas.2102050118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126782PMC
May 2021

CD8 T cells inhibit metastasis and CXCL4 regulates its function.

Br J Cancer 2021 07 1;125(2):176-189. Epub 2021 Apr 1.

Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Background: The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival.

Methods: We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout (CD8 and CD4) models and administered CXCL4. Tumours and lungs were analysed for cancer cells by bioluminescence, and circulating tumour cells were isolated from blood. Immunohistochemistry on the mouse tumours was performed to confirm cell type, and on a tissue microarray with 180 TNBCs for human relevance. TCGA data from over 10,000 patients were analysed as well.

Results: We reveal that intratumoral immune infiltration differs between metastatic and non-metastatic tumours. The non-metastatic tumours harbour high levels of CD8 T cells and low levels of platelets, which is reverse in metastatic tumours. During tumour progression, platelets and CXCL4 induce differentiation of monocytes into myeloid-derived suppressor cells (MDSCs), which inhibit CD8 T-cell function. TCGA pan-cancer data confirmed that CD8Platelet patients have a significantly lower survival probability compared to CD8Platelet.

Conclusions: CD8 T cells inhibit metastasis. When the balance between CD8 T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8 T-cell function.
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http://dx.doi.org/10.1038/s41416-021-01338-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292398PMC
July 2021

EMTome: a resource for pan-cancer analysis of epithelial-mesenchymal transition genes and signatures.

Br J Cancer 2021 01 10;124(1):259-269. Epub 2020 Dec 10.

Department of Translational Molecular Pathology, Division of Pathology/Lab Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.

Background: The epithelial-mesenchymal transition (EMT) enables dissociation of tumour cells from the primary tumour mass, invasion through the extracellular matrix, intravasation into blood vessels and colonisation of distant organs. Cells that revert to the epithelial state via the mesenchymal-epithelial transition cause metastases, the primary cause of death in cancer patients. EMT also empowers cancer cells with stem-cell properties and induces resistance to chemotherapeutic drugs. Understanding the driving factors of EMT is critical for the development of effective therapeutic interventions.

Methods: This manuscript describes the generation of a database containing EMT gene signatures derived from cell lines, patient-derived xenografts and patient studies across cancer types and multiomics data and the creation of a web-based portal to provide a comprehensive analysis resource.

Results: EMTome incorporates (i) EMT gene signatures; (ii) EMT-related genes with multiomics features across different cancer types; (iii) interactomes of EMT-related genes (miRNAs, transcription factors, and proteins); (iv) immune profiles identified from The Cancer Genome Atlas (TCGA) cohorts by exploring transcriptomics, epigenomics, and proteomics, and drug sensitivity and (iv) clinical outcomes of cancer cohorts linked to EMT gene signatures.

Conclusion: The web-based EMTome portal is a resource for primary and metastatic tumour research publicly available at www.emtome.org .
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http://dx.doi.org/10.1038/s41416-020-01178-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782839PMC
January 2021

The Epithelial to Mesenchymal Transition Promotes Glutamine Independence by Suppressing Expression.

Cancers (Basel) 2019 Oct 22;11(10). Epub 2019 Oct 22.

Department of Translational Molecular Pathology, MD Anderson Cancer Center, Houston, TX 77030, USA.

Identifying bioenergetics that facilitate the epithelial to mesenchymal transition (EMT) in breast cancer cells may uncover targets to treat incurable metastatic disease. Metastasis is the number one cause of cancer-related deaths; therefore, it is urgent to identify new treatment strategies to prevent the initiation of metastasis. To characterize the bioenergetics of EMT, we compared metabolic activities and gene expression in cells induced to differentiate into the mesenchymal state with their epithelial counterparts. We found that levels of , which encodes a glutaminase, are inversely associated with EMT. down-regulation was correlated with reduced mitochondrial activity and glutamine independence even in low-glucose conditions. Restoration of expression in -negative breast cancer cells rescued mitochondrial activity, enhanced glutamine utilization, and inhibited stem-cell properties. Additionally, inhibition of expression of the transcription factor FOXC2, a critical regulator of EMT in -negative cells, restored GLS2 expression and glutamine utilization. Furthermore, in breast cancer patients, high expression is associated with improved survival. These findings suggest that epithelial cancer cells rely on glutamine and that cells induced to undergo EMT become glutamine independent. Moreover, the inhibition of EMT leads to a GLS2-directed metabolic shift in mesenchymal cancer cells, which may make these cells susceptible to chemotherapies.
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http://dx.doi.org/10.3390/cancers11101610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826439PMC
October 2019

UDP-glucose 6-dehydrogenase regulates hyaluronic acid production and promotes breast cancer progression.

Oncogene 2020 04 15;39(15):3089-3101. Epub 2019 Jul 15.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, 77030, USA.

An improved understanding of the biochemical alterations that accompany tumor progression and metastasis is necessary to inform the next generation of diagnostic tools and targeted therapies. Metabolic reprogramming is known to occur during the epithelial-mesenchymal transition (EMT), a process that promotes metastasis. Here, we identify metabolic enzymes involved in extracellular matrix remodeling that are upregulated during EMT and are highly expressed in patients with aggressive mesenchymal-like breast cancer. Activation of EMT significantly increases production of hyaluronic acid, which is enabled by the reprogramming of glucose metabolism. Using genetic and pharmacological approaches, we show that depletion of the hyaluronic acid precursor UDP-glucuronic acid is sufficient to inhibit several mesenchymal-like properties including cellular invasion and colony formation in vitro, as well as tumor growth and metastasis in vivo. We found that depletion of UDP-glucuronic acid altered the expression of PPAR-gamma target genes and increased PPAR-gamma DNA-binding activity. Taken together, our findings indicate that the disruption of EMT-induced metabolic reprogramming affects hyaluronic acid production, as well as associated extracellular matrix remodeling and represents pharmacologically actionable target for the inhibition of aggressive mesenchymal-like breast cancer progression.
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http://dx.doi.org/10.1038/s41388-019-0885-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960374PMC
April 2020

GSK3β regulates epithelial-mesenchymal transition and cancer stem cell properties in triple-negative breast cancer.

Breast Cancer Res 2019 03 7;21(1):37. Epub 2019 Mar 7.

Department of Translational Molecular Pathology, UT MD Anderson Cancer Center, Houston, TX, USA.

Background: Triple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC.

Methods: A library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3β inhibitors were identified as EMT inhibitors. The effects of GSK3β inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3β correlates with the overall survival of breast cancer patients.

Results: We identified a GSK3β inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3β inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3β reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3β inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24- in cells after exposure to GSK3β inhibitors. We found that GSK3β inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3β inhibitors and found that GSK3β inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3β correlated with poorer overall patient survival.

Conclusions: Taken together, our data demonstrate that GSK3β is a potential target for TNBCs and suggest that GSK3β inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3β inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.
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http://dx.doi.org/10.1186/s13058-019-1125-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407242PMC
March 2019

Distinguishing mechanisms underlying EMT tristability.

Cancer Converg 2017 1;1(1). Epub 2017 Nov 1.

1Center for Theoretical Biological Physics, Rice University, Houston, TX 77005 USA.

Background: The Epithelial-Mesenchymal Transition (EMT) endows epithelial-looking cells with enhanced migratory ability during embryonic development and tissue repair. EMT can also be co-opted by cancer cells to acquire metastatic potential and drug-resistance. Recent research has argued that epithelial (E) cells can undergo either a partial EMT to attain a hybrid epithelial/mesenchymal (E/M) phenotype that typically displays collective migration, or a complete EMT to adopt a mesenchymal (M) phenotype that shows individual migration. The core EMT regulatory network - miR-34/SNAIL/miR-200/ZEB1 - has been identified by various studies, but how this network regulates the transitions among the E, E/M, and M phenotypes remains controversial. Two major mathematical models - ternary chimera switch (TCS) and cascading bistable switches (CBS) - that both focus on the miR-34/SNAIL/miR-200/ZEB1 network, have been proposed to elucidate the EMT dynamics, but a detailed analysis of how well either or both of these two models can capture recent experimental observations about EMT dynamics remains to be done.

Results: Here, via an integrated experimental and theoretical approach, we first show that both these two models can be used to understand the two-step transition of EMT - E→E/M→M, the different responses of SNAIL and ZEB1 to exogenous TGF-β and the irreversibility of complete EMT. Next, we present new experimental results that tend to discriminate between these two models. We show that ZEB1 is present at intermediate levels in the hybrid E/M H1975 cells, and that in HMLE cells, overexpression of SNAIL is not sufficient to initiate EMT in the absence of ZEB1 and FOXC2.

Conclusions: These experimental results argue in favor of the TCS model proposing that miR-200/ZEB1 behaves as a three-way decision-making switch enabling transitions among the E, hybrid E/M and M phenotypes.
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http://dx.doi.org/10.1186/s41236-017-0005-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876698PMC
November 2017

Analysis of phosphatases in ER-negative breast cancers identifies DUSP4 as a critical regulator of growth and invasion.

Breast Cancer Res Treat 2016 08 8;158(3):441-54. Epub 2016 Jul 8.

Department of Clinical Cancer Prevention, The University of Texas M.D. Anderson Cancer Center, Houston, TX, 77030, USA.

Estrogen receptor (ER)-negative cancers have a poor prognosis, and few targeted therapies are available for their treatment. Our previous analyses have identified potential kinase targets critical for the growth of ER-negative, progesterone receptor (PR)-negative and HER2-negative, or "triple-negative" breast cancer (TNBC). Because phosphatases regulate the function of kinase signaling pathways, in this study, we investigated whether phosphatases are also differentially expressed in ER-negative compared to those in ER-positive breast cancers. We compared RNA expression in 98 human breast cancers (56 ER-positive and 42 ER-negative) to identify phosphatases differentially expressed in ER-negative compared to those in ER-positive breast cancers. We then examined the effects of one selected phosphatase, dual specificity phosphatase 4 (DUSP4), on proliferation, cell growth, migration and invasion, and on signaling pathways using protein microarray analyses of 172 proteins, including phosphoproteins. We identified 48 phosphatase genes are significantly differentially expressed in ER-negative compared to those in ER-positive breast tumors. We discovered that 31 phosphatases were more highly expressed, while 11 were underexpressed specifically in ER-negative breast cancers. The DUSP4 gene is underexpressed in ER-negative breast cancer and is deleted in approximately 50 % of breast cancers. Induced DUSP4 expression suppresses both in vitro and in vivo growths of breast cancer cells. Our studies show that induced DUSP4 expression blocks the cell cycle at the G1/S checkpoint; inhibits ERK1/2, p38, JNK1, RB, and NFkB p65 phosphorylation; and inhibits invasiveness of TNBC cells. These results suggest that that DUSP4 is a critical regulator of the growth and invasion of triple-negative breast cancer cells.
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http://dx.doi.org/10.1007/s10549-016-3892-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963453PMC
August 2016

Phosphatase PTP4A3 Promotes Triple-Negative Breast Cancer Growth and Predicts Poor Patient Survival.

Cancer Res 2016 04 26;76(7):1942-53. Epub 2016 Feb 26.

Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, Texas. Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas.

Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and women diagnosed with TNBC currently lack targeted treatment options. To identify novel targets for TNBC, we evaluated phosphatase expression in breast tumors and characterized their contributions to in vitro and in vivo growth of TNBC. Using Affymetrix microarray analysis of 102 breast cancers, we identified 146 phosphatases that were significantly differentially expressed in TNBC compared with estrogen receptor (ER)-positive tumors. Of these, 19 phosphatases were upregulated (0.66-fold; FDR = 0.05) in TNBC compared with ER-positive breast cancers. We knocked down 17 overexpressed phosphatases in four triple-negative and four ER-positive breast cancer lines using specific siRNAs and found that depletion of six of these phosphatases significantly reduced growth and anchorage-independent growth of TNBC cells to a greater extent than ER-positive cell lines. Further analysis of the phosphatase PTP4A3 (also known as PRL-3) demonstrated its requirement for G1-S cell-cycle progression in all breast cancer cells, but PTP4A3 regulated apoptosis selectively in TNBC cells. In addition, PTP4A3 inhibition reduced the growth of TNBC tumors in vivo Moreover, in silico analysis revealed the PTP4A3 gene to be amplified in 29% of basal-like breast cancers, and high expression of PTP4A3 could serve as an independent prognostic indicator for worse overall survival. Collectively, these studies define the importance of phosphatase overexpression in TNBC and lay the foundation for the development of new targeted therapies directed against phosphatases or their respective signaling pathways for TNBC patients. Cancer Res; 76(7); 1942-53. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-0673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4873402PMC
April 2016

Targeted therapy for breast cancer prevention.

Front Oncol 2013 Sep 23;3:250. Epub 2013 Sep 23.

Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center , Houston, TX , USA.

With a better understanding of the etiology of breast cancer, molecularly targeted drugs have been developed and are being testing for the treatment and prevention of breast cancer. Targeted drugs that inhibit the estrogen receptor (ER) or estrogen-activated pathways include the selective ER modulators (tamoxifen, raloxifene, and lasofoxifene) and aromatase inhibitors (AIs) (anastrozole, letrozole, and exemestane) have been tested in preclinical and clinical studies. Tamoxifen and raloxifene have been shown to reduce the risk of breast cancer and promising results of AIs in breast cancer trials, suggest that AIs might be even more effective in the prevention of ER-positive breast cancer. However, these agents only prevent ER-positive breast cancer. Therefore, current research is focused on identifying preventive therapies for other forms of breast cancer such as human epidermal growth factor receptor 2 (HER2)-positive and triple-negative breast cancer (TNBC, breast cancer that does express ER, progesterone receptor, or HER2). HER2-positive breast cancers are currently treated with anti-HER2 therapies including trastuzumab and lapatinib, and preclinical and clinical studies are now being conducted to test these drugs for the prevention of HER2-positive breast cancers. Several promising agents currently being tested in cancer prevention trials for the prevention of TNBC include poly(ADP-ribose) polymerase inhibitors, vitamin D, and rexinoids, both of which activate nuclear hormone receptors (the vitamin D and retinoid X receptors). This review discusses currently used breast cancer preventive drugs, and describes the progress of research striving to identify and develop more effective preventive agents for all forms of breast cancer.
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http://dx.doi.org/10.3389/fonc.2013.00250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3780469PMC
September 2013

Growth of triple-negative breast cancer cells relies upon coordinate autocrine expression of the proinflammatory cytokines IL-6 and IL-8.

Cancer Res 2013 Jun 30;73(11):3470-80. Epub 2013 Apr 30.

Department of Clinical Cancer Prevention and Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

Triple-negative breast cancers (TNBC) are aggressive with no effective targeted therapies. A combined database analysis identified 32 inflammation-related genes differentially expressed in TNBCs and 10 proved critical for anchorage-independent growth. In TNBC cells, an LPA-LPAR2-EZH2 NF-κB signaling cascade was essential for expression of interleukin (IL)-6, IL-8, and CXCL1. Concurrent inhibition of IL-6 and IL-8 expression dramatically inhibited colony formation and cell survival in vitro and stanched tumor engraftment and growth in vivo. A Cox multivariable analysis of patient specimens revealed that IL-6 and IL-8 expression predicted patient survival times. Together these findings offer a rationale for dual inhibition of IL-6/IL-8 signaling as a therapeutic strategy to improve outcomes for patients with TNBCs.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-4524-TDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853111PMC
June 2013

Long-range massively parallel mate pair sequencing detects distinct mutations and similar patterns of structural mutability in two breast cancer cell lines.

Cancer Genet 2011 Aug;204(8):447-57

Graduate Program in Structural and Computational Biology and Molecular Biophysics, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.

Cancer genomes frequently undergo genomic instability resulting in accumulation of chromosomal rearrangement. To date, one of the main challenges has been to confidently and accurately identify these rearrangements by using short-read massively parallel sequencing. We were able to improve cancer rearrangement detection by combining two distinct massively parallel sequencing strategies: fosmid-sized (36 kb on average) and standard 5 kb mate pair libraries. We applied this combined strategy to map rearrangements in two breast cancer cell lines, MCF7 and HCC1954. We detected and validated a total of 91 somatic rearrangements in MCF7 and 25 in HCC1954, including genomic alterations corresponding to previously reported transcript aberrations in these two cell lines. Each of the genomes contains two types of breakpoints: clustered and dispersed. In both cell lines, the dispersed breakpoints show enrichment for low copy repeats, while the clustered breakpoints associate with high copy number amplifications. Comparing the two genomes, we observed highly similar structural mutational spectra affecting different sets of genes, pointing to similar histories of genomic instability against the background of very different gene network perturbations.
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http://dx.doi.org/10.1016/j.cancergen.2011.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185296PMC
August 2011

A sequence-level map of chromosomal breakpoints in the MCF-7 breast cancer cell line yields insights into the evolution of a cancer genome.

Genome Res 2009 Feb 3;19(2):167-77. Epub 2008 Dec 3.

Bioinformatics Research Laboratory, Baylor College of Medicine, Houston, Texas 77030, USA.

By applying a method that combines end-sequence profiling and massively parallel sequencing, we obtained a sequence-level map of chromosomal aberrations in the genome of the MCF-7 breast cancer cell line. A total of 157 distinct somatic breakpoints of two distinct types, dispersed and clustered, were identified. A total of 89 breakpoints are evenly dispersed across the genome. A majority of dispersed breakpoints are in regions of low copy repeats (LCRs), indicating a possible role for LCRs in chromosome breakage. The remaining 68 breakpoints form four distinct clusters of closely spaced breakpoints that coincide with the four highly amplified regions in MCF-7 detected by array CGH located in the 1p13.1-p21.1, 3p14.1-p14.2, 17q22-q24.3, and 20q12-q13.33 chromosomal cytobands. The clustered breakpoints are not significantly associated with LCRs. Sequences flanking most (95%) breakpoint junctions are consistent with double-stranded DNA break repair by nonhomologous end-joining or template switching. A total of 79 known or predicted genes are involved in rearrangement events, including 10 fusions of coding exons from different genes and 77 other rearrangements. Four fusions result in novel expressed chimeric mRNA transcripts. One of the four expressed fusion products (RAD51C-ATXN7) and one gene truncation (BRIP1 or BACH1) involve genes coding for members of protein complexes responsible for homology-driven repair of double-stranded DNA breaks. Another one of the four expressed fusion products (ARFGEF2-SULF2) involves SULF2, a regulator of cell growth and angiogenesis. We show that knock-down of SULF2 in cell lines causes tumorigenic phenotypes, including increased proliferation, enhanced survival, and increased anchorage-independent growth.
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http://dx.doi.org/10.1101/gr.080259.108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652200PMC
February 2009

Dynein light chain 1 peptide inhibits human immunodeficiency virus infection in eukaryotic cells.

Biochem Biophys Res Commun 2007 Nov 21;363(4):901-7. Epub 2007 Sep 21.

University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

Human immunodeficiency virus (HIV) uses kinases such as Pak1 and macropinocytosis for a productive infection. Recently dynein light chain 1 (DLC1), a component of the dynein motor, was identified as a Pak1 substrate and interacted with the C-terminal region of DLC1 (aa 61-89). The dynein motor is implicated in retrograde transport, also of HIV, to the nucleus. It is known that DLC1 is important in macropinocytosis, and anti-dynein antibodies inhibit a productive HIV infection. Here, we show that in Hela-beta-gal cells macropinocytosis was effectively blocked by a peptide spanning the C-terminal 19 amino acids of DLC1. We also found that the DLC1 peptide was capable of inhibiting the early entry steps of HIV, and the DLC1 peptide efficiently inhibited a productive HIV infection, and cooperated with the anti-HIV activity of CD4 antibodies. Taken together, the potentially therapeutic DLC1 peptide represents an interesting class of HIV inhibitors, targeting an essential cellular component for HIV infection. Our findings raise the possibility that the use of a DLC1 peptide in combination with currently used anti-HIV agents, might offer additional arsenal against HIV infection in human cells.
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http://dx.doi.org/10.1016/j.bbrc.2007.09.046DOI Listing
November 2007

Ciz1, a Novel DNA-binding coactivator of the estrogen receptor alpha, confers hypersensitivity to estrogen action.

Cancer Res 2006 Nov;66(22):11021-9

Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

The transcriptional activity of the estrogen receptor (ER) is affected by regulatory cofactors, including chromatin-remodeling complexes, coactivators, and corepressors. Coregulators are recruited to target gene promoters through protein-protein interactions with ER and function as linker molecules between the DNA, DNA-binding proteins, and DNA-modifying enzymes. We recently showed that Cip-interacting zinc finger protein 1 (Ciz1) participates in the regulation of the cell cycle in estrogen-stimulated breast cancer cells. Despite the emerging significance of Ciz1 in the biology of breast cancer cells, regulation of endogenous Ciz1 in hormone-responsive cancer cells remains unknown. To shed light on the role of Ciz1 in breast tumorigenesis, we defined the regulation of Ciz1 by the ER pathway and found that Ciz1 is an estrogen-responsive gene. We also discovered that Ciz1 protein, a DNA-binding factor, coregulates ER by enhancing ER transactivation activity by promoting the recruitment of the ER complex to the target gene chromatin. In addition, we found that Ciz1 overexpression confers estrogen hypersensitivity to breast cancer cells and promotes the growth rate, anchorage independency, and tumorigenic properties of breast cancer cells. These findings revealed the inherent role of Ciz1, a novel DNA binding and ER coactivator, in amplifying estrogenic responses and promoting breast cancer tumorigenesis.
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http://dx.doi.org/10.1158/0008-5472.CAN-06-2336DOI Listing
November 2006

Dynein light chain 1 contributes to cell cycle progression by increasing cyclin-dependent kinase 2 activity in estrogen-stimulated cells.

Cancer Res 2006 Jun;66(11):5941-9

Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center and The University of Texas School of Biomedical Sciences at Houston Program of Cancer Biology, Houston, Texas, USA.

Progression of hormone-responsive cancers is characterized by deregulation of the cell cycle and cytoskeleton signaling. In addition, development of breast and endometrial cancer is influenced by the stimulatory action of estrogen. Up-regulation of dynein light chain 1 (DLC1), a component of cytoskeleton signaling, was recently found to promote tumorigenesis. The purpose of our study was to determine the role that DLC1 up-regulation plays in cell cycle progression. To achieve this goal, we used human breast ductal carcinoma ZR-75 cells overexpressing DLC1 as a model system. We found that ZR-75 cells with up-regulated DLC1 were hypersensitive to estrogen-dependent growth stimulation and that DLC1 had an accelerating effect on the G(1)-S transition and stimulated cyclin-dependent kinase 2 (Cdk2) activity. To better understand the promotion of the G(1)-S transition by DLC1, we sought to identify new DLC1-interacting proteins with roles in cell cycle regulation. Using a modified proteomic strategy, we identified two such DLC1-interacting proteins: Cdk2 and Cip-interacting zinc finger protein 1 (Ciz1). DLC1 was verified to interact with Cdk2 and Ciz1 in vivo. We also showed that down-regulation of DLC1 and Ciz1 reduced both Cdk2 activity and cell cycle progression of breast cancer ZR-75 and MCF-7 and endometrial Ishikawa cancer cells. Further, we showed that overexpression of DLC1 is accompanied by a reduction of nuclear p21(WAF1). These findings suggest that interactions among DLC1, Cdk2, and Ciz1 play a regulatory role in cell cycle progression of cancer cells presumably by influencing the levels of nuclear p21(WAF1).
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http://dx.doi.org/10.1158/0008-5472.CAN-05-3480DOI Listing
June 2006

Essential role of KIBRA in co-activator function of dynein light chain 1 in mammalian cells.

J Biol Chem 2006 Jul 9;281(28):19092-9. Epub 2006 May 9.

Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.

Recently dynein light chain 1 (DLC1), a cytoskeleton signaling component, has been shown to interact with and transactivate estrogen receptor-alpha (ER), leading to increased expression of ER target genes and growth stimulation of breast cancer cells. However, the molecular mechanism by which DLC1 regulates the ER pathway remains poorly understood. To gain insights into the putative mechanism, here we set out to identify novel DLC1-interacting proteins. We identified KIBRA, a WW domain- and a glutamic acid stretch-containing protein, as a DLC1-binding protein and showed that it interacts with DLC1 both in vitro and in vivo. We found that KIBRA-DLC1 complex is recruited to ER-responsive promoters. We also found that KIBRA-DLC1 interaction is mandatory for the recruitment and transactivation functions of ER or DLC1 to the target chromatin. Finally we found that KIBRA interacts with histone H3 via its glutamic acid-rich region and that such interaction might play a mechanistic role in conferring an optimal ER transactivation function as well as the proliferation of ligand-stimulated breast cancer cells. Together these findings indicate that DLC1-KIBRA interaction is essential for ER transactivation in breast cancer cells.
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http://dx.doi.org/10.1074/jbc.M600021200DOI Listing
July 2006

MTA1, a transcriptional activator of breast cancer amplified sequence 3.

Proc Natl Acad Sci U S A 2006 Apr 14;103(17):6670-5. Epub 2006 Apr 14.

Department of Molecular and Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.

Here we define a function of metastasis-associated protein 1 (MTA1), a presumed corepressor of estrogen receptor alpha (ERalpha), as a transcriptional activator of Breast Cancer Amplified Sequence 3 (BCAS3), a gene amplified and overexpressed in breast cancers. We identified BCAS3 as a MTA1 chromatin target in a functional genomic screen. MTA1 stimulation of BCAS3 transcription required ERalpha and involved a functional ERE half-site in BCAS3. Furthermore, we discovered that MTA1 is acetylated on lysine 626, and that this acetylation is necessary for a productive transcriptional recruitment of RNA polymerase II complex to the BCAS3 enhancer sequence. BCAS3 expression was elevated in mammary tumors from MTA1 transgenic mice and 60% of the human breast tumors, and correlated with the coexpression of MTA1 as well as with tumor grade and proliferation of primary breast tumor samples. These findings reveal a previously unrecognized function of MTA1 in stimulating BCAS3 expression and suggest an important role for MTA1-BCAS3 pathway in promoting cancerous phenotypes in breast tumor cells.
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http://dx.doi.org/10.1073/pnas.0601989103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1458939PMC
April 2006

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) interacts with PELP1 and activates MAPK.

J Biol Chem 2006 Feb 12;281(7):4395-403. Epub 2005 Dec 12.

Department of Molecular and Cellular Oncology, the University of Texas MD Anderson Cancer Center, Houston, 77030, USA.

PELP1 (proline-, glutamic acid-, and leucine-rich protein-1) (also known as the modulator of nongenomic activity of estrogen receptor) plays a role in genomic functions of the estrogen receptor via histone interactions and in nongenomic functions via its influence on the MAPK-Src pathway. However, recent studies have shown that differential compartmentalization of PELP1 could play a crucial role in modulating the status of nongenomic signaling by using molecular mechanisms that remain poorly understood. Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is an early endosomal protein that plays a role in regulating the trafficking of growth factor-receptor complexes through early endosomes. By using a yeast two-hybrid screen, we identified HRS as a novel PELP1-binding protein providing evidence of a physiologic interaction between HRS and PELP1. The noted HRS-PELP1 interaction was accompanied by inhibition of the basal coactivator function of PELP1 upon estrogen receptor transactivation. HRS was found to sequester PELP1 in the cytoplasm, leading to the activation of MAPK in a manner that is dependent on the epidermal growth factor receptor but independent of the estrogen receptor, Shc, and Src. In addition, stimulation of MAPK and the subsequent activation of its downstream effector pathway, Elk-1, by HRS or PELP1 were found to depend on the presence of endogenous PELP1 or HRS. Furthermore, HRS was overexpressed and correlated well with the cytoplasmic PELP1, increased MAPK, and EGFR status in breast tumors. These findings highlight a novel role of HRS in up-regulating MAPK, presumably involving interaction with PELP1.
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http://dx.doi.org/10.1074/jbc.M510368200DOI Listing
February 2006

Functional regulation of oestrogen receptor pathway by the dynein light chain 1.

EMBO Rep 2005 Jun;6(6):538-44

Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.

Overexpression and phosphorylation of dynein light chain 1 (DLC1) have been shown to promote the growth of breast cancer cells. However, the role of DLC1 in the action of the oestrogen receptor (ER) remains unknown. Here, we found that oestrogen induces the transcription and expression of DLC1. DLC1 facilitated oestrogen-induced ER transactivation and anchorage-independent growth of breast cancer cells. We show that DLC1 interacts with ER, and such interaction is required for the transactivation-promoting activity of DLC1. Further, DLC1 expression led to enhanced recruitment of the DLC1-ER complex to the ER-target gene chromatin. Conversely, DLC1 downregulation compromised the ER-transactivation activity and also its nuclear accumulation, suggesting a potential chaperone-like activity of DLC1 in the nuclear translocation of ER. Together, these data define an unexpected upregulation of DLC1 by oestrogen and a previously unrecognized DLC1-ER interaction in supporting and amplifying ER-initiated cellular responses in breast cancer cells.
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http://dx.doi.org/10.1038/sj.embor.7400417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1369089PMC
June 2005

Dynein light chain 1, a p21-activated kinase 1-interacting substrate, promotes cancerous phenotypes.

Cancer Cell 2004 Jun;5(6):575-85

Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

We identified dynein light chain 1 (DLC1) as a physiologic substrate of p21-activated kinase 1 (Pak1). Pak1-DLC1 interaction plays an essential role in cell survival, which depends on Pak1's phosphorylation of DLC1 on Ser88. Pak1 associates with the complex of DLC1 and BimL, a proapoptotic BH3-only protein, and phosphorylates both proteins. Phosphorylation of BimL by Pak1 prevents it from interacting with and inactivation of Bcl-2, an antiapoptotic protein. Overexpression of DLC1 but not DLC1-Ser88Ala mutant promotes cancerous properties of breast cancer cells. DLC1 protein level is elevated in more than 90% of human breast tumors. The regulation of cell survival functions by Pak1-DLC1 interaction represents a novel mechanism by which a signaling kinase might regulate the cancerous phenotypes.
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http://dx.doi.org/10.1016/j.ccr.2004.05.022DOI Listing
June 2004

Cyclin D1 genotype, response to biochemoprevention, and progression rate to upper aerodigestive tract cancer.

J Natl Cancer Inst 2003 Feb;95(3):198-205

Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA.

Background: Altered cyclin D1 expression in advanced preinvasive lesions of the upper aerodigestive tract (UADT) is associated with an increased risk of developing cancer and histologic progression during and after combination biochemopreventive therapy (13-cis-retinoic acid, alpha-interferon, and alpha-tocopherol). Both alleles of the adenine (A)/guanine (G) cyclin D1 polymorphism located at nucleotide 870 encode two alternatively spliced transcripts, but the A allele preferentially encodes a protein with an extended half-life. We investigated whether the cyclin D1 genotype at nucleotide 870 was associated with baseline levels of cyclin D1 protein, post-treatment modulation of cyclin D1 protein levels, histologic response to treatment, and the outcome for subjects with preinvasive UADT lesions after biochemopreventive therapy.

Methods: UADT tissue biopsy samples were obtained before and 6 and 12 months after biochemopreventive treatment from 31 individuals with advanced preinvasive UADT lesions. Tissues were examined for cyclin D1 genotype (by DNA single-strand conformation polymorphism analysis), for cyclin D1 protein expression (by immunohistochemistry), and for cyclin D1 gene copy number (by fluorescence in situ hybridization). Associations of cyclin D1 genotype with histologic response to therapy and time to progression to a higher degree of dysplasia or invasive cancer were investigated. All statistical tests were two-sided.

Results: The A allele was associated with increased baseline cyclin D1 expression in the parabasal epithelial layer (16 of 18 AA/AG subjects versus four of nine GG subjects; P =.02), decreased histologic response to biochemopreventive treatment (six of 21 AA/AG subjects versus four of 10 GG subjects; P =.70), decreased favorable modulation of cyclin D1 expression by the treatment (seven of 18 AA/AG subjects versus eight of nine GG subjects; P =.02), and shorter progression-free survival (P =.05).

Conclusions: The cyclin D1 A allele was associated with a diminished modulation of normal physiologic and treatment-induced decreased expression of cyclin D1, a decreased likelihood of response to biochemopreventive intervention, and an increased rate of progression to cancer development, findings that require validation in a larger cohort.
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http://dx.doi.org/10.1093/jnci/95.3.198DOI Listing
February 2003
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