Publications by authors named "Heide L Ford"

57 Publications

VEGF-C mediates tumor growth and metastasis through promoting EMT-epithelial breast cancer cell crosstalk.

Oncogene 2021 Feb 9;40(5):964-979. Epub 2020 Dec 9.

Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

It is well established that a subset of cells within primary breast cancers can undergo an epithelial-to-mesenchymal transition (EMT), although the role of EMT in metastasis remains controversial. We previously demonstrated that breast cancer cells that had undergone an oncogenic EMT could increase metastasis of neighboring cancer cells via non-canonical paracrine-mediated activation of GLI activity that is dependent on SIX1 expression in the EMT cancer cells. However, the mechanism by which these SIX1-expressing EMT cells activate GLI signaling remained unclear. In this study, we demonstrate a novel mechanism for activation of GLI-mediated signaling in epithelial breast tumor cells via EMT cell-induced production and secretion of VEGF-C. We show that VEGF-C, secreted by breast cancer cells that have undergone an EMT, promotes paracrine-mediated increases in proliferation, migration, and invasion of epithelial breast cancer cells, via non-canonical activation of GLI-signaling. We further show that the aggressive phenotypes, including metastasis, imparted by EMT cells on adjacent epithelial cancer cells can be disrupted by either inhibiting VEGF-C in EMT cells or by knocking down NRP2, a receptor which interacts with VEGF-C, in neighboring epithelial cancer cells. Interrogation of TCGA and GEO public datasets supports the relevance of this pathway in human breast cancer, demonstrating that VEGF-C strongly correlates with activation of Hedgehog signaling and EMT in the human disease. Our study suggests that the VEGF-C/NRP2/GLI axis is a novel and conserved paracrine means by which EMT cells enhance metastasis, and provides potential targets for therapeutic intervention in this heterogeneous disease.
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http://dx.doi.org/10.1038/s41388-020-01539-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867573PMC
February 2021

Corrigendum to "The Eya phosphatase: Its unique role in cancer" [Int. J. Biochem. Cell Biol. 96 (2018) 165-170].

Int J Biochem Cell Biol 2020 Oct 12;127:105847. Epub 2020 Sep 12.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver Anschutz Medical Campus, United States. Electronic address:

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http://dx.doi.org/10.1016/j.biocel.2020.105847DOI Listing
October 2020

Cellular Plasticity in Breast Cancer Progression and Therapy.

Front Mol Biosci 2020 24;7:72. Epub 2020 Apr 24.

Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.

With the exception of non-melanoma skin cancer, breast cancer is the most frequently diagnosed malignant disease among women, with the majority of mortality being attributable to metastatic disease. Thus, even with improved early screening and more targeted treatments which may enable better detection and control of early disease progression, metastatic disease remains a significant problem. While targeted therapies exist for breast cancer patients with particular subtypes of the disease (Her2+ and ER/PR+), even in these subtypes the therapies are often not efficacious once the patient's tumor metastasizes. Increases in stemness or epithelial-to-mesenchymal transition (EMT) in primary breast cancer cells lead to enhanced plasticity, enabling tumor progression, therapeutic resistance, and distant metastatic spread. Numerous signaling pathways, including MAPK, PI3K, STAT3, Wnt, Hedgehog, and Notch, amongst others, play a critical role in maintaining cell plasticity in breast cancer. Understanding the cellular and molecular mechanisms that regulate breast cancer cell plasticity is essential for understanding the biology of breast cancer progression and for developing novel and more effective therapeutic strategies for targeting metastatic disease. In this review we summarize relevant literature on mechanisms associated with breast cancer plasticity, tumor progression, and drug resistance.
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http://dx.doi.org/10.3389/fmolb.2020.00072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7194153PMC
April 2020

Identification of a Small-Molecule Inhibitor That Disrupts the SIX1/EYA2 Complex, EMT, and Metastasis.

Cancer Res 2020 06 27;80(12):2689-2702. Epub 2020 Apr 27.

Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFβ signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFβ signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510951PMC
June 2020

Guidelines and definitions for research on epithelial-mesenchymal transition.

Nat Rev Mol Cell Biol 2020 06 16;21(6):341-352. Epub 2020 Apr 16.

International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan.

Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.
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http://dx.doi.org/10.1038/s41580-020-0237-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250738PMC
June 2020

Structural and Functional Analyses of an Allosteric EYA2 Phosphatase Inhibitor That Has On-Target Effects in Human Lung Cancer Cells.

Mol Cancer Ther 2019 09 8;18(9):1484-1496. Epub 2019 Jul 8.

Experimental Drug Discovery Centre, A*STAR, Singapore, Singapore.

EYA proteins (EYA1-4) are critical developmental transcriptional cofactors that contain an EYA domain (ED) harboring Tyr phosphatase activity. EYA proteins are largely downregulated after embryogenesis but are reexpressed in cancers, and their Tyr phosphatase activity plays an important role in the DNA damage response and tumor progression. We previously identified a class of small-molecule allosteric inhibitors that specifically inhibit the Tyr phosphatase activity of EYA2. Herein, we determined the crystal structure of the EYA2 ED in complex with NCGC00249987 (a representative compound in this class), revealing that it binds to an induced pocket distant from the active site. NCGC00249987 binding leads to a conformational change of the active site that is unfavorable for Mg binding, thereby inhibiting EYA2's Tyr phosphatase activity. We demonstrate, using genetic mutations, that migration, invadopodia formation, and invasion of lung adenocarcinoma cells are dependent on EYA2 Tyr phosphatase activity, whereas growth and survival are not. Further, we demonstrate that NCGC00249987 specifically targets migration, invadopodia formation, and invasion of lung cancer cells, but that it does not inhibit cell growth or survival. The compound has no effect on lung cancer cells carrying an EYA2 F290Y mutant that abolishes compound binding, indicating that NCGC00249987 is on target in lung cancer cells. These data suggest that the NCGC00249987 allosteric inhibitor can be used as a chemical probe to study the function of the EYA2 Tyr phosphatase activity in cells and may have the potential to be developed into an antimetastatic agent for cancers reliant on EYA2's Tyr phosphatase activity.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-1239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726557PMC
September 2019

SIX2 Mediates Late-Stage Metastasis via Direct Regulation of and Induction of a Cancer Stem Cell Program.

Cancer Res 2019 02 3;79(4):720-734. Epub 2019 Jan 3.

Integrated Physiology Program, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

The capacity for tumor cells to metastasize efficiently is directly linked to their ability to colonize secondary sites. Here we identify Six2, a developmental transcription factor, as a critical regulator of a breast cancer stem cell program that enables metastatic colonization. In several triple-negative breast cancer (TNBC) models, Six2 enhanced the expression of genes associated with embryonic stem cell programs. Six2 directly bound the Srr2 enhancer, promoting expression and downstream expression of , which are both key pluripotency factors. Regulation of by Six2 enhanced cancer stem cell properties and increased metastatic colonization. and expression correlated highly in breast cancers including TNBC, where a Six2 expression signature was predictive of metastatic burden and poor clinical outcome. Our findings demonstrate that a SIX2/SOX2 axis is required for efficient metastatic colonization, underscoring a key role for stemness factors in outgrowth at secondary sites. SIGNIFICANCE: These findings provide novel mechanistic insight into stemness and the metastatic outgrowth of triple-negative breast cancer cells. http://cancerres.aacrjournals.org/content/canres/79/4/720/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586234PMC
February 2019

Publisher Correction: EMT cells increase breast cancer metastasis via paracrine GLI activation in neighbouring tumour cells.

Nat Commun 2018 11 12;9(1):4720. Epub 2018 Nov 12.

Department of Pharmacology, University of Colorado-Denver, 12800 East 19th Avenue, Room P18-6115, Aurora, Colorado, 80045, USA.

This Article contains an error in Figure 2. In panel a, the second lane of the western blot should have been labelled 'siNT'. A correct version of Figure 2a appears in the Author Correction associated with this Article; the error has not been fixed in the original Article.
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http://dx.doi.org/10.1038/s41467-018-07168-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232083PMC
November 2018

Author Correction: Eya3 partners with PP2A to induce c-Myc stabilization and tumor progression.

Nat Commun 2018 09 17;9(1):3830. Epub 2018 Sep 17.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver Anschutz Medical Campus, Aurora 80045, CO, USA.

In the original version of this Article, the title of the legend to Fig. 7 incorrectly read 'Knockdown of B55α increases breast cancer metastasis' instead of 'Knockdown of B55α decreases breast cancer metastasis'. This has now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-018-06265-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141458PMC
September 2018

Eya3 promotes breast tumor-associated immune suppression via threonine phosphatase-mediated PD-L1 upregulation.

J Clin Invest 2018 06 14;128(6):2535-2550. Epub 2018 May 14.

Department of Pharmacology, University of Colorado Denver, Aurora, Colorado, USA.

Eya proteins are critical developmental regulators that are highly expressed in embryogenesis but downregulated after development. Amplification and/or re-expression of Eyas occurs in many tumor types. In breast cancer, Eyas regulate tumor progression by acting as transcriptional cofactors and tyrosine phosphatases. Intriguingly, Eyas harbor a separate threonine (Thr) phosphatase activity, which was previously implicated in innate immunity. Here we describe what we believe to be a novel role for Eya3 in mediating triple-negative breast cancer-associated immune suppression. Eya3 loss decreases tumor growth in immune-competent mice and is associated with increased numbers of infiltrated CD8+ T cells, which, when depleted, reverse the effects of Eya3 knockdown. Mechanistically, Eya3 utilizes its Thr phosphatase activity to dephosphorylate Myc at pT58, resulting in a stabilized form. We show that Myc is required for Eya3-mediated increases in PD-L1, and that rescue of PD-L1 in Eya3-knockdown cells restores tumor progression. Finally, we demonstrate that Eya3 significantly correlates with PD-L1 in human breast tumors, and that tumors expressing high levels of Eya3 have a decreased CD8+ T cell signature. Our data uncover a role for Eya3 in mediating tumor-associated immune suppression, and suggest that its inhibition may enhance checkpoint therapies.
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http://dx.doi.org/10.1172/JCI96784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983346PMC
June 2018

Eya3 partners with PP2A to induce c-Myc stabilization and tumor progression.

Nat Commun 2018 03 13;9(1):1047. Epub 2018 Mar 13.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver Anschutz Medical Campus, Aurora, 80045, CO, USA.

Eya genes encode a unique family of multifunctional proteins that serve as transcriptional co-activators and as haloacid dehalogenase-family Tyr phosphatases. Intriguingly, the N-terminal domain of Eyas, which does not share sequence similarity to any known phosphatases, contains a separable Ser/Thr phosphatase activity. Here, we demonstrate that the Ser/Thr phosphatase activity of Eya is not intrinsic, but arises from its direct interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme. Importantly, Eya3 alters the regulation of c-Myc by PP2A, increasing c-Myc stability by enabling PP2A-B55α to dephosphorylate pT58, in direct contrast to the previously described PP2A-B56α-mediated dephosphorylation of pS62 and c-Myc destabilization. Furthermore, Eya3 and PP2A-B55α promote metastasis in a xenograft model of breast cancer, opposing the canonical tumor suppressive function of PP2A-B56α. Our study identifies Eya3 as a regulator of PP2A, a major cellular Ser/Thr phosphatase, and uncovers a mechanism of controlling the stability of a critical oncogene, c-Myc.
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http://dx.doi.org/10.1038/s41467-018-03327-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849647PMC
March 2018

The Eya phosphatase: Its unique role in cancer.

Int J Biochem Cell Biol 2018 03 5;96:165-170. Epub 2017 Sep 5.

Department of Biochemistry and Molecular Genetics, University of Colorado Denver Anschutz Medical Campus, United States. Electronic address:

The Eya proteins were originally identified as essential transcriptional co-activators of the Six family of homeoproteins. Subsequently, the highly conserved C-terminal domains of the Eya proteins were discovered to act as a Mg-dependent Tyr phosphatases, making Eyas the first transcriptional activators to harbor intrinsic phosphatase activity. Only two direct targets of the Eya Tyr phosphatase have been identified: H2AX, whose dephosphorylation directs cells to the DNA repair instead of the apoptotic pathway upon DNA damage, and ERβ, whose dephosphorylation inhibits its anti-tumor transcriptional activity. The Eya Tyr phosphatase mediates breast cancer cell transformation, migration, invasion, as well as metastasis, through targets not yet identified. Intriguingly, the N-terminal domain of Eya contains a separate Ser/Thr phosphatase activity implicated in innate immunity and in regulating c-Myc stability. Thus, Eya proteins are highly complex, containing two separable phosphatase domains and a transcriptional activation domain, thereby influencing tumor progression through multiple mechanisms.
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http://dx.doi.org/10.1016/j.biocel.2017.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808901PMC
March 2018

EMT cells increase breast cancer metastasis via paracrine GLI activation in neighbouring tumour cells.

Nat Commun 2017 06 12;8:15773. Epub 2017 Jun 12.

Department of Pharmacology, University of Colorado-Denver, 12800 East 19th Avenue, Room P18-6115, Aurora, Colorado 80045, USA.

Recent fate-mapping studies concluded that EMT is not required for metastasis of carcinomas. Here we challenge this conclusion by showing that these studies failed to account for possible crosstalk between EMT and non-EMT cells that promotes dissemination of non-EMT cells. In breast cancer models, EMT cells induce increased metastasis of weakly metastatic, non-EMT tumour cells in a paracrine manner, in part by non-cell autonomous activation of the GLI transcription factor. Treatment with GANT61, a GLI1/2 inhibitor, but not with IPI 926, a Smoothened inhibitor, blocks this effect and inhibits growth in PDX models. In human breast tumours, the EMT-transcription factors strongly correlate with activated Hedgehog/GLI signalling but not with the Hh ligands. Our findings indicate that EMT contributes to metastasis via non-cell autonomous effects that activate the Hh pathway. Although all Hh inhibitors may act against tumours with canonical Hh/GLI signalling, only GLI inhibitors would act against non-canonical EMT-induced GLI activation.
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http://dx.doi.org/10.1038/ncomms15773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472791PMC
June 2017

Negative regulation of endothelin signaling by SIX1 is required for proper maxillary development.

Development 2017 06 28;144(11):2021-2031. Epub 2017 Apr 28.

Department of Craniofacial Biology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA

Jaw morphogenesis is a complex event mediated by inductive signals that establish and maintain the distinct developmental domains required for formation of hinged jaws, the defining feature of gnathostomes. The mandibular portion of pharyngeal arch 1 is patterned dorsally by Jagged-Notch signaling and ventrally by endothelin receptor A (EDNRA) signaling. Loss of EDNRA signaling disrupts normal ventral gene expression, the result of which is homeotic transformation of the mandible into a maxilla-like structure. However, loss of Jagged-Notch signaling does not result in significant changes in maxillary development. Here we show in mouse that the transcription factor SIX1 regulates dorsal arch development not only by inducing dorsal expression but also by inhibiting endothelin 1 () expression in the pharyngeal endoderm of the dorsal arch, thus preventing dorsal EDNRA signaling. In the absence of SIX1, but not JAG1, aberrant EDNRA signaling in the dorsal domain results in partial duplication of the mandible. Together, our results illustrate that SIX1 is the central mediator of dorsal mandibular arch identity, thus ensuring separation of bone development between the upper and lower jaws.
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http://dx.doi.org/10.1242/dev.145144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482985PMC
June 2017

A Genome-Wide Loss-of-Function Screen Identifies SLC26A2 as a Novel Mediator of TRAIL Resistance.

Mol Cancer Res 2017 04 20;15(4):382-394. Epub 2017 Jan 20.

Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

TRAIL is a potent death-inducing ligand that mediates apoptosis through the extrinsic pathway and serves as an important endogenous tumor suppressor mechanism. Because tumor cells are often killed by TRAIL and normal cells are not, drugs that activate the TRAIL pathway have been thought to have potential clinical value. However, to date, most TRAIL-related clinical trials have largely failed due to the tumor cells having intrinsic or acquired resistance to TRAIL-induced apoptosis. Previous studies to identify resistance mechanisms have focused on targeted analysis of the canonical apoptosis pathway and other known regulators of TRAIL receptor signaling. To identify novel mechanisms of TRAIL resistance in an unbiased way, we performed a genome-wide shRNA screen for genes that regulate TRAIL sensitivity in sublines that had been selected for acquired TRAIL resistance. This screen identified previously unknown mediators of TRAIL resistance including angiotensin II receptor 2, Crk-like protein, T-Box Transcription Factor 2, and solute carrier family 26 member 2 (SLC26A2). SLC26A2 downregulates the TRAIL receptors, DR4 and DR5, and this downregulation is associated with resistance to TRAIL. Its expression is high in numerous tumor types compared with normal cells, and in breast cancer, is associated with a significant decrease in relapse-free survival. Our results shed light on novel resistance mechanisms that could affect the efficacy of TRAIL agonist therapies and highlight the possibility of using these proteins as biomarkers to identify TRAIL-resistant tumors, or as potential therapeutic targets in combination with TRAIL. .
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380511PMC
April 2017

CD147: a small molecule transporter ancillary protein at the crossroad of multiple hallmarks of cancer and metabolic reprogramming.

Oncotarget 2017 Jan;8(4):6742-6762

Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, CO, USA.

Increased expression of CD147 in pancreatic cancer has been proposed to play a critical role in cancer progression via CD147 chaperone function for lactate monocarboxylate transporters (MCTs). Here, we show for the first time that CD147 interacts with membrane transporters beyond MCTs and exhibits a protective role for several of its interacting partners. CD147 prevents its interacting partner's proteasome-dependent degradation and incorrect plasma membrane localization through the CD147 transmembrane (TM) region. The interactions with transmembrane small molecule and ion transporters identified here indicate a central role of CD147 in pancreatic cancer metabolic reprogramming, particularly with respect to amino acid anabolism and calcium signaling. Importantly, CD147 genetic ablation prevents pancreatic cancer cell proliferation and tumor growth in vitro and in vivo in conjunction with metabolic rewiring towards amino acid anabolism, thus paving the way for future combined pharmacological treatments.
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http://dx.doi.org/10.18632/oncotarget.14272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341751PMC
January 2017

Non-cell-autonomous Effects of Autophagy Inhibition in Tumor Cells Promote Growth of Drug-resistant Cells.

Mol Pharmacol 2017 Jan 9;91(1):58-64. Epub 2016 Nov 9.

Department. of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado (J.T., L.S., M.L.G., L.D., H.L.F., A.T.); and Department of Medicine, University of Texas Southwest Medical School, Dallas, Texas (A.E.F.)

Autophagy, the mechanism by which cells deliver material to the lysosome, has been associated with resistance to anticancer drugs, leading autophagy inhibition to be widely studied as a potential chemosensitization strategy for cancer cells. This strategy is based on the idea that inhibition of autophagy will increase drug sensitivity and kill more cancer cells. Here we report an unintended negative effect of this strategy. When modeling the effect of drug resistance in a heterogeneous cancer cell population, we found that autophagy inhibition in drug-sensitive tumor cells causes increased growth of drug-resistant cells in the population through a mechanism involving caspase activation and prostaglandin E signaling. These results emphasize the importance of understanding how autophagy manipulation in a tumor cell can have both cell-autonomous and nonautonomous effects and suggest that attempts to chemosensitize by inhibiting autophagy could be enhanced by adopting methods aimed at reducing tumor repopulation.
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http://dx.doi.org/10.1124/mol.116.106070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5198514PMC
January 2017

Clonal cooperativity in heterogenous cancers.

Semin Cell Dev Biol 2017 04 28;64:79-89. Epub 2016 Aug 28.

Program in Cancer Biology, University of Colorado School of Medicine, 12800 East 19th Avenue, Aurora, CO 80045, United States; Program in Molecular Biology, University of Colorado School of Medicine, 12800 East 19th Avenue, Aurora, CO 80045, United States; Department of Pharmacology, University of Colorado School of Medicine, 12800 East 19th Avenue, Aurora, CO 80045, United States. Electronic address:

Tumor heterogeneity is a major obstacle to the development of effective therapies and is thus an important focus of cancer research. Genetic and epigenetic alterations, as well as altered tumor microenvironments, result in tumors made up of diverse subclones with different genetic and phenotypic characteristics. Intratumor heterogeneity enables competition, but also supports clonal cooperation via cell-cell contact or secretion of factors, resulting in enhanced tumor progression. Here, we summarize recent findings related to interclonal interactions within a tumor and the therapeutic implications of such interactions, with an emphasis on how different subclones collaborate with each other to promote proliferation, metastasis and therapy-resistance. Furthermore, we propose that disruption of clonal cooperation by targeting key factors (such as Wnt and Hedgehog, amongst others) can be an alternative approach to improving clinical outcomes.
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http://dx.doi.org/10.1016/j.semcdb.2016.08.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330947PMC
April 2017

A tale of two ends.

Cell Cycle 2016 06 21;15(12):1523-4. Epub 2016 Apr 21.

a Department of Pharmacology , University of Colorado, Denver, Anschutz Medical Campus , Aurora , CO , USA.

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http://dx.doi.org/10.1080/15384101.2016.1171652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4934064PMC
June 2016

The Six1 oncoprotein downregulates p53 via concomitant regulation of RPL26 and microRNA-27a-3p.

Nat Commun 2015 Dec 21;6:10077. Epub 2015 Dec 21.

Program in Molecular Biology, University of Colorado, Denver, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, Colorado 80045, USA.

TP53 is mutated in 50% of all cancers, and its function is often compromised in cancers where it is not mutated. Here we demonstrate that the pro-tumorigenic/metastatic Six1 homeoprotein decreases p53 levels through a mechanism that does not involve the negative regulator of p53, MDM2. Instead, Six1 regulates p53 via a dual mechanism involving upregulation of microRNA-27a and downregulation of ribosomal protein L26 (RPL26). Mutation analysis confirms that RPL26 inhibits miR-27a binding and prevents microRNA-mediated downregulation of p53. The clinical relevance of this interaction is underscored by the finding that Six1 expression strongly correlates with decreased RPL26 across numerous tumour types. Importantly, we find that Six1 expression leads to marked resistance to therapies targeting the p53-MDM2 interaction. Thus, we identify a competitive mechanism of p53 regulation, which may have consequences for drugs aimed at reinstating p53 function in tumours.
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http://dx.doi.org/10.1038/ncomms10077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703841PMC
December 2015

TWIST1-Induced miR-424 Reversibly Drives Mesenchymal Programming while Inhibiting Tumor Initiation.

Cancer Res 2015 May 25;75(9):1908-21. Epub 2015 Feb 25.

Program in Molecular Biology, University of Colorado Anschutz Medical Campus, Aurora, Colorado. Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, Colorado. Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Epithelial-to-mesenchymal transition (EMT) is a dynamic process that relies on cellular plasticity. Recently, the process of an oncogenic EMT, followed by a reverse mesenchymal-to-epithelial transition (MET), has been implicated as critical in the metastatic colonization of carcinomas. Unlike governance of epithelial programming, regulation of mesenchymal programming is not well understood in EMT. Here, we describe and characterize the first microRNA that enhances exclusively mesenchymal programming. We demonstrate that miR-424 is upregulated early during a TWIST1 or SNAI1-induced EMT, and that it causes cells to express mesenchymal genes without affecting epithelial genes, resulting in a mixed/intermediate EMT. Furthermore, miR-424 increases motility, decreases adhesion, and induces a growth arrest, changes associated with a complete EMT that can be reversed when miR-424 expression is lowered, concomitant with an MET-like process. Breast cancer patient miR-424 levels positively associate with TWIST1/2 and EMT-like gene signatures, and miR-424 is increased in primary tumors versus matched normal breast. However, miR-424 is downregulated in patient metastases versus matched primary tumors. Correspondingly, miR-424 decreases tumor initiation and is posttranscriptionally downregulated in macrometastases in mice, suggesting the need for biphasic expression of miR-424 to transit the EMT-MET axis. Next-generation RNA sequencing revealed miR-424 regulates numerous EMT and cancer stemness-associated genes, including TGFBR3, whose downregulation promotes mesenchymal phenotypes, but not tumor-initiating phenotypes. Instead, we demonstrate that increased MAPK-ERK signaling is critical for miR-424-mediated decreases in tumor-initiating phenotypes. These findings suggest miR-424 plays distinct roles in tumor progression, potentially facilitating earlier, but repressing later, stages of metastasis by regulating an EMT-MET axis.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-2394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417413PMC
May 2015

The SIX1-EYA transcriptional complex as a therapeutic target in cancer.

Expert Opin Ther Targets 2015 Feb 2;19(2):213-25. Epub 2015 Jan 2.

University of Colorado Anschutz Medical Campus, Department of Biochemistry and Molecular Genetics , Aurora, CO 80045 , USA ,

Introduction: The SIX homeodomain proteins and the eyes absent (EYA) family of co-activators form a bipartite transcription factor complex that promotes the proliferation and survival of progenitor cells during organogenesis and is down-regulated in most adult tissues. Abnormal over-expression of SIX1 and EYA in adult tissue is associated with the initiation and progression of diverse tumor types. Importantly, SIX1 and EYA are often co-overexpressed in tumors, and the SIX1-EYA2 interaction has been shown to be critical for metastasis in a breast cancer model. The EYA proteins also contain protein tyrosine phosphatase activity, which plays an important role in breast cancer growth and metastasis as well as directing cells to the repair pathway upon DNA damage.

Areas Covered: This review provides a summary of the SIX1/EYA complex as it relates to development and disease and the current efforts to therapeutically target this complex.

Expert Opinion: Recently, there have been an increasing number of studies suggesting that targeting the SIX1/EYA transcriptional complex will potently inhibit tumor progression. Although current attempts to develop inhibitors targeting this complex are still in the early stages, continued efforts toward developing better compounds may ultimately result in effective anti-cancer therapies.
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http://dx.doi.org/10.1517/14728222.2014.978860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336540PMC
February 2015

Intratumoral heterogeneity: Clonal cooperation in epithelial-to-mesenchymal transition and metastasis.

Cell Adh Migr 2015 16;9(4):265-76. Epub 2014 Oct 16.

a Department of Pharmacology ; University of Colorado; School of Medicine ; Aurora, CO USA.

Although phenotypic intratumoral heterogeneity was first described many decades ago, the advent of next-generation sequencing has provided conclusive evidence that in addition to phenotypic diversity, significant genotypic diversity exists within tumors. Tumor heterogeneity likely arises both from clonal expansions, as well as from differentiation hierarchies existent in the tumor, such as that established by cancer stem cells (CSCs) and non-CSCs. These differentiation hierarchies may arise due to genetic mutations, epigenetic alterations, or microenvironmental influences. An additional differentiation hierarchy within epithelial tumors may arise when only a few tumor cells trans-differentiate into mesenchymal-like cells, a process known as epithelial-to-mesenchymal transition (EMT). Again, this process can be influenced by both genetic and non-genetic factors. In this review we discuss the evidence for clonal interaction and cooperation for tumor maintenance and progression, particularly with respect to EMT, and further address the far-reaching effects that tumor heterogeneity may have on cancer therapy.
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http://dx.doi.org/10.4161/19336918.2014.972761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594578PMC
May 2016

Vascular endothelial growth factor C promotes breast cancer progression via a novel antioxidant mechanism that involves regulation of superoxide dismutase 3.

Breast Cancer Res 2014 Oct 30;16(5):462. Epub 2014 Oct 30.

Introduction: Triple-negative breast cancers, particularly the claudin-low subtype, are highly aggressive and exhibit increased tumor-initiating cell (TIC) characteristics. In this study, we demonstrate that vascular endothelial growth factor C (VEGF-C) is highly expressed in the claudin-low breast cancer subtype and also that it mediates tumor progression, not only through its role in lymphangiogenesis but also through regulating TIC characteristics and the response to reactive oxygen species (ROS).

Methods: VEGF C expression was examined in breast cancer subtypes, and a VEGF C expression signature was derived. VEGF C expression and/or its associated signature was correlated with TIC and chemoresistance signatures. In vitro and in vivo assays were performed to determine whether VEGF-C expression alters TIC characteristics and the response of breast cancer cells to chemotherapy and oxidative stress. Array analysis was used to identify a downstream effector of VEGF-C, superoxide dismutase 3 (Sod3), which was tested for its involvement in VEGF-C-mediated resistance to oxidative stress and enhancement of in vivo metastasis. The VEGF-C-associated receptor neuropilin 2 (Nrp2) was knocked down to determine whether it is required for the observed effects of VEGF-C. Expression of VEGF C and Sod3 was assessed in human breast cancers.

Results: VEGF C is highly expressed in claudin-low breast cancers, and VEGF C and the VEGF C signature are associated with TIC-related gene signatures. VEGF-C-knockdown in mammary carcinoma cells decreases TIC properties in vitro and in vivo, sensitizing cells to oxidative stress and chemotherapy. We identified Sod3 as a target of VEGF-C in breast cancer cells by demonstrating that it is required for VEGF-C-mediated cell survival in response to oxidative stress and for VEGF-C-mediated metastasis. We demonstrate that Nrp2 is the VEGF-C-associated receptor that mediates alterations in Sod3 expression and the response of tumor cells to oxidative stress. We show that VEGF C and Sod3 are positively associated in human breast cancer.

Conclusions: We describe a novel mechanism by which VEGF-C contributes to metastasis via its ability to enhance TIC-associated characteristics, particularly the response to ROS. We identified Sod3 as a critical mediator of VEGF-C-induced metastasis, and we provide evidence that the VEGF-C-Sod3 axis plays a role in human breast cancers.
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http://dx.doi.org/10.1186/s13058-014-0462-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303136PMC
October 2014

Homeoprotein Six2 promotes breast cancer metastasis via transcriptional and epigenetic control of E-cadherin expression.

Cancer Res 2014 Dec 27;74(24):7357-70. Epub 2014 Oct 27.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado. Program in Molecular Biology, University of Colorado School of Medicine, Aurora, Colorado. Program in Cancer Biology, University of Colorado School of Medicine, Aurora, Colorado. Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado.

Misexpression of developmental transcription factors occurs often in human cancers, where embryonic programs may be reinstated in a context that promotes or sustains malignant development. In this study, we report the involvement of the kidney development transcription factor Six2 in the metastatic progression of human breast cancer. We found that Six2 promoted breast cancer metastasis by a novel mechanism involving both transcriptional and epigenetic regulation of E-cadherin. Downregulation of E-cadherin by Six2 was necessary for its ability to increase soft agar growth and in vivo metastasis in an immunocompetent mouse model of breast cancer. Mechanistic investigations showed that Six2 represses E-cadherin expression by upregulating Zeb2, in part, through a microRNA-mediated mechanism and by stimulating promoter methylation of the E-cadherin gene (Cdh1). Clinically, SIX2 expression correlated inversely with CDH1 expression in human breast cancer specimens, corroborating the disease relevance of their interaction. Our findings establish Six2 as a regulator of metastasis in human breast cancers and demonstrate an epigenetic function for SIX family transcription factors in metastatic progression through the regulation of E-cadherin.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-0666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4268359PMC
December 2014

Allosteric inhibitors of the Eya2 phosphatase are selective and inhibit Eya2-mediated cell migration.

J Biol Chem 2014 Jun 22;289(23):16349-61. Epub 2014 Apr 22.

From the Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045,

Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg(2+). Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.
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http://dx.doi.org/10.1074/jbc.M114.566729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047403PMC
June 2014

MicroRNA-30a regulates zebrafish myogenesis through targeting the transcription factor Six1.

J Cell Sci 2014 May 14;127(Pt 10):2291-301. Epub 2014 Mar 14.

Department of Pharmacology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA

Precise spatiotemporal regulation of the SIX1 homeoprotein is required to coordinate vital tissue development, including myogenesis. Whereas SIX1 is downregulated in most tissues following embryogenesis, it is re-expressed in numerous cancers, including tumors derived from muscle progenitors. Despite crucial roles in development and disease, the upstream regulation of SIX1 expression has remained elusive. Here, we identify the first direct mechanism for Six1 regulation in embryogenesis, through microRNA30a (miR30a)-mediated repression. In zebrafish somites, we show that miR30a and six1a and six1b (hereafter six1a/b) are expressed in an inverse temporal pattern. Overexpression of miR30a leads to a reduction in six1a/b levels, and results in increased apoptosis and altered somite morphology, which phenocopies six1a/b knockdown. Conversely, miR30a inhibition leads to increased Six1 expression and abnormal somite morphology, revealing a role for endogenous miR30a as a muscle-specific miRNA (myomiR). Importantly, restoration of six1a in miR30a-overexpressing embryos restores proper myogenesis. These data demonstrate a new role for miR30a at a key node in the myogenic regulatory gene network through controlling Six1 expression.
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http://dx.doi.org/10.1242/jcs.143677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4021474PMC
May 2014

CDK6 binds and promotes the degradation of the EYA2 protein.

Cell Cycle 2014 12;13(1):62-71. Epub 2013 Oct 12.

Department of Biology; Connecticut College; New London, CT USA.

Cyclin-dependent kinase 6 (Cdk6) is a D-Cyclin-activated kinase that is directly involved in driving the cell cycle through inactivation of pRB in G₁ phase. Increasingly, evidence suggests that CDK6, while directly driving the cell cycle, may only be essential for proliferation of specialized cell types, agreeing with the notion that CDK6 also plays an important role in differentiation. Here, evidence is presented that CDK6 binds to and promotes degradation of the EYA2 protein. The EYA proteins are a family of proteins that activate genes essential for the development of multiple organs, regulate cell proliferation, and are misregulated in several types of cancer. This interaction suggests that CDK6 regulates EYA2 activity, a mechanism that could be important in development and in cancer.
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http://dx.doi.org/10.4161/cc.26755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3925736PMC
September 2014

Epithelial-mesenchymal transition and tumor suppression are controlled by a reciprocal feedback loop between ZEB1 and Grainyhead-like-2.

Cancer Res 2013 Oct 13;73(20):6299-309. Epub 2013 Aug 13.

Authors' Affiliations: Mary Babb Randolph Cancer Center and Department of Biochemistry, West Virginia University, Morgantown; Department of Biochemistry and Microbiology, Marshall University, Huntington, West Virginia; and Departments of Biochemistry/Molecular Genetics and Obstetrics/Gynecology, University of Colorado, Denver, Colorado.

Epithelial-mesenchymal transition (EMT) in carcinoma cells enhances malignant progression by promoting invasion and survival. EMT is induced by microenvironmental factors, including TGF-β and Wnt agonists, and by the E-box-binding transcription factors Twist, Snail, and ZEB. Grainyhead-like-2 (GRHL2), a member of the mammalian Grainyhead family of wound-healing regulatory transcription factors, suppresses EMT and restores sensitivity to anoikis by repressing ZEB1 expression and inhibiting TGF-β signaling. In this study, we elucidate the functional relationship between GRHL2 and ZEB1 in EMT/MET and tumor biology. At least three homeodomain proteins, Six1, LBX1, and HoxA5, transactivated the ZEB1 promoter, in the case of Six1, through direct protein-promoter interaction. GRHL2 altered the Six1-DNA complex, inhibiting this transactivation. Correspondingly, GRHL2 expression prevented tumor initiation in xenograft assays, sensitized breast cancer cells to paclitaxel, and suppressed the emergence of CD44(high)CD24(low) cells (defining the cancer stem cell phenotype in the cell type studied). GRHL2 was downregulated in recurrent mouse tumors that had evolved to an oncogene-independent, EMT-like state, supporting a role for GRHL2 downregulation in this phenotypic transition, modeling disease recurrence. The combination of TGF-β and Wnt activation repressed GRHL2 expression by direct interaction of ZEB1 with the GRHL2 promoter, inducing EMT. Together, our observations indicate that a reciprocal feedback loop between GRHL2 and ZEB1 controls epithelial versus mesenchymal phenotypes and EMT-driven tumor progression.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-4082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3806457PMC
October 2013

Transcriptional differences between normal and glioma-derived glial progenitor cells identify a core set of dysregulated genes.

Cell Rep 2013 Jun 30;3(6):2127-41. Epub 2013 May 30.

Center for Translational Neuromedicine, University of Rochester Medical Center, Rochester, NY 14642, USA.

Glial progenitor cells (GPCs) are a potential source of malignant gliomas. We used A2B5-based sorting to extract tumorigenic GPCs from human gliomas spanning World Health Organization grades II-IV. Messenger RNA profiling identified a cohort of genes that distinguished A2B5+ glioma tumor progenitor cells (TPCs) from A2B5+ GPCs isolated from normal white matter. A core set of genes and pathways was substantially dysregulated in A2B5+ TPCs, which included the transcription factor SIX1 and its principal cofactors, EYA1 and DACH2. Small hairpin RNAi silencing of SIX1 inhibited the expansion of glioma TPCs in vitro and in vivo, suggesting a critical and unrecognized role of the SIX1-EYA1-DACH2 system in glioma genesis or progression. By comparing the expression patterns of glioma TPCs with those of normal GPCs, we have identified a discrete set of pathways by which glial tumorigenesis may be better understood and more specifically targeted.
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http://dx.doi.org/10.1016/j.celrep.2013.04.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293199PMC
June 2013