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1.
ACS Nano ; 15(7): 11789-11805, 2021 Jul 27.
Article in English | MEDLINE | ID: mdl-34189924

ABSTRACT

| Several diseases exhibit a high degree of heterogeneity and diverse reprogramming of cellular pathways. To address this complexity, additional strategies and technologies must be developed to define their scope and variability with the goal of improving current treatments. Nanomedicines derived from viruses are modular systems that can be easily adapted for combinatorial approaches, including imaging, biomarker targeting, and intracellular delivery of therapeutics. Here, we describe a "designer nanoparticle" system that can be rapidly engineered in a tunable and defined manner. Phage-like particles (PLPs) derived from bacteriophage lambda possess physiochemical properties compatible with pharmaceutical standards, and in vitro particle tracking and cell targeting are accomplished by simultaneous display of fluorescein-5-maleimide (F5M) and trastuzumab (Trz), respectively (Trz-PLPs). Trz-PLPs bind to the oncogenically active human epidermal growth factor receptor 2 (HER2) and are internalized by breast cancer cells of the HER2 overexpression subtype, but not by those lacking the HER2 amplification. Compared to treatment with Trz, robust internalization of Trz-PLPs results in higher intracellular concentrations of Trz, prolonged inhibition of cell growth, and modulated regulation of cellular programs associated with HER2 signaling, proliferation, metabolism, and protein synthesis. Given the implications to cancer pathogenesis and that dysregulated signaling and metabolism can lead to drug resistance and cancer cell survival, the present study identifies metabolic and proteomic liabilities that could be exploited by the PLP platform to enhance therapeutic efficacy. The lambda PLP system is robust and rapidly modifiable, which offers a platform that can be easily "tuned" for broad utility and tailored functionality.


Subject(s)
Breast Neoplasms , Nanoparticles , Humans , Female , Trastuzumab/pharmacology , Bacteriophage lambda , Proteomics , Breast Neoplasms/drug therapy , Receptor, ErbB-2/metabolism , Nanoparticles/chemistry , Cell Line, Tumor
2.
JCI Insight ; 5(18)2020 09 17.
Article in English | MEDLINE | ID: mdl-32841218

ABSTRACT

ETV6 is an ETS family transcription factor that plays a key role in hematopoiesis and megakaryocyte development. Our group and others have identified germline mutations in ETV6 resulting in autosomal dominant thrombocytopenia and predisposition to malignancy; however, molecular mechanisms defining the role of ETV6 in megakaryocyte development have not been well established. Using a combination of molecular, biochemical, and sequencing approaches in patient-derived PBMCs, we demonstrate abnormal cytoplasmic localization of ETV6 and the HDAC3/NCOR2 repressor complex that led to overexpression of HDAC3-regulated interferon response genes. This transcriptional dysregulation was also reflected in patient-derived platelet transcripts and drove aberrant proplatelet formation in megakaryocytes. Our results suggest that aberrant transcription may predispose patients with ETV6 mutations to bone marrow inflammation, dysplasia, and megakaryocyte dysfunction.


Subject(s)
Bone Marrow Diseases/pathology , Germ-Line Mutation , Histone Deacetylases/metabolism , Interferon Regulatory Factors/metabolism , Nuclear Receptor Co-Repressor 2/metabolism , Proto-Oncogene Proteins c-ets/genetics , Repressor Proteins/genetics , Thrombocytopenia/pathology , Bone Marrow Diseases/etiology , Bone Marrow Diseases/metabolism , Child , Cohort Studies , Genetic Predisposition to Disease , Histone Deacetylases/genetics , Humans , Interferon Regulatory Factors/genetics , Megakaryocytes/metabolism , Megakaryocytes/pathology , Nuclear Receptor Co-Repressor 2/genetics , Protein Transport , Thrombocytopenia/etiology , Thrombocytopenia/metabolism , ETS Translocation Variant 6 Protein
3.
Breast Cancer Res Treat ; 182(3): 601-612, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32562116

ABSTRACT

PURPOSE: The ETS transcription factor ESE-1 has been shown to be important in HER2+ breast cancer and ESE-1 mRNA expression has been shown to associate with prognostic outcomes in the HER2+ subtype, as well as in ER+, HER2+ luminal B patients. However, the clinical significance of ESE-1 protein expression remains unknown. The purpose of the current exploratory study is to evaluate the prognostic value of ESE-1 protein expression in molecular breast cancer subtypes with special emphasis on hormone receptor positive HER2+(HR+ HER2+) and the HER2 positive (HER2+-only) breast cancer patients. METHODS: We developed a mouse monoclonal anti-ESE-1 antibody, verified its specificity, epitope, and used immunohistochemical staining to assess ESE-1 expression in an IBC approved archive of 957 breast tumor samples. Using Pearson product correlation, contingency analysis, and long rank P value testing, we analyzed the association of ESE-1 expression with clinicopathological features and survival outcomes in HR+HER2-; HR+HER2+; HR- HER2- (Triple negative) and HR-HER2+ (HER2 subtype) patients. RESULTS: ESE-1, nuclear or cytoplasmic, was not significantly associated with survival outcomes in HR+HER2-, triple-negative, or HER2+-only breast cancer patients. However, high nuclear ESE-1 was associated with poor survival outcomes in hormone receptor positive (ERα+, PR+) HER2+ patients and was an independent prognostic marker for that group. CONCLUSIONS: This study provides evidence for prognostic significance of nuclear ESE-1 in ERalpha positive breast cancers patients also positive for HER2 indicating that crosstalk between ERalpha and ESE-1 in HER2+ tumors could be important for prognostic outcomes. Further studies regarding the nature of interaction between ESE-1 and ERalpha in these tumors are warranted.


Subject(s)
Breast Neoplasms/metabolism , DNA-Binding Proteins/metabolism , Proto-Oncogene Proteins c-ets/metabolism , Receptor, ErbB-2/metabolism , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Transcription Factors/metabolism , Biomarkers, Tumor/metabolism , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Breast Neoplasms/surgery , Female , Follow-Up Studies , Humans , Immunohistochemistry , Middle Aged , Prognosis , Survival Rate
4.
Nanotheranostics ; 3(2): 212-222, 2019.
Article in English | MEDLINE | ID: mdl-31183315

ABSTRACT

The highly tunable, noninvasive and spatially targeted nature of microbubble-enhanced, ultrasound-guided (MB+US) drug delivery makes it desirable for a wide variety of therapies. In breast cancer, both HER2+ and HER2- type neoplasms pose significant challenges to conventional therapeutics in greater than 40% of breast cancer patients, even with the widespread application of biologics such as trastuzumab. To address this therapeutic challenge, we examined the novel combination of tumor-injected microbubble-bound siRNA complexes and monodisperse size-isolated microbubbles (4-µm diameter) to attenuate tumor growth in vivo, as well as MB+US-facilitated shRNA and siRNA knockdown of ESE-1, an effector linked to dysregulated HER2 expression in HER2+/- cell line propagation. We first screened six variants of siESE and shESE for efficient knockdown of ESE in breast cancer cell lines. We demonstrated efficient reduction of BT-474 (PR+, ER+, HER2+; luminal B) and MDA-MB-468 (PR-, ER-, HER2-; triple-negative) clonogenicity and non-adherent growth after knockdown of ESE-1. A significant reduction in proliferative potential was seen for both cell lines using MB+US to deliver shESE and siESE. We then demonstrated significant attenuation of BT-474 xenograft tumor growth in Nod/SCID female mice using direct injection of microbubble-adsorbed siESE to the tumor and subsequent sonication. Our results suggest a positive effect on drug delivery from MB+US, and highlights the feasibility of using RNAi and MB+US for breast cancer pathologies. RNAi coupled with MB+US may also be an effective theranostic approach to treat other acoustically accessible tumors, such as melanoma, thyroid, parotid and skin cancer.


Subject(s)
Breast Neoplasms , Microbubbles , Receptor, ErbB-2/metabolism , Ultrasonic Waves , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Trastuzumab/pharmacology , Xenograft Model Antitumor Assays
5.
Endocrinology ; 159(6): 2421-2434, 2018 06 01.
Article in English | MEDLINE | ID: mdl-29726995

ABSTRACT

Prolactin-secreting adenomas, or prolactinomas, cause hypogonadism, osteoporosis, and infertility. Although dopamine agonists (DAs) are used clinically to treat prolactinoma and reduce prolactin secretion via cAMP inhibition, the precise mechanism by which DAs inhibit lactotrope proliferation has not been defined. In this study, we report that phosphatidylinositol 3-kinase (PI3K) signals through AKT and mTOR to drive proliferation of pituitary somatolactotrope GH4T2 cells. We demonstrate that the DA cabergoline reduces activity of the mTOR effector s6K and diminishes GH4T2 cell proliferation primarily via activation of the long isoform of the dopamine D2 receptor (D2R). Dysfunctional D2R-mediated signaling and/or downregulated D2R expression is thought be the primary mechanism of DA resistance, which is observed in 10% to 20% of prolactinoma tumors. Dopamine-mediated D2R activation results in ERK stimulation and PI3K inhibition, suggesting that these two pathways act in an inverse manner to maintain lactotrope homeostasis. In this study, we found that ERK1/2-mediated prolactin transcription is inhibited by PI3K/CDK4-driven cell cycle progression, emphasizing that the ERK and PI3K signaling pathways oppose one another in lactotrope cells under homeostatic conditions. Lastly, we show that both ERK1/2 and AKT are activated in prolactinoma, demonstrating that the balance of ERK and AKT is dysregulated in human prolactinoma. Our findings reveal a potential use for dual pharmacological inhibitors of ERK and AKT as an alternative treatment strategy for DA-resistant prolactinomas.


Subject(s)
Dopamine/pharmacology , MAP Kinase Signaling System/drug effects , Phosphatidylinositol 3-Kinase/metabolism , Pituitary Neoplasms/metabolism , Prolactinoma/metabolism , Animals , Cabergoline/pharmacology , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cells, Cultured , Down-Regulation/drug effects , Down-Regulation/genetics , HEK293 Cells , Homeostasis/drug effects , Homeostasis/physiology , Humans , MAP Kinase Signaling System/genetics , Phosphatidylinositol 3-Kinase/genetics , Phosphoinositide-3 Kinase Inhibitors , Pituitary Neoplasms/genetics , Pituitary Neoplasms/pathology , Prolactinoma/genetics , Prolactinoma/pathology , Rats , Signal Transduction/drug effects , Signal Transduction/genetics
6.
Mol Cell Endocrinol ; 476: 165-172, 2018 11 15.
Article in English | MEDLINE | ID: mdl-29753028

ABSTRACT

Distinct cell types have been shown to respond to activated Ras signaling in a cell-specific manner. In contrast to its pro-tumorigenic role in some human epithelial cancers, oncogenic Ras triggers differentiation of pheochromocytoma cells and medullary thyroid carcinoma cells. Furthermore, we have previously demonstrated that in pituitary somatolactotropes, activated Ras promotes differentiation and is not sufficient to drive tumorigenesis. These findings demonstrate that lactotrope cells have the ability to evade the tumorigenic fate that is often associated with persistent activation of Ras/ERK signaling, and suggest that there may be differential expression of inhibitory signaling molecules or negative cell cycle regulators that act as a brake to prevent the tumorigenic effects of sustained Ras signaling. Here we aim to gain further insight into the mechanisms that allow GH4T2 cells to evade an oncogenic response to Ras. We show that Ral, but likely not menin, plays a key role in directing Ras-mediated differentiation of somatolactotropes, which may allow these cells to escape the tumorigenic fate that is often associated with activated Ras signaling. We also show that dominant negative Ras expression results in reduced GH4T2 cell proliferation and transformation, but does not influence differentiation. Taken together, the data presented here begin to shed light on the mechanisms by which pituitary somatolactotropes evade an oncogenic response to persistently activated Ras signaling and suggest that the architecture of the Ras signaling cascade in some endocrine cell types may be distinct from that of cells that respond to Ras in an oncogenic manner.


Subject(s)
Carcinogenesis/metabolism , Carcinogenesis/pathology , Lactotrophs/metabolism , Oncogenes , Pituitary Gland/metabolism , ras Proteins/metabolism , Animals , Cell Proliferation , Prolactin/genetics , Prolactin/metabolism , Promoter Regions, Genetic/genetics , Rats , Transcription Factors/metabolism
7.
Mol Cell Endocrinol ; 463: 87-96, 2018 03 05.
Article in English | MEDLINE | ID: mdl-28445712

ABSTRACT

Conserved signaling pathways are critical regulators of pituitary homeostasis and, when dysregulated, contribute to adenoma formation. Pituitary adenomas are typically benign and rarely progress to malignant cancer. Pituitary and other neuroendocrine cell types often display non-proliferative responses to ERK and PI3K, in contrast to non-endocrine cell types which typically proliferate in response to ERK and PI3K activation. These differences likely contribute to the infrequent progression to malignancy in many endocrine tumors. In this review, we highlight the Ras/ERK and PI3K/AKT/mTOR signaling pathways in each pituitary cell type, as well as in other endocrine tissues. Furthermore, we provide evidence that a balance of ERK and PI3K signaling is required to maintain pituitary homeostasis. It is unlikely that one sole oncogene will be identified as being responsible for sporadic pituitary adenoma formation. This review emphasizes the necessity to consider endocrine cell-specific contexts and the interplay of signaling pathways to define the mechanisms underlying pituitary tumorigenesis.


Subject(s)
Pituitary Gland/metabolism , Pituitary Gland/pathology , Signal Transduction , Animals , Carcinogenesis/metabolism , Carcinogenesis/pathology , Humans , Models, Biological , Neurosecretory Systems/metabolism , Pituitary Neoplasms/metabolism , Pituitary Neoplasms/pathology
8.
Anticancer Res ; 37(12): 6583-6591, 2017 12.
Article in English | MEDLINE | ID: mdl-29187433

ABSTRACT

BACKGROUND/AIM: ESE-1/Elf3 controls transformation properties in mammary epithelial cells, and is most clinically relevant in HER2+ breast cancer. Herein we showed that ESE-1 knockdown inhibits tumorigenic growth in HER2+, trastuzumab-resistant HR20 (derived from HER2+ ER+ BT474) and Pool2 (derived from HER2+ ER- SKBR3 cells) cell lines. MATERIALS AND METHODS: We used cell proliferation, clonogenicity, viability, and soft agar assays to measure the effects of ESE-1 knockdown in cell lines. RESULTS: ESE-1 knockdown in the resistant cell lines inhibited HER2 and other downstream effectors in a cell-type specific manner, but caused down-regulation of pAkt and cyclin D1 in both sublines. In parental BT474 and SKBR3 ESE-1 silencing revealed a potent anti-proliferative effect that mimics the trastuzumab-mediated growth inhibition but did not enhance trastuzumab sensitivity in the resistant sublines. CONCLUSION: This study provides rationale to study ESE-1 as a novel mean to treat HER2+ patients who show resistance to anti-HER2 therapy.


Subject(s)
Cell Proliferation/drug effects , DNA-Binding Proteins/metabolism , Proto-Oncogene Proteins c-ets/metabolism , Receptor, ErbB-2/metabolism , Transcription Factors/metabolism , Trastuzumab/pharmacology , Antineoplastic Agents, Immunological/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Cyclin D1/metabolism , DNA-Binding Proteins/genetics , Drug Resistance, Neoplasm/genetics , Female , Humans , Immunoblotting , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-ets/genetics , RNA Interference , Transcription Factors/genetics
9.
Oncotarget ; 8(41): 69622-69640, 2017 Sep 19.
Article in English | MEDLINE | ID: mdl-29050229

ABSTRACT

ESE-1/Elf3 and HER2 appear to establish a positive feedback regulatory loop, but the precise role of ESE-1 in HER2+ breast tumorigenesis remains unknown. Analyzing public repositories, we found that luminal B and HER2 subtype patients with high ESE-1 mRNA levels displayed worse relapse free survival. We stably knocked down ESE-1 in HER2+ luminal B BT474 cells and HER2 subtype SKBR3 cells, which resulted in decreased cell proliferation, colony formation, and anchorage-independent growth in vitro. Stable ESE-1 knockdown inhibited HER2-dependent signaling in BT474 cells and inhibited mTOR activation in SKBR3 cells, but reduced Akt signaling in both cell types. Expression of a constitutively-active Myr-Akt partially rescued the anti-proliferative effect of ESE-1 knockdown in both cell lines. Furthermore, ESE-1 knockdown inhibited cyclin D1, resulting in a G1 delay in both cell lines. Finally, ESE-1 knockdown completely inhibited BT474 cell xenograft tumors in NOD/SCID female mice, which correlated with reduced in vitro tumorsphere formation. Taken together, these results reveal the ESE-1 controls transformation via distinct upstream signaling mechanisms in SKBR3 and BT474 cells, which ultimately impinge on Akt and cyclin D1 in both cell types to regulate cell proliferation. Particularly significant is that ESE-1 controls tumorigenesis and is associated with worse clinical outcomes in HER2 breast cancer.

10.
Oncotarget ; 8(10): 16951-16963, 2017 Mar 07.
Article in English | MEDLINE | ID: mdl-28199976

ABSTRACT

Transcription factors are master switches for various biochemical pathways. However, transcription factors involved in the pathogenesis of ovarian cancer have yet to be explored thoroughly. Therefore, in the present study, we assessed the prognostic value of the transcription factor E74-like factor 3 (ELF3) identified via transcriptome profiling of the epithelial components of microdissected ovarian tumor samples isolated from long- and short-term survivors and determined its roles in ovarian cancer pathogenesis. Immunohistochemical analysis of ELF3 in tumor tissue sections suggested that ELF3 was exclusively expressed by epithelial ovarian cancer cells. Furthermore, using 112 high-grade ovarian cancer samples isolated from patients and The Cancer Genome Atlas (TCGA) data, we found that downregulation of ELF3 expression was markedly associated with reduced survival. Functional studies demonstrated that overexpression of ELF3 in ovarian cancer cells suppressed proliferation and anchorage-dependent growth of the cells and that ELF3 silencing increased cell proliferation. Furthermore, upregulation of ELF3 increased expression of epithelial markers, decreased expression of mesenchymal markers, and mediated translocation of epithelial-mesenchymal transition (EMT) signaling molecules in ovarian cancer cells. Finally, we validated the tumor-inhibitory roles of ELF3 using animal models. In conclusion, ELF3 is a favorable prognostic marker for ovarian cancer. As a negative regulator of EMT, ELF3-modulated reversal of EMT may be a new effective modality in the treatment of ovarian cancer.


Subject(s)
DNA-Binding Proteins/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Ovarian Neoplasms/genetics , Proto-Oncogene Proteins c-ets/genetics , Transcription Factors/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Cell Line, Tumor , Cell Proliferation/genetics , DNA-Binding Proteins/metabolism , Female , Gene Expression Profiling/statistics & numerical data , Humans , Kaplan-Meier Estimate , Microscopy, Confocal , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Prognosis , Proportional Hazards Models , Proto-Oncogene Proteins c-ets/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Survivors , Transcription Factors/metabolism , Transplantation, Heterologous
11.
Theranostics ; 5(12): 1419-27, 2015.
Article in English | MEDLINE | ID: mdl-26681986

ABSTRACT

Microbubbles interact with ultrasound to induce transient microscopic pores in the cellular plasma membrane in a highly localized thermo-mechanical process called sonoporation. Theranostic applications of in vitro sonoporation include molecular delivery (e.g., transfection, drug loading and cell labeling), as well as molecular extraction for measuring intracellular biomarkers, such as proteins and mRNA. Prior research focusing mainly on the effects of acoustic forcing with polydisperse microbubbles has identified a "soft limit" of sonoporation efficiency at 50% when including dead and lysed cells. We show here that this limit can be exceeded with the judicious use of monodisperse microbubbles driven by a physiotherapy device (1.0 MHz, 2.0 W/cm(2), 10% duty cycle). We first examined the effects of microbubble size and found that small-diameter microbubbles (2 µm) deliver more instantaneous power than larger microbubbles (4 & 6 µm). However, owing to rapid fragmentation and a short half-life (0.7 s for 2 µm; 13.3 s for 6 µm), they also deliver less energy over the sonoporation time. This translates to a higher ratio of FITC-dextran (70 kDa) uptake to cell death/lysis (4:1 for 2 µm; 1:2 for 6 µm) in suspended HeLa cells after a single sonoporation. Sequential sonoporations (up to four) were consequently employed to increase molecular delivery. Peak uptake was found to be 66.1 ± 1.2% (n=3) after two sonoporations when properly accounting for cell lysis (7.0 ± 5.6%) and death (17.9 ± 2.0%), thus overcoming the previously reported soft limit. Substitution of TRITC-dextran (70 kDa) on the second sonoporation confirmed the effects were multiplicative. Overall, this study demonstrates the possibility of utilizing monodisperse small-diameter microbubbles as a means to achieve multiple low-energy sonoporation bursts for efficient in vitro cellular uptake and sequential molecular delivery.


Subject(s)
Drug Delivery Systems/methods , Epithelial Cells/drug effects , Epithelial Cells/radiation effects , Microbubbles , Ultrasonography/methods , Dextrans/analysis , Dextrans/pharmacokinetics , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/analysis , Fluorescein-5-isothiocyanate/pharmacokinetics , HeLa Cells , Humans , Rhodamines/analysis , Rhodamines/pharmacokinetics
12.
Nat Genet ; 47(5): 535-538, 2015 May.
Article in English | MEDLINE | ID: mdl-25807284

ABSTRACT

Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia. We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV) and two occurrences of B cell-precursor acute lymphoblastic leukemia (ALL). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 (ets variant 6), c.641C>T, encoding a p.Pro214Leu substitution in the central domain, segregating with thrombocytopenia and elevated MCV. A screen of 23 families with similar phenotypes identified 2 with ETV6 mutations. One family also had a mutation encoding p.Pro214Leu and one individual with ALL. The other family had a c.1252A>G transition producing a p.Arg418Gly substitution in the DNA-binding domain, with alternative splicing and exon skipping. Functional characterization of these mutations showed aberrant cellular localization of mutant and endogenous ETV6, decreased transcriptional repression and altered megakaryocyte maturation. Our findings underscore a key role for ETV6 in platelet formation and leukemia predisposition.


Subject(s)
Hematologic Diseases/genetics , Proto-Oncogene Proteins c-ets/genetics , Repressor Proteins/genetics , Thrombocytopenia/genetics , Adult , Child, Preschool , DNA Mutational Analysis , Erythrocytes, Abnormal , Exome , Female , Genetic Association Studies , Genetic Predisposition to Disease , Germ-Line Mutation , HEK293 Cells , Humans , Male , Mutation, Missense , Pedigree , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , ETS Translocation Variant 6 Protein
13.
Adv Exp Med Biol ; 846: 37-59, 2015.
Article in English | MEDLINE | ID: mdl-25472533

ABSTRACT

Dysregulation of the signaling pathways that govern lactotrope biology contributes to tumorigenesis of prolactin (PRL)-secreting adenomas, or prolactinomas, leading to a state of pathological hyperprolactinemia. Prolactinomas cause hypogonadism, infertility, osteoporosis, and tumor mass effects, and are the most common type of neuroendocrine tumor. In this review, we highlight signaling pathways involved in lactotrope development, homeostasis, and physiology of pregnancy, as well as implications for signaling pathways in pathophysiology of prolactinoma. We also review mutations found in human prolactinoma and briefly discuss animal models that are useful in studying pituitary adenoma, many of which emphasize the fact that alterations in signaling pathways are common in prolactinomas. Although individual mutations have been proposed as possible driving forces for prolactinoma tumorigenesis in humans, no single mutation has been clinically identified as a causative factor for the majority of prolactinomas. A better understanding of lactotrope-specific responses to intracellular signaling pathways is needed to explain the mechanism of tumorigenesis in prolactinoma.


Subject(s)
Carcinogenesis , Lactotrophs/pathology , Lactotrophs/physiology , Animals , Carcinogenesis/genetics , Cell Differentiation/genetics , Female , Homeostasis/genetics , Humans , Pituitary Neoplasms/genetics , Pregnancy , Prolactinoma/genetics , Signal Transduction
14.
Mol Endocrinol ; 28(12): 1999-2011, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25361391

ABSTRACT

The signaling pathways that govern the lactotrope-specific differentiated phenotype, and those that control lactotrope proliferation in both physiological and pathological lactotrope expansion, are poorly understood. Moreover, the specific role of MAPK signaling in lactotrope proliferation vs differentiation, whether activated phosphorylated MAPK is sufficient for prolactinoma tumor formation remain unknown. Given that oncogenic Ras mutations and persistently activated phosphorylated MAPK are found in human tumors, including prolactinomas and other pituitary tumors, a better understanding of the role of MAPK in lactotrope biology is required. Here we directly examined the role of persistent Ras/MAPK signaling in differentiation, proliferation, and tumorigenesis of rat pituitary somatolactotrope GH4 cells. We stimulated Ras/MAPK signaling in a persistent, long-term manner (over 6 d) in GH4 cells using two distinct approaches: 1) a doxycycline-inducible, oncogenic V12Ras expression system; and 2) continuous addition of exogenous epidermal growth factor. We find that long-term activation of the Ras/MAPK pathway over 6 days promotes differentiation of the bihormonal somatolactotrope GH4 precursor cell into a prolactin-secreting, lactotrope cell phenotype in vitro and in vivo with GH4 cell xenograft tumors. Furthermore, we show that persistent activation of the Ras/MAPK pathway not only fails to promote cell proliferation, but also diminishes tumorigenic characteristics in GH4 cells in vitro and in vivo. These data demonstrate that activated MAPK promotes differentiation and is not sufficient to drive tumorigenesis, suggesting that pituitary lactotrope tumor cells have the ability to evade the tumorigenic fate that is often associated with Ras/MAPK activation.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , ras Proteins/metabolism , Animals , Blotting, Western , Cell Differentiation/physiology , Cell Line , Cell Line, Tumor , Electrophoresis, Polyacrylamide Gel , Female , Humans , Mice , Mice, Nude , Mitogen-Activated Protein Kinases/genetics , Prolactinoma/metabolism , Rats , Signal Transduction/genetics , Signal Transduction/physiology , ras Proteins/genetics
15.
Crit Rev Biochem Mol Biol ; 48(6): 522-43, 2013.
Article in English | MEDLINE | ID: mdl-24066765

ABSTRACT

The E26 transformation-specific (ETS) family of transcription factors is critical for development, differentiation, proliferation and also has a role in apoptosis and tissue remodeling. Changes in expression of ETS proteins therefore have a significant impact on normal physiology of the cell. Transcriptional consequences of ETS protein deregulation by overexpression, gene fusion, and modulation by RAS/MAPK signaling are linked to alterations in normal cell functions, and lead to unlimited increased proliferation, sustained angiogenesis, invasion and metastasis. Existing data show that ETS proteins control pathways in epithelial cells as well as stromal compartments, and the crosstalk between the two is essential for normal development and cancer. In this review, we have focused on ETS factors with a known contribution in cancer development. Instead of focusing on a prototype, we address cancer associated ETS proteins and have highlighted the diverse mechanisms by which they affect carcinogenesis. Finally, we discuss strategies for ETS factor targeting as a potential means for cancer therapeutics.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Proto-Oncogene Proteins c-ets/metabolism , Apoptosis , Cell Differentiation , Cell Proliferation , DNA-Binding Proteins/metabolism , Humans , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Neovascularization, Pathologic , Protein Binding , Proto-Oncogene Proteins c-ets/genetics , Signal Transduction/genetics
16.
DNA Cell Biol ; 31(8): 1403-11, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22553926

ABSTRACT

MicroRNAs (miRs) are important regulators of gene expression in normal physiology and disease, and are widely misexpressed in cancer. A number of studies have identified miR-21 as an important promoter of oncogenesis. However, as is true of most miRs, the mechanisms behind the aberrant expression of miR-21 in cancer are poorly understood. Herein, we examine the regulation of miR-21 expression in colorectal cancer (CRC) cells by the oncogenic epidermal growth factor (EGF)/Ras pathway and by Ets transcription factors, modulators of epithelial oncogenesis that are frequently misexpressed in CRC. We show that EGF/Ras efficiently induces the miR-21 primary transcript, but this does not rapidly and simply translate into higher mature miR-21 levels. Rather, induction of mature miR-21 by constitutive activation of this pathway is slow, is associated with only minimal activation of mitogen-activated protein kinase, and may involve stimulation of post-transcriptional processing by mechanisms other than Dicer stabilization. We further identify Ets transcription factors as modifiers of miR-21 expression in CRC. The effects of Ets factors on miR-21 expression are cell context-dependent, and appear to involve both direct and indirect mechanisms. The Ets factor Pea3 emerges from our studies as a consistent repressor of miR-21 transcription. Overall, our studies identify a complex relationship between oncogenic pathways and steady-state miR-21 levels in CRC, and highlight the need for greater understanding of the control of miR expression in cancer and other disease states.


Subject(s)
Colorectal Neoplasms/genetics , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , Proto-Oncogene Proteins c-ets/metabolism , Signal Transduction , Cell Line, Tumor , Colorectal Neoplasms/metabolism , Epidermal Growth Factor/metabolism , Humans , MicroRNAs/metabolism , Oncogene Protein p21(ras)/genetics , Oncogene Protein p21(ras)/metabolism , Transcription, Genetic
17.
Mol Endocrinol ; 25(11): 1880-91, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21980073

ABSTRACT

Hyperprolactinemia, usually caused by a pituitary lactotroph tumor, leads to galactorrhea and infertility. Increased prolactin (PRL) levels may be due to enhanced PRL expression or proliferation of PRL-secreting cells. We hypothesize that PRL expression and PRL-secreting cell proliferation are linked. Using microarray-based gene expression profiling, we identified CCAAT-enhancer-binding protein δ (CEBPD) transcription factor as a critical gene that regulates both PRL expression and lactotroph cell proliferation. CEBPD expression levels are decreased approximately 7-fold in experimental rat prolactinoma cells. Forced expression of this transcription factor in PRL-secreting cells (GH3 and MMQ) inhibited PRL expression and cellular proliferation, and CEBPD knockdown by small interfering RNA leads to increased PRL expression in both cell lines. To determine mechanisms underlying this observation, we determined binding of CEBPD to the PRL promoter and also showed marked suppression (96%) of PRL promoter activity. CEBPD and Pit1 interact and attenuate each other's binding to the PRL promoter. CEBPD also suppresses expression of proliferation-related genes, including c-Myc, survivin, as well as cyclins B1, B2, and D1. These results show that PRL expression and cell proliferation are controlled in part by CEBPD.


Subject(s)
Prolactin/metabolism , Prolactinoma/metabolism , Animals , Blotting, Western , CCAAT-Enhancer-Binding Protein-delta , Cell Line, Tumor , Cell Proliferation , Chromatin Immunoprecipitation , Cyclin B1/genetics , Cyclin B1/metabolism , Cyclin B2/genetics , Cyclin B2/metabolism , Cyclin D1/genetics , Cyclin D1/metabolism , Estrogens/pharmacology , Female , Fluorescent Antibody Technique , Growth Hormone/metabolism , Mice , Mice, Nude , Oligonucleotide Array Sequence Analysis , Prolactin/genetics , Prolactinoma/chemically induced , Prolactinoma/pathology , Protein Binding , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA, Small Interfering , Radioimmunoassay , Rats , Transcription Factor Pit-1/genetics , Transcription Factor Pit-1/metabolism
18.
Mol Cancer ; 10: 103, 2011 Aug 28.
Article in English | MEDLINE | ID: mdl-21871131

ABSTRACT

BACKGROUND: The ETS family transcription factor ESE-1 is often overexpressed in human breast cancer. ESE-1 initiates transformation of MCF-12A cells via a non-transcriptional, cytoplasmic process that is mediated by a unique 40-amino acid serine and aspartic acid rich (SAR) subdomain, whereas, ESE-1's nuclear transcriptional property is required to maintain the transformed phenotype of MCF7, ZR-75-1 and T47D breast cancer cells. RESULTS: To map the minimal functional nuclear localization (NLS) and nuclear export (NES) signals, we fused in-frame putative NLS and NES motifs between GFP and the SAR domain. Using these GFP constructs as reporters of subcellular localization, we mapped a single NLS to six basic amino acids (242 HGKRRR 247) in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102 LCNCALEELRL 112) and another to the DNA binding domain (DBD), (NES2: 275 LWEFIRDILI 284). Moreover, analysis of a putative NLS located in the DBD (316 GQKKKNSN 323) by a similar GFP-SAR reporter or by internal deletion of the DBD, revealed this sequence to lack NLS activity. To assess the role of NES2 in regulating ESE-1 subcellular localization and subsequent transformation potency, we site-specifically mutagenized NES2, within full-length GFP-ESE-1 and GFP-NES2-SAR reporter constructs. These studies show that site-specific mutation of NES2 completely abrogates ESE-1 transforming activity. Furthermore, we show that exclusive cytoplasmic targeting of the SAR domain is sufficient to initiate transformation, and we report that an intact SAR domain is required, since block mutagenesis reveals that an intact SAR domain is necessary to maintain its full transforming potency. Finally, using a monoclonal antibody targeting the SAR domain, we demonstrate that the SAR domain contains a region accessible for protein - protein interactions. CONCLUSIONS: These data highlight that ESE-1 contains NLS and NES signals that play a critical role in regulating its subcellular localization and function, and that an intact SAR domain mediates MEC transformation exclusively in the cytoplasm, via a novel nontranscriptional mechanism, whereby the SAR motif is accessible for ligand and/or protein interactions. These findings are significant, since they provide novel molecular insights into the functions of ETS transcription factors in mammary cell transformation.


Subject(s)
Cell Transformation, Neoplastic , Cytoplasm/metabolism , DNA-Binding Proteins/metabolism , Epithelial Cells/metabolism , Mammary Glands, Human/pathology , Peptide Fragments/metabolism , Proto-Oncogene Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution , Cell Line , DNA-Binding Proteins/chemistry , Humans , Mammary Glands, Human/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Export Signals , Nuclear Localization Signals , Peptide Fragments/chemistry , Peptide Mapping , Protein Structure, Tertiary , Protein Transport , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins c-ets , Recombinant Fusion Proteins/chemistry , Transcription Factors/chemistry
19.
Mol Cancer ; 10: 75, 2011 Jun 18.
Article in English | MEDLINE | ID: mdl-21682918

ABSTRACT

BACKGROUND: LIM kinase 1 (LIMK1) is expressed in both cytoplasmic and nuclear compartments, and is a key regulator of cytoskeletal organization involved in cell migration and proliferation. LIMK1 levels are increased in several human cancers, with LIMK1 over-expression in prostate and breast cancer cells leading to tumor progression. While it has been presumed that the mechanism by which LIMK1 promotes cancer progression is via its cytoplasmic effects, the role of nuclear vs cytoplasmic LIMK1 in the tumorigenic process has not been examined. RESULTS: To determine if cytoplasmic or nuclear LIMK1 expression correlated with breast cancer, we performed immunohistochemical (IHC) analysis of breast tissue microarrays (TMAs), The IHC analysis of breast TMAs revealed that 76% of malignant breast tissue samples strongly expressed LIMK1 in the cytoplasm, with 52% of these specimens also expressing nuclear LIMK1. Only 48% of benign breast samples displayed strong cytoplasmic LIMK1 expression and 27% of these expressed nuclear LIMK1. To investigate the respective roles of cytoplasmic and nuclear LIMK1 in breast cancer progression, we targeted GFP-LIMK1 to cytoplasmic and nuclear subcellular compartments by fusing nuclear export signals (NESs) or nuclear localization sequences (NLS), respectively, to the amino-terminus of GFP-LIMK1. Stable pools of MDA-MB-231 cells were generated by retroviral transduction, and fluorescence microscopy revealed that GFP alone (control) and GFP-LIMK1 were each expressed in both the cytoplasm and nucleus of MDA-MB-231 cells, whereas NLS-GFP-LIMK1 was expressed in the nucleus and NES-GFP-LIMK1 was expressed in the cytoplasm. Western blot analyses revealed equal expression of GFP-LIMK1 and NES-GFP-LIMK1, with NLS-GFP-LIMK1 expression being less but equal to endogenous LIMK1. Also, Western blotting revealed increased levels of phospho-cofilin, phospho-FAK, phospho-paxillin, phospho-Src, phospho-AKT, and phospho-Erk1/2 in cells expressing all GFP-LIMK1 fusions, compared to GFP alone. Invasion assays revealed that all GFP-LIMK1 fusions increased MDA-MB-231 cell invasion ~1.5-fold, compared to GFP-only control cells. Tumor xenograft studies in nude mice revealed that MDA-MB-231 cells stably expressing GFP-LIMK, NLS-GFP-LIMK1 and NES-GFP-LIMK1 enhanced tumor growth 2.5-, 1.6- and 4.7-fold, respectively, compared to GFP-alone. CONCLUSION: Taken together, these data demonstrate that LIMK1 activity in both the cytoplasmic and nuclear compartments promotes breast cancer progression, underscoring that nuclear LIMK1 contributes to the transforming function of LIMK1.


Subject(s)
Breast Neoplasms/physiopathology , Cell Nucleus/metabolism , Cytoplasm/metabolism , Lim Kinases/metabolism , Actin Depolymerizing Factors/metabolism , Animals , Cell Line, Tumor , Cell Movement/genetics , Disease Progression , Female , Focal Adhesion Kinase 1/metabolism , Gene Order , Humans , Lim Kinases/genetics , Mice , Mice, Nude , Nuclear Export Signals/genetics , Nuclear Localization Signals/genetics , Nuclear Localization Signals/metabolism , Phosphorylation , Protein Transport , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Tumor Burden/genetics , Xenograft Model Antitumor Assays
20.
J Biol Chem ; 285(45): 34718-28, 2010 Nov 05.
Article in English | MEDLINE | ID: mdl-20807761

ABSTRACT

To understand the role of microRNAs (miRNAs) in pituitary development, a group of pituitary-specific miRNAs were identified, and Dicer1 was then conditionally knocked out using the Pitx2-Cre mouse, resulting in the loss of mature miRNAs in the anterior pituitary. The Pitx2-Cre/Dicer1 mutant mice demonstrate growth retardation, and the pituitaries are hypoplastic with an abnormal branching of the anterior lobe, revealing a role for microRNAs in pituitary development. Growth hormone, prolactin, and thyroid-stimulating hormone ß-subunit expression were decreased in the Dicer1 mutant mouse, whereas proopiomelanocortin and luteinizing hormone ß-subunit expression were normal in the mutant pituitary. Further analyses revealed decreased Pit-1 and increased Lef-1 expression in the mutant mouse pituitary, consistent with the repression of the Pit-1 promoter by Lef-1. Lef-1 directly targets and represses the Pit-1 promoter. miRNA-26b (miR-26b) was identified as targeting Lef-1 expression, and miR-26b represses Lef-1 in pituitary and non-pituitary cell lines. Furthermore, miR-26b up-regulates Pit-1 and growth hormone expression by attenuating Lef-1 expression in GH3 cells. This study demonstrates that microRNAs are critical for anterior pituitary development and that miR-26b regulates Pit-1 expression by inhibiting Lef-1 expression and may promote Pit-1 lineage differentiation during pituitary development.


Subject(s)
Gene Expression Regulation, Developmental/physiology , Lymphoid Enhancer-Binding Factor 1/metabolism , MicroRNAs/biosynthesis , Pituitary Gland, Anterior/embryology , Promoter Regions, Genetic/physiology , Transcription Factor Pit-1/biosynthesis , Animals , Cell Differentiation/physiology , Cell Line , Cell Lineage/physiology , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Endoribonucleases/genetics , Endoribonucleases/metabolism , Growth Disorders/genetics , Growth Disorders/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Luteinizing Hormone, beta Subunit/biosynthesis , Luteinizing Hormone, beta Subunit/genetics , Lymphoid Enhancer-Binding Factor 1/genetics , Mice , Mice, Mutant Strains , Mice, Transgenic , MicroRNAs/genetics , Pituitary Gland, Anterior/growth & development , Pro-Opiomelanocortin/biosynthesis , Pro-Opiomelanocortin/genetics , Prolactin/biosynthesis , Prolactin/genetics , Ribonuclease III , Thyrotropin, beta Subunit/biosynthesis , Thyrotropin, beta Subunit/genetics , Transcription Factor Pit-1/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Homeobox Protein PITX2
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