ETS transcription factor ESE-1/Elf3 is an independent prognostic factor of survival in HR+HER2+ breast cancer patients.

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.

Molecular mechanisms of ETS transcription factor-mediated tumorigenesis.

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.

Signaling pathways regulating pituitary lactotrope homeostasis and tumorigenesis.

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.

Carotid Artery Stenosis as a Predictor for Stroke Following Transcatheter Aortic Valve Implantation.

Stroke remains a clinical risk in the long-term follow-up of patients after transcatheter aortic valve implantation (TAVI). We aimed to investigate the association between existing carotid artery stenosis (CS) and the risk of stroke after TAVI. The study participants were consecutive patients who underwent TAVI between 2008 and 2020 and were screened for CS at baseline and at the 30-day follow-up. Hemodynamically relevant CS was defined as a reduction of the carotid artery caliber by >50% in relation to the distal segment. The primary endpoint of this study was ischemic stroke or transient ischemic attack (TIA) within 30 days of TAVI. Stroke-related death was also assessed. Of the 1,537 patients included, 220 (14.3%) patients had CS ≥50%. Within 30 days following TAVI, 41 patients (2.7%) experienced an ischemic stroke or TIA, and seven had stroke-related death. The median time from TAVI to stroke/TIA was 3.0 days. Patients with CS ≥50% had a 2.56-fold increased risk of stroke/TIA after TAVI (95%CI: 1.29-5.10, P = .006). The association was consistent in the multivariable model after adjusting for sex, atrial fibrillation, and estimated glomerular filtration rate. Investigations are needed to mitigate the risk of stroke in patients with CS ≥50%.

Changes in body composition, hematologic parameters, and serum biochemistry after rapid intravenous infusion or oral intake of 2 liters of 0.9% saline solution in young healthy volunteers: randomized crossover study.

BACKGROUND: The perioperative infusion of 2 L of saline is associated with weight gain and decreased serum albumin and hematocrit. We hypothesized that these parameters would respond differently to oral administration and intravenous infusion of saline solution. METHODS: This was a crossover study that included 10 healthy young men (ages 18-26 years). At two times, 8 weeks apart, the participants were randomized to receive 2 L of 0.9% saline over 1 h by intravenous (IV) administration to a forearm vein or by oral intake. The participants were weighed and body masses were calculated. Bioelectrical impedance analysis was performed with a single-frequency device using tetrapolar distal limb electrodes. Blood samples were collected 1 h after the administration period for laboratory assays: hematocrit, hemoglobin, blood glucose, serum electrolytes, albumin, creatinine, osmolality. RESULTS: There was an increase in body weight (p<0.01), total body water (p<0.01), and lean body mass (p<0.01) after the experiment in both groups, with no difference between them. The volume of urine output was similar in the two experiments. The hemoglobin (oral group from 14.4±0.8 g/dl to 13.8±0.8 g/dl; IV group from 14.4±0.6 g/dl to 12.6±0.6 g/dl) and hematocrit (oral group from 43.2±1.8% to 43.2±2.8%; IV group from 43.6±2.2% to 40.0±2.6%) significantly decreased (p<0.01) with IV saline. Serum albumin remained stable after oral intake but significantly decreased (p=0.04) after IV infusion. CONCLUSIONS: Oral intake of 2 L of 0.9% saline results in minimal variations in serum albumin, hemoglobin, and hematocrit when compared to IV infusion of the same volume.

MicroRNAs regulate pituitary development, and microRNA 26b specifically targets lymphoid enhancer factor 1 (Lef-1), which modulates pituitary transcription factor 1 (Pit-1) expression.

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.

Nuclear and cytoplasmic LIMK1 enhances human breast cancer progression.

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.

Mapping of ESE-1 subdomains required to initiate mammary epithelial cell transformation via a cytoplasmic mechanism.

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.

Targeted Intracellular Delivery of Trastuzumab Using Designer Phage Lambda Nanoparticles Alters Cellular Programs in Human Breast Cancer Cells.

| 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.

Ets transcription factors control epithelial maturation and transit and crypt-villus morphogenesis in the mammalian intestine.

Members of the Ets transcription factor family are widely expressed in both the developing and mature mammalian intestine, but their biological functions remain primarily uncharacterized. We used a dominant repressor transgene approach to probe the function of epithelial Ets factors in the homeostasis of the crypt-villus unit, the functional unit of the small intestine. We show that targeted expression in small intestinal epithelium of a fusion protein composed of the Engrailed repressor domain and the Erm DNA-binding domain (En/Erm) results in marked disruption of normal crypt-villus homeostasis, including a cell-autonomous disturbance of epithelial maturation, increased epithelial transit, severe villus dysmorphogenesis, and crypt dysmorphogenesis. The epithelial maturation disturbance is independent of the regulation of TGFbetaRII levels, in contrast to Ets-mediated epithelial differentiation during development; rather, regulation of Cdx2 expression may play a role. The villus dysmorphogenesis is independent of alterations in the crypt-villus boundary and inappropriate beta-catenin activation, and thus appears to represent a new mechanism controlling villus architectural organization. An Analysis of animals mosaic for En/Erm expression suggests that crypt nonautonomous mechanisms underlie the crypt dysmorphogenesis phenotype. Our studies thus uncover novel Ets-regulated pathways of intestinal homeostasis in vivo. Interestingly, the overall En/Erm phenotype of disturbed crypt-villus homeostasis is consistent with recently identified Ets function(s) in the restriction of intestinal epithelial tumorigenesis.

Benign mammary epithelial cells enhance the transformed phenotype of human breast cancer cells.

BACKGROUND: Recent research has yielded a wealth of data underscoring the key role of the cancer microenvironment, especially immune and stromal cells, in the progression of cancer and the development of metastases. However, the role of adjacent benign epithelial cells, which provide initial cell-cell contacts with cancer cells, in tumor progression has not been thoroughly examined. In this report we addressed the question whether benign MECs alter the transformed phenotype of human breast cancer cells. METHODS: We used both in vitro and in vivo co-cultivation approaches, whereby we mixed GFP-tagged MCF-10A cells (G2B-10A), as a model of benign mammary epithelial cells (MECs), and RFP-tagged MDA-MB-231-TIAS cells (R2-T1AS), as a model of breast cancer cells. RESULTS: The in vitro studies showed that G2B-10A cells increase the colony formation of R2-T1AS cells in both soft agar and clonogenicity assays. Conditioned media derived from G2B-10A cells enhanced colony formation of R2-T1AS cells, whereas prior paraformaldehyde (PFA) fixation of G2B-10A cells abrogated this enhancement effect. Moreover, two other models of benign MECs, MCF-12A and HuMECs, also enhanced R2-T1AS colony growth in soft agar and clonogenicity assays. These data reveal that factors secreted by benign MECs are responsible for the observed enhancement of the R2-T1AS transformed phenotype. To determine whether G2B-10A cells enhance the tumorigenic growth of co-injected R2-T1AS cells in vivo, we used the nude mouse xenograft assay. Co-injecting R2-T1AS cells with G2B-10A cells +/- PFA-fixation, revealed that G2B-10A cells promoted a ~3-fold increase in tumor growth, irrespective of PFA pre-treatment. These results indicate that soluble factors secreted by G2B-10A cells play a less important role in promoting R2-T1AS tumorigenesis in vivo, and that additional components are operative in the nude mouse xenograft assay. Finally, using array analysis, we found that both live and PFA-fixed G2B-10A cells induced R2-T1AS cells to secrete specific cytokines (IL-6 and GM-CSF), suggesting that cell-cell contact activates R2-T1AS cells. CONCLUSIONS: Taken together, these data shift our understanding of adjacent benign epithelial cells in the cancer process, from passive, noncontributory cells to an active and tumor-promoting vicinal cell population that may have significant effects early, when benign cells outnumber malignant cells.

ETV6 germline mutations cause HDAC3/NCOR2 mislocalization and upregulation of interferon response genes.

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.

The Ets dominant repressor En/Erm enhances intestinal epithelial tumorigenesis in ApcMin mice.

BACKGROUND: Ets transcription factors have been widely implicated in the control of tumorigenesis, with most studies suggesting tumor-promoting roles. However, few studies have examined Ets tumorigenesis-modifying functions in vivo using model genetic systems. METHODS: Using mice expressing a previously characterized Ets dominant repressor transgene in the intestinal epithelium (Villin-En/Erm), we examined the consequences of blocking endogenous Ets-mediated transcriptional activation on tumorigenesis in the ApcMin model of intestinal carcinoma. RESULTS: En/Erm expression in the intestine, at levels not associated with overt crypt-villus dysmorphogenesis, results in a marked increase in tumor number in ApcMin animals. Moreover, when examined histologically, tumors from En/Erm-expressing animals show a trend toward greater stromal invasiveness. Detailed analysis of crypt-villus homeostasis in these En/Erm transgenic animals suggests increased epithelial turnover as one possible mechanism for the enhanced tumorigenesis. CONCLUSION: Our findings provide in vivo evidence for a tumor-restricting function of endogenous Ets factors in the intestinal epithelium.

Ultrasound-mediated delivery of siESE complexed with microbubbles attenuates HER2+/- cell line proliferation and tumor growth in rodent models of breast cancer.

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.

ETS transcription factors in endocrine systems.

E26 transformation-specific (ETS) transcription factors have become increasingly recognized as key regulators of differentiation, hormone responses and tumorigenesis in endocrine organs and target tissues. The ETS family is highly diverse, consisting of both transcription activators and repressors that mediate growth factor signaling and regulate gene expression through combinatorial interactions with multiple protein partners on composite DNA elements. ETS proteins have a role in the endocrine system in establishing pituitary-specific gene expression, mammary gland development and cancers of the breast, prostate and reproductive organs.

Differential utilization of transcription activation subdomains by distinct coactivators regulates Pit-1 basal and Ras responsiveness.

The POU-homeodomain transcription factor Pit-1 governs ontogeny and cell-specific gene expression of pituitary lactotropes, somatotropes, and thyrotropes. The splice isoform, Pit-1beta, inserts a 26-amino acid (AA) repressor at AA48 in the Pit-1 transcription activation domain (TAD). The Pit-1 TAD contains a basal regulatory subregion, R1 (AA1-45), and a basal and Ras-responsive region, R2 (AA46-80). To precisely map these activities, we generated GAL4-Pit-1/Pit-1betaTAD fusions and, in full-length HA-Pit-1, a series of substitution mutants of R2. Analysis in GH4 cells identified an activation domain at AA50-70, followed by an overlapping, dual-function, Ras-responsive-inhibitory domain, located from AA60-80. In contrast, GAL4-Pit-1betaTAD repressed both basal and Ras-mediated TAD activity. To determine the functional interplay between TAD subregions and the beta-domain, we inserted the beta-domain every 10 AA across the 80-AA Pit-1 TAD. Like wild-type Pit-1beta, each construct retained transcriptional activity in HeLa cells and repressed the Ras response in GH4 cells. However, beta-domain insertion at AA61 and 71 resulted in greater repression of Ras responsiveness, defining a critical R2 TAD spanning AA61-71 of Pit-1. Furthermore, Ras activation is augmented by steroid receptor coactivator 1, whereas cAMP response element binding protein-binding protein is not a Ras mediator in this system. In summary, the Pit-1/Pit-1beta TADs are composed of multiple, modular, and transferable subdomains, including a regulatory R1 domain, a basal activation region, a selective inhibitory-Ras-responsive segment, and a beta-specific repressor domain. These data provide novel insights into the mechanisms by which the Pit-1 TAD integrates DNA binding, protein partner interactions, and distinct signaling pathways to fine-tune Pit-1 activity.

Consider the context: Ras/ERK and PI3K/AKT/mTOR signaling outcomes are pituitary cell type-specific.

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.

Pituitary somatolactotropes evade an oncogenic response to Ras.

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.

The Balance of PI3K and ERK Signaling Is Dysregulated in Prolactinoma and Modulated by Dopamine.

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.