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.

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.

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.

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.

High Efficiency Molecular Delivery with Sequential Low-Energy Sonoporation Bursts.

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.

Persistent ERK/MAPK activation promotes lactotrope differentiation and diminishes tumorigenic phenotype.

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.

An extended tyrosine-targeting motif for endocytosis and recycling of the dense-core vesicle membrane protein phogrin.

Integral membrane proteins of neuroendocine dense-core vesicles (DCV) appear to undergo multiple rounds of exocytosis; however, their trafficking and site of incorporation into nascent DCVs is unclear. Previous studies with phogrin (IA-2beta) identified sorting signals in the luminal domain that is cleaved post-translationally; we now describe an independent DCV targeting motif in the cytosolic domain that may function at the level of endocytosis and recycling. Pulse-chase radiolabeling and cell surface biotinylation experiments in the pituitary corticotroph cell line AtT20 showed that the mature 60/65 kDa form that resides in the DCV is generated by limited proteolysis in a post-trans Golgi network compartment with similar kinetics to the formation of the principal cargo, ACTH. Phogrin is exposed on the cell surface in response to stimuli and progressively internalized to a perinuclear compartment that overlaps with recycling endosomes marked by transferrin. Chimeric molecules of phogrin transmembrane and cytosolic sequences with the interleukin-2 receptor alpha chain (Tac) were sorted to DCVs through the action of an extended tyrosine-based motif Y(654)QELCRQRMA located in a 27aa sequence adjacent to the membrane-spanning domain. A 36aa domain terminating in this sequence conferred DCV localization to Tac in the absence of any other cytosolic or luminal phogrin components. The endocytosis and DCV targeting of phogrin Y(654) > A mutants correlated with the impaired binding of the phogrin cytosolic tail to the micro-subunit of the AP2 adaptor complex in vitro.