Dopamine transporter membrane mobility is bidirectionally regulated by phosphorylation and palmitoylation.

The primary regulator of dopamine availability in the brain is the dopamine transporter (DAT), a plasma membrane protein that drives reuptake of released dopamine from the extracellular space into the presynaptic neuron. DAT activity is regulated by post-translational modifications that establish clearance capacity through impacts on transport kinetics, and dysregulation of these events may underlie dopaminergic imbalances in mood and psychiatric disorders. Here, using fluorescence recovery after photobleaching, we show that phosphorylation and palmitoylation induce opposing effects on DAT lateral membrane mobility, which may influence functional outcomes by regulating subcellular localization and binding partner interactions. Membrane mobility was also impacted by amphetamine and in polymorphic variant A559V in directions consistent with enhanced phosphorylation. These findings grow the list of DAT properties controlled by these post-translational modifications and highlight their role in establishment of dopaminergic tone in physiological and pathophysiological states.

Inhibition of Tumor Microenvironment Cytokine Signaling Sensitizes Ovarian Cancer Cells to Antiestrogen Therapy.

Antiestrogen therapy (AET) is an alternative to cytotoxic chemotherapy for recurrent ovarian cancer, yet the often short duration of response suggests mechanisms of resistance. We previously demonstrated that tumor microenvironment interleukin-6/leukemia inhibitory factor (IL6/LIF) cytokines induce tumor cell JAK-STAT signaling to promote cancer growth. Crosstalk between estrogen signaling and cytokine signaling has been reported. Therefore, we sought to characterize the impact of IL6/LIF signaling on estrogen signaling in epithelial ovarian cancer and investigate the efficacy of combination therapy. We first assessed patient tumors for cytokine expression and compared it with response to AET to determine clinical relevance. In vitro, we determined the effect of IL6/LIF on estrogen receptor expression and signaling. Cell viability assays were used to determine the efficacy and potential synergy of cytokine blockade and AET. We then extended studies to animal models, incorporating patient-derived stromal cells. Our results demonstrated shorter progression-free interval on AET in patients with stromal IL6/LIF expression. In vitro, IL6/LIF increased tumor cell estrogen receptor expression and signaling, and combination cytokine blockade and AET resulted in synergistic inhibition of tumor cell growth. The anticancer effect was verified in a mouse model. In conclusion, due to crosstalk between IL6/LIF cytokine signaling and estrogen signaling, dual blockade is a potential new treatment approach for ovarian cancer.

Sodium hydrogen exchanger (NHE1) palmitoylation and potential functional regulation.

AIMS: Determine the effect of palmitoylation on the sodium hydrogen exchanger isoform 1 (NHE1), a member of the SLC9 family. MAIN METHODS: NHE1 expressed in native rat tissues or in heterologous cells was assessed for palmitoylation by acyl-biotinyl exchange (ABE) and metabolic labeling with [3H]palmitate. Cellular palmitoylation was inhibited using 2-bromopalmitate (2BP) followed by determination of NHE1 palmitoylation status, intracellular pH, stress fiber formation, and cell migration. In addition, NHE1 was activated with LPA treatment followed by determination of NHE1 palmitoylation status and LPA-induced change in intracellular pH was determined in the presence and absence of preincubation with 2BP. KEY FINDINGS: In this study we demonstrate for the first time that NHE1 is palmitoylated in both cells and rat tissue, and that processes controlled by NHE1 including intracellular pH (pHi), stress fiber formation, and cell migration, are regulated in concert with NHE1 palmitoylation status. Importantly, LPA stimulates NHE1 palmitoylation, and 2BP pretreatment dampens LPA-induced increased pHi which is dependent on the presence of NHE1. SIGNIFICANCE: Palmitoylation is a reversible lipid modification that regulates an array of critical protein functions including activity, trafficking, membrane microlocalization and protein-protein interactions. Our results suggest that palmitoylation of NHE1 and other control/signaling proteins play a major role in NHE1 regulation that could significantly impact multiple critical cellular functions.

Palmitoylation by Multiple DHHC Enzymes Enhances Dopamine Transporter Function and Stability.

The dopamine transporter (DAT) is a plasma membrane protein that mediates the reuptake of extracellular dopamine (DA) and controls the spatiotemporal dynamics of dopaminergic neurotransmission. The transporter is subject to fine control that tailors clearance of transmitter to physiological demands, and dysregulation of reuptake induced by psychostimulant drugs, transporter polymorphisms, and signaling defects may impact transmitter tone in disease states. We previously demonstrated that DAT undergoes complex regulation by palmitoylation, with acute inhibition of the modification leading to rapid reduction of transport activity and sustained inhibition of the modification leading to transporter degradation and reduced expression. Here, to examine mechanisms and outcomes related to increased modification, we coexpressed DAT with palmitoyl acyltransferases (PATs), also known as DHHC enzymes, which catalyze palmitate addition to proteins. Of 12 PATs tested, DAT palmitoylation was stimulated by DHHC2, DHHC3, DHHC8, DHHC15, and DHHC17, with others having no effect. Increased modification was localized to previously identified palmitoylation site Cys580 and resulted in upregulation of transport kinetics and elevated transporter expression mediated by reduced degradation. These findings confirm palmitoylation as a regulator of multiple DAT properties crucial for appropriate DA homeostasis and identify several potential PAT pathways linked to these effects. Defects in palmitoylation processes thus represent possible mechanisms of transport imbalances in DA disorders.

Leukemia inhibitory factor functions in parallel with interleukin-6 to promote ovarian cancer growth.

Ovarian carcinoma-associated mesenchymal stem cells (CA-MSC) produce not only high levels of interleukin-6 (IL6) but also the related cytokine leukemia inhibitory factor (LIF). IL6-mediated activation of STAT3 is implicated as a critical therapeutic target for cancer therapy. Less is known about the role of LIF, which can similarly activate STAT3, in ovarian cancer. We therefore sought to evaluate the tumorigenic effects of CA-MSC paracrine LIF signaling and the redundancy of IL6 and LIF in activating ovarian cancer STAT3 mediated cancer growth. As expected, we found that both IL6 and LIF induce STAT3 phosphorylation in tumor cells. In addition, both IL6 and LIF increased the percentage of ALDH+ ovarian cancer stem-like cells (CSC). Supporting redundancy of function by the two cytokines, CA-MSC induced STAT3 phosphorylation and increased cancer cell "stemness". This effect was not inhibited by LIF or IL6 blocking antibodies alone, but was prevented by dual IL6/LIF blockade or JAK2 inhibition. Similarly, small hairpin RNA (shRNA)-mediated reduction of IL6 or LIF in CA-MSC partially decreased but could not completely abrograte the ability of CA-MSC to induce STAT3 phosphorylation and stemness. Importantly, the in vivo pro-tumorigenic effect of CA-MSC is abrogated by dual blockade with the JAK2 inhibitor ruxolitinib to a much greater extent than treatment with anti-IL6 or anti-LIF antibody alone. Ruxolitinib treatment also improves survival in the immunocompetent ovarian cancer mouse model system with ID8 tumor cells plus MSC. Ruxolitinib-treated tumors in both the immunocompromised and immunocompetent animal models demonstrate decreased phospho-STAT3, indicating on-target activity. In conclusion, CA-MSC activate ovarian cancer cell STAT3 signaling via IL6 and LIF and increase tumor cell stemness. This functional redundancy suggests that therapeutic targeting of a single cytokine may be less effective than strategies such as dual inhibitor therapy or targeting shared downstream factors of the JAK/STAT pathway.

The DEK Oncoprotein Functions in Ovarian Cancer Growth and Survival.

DNA damage repair alterations play a critical role in ovarian cancer tumorigenesis. Mechanistic drivers of the DNA damage response consequently present opportunities for therapeutic targeting. The chromatin-binding DEK oncoprotein functions in DNA double-strand break repair. We therefore sought to determine the role of DEK in epithelial ovarian cancer. DEK is overexpressed in both primary epithelial ovarian cancers and ovarian cancer cell lines. To assess the impact of DEK expression levels on cell growth, small interfering RNA and short hairpin RNA approaches were utilized. Decreasing DEK expression in ovarian cancer cell lines slows cell growth and induces apoptosis and DNA damage. The biologic effects of DEK depletion are enhanced with concurrent chemotherapy treatment. The in vitro effects of DEK knockdown are reproduced in vivo, as DEK depletion in a mouse xenograft model results in slower tumor growth and smaller tumors compared to tumors expressing DEK. These findings provide a compelling rationale to target the DEK oncoprotein and its pathways as a therapeutic strategy for treating epithelial ovarian cancer.