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The hormone prolactin (PRL) and its receptor (hPRLr) are significantly involved in breast cancer pathogenesis. The intermediate hPRLr (hPRLrI) is an alternatively-spliced isoform, capable of stimulating cellular viability and proliferation. An analogous truncated mouse PRLr (mPRLr) was recently found to be oncogenic when co-expressed with wild-type mPRLr. The goal of this study was to determine if a similar transforming event occurs with the hPRLr in human breast epithelial cells and to better understand the mechanism behind such transformation. hPRLrL+I co-expression in MCF10AT cells resulted in robust in vivo and in vitro transformation, while hPRLrI knock-down in MCF7 cells significantly decreased in vitro malignant potential. hPRLrL+I heterodimers displayed greater stability than hPRLrL homodimers, and while being capable of activating Jak2, Ras, and MAPK, they were unable to induce Stat5a tyrosine phosphorylation. Both immunohistochemical breast cancer tissue microarray data and RNA sequencing analyses using The Cancer Genome Atlas (TCGA) identified that higher hPRLrI expression associates with triple-negative breast cancer. These studies indicate the hPRLrI, when expressed alongside hPRLrL, participates in mammary transformation, and represents a novel oncogenic mechanism.

The neuroendocrine hormone prolactin (PRL) and its cognate receptor (PRLr) have been implicated in the pathogenesis of breast cancer. PRL signaling relies on activating kinases such as the tyrosine kinase Jak2 and serine/threonine kinases ERK1/2, Nek3, PI3K, and AKT. In the canonical pathway of PRL signaling, Jak2 phosphorylates the transcription factor Stat5a at tyrosine residue 694 (pY694-Stat5a), preceding Stat5a nuclear translocation and transcriptional activity. However, Stat5a exists with functional duality as a transcription factor, having both pro-differentiative and pro-proliferative target genes. Other Stat family members (Stats 1, 3, and 6) have been shown to have transcriptional activity in the un-phosphorylated (upY) state, distinct from that of pY-Stat activity. This distinction (upY vs. pY) may underlie the duality of Stat5a, coupled with additional regulatory non-canonical post-translational modifications. Within this notion, Stat5a contains two serine residues, S726 and S780, whose phosphorylation are necessary for hematopoietic transformation. However, their functions in PRL-mediated breast cancer pathogenesis have not been examined. We hypothesize that Stat5a serine phosphorylation regulates Stat5a nuclear activity in a non-canonical fashion, contributing to its role in mammary oncogenesis. As shown in a tissue microarray (TMA), human breast cancer tissues express both pS726- and pS780-Stat5a. Nuclear Allred score for pS726-Stat5a increases two-fold with increasing tumor grade, with no difference in staining associated with estrogen or progesterone receptor (ER, PR) status, nor other clinical characteristics. Likewise, patient derived xenograft (PDX) tumors of various molecular subtypes express pS726- and pS780-Stat5a. Phosphorylation of S726-Stat5a is PRL-responsive in vitro. Pharmacologic inhibition of ERK1/2 prevents this phosphorylation, uncovering a novel pathway in which ERK1/2 mediates Stat5a activity in response to PRL in breast cancer. To examine the functional significance of Stat5a serine phosphorylation in vitro, we have performed Stat5a knockdown (KD) in the breast cancer cell line MCF7. Following Stat5a KD, cells were rescued with phospho-site specific Stat5a mutant constructs. Characteristics of breast cancer examined in these mutation-carrying cells, including anchorage-independent growth and proliferation, show distinct phenotypes compared to controls. PRL-induced expression of the CISH gene is significantly decreased up to 65% in the mutation-carrying cells compared to wild type Stat5a. Mechanistic studies will examine the ability of these Stat5a mutants to undergo nuclear translocation and interact with other transcription factors. Collectively, these studies have the potential to provide novel insights into the role of the non-canonical pathway of Stat5a activation in breast cancer pathogenesis.

Introduction Metabotropic glutamate subtype receptor 1 (mGluR1) is implicated in several neuropsychiatric disorders and is a target for drug development. [ 18 F]FIMX ([ 18 F]4-fluoro-- N -methyl- N --(4-(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide) is an effective radioligand for imaging brain mGluR1 with PET. A similarly effective radioligand with a shorter half-life would usefully allow PET studies of mGluR1 at baseline and after pharmacological or other challenge on the same day. Here we describe the preparation of [ 11 C]FIMX for evaluation in monkey with PET. Methods [ 11 C]FIMX was prepared via Pd-promoted carbonylation of 1-fluoro-4-iodobenzene with [ 11 C]carbon monoxide, aminolysis of the [ 11 C]acyl-palladium complex with the requisite Boc-protected amine, and deprotection with HCl in THF. PET scans of [ 11 C]FIMX injected into a monkey were performed at baseline and after preblock of mGluR1 with measurement of the arterial input function. Results The radiosynthesis required 42min and gave [ 11 C]FIMX in about 5% overall decay-corrected radiochemical yield and with a specific activity of about 100GBq/μmol. PET in rhesus monkey at baseline showed that radioactivity peaked high in receptor-rich cerebellum and much lower in receptor-poor occipital cortex. Radioactivity in cerebellum declined to 32% of peak at 85min. V T at baseline appeared stable in all brain regions after 60min. Under mGluR1 pre-blocked condition, radioactivity uptake in all regions declined more rapidly to a low level. Receptor pre-block reduced V T from 13.0 to 1.5 in cerebellum and from 2.9 to 1.4 in occipital cortex. Conclusion [ 11 C]FIMX is an effective radioligand for imaging mGluR1 in monkey with PET.

The permeability-glycoprotein (P-gp) efflux transporter is densely expressed at the blood–brain barrier, and its resultant spare capacity requires substantial blockade to increase the uptake of avid substrates, blunting the ability of investigators to measure clinically meaningful alterations in P-gp function. This study, conducted in humans, examined 2 P-gp inhibitors (tariquidar, a known inhibitor, and disulfiram, a putative inhibitor) and 2 routes of administration (intravenous and oral) to maximally increase brain uptake of the avid and selective P-gp substrate 11C-N-desmethyl-loperamide (dLop) while avoiding side effects associated with high doses of tariquidar. Methods: Forty-two 11C-dLop PET scans were obtained from 37 healthy volunteers. PET was performed with 11C-dLop under the following 5 conditions: injected under baseline conditions without P-gp inhibition, injected 1 h after intravenous tariquidar infusion, injected during intravenous tariquidar infusion, injected after oral tariquidar, and injected after disulfiram. 11C-dLop uptake was quantified with kinetic modeling using metabolite-corrected arterial input function or by measuring the area under the time–activity curve in the brain from 10 to 30 min. Results: Neither oral tariquidar nor oral disulfiram increased brain uptake of 11C-dLop. Injecting 11C-dLop during tariquidar infusion, when plasma tariquidar concentrations reach their peak, resulted in a brain uptake of the radioligand approximately 5-fold greater than baseline. Brain uptake was similar with 2 and 4 mg of intravenous tariquidar per kilogram; however, the lower dose was better tolerated. Injecting 11C-dLop after tariquidar infusion also increased brain uptake, though higher doses (up to 6 mg/kg) were required. Brain uptake of 11C-dLop increased fairly linearly with increasing plasma tariquidar concentrations, but we are uncertain whether maximal uptake was achieved. Conclusion: We sought to increase the dynamic range of P-gp function measured after blockade. Performing 11C-dLop PET during peak plasma concentrations of tariquidar, achieved with concurrent administration of intravenous tariquidar, resulted in greater P-gp inhibition at the human blood–brain barrier than delayed administration and allowed the use of a lower, more tolerable dose of tariquidar. On the basis of prior monkey studies, we suspect that plasma concentrations of tariquidar did not fully block P-gp; however, higher doses of tariquidar would likely be associated with unacceptable side effects.

We sought to develop a PET radioligand that would be useful for imaging human brain metabotropic subtype 1 receptors (mGluR1) in neuropsychiatric disorders and in drug development. 4-Fluoro-N-methyl-N-(4-(6-(methylamino)pyrimidin-4-yl)thiazol-2-yl)benzamide (FIMX, 11) was identified as having favorable properties for development as a PET radioligand. We developed a method for preparing [(18)F]11 in useful radiochemical yield and in high specific activity from [(18)F]fluoride ion and an N-Boc-protected (phenyl)aryliodonium salt precursor (15). In baseline experiments in rhesus monkey, [(18)F]11 gave high brain radioactivity uptake, reflecting the expected distribution of mGluR1 with notably high uptake in cerebellum, which became 47% lower by 120 min after radioligand injection. Pharmacological challenges demonstrated that a very high proportion of the radioactivity in monkey brain was bound specifically and reversibly to mGluR1. [(18)F]11 is concluded to be an effective PET radioligand for imaging mGluR1 in monkey brain and therefore merits further evaluation in human subjects.