Citations

Abstract Background: The primary mechanism by which tamoxifen (Tam) and its metabolites exert their biologic effects is through estrogen receptor (ER) binding and inhibition of ER signaling. We and others demonstrated that endoxifen (Endx) has greater antitumor activity in vitro and in vivo compared to Tam and the first-in-human Endx phase I study demonstrated its antitumor activity in patients with prior progression on Tam (Goetz SABC 2015). PKCs are a family of serine/threonine-specific protein kinases that regulate signaling pathways involved in cell proliferation and tumorigenic transformation. Our prior protein docking studies suggested endoxifen may be a substrate for PKCs. Here we report the effects of Tam and Endx on PKCβ1 binding, kinase activity, as well as interactions between PKCβ1 and ERα. Methods: Surface Plasmon Resonance (SPR, Biacore T200, GE Healthcare) was used to evaluate binding of Tam, N-desmethyl Tam (NDMT), 4-HT, and Endx to PKCβ1 and PKCβ2. The effects of Tam and Endx on PKCβ1 kinase activity were determined. Proliferation and colony formation in MCF7 parental and PKCβ1 overexpressing cells were evaluated. siRNA silencing was used to knockdown PKCβ1 expression in the following cells: MCF7 aromatase expressing cells that were either sensitive (MCF7/AC1) or resistant to letrozole (MCF7/AC1 L-resistant); T47D; and MDA-MB-361. Coimmunoprecipitation assay and DUOlink in situ proximity ligation were used to investigate the interaction between PKCβ1 and ERα. Results: Endx more potently inhibited PKCβ1 kinase activity compared to Tam ( IC50 350 nM vs 47.8 μM ) with KDs for PKCβ1 binding as follows: Endx (100 nM ), Tam ( 2 μM ), 4-HT ( 2 μM ) and NDMT (> 7 μM ). None of the SERMs exhibited PKCβ2 binding. In the MCF7/AC1 and MCF7/AC1 L-resistant cells, PKCβ1 knockdown resulted in ERα degradation and potently inhibited cell proliferation. These results were confirmed in T47D and MDA-MB-361 cells. Notably, PKCβ1 knockdown in MCF7/AC1 cells resulted in significantly greater E2 induced proliferation comparing siRNA knockdown vs. control. To further explore these effects, we evaluated the effects of PKCβ1 overexpression in MCF7 cells and demonstrated that PKCβ1 overexpression reduced cell proliferation and colony formation compared to parental MCF-7 cells without affecting ERα protein stability. Coimmunoprecipitation assays in transient transfected MCF-7 cells with exogenous PKCβ1 as well as PKCβ1 expressing MDA-MB-231 cells transiently or stably transfected with ERα demonstrated PKCβ1 and ERα interaction, with confirmation by Duolink assay that this interaction occurs in the cytoplasm. Conclusions: Our findings demonstrated that endoxifen binds and inhibits PKCβ1 at relevant concentrations achieved in the endoxifen clinical trial studies. PKCβ1 interacts with cytoplasmic ERα and PKCβ1 knockdown inhibits cell proliferation and enhances ERα turnover. However, in PKCβ1 overexpressing cells, PKCβ1 may exhibit tumor suppressive effects. These data suggest a complex interaction between PKCβ1 and ERα and that endoxifen's effects on PKCβ1 may alter drug response of endocrine therapy. Further studies are ongoing to characterize the role of PKCβ1 and its role in ER biology and response to endoxifen. Citation Format: Guo C, Kuffel MJ, Kudgus RA, Huang Z, Bode AM, Cheng J, Suman VJ, Reid JM, Bruinsma ES, Subramaniam M, Ames MM, Hawse JR, Goetz MP. Identification and characterization of a novel endoxifen substrate, PKCβ1, and its interaction with the estrogen receptor [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-03.

Amicrobial pustulosis of the folds (APF) is a rare disease characterized by aseptic pustular lesions involving cutaneous folds, typically occurring in the context of an autoimmune disorder. We present 4 patients with APF, focusing on clinical and histopathologic characteristics to improve the recognition of this entity. All 4 patients had intertriginous and extra‐intertriginous involvement. Common histopathologic features of skin biopsies in these patients were intracorneal, subcorneal, intraepidermal, perivascular, perifollicular and interstitial neutrophilic inflammation. Pustules overlying adnexal ostium and papillary dermal edema were consistently observed. The pustules were negative for microorganisms on stain testing. In these cases, associated conditions were undifferentiated connective tissue disease, systemic lupus erythematosus, antiphospholipid syndrome, Crohn disease and Hashimoto thyroiditis. The aforementioned clinical and histopathologic characteristics of patients with suspected or diagnosed connective tissue disorders should lead to suspicion for APF.

Background Periodic acid Schiff‐diastase ( PAS ‐D) staining is used routinely in dermatopathology. Conventionally, parakeratosis or intracorneal neutrophils justify use of PAS ‐D stain to identify dermatophytosis. We aimed to investigate the histopathological features of superficial dermatophytosis. Methods Skin biopsies with PAS ‐D stain were retrospectively reviewed by a blinded dermatopathologist. Histopathologic findings in cases with dermatophytosis were compared with those without. Results We studied 110 cases, of which 63 were PAS ‐D positive for dermatophytes. Dermatophytes were identified on hematoxylin and eosin (H&E) staining in 49 (77.8%) of the PAS ‐D‐positive cases. Diffuse compact orthokeratosis (79.37%, p < 0.001) and focal parakeratosis (65.08%, p = 0.03) were significantly more frequent in dermatophytosis. Intracorneal neutrophils and spongiosis were not significant findings. Fungal elements were frequently observed within compact orthokeratosis (62.0%) and parakeratosis (31.8%) but rarely within intracorneal pustules (12.7%). In some dermatophyte cases, intracorneal neutrophils (47.6%) or parakeratosis (17.5%) were absent. Conclusions Compact orthokeratosis or focal parakeratosis justifies PAS ‐D staining, whereas the absence of intracorneal neutrophils or parakeratosis is insufficient to exclude dermatophytosis. Results indicate that having a low threshold to perform PAS‐D stain is justified. PAS‐D stain may be deferred until H&E slides have been evaluated.

Background: The spectrum of central nervous system–idiopathic inflammatory demyelinating disease (CNS-IIDD) in the elderly is uncertain. Objective: To describe the clinical, radiological, and pathological features of a cohort of 30 pathologically proven CNS-IIDD patients ⩾65 years. Methods: Elderly multiple sclerosis (MS)/clinically isolated syndrome (CIS) patients were compared to a cohort of 125 patients with pathologically proven MS/CIS and symptom onset <65 years. Results: Median age at symptom onset was 69 years (interquartile range (IQR) = 68–75). Median follow-up was 1.9 years (IQR = 1.0–5.6). Diagnoses were MS (14/30), CIS (11/30), neuromyelitis optica (NMO; 4/30), and acute disseminated encephalomyelitis (ADEM; 1/30). Disability was higher in patients with MS/CIS ⩾65 compared to patients <65 (median Expanded Disability Status Scale (EDSS) 4 (IQR = 2.5–7) vs 2.5 (IQR = 1.5–4); p = 0.002). When compared to patients <65 years, there was no difference in the lesion size, number of patients fulfilling Barkhof’s criteria, edema, or mass effect. Confluent demyelination was observed in 27 patients (MS/CIS (23/25), NMO (4)), 2 had a mixed perivenular/confluent pattern (MS (1), ADEM (1)), and 1 patient with MS had a mixed confluent/perivenular/coalescent pattern. Early active lesions were found in 19/30 patients ((MS (4), CIS (13), NMO (2); 53%). Cortical demyelination was present in 7/12 (58%) patients (MS (3), CIS(3), ADEM (1)). Conclusion: A spectrum of CNS-IIDD can develop in the elderly, with presenting symptoms similar to younger patients. Early diagnosis of CNS demyelinating disease is essential to avoid invasive and disabling procedures.

ABSTRACT Inflammatory response of blood–brain barrier (BBB) endothelial cells plays an important role in pathogenesis of many central nervous system inflammatory diseases, including multiple sclerosis; however, the molecular mechanism mediating BBB endothelial cell inflammatory response remains unclear. In this study, we first observed that knockdown of neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, suppressed interferon-γ (IFNγ)-induced C-X-C motif chemokine 10 expression and activation of STAT1 in brain microvascular endothelial cells in a Rac1-dependent manner. Moreover, endothelial-specific NRP1-knockout mice, VECadherin-Cre-ERT2/NRP1flox/flox mice, showed attenuated disease progression during experimental autoimmune encephalomyelitis, a mouse neuroinflammatory disease model. Detailed analysis utilizing histological staining, quantitative PCR, flow cytometry and magnetic resonance imaging demonstrated that deletion of endothelial NRP1 suppressed neuron demyelination, altered lymphocyte infiltration, preserved BBB function and decreased activation of the STAT1–CXCL10 pathway. Furthermore, increased expression of NRP1 was observed in endothelial cells of acute multiple sclerosis lesions. Our data identify a new molecular mechanism of brain microvascular endothelial inflammatory response through NRP1–IFNγ crosstalk that could be a potential target for intervention of endothelial cell dysfunction in neuroinflammatory diseases.

AbstractScaffolds with porous structures are highly applicable for tissue engineering and regenerative medicine. In the present study, 3‐dimensional poly(propylene fumarate‐co‐caprolactone) [P(PF‐co‐CL)] scaffolds were fabricated from a P(PF‐co‐CL)–dioxane‐water ternary system through thermally induced phase separation (TIPS). Cloud points of P(PF‐co‐CL) in dioxane‐water solutions increased with increased solute concentration, but increased dioxane composition decreased cloud point. Among 3 polymer concentrations (4, 8, and 12 wt%), 8 wt% P(PF‐co‐CL) scaffolds exhibited the best pore interconnectivity, with large, regular sized pores. Scaffolds were formed in 3 solutions with different dioxane‐water ratios (74/26, 78/22, and 82/18 wt/wt); the 78/22 wt/wt scaffold had finger‐shaped patterns with better interconnectivity than scaffolds from the other two ratios. Higher dioxane‐water ratios resulted in a larger contact angle and thus less wettability for the fabricated scaffold, while scaffolds fabricated from higher concentrations of P(PF‐co‐CL) or high dioxane‐water ratios had better biomineralization after soaking in simulated body fluid. In vitro cell viability testing showed the scaffolds had good biocompatibility with both bone and nerve cells. The results indicate that the polymer concentration and solvents ratio significantly affect the formation of porous structures, and optimum processing parameters were found to be 8% polymer concentration and 22% to 24% water content. These porous P(PF‐co‐CL) scaffolds fabricated via TIPS may be useful in various tissue engineering applications © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 226–235, 2017.

Abstract Esophageal adenocarcinoma is a deadly cancer with increasing incidence in the United States, but mechanisms underlying pathogenesis are still mostly elusive. In addressing this question, we assessed gene fusion landscapes by comprehensive RNA sequencing (RNAseq) of 55 pretreatment esophageal adenocarcinoma and 49 nonmalignant biopsy tissues from patients undergoing endoscopy for Barrett's esophagus. In this cohort, we identified 21 novel candidate esophageal adenocarcinoma–associated fusions occurring in 3.33% to 11.67% of esophageal adenocarcinomas. Two candidate fusions were selected for validation by PCR and Sanger sequencing in an independent set of pretreatment esophageal adenocarcinoma (N = 115) and nonmalignant (N = 183) biopsy tissues. In particular, we observed RPS6KB1–VMP1 gene fusion as a recurrent event occurring in approximately 10% of esophageal adenocarcinoma cases. Notably, esophageal adenocarcinoma cases harboring RPS6KB1–VMP1 fusions exhibited significantly poorer overall survival as compared with fusion-negative cases. Mechanistic investigations established that the RPS6KB1–VMP1 transcript coded for a fusion protein, which significantly enhanced the growth rate of nondysplastic Barrett's esophagus cells. Compared with the wild-type VMP1 protein, which mediates normal cellular autophagy, RPS6KB1–VMP1 fusion exhibited aberrant subcellular localization and was relatively ineffective in triggering autophagy. Overall, our findings identified RPS6KB1–VMP1 as a genetic fusion that promotes esophageal adenocarcinoma by modulating autophagy-related processes, offering new insights into the molecular pathogenesis of esophageal adenocarcinomas. Cancer Res; 76(19); 5628–33. ©2016 AACR.