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Clinical management of castration-resistant prostate cancer (CRPC) resulting from androgen deprivation therapy (ADT) remains challenging. Many studies indicate that androgen receptor splice variants (ARVs) play a critical role in the development of CRPC, including resistance to the new generation of inhibitors of androgen receptor (AR) action. ARVs are constitutively active and lack the ligand-binding domain (LBD), thereby allowing prostate cancer (PC) to maintain AR activity despite therapies that target the AR (full-length AR; AR-FL). Previously, we have reported that long-term ADT increases the neuroendocrine (NE) hormone - Gastrin Releasing Peptide (GRP) and its receptor (GRP-R) expression in PC cells. Further, we demonstrated that activation of GRP/GRP-R signaling increases ARVs expression by activating NF-κB signaling, thereby promoting cancer progression to CRPC. Most importantly, as a cell surface protein, GRP-R is easily targeted by drugs to block GRP/GRP-R signaling. In this study, we tested if blocking GRP/GRP-R signaling by targeting GRP-R using GRP-R antagonist is sufficient to control CRPC progression. Our studies show that blocking GRP/GRP-R signaling by targeting GRP-R using RC-3095, a selective GRP-R antagonist, efficiently inhibits NF-κB activity and ARVs (AR-V7) expression in CRPC and therapy-induced NEPC (tNEPC) cells. In addition, blocking of GRP/GRP-R signaling by targeting GRP-R can sensitize CRPC cells to anti-androgen treatment (such as MDV3100). Further, preclinical animal studies indicate combination of GRP-R antagonist (targeting ARVs) with anti-androgen (targeting AR-FL) is sufficient to inhibit CRPC and tNEPC tumor growth., (Copyright © 2021. Published by Elsevier Inc.)

Castration-resistant prostate cancer can be treated with the antiandrogen enzalutamide, but responses and duration of response are variable. To identify genes that support enzalutamide resistance, we performed a short hairpin RNA (shRNA) screen in the bone-homing, castration-resistant prostate cancer cell line, C4-2B. We identified 11 genes ( TFAP2C, CAD, SPDEF, EIF6, GABRG2, CDC37, PSMD12, COL5A2, AR, MAP3K11, and ACAT1 ) whose loss resulted in decreased cell survival in response to enzalutamide. To validate our screen, we performed transient knockdowns in C4-2B and 22Rv1 cells and evaluated cell survival in response to enzalutamide. Through these studies, we validated three genes ( ACAT1, MAP3K11, and PSMD12 ) as supporters of enzalutamide resistance in vitro Although ACAT1 expression is lower in metastatic castration-resistant prostate cancer samples versus primary prostate cancer samples, knockdown of ACAT1 was sufficient to reduce cell survival in C4-2B and 22Rv1 cells. MAP3K11 expression increases with Gleason grade, and the highest expression is observed in metastatic castration-resistant disease. Knockdown of MAP3K11 reduced cell survival, and pharmacologic inhibition of MAP3K11 with CEP-1347 in combination with enzalutamide resulted in a dramatic increase in cell death. This was associated with decreased phosphorylation of AR-Serine650, which is required for maximal AR activation. Finally, although PSMD12 expression did not change during disease progression, knockdown of PSMD12 resulted in decreased AR and AR splice variant expression, likely contributing to the C4-2B and 22Rv1 decrease in cell survival. Our study has therefore identified at least three new supporters of enzalutamide resistance in castration-resistant prostate cancer cells in vitro ., (©2020 American Association for Cancer Research.)

Background: Male lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often uses LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a proinflammatory and profibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to proinflammatory stimuli and identified downstream targets of OPN in prostate stromal cells., Methods: Immunohistochemistry was performed on prostate sections obtained from the transition zone of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS (surgical BPH, S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging System and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot analysis. The ability of prostate cells to secrete osteopontin in response to IL-1β and TGF-β1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by enzyme-linked immunosorbent assay. Quantitative polymerase chain reaction was used to measure gene expression changes in these cells in response to OPN., Results: OPN immunostaining and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with the highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-β1 stimulated OPN secretion by NHPrE-1 cells and both IL-1β and TGF-β1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression of CXCL1, CXCL2, CXCL8, PTGS2, and IL6 in BHPrS-1, but not in epithelial cell lines., Conclusions: OPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by proinflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of proinflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways., (© 2020 Wiley Periodicals, Inc.)

Numerous studies indicate that androgen receptor splice variants (ARVs) play a critical role in the development of castration-resistant prostate cancer (CRPC), including the resistance to the new generation of inhibitors of androgen receptor (AR) action. Previously, we demonstrated that activation of NF-κB signaling increases ARVs expression in prostate cancer (PC) cells, thereby promoting progression to CRPC. However, it is unclear how NF-κB signaling is activated in CRPC. In this study, we report that long-term treatment with anti-androgens increases a neuroendocrine (NE) hormone - gastrin-releasing peptide (GRP) and its receptor (GRP-R) expression in PC cells. In addition, activation of GRP/GRP-R signaling increases ARVs expression through activating NF-κB signaling. This results in an androgen-dependent tumor progressing to a castrate resistant tumor. The knock-down of AR-V7 restores sensitivity to antiandrogens of PC cells over-expressing the GRP/GRP-R signaling pathway. These findings strongly indicate that the axis of Androgen-Deprivation Therapy (ADT) induces GRP/GRP-R activity, activation NF-κB and increased levels of AR-V7 expression resulting in progression to CRPC. Both prostate adenocarcinoma and small cell NE prostate cancer express GRP-R. Since the GRP-R is clinically targetable by analogue-based approach, this provides a novel therapeutic approach to treat advanced CRPC., Competing Interests: The authors declare no conflicts of interest.

A functional complex consisting of androgen receptor (AR) and forkhead box A1 (FOXA1) proteins supports prostatic development, differentiation, and disease. In addition, the interaction of FOXA1 with cofactors such as nuclear factor I (NFI) family members modulates AR target gene expression. However, the global role of specific NFI family members has yet to be described in the prostate. In these studies, chromatin immunoprecipitation followed by DNA sequencing in androgen-dependent LNCaP prostate cancer cells demonstrated that 64.3% of NFIB binding sites are associated with AR and FOXA1 binding sites. Interrogation of published data revealed that genes associated with NFIB binding sites are predominantly induced after dihydrotestosterone treatment of LNCaP cells, whereas NFIB knockdown studies demonstrated that loss of NFIB drives increased AR expression and superinduction of a subset of AR target genes. Notably, genes bound by NFIB only are associated with cell division and cell cycle. To define the role of NFIB in vivo, mouse Nfib knockout prostatic tissue was rescued via renal capsule engraftment. Loss of Nfib expression resulted in prostatic hyperplasia, which did not resolve in response to castration, and an expansion of an intermediate cell population in a small subset of grafts. In human benign prostatic hyperplasia, luminal NFIB loss correlated with more severe disease. Finally, some areas of intermediate cell expansion were also associated with NFIB loss. Taken together, these results show a fundamental role for NFIB as a coregulator of AR action in the prostate and in controlling prostatic hyperplasia.

The forkhead box (Fox) superfamily of transcription factors has essential roles in organogenesis and tissue differentiation. Foxa1 and Foxa2 are expressed during prostate budding and ductal morphogenesis, whereas Foxa1 expression is retained in adult prostate epithelium. Previous characterization of prostatic tissue rescued from embryonic Foxa1 knockout mice revealed Foxa1 to be essential for ductal morphogenesis and epithelial maturation. However, it is unknown whether Foxa1 is required to maintain the differentiated status in adult prostate epithelium. Here, we employed the PBCre4 transgenic system and determined the impact of prostate-specific Foxa1 deletion in adult murine epithelium. PBCre4/Foxa1(loxp/loxp) mouse prostates showed progressive florid hyperplasia with extensive cribriform patterning, with the anterior prostate being most affected. Immunohistochemistry studies show mosaic Foxa1 KO consistent with PBCre4 activity, with Foxa1 KO epithelial cells specifically exhibiting altered cell morphology, increased proliferation, and elevated expression of basal cell markers. Castration studies showed that, while PBCre4/Foxa1(loxp/loxp) prostates did not exhibit altered sensitivity in response to hormone ablation compared with control prostates, the number of Foxa1-positive cells in mosaic Foxa1 KO prostates was significantly reduced compared with Foxa1-negative cells following castration. Unexpectedly, gene expression profile analyses revealed that Foxa1 deletion caused abnormal expression of seminal vesicle-associated genes in KO prostates. In summary, these results indicate Foxa1 expression is required for the maintenance of prostatic cellular differentiation.

Purpose: Metastasis, the main cause of death from cancer, remains poorly understood at the molecular level., Experimental Design: Based on a pattern of reduced expression in human prostate cancer tissues and tumor cell lines, a candidate suppressor gene (SPARCL1) was identified. We used in vitro approaches to determine whether overexpression of SPARCL1 affects cell growth, migration, and invasiveness. We then employed xenograft mouse models to analyze the impact of SPARCL1 on prostate cancer cell growth and metastasis in vivo., Results: SPARCL1 expression did not inhibit tumor cell proliferation in vitro. By contrast, SPARCL1 did suppress tumor cell migration and invasiveness in vitro and tumor metastatic growth in vivo, conferring improved survival in xenograft mouse models., Conclusions: We present the first in vivo data suggesting that SPARCL1 suppresses metastasis of prostate cancer., (Copyright © 2013 Federation of European Biochemical Societies. All rights reserved.)

Background: Neuronal synaptic junction protein δ-catenin (CTNND2) is often overexpressed in prostatic adenocarcinomas but the mechanisms of its activation are unknown. To address this question, we studied the hypothesis that Hes1, human homolog of Drosophila Hairy and enhancer of split (Hes) 1, is a transcriptional repressor of δ-catenin expression and plays an important role in molecular carcinogenesis., Results: We identified that, using a δ-catenin promoter reporter assay, Hes1, but not its inactive mutant, significantly repressed the upregulation of δ-catenin-luciferase activities induced by E2F1. Hes1 binds directly to the E-boxes on δ-catenin promoter and can reduce the expression of δ-catenin in prostate cancer cells. In prostate cancer CWR22-Rv1 and PC3 cell lines, which showed distinct δ-catenin overexpression, E2F1 and Hes1 expression pattern was altered. The suppression of Hes1 expression, either by γ-secretase inhibitors or by siRNA against Hes1, increased δ-catenin expression. γ-Secretase inhibition delayed S/G2-phase transition during cell cycle progression and induced cell shape changes to extend cellular processes in prostate cancer cells. In neuroendocrine prostate cancer mouse model derived allograft NE-10 tumors, δ-catenin showed an increased expression while Hes1 expression was diminished. Furthermore, E2F1 transcription was very high in subgroup of NE-10 tumors in which Hes1 still displayed residual expression, while its expression was only moderately increased in NE-10 tumors where Hes1 expression was completely suppressed., Conclusion: These studies support coordinated regulation of δ-catenin expression by both the activating transcription factor E2F1 and repressive transcription factor Hes1 in prostate cancer progression.

Background: The androgen-regulated probasin (PB) promoter has been used extensively to target transgenes to the prostate in transgenic mice; however, limited data exist on the mechanism that dictates prostate-specific gene expression. Tissue-specific gene expression involves synergistic effects among transcription factors associated in a complex bound to cis-acting DNA elements., Methods: Using comprehensive linker scan mutagenesis, enzyme mobility shift and supershift assays, chromatin immunoprecipitation, and transgenic animal studies, we have extensively characterized the prostate-specific PB promoter., Results: We identified a series of nonreceptor transcription factors that are bound to the prostate-specific rat PB promoter. These factors include several ubiquitously distributed proteins known to participate in steroid receptor-mediated transcription. In addition, we identified two tissue-specific DNA elements that are crucial in directing prostate-specific PB expression, and confirmed the functional importance of both elements in transgenic animal studies. These two elements are functionally interchangeable and can be bound by multiple protein complexes, including the forkhead transcription factor FoxA1, a "pioneer factor" that has a restricted distribution to some cells type that are ectoderm and endoderm in origin. Using transgenic mice, we further demonstrate that the minimal PB promoter region (-244/-96 bp) that encompasses these tissue-specific elements results in prostate-specific gene expression in transgenic mice, contains androgen receptor and FoxA1-binding sites, as well as ubiquitous transcription factor binding sites., Conclusion: We propose that these sequence-specific DNA-binding proteins, including tissue-restricted and ubiquitous factors, create the first level of transcriptional control, which responds to intracellular pathways that directs prostate-specific gene expression.

Typically, the initial response of a prostate cancer patient to androgen ablation therapy is regression of the disease. However, the tumor will progress to an "androgen-independent" stage that results in renewed growth and spread of the cancer. Both nuclear factor-kappaB (NF-kappaB) expression and neuroendocrine differentiation predict poor prognosis, but their precise contribution to prostate cancer progression is unknown. This report shows that secretory proteins from neuroendocrine cells will activate the NF-kappaB pathway in LNCaP cells, resulting in increased levels of active androgen receptor (AR). By blocking NF-kappaB signaling in vitro, AR activation is inhibited. In addition, the continuous activation of NF-kappaB signaling in vivo by the absence of the IkappaBalpha inhibitor prevents regression of the prostate after castration by sustaining high levels of nuclear AR and maintaining differentiated function and continued proliferation of the epithelium. Furthermore, the NF-kappaB pathway was activated in the ARR(2)PB-myc-PAI (Hi-myc) mouse prostate by cross-breeding into a IkappaBalpha(+/-) haploid insufficient line. After castration, the mouse prostate cancer continued to proliferate. These results indicate that activation of NF-kappaB is sufficient to maintain androgen-independent growth of prostate and prostate cancer by regulating AR action. Thus, the NF-kappaB pathway may be a potential target for therapy against androgen-independent prostate cancer.

Androgen receptor (AR) within prostatic mesenchymal cells, with the absence of AR in the epithelium, is still sufficient to induce prostate development. AR in the luminal epithelium is required to express the secretory markers associated with differentiation. Nkx3.1 is expressed in the epithelium in early prostatic embryonic development and expression is maintained in the adult. Induction of the mouse prostate gland by the embryonic mesenchymal cells results in the organization of a sparse basal layer below the luminal epithelium with rare neuroendocrine cells that are interdispersed within this basal layer. The human prostate shows similar glandular organization; however, the basal layer is continuous. The strong inductive nature of embryonic prostatic and bladder mesenchymal cells is demonstrated in grafts where embryonic stem (ES) cells are induced to differentiate and organize as a prostate and bladder, respectively. Further, the ES cells can be driven by the correct embryonic mesenchymal cells to form epithelium that differentiates into secretory prostate glands and differentiated bladders that produce uroplakin. This requires the ES cells to mature into endoderm that gives rise to differentiated epithelium. This process is control by transcription factors in both the inductive mesenchymal cells (AR) and the responding epithelium (FoxA1 and Nkx3.1) that allows for organ development and differentiation. In this review, we explore a molecular mechanism where the pattern of transcription factor expression controls cell determination, where the cell is assigned a developmental fate and subsequently cell differentiation, and where the assigned cell now emerges with it's own unique character.

Androgens and mesenchymal factors are essential extracellular signals for the development as well as the functional activity of the prostate epithelium. Little is known of the intraepithelial determinants that are involved in prostatic differentiation. Here we found that hepatocyte nuclear factor-3 alpha (HNF-3 alpha), an endoderm developmental factor, is essential for androgen receptor (AR)-mediated prostatic gene activation. Two HNF-3 cis-regulatory elements were identified in the rat probasin (PB) gene promoter, each immediately adjacent to an androgen response element. Remarkably, similar organization of HNF-3 and AR binding sites was observed in the prostate-specific antigen (PSA) gene core enhancer, suggesting a common functional mechanism. Mutations that disrupt these HNF-3 motifs significantly abolished the maximal androgen induction of PB and PSA activities. Overexpressing a mutant HNF-3 alpha deleted in the C-terminal region inhibited the androgen-induced promoter activity in LNCaP cells where endogenous HNF-3 alpha is expressed. Chromatin immunoprecipitation revealed in vivo that the occupancy of HNF-3 alpha on PSA enhancer can occur in an androgen-depleted condition, and before the recruitment of ligand-bound AR. A physical interaction of HNF-3 alpha and AR was detected through immunoprecipitation and confirmed by glutathione-S-transferase pull-down. This interaction is directly mediated through the DNA-binding domain/hinge region of AR and the forkhead domain of HNF-3 alpha. In addition, strong HNF-3 alpha expression, but not HNF-3 beta or HNF-3 gamma, is detected in both human and mouse prostatic epithelial cells where markers (PSA and PB) of differentiation are expressed. Taken together, these data support a model in which regulatory cues from the cell lineage and the extracellular environment coordinately establish the prostatic differentiated response.

Background: Investigation into filarial lymphedema has been hampered by the lack of a simple, safe, and easily repeated test to image the peripheral lymphatic system. Recent refinements in radionuclide lymphangioscintigraphy have established this noninvasive technique as the initial procedure of choice for visualizing lymphatics. Accordingly, we applied lymphangioscintigraphy to patients with filariasis and, for purposes of interpretation, compared the findings with those in patients with non-filarial lymphedema., Methods: Thirty-three patients with classic symptoms or signs consistent with acute or chronic filariasis underwent lymphangioscintigraphy, and the findings were compared with those in five patients without lymphatic dysfunction and in 50 other patients with primary or secondary lymphedema without exposure to filariasis., Results: As in patients with nonfilarial lymphedema, scintigraphic abnormalities in the 33 patients with filariasis included delayed or absent tracer transport of the radiotracer (25 patients), tortuous and bizarre deep lymphatics (seven patients), dermal diffusion (15 patients), retrograde tracer flow (six patients), and faint or absent regional nodal visualization (14 patients). Even in patients with long-standing filarial lymphedema, peripheral trunks were often visualized (at least in part), and regional nodes and more central lymphatics sometimes filled after light exercise. In some of the latter patients, however, discrete lymphatic trunks were not detected., Conclusion: Lymphangioscintigraphy is a simple, safe, reliable, noninvasive method with which to examine the peripheral lymphatic system, including truncal and nodal abnormalities, in endemic populations with occult and overt lymphatic filariasis.

Rationale and Objectives: To evaluate acquired lymphatic abnormalities caused by filariasis, the authors examined the peripheral lymphatic system in normal ferrets and those chronically infected with Brugia malayi using magnetic resonance imaging (MRI). The findings were compared with previously obtained lymphangioscintigraphic (LAS) images in ferrets both with and without experimental filariasis., Methods: Fifteen ferrets (11 infected with B. malayi and four noninfected controls) underwent whole body coronal MRI using a quadrature transmission-receive head coil at 0.5 Tesla operating at a resonant frequency of 21.5 mHz for protons with a 25-cm field of view., Results: In contrast to normal animals, infected ferrets showed dilated hindlimb dermal lymphatic collaterals, enlarged high-signal intensity groin lymph nodes with punctate low-signal intensity centers and separate low-signal intensity spots with irregular thin channels, suggestive of nests of viable adult nematodes within tortuous lymphatics and nodes. MRI correlated with the LAS findings, and the interpretations were supported by light, scanning electron, and video microscopy., Conclusions: T2-weighted MRI in conjunction with LAS accurately depicts the peripheral lymphatic system in filarial-infected ferrets. These two modalities are useful complementary techniques to examine disorders characterized by lymphatic insufficiency.

Magnetic resonance (MR) imaging and isotope lymphography (lymphangioscintigraphy, LAS) was done in 32 patients with peripheral lymphedema (19 primary and 13 secondary). MRI characteristically showed diffuse dermal and subcutaneous edema, a nonedematous, occasionally hypertrophied skeletal muscle compartment, variability in regional lymph node size and appearance depending on the underlying clinical disorder, serpiginous "channels" or "lakes" consistent with dermal collateral lymphangiectasis and sequestered lymph, and increased subcutaneous fat. In contrast, LAS showed dermal diffusion ("backflow"), cross-over with retrograde tracer backflow (reflux), delayed tracer transport, and depending on the cause of lymphedema (i.e., primary or secondary), discrete or poorly defined lymph trunks (tracer "bands") and delayed or nonvisualization of regional lymph nodes. Although not a first-line clinical test, MR particularly in conjunction with LAS noninvasively provides accurate anatomical definition of the peripheral lymphatic system. In contradistinction to LAS, MR can visualize lymph trunks, nodes, and soft tissues proximal to sites of lymphatic obstruction. Together these imaging modalities may substitute for conventional oil contrast lymphography in the evaluation of the pathogenesis and evolution of most lymphologic disorders.

The tripeptide, L-prolyl-L-leucyl-glycinamide (PLG) has been shown to facilitate dopaminergic mechanisms in the brain. In the present study, we evaluated the interaction of PLG and its synthetic analogs with levodopa in two animal models of Parkinson's disease. In one experiment using rats with chronic unilateral lesions of the nigrostriatal dopamine pathway, PLG and Z-PLG potentiated the contraversive rotation elicited by levodopa with carbidopa (L/C). In a second experiment using reserpinized rats, PLG, Z-PLG and cyclo-LG potentiated L/C reversal of hypokinesia. Further studies of the PLG analogs, Z-PLG and cyclo-LG as adjunctive drugs with levodopa in the treatment of parkinsonism are warranted.

A 66-year-old male patient who had undergone repeated operations for peptic ulcer disease involving the right upper abdominal quadrant, developed cholecystitis with calculous obstruction of the common bile duct. The gallbladder was removed. Later, an operation was performed for removal of a residual stone from the common duct. At this time an anomalous arterial structure was noted about the duct. Hemorrhage occurred ten days postoperatively, and the anomalous hepatic artery was found to be eroded. The bleeding was controlled. During the succeeding two weeks there were four episodes of bleeding (involving erosion of the hepatic artery and adjacent tissues), three of which were controlled. The fourth episode ended in the death of the patient from exsanguination secondary to bleeding from stress ulcers in the gastric remnant. At no time did the laboratory data unequivocally indicate an abnormality of blood coagulation. Erosion of the anomalous cystic artery apparently precipitated the fatal chain of events.

A case is presented of transitional-cell cloacogenic carcinoma of the anal canal in a 66-year-old woman. Eight years previously she had received radiation therapy for early carcinoma of the uterine cervix, but there was no evidence of recurrence. The rectal carcinoma was therefore regarded as a second primary malignant tumor. This point in differential diagnosis was considered important because of its bearing on the plan of treatment. After abdominoperineal resection of the tumor, the prognosis seemed favorable.

A case is presented of bilateral lymphosarcoma of the breast in a 77-year-old woman. The pre-admission surgical history recorded a mastectomy in July 1972, for lymphosarcoma of the right breast. She had been well until shortly before admission in March 1974 because of a tumor in her left breast. This proved to be lymphosarcoma. Treatment consisted of a left mastectomy. At that time there were no signs of axillary or systemic involvement. Later, evidence of systemic lymphosarcomatosis necessitated starting chemotherapy.