Prostatic Intraepithelial Neoplasia Research Articles

Abstract 815: Regulation of p53 by INPP4B and high fat diet in mouse prostate

Abstract Obesity is one of the risk factors for prostate neoplasia development, however the underlying molecular mechanisms that link obesity and prostate cancer remain elusive. The roles of AR, p53, EZH2, INPP4B, and PTEN are implicated in prostate cancer etiology. Androgen receptor (AR) signaling interacts functionally with PTEN, INPP4B, EZH2, and p53 signaling in prostate epithelium, and its transcriptional activity is reprogrammed by changes in the levels of these proteins during prostate cancer progression. Phosphatase and tensin homolog (PTEN) and Inositol Polyphosphate 4-Phosphatase Type-II B (INPP4B) are dual specificity phosphatases which are tumor suppressors in prostate cancer and are lost in nearly half of advanced castration resistant prostate cancers (CRPC). p53 is also a tumor suppressor gene that is frequently inactivated in prostate cancers. Enhancer of Zeste 2 (EZH2) is an epigenetic silencer that increases methylation of histone H3 on K27 and K9 residues and acts as an oncogene in CRPC. We have previously shown that INPP4B inhibits the Akt and PKC signaling pathways, which are activated in obesity. Using an Inpp4b-deficient mouse model, we showed that three-month-old Inpp4b-/- males developed prostatic intraepithelial neoplasia (PIN) when fed a high fat diet (HFD). While the AR protein levels were not altered, the transcriptional activity of AR was reduced in the prostates of Inpp4b-/- males. Furthermore, the prostates of HFD-fed Inpp4b-/- males exhibited increased expression of pro-inflammatory cytokines and macrophage infiltration. It was previously reported that indolent nature of the prostate neoplasia, which is caused by prostate specific Pten knockout, is due to the compensatory increase in other tumor suppressor proteins, such as p53, SMAD4, and PML. Concomitant with Pten, knockout of these individual tumor suppressors led to drastic acceleration of prostate neoplasia. Similar to prostates of Pten-/- mice, p53 levels increased in Inpp4b-/- males fed a low fat diet (LFD). Unlike males fed LFD, p53 protein levels decreased in HFD fed Inpp4b-/- mice, consistent with the development of PIN in this group. EZH2 levels as well as the levels of its targets, H3K27me3 and H3K9me2, decreased in ventral prostates of the Inpp4b-/- males and that decrease was exacerbated by the HFD. Similar to our mouse model, we observed significant positive correlation between the INPP4B and EZH2 expression in the prostates of healthy men and prostate cancer patients. In summary, we showed that loss of INPP4B synergizes with the HFD in reprogramming AR activity, reduction of EZH2 levels, and increases inflammation. Importantly, HFD reverses compensatory increase in p53 protein levels in prostates of Inpp4b-/- males. These alterations contribute to the development of PIN in the prostates of HFD-fed Inpp4b-/- males. Citation Format: Yasemin Ceyhan, Manqi Zhang, Irina U. Agoulnik. Regulation of p53 by INPP4B and high fat diet in mouse prostate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 815.

Abstract 3816: Functional atlas of prostate mesenchyme

Abstract Prostate cancer has a heterogeneous prognosis, and genetic alterations alone do not fully explain clinical behavior. We previously characterized the stroma of localized human prostates by Laser Capture Microdissection, and found that stroma was substantially different in prostates with and without tumor. Furthermore, a stromal gene signature reflecting bone remodeling was upregulated in high compared to low Gleason grade cases. To determine how stromal cells contribute to carcinogenesis and progression we study whether specific genetic alterations in the epithelium induce unique stromal changes. To do this, we utilized Genetically Engineered Mouse Models (GEMMs) representing common prostate cancer mutations and compared these to their wild-type conterparts: the Tmprss2-ERG fusion knock-in murine model induces histological alterations in the stroma in the absence of an epithelial phenotype; the Pten deletion mouse model (PtenKO) results in prostate intraepithelial neoplasia (PIN) but not invasive cancer; the Hi-Myc GEMM, leads to PIN and subsequently invasion; and the Pb4-Cre +/-;Pten f/f; LSL-MYCN +/+; Rb1 f/f (MNRPDKO) mouse model that leads to neuroendocrine prostate cancer (NEPC). We generated a comprehensive single-cell transcriptomic atlas of the mouse prostate cancer mesenchyme in these models. Using deep generative modeling followed by graph-based clustering and gene regulatory network inference, six (6) distinct subsets of fibroblasts and two (2) subsets of smooth muscle cells (myofibroblasts and pericytes) were identified. Notably, some subsets were common across all GEMMs and WT mice, while others aligned with specific genotypes. Moreover, we found a variable pattern of positive and negative Ar expressing cells between genotypes. Analysis by CellphoneDB of mesenchymal-epithelial communications revealed the complex cross-talk between mutated epithelial cells and the tumor microenvironment. Multiplex immunofluorescence phenotyping of mesenchymal cell confirmed the cluster subtypes by both expression and spatial location. Finally, stromal transcripts defining mesenchymal cluster subtypes associated with Tmprss2-ERG were conserved between mouse and human genotypes.This study lays the groundwork for understanding and ultimately targeting stromal-epithelial interactions in prostate cancer. Citation Format: Hubert Pakula, Ryan Carelli, Nicolo Fanelli, Madhavi Jere, Caitlin Unkenholz, Mohamed Omar, Caroline Ribeiro- Fidalgo, Filippo Pederzoli, Cory Abate-Shen, David S. Rickman, Brian Robinson, Luigi Marchionni, Massimo Loda. Functional atlas of prostate mesenchyme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3816.

Abstract 5056: Automated Gleason grading of digitized frozen section prostate tissue slide images

Abstract Frozen sections are used in intraoperative consultations which need rapid microscopic analysis and in certain pathological procedures that require fresh tissue. Due to the various artifacts present on frozen section tissue slides, it is difficult to automatically analyze them. We tried automatic analysis of frozen sections. Two types of segmentation deep learning models detecting Gleason patterns were used. Both models were trained from hematoxylin and eosin (H&E) stained formalin-fixed, paraffin-embedded prostate tissue slide images. Each slide image was reviewed by an experienced pathologist, and all prostate cancer lesions were annotated with corresponding Gleason grades. One model was trained with H&E stained images (RS model), and the other model was trained with Hematoxylin-only (H-only) stained images (H-only model). A color deconvolution method was used to generate H-only stained images from original H&E stained images. In evaluation, frozen section prostate cancer slide images from The Cancer Genome Atlas (TCGA) were used. To diagnose the slide, we splitted whole slide images (WSI) into patches and analyze those patches to segment the Gleason pattern area. For the WSI result, we reconstructed the result as a whole size heatmap, then we counted the Gleason pattern to diagnose the slide as the prostate grade group. In detecting malignant cases, the RS model achieved a sensitivity of 98% and the H only model of 97%, respectively. In detecting clinically significant risk cases (grade group 2 or over), sensitivities of 99% and 96% were achieved, respectively. Most of the errors were all false-positive cases. In particular, the RS model mainly had false positives because of the ice crystal artifact, and the H-only model had many false positives for the folded part and the part suspected of being a prostatic intraepithelial neoplasia (PIN). False negatives were common in the compression artifacts for both models. We confirmed that both models showed high performance in cancer detection. In addition, they showed high accuracy in the classification of high-risk and low-risk groups of cancer. Further performance improvement can be expected with additional training of data related to artifacts or PIN. Citation Format: Joonyoung Cho, Tae-Yeong Kwak, Sun Woo Kim, Hyeyoon Chang. Automated Gleason grading of digitized frozen section prostate tissue slide images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5056.

Abstract 3772: Increased mitochondrial gene expression in prostate cancer

Abstract Warburg postulated that aerobic glycolysis, which is commonly observed in cancer, results from crippled mitochondrial function. Yet, most cancers and other rapidly proliferating cells rely on a functioning mitochondrial TCA cycle to generate important biosynthetic intermediates required for cell growth, and on the electron transport chain for pyrimidine synthesis and to recycle electron acceptors. Thus, increases in a number of mitochondrial activities have been proposed to be required for robust proliferation in most cancers. Mitochondrial function is regulated in part through mitochondrial biogenesis, which is influenced by mitochondrial DNA copy number (mtDNAcn). We previously developed an in situ hybridization method to quantify mtDNAcn in specific cell compartments in relation to normal physiological functioning and disease processes. We reported marked cell type heterogeneity across normal tissues including relatively higher mtDNAcn in a number of stem/proliferative compartments in humans and mice (PMID: 32304697). More recently, we combined this in situ approach with immunohistochemistry and reported increased mtDNAcn along with widespread heterogeneity in prostate cancer (DOI: 10.1158/1538-7445.AM2021-2404). We also found increased mtDNAcn in high grade prostatic intraepithelial neoplasia, the main precursor to prostate cancer, as well as precursor lesions in the large intestine and pancreas. To interrogate the functional significance of this mtDNAcn alteration, we sought to determine whether increased and heterogeneous mtDNAcn levels are accompanied by corresponding changes in its gene expression (mtRNA). To establish an initial survey of the landscape of steady state mtRNA levels, we used the RNAscope HiPlex system for multiplex in situ hybridization for 4 different mtRNAs along with in situ hybridization for mtDNA and combined this with immunofluorescence. In normal prostatic epithelium, we observed that the overall mtRNA expression pattern correlated with mtDNAcn, with higher levels in the basal cell layer compared with the luminal cell layer. We also found that, similar to changes in mtDNAcn in invasive prostate cancer, the 4 mtRNAs were upregulated yet heterogeneous in cancer lesions. Among the invasive tumors, cribriform prostate cancer showed distinct mtDNA and mtRNAs expression patterns, with higher signals in the peripheral cells than central cells, suggesting underlying biological differences in this unique histologic pattern. This spatial pattern in cribriform lesions correlated with the pattern of MYC protein expression, which is known to regulate mitochondrial biogenesis. Overall, we present a novel approach that advances the ability to quantify mtDNAcn and mtRNAs simultaneously in specific cell types while preserving the tissue spatial context. These findings suggest that increased mitochondrial function accompanies prostate cancer development and progression. Citation Format: Jiayu Chen, Qizhi Zheng, Jessica L. Hicks, Levent Trabzonlu, Ibrahim Kulac, Alan K. Meeker, Srinivasan Yegnasubramanian, Angelo M. De Marzo. Increased mitochondrial gene expression in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3772.

Abstract 5295: Combined loss of tristetraprolin (TTP) and PTEN leads to aggressive prostate cancer and hypersensitivity to nuclear factor kappa B (NFkB) inhibition

Abstract We have reported that loss of tristetraprolin (TTP, gene name ZFP36), an RNA binding protein which modulates mRNA stability increased NFκB activation and is associated with higher rates of disease recurrence in prostatectomy series. Given TTP loss occurs in early prostate cancer we sought to examine the clinical and biological impact of TTP loss combined with PTEN loss, which also occurs in early prostate cancer. IHC revealed an increased risk of relapse with loss of one gene alone and a significantly higher risk of relapse with loss of both TTP and PTEN, (HR 4.50, p=0.04 on MVA adjusting for standard clinicopathologic factors). These findings were confirmed by analysis of RNA profiles of two publicly available prostatectomy series. Engineering ZFP36 deletion specific to prostate luminal cells in vivo induced high-grade prostatic intraepithelial neoplasia (HG-PIN), and when combined with PTEN deletion (P:Z) resulted in rapid progression to invasive adenocarcinoma associated with increased proliferation and development of micrometastases. Combined loss of PTEN and ZFP36 in mice significantly reduced overall survival (53.4 (P:Z+/-) and 49.6 (P:Z-/-) weeks versus 66.9 weeks in PTEN-/- (p<0.0001). Further, surgical castration of mice demonstrated that combined loss of ZFP36 and PTEN significantly accelerated progression to castration resistance versus PTEN alone (median survival, 56.1 (P:Z+/-) and 53.9 (P:Z-/-) weeks versus all PTEN-/- mice still alive by 70 weeks). Ex vivo analysis of organoid growth of the tumors revealed presence of budding with PTEN-/- with ZFP36-/- and almost none with PTEN-/-. These findings were recapitulated with GEMM-derived allograft tumor growth with tumor sizes 20 weeks after castration being 200mm3 in PTEN-/- versus 900mm3 with [PTEN-/- + TTP-/-] versus 600mm3 for PTEN-/- and 1200mm3 for [PTEN-/- + TTP-/-] after sham castration (all comparisons p < 0.05). When the PTEN/ZFP36 null allograft was treated with the NFκB inhibitor dimethylaminoparthenolide (DMAPT, D), tumor sizes 36 days after treatment were: vehicle - 2000mm3, castration (C) - 1500mm3; D alone - 600mm3; C + D - 500mm3 (all comparisons p<0.05; except D vs C + D; p=0.07). These findings were supported by cell death assays of organoid models for the GEMMs. The hypersensitivity of PTEN/ZFP36 null cell line to DMAPT was seen with dose response curves with DMAPT LD50 being 4.52 μM PTEN-/-; 0.76 μM [PTEN -/- + TTP-/-] and 8.66 μM for [PTEN-/- + TTP-/- + silenced p65]. In conclusion, combined loss of PTEN and TTP results in prostate cancer that is clinically more aggressive than PTEN loss alone and this was corroborated by GEMM models. Namely, PTEN/TTP null tumors were more aggressive, minimally responsive to castration, demonstrated activation of NFκB and hypersensitivity to DMAPT. This work supports the planned clinical trials of DMAPT in castration resistant prostate cancer Citation Format: Christopher J. Sweeney, Katherine Morel, Anis Hamid, Leigh Ellis. Combined loss of tristetraprolin (TTP) and PTEN leads to aggressive prostate cancer and hypersensitivity to nuclear factor kappa B (NFkB) inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5295.

Abstract 924: Modelling the metastatic castration-resistant prostate cancer in mice by orthotopic delivering multiplexed gRNAs of CRISPR/Cas9 based on AAV system

Abstract Prostate cancer (PCa) is a most common cause of cancer-related death in men. While it develops to metastatic stage, the treatment options are limited. Hence, it desires in vivo models to test novel strategies for treating PCa. Current murine models of prostate cancer need long time for the diseases to progress to an invasive and metastatic cancer. Here, we combined AAV delivery and CRISPR-Cas9 technology to mutate several genes simultaneously in the mouse prostate. Four weeks after delivering of engineered virus, benign prostatic intraepithelial neoplasia (PIN) can be achieved and within eight weeks, the tumor developed to an extremely aggressive cancer. Surprisingly, a basic virus, engineering quintuple gRNAs of Cas9 including Pten, Trp53 and Rb1 induced a primary tumor without formation of metastasis. While adding triple gRNAs targeting epigenetic factors to the basic viral vector, all mice developed lung metastasis with metastasis started to appear after 6 weeks. Castration of the tumor baring mice 5 weeks after initiation did not alter primary tumor and metastasis formation, indicating the metastatic castration-resistant prostate cancer (mCRPC) nature of the model. To reveal the molecular mechanism for cancer progression and metastasis formation, whole genome sequencing (WGS) were performed on lung metastasis samples in combination with mRNA-seq on primary and secondary tumors from the two groups and PBS-injected mice control. Up to 5,000 mutations were accumulated in the genome, although the number varied across the metastatic samples, which were mainly due to chromosome 6 and 8 and revealed heterogeneity of the tumors. Among it, novel key mutations were identified, which will be addressed in the future. Tumor cell plasticity, including epithelial-mesenchymal transition and de-differentiation, were conformed. A short 100kb genomic window at mouse chr5 was captured and found to be sensitive to mutation status of an epigenetic factor. This window is highly conserved to a region at human chr4 and in vitro work confirm evolutionary preserved mechanism. Finally, p-Src/p-Lyn-cMyc related pathways showed clear difference between the two primary tumors with and without metastasis potency. Overall, our model of mCRPC can provide very diverse angles to the initiation and progression of prostate cancer. Furthermore, this model can be used to rapidly verify ideas and hypothesis regarding the mechanism behind the disease, and to test the treatment strategies in vivo in both pre-clinic studies and pharmacy field. Citation Format: Huiqiang Cai, Bin Zhang, Johanne Ahrenfeldt, Nicolai Birkbak, Martin Thomsen. Modelling the metastatic castration-resistant prostate cancer in mice by orthotopic delivering multiplexed gRNAs of CRISPR/Cas9 based on AAV system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 924.

Abstract 1: Transcriptional profiling of prostatic Skp2 knock-in mouse model and development of the associated prostate organoids for testing Skp2 targeting agents

Abstract S-phase kinase associated protein 2 (Skp2) is a promising drug target as studies have demonstrated that Skp2 is required for spontaneous tumor development that occurs in the retinoblastoma protein (pRb), Pten, or p53 deficient mice. We, therefore, aim to establish CRISPR human Skp2 (hSkp2) knock-in in the prostates of mice to study the molecular profiling features in prostate carcinogenesis. Prostate weights increased in transgenic mice and overexpression of hSkp2 induced prostatic lesions including hyperplasia, mouse prostate intraepithelial neoplasia (mPIN), and carcinoma, which suggested the initial role of hSkp2 in early prostate carcinogenesis. RNAseq results revealed Myl3 and Ctgf as the top down-expressed and up-expressed genes respectively in hSkp2 mice prostates. Gene set enrichment analysis (GSEA) demonstrated the significant upregulations of ribosome and proteasome pathways which had similar alterations in the human prostate cancer dataset with Skp2 overexpression, suggesting the potential role of ribosome biogenesis in prostate tumorigenesis and for drug development. In addition, Skp2 organoids derived from transgenic mice prostates were being developed for convenient and efficient screening of specific Skp2 inhibitors. Flavokawain A (FKA) and Skp2 inhibitor C1 both selectively decreased the viability and altered the morphologies of hSkp2 organoids, meanwhile FKA exhibited less toxicity on wild-type organoids. Citation Format: Liankun Song, Vyvyan Nguyen, Shan Xu, Jana Yamak, Kia Arabzadehkaffash, Ali Fazelpour, Merci Mino, Matthew Tippin, Shuang Meng, Xiaolin Zi. Transcriptional profiling of prostatic Skp2 knock-in mouse model and development of the associated prostate organoids for testing Skp2 targeting agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1.

Abstract 706: Efficacy evaluation of BR-9001, a SMEDDS formulation of 3,3’-diindolylmethane, as an inhibitor of preneoplastic and neoplastic lesion development in the prostate of Hi-Myc mice

Abstract Self-Microemulsifying Drug Delivery Systems (SMEDDS) can greatly increase the bioavailability of agents with limited solubility. Studies were performed to characterize the efficacy of BR-9001, a SMEDDS formulation of 3,3’-diindolylmethane (DIM), in preventing preneoplastic and neoplastic prostate lesion development in Hi-Myc mice. Beginning at age 8 weeks, groups of 105 male Hi-Myc mice received twice daily oral (gavage) administration of BR-9001 (BioResponse LLC, Boulder, CO) at DIM-equivalent doses of 0 (vehicle control), 200, or 400 mg/kg/dose. At ages 4 months, 5.5 months, and 7 months, cohorts of 35 mice/group were euthanized and necropsied; at each time point, tissues were collected from 25 mice/group for pathology; 5 mice/group for analysis of DIM levels in the plasma and prostate; and 5 mice/group for biomarker analysis. Prostate lesion development in pathology cohorts was determined by microscopic evaluation of step sections through the entire accessory sex gland block. Plasma and prostate levels of DIM were quantitated by LC-MS/MS; biomarkers were analyzed by RT-PCR. Administration of BR-9001 produced dose-related levels of DIM in the plasma and prostate at all time points. However, BR-9001 provided at most limited protection against prostate lesion development. Most lesions were identified in the lateral prostate (LP), where statistically significant reductions in incidences of murine prostatic intraepithelial neoplasias (mPIN) were seen in 4-month-old mice receiving the high dose of BR-9001 and in 5.5-month-old and 7-month-old mice receiving the low dose of BR-9001. Reductions in mPIN incidence in groups receiving BR-9001 were due to lower incidences of small mPIN lesions; incidences of large mPIN were significantly increased at 5.5 months in both groups receiving BR-9001. BR-9001 had no statistically significant effect on % occupancy of the LP by mPIN, the incidence of microinvasive mPIN in the LP, or cancer incidence in the LP. In the anterior prostate (AP), incidences of atypical hyperplasia were similar in all groups. However, at all necropsy time points, mice receiving the high dose of BR-9001 demonstrated significant increases in the incidences of moderate to marked AP atypia; these increases were accompanied by decreases in the incidence of mice with slight to moderate AP atypia. None of 22 potential molecular biomarkers were found to be clearly linked to development and progression of prostate lesions in Hi-Myc mice. Despite the presence of measurable concentrations of DIM in the plasma and prostate in mice in both treated groups at all time points, BR-9001 demonstrated equivocal efficacy in preventing the development of preneoplastic and neoplastic lesions in the Hi-Myc mouse model of prostate cancer. [Supported by NCI HHSN261201500042I/HHSN26100003.] Citation Format: Genoveva Murillo, Gina Qualter, Xinjian Peng, Miguel Muzzio, Elizabeth R. Glaze, Altaf Mohammed, Maarten C. Bosland, David L. McCormick. Efficacy evaluation of BR-9001, a SMEDDS formulation of 3,3’-diindolylmethane, as an inhibitor of preneoplastic and neoplastic lesion development in the prostate of Hi-Myc mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 706.

The Biochemical Effects of Silver Nanoparticles and Spirulina Extract on Experimentally Induced Prostatic Cancer in Rats

Prostate cancer (PCa) is the most diagnosed cancer in 112 countries and the second leading cause of death in men in 48 countries. We studied the outstanding agents silver nanoparticles (AgNPs) and Spirulina algae (Sp) for the management of PCa once as monotherapy or last as a combination. PCa in rats was induced using bicalutamide (Casodex®) and testosterone, followed by (7, 12-dimethylbenz[a]anthracene). Then, testosterone was injected s.c. for 3 months. Rats were divided into six groups, with 12 rats in each group. Group I was assigned as the control (co), group II as the PCa model, group III treated with AgNPs, group IV treated with Spirulina extract, group V treated with a combination of AgNPs plus Spirulina, and group VI treated with bicalutamide. The results show that AgNPs could normalize IL-6 levels and could overcome the hormonal disturbance induced in PCa rats along the hypothalamic–pituitary–testis axis. Spirulina revealed a significant reduction in the level of total and free prostatic specific antigen (PSA) to the same level as bicalutamide treatment, which was the same as the control group. Histopathological study revealed regression (75%) of the histological pattern of high-grade prostatic intraepithelial neoplasia (HGPIN) for Spirulina alone, and (50%) for bicalutamide. The best effect on IL-6 decline was reached with the AgNPs/Spirulina combination as well as bicalutamide treatment compared with the PCa group. Bicalutamide treatment significantly decreased the PSA concentration relative to the PCa group and reached the normal level. Adding Spirulina to AgNPs as a combination enhanced its effect on all mentioned drawbacks associated with PCa except hormonal imbalance that needs more adjustments.

Cut-off Point of Ki-67 Proliferation Marker in Differentiating Premalignant and Malignant Prostatic Lesions

Immunohistochemistry Ki-67 is a marker of cell proliferation. Using immunohistochemical markers can be helpful to determine and diagnose prostate intraepithelial neoplasia (PIN) and carcinoma prostate. There are many studies about Ki67 as a prognostic tool, a diagnostic tool, and a potential therapeutic target for cancer therapy, but there are no standard criteria to define the cut-off point for the value of Ki-67 in premalignant and malignant prostate lesions.

Copy number variation of urine exfoliated cells by low-coverage whole genome sequencing for diagnosis of prostate adenocarcinoma: a prospective cohort study.

BackgroundNon-invasive, especially the urine-based diagnosis of prostate cancer (PCa) remains challenging. Although prostate cancer antigen (PSA) is widely used in prostate cancer screening, the false positives may result in unnecessary invasive procedures. PSA elevated patients are triaged to further evaluation of free/total PSA ratio (f/t PSA), to find out potential clinically significant PCa before undergoing invasive procedures. Genomic instability, especially chromosomal copy number variations (CNVs) were proved much more tumor specific. Here we performed a prospective study to evaluate the diagnostic value of CNV via urine-exfoliated cell DNA analysis in PCa.MethodsWe enrolled 28 PSA elevated patients (≥ 4 ng/ml), including 16 PCa, 9 benign prostate hypertrophy (BPH) and 3 prostatic intraepithelial neoplasia (PIN). Fresh initial portion urine was collected after hospital admission. Urine exfoliated cell DNA was analyzed by low coverage Whole Genome Sequencing, followed by CNV genotyping by the prostate cancer chromosomal aneuploidy detector (ProCAD). CNVs were quantified in absolute z-score (|Z|). Serum free/total PSA ratio (f/t PSA) was reported altogether.ResultsIn patients with PCa, the most frequent CNV events were chr3q gain (n = 2), chr8q gain (n = 2), chr2q loss (n = 4), and chr18q loss (n = 3). CNVs were found in 81.2% (95% Confidence Interval (CI) 53.7–95.0%) PCa. No CNV was identified in BPH patients. A diagnosis model was established by incorporating all CNVs. At the optimal cutoff of |Z|≥ 2.50, the model reached an AUC of 0.91 (95% CI 0.83–0.99), a sensitivity of 81.2% and a specificity of 100%. The CNV approach significantly outperformed f/t PSA (AUC = 0.62, P = 0.012). Further analyses showed that the CNV positive rate was significantly correlated with tumor grade. CNVs were found in 90.9% (95% CI 57.1–99.5%) high grade tumors and 60.0% (95% CI 17.0–92.7%) low grade tumors. No statistical significance was found for patient age, BMI, disease history and family history.ConclusionsUrine exfoliated cells harbor enriched CNV features in PCa patients. Urine detection of CNV might be a biomarker for PCa diagnosis, especially in terms of the clinically significant high-grade tumors.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12920-022-01253-5.

PGC1 alpha coactivates ERG fusion to drive antioxidant target genes under metabolic stress

The presence of ERG gene fusion; from developing prostatic intraepithelial neoplasia (PIN) lesions to hormone resistant high grade prostate cancer (PCa) dictates disease progression, altered androgen metabolism, proliferation and metastasis1–3. ERG driven transcriptional landscape may provide pro-tumorigenic cues in overcoming various strains like hypoxia, nutrient deprivation, inflammation and oxidative stress. However, insights on the androgen independent regulation and function of ERG during stress are limited. Here, we identify PGC1α as a coactivator of ERG fusion under various metabolic stress. Deacetylase SIRT1 is necessary for PGC1α-ERG interaction and function. We reveal that ERG drives the expression of antioxidant genes; SOD1 and TXN, benefitting PCa growth. We observe increased expression of these antioxidant genes in patients with high ERG expression correlates with poor survival. Inhibition of PGC1α-ERG axis driven transcriptional program results in apoptosis and reduction in PCa xenografts. Here we report a function of ERG under metabolic stress which warrants further studies as a therapeutic target for ERG fusion positive PCa.

Periprostatic Adipose Tissue Thromboinflammation Drives Early Prostatic Neoplastic Alterations in a Rat Model of Mild Metabolic Dysfunction

Emerging evidence links periprostatic adipose tissue (PPAT) inflammation, a consequence of metabolic impairment, and certain features of prostate cancer (PCa) aggressiveness. AT thromboinflammation, mediated by the proteolytic activation of protease‐activated receptors (PARs) particularly by thrombin and Factor Xa (FXa), drives AT dysfunction. Nevertheless, the contribution of PPAT thromboinflammation to early prostatic neoplastic alterations has not been investigated. Here, we utilized a non‐obese prediabetic rat model to assess prostatic neoplasia in association with PPAT thromboinflammation. Mild hypercaloric diet (MHC)‐fed rats exhibited hyperinsulinemia, hypertriglyceridemia, a lower metabolic efficiency, and a higher fat/lean ratio in comparison to regular chow‐fed rats. When compared to regular chow‐fed rats, MHC‐fed rats exhibited PPAT with larger adipocytes, enhanced oxidative stress and increased immune cell influx. Concurrently, prostates from MHC‐fed rats displayed higher number of prostatic intraepithelial neoplasia (PIN), enhanced expression of the proliferation markers Ki67 and PCNA, enhanced expression of cytokeratin 8 (CK8), pronounced fibrosis and increased immune cell influx. Further profiling of macrophage polarization in PPAT and the prostate is underway. Importantly, these prostatic and adipocytic manifestations were attenuated in MHC‐fed rats treated with non‐hemorrhagic doses of rivaroxaban, a direct FXa inhibitor. Ongoing in vitro investigation demonstrates that rivaroxaban reduces the migratory potential of murine prostate cancer cells (Plum‐AD); and the effect of rivaroxaban on the colony forming and sphere forming capacities of Plum‐AD cells are being studied. Further investigation is ongoing to delineate the molecular underpinnings of rivaroxaban‐associated amelioration of PPAT inflammation and the associated prostatic neoplastic alterations.

Macrophages Cytokine Spp1 Increases Growth of Prostate Intraepithelial Neoplasia to Promote Prostate Tumor Progression.

Prostate cancer development and progression are associated with increased infiltrating macrophages. Prostate cancer is derived from prostatic intraepithelial neoplasia (PIN) lesions. However, the effects macrophages have on PIN progression remain unclear. Here, we showed that the recruited macrophages adjacent to PIN expressed M2 macrophage markers. In addition, high levels of Spp1 transcripts, also known as osteopontin, were identified in these macrophages. Extraneously added Spp1 accelerated PIN cell proliferation through activation of Akt and JNK in a 3D culture setting. We also showed that PIN cells expressed CD44, integrin αv, integrin β1, and integrin β3, all of which have been previously reported as receptors for Spp1. Finally, blockade of Akt and JNK activation through their specific inhibitor completely abolished macrophage Spp1-induced cell proliferation of PIN. Hence, our data revealed Spp1 as another macrophage cytokine/growth factor and its mediated mechanism to upregulate PIN cell growth, thus promoting prostate cancer development.

Estrogen receptor expression is modulated in human and mouse prostate epithelial cells during cancer progression

Substantial data posit estrogen receptors (ERs) as promising targets for prostate cancer (PCa) therapeutics. However, the trials on assessing the chemo-preventive or therapeutic potential of ER targeting drugs or selective estrogen receptor modulators (SERMs) have not yet established their clinical benefits. This could be ascribed to a possible modulation in the ER expression during PCa progression. Further it is warranted to test various ER targeting drugs in appropriate preclinical models that simulate human ER expression pattern during PCa progression. The study was undertaken to revisit the existing data on the epithelial ER expression pattern in human cancerous prostates and experimentally determine whether these patterns are replicated in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice, a model for human PCa. Estradiol (E2) binding to the plasma membrane of the epithelial cells and its modulation during the PCa progression in TRAMP were also investigated.A reassessment of the existing data revealed a trend towards downregulation in the epithelial expression of wild-type ESR1 transcripts in high-grade PCa, compared to non-cancerous prostate in humans. Next, epithelial cell-enriched populations from TRAMP prostates (TP) displaying low-grade prostatic intraepithelial neoplasia (LGPIN), high-grade PIN (HGPIN), HGPIN with well-differentiated carcinoma (PIN + WDC), WDC (equivalent to grade 2/3 human PCa), and poorly-differentiated carcinoma (PDC-equivalent to grade 4/5 human PCa) revealed significantly higher Esr1 and Esr2 levels in HGPIN and significantly reduced levels in WDC, compared to respective age-matched control prostates. These patterns for the nuclear ERs were similar to the trend shown by E2 binding to the plasma membrane of the epithelial cells during PCa progression in TRAMP. E2 binding to epithelial cells (EpCAM+), though significantly higher in TPs displaying LGPIN, decreased significantly as the disease progressed to WDC. The study highlights a reduction in the epithelial ESR level with the PCa progression and this pattern was evident in both humans and TRAMP. These observations may have major implications in refining PCa therapeutics targeting ER.

ETS-related gene (ERG) undermines genome stability in mouse prostate progenitors via Gsk3β dependent Nkx3.1 degradation

21q22.2–3 deletion is the most common copy number alteration in prostate cancer (PCa). The genomic rearrangement results in the androgen-dependent de novo expression of ETS-related gene (ERG) in prostate cancer cells, a condition promoting tumor progression to advanced stages of the disease.Interestingly, ERG expression characterizes 5–30% of tumor precursor lesions – High Grade Prostatic Intraepithelial Neoplasia (HGPIN) - where its role remains unclear.Here, by combining organoids technology with Click-chemistry coupled Mass Spectrometry, we demonstrate a prominent role of ERG in remodeling the protein secretome of prostate progenitors. Functionally, by lowering autocrine Wnt-4 signaling, ERG represses canonical Wnt pathway in prostate progenitors, and, in turn, promotes the accumulation of DNA double strand breaks via Gsk3β-dependent degradation of the tumor suppressor Nkx3.1. On the other hand, by shaping extracellular paracrine signals, ERG strengthens the pro-oxidative transcriptional signature of inflammatory macrophages, which we demonstrate to infiltrate pre-malignant ERG positive prostate lesions.These findings highlight previously unrecognized functions of ERG in undermining adult prostate progenitor niche through cell autonomous and non-autonomous mechanisms. Overall, by supporting the survival and proliferation of prostate progenitors in the absence of growth stimuli and promoting the accumulation of DNA damage through destabilization of Nkx3.1, ERG could orchestrate the prelude to neoplastic transformation.

ZEB1 is Negatively Correlated with E-Cadherin in Prostatic Anomaly Tissue

Background: Prostatic anomalies are common in tumor or infection condition. The enlargement of prostate gland affects the epithelial cell polarity that involves epithelial-mesenchymal transition (EMT). Transition into mesenchymal is mediated by transcription factor ZEB1 and E-cadherin protein. Upregulation of ZEB1 and loss of E-Cadherin expression were associated to proliferation and metastasis of malignancy cells. This study aims to describe the correlation of ZEB1 and E-cadherin expression in prostatic anomaly.Materials and method: Samples were Formalin Fixed Paraffin Embedded (FFPE) block consist of 8 block Benign Prostatic Hyperplasia (BPH), 6 blocks High Grade Prostatic Intraepithelial Neoplasia (HGPIN) and 6 blocks Prostate Carcinoma (PCA). The blocks then sliced into 5 sections to be prepared for RNA extraction procedures. ZEB1 and E-Cadherin expression was analyzed by semi-quantitative procedures using PCR and electrophoresis. Correlation between ZEB1 and E-Cadherin espression was analyzed using Spearman’s rank correlation.Results: Relative expression of ZEB1 and E-cadherin mRNA in each group of prostatic anomaly were not significantly different (p>0.05). ZEB1 and E-Cadherin mRNA expression showed a significant and moderate level of negative correlation (p<0.05; 0.40 < r < 0.59). Increasing of ZEB1 mRNA expression will be followed by decreasing of E-Cadherin mRNA expression.Conclusion: ZEB1 negatively correlates with E-cadherin due to EMT process in prostatic anomaly. High expression of ZEB1 induced down-regulation of E-cadherin and vise versa. Various studies can be developed, especially the development of targeted therapy against ZEB1 to suppress the EMT process by increasing the expression of E-cadherin.Keywords: epithelial-mesenchymal transition (EMT), ZEB1, E-Cadherin, BPH, HGPIN, PCA

Diagnosis codes overestimate the burden of prostate cancer cases.

72 Background: Identifying cancer cases within the electronic health record (EHR) or claims data can be challenging because diagnosis codes are often entered into patient records during routine screenings or as “rule out” diagnosis codes when the patient is referred to a procedure. To improve accuracy of prostate cancer (PCa) case ascertainment, we compared algorithms that used diagnoses codes to natural language processing (NLP) tools applied to clinical notes and pathology reports to identify Veterans with prostate cancer (PCa). Methods: This is a retrospective observational cohort study using VA EHR data to identify veterans diagnosed with PCa between 2000 and 2020. Using International Classification of Diseases (ICD-10 CM or ICD-9 CM) diagnosis and procedure codes, we identified veterans who may have PCa. We deployed validated NLP tools to identify the presence of Gleason score, metastatic PCa, and castration sensitivity to identify evidence of PCa within the notes. We conducted a descriptive analysis to compare the results of algorithms that relied exclusively on diagnosis codes compared to use of NLP tools. Results: From 2000 through 2020,1,031,296 veterans had one or more PCa diagnosis code. This number decreased by 11% for each additional PCa diagnosis code required. When we required 4 or more PCa diagnosis codes to be present, only 746,350 veterans had PCa. When we deployed NLP tools to identify mention of a Gleason score or an indicator of mPCa, only 685,847 Veterans had these indicators of PCa, a 35% decrease in the number of PCa cases with a single diagnosis code. Chart review of patients with their first PCa diagnosis codes in 2019 and 4 or more codes in their records illustrated no evidence of Gleason score or mPCa disease in their EHR. Analysis of their pathology reports revealed that these patients had prostatic intraepithelial neoplasia or atypical small acinar proliferation and had not yet developed prostate cancer. Conclusions: Accurate ascertainment of PCa using EHR and claims data requires using NLP tools and clinical notes combined with structured data sources such as diagnosis codes. Relying on ICD diagnosis codes alone will overestimate the burden of PCa up to 30%.

Prostatic adenocarcinoma with a peculiar morphology \u2013 a rare case of pseudohyperplastic variant with inverted polarity

BackgroundThe inverted (hobnail) variant of high-grade prostatic intraepithelial neoplasia (HGPIN) has been reported in two previous series and one case of inverted polarity in invasive adenocarcinoma has been reported. We reported an additional case of invasive carcinoma with this peculiar morphology.Case presentationWe reported an additional case of invasive carcinoma with this peculiar morphology. A prostatectomy specimen of a 64-year-old patient showed a GG2 adenocarcinoma with extensive intraprostatic perineural infiltration and extraprostatic extension. Half of the entire tumor showed a distinctive inverted morphology.ConclusionAlthough pseudohyperplastic adenocarcinoma is believed to be a low-grade tumor to be graded as Gleason pattern 3, awareness of this morphology is important to collect more information on its biologic behavior and clinical implication.

Dietary Pterostilbene for MTA1-Targeted Interception in High-Risk Premalignant Prostate Cancer.

Prostate cancer remains one of the most prevalent cancers in aging men. Active surveillance subpopulation of patients with prostate cancer includes men with varying cancer risk categories of precancerous disease due to prostatic intraepithelial neoplasia (PIN) heterogeneity. Identifying molecular alterations associated with PIN can provide preventable measures through finding novel pharmacologic targets for cancer interception. Targeted nutritional interception may prove to be the most appropriate chemoprevention for intermediate- and high-risk active surveillance patients. Here, we have generated two prostate-specific transgenic mouse models, one overexpressing MTA1 (R26MTA1 ) and the other overexpressing MTA1 on the background of Pten heterozygosity (R26MTA1 ; Pten+/f ), in which we examined the potential chemopreventive efficacy of dietary pterostilbene. We show that MTA1 promotes neoplastic transformation of prostate epithelial cells by activating cell proliferation and survival, leading to PIN development. Moreover, MTA1 cooperates with PTEN deficiency to accelerate PIN development by increasing cell proliferation and MTA1-associated signaling. Further, we show that mice fed with a pterostilbene-supplemented diet exhibited more favorable histopathology with decreased severity and number of PIN foci accompanied by reduced proliferation, angiogenesis, and inflammation concomitant to reduction in MTA1 and MTA1-associated CyclinD1, Notch2, and oncogenic miR-34a and miR-22 levels. PREVENTION RELEVANCE: Developing novel interceptive strategies for prostate cancer chemoprevention is a paramount goal in clinical oncology. We offer preclinical evidence for the potential of pterostilbene as a promising natural agent for MTA1-targeted interceptive strategy in future cancer prevention trials towards protecting select patients with prostate cancer under active surveillance from developing cancer.