Search

The discovery that androgens play an important role in the progression of prostate cancer led to the development of androgen deprivation therapy (ADT) as a first line of treatment. However, paradoxical growth inhibition has been observed in a subset of prostate cancer upon administration of supraphysiologic levels of testosterone (SupraT), both experimentally and clinically. Here we report that SupraT activates cytoplasmic nucleic acid sensors and induces growth inhibition of SupraT-sensitive prostate cancer cells. This was initiated by the induction of two parallel autophagy-mediated processes, namely, ferritinophagy and nucleophagy. Consequently, autophagosomal DNA activated nucleic acid sensors converge on NFκB to drive immune signaling pathways. Chemokines and cytokines secreted by the tumor cells in response to SupraT resulted in increased migration of cytotoxic immune cells to tumor beds in xenograft models and patient tumors. Collectively, these findings indicate that SupraT may inhibit a subset of prostate cancer by activating nucleic acid sensors and downstream immune signaling.Significance:This study demonstrates that supraphysiologic testosterone induces two parallel autophagy-mediated processes, ferritinophagy and nucleophagy, which then activate nucleic acid sensors to drive immune signaling pathways in prostate cancer.

Supplementary Figures S1-S9

Oligo sequences

Increased mitochondrial function may render some cancers vulnerable to mitochondrial inhibitors. Since mitochondrial function is regulated partly by mitochondrial DNA copy number (mtDNAcn), accurate measurements of mtDNAcn could help reveal which cancers are driven by increased mitochondrial function and may be candidates for mitochondrial inhibition. However, prior studies have employed bulk macrodissections that fail to account for cell type-specific or tumor cell heterogeneity in mtDNAcn. These studies have often produced unclear results, particularly in prostate cancer. Herein, we developed a multiplexin situmethod to spatially quantify cell type specific mtDNAcn. We show that mtDNAcn is increased in luminal cells of high-grade prostatic intraepithelial neoplasia (HGPIN), is increased in prostatic adenocarcinomas (PCa), and is further elevated in metastatic castration-resistant prostate cancer. Increased PCa mtDNAcn was validated by two orthogonal methods and is accompanied by increases in mtRNAs and enzymatic activity. Mechanistically, MYC inhibition in prostate cancer cells decreases mtDNA replication and expression of several mtDNA replication genes, and MYC activation in the mouse prostate leads to increased mtDNA levels in the neoplastic prostate cells. Ourin situapproach also revealed elevated mtDNAcn in precancerous lesions of the pancreas and colon/rectum, demonstrating generalization across cancer types using clinical tissue samples.

In this study, the compostability of treatment sludge sourced from the food industry (TS) and cattle manure (CM) was investigated. For this purpose, sunflower stalks (SS) were added as a bulking agent and composting experiments were performed. Different mixtures (R1, R2, R3) were prepared in order to determine the individual and co-compostability of the raw materials used. In the R1 reactor, TS and SS in the ratio of 70:30, in the R2 reactor, CM and SS in the ratio of 70:30, and in the R3 reactor, TS, CM and SS in the ratio of 35:35:30 were mixed, respectively. Temperature, organic matter, pH, electrical conductivity (EC) and C/N ratio parameters were monitored during the 21-day process. The highest temperatures during the composting process were determined as 43.9°C, 43.9°C and 53.7°C in the R1, R2 and R3 reactors, respectively. When the C/N ratios are examined, R3, in which the C/N ratio had decreased at a rate of 18.5% by the end of the process and which had the highest temperature rise, can be defined as the best reactor. It has been found that the efficiency of the composting process of food industry treatment sludge and cattle manure is high.

Understanding the intricacies of lethal prostate cancer poses specific challenges due to difficulties in accurate modeling of metastasis in vivo. Here we show that NPKEYFP mice (for Nkx3.1CreERT2/+; Ptenflox/flox; KrasLSL-G12D/+; R26R-CAG-LSL-EYFP/+) develop prostate cancer with a high penetrance of metastasis to bone, thereby enabling detection and tracking of bone metastasis in vivo and ex vivo. Transcriptomic and whole-exome analyses of bone metastasis from these mice revealed distinct molecular profiles conserved between human and mouse and specific patterns of subclonal branching from the primary tumor. Integrating bulk and single-cell transcriptomic data from mouse and human datasets with functional studies in vivo unravels a unique MYC/RAS co-activation signature associated with prostate cancer metastasis. Finally, we identify a gene signature with prognostic value for time to metastasis and predictive of treatment response in human patients undergoing androgen receptor therapy across clinical cohorts, thus uncovering conserved mechanisms of metastasis with potential translational significance. Using lineage tracing and molecular profiling, Abate-Shen and colleagues identify a Ras and Myc co-activation signature that predicts metastasis and castration resistance in localized prostate cancer.

Mitochondria regulate ATP production, metabolism, and cell death. Alterations in mitochondrial DNA (mtDNA) sequence and copy number are implicated in aging and organ dysfunction in diverse inherited and sporadic diseases. Because most measurements of mtDNA use homogenates of complex tissues, little is known about cell-type–specific mtDNA copy number heterogeneity in normal physiology, aging, and disease. Thus, the precise cell types whose loss of mitochondrial activity and altered mtDNA copy number that result in organ dysfunction in aging and disease have often not been clarified. Here, an in situ hybridization approach to generate a single-cell–resolution atlas of mtDNA content in mammalian tissues was validated. In hierarchically organized self-renewing tissues, higher levels of mtDNA were observed in stem/proliferative compartments compared with differentiated compartments. Striking zonal patterns of mtDNA levels in the liver reflected the known oxygen tension gradient. In the kidney, proximal and distal tubules had markedly higher mtDNA levels compared with cells within glomeruli and collecting duct epithelial cells. In mice, decreased mtDNA levels were visualized in renal tubules as a function of aging, which was prevented by calorie restriction. This study provides a novel approach for quantifying species- and cell-type–specific mtDNA copy number and dynamics in any normal or diseased tissue that can be used for monitoring the effects of interventions in animal and human studies.

Background Most prostate cancers are "immune cold" and poorly responsive to immune checkpoint inhibitors. However, the mechanisms responsible for the lack of a robust antitumor adaptive immune response in the prostate are poorly understood, which hinders the development of novel immunotherapeutic approaches. Aims Most inflammatory infiltrates in the prostate are centered around benign glands and stroma, which can confound the molecular characterization of the antitumor immune response. We sought to analytically validate a chromogenic-based multiplex immunohistochemistry (IHC) approach applicable to whole slide digital image analysis to quantify T cell subsets from the tumor microenvironment of primary prostatic adenocarcinomas. As an initial application, we tested the hypothesis that PTEN loss leads to an altered antitumor immune response by comparing matched regions of tumors within the same individual with and without PTEN loss. Materials & Methods Using the HALO Image Analysis Platform (Indica Labs), we trained a classifier to quantify the densities of eight T cell phenotypes separately in the tumor epithelial and stromal subcompartments. Results The iterative chromogenic approach using 7 different antibodies on the same slide provides highly similar findings to results using individually stained slides with single antibodies. Our main findings in carcinomas (benign removed) include the following: i) CD4+ T cells are present at higher density than CD8+ T cells; ii) all T cell subsets are present at higher densities in the stromal compartment compared to the epithelial tumor compartment; iii) most CD4+ and CD8+ T cells are PD1+; iv) cancer foci with PTEN loss harbored increased numbers of T cells compared to regions without PTEN loss, in both stromal and epithelial compartments; and v) the increases in T cells in PTEN loss regions were associated with ERG gene fusion status. Discussion This modular approach can apply to any IHC-validated antibody combination and sets the groundwork for more detailed spatial analyses. Conclusion Iterative chromogenic IHC can be used for whole slide analysis of prostate tissue samples and can complement transcriptomic results including those using single cell and spatial genomic approaches.

Prostate adenocarcinoma is the second most commonly diagnosed cancer in men worldwide, and the initiating factors are unknown. Oncogenic TMPRSS2:ERG (ERG+) gene fusions are facilitated by DNA breaks and occur in up to 50% of prostate cancers. Infection-driven inflammation is implicated in the formation of ERG+ fusions, and we hypothesized that these fusions initiate in early inflammation-associated prostate cancer precursor lesions, such as proliferative inflammatory atrophy (PIA), prior to cancer development. We investigated whether bacterial prostatitis is associated with ERG+ precancerous lesions in unique cases with active bacterial infections at the time of radical prostatectomy. We identified a high frequency of ERG+ non–neoplastic-appearing glands in these cases, including ERG+ PIA transitioning to early invasive cancer. These lesions were positive for ERG protein by immunohistochemistry and ERG messenger RNA by in situ hybridization. We additionally verified TMPRSS2:ERG genomic rearrangements in precursor lesions using tricolor fluorescence in situ hybridization. Identification of rearrangement patterns combined with whole-prostate mapping in three dimensions confirmed multiple (up to eight) distinct ERG+ precancerous lesions in infected cases. We further identified the pathogen-derived genotoxin colibactin as a potential source of DNA breaks in clinical cases as well as cultured prostate cells. Overall, we provide evidence that bacterial infections can initiate driver gene alterations in prostate cancer. In addition, our observations indicate that infection-induced ERG+ fusions are an early alteration in the carcinogenic process and that PIA may serve as a direct precursor to prostate cancer.

The discovery that androgens play an important role in the progression of prostate cancer (PCa) has led to the development of androgen deprivation therapy as a first line of treatment against PCa. However, paradoxical growth inhibition has been observed, both experimentally and clinically, in a subset of PCa upon administration of supraphysiological levels of testosterone (SupraT). Here we report that SupraT activates cytoplasmic nucleic acid sensors and induces growth inhibition of SupraT-sensitive PCa cells. This is initiated by induction of two parallel autophagy-mediated processes, namely, ferritinophagy and nucleophagy. Consequently, autophagosomal DNA activates nucleic acid sensors that converge on NF-kappaB to drive immune signaling pathways. Chemokines and cytokines secreted by the tumor cells in response to SupraT results in increased migration of cytotoxic immune cells to tumor beds of animal xenografts and patient tumors. Collectively, our findings indicate that SupraT may inhibit a subset of PCa by activating nucleic acid sensors and downstream immune signaling.

Prostate adenocarcinoma is the second most commonly diagnosed cancer in men worldwide and the initiating factors are unknown. Oncogenic TMPRSS2:ERG (ERG+) gene fusions are facilitated by DNA breaks and occur in up to 50% of prostate cancers1,2. Infection-driven inflammation is implicated in the formation of ERG+ fusions3, and we hypothesized that these fusions initiate in early inflammation-associated prostate cancer precursor lesions, such as proliferative inflammatory atrophy (PIA), prior to cancer development. We investigated whether bacterial prostatitis is associated with ERG+ precancerous lesions in unique cases with active bacterial infections at time of radical prostatectomy. We identified a high frequency of ERG+ non-neoplastic-appearing glands in these cases, including ERG+ PIA transitioning to early invasive cancer. We verified TMPRSS2:ERG genomic rearrangements in precursor lesions using tri-color fluorescence in situ hybridization. Identification of rearrangement patterns combined with whole prostate mapping in 3 dimensions confirmed multiple (up to 8) distinct ERG+ precancerous lesions in infected cases. Finally, we identified the pathogen-derived genotoxin colibactin as a potential source of DNA breaks in clinical cases as well as cultured prostate cells. Overall, we provide evidence that bacterial infections initiate driver gene alterations in prostate cancer. Furthermore, infection-induced ERG+ fusions are an early alteration in the carcinogenic process and PIA may serve as a direct precursor to prostate cancer.

Mitochondria regulate ATP production, metabolism and cell death. Alterations in mitochondrial DNA (mtDNA) sequence and copy number are implicated in aging and organ dysfunction in diverse inherited and sporadic diseases. Since most measurements of mtDNA use homogenates of complex tissues, little is known about cell type-specific mtDNA copy number heterogeneity in normal physiology, aging and disease. Thus, the precise cell types whose loss of mitochondrial activity and altered mtDNA copy number that result in organ dysfunction in aging and disease have often not been clarified. Here, we validated an in situ hybridization approach to generate a single cell resolution atlas of mtDNA content in mammalian tissues. In hierarchically organized self-renewing tissues, higher levels of mtDNA were observed in stem/proliferative compartments compared to differentiated compartments. Striking zonal patterns of mtDNA levels in the liver reflected the known oxygen tension gradient. In the kidney, proximal and distal tubules had markedly higher mtDNA levels compared to cells within glomeruli and collecting duct epithelial cells. Decreased mtDNA levels were visualized in renal tubules as a function of aging, which was prevented by calorie restriction. We provide a novel approach for quantifying species- and cell type-specific mtDNA copy number and dynamics in any normal or diseased tissue and can be used for monitoring the effects of interventions in animal and human studies.

Accurately measuring mitochondrial DNA copy number (mtDNAcn) is important for improving our understanding of cancer biology. Prior studies have revealed differences in mtDNAcn between matched bulk tumor-normal pairs from a range of different organs. However, there is little consistency across tumor types; compared to their normal tissue counterparts, some cancers appear to have increased mtDNAcn, others appear to have decreased mtDNAcn, and in others, such as prostate cancer, studies have shown contradictory results. The lack of consistency of mtDNAcn changes in tumor-normal pairs may reflect a failure to address the marked heterogeneity in mtDNAcn across different cell types within normal tissues. In addition, tumor cell heterogeneity in mtDNAcn has also not been taken into account in most prior studies, which can further confound tumor-normal comparisons. Finally, little is known about mtDNAcn alterations in cancer precursor lesions. To address these technical limitations, we combined a recently introduced quantitative in situ hybridization method with immunohistochemistry in a multiplex assay. This enabled us to measure mtDNAcn using digital image analysis in specific cell populations of interest on formalin fixed paraffin embedded (FFPE) and frozen tissue samples. We determined that normal prostate basal cells have approximately 3-fold higher mtDNA levels than normal prostate luminal cells. Compared to the adjacent normal prostate glands, we found markedly increased levels of mtDNA in high-grade prostatic intraepithelial neoplasia (HGPIN), the presumptive precursor lesions to most prostate cancers. We also found increased levels as well as heterogeneity of mtDNAcn in invasive prostate cancer lesions, and more consistently elevated levels in castration-resistant metastatic prostate cancer (CRPC). Increased mtDNA copy number was also identified by whole genome sequencing and quantitative PCR from laser capture micro-dissected human prostate tumor-normal pairs. We also observed higher mtDNAcn in two other precancer lesion types, pancreatic intraepithelial neoplasia and colorectal adenomas. Our approach represents a technological advance as it facilitates mtDNAcn measurements in a manner that preserves morphology, allowing for the evaluation of specific cell populations of interest. These findings raise the hypothesis that increased mitochondrial mass and function may drive the development of precancerous lesions in the prostate, pancreas and colon, as well as invasive prostate cancer development and progression. Citation Format: Jiayu Chen, Qizhi Zheng, Jessica L. Hicks, Levent Trabzonlu, Emmanuel S. Antonarakis, Mark C. Markowski, Samuel R. Denmeade, Busra Ozbek, Anuj Gupta, Tatianna C. Larman, Ralph H. Hruban, Sarah Wheelan, Srinivasan Yegnasubramanian, Angelo M. De Marzo. Increased mitochondrial DNA copy number occurs during prostate cancer progression and in cancer precursor lesions across multiple organs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2404.

Mitochondria are critical regulators of energy production, metabolism and cell death. Warburg observed that in the presence of oxygen, many cancer cells take up abundant amounts of glucose, and excrete the carbon mainly as lactate. Warburg postulated that this aerobic glycolysis was essential and universal in cancer, and was caused by mitochondrial dysfunction. Yet, more recent findings suggest that aerobic glycolysis also occurs in rapidly dividing normal cells to support anabolic reactions needed for cell growth and replication. Furthermore, a number of metabolites generated by the tricarboxylic acid cycle and the electron transport chain are also used for macromolecular synthesis in proliferating cancer and normal cells. This suggests that an increase in mitochondrial activity, especially those functions related to anabolic metabolism, may be required for cancer development and progression. One mechanism to increase mitochondrial function is through mitochondrial biogenesis, which is partly regulated by regulating mitochondrial DNA (mtDNA) copy number. Alterations in mtDNA sequence and copy number have been implicated in various cancers. Since most prior measurements of mtDNA copy number have been performed using homogenates of complex tissues composed of multiple cell types, very little is known about cell type-specific mtDNA copy number heterogeneity. Here, we validated a quantitative in situ hybridization approach to study mtDNA levels at the single cell level in tissue sections. In hierarchically organized self-renewing tissues, higher levels of mtDNA were observed in stem/proliferative compartments than in differentiated compartments. Also, we found zonal heterogeneity of mtDNA levels in the liver that mirrors the known oxygen tension gradient. In the kidney, we observed markedly lower mtDNA levels in glomeruli and the collecting ducts compared to cortical proximal and distal tubules. This work provides visualization of mtDNA copy number in distinct cell populations in situ for the first time across normal mammalian tissues, and can serve as a foundation for quantifying cell type-specific mtDNA copy number dynamics in any relevant diseases, especially in cancers and their precursor lesions. Citation Format: JIAYU CHEN, Qizhi Zheng, Lauren B. Peiffer, Jessica L. Hicks, Michael C. Haffner, Avi Z. Rosenberg, Moshe Levi, Xiaoxin Wang, Busra Ozbek, Srinivasan Yegnasubramanian, Angelo M. De Marzo. An in situ hybridization assay reveals high mitochondrial DNA copy number in stem cell/proliferative cell compartments [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4790.

Over the last few decades, fine needle aspiration cytology (FNA) has emerged as a SAFE (Simple, Accurate, Fast, Economical) diagnostic tool based on the morphologic evaluation of cells. The first and most important step in obtaining accurate results from FNA is to procure sufficient and representative material from the lesion and to appropriately transfer this material to the laboratory. Unfortunately, the most important aspect of this task occurs beyond the control of the cytopathologist, a key reason for obtaining unsatisfactory results with FNA. There is growing interest in the field of cytology in “cytopathologist-performed ultrasound (US)-guided FNA,” which has been reported to yield accurate results. The first author has been applying FNA in his own private cytopathology practice with a radiologist and under the guidance of US for more than 20 years. This study retrospectively reviews the utility of this practice. We present a selection of didactic examples under different headings that highlight the application of FNA by a cytopathologist, accompanied by US, under the guidance of a radiologist, in the form of an “outpatient FNA clinic.” The use of this technique enhances diagnostic accuracy and prevents pitfalls. The highlights of each case are also outlined as “take-home messages.”