Characterization of prostate macrophage heterogeneity, foam cell markers, and CXCL17 upregulation in a mouse model of steroid hormone imbalance.

Benign prostatic hyperplasia (BPH) is a prevalent age-related condition often characterized by debilitating urinary symptoms. Its etiology is believed to stem from hormonal imbalance, particularly an elevated estradiol-to-testosterone ratio and chronic inflammation. Our previous studies using a mouse steroid hormone imbalance model identified a specific increase in macrophages that migrated and accumulated in the prostate lumen where they differentiated into lipid-laden foam cells in mice implanted with testosterone and estradiol pellets, but not in sham animals. The current study focused on further characterizing the cellular heterogeneity of the prostate in this model as well as identifying the specific transcriptomic signature of the recruited foam cells. Moreover, we aimed to identify epithelia-derived signals that drive macrophage infiltration and luminal translocation. Male C57BL/6J mice were implanted with slow-release testosterone and estradiol pellets (T + E2) or sham surgery was performed and the ventral prostates were harvested two weeks later for scRNA-seq analysis. We identified Ear2 + and Cd72 + macrophages that were elevated in response to steroid hormone imbalance, whereas a Mrc1 + resident macrophage population did not change. In addition, an Spp1 + foam cell cluster was almost exclusively found in T + E2 mice. Further markers of foam cells were also identified, including Gpnmb and Trem2, and GPNMB was confirmed as a novel histological marker with immunohistochemistry. Foam cells were also shown to express known pathological factors Vegf, Tgfb1, Ccl6, Cxcl16 and Mmp12. Intriguingly, a screen for chemokines identified the upregulation of epithelia-derived Cxcl17, a known monocyte attractant, in T + E2 prostates suggesting that it might be responsible for the elevated macrophage number as well as their translocation to the lumen. Our study identified macrophage subsets that responded to steroid hormone imbalance as well as further confirmed a potential pathological role of luminal foam cells in the prostate. These results underscore a potential pathological role of the identified prostate foam cells and suggests CXCL17-mediated macrophage migration as a critical initiating event.

PROSTATE CELL HETEROGENEITY AND CXCL17 UPREGULATION IN MOUSE STEROID HORMONE IMBALANCE.

Benign prostatic hyperplasia (BPH) is a prevalent age-related condition often characterized by debilitating urinary symptoms. Its etiology is believed to stem from hormonal imbalance, particularly an elevated estradiol-to-testosterone ratio and chronic inflammation. Our previous studies using a mouse steroid hormone imbalance model identified a specific increase in macrophages that migrate and accumulate in the prostate lumen where they differentiate into lipid-laden foam cells in mice implanted with testosterone and estradiol pellets, but not in sham animals. The current study focused on further characterizing the cellular heterogeneity of the prostate in this model as well as identifying the specific transcriptomic signature of the recruited foam cells. Moreover, we aimed to identify the epithelia-derived signals that drive macrophage infiltration and luminal translocation. Male C57BL/6J mice were implanted with slow-release testosterone and estradiol pellets (T+E2) and harvested the ventral prostates two weeks later for scRNA-seq analysis, or performed sham surgery. We identified Ear2+ and Cd72+ macrophages that were elevated in response to steroid hormone imbalance, whereas a Mrc1+ resident macrophage population did not change. In addition, an Spp1+ foam cell cluster was almost exclusively found in T+E2 mice. Further markers of foam cells were also identified, including Gpnmb and Trem2, and GPNMB was confirmed as a novel histological marker with immunohistochemistry. Foam cells were also shown to express known pathological factors Vegf, Tgfb1, Ccl6, Cxcl16 and Mmp12. Intriguingly, a screen for chemokines identified the upregulation of epithelial-derived Cxcl17, a known monocyte attractant, in T+E2 prostates suggesting that it might be responsible for the elevated macrophage number as well as their translocation to the lumen. Our study identified macrophage subsets that respond to steroid hormone imbalance as well as further confirmed a potential pathological role of luminal foam cells in the prostate. These results underscore a pathological role of the identified prostate foam cells and suggests CXCL17-mediated macrophage migration as a critical initiating event.

miR-497 Target Gene Regulatory Network in Angiosarcoma.

Angiosarcoma is a vascular sarcoma that is highly aggressive and metastatic. Because of its rarity, treatment options for patients are limited. Therefore, more research is needed to identify possible therapeutic vulnerabilities. We previously found that conditional deletion of Dicer1 drives angiosarcoma development in mice. Given the role of DICER1 in canonical miRNA biogenesis, this suggests that miRNA loss is important in angiosarcoma development. After testing miRNAs previously suggested to have a tumor-suppressive role in angiosarcoma, miRNA-497-5p (miR-497) suppressed cell viability most significantly. We also found that miR-497 overexpression led to significantly reduced cell migration and tumor formation. To understand the mechanism of miR-497 in tumor suppression, we identified clinically relevant target genes using a combination of RNA-sequencing data in an angiosarcoma cell line, expression data from patients with angiosarcoma, and target prediction algorithms. We validated miR-497 direct regulation of cyclin-D2, cyclin-dependent kinase 6, and vesicle amine transport protein 1 (VAT1). One of these genes, VAT1, is an understudied protein that has been suggested to promote cell migration and metastasis in other cancers. Indeed, we find that pharmacologic inhibition of VAT1 with the natural product neocarzilin A reduces angiosarcoma migration. Implications: This work supports the potent tumor-suppressive abilities of miR-497 in angiosarcoma, providing evidence for its potential as a therapeutic agent, and provides insight into the mechanisms of tumor suppression through analysis of the target gene regulatory network of miR-497.

synNotch-programmed iPSC-derived NK cells usurp TIGIT and CD73 activities for glioblastoma therapy.

Severe heterogeneity within glioblastoma has spurred the notion that disrupting the interplay between multiple elements on immunosuppression is at the core of meaningful anti-tumor responses. T cell immunoreceptor with Ig and ITIM domains (TIGIT) and its glioblastoma-associated antigen, CD155, form a highly immunosuppressive axis in glioblastoma and other solid tumors, yet targeting of TIGIT, a functionally heterogeneous receptor on tumor-infiltrating immune cells, has largely been ineffective as monotherapy, suggesting that disruption of its inhibitory network might be necessary for measurable responses. It is within this context that we show that the usurpation of the TIGIT - CD155 axis via engineered synNotch-mediated activation of induced pluripotent stem cell-derived natural killer (NK) cells promotes transcription factor-mediated activation of a downstream signaling cascade that results in the controlled, localized blockade of CD73 to disrupt purinergic activity otherwise resulting in the production and accumulation of immunosuppressive extracellular adenosine. Such "decoy" receptor engages CD155 binding to TIGIT, but tilts inhibitory TIGIT/CD155 interactions toward activation via downstream synNotch signaling. Usurping activities of TIGIT and CD73 promotes the function of adoptively transferred NK cells into intracranial patient-derived models of glioblastoma and enhances their natural cytolytic functions against this tumor to result in complete tumor eradication. In addition, targeting both receptors, in turn, reprograms the glioblastoma microenvironment via the recruitment of T cells and the downregulation of M2 macrophages. This study demonstrates that TIGIT/CD155 and CD73 are targetable receptor partners in glioblastoma. Our data show that synNotch-engineered pluripotent stem cell-derived NK cells are not only effective mediators of anti-glioblastoma responses within the setting of CD73 and TIGIT/CD155 co-targeting, but represent a powerful allogeneic treatment option for this tumor.

TIGIT contributes to the regulation of 4-1BB and does not define NK cell dysfunction in glioblastoma.

TIGIT is a receptor on human natural killer (NK) cells. Here, we report that TIGIT does not spontaneously induce inhibition of NK cells in glioblastoma (GBM), but rather acts as a decoy-like receptor, by usurping binding partners and regulating expression of NK activating ligands and receptors. Our data show that in GBM patients, one of the underpinnings of unresponsiveness to TIGIT blockade is that by targeting TIGIT, NK cells do not lose an inhibitory signal, but gains the potential for new interactions with other, shared, TIGIT ligands. Therefore, TIGIT does not define NK cell dysfunction in GBM. Further, in GBM, TIGIT+ NK cells are hyperfunctional. In addition, we discovered that 4-1BB correlates with TIGIT expression, the agonism of which contributes to TIGIT immunotherapy. Overall, our data suggest that in GBM, TIGIT acts as a regulator of a complex network, and provide new clues about its use as an immunotherapeutic target.

Target gene regulatory network of miR-497 in angiosarcoma.

Angiosarcoma (AS) is a vascular sarcoma that is highly aggressive and metastatic. Due to its rarity, treatment options for patients are limited, therefore more research is needed to identify possible therapeutic vulnerabilities. We previously found that conditional deletion of Dicer1 drives AS development in mice. Given the role of DICER1 in canonical microRNA (miRNA) biogenesis, this suggests that miRNA loss is important in AS development. After testing miRNAs previously suggested to have a tumor-suppressive role in AS, microRNA-497-5p (miR-497) suppressed cell viability most significantly. We also found that miR-497 overexpression led to significantly reduced cell migration and tumor formation. To understand the mechanism of miR-497 in tumor suppression, we identified clinically relevant target genes using a combination of RNA-sequencing data in an AS cell line, expression data from AS patients, and target prediction algorithms. We validated miR-497 direct regulation of CCND2, CDK6, and VAT1. One of these genes, VAT1, is an understudied protein that has been suggested to promote cell migration and metastasis in other cancers. Indeed, we find that pharmacologic inhibition of VAT1 with the natural product Neocarzilin A reduces AS migration. This work provides insight into the mechanisms of miR-497 and its target genes in AS pathogenesis.

A first-in-class fully modified version of miR-34a with outstanding stability, activity, and anti-tumor efficacy.

Altered by defects in p53, epigenetic silencing, and genomic loss, the microRNA miR-34a represents one of the most clinically relevant tumor-suppressive microRNAs. Without question, a striking number of patients with cancer would benefit from miR-34a replacement, if poor miR-34a stability, non-specific delivery, and delivery-associated toxicity could be overcome. Here, we highlight a fully modified version of miR-34a (FM-miR-34a) that overcomes these hurdles when conjugated to a synthetically simplistic ligand. FM-miR-34a is orders of magnitude more stable than a partially modified version, without compromising its activity, leading to stronger repression of a greater number of miR-34a targets. FM-miR-34a potently inhibited proliferation and invasion, and induced sustained downregulation of endogenous target genes for >120 h following in vivo delivery. In vivo targeting was achieved through conjugating FM-miR-34a to folate (FM-FolamiR-34a), which inhibited tumor growth leading to complete cures in some mice. These results have the ability to revitalize miR-34a as an anti-cancer agent, providing a strong rationale for clinical testing.

cTULIP: application of a human-based RNA-seq primary tumor classification tool for cross-species primary tumor classification in canine.

Introduction: The domestic dog, Canis familiaris, is quickly gaining traction as an advantageous model for use in the study of cancer, one of the leading causes of death worldwide. Naturally occurring canine cancers share clinical, histological, and molecular characteristics with the corresponding human diseases. Methods: In this study, we take a deep-learning approach to test how similar the gene expression profile of canine glioma and bladder cancer (BLCA) tumors are to the corresponding human tumors. We likewise develop a tool for identifying misclassified or outlier samples in large canine oncological datasets, analogous to that which was developed for human datasets. Results: We test a number of machine learning algorithms and found that a convolutional neural network outperformed logistic regression and random forest approaches. We use a recently developed RNA-seq-based convolutional neural network, TULIP, to test the robustness of a human-data-trained primary tumor classification tool on cross-species primary tumor prediction. Our study ultimately highlights the molecular similarities between canine and human BLCA and glioma tumors, showing that protein-coding one-to-one homologs shared between humans and canines, are sufficient to distinguish between BLCA and gliomas. Discussion: The results of this study indicate that using protein-coding one-to-one homologs as the features in the input layer of TULIP performs good primary tumor prediction in both humans and canines. Furthermore, our analysis shows that our selected features also contain the majority of features with known clinical relevance in BLCA and gliomas. Our success in using a human-data-trained model for cross-species primary tumor prediction also sheds light on the conservation of oncological pathways in humans and canines, further underscoring the importance of the canine model system in the study of human disease.

Human prostate organoid generation and the identification of prostate development drivers using inductive rodent tissues.

The reactivation of developmental genes and pathways during adulthood may contribute to pathogenesis of diseases such as prostate cancer. Analysis of the mechanistic links between development and disease could be exploited to identify signalling pathways leading to disease in the prostate. However, the mechanisms underpinning prostate development require further characterisation to interrogate fully the link between development and disease. Previously, our group developed methods to produce prostate organoids using induced pluripotent stem cells (iPSCs). Here, we show that human iPSCs can be differentiated into prostate organoids using neonatal rat seminal vesicle mesenchyme in vitro. The organoids can be used to study prostate development or modified to study prostate cancer. We also elucidated molecular drivers of prostate induction through RNA-sequencing analyses of the rat urogenital sinus and neonatal seminal vesicles. We identified candidate drivers of prostate development evident in the inductive mesenchyme and epithelium involved with prostate specification. Our top candidates included Spx, Trib3, Snai1, Snai2, Nrg2 and Lrp4. This work lays the foundations for further interrogation of the reactivation of developmental genes in adulthood, leading to prostate disease.

Shared Inherited Genetics of Benign Prostatic Hyperplasia and Prostate Cancer.

Background: The association between benign prostatic hyperplasia (BPH) and prostate cancer (PCa) remains controversial, largely due to a detection bias in traditional observational studies. Objective: To assess the association between BPH and PCa using inherited single nucleotide polymorphisms (SNPs). Design setting and participants: The participants were White men from the population-based UK Biobank (UKB). Outcome measurements and statistical analysis: The association between BPH and PCa was tested for (1) phenotypic correlation using chi-square, (2) genetic correlation (r g) based on genome-wide SNPs using linkage disequilibrium score regression, and (3) cross-disease genetic associations based on known risk-associated SNPs (15 for BPH and 239 for PCa), individually and cumulatively using genetic risk score (GRS). Results and limitations: Among 214 717 White men in the UKB, 24 623 (11%) and 14 311 (6.7%) had a diagnosis of BPH and PCa, respectively. Diagnoses of these two diseases were significantly correlated (χ2 = 1862.80, p < 0.001). A significant genetic correlation was found (r g = 0.16; 95% confidence interval 0.03-0.28, p = 0.01). In addition, significant cross-disease genetic associations for established risk-associated SNPs were also found. Among the 250 established genome-wide association study-significant SNPs of PCa or BPH, 49 were significantly associated with the risk of the other disease at p < 0.05, significantly more than expected by chance (N = 12, p < 0.001; χ2 test). Furthermore, significant cross-disease GRS associations were also found; GRSBPH was significantly associated with PCa risk (odds ratio [OR] = 1.26 [1.18-1.36], p < 0.001), and GRSPCa was significantly associated with BPH risk (OR = 1.03 [1.02-1.04], p < 0.001). Moreover, GRSBPH was significantly and inversely associated with lethal PCa risk in a PCa case-case analysis (OR = 0.58 [0.41-0.81], p = 0.002). Only White men were studied. Conclusions: BPH and PCa share common inherited genetics, which suggests that the phenotypic association of these two diseases in observational studies is not entirely caused by the detection bias. Patient summary: For the first time, we found that benign prostatic hyperplasia and prostate cancer are genetically related. This finding may have implications in disease etiology and risk stratification.

1α,25-dihydroxyvitamin D reduction of MCF10A-ras cell viability in extracellular matrix detached conditions is dependent on regulation of pyruvate carboxylase.

An emerging hallmark of cancer is cellular metabolic reprogramming to adapt to varying cellular environments. Throughout the process of metastasis cancer cells gain anchorage independence which confers survival characteristics when detached from the extracellular matrix (ECM). Previous work demonstrates that the bioactive metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25[OH]2D), suppresses cancer progression, potentially by suppressing the ability of cells to metabolically adapt to varying cellular environments such as ECM detachment. The purpose of the present study was to determine the mechanistic bases of the effects of 1,25(OH)2D on cell survival in ECM-detached conditions. Pretreatment of MCF10A-ras breast cancer cells for 3 d with 1,25(OH)2D reduced the viability of cells in subsequent detached conditions by 11%. Enrichment of 13C5-glutamine was reduced in glutamate (21%), malate (30%), and aspartate (23%) in detached compared to attached MCF10A-ras cells. Pretreatment with 1,25(OH)2D further reduced glutamine flux into downstream metabolites glutamate (5%), malate (6%), and aspartate (10%) compared to detached vehicle treated cells. Compared to attached cells, detachment increased pyruvate carboxylase (PC) mRNA abundance and protein expression by 95% and 190%, respectively. Consistent with these results, 13C6-glucose derived M+3 labelling was shown to preferentially replenish malate and aspartate, but not citrate pools, demonstrating increased PC activity in detached cells. In contrast, 1,25(OH)2D pretreatment of detached cells reduced PC mRNA abundance and protein expression by 63% and 56%, respectively, and reduced PC activity as determined by decreased 13C6-glucose derived M+3 labeling in citrate (8%) and aspartate (50%) pools, relative to vehicle-treated detached cells. While depletion of PC with doxycycline-inducible shRNA reduced detached cell viability, PC knockdown in combination with 1,25(OH)2D treatment did not additionally affect the viability of detached cells. Further, PC overexpression improved detached cell viability, and inhibited the effect of 1,25(OH)2D on detached cell survival, suggesting that 1,25(OH)2D mediates its effects in detachment through regulation of PC expression. These results suggest that inhibition of PC by 1,25(OH)2D suppresses cancer cell anchorage independence.

GSTM2 is a key molecular determinant of resistance to SG-ARIs.

Prostate cancer (PCa) continues to threaten men's health, and treatment targeting the androgen receptor (AR) pathway is the major therapy for PCa patients. Several second-generation androgen receptor inhibitors (SG-ARIs), including enzalutamide (ENZ), apalutamide (APA) and darolutamide (DARO), have been developed to better block the activity of AR. Unavoidably, emergence of resistance to these novel drugs still persists. Herein, we identified glutathione S-transferase Mu 2 (GSTM2) as an important determinant in the acquisition of resistance to SG-ARIs. Elevated GSTM2 was detected in enzalutamide-resistant (ENZ-R) PCa, and overexpression of GSTM2 in naïve enzalutamide-sensitive (ENZ-S) cells effectively transformed them to ENZ-R PCa. Aryl hydrocarbon receptor (AhR), the upstream transcription factor, was implicated in the overexpression of GSTM2 in ENZ-R cells. Mechanistically, GSTM2 antagonized the effect of ENZ by rescuing cells from oxidative stress-associated damage and activation of p38 MAPK pathway. Surprisingly, high GSTM2 levels also associated with cross-resistance to APA and DARO. Taking together, these results provide new insight to ameliorate resistance to SG-ARIs and improve treatment outcome.

Weight Loss and/or Sulindac Mitigate Obesity-associated Transcriptome, Microbiome, and Protumor Effects in a Murine Model of Colon Cancer.

Obesity is associated with an increased risk of colon cancer. Our current study examines whether weight loss and/or treatment with the NSAID sulindac suppresses the protumor effects of obesity in a mouse model of colon cancer. Azoxymethane-treated male FVB/N mice were fed a low-fat diet (LFD) or high-fat diet (HFD) for 15 weeks, then HFD mice were randomized to remain on HFD (obese) or switch to LFD [formerly obese (FOb-LFD)]. Within the control (LFD), obese, and FOb-LFD groups, half the mice started sulindac treatment (140 ppm in the diet). All mice were euthanized 7 weeks later. FOb-LFD mice had intermediate body weight levels, lower than obese but higher than control (P < 0.05). Sulindac did not affect body weight. Obese mice had greater tumor multiplicity and burden than all other groups (P < 0.05). Transcriptomic profiling indicated that weight loss and sulindac each modulate the expression of tumor genes related to invasion and may promote a more antitumor immune landscape. Furthermore, the fecal microbes Coprobacillus, Prevotella, and Akkermansia muciniphila were positively correlated with tumor multiplicity and reduced by sulindac in obese mice. Coprobacillus abundance was also decreased in FOb-LFD mice. In sum, weight loss and sulindac treatment, alone and in combination, reversed the effects of chronic obesity on colon tumor multiplicity and burden. Our findings suggest that an investigation regarding the effects of NSAID treatment on colon cancer risk and/or progression in obese individuals is warranted, particularly for those unable to achieve moderate weight loss. PREVENTION RELEVANCE: Obesity is a colon cancer risk and/or progression factor, but the underlying mechanisms are incompletely understood. Herein we demonstrate that obesity enhances murine colon carcinogenesis and expression of numerous tumoral procancer and immunosuppressive pathways. Moreover, we establish that weight loss via LFD and/or the NSAID sulindac mitigate procancer effects of obesity.

A single local delivery of paclitaxel and nucleic acids via an immunoactive polymer eliminates tumors and induces antitumor immunity.

Despite recent advances in cancer therapy, hard-to-reach, unidentified tumors remain a significant clinical challenge. A promising approach is to treat locatable and accessible tumors locally and stimulate antitumor immunity in situ to exert systemic effects against distant tumors. We hypothesize that a carrier of immunotherapeutics can play a critical role in activating antitumor immunity as an immunoadjuvant and a local retainer of drug combinations. Here, we develop a polyethyleneimine-lithocholic acid conjugate (2E'), which forms a hydrophobic core and cationic surface to codeliver hydrophobic small molecules and anionic nucleic acids and activates antigen-presenting cells via the intrinsic activities of 2E' components. 2E' delivers paclitaxel and small-interfering RNA (siRNA) targeting PD-L1 (or cyclic dinucleotide, [CDN]) to induce the immunogenic death of tumor cells and maintain the immunoactive tumor microenvironment, and further activates dendritic cells and macrophages, leveraging the activities of loaded drugs. A single local administration of 2E' or its combination with paclitaxel and PD-L1­targeting siRNA or CDN induces strong antitumor immunity, resulting in immediate regression of large established tumors, tumor-free survival, an abscopal effect on distant tumors, and resistance to rechallenge and metastasis in multiple models of murine tumors, including CT26 colon carcinoma, B16F10 melanoma, and 4T1 breast cancer. This study supports the finding that local administration of immunotherapeutics, when accompanied by the rationally designed carrier, can effectively protect the host from distant and recurrent diseases.

TNF is a potential therapeutic target to suppress prostatic inflammation and hyperplasia in autoimmune disease.

Autoimmune (AI) diseases can affect many organs; however, the prostate has not been considered to be a primary target of these systemic inflammatory processes. Here, we utilize medical record data, patient samples, and in vivo models to evaluate the impact of inflammation, as seen in AI diseases, on prostate tissue. Human and mouse tissues are used to examine whether systemic targeting of inflammation limits prostatic inflammation and hyperplasia. Evaluation of 112,152 medical records indicates that benign prostatic hyperplasia (BPH) prevalence is significantly higher among patients with AI diseases. Furthermore, treating these patients with tumor necrosis factor (TNF)-antagonists significantly decreases BPH incidence. Single-cell RNA-seq and in vitro assays suggest that macrophage-derived TNF stimulates BPH-derived fibroblast proliferation. TNF blockade significantly reduces epithelial hyperplasia, NFκB activation, and macrophage-mediated inflammation within prostate tissues. Together, these studies show that patients with AI diseases have a heightened susceptibility to BPH and that reducing inflammation with a therapeutic agent can suppress BPH.

Loss of KMT5C Promotes EGFR Inhibitor Resistance in NSCLC via LINC01510-Mediated Upregulation of MET.

EGFR inhibitors (EGFRi) are standard-of-care treatments administered to patients with non-small cell lung cancer (NSCLC) that harbor EGFR alterations. However, development of resistance posttreatment remains a major challenge. Multiple mechanisms can promote survival of EGFRi-treated NSCLC cells, including secondary mutations in EGFR and activation of bypass tracks that circumvent the requirement for EGFR signaling. Nevertheless, the mechanisms involved in bypass signaling activation are understudied and require further elucidation. In this study, we identify that loss of an epigenetic factor, lysine methyltransferase 5C (KMT5C), drives resistance of NSCLC to multiple EGFRis, including erlotinib, gefitinib, afatinib, and osimertinib. KMT5C catalyzed trimethylation of histone H4 lysine 20 (H4K20), a modification required for gene repression and maintenance of heterochromatin. Loss of KMT5C led to upregulation of an oncogenic long noncoding RNA, LINC01510, that promoted transcription of the oncogene MET, a component of a major bypass mechanism involved in EGFRi resistance. These findings underscore the loss of KMT5C as a critical event in driving EGFRi resistance by promoting a LINC01510/MET axis, providing mechanistic insights that could help improve NSCLC treatment. SIGNIFICANCE: Dysregulation of the epigenetic modifier KMT5C can drive MET-mediated EGFRi resistance, implicating KMT5C loss as a putative biomarker of resistance and H4K20 methylation as a potential target in EGFRi-resistant lung cancer.

Genetic context of oncogenic drivers dictates vascular sarcoma development in aP2-Cre mice.

Angiosarcomas are aggressive vascular sarcomas that arise from endothelial cells and have an extremely poor prognosis. Because of the rarity of angiosarcomas, knowledge of molecular drivers and optimized treatment strategies is lacking, highlighting the need for in vivo models to study the disease. Previously, we generated genetically engineered mouse models of angiosarcoma driven by aP2-Cre-mediated biallelic loss of Dicer1 or conditional activation of KrasG12D with Cdkn2a loss that histologically and genetically resemble human tumors. In the present study, we found that DICER1 functions as a potent tumor suppressor and its deletion, in combination with either KRASG12D expression or Cdkn2a loss, is associated with angiosarcoma development. Independent of the genetic driver, the mTOR pathway was activated in all murine angiosarcoma models. Direct activation of the mTOR pathway by conditional deletion of Tsc1 with aP2-Cre resulted in tumors that resemble intermediate grade human kaposiform hemangioendotheliomas, indicating that mTOR activation was not sufficient to drive the malignant angiosarcoma phenotype. Genetic dissection of the spectrum of vascular tumors identified genes specifically regulated in the aggressive murine angiosarcomas that are also enriched in human angiosarcoma. The genetic dissection driving the transition across the malignant spectrum of endothelial sarcomas provides an opportunity to identify key determinants of the malignant phenotype, novel therapies for angiosarcoma, and novel in vivo models to further explore angiosarcoma pathogenesis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Inhibition of the erythropoietin-producing receptor EPHB4 antagonizes androgen receptor overexpression and reduces enzalutamide resistance.

Prostate cancer (PCa) cells heavily rely on an active androgen receptor (AR) pathway for their survival. Enzalutamide (MDV3100) is a second-generation antiandrogenic drug that was approved by the Food and Drug Administration in 2012 to treat patients with castration-resistant prostate cancer (CRPC). However, emergence of resistance against this drug is inevitable, and it has been a major challenge to develop interventions that help manage enzalutamide-resistant CRPC. Erythropoietin-producing human hepatocellular (Eph) receptors are targeted by ephrin protein ligands and have a broad range of functions. Increasing evidence indicates that this signaling pathway plays an important role in tumorigenesis. Overexpression of EPH receptor B4 (EPHB4) has been observed in multiple types of cancer, being closely associated with proliferation, invasion, and metastasis of tumors. Here, using RNA-Seq analyses of clinical and preclinical samples, along with several biochemical and molecular methods, we report that enzalutamide-resistant PCa requires an active EPHB4 pathway that supports drug resistance of this tumor type. Using a small kinase inhibitor and RNAi-based gene silencing to disrupt EPHB4 activity, we found that these disruptions re-sensitize enzalutamide-resistant PCa to the drug both in vitro and in vivo Mechanistically, we found that EPHB4 stimulates the AR by inducing proto-oncogene c-Myc (c-Myc) expression. Taken together, these results provide critical insight into the mechanism of enzalutamide resistance in PCa, potentially offering a therapeutic avenue for enhancing the efficacy of enzalutamide to better manage this common malignancy.

Oct4-Mediated Inhibition of Lsd1 Activity Promotes the Active and Primed State of Pluripotency Enhancers.

An aberrant increase in pluripotency gene (PpG) expression due to enhancer reactivation could induce stemness and enhance the tumorigenicity of cancer stem cells. Silencing of PpG enhancers (PpGe) during embryonic stem cell differentiation involves Lsd1-mediated H3K4me1 demethylation and DNA methylation. Here, we observed retention of H3K4me1 and DNA hypomethylation at PpGe associated with a partial repression of PpGs in F9 embryonal carcinoma cells (ECCs) post-differentiation. H3K4me1 demethylation in F9 ECCs could not be rescued by Lsd1 overexpression. Given our observation that H3K4me1 demethylation is accompanied by strong Oct4 repression in P19 ECCs, we tested if Oct4 interaction with Lsd1 affects its catalytic activity. Our data show a dose-dependent inhibition of Lsd1 activity by Oct4 and retention of H3K4me1 at PpGe in Oct4-overexpressing P19 ECCs. These data suggest that Lsd1-Oct4 interaction in cancer stem cells could establish a "primed" enhancer state that is susceptible to reactivation, leading to aberrant PpG expression.

Heterogeneity of human prostate carcinoma-associated fibroblasts implicates a role for subpopulations in myeloid cell recruitment.

BACKGROUND: Carcinoma-associated fibroblasts (CAF) are a heterogeneous group of cells within the tumor microenvironment (TME) that can promote tumorigenesis in the prostate. By understanding the mechanism(s) by which CAF contributes to tumor growth, new therapeutic targets for the management of this disease may be identified. These studies determined whether unique sub-populations of human prostate CAF can be identified and functionally characterized. METHODS: Single-cell RNA-seq of primary human prostate CAF followed by unsupervised clustering was utilized to generate cell clusters based on differentially expressed (DE) gene profiles. Potential communication between CAF and immune cells was analyzed using in vivo tissue recombination by combining CAF or normal prostate fibroblasts (NPF) with non-tumorigenic, initiated prostate epithelial BPH-1 cells. Resultant grafts were assessed for inflammatory cell recruitment. RESULTS: Clustering of 3321 CAF allows for visualization of six subpopulations, demonstrating heterogeneity within CAF. Sub-renal capsule recombination assays show that the presence of CAF significantly increases myeloid cell recruitment to resultant tumors. This is supported by significantly increased expression of chemotactic chemokines CCL2 and CXCL12 in large clusters compared to other subpopulations. Bayesian analysis topologies also support differential communication signals between chemokine-related genes of individual clusters. Migration of THP-1 monocyte cells in vitro is stimulated in the presence of CAF conditioned medium (CM) compared with NPF CM. Further in vitro analyses suggest that CAF-derived chemokine CCL2 may be responsible for CAF-stimulated migration of THP-1 cells, since neutralization of this chemokine abrogates migration capacity. CONCLUSIONS: CAF clustering based on DE gene expression supports the concept that clusters have unique functions within the TME, including a role in immune/inflammatory cell recruitment. These data suggest that CCL2 produced by CAF may be involved in the recruitment of inflammatory cells, but may also directly regulate the growth of the tumor. Further studies aimed at characterizing the subpopulation(s) of CAF which promote immune cell recruitment to the TME and/or stimulate prostate cancer growth and progression will be pursued.