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Supplementary Figure S1. Generation of ENZR Xenografts and Cell Lines. Supplementary Figure S2. AR non-driven ENZR cells display a NE differentiation signature. Supplementary Figure S3. BRN2 expression is associated with PCa progression and a NE phenotype in human tumors. Supplementary Figure S4. BRN2 expression is inversely related to AR activity and is required for ENZ-induced NE marker expression in CRPC. Supplementary Figure S5. BRN2 overexpression induces NE marker expression and reciprocally regulates AR. Supplementary Figure S6. BRN2 activity and markers of NE differentiation are suppressed by androgens in CRPC and ENZR cells. Supplementary Figure S7. SOX2 expression is induced by ENZ and suppressed by androgens. Supplementary Figure S8. BRN2 is required for neuroendocrine marker expression and aggressive growth of CRPC cells in vitro.

Supplementary methods, figure legends, and primer lists.

RNAseq of ENZ-resistant cells.

NEPC signature for GSEA analysis.

Gene Profiling data of CRPC cells with BRN2 over-expression.

Supplementary Table S1. Summary of ENZ resistant cells' characterization

Supplementary Table S2. Cox regression analyses of FOXM1 as a prognostic factor for patients

Supplementary Table S3. Up and downregulated genes in response to Mon in 42DENZR cells assessed by gene expression profiling.

Supplementary Figure S1. GSEA analyses of subtype-specific genes (PCS1, PCS2 and PCS3) in 42DENZR and 42FENZR cells. Supplementary Figure S2. A) Expression of FOXM1 in different cell lines. Total proteins were extracted from LNCaP, 16DCRPC and ENZR 42D and 42F cells and western blot was performed using FOXM1 antibody (Abcam, 1:1000). Vinculin (1:5000) was used as a loading control. FOXM1 band densitometry was normalized to vinculin. B) Upstream regulator analysis of FOXM1 in PCS1 signature and 42DENZR and 42FENZR cells assessed by IPA (red: target gene overexpression, green: target gene downregulation, orange arrow: target gene is known as induced and blue arrow: target gene known as reduced by pathway activation). C) Expression of FOXM1 pathway expression in metastatic prostate cancer patients analysed using Genesapiens database (20). Supplementary Figure S3. A) Effect of Mon and Thiostrepton effect on FOXM1 transcriptional activity, 42DENZR cells were treated with 100 nM of Mon and Thiostrepton for 24 hours and FOXM1 luciferase activity was performed. B) Monensin reduces FOXM1 protein level. Monensin effect (0, 1, 10, 50 and 100 nM) on FOXM1 protein was addressed using Western blot in 42DENZR cells. Total protein were extracted and western blot was performed using FOXM1 antibody (1:1000). Vinculin (1:5000) was used as a loading control. C) Effect of Mon on cell cycle. Flow cytometry was performed to analyse cell cycle population after Mon treatment for 0 to 72h, percentage of sub-G1 cells in response to Mon in 16DCRPC, 42DENZR and 42FENZR cells was evaluated. D) DARTS assay in presence and absence of Mon in pronase treated samples indicated Mon binding to FOXM1. GAPDH was used as a control. E) Analysis of the effect of Mon on FOXM1 pathway by IPA Upstream regulator analysis of Mon exposed 42DENZR cells (green: target gene downregulation, blue arrow: target gene known as reduced by pathway inactivation). Supplementary Figure S4. Portion of high CD49b (CD49b+/CD24-) cells in 16DENZR, 42DENZR and 42FENZR cells. B) Effect of Mon and C) FOXM1 silencing by FOXM1 siRNA on portion of CD49b+/CD24- cells. Supplementary Figure S5. The effect of FOXM1 inhibition on ALDH activity. A) Portion of ALDH active cells in 16DENZR, 42DENZR and 42FENZR cells. B) The effect of Mon and C) FOXM1 silencing on ALDH activity in 42DENZR and 42FENZR cells. D) Cell proliferation of ALDHHigh and ALDHLow cells (separated from 42DENZR and 42FENZR cells using FACS and Aldefluor ALDH assay) in response to Mon measured by CTG cell proliferation assay. E) FOXM1 protein expression in 2 ALDHHigh and ALDHLow 42DENZR and 42FENZR cells assessed by western blot of FOXM1 and vinculin (loading control). Supplementary Figure S6. A) Mouse body weight (g) in response to Vehicle and Monensin (Mo) treatments for 3.5 weeks. B) Effect of Mon on ALDH activity in 42DENZR tumor xenografts in vivo assessed by Aldefluor ALDH assay and FACS. Supplementary Figure S7. A) The expression of PCS1 signature is enriched in CRPCNeuroendocrine patients. Fold changes of genes in PCS1 signature in CRPC-NE compared to CRPC-Adeno prostate cancer patients are presented as a heatmap. B) The expression of PCS1 signature is enriched in Trp53 and Pten (NPp53 mice) that fail to respond to abiraterone and differentiate to neuroendocrine prostate cancer. Fold changes of genes in PCS1 signature in NPp53 abiraterone-"exceptional non-responders" compared to NPp53 vehicle are presented as a Heatmap. C) PCS1 signature in wild type (WT), single (SKO, (PBCre4:Pten f/f :Rb1 f/+ )), double (DKO, PBCre4:Pten f/f :Rb1 f/f ) and triple knock out (TKO, PBCre4:Pten f/f :Rb1 f/f :Trp53 f/f ) mice . Fold changes of genes in PCS1 signature in SKO, DKO and TKO mice compared to WT mice are presented as a heatmap.

Purpose: Prostate cancer was recently classified to three clinically relevant subtypes (PCS) demarcated by unique pathway activation and clinical aggressiveness. In this preclinical study, we investigated molecular targets and therapeutics for PCS1, the most aggressive and lethal subtype, with no treatment options available in the clinic.Experimental Design: We utilized the PCS1 gene set and our model of enzalutamide (ENZR) castration-resistant prostate cancer (CRPC) to identify targetable pathways and inhibitors for PCS1. The findings were evaluated in vitro and in the ENZR CRPC xenograft model in vivo.Results: The results revealed that ENZR CRPC cells are enriched with PCS1 signature and that Forkhead box M1 (FOXM1) pathway is the central driver of this subtype. Notably, we identified Monensin as a novel FOXM1-binding agent that selectively targets FOXM1 to reverse the PCS1 signature and its associated stem-like features and reduces the growth of ENZR CRPC cells and xenograft tumors.Conclusions: Our preclinical data indicate FOXM1 pathway as a master regulator of PCS1 tumors, namely in ENZR CRPC, and targeting FOXM1 reduces cell growth and stemness in ENZR CRPC in vitro and in vivo. These preclinical results may guide clinical evaluation of targeting FOXM1 to eradicate highly aggressive and lethal PCS1 prostate cancer tumors. Clin Cancer Res; 23(22); 6923–33. ©2017 AACR.

PDF file - 48K, Hsp27 knockdown reduces cell invasion through matrigel.

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PDF file - 61K, Hsp27 targeting siRNA has similar effects on EMT as stable Hsp27 knockdown.

PDF file - 425K, Hsp27 knockdown decreases cell migration in PC-3M cells.

PDF file - 47K, Effect of OGX-427 on circulating tumor cells.

PDF file - 70K, Hsp27 knockdown reduces cell invasion and MMP activity.

Cancers adapt to increasingly potent targeted therapies by reprogramming their phenotype. Here we investigated such a phenomenon in prostate cancer, in which tumours can escape epithelial lineage confinement and transition to a high-plasticity state as an adaptive response to potent androgen receptor (AR) antagonism. We found that AR activity can be maintained as tumours adopt alternative lineage identities, with changes in chromatin architecture guiding AR transcriptional rerouting. The epigenetic regulator enhancer of zeste homologue 2 (EZH2) co-occupies the reprogrammed AR cistrome to transcriptionally modulate stem cell and neuronal gene networks-granting privileges associated with both fates. This function of EZH2 was associated with T350 phosphorylation and establishment of a non-canonical polycomb subcomplex. Our study provides mechanistic insights into the plasticity of the lineage-infidelity state governed by AR reprogramming that enabled us to redirect cell fate by modulating EZH2 and AR, highlighting the clinical potential of reversing resistance phenotypes.

IL-12 is a pleiotropic cytokine that potently stimulates anti-tumor cytotoxic T and NK cell mediated immunity. Recombinant IL-12 reduces tumor growth in multiple mouse models, but its therapeutic application has been limited by severe toxicities. Protease dependent activation of therapeutics with high on-target, off-tumor toxicities may be used to localize activity to the tumor but achieving sufficient exposure of the activated therapeutic in the tumor microenvironment remains a challenge. We have previously shown our design strategy for masked, protease activated IL-12Fc that included optimization of blocking modules, fusion geometry, and linker design. Here we have further expanded our mechanistic evaluations by combining masked, protease activated IL-12Fc with attenuated potency engineering, and selected a lead candidate (ZW270) based on anti-tumor activity and non-human primate (NHP) tolerability. The relative activity of masked and unmasked IL-12Fc variants comprising wild type (wt) or attenuated (att) IL-12 potency was evaluated in CD8 T cell activity assays. The wt IL-12Fc displayed comparable potency to recombinant IL-12 and up to 100x reduced potency when masked. Introduction of attenuating mutations to the p40 subunit reduced the potency of the IL-12Fc by up to 20x and addition of the protease cleavable mask further reduced the potency to up to 5000x compared to wt IL-12Fc. In a single dose NHP study, masked att IL-12Fc was well tolerated up to 31.8 mg/kg, while wt IL-12Fc demonstrated a maximum tolerated dose of below 1.3 mg/kg. Despite the reduced potency of att IL-12Fc in vitro, in in vivo efficacy studies in a human PBMC engrafted xenograft model the masked att IL-12Fc was able to control tumor growth whereas masked wt IL-12Fc and wt IL-12Fc showed limited anti-tumor activity, suggesting the approach of masked, protease activated att IL-12Fc might be able to achieve a higher exposure of active cytokine in the tumor in comparison to the masked wt IL-12Fc and wt IL-12 comparator. To further investigate the potentially superior exposure response relationship of masked att IL-12Fc, we developed a quantitative systems pharmacology (QSP) model based on our experimental data and literature data. In this model the predicted ratio of IL-12-receptor occupancy by active IL-12Fc in the tumor vs. blood was 18x greater for masked att IL-12Fc than for wt IL-12Fc, whereas the ratio was under 10x greater for masked wt IL-12Fc vs. wt IL-12Fc. In summary, the affinity attenuated, masked IL-12Fc lead ZW270 has potent and superior anti-tumor activity to wt IL-12Fc and masked wt IL-12Fc, and is well tolerated in NHPs to >30 mg/kg. Our data suggests that the combined strategy of masked, protease activated IL-12Fc and attenuated IL-12 potency has the potential to widen the therapeutic index and to have superior activity to masked, protease cleavable wt IL-12 and unmasked wt IL-12 fusions. Citation Format: Maya C. Poffenberger, Jennifer L. Bishop, Ryan J. Blackler, Kevin G. Haworth, Steven Booth, Shalla Hanson, Jeff R. Proctor, I-Ting Shao, Nichole K. Escalante, Dayananda Siddappa, Joel Smith, Gursev Anmole, Saki Konomura, Nicholas A. Dawson, Sifa Arrafi, Desmond Lau, Gerry Rowse, Rupert H. Davies, Thomas Spreter von Kreudenstein. ZW270, a conditionally masked IL-12 cytokine fusion protein displaying potent anti-tumor activity absent systemic toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2935.

Given the large body of evidence linking attachment insecurity to psychological distress (e.g., depression) in the transition from adolescence to emerging adulthood and across the lifespan, there is a need to better understand how attachment dimensions (e.g., anxious, avoidant) influence depressive symptoms during this developmental period. Based on limited findings that difficulties with role balance may uniquely contribute to depression among college students (Lopez and Fons-Scheyd in J Coll Couns 11:133–147, 2008; Marks and McDermid in J Marriage Family 58:417–432, 1996) and may be associated with individual factors such as internal working models of attachment, this study tested the indirect effects of anxious and avoidant attachment on depression through role balance using a college student sample (n = 299). Structural equation modeling indicated that role balance partially mediated the relationship between attachment anxiety and depression. Results of this study suggest that difficulty in balancing individual (i.e., autonomy-focused) and relational (i.e., intimacy-focused) role commitments might help to explain depressive symptoms above and beyond insecure attachment for anxiously attached emerging adults and serve as a mechanism by which anxious attachment influences depressive symptoms during this developmental period. Implications for theory and practice are discussed.

Purpose: Prostate cancer was recently classified to three clinically relevant subtypes (PCS) demarcated by unique pathway activation and clinical aggressiveness. In this preclinical study, we investigated molecular targets and therapeutics for PCS1, the most aggressive and lethal subtype, with no treatment options available in the clinic. Experimental Design: We utilized the PCS1 gene set and our model of enzalutamide (ENZR) castration-resistant prostate cancer (CRPC) to identify targetable pathways and inhibitors for PCS1. The findings were evaluated in vitro and in the ENZR CRPC xenograft model in vivo. Results: The results revealed that ENZR CRPC cells are enriched with PCS1 signature and that Forkhead box M1 (FOXM1) pathway is the central driver of this subtype. Notably, we identified Monensin as a novel FOXM1-binding agent that selectively targets FOXM1 to reverse the PCS1 signature and its associated stem-like features and reduces the growth of ENZR CRPC cells and xenograft tumors. Conclusions: Our preclinical data indicate FOXM1 pathway as a master regulator of PCS1 tumors, namely in ENZR CRPC, and targeting FOXM1 reduces cell growth and stemness in ENZR CRPC in vitro and in vivo. These preclinical results may guide clinical evaluation of targeting FOXM1 to eradicate highly aggressive and lethal PCS1 prostate cancer tumors. Clin Cancer Res; 23(22); 6923–33. ©2017 AACR.

While the relationship between individual factors, including personality dimensions, and tertiary academic outcomes is well established, the mechanisms by which these factors influence academic success have been less fully explored. This study tested one potential mechanism, the developmental task of role balance, by which personality may influence college success. During emerging adulthood, young people simultaneously explore and establish both individual and relational role identities. As part of this process, these emerging adults work to balance their individual roles with their relational roles in order to maximize success and satisfaction across domains. Using a college student sample ( N = 299), this study used Structural Equation Modeling (SEM) to test role balance as a mediator between personality dimensions and multiple indicators of college success. Tests indicated that role balance partially mediates the relationship between conscientiousness and neuroticism and college satisfaction but is not associated with other traditional measures of college achievement (grade point average, intention to withdraw).

The acquisition of an invasive phenotype by epithelial cells occurs through a loss of cellular adhesion and polarity, heralding a multistep process that leads to metastatic dissemination. Since its characterization in 1995, epithelial-mesenchymal transition (EMT) has been closely linked to the metastatic process. As a defining aspect of EMT, loss of cell adhesion through downregulation of E-cadherin is carried out by several transcriptional repressors; key among them the SNAI family of transcription factors. Here we identify for the first time that Lyn kinase functions as a key modulator of SNAI family protein localization and stability through control of the Vav-Rac1-PAK1 (Vav-Rac1-p21-activated kinase) pathway. Accordingly, targeting Lyn in vitro reduces EMT and in vivo reduces metastasis of primary tumors. We also demonstrate the clinical relevance of targeting Lyn as a key player controlling EMT; patient samples across many cancers revealed a strong negative correlation between Lyn and E-cadherin, and high Lyn expression in metastatic tumors as well as metastasis-prone primary tumors. This work reveals a novel pancancer mechanism of Lyn-dependent control of EMT and further underscores the role of this kinase in tumor progression.

Mechanisms controlling the emergence of lethal neuroendocrine prostate cancer (NEPC), especially those that are consequences of treatment-induced suppression of the androgen receptor (AR), remain elusive. Using a unique model of AR pathway inhibitor–resistant prostate cancer, we identified AR-dependent control of the neural transcription factor BRN2 (encoded by POU3F2) as a major driver of NEPC and aggressive tumor growth, both in vitro and in vivo. Mechanistic studies showed that AR directly suppresses BRN2 transcription, which is required for NEPC, and BRN2-dependent regulation of the NEPC marker SOX2. Underscoring its inverse correlation with classic AR activity in clinical samples, BRN2 expression was highest in NEPC tumors and was significantly increased in castration-resistant prostate cancer compared with adenocarcinoma, especially in patients with low serum PSA. These data reveal a novel mechanism of AR-dependent control of NEPC and suggest that targeting BRN2 is a strategy to treat or prevent neuroendocrine differentiation in prostate tumors.Significance: Understanding the contribution of the AR to the emergence of highly lethal, drug-resistant NEPC is critical for better implementation of current standard-of-care therapies and novel drug design. Our first-in-field data underscore the consequences of potent AR inhibition in prostate tumors, revealing a novel mechanism of AR-dependent control of neuroendocrine differentiation, and uncover BRN2 as a potential therapeutic target to prevent emergence of NEPC. Cancer Discov; 7(1); 54–71. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1

Clusterin (CLU) is a stress‐activated molecular chaperone that confers treatment resistance to taxanes when highly expressed. While CLU inhibition potentiates activity of taxanes and other anti‐cancer therapies in preclinical models, progression to treatment‐resistant disease still occurs implicating additional compensatory survival mechanisms. Taxanes are believed to selectively target cells in mitosis, a complex mechanism controlled in part by balancing antagonistic roles of Cdc25C and Wee1 in mitosis progression. Our data indicate that CLU silencing induces a constitutive activation of Cdc25C, which delays mitotic exit and hence sensitizes cancer cells to mitotic‐targeting agents such as taxanes. Unchecked Cdc25C activation leads to mitotic catastrophe and cell death unless cells up‐regulate protective mechanisms mediated through the cell cycle regulators Wee1 and Cdk1. In this study, we show that CLU silencing induces a constitutive activation of Cdc25C via the phosphatase PP2A leading to relief of negative feedback inhibition and activation of Wee1‐Cdk1 to promote survival and limit therapeutic efficacy. Simultaneous inhibition of CLU‐regulated cell cycle effector Wee1 may improve synergistic responses of biologically rational combinatorial regimens using taxanes and CLU inhibitors.

Most prostate cancer patients will progress to a castration-resistant state (CRPC) after androgen ablation therapy and despite the development of new potent anti-androgens, like enzalutamide (ENZ), which prolong survival in CRPC, ENZ-resistance (ENZR) rapidly occurs. Re-activation of the androgen receptor (AR) is a major mechanism of resistance. Interrogating our in vivo derived ENZR model, we discovered that transcription factor STAT3 not only displayed increased nuclear localization but also bound to and facilitated AR activity. We observed increased STAT3 S727 phosphorylation in ENZR cells, which has been previously reported to facilitate AR binding. Strikingly, ENZR cells were more sensitive to inhibition with STAT3 DNA-binding inhibitor galiellalactone (GPA500) compared to CRPC cells. Treatment with GPA500 suppressed AR activity and significantly reduced expression of Cyclin D1, thus reducing cell cycle progression into S phase and hindering cell proliferation. In vivo, GPA500 reduced tumor volume and serum PSA in ENZR xenografts. Lastly, the combination of ENZ and GPA500 was additive in the inhibition of AR activity and proliferation in LNCaP and CRPC cells, providing rationale for combination therapy. Overall, these results suggest that STAT3 inhibition is a rational therapeutic approach for ENZR prostate cancer, and could be valuable in CRPC in combination with ENZ.

Compression apparel is popular in both medical and sport performance settings. Perceived benefits are suggested to include changes in sensory feedback transmission caused by activation of mechanoreceptors. However, little is known about effects of compression apparel on sensorimotor control. Our purpose was to mechanistically examine whether compression apparel modulates sensory feedback transmission and reaching accuracy in the upper limb. Two experiments were completed under CONTROL and COMPRESSION (sleeve applied across the elbow joint) conditions. M-waves and H-reflexes were elicited by stimulating the median nerve and were recorded via surface electromyography (EMG). In experiment 1, H-reflexes and M-H recruitment curves were assessed at REST, during wrist flexion (10% EMGmax), and during a cutaneous conditioning of the superficial radial (SR) or distal median (MED) nerve. Cutaneous reflexes were elicited during 10% wrist flexion via stimulation of SR or MED. In experiment 2, unconditioned H-reflex measures were assessed at rest, during arm cycling, and during a discrete reaching task. Results indicate that compression apparel modulates spinal cord excitability across multiple sensory pathways and movement tasks. Interestingly, there was a significant improvement in reaching accuracy while wearing the compression sleeve. Taken together, the compression sleeve appears to increase precision and sensitivity around the joint where the sleeve is applied. Compression apparel may function as a “filter” of irrelevant mechanoreceptor information allowing for optimal task-related sensory information to enhance proprioception. NEW & NOTEWORTHY Wearing a customized compression sleeve was shown to alter the excitability of multiple pathways within the central nervous system regardless of conditioning input or movement task and was accompanied by improved accuracy of reaching movements and determination of movement end point. Compression apparel may assist as a type of “filter function” of tonic and nonspecific mechanoreceptor information leading to increased precision and movement sensitivity around the joint where compression is applied.

Prostate cancer (PCa) has become the most common form of cancer in men in the developed world, and it ranks second in cancer-related deaths. Men that succumb to PCa have a disease that is resistant to hormonal therapies that suppress androgen receptor (AR) signaling, which plays a central role in tumor development and progression. Although AR continues to be a clinically relevant therapeutic target in PCa, selection pressures imposed by androgen-deprivation therapies promote the emergence of heterogeneous cell populations within tumors that dictate the severity of disease. This cellular plasticity, which is induced by androgen deprivation, is the focus of this review. More specifically, we address the emergence of cancer stem-like cells, epithelial–mesenchymal or myeloid plasticity, and neuroendocrine transdifferentiation as well as evidence that demonstrates how each is regulated by the AR. Importantly, because all of these cell phenotypes are associated with aggressive PCa, we examine novel therapeutic approaches for targeting therapy-induced cellular plasticity as a way of preventing PCa progression.

Men with advanced prostate cancer are typically treated with hormonal therapy, which results in tumor shrinkage. However, tumors relapse and develop into a highly aggressive and lethal form of the disease, termed castration-resistant prostate cancer (CRPC). Evidence suggests that tumor cells acquire new genetic and epigenetic alterations that enable them to survive in the castrated state. Yet, it has recently emerged that immune cells in the tumor microenvironment and a population of rare, pre-existing cancer stem cells can also facilitate the outgrowth of CRPC following hormonal therapy. Targeting the cells that survive therapy is principal to prevent the development of CRPC. This chapter will describe the molecular identity of CRPC and the cellular mechanisms employed by prostate cancer cells that drive progression to this lethal phase of prostate cancer. The treatment of CRPC is reviewed, with a focus on emerging therapeutic strategies targeting critical pathways in castration-resistant cancer cells.

Increased expression of the molecular chaperone Hsp27 is associated with the progression of prostate cancer (PCa) to castration-resistant disease, which is lethal due to metastatic spread of the prostate tumor. Metastasis requires epithelial to mesenchymal transition (EMT), which endows cancer cells with the ability to disseminate from the primary tumor and colonize new tissue sites. A wide variety of secreted factors promote EMT, and while overexpression and constitutive activation of epidermal growth factor (EGF) signaling is associated with poor prognosis of PCa, a precise role of EGF in PCa progression to metastasis remains unclear. Here, we show that Hsp27 is required for EGF-induced cell migration, invasion and MMPs activity as well as the expression of EMT markers including Fibronectin, Vimentin and Slug with concomitant decrease of E-cadherin. Mechanistically, we found that Hsp27 is required for EGF-induced AKT and GSK3β phosphorylation and β-catenin nuclear translocation. Moreover, silencing Hsp27 decreases EGF dependent phosphorylation of β-catenin on tyrosine 142 and 654, enhances β-catenin ubiquitination and degradation, prevents β-catenin nuclear translocation and binding to the Slug promoter. These data suggest that Hsp27 is required for EGF-mediated EMT via modulation of the β-catenin/Slug signaling pathway. Together, our findings underscore the importance of Hsp27 in EGF induced EMT in PCa and highlight the use of Hsp27 knockdown as a useful strategy for patients with advanced disease.

Ankle injuries are common. These injuries are often the result of excessive inversion at the subtalar joint. Previous research measuring shoe motion suggests that inversion at the ankle during cutting is as high as 40 degrees. However, this does not seem possible without injury. Some researchers have cut holes in shoes to view foot motion directly; however, this compromises the integrity of the shoe and potentially changes ankle kinematics. During barefoot cutting, ankle inversion has been measured directly showing that there is little or no ankle inversion. It is not known how footwear affects ankle inversion inside the shoe during cutting. The purpose of this study was to determine the effect of footwear on ankle inversion during aggressive lateral cutting without compromising shoe integrity. This was done using a dynamic x-ray imaging technique to measure ankle and shoe inversion while barefoot and shod. It was hypothesised that during aggressive cutting: 1) maximum ankle inversion would be the same in...

Ankle strains and sprains are one of the most common basketball injuries. These injuries are often the result of excessive ankle inversion. High-top shoes are worn because it is thought that they limit inversion and, therefore, reduce the risk of inversion injuries. However, it is difficult to quantify inversion of the ankle inside the shoe with traditional measurement techniques, making comparison between footwear conditions difficult. The purpose of this study was to compare inversion of the ankle and shoe in a high-top and a low-top shoe during aggressive lateral cutting. It was hypothesised that ankle inversion would be lower than shoe inversion in both a high-top shoe and a low-top shoe. Six male basketball players were analysed over three dynamic cutting trials in each condition. As hypothesised, shoe inversion during aggressive cutting was higher than ankle inversion in the high-top shoe (17°) and the low-top shoe (25.5°). Future research should focus on determining the relative contribution of ank...

Resistance to antiandrogen drugs, like MDV3100, occurs in patients with castration-resistant prostate cancer (CRPC). Thus, preventing or treating antiandrogen resistance is a major clinical challenge. We identified a novel antiandrogen, Compound 30, and compared its efficacy with MDV3100. We found that Compound 30 inhibits androgen receptor (AR) activity in LNCaP cells, C4-2 cells, as well as MDV3100-resistant cell lines. Compared with MDV3100, Compound 30 treatment induces greater reduction in AR, prostate-specific antigen (PSA), and AR transcriptional activity, and prevents AR nuclear translocation in AR-sensitive LNCaP cells. Compound 30 has antiproliferative effects in LNCaP cells, in castrate-resistant C4-2 cells, and those resistant to MDV3100. Compound 30 was equally as effective as MDV3100 in reducing tumor volume and PSA in vivo. More importantly, Compound 30 is effective at inhibiting AR activity in MDV3100-resistant cell lines and significantly prevented tumor growth and PSA increases in mice bearing MDV3100-resistant xenografts. Together, our data show that Compound 30 strongly inhibited AR activity and suppressed castration-resistant LNCaP growth as well as MDV3100-resistant cell growth in vitro and in vivo. These data provide a preclinical proof-of-principle that Compound 30 could be a promising next generation anti-AR agent, especially in the context of antiandrogen-resistant tumors. Mol Cancer Ther; 12(5); 567–76. ©2013 AACR.

Cancer is a complex, often aggressive disease. As such, cancer treatment requires a diverse approach that often includes surgery, chemotherapy, radiotherapy, targeted therapy, or immunotherapy. Despite the potency of these treatments, cancer cells adapt to escape killing and survive either in their original microenvironmental niche, or as disseminated cancer cells in distant organs. Depending on tumour type and treatment modality, tumours display a variety of growth patterns, from rapid proliferation and invasion to a more controlled dormant phenotype. This dormant phenotype is characterized clinically as the asymptomatic period post therapy before relapse, and biologically by an enrichment in cancer cells that are not dividing but survive in a quiescent state, arrested in G0-G1 phase of cell cycle. Dormancy is a tumour intrinsic characteristic that corresponds to the equilibrium phase of the immune-editing hypothesis, in which tumour cells neither proliferate nor are eliminated by the immune response. In this chapter we provide an overview of anti-tumour immunity and ways in which the immune response may shape tumour dormancy.

BACKGROUND: B7-H3 (CD276), part of the B7 superfamily of immune checkpoint molecules, has been shown to have an immunomodulatory role. Its regulation, receptor and mechanism of action remain unclear. B7-H3 protein expression correlates with prostate cancer outcomes, and humanized monoclonal antibodies (that is, enoblituzumab) are currently being investigated for therapeutic use. Here we used genomic expression data to examine the relationship between B7-H3 mRNA expression and prostate cancer. METHODS: Prostatectomy tissue from 2781 patients were profiled using the Affymetrix HuEx 1.0 ST microarray. Pairwise comparisons were used to identify significant associations between B7-H3 expression and clinicopathologic variables, and survival analyses were used to evaluate the prognostic significance of B7-H3. Pearson’s correlation analyses were performed to assess the relationship of B7-H3 expression with molecular subtypes and individual transcripts. Androgen receptor (AR) occupancy at the B7-H3 locus was determined using chromatin immunoprecipitation (ChIP), and androgen-dependent expression changes in B7-H3 was evaluated by quantitative reverse transcription PCR in LNCaP cell lines. Oncomine was queried to evaluate B7-H3 expression in metastatic disease. RESULTS: B7-H3 mRNA expression was positively associated with higher Gleason score (P

Treatment-induced neuroendocrine transdifferentiation (NEtD) complicates therapies for metastatic prostate cancer (PCa). Based on evidence that PCa cells can transdifferentiate to other neuroectodermally-derived cell lineages in vitro, we proposed that NEtD requires first an intermediary reprogramming to metastable cancer stem-like cells (CSCs) of a neural class and we demonstrate that several different AR+/PSA+ PCa cell lines were efficiently reprogrammed to, maintained and propagated as CSCs by growth in androgen-free neural/neural crest (N/NC) stem medium. Such reprogrammed cells lost features of prostate differentiation; gained features of N/NC stem cells and tumor-initiating potential; were resistant to androgen signaling inhibition; and acquired an invasive phenotype in vitro and in vivo. When placed back into serum-containing mediums, reprogrammed cells could be re-differentiated to N-/NC-derived cell lineages or return back to an AR+ prostate-like state. Once returned, the AR+ cells were resistant to androgen signaling inhibition. Acute androgen deprivation or anti-androgen treatment in serum-containing medium led to the transient appearance of a sub-population of cells with similar characteristics. Finally, a 132 gene signature derived from reprogrammed PCa cell lines distinguished tumors from PCa patients with adverse outcomes. This model may explain neural manifestations of PCa associated with lethal disease. The metastable nature of the reprogrammed stem-like PCa cells suggests that cycles of PCa cell reprogramming followed by re-differentiation may support disease progression and therapeutic resistance. The ability of a gene signature from reprogrammed PCa cells to identify tumors from patients with metastasis or PCa-specific mortality implies that developmental reprogramming is linked to aggressive tumor behaviors.

Heat shock protein 27 (Hsp27) is a molecular chaperone highly expressed in aggressive cancers, where it is involved in numerous pro-tumorigenic signaling pathways. Using functional genomics we identified for the first time that Hsp27 regulates the gene signature of transcriptional co-activators YAP and TAZ, which are negatively regulated by the Hippo Tumor Suppressor pathway. The Hippo pathway inactivates YAP by phosphorylating and increasing its cytoplasmic retention with the 14.3.3 proteins. Gain and loss of function experiments in prostate, breast and lung cancer cells showed that Hsp27 knockdown induced YAP phosphorylation and cytoplasmic localization while overexpression of Hsp27 displayed opposite results. Mechanistically, Hsp27 regulates the Hippo pathway by accelerating the proteasomal degradation of ubiquitinated MST1, the core Hippo kinase, resulting in reduced phosphorylation/activity of LATS1 and MOB1, its downstream effectors. Importantly, our in vitro results were supported by data from human tumors; clinically, high expression of Hsp27 in prostate tumors is correlated with increased expression of YAP gene signature and reduced phosphorylation of YAP in lung and invasive breast cancer clinical samples. This study reveals for the first time a link between Hsp27 and the Hippo cascade, providing a novel mechanism of deregulation of this tumor suppressor pathway across multiple cancers.

PurposeSchool principals make sense of multiple messages, policies, and contexts within their school environments. The purpose of this paper is to examine specifically how school leaders make sense of hiring and subjective evaluation of on‐the‐job teacher performance.Design/methodology/approachThis qualitative study drew from 42 interviews with 21 principals from a mid‐sized Florida school district. Two rounds of semi‐structured interviews (one to two hours each) were conducted with the informants over two summers (2005‐2006). The multi‐year study allows the authors to assess the consistency across principal participants.FindingsPrincipals' personal beliefs, background, and experiences were found to shape their conceptions and preferences for teacher characteristics. School type (e.g. elementary, secondary, levels of poverty) also influenced principals' perceptions of and preferences for specific applicant and teacher characteristics. Principals in the sample, however, showed surprising consistency towards certain characteristics (caring, subject matter knowledge, strong teaching skills) and job fit (person‐job). Sampled principals reported that each vacancy is different and is highly dependent on the position, team, and individuals. Regardless of the position or school setting, federal, state, and district mandates strongly influenced how principals made sense of the hiring process and on‐the‐job performance.Practical implicationsThe findings underscore the complexity of the human resource functions in education and raise important questions of how school leaders reconcile personal preferences and building‐level needs with demands from the district, state, and federal levels.Originality/valueThe authors' findings offer important insight into the complex conceptualizations that principals hold and the balances that must be struck in the face of policy and hiring constraints. How principals make sense of teacher quality, however, has not been examined. This study contributes to the extant research and makes a theoretical contribution to studies using a cognitive frame to understand school leadership.

Background: Potent targeting of the androgen receptor (AR) in castration-resistant prostate cancer has altered the archetypal course of the disease, fueling the emergence of aggressive and incurable neuroendocrine prostate cancer (NEPC). Recent evidence suggests that these tumors can arise from non-neuroendocrine cells in response to AR pathway inhibitors (ARPIs), such as enzalutamide (ENZ), an observation consistent with lineage plasticity. What regulates this plasticity that allows cells to shed their dependence on the AR and re-emerge as “AR-indifferent” NEPC? Sequencing studies have uncovered that the evolution toward a NEPC phenotype is aligned with dynamic epigenetic reprogramming, but the molecular basis underlying this phenomenon remains poorly understood. Methods: We developed an in vivo model of acquired ENZ resistance to (a) identify reprogramming factors that facilitate lineage plasticity, and (b) determine how to best capitalize on therapeutic strategies aimed at blocking or reversing lineage transformation. Cell lines derived from ENZ-resistant tumors were profiled by RNA-seq and ChIP-seq, and functionally assessed for stem cell-associated properties. Our findings were validated across NEPC cell lines (NCI-H660), genetically engineered mouse models (PBCre4:Ptenf/f:Rb1f/f), and patient tumors and organoids. CRISPR/Cas9-mediated genomic editing allowed us to assess the effect of knocking out reprogramming factors on therapy-induced neuroendocrine transdifferentiation. Results: AR-indifferent ENZ-resistant tumors were enriched for a Polycomb/EZH2 signature; in particular, we identified EZH2 to be phosphorylated at threonine-350 (pEZH2-T350) by CDK1 in NEPC cell lines, mouse models, and patient tumors. Accordingly, RB1 loss was sufficient to enhance pEZH2-T350, which was required for prostate cancer cells to convert to a metastable stem-like state and, in turn, acquire neuroendocrine features under the pressure of ARPIs both in vitro and in patient-derived xenografts. This therapy-induced NEPC transdifferentation was associated with a marked redistribution of EZH2 and H3K27me3, specifically to a core set of genes governing lineage identity. AR colocalized at the reprogrammed EZH2 binding sites, and was found to be part of the same complex with EZH2. Treating AR-indifferent/NEPC cell lines with clinically relevant EZH2 inhibitors reversed the lineage switch and mitigated ENZ resistance. Conclusions: This research establishes the centrality of epigenetic reprogramming in driving the insurgence of a neuroendocrine phenotype in response to ARPIs, and posits that drugging the epigenome via EZH2 inhibition may reverse or delay lineage transformation to extend the durability of clinically beneficial ARPIs. Citation Format: Alastair Davies, Chiara Bostock, Musaddeque Ahmed, Yen-Yi Lin, Fraser Johnson, Ka Mun Nip, Kirsi Ketola, Jennifer Bishop, Ladan Fazli, David Goodrich, Faraz Hach, Hansen He, Himisha Beltran, Amina Zoubeidi. Identity fraud: Lineage plasticity as a mechanism of antiandrogen resistance and target for therapy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A033.

Introduction: Resistance to newly developed androgen receptor pathway inhibitors (ARPIs), such as Enzalutamide (ENZ), rapidly emerges and patients generally die within two years. In particular, a subset of patients who relapse following ARPI therapy exhibit lineage switching whereby tumours shed their dependence on AR signaling and emerge with neuroendocrine features. These tumours, termed treatment induced neuroendocrine prostate cancer (t-NEPC), carry an extremely poor prognosis and, to date, treatment remains decades old cytotoxic chemotherapy which carries a short-lived response at the cost of significant toxicity. Therefore, targeted therapies for this deadly disease are desperately needed. Thus, the need to develop targeted treatments for this devastating disease is of paramount importance. Recently our group identified the neural transcription factor BRN2 as a major clinically relevant driver of NEPC and aggressive tumor growth, both in vitro and in vivo, suggesting targeting BRN2 is a promising strategy to prevent neuroendocrine differentiation or treat NEPC. Methods: Study the effects of BRN2 inhibition using siRNA and CRISPR K/O models. Results: Inhibition of BRN2 by siRNA and by CRISPR/Cas9 knockout drastically reduced cell proliferation in 42DENZR (NEPC) cell lines. This data was re-capitulated in human NEPC NCI-H660 cells. Loss of BRN2 initiated drastic epigenetic changes in NEPC cell lines as well as in G1 arrest through up-regulation of CDKN1A/1B. This was confirmed using our first in field BRN2 inhibitors. Targeting BRN2 also lead to downregulation several known targets in NEPC like EZH2, AURKA, SOX2 and Peg10. Conclusion: No therapies exist for highly lethal NEPC. Hence, the described work aims to verify BRN2 as a central driver of NEPC, and lay the pre-clinical foundation for the integration of targeted therapies into the treatment landscape to improve survival and quality of life for patients suffering from deadly form of prostate cancer. Citation Format: Daksh Thaper, Ravi Munuganti, Shaghayegh Norouzi, Sahil Kumar, Soojin Kim, Kriti Singh, Sepideh Vahid, Jennifer Bishop, Amina Zoubeidi. Targeting master neuronal transcription factor BRN2 in neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1931.

Glenoid inclination has been associated with rotator cuff tears and superior humeral translation, but the relationship between glenoid inclination and superior humeral translation has not been assessed in-vivo. The objective of this study was to compare glenoid inclination between repaired and contralateral shoulders of unilateral rotator cuff repair patients. As a secondary analysis, we assessed the relationship between glenoid inclination and in-vivo superior humeral translation. Glenoid inclination was measured from patient-specific, CT-based bone models. Glenohumeral joint motion was measured from biplane x-ray images collected during coronal-plane abduction of 21 rotator cuff repair patients. Glenoid inclination was significantly lower for the rotator cuff tear shoulders (90.7°) than the asymptomatic, contralateral shoulders (92.3°, p=0.04). There was no significant correlation between increased glenoid inclination and superior/inferior translation of the uninjured shoulder (p>0.30). This study failed to support the theory that glenoid inclination is responsible for superior humeral translation and the development of subacromial impingement.

In an age of antibiotic-resistant pathogens, antimicrobial peptides have emerged as novel therapeutics hailed for their bactericidal and immunomodulatory properties. However, a recent paper by Bader et al. demonstrates that these molecules also trigger bacteria to arm themselves against host immune responses. The authors show that the two-component regulatory system PhoP-PhoQ of Salmonella is activated not only in cation-deficient environments as previously thought, but also by binding to antimicrobial peptides, thus promoting gene transcription necessary for Salmonella survival within the host. Thus, the antimicrobial peptide might be a double-edged sword, promoting antibacterial immunity while simultaneously triggering pathogen virulence.

Individual characteristics (i.e., teacher-rated aggression and popularity) and peer group membership type in 7th grade was examined in relation to school dropout. Peer group type was characterized according to the proportion of group members who were high on teacher-rated aggression and popularity. Both aggressive and popular group types were linked to dropping out. Being a member of an aggressive group was associated with increased rates of dropout for aggressive, but not nonaggressive, youth. Membership in popular (i.e., majority of members were popular) and zero-popular (i.e., no popular members) groups was linked to dropping out, while membership in a nonpopular group (i.e., a few popular members) appeared to be protective for aggressive youth. Both popular and nonpopular youth who affiliated with aggressive peers had elevated rates of school dropout. All aggressive participants who were socially isolated dropped out, while nonaggressive youth who were socially isolated tended to complete school.

Teacher assessments of interpersonal characteristics were used to identify subtypes of rural African American early adolescents (161 boys and 258 girls). Teacher ratings of interpersonal characteristics were used to identify popular and unpopular aggressive subtypes for both boys and girls. Unpopular aggressive youths did not have elevated levels of rejected sociometric status but were more likely to have lower levels of peer-perceived social prominence and social skills. Conversely, popular aggressive youths were more likely to be disliked by peers even though they were perceived by peers as socially prominent and socially skilled and were identified by teachers as highly involved in extracurricular activities. Both popular and unpopular aggressive youths tended to associate with others who had similar levels of peer-perceived popularity.

Incurable castration-resistant prostate cancer (CRPC) is driven by androgen receptor (AR) activation. Potent therapies that prevent AR signaling, such as Enzalutamide (ENZ), are mainstay treatments for CRPC; however patients eventually progress with ENZ resistant (ENZR) disease. In this study, we investigated one mechanism of ENZ resistance, and tried to improve therapeutic efficiency of ENZ. We found HER2 expression is increased in ENZR tumors and cell lines, and is induced by ENZ treatment of LNCaP cells. ENZ-induced HER2 overexpression was dependent on AKT-YB1 activation and modulated AR activity. HER2 dependent AR activation in LNCaP and ENZR cells was effectively blocked by treatment with the EGFR/HER2 inhibitor Lapatinib, which reduced cell viability and increased apoptosis. Despite efficacy in vitro, in vivo monotherapy with Lapatinib did not prevent ENZR tumor growth. However, combination treatment of Lapatinib with ENZ most effectively induced cell death in LNCaP cells in vitro and was more effective than ENZ alone in preventing tumor growth in an in vivo model of CRPC. These results suggest that while HER2 overexpression and subsequent AR activation is a targetable mechanism of resistance to ENZ, therapy using Lapatinib is only a rational therapeutic approach when used in combination with ENZ in CRPC.

Introduction: Potent targeting of the androgen receptor (AR) in castration-resistant prostate cancer (CRPC) has altered the archetypal course of the disease, fueling the emergence of aggressive and incurable neuroendocrine prostate cancer (NEPC). Alarmingly, no targeted therapies exist for NEPC, which stems from our poor molecular understanding of the disease. What regulates the plasticity that allows cells to shed their dependence on the AR and re-emerge as “AR-indifferent” NEPC, especially under the pressure of modern AR pathway inhibitors (ARPIs) such as enzalutamide (ENZ)? Recent data suggest that neuroendocrine transdifferentiation is aligned with dynamic reprogramming of the epigenome by developmental regulators, like EZH2, making them attractive therapeutic targets. Method: To uncover reprogramming factors that are activated in response to AR pathway inhibition, we interrogated a panel of cell lines derived from ENZ-resistant prostate tumors by RNA-seq. Mirroring what is observed in a subset of patients who progress on ENZ, 25% of ENZ-resistant tumors and matched cell lines displayed reduction in canonical AR pathway activity and a NEPC phenotype. Gene set enrichment analysis revealed that these cells are enriched for a Polycomb/EZH2 signature and share a conserved transcriptional program with embryonic stem cells. In particular, we identified a distinct phosphorylated form of EZH2 (EZH2-T350), upregulated in AR-indifferent/NEPC cell lines and patient tumors, that was found to be indispensable for the emergence and maintenance of a stem-like state. Results: Blocking EZH2-T350 phosphorylation in AR-indifferent/NEPC cell lines yielded a marked reduction in expression of pluripotency transcription factors and stem-like features, including ALDH activity and spheroid formation capacity. Notably, EZH2-T350 was found to be sufficient and required for cells to enter a transient stem-like state, a pre-requisite for neuroendocrine transdifferentiation under the pressure of ARPIs both in vitro and in patient-derived prostate tumor xenografts. As our data revealed a strong association between EZH2 and AR in response to ENZ we performed ChIP-seq and observed extensive reprogramming of the AR cistrome, specifically at a core set of genes governing stem cell identity. EZH2 colocalized at the reprogrammed AR binding sites. Accordingly, treating AR-indifferent/NEPC cell lines with the EZH2 inhibitor GSK126 yielded a molecular subtype shift from PCS1 to AR-driven PCS2, and re-sensitized cells to ARPIs. Conclusion: Our findings establish the centrality of epigenetic reprogramming in driving the insurgence of a clinically aggressive neuroendocrine phenotype in response to AR pathway inhibition. Drugging the epigenome via EZH2 inhibition to reverse the NEPC state and re-sensitize tumors to our powerful arsenal of ARPIs has the potential to transform the treatment of prostate cancer. Citation Format: Alastair Davies, Musa Ahmed, Chiara Bostock, Anna Gleave, Kirsi Ketola, Fraser Johnson, Jennifer Bishop, Ladan Fazli, Haojie Huang, Hansen He, Amina Zoubeidi. EZH2 reprogramming confers intrinsic stem cell properties and developmental plasticity driving neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5025. doi:10.1158/1538-7445.AM2017-5025

The treatment effects of the anti-androgen Enzalutamide (ENZ) in patients with castration resistant prostate cancer (CRPC) are short lived. Immunotherapy may improve patient survival, however how efficacious these treatments are for CRPC, particularly those that inhibit T cell checkpoint molecules, remains questionable. Indeed, the lack of PD-L1 expression on CRPC tumors has made rationalizing the use of PD-1 blockade difficult for CRPC patients, however whether patients with ENZ resistant CRPC may be a more relevant cohort to examine the efficacy of anti-PD1 therapies remains unknown. In this study, we show that compared to CRPC, ENZ resistant CRPC expresses more PD-L1 in vitro and expresses high levels of both PD-L1 and 2 in vivo, suggesting that up-regulation of immune checkpoint molecules may be one unique mechanism of ENZ resistance that is not observed in CRPC. Our results also suggest that that ENZ resistant CRPC may not only be able to suppress immune responses via intrinsic PD-L1 expression, but also through the induction of PDL-1 and 2 on innate immune subsets in the circulation. This hypothesis was supported by our data showing an increased frequency of PD-L1/2 expressing dendritic cells (DC) and myeloid derived suppressor (MDSC) cells in the blood of ENZ resistant tumor bearing mice compared to those with CRPC. We also found that in vivo compared to CRPC, ENZ resistant CRPC was able to prevent DC infiltration into tumors and suppress DC activation, as marked by the reduced frequency of CD80/86 and PD-L1/2 expressing DC in the tumors. Moreover, we show for the first time that CRPC patients progressing on ENZ treatment have high frequencies of PD-L1 + DCs and PD-1 + CD8 T cells in their blood. Taken together, our work suggests that ENZ resistant CRPC in both mouse models and patients is associated with strong expression of the targets for anti-PD1 therapy. These data provide impetus for future studies that examine the relative contribution of tumor vs. immune cell PD-L1 in the progression of CRPC to anti-androgen resistance and the utility of monitoring circulating cell PD-L1 pathway activity in CRPC patients to predict responsiveness to checkpoint blockade immunotherapy.

Efficacy of Enzalutamide (ENZ) in castration resistant prostate cancer (CRPC) patients is short-lived. Immunotherapy like T cell checkpoint blockade may improve patient survival. However, when and where checkpoint molecules are expressed in CRPC and whether immune evasion is a mechanism of ENZ resistance remains unclear. Thus, we investigated whether clinically relevant immunotherapy targets, specifically PD-L1/2 , PD-1 and CTLA-4, are upregulated in ENZ resistant (ENZR) patients and in a pre-clinical model of ENZ resistance. We show for the first time that patients progressing on ENZ had significantly increased PD-L1/2+ dendritic cells (DC) in blood compared to those naive or responding to treatment, and a high frequency of PD-1+T cells. These data supported our pre-clinical results, in which we found significantly increased circulating PD-L1/2+ DCs in mice bearing ENZR tumors compared to CRPC, and ENZR tumors expressed significantly increased levels of tumor-intrinsic PD-L1. Importantly, the expression of PD-L1 on ENZR cells, or the ability to modulate PD-L1/2+ DC frequency, was unique to ENZR cell lines and xenografts that did not show classical activation of the androgen receptor. Overall, our results suggest that ENZ resistance is associated with the strong expression of anti-PD-1 therapy targets in circulating immune cells both in patients and in a pre-clinical model that is non-AR driven. Further evaluation of the contribution of tumor vs. immune cell PD-L1 expression in progression of CRPC to anti-androgen resistance and the utility of monitoring circulating cell PD-L1 pathway activity in CRPC patients to predict responsiveness to checkpoint immunotherapy, is warranted.

The Lyn tyrosine kinase governs the development and function of various immune cells, and its dysregulation has been linked to malignancy and autoimmunity. Using models of chemically induced colitis and enteric infection, we show that Lyn plays a critical role in regulating the intestinal microbiota and inflammatory responses as well as protection from enteric pathogens. Lyn−/− mice were highly susceptible to dextran sulfate sodium (DSS) colitis, characterized by significant wasting, rectal bleeding, colonic pathology, and enhanced barrier permeability. Increased DSS susceptibility in Lyn−/− mice required the presence of T but not B cells and correlated with dysbiosis and increased IFN-γ+ and/or IL-17+ colonic T cells. This dysbiosis was characterized by an expansion of segmented filamentous bacteria, associated with altered intestinal production of IL-22 and IgA, and was transmissible to wild-type mice, resulting in increased susceptibility to DSS. Lyn deficiency also resulted in an inability to control infection by the enteric pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium. Lyn−/− mice exhibited profound cecal inflammation, bacterial dissemination, and morbidity following S. Typhimurium challenge and greater colonic inflammation throughout the course of C. rodentium infection. These results identify Lyn as a key regulator of the mucosal immune system, governing pathophysiology in multiple models of intestinal disease.