β-TrCP Inhibition Reduces Prostate Cancer Cell Growth via Upregulation of the Aryl Hydrocarbon Receptor

Prostate cancer is a common and heterogeneous disease, where androgen receptor (AR) signaling plays a pivotal role in development and progression. The initial treatment for advanced prostate cancer is suppression of androgen signaling. Later on, essentially all patients develop an androgen independent stage which does not respond to anti hormonal treatment. Thus, alternative strategies targeting novel molecular mechanisms are required. beta-TrCP is an E3 ligase that targets various substrates essential for many aspects of tumorigenesis.Here we show that beta-TrCP depletion suppresses prostate cancer and identify a relevant growth control mechanism. shRNA targeted against beta-TrCP reduced prostate cancer cell growth and cooperated with androgen ablation in vitro and in vivo. We found that beta-TrCP inhibition leads to upregulation of the aryl hydrocarbon receptor (AhR) mediating the therapeutic effect. This phenomenon could be ligand independent, as the AhR ligand 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) did not alter prostate cancer cell growth. We detected high AhR expression and activation in basal cells and atrophic epithelial cells of human cancer bearing prostates. AhR expression and activation is also significantly higher in tumor cells compared to benign glandular epithelium.Together these observations suggest that AhR activation may be a cancer counteracting mechanism in the prostate. We maintain that combining beta-TrCP inhibition with androgen ablation could benefit advanced prostate cancer patients

Spermatogenesis rescue in a mouse deficient for the ubiquitin ligase SCFβ-TrCP by single substrate depletion

β-TrCP, the substrate recognition subunit of a Skp1–Cul1–F-box (SCF) ubiquitin ligase, is ubiquitously expressed from two distinct paralogs, targeting many regulatory proteins for proteasomal degradation. We generated inducible β-TrCP hypomorphic mice and found that they are surprisingly healthy, yet have a severe testicular defect. We show that the two β-TrCP paralogs have a nonredundant role in spermatogenesis. The testicular defect is tightly associated with cell adhesion failure within the seminiferous tubules and is fully reversible upon β-TrCP restoration. Remarkably, testicular depletion of a single β-TrCP substrate, Snail1, rescued the adhesion defect and restored spermatogenesis. Our studies highlight an unexpected functional reserve of this central E3, as well as a bottleneck in a specific tissue: a single substrate whose stabilization is incompatible with testicular differentiation

Pathways of immune exclusion in metastatic osteosarcoma are associated with inferior patient outcomes

Background Current therapy for osteosarcoma pulmonary metastases (PMs) is ineffective. The mechanisms that prevent successful immunotherapy in osteosarcoma are incompletely understood. We investigated the tumor microenvironment of metastatic osteosarcoma with the goal of harnessing the immune system as a therapeutic strategy.Methods 66 osteosarcoma tissue specimens were analyzed by immunohistochemistry (IHC) and immune markers were digitally quantified. Tumor-infiltrating lymphocytes (TILs) from 25 specimens were profiled by functional cytometry. Comparative transcriptomic studies of distinct tumor-normal lung ‘PM interface’ and ‘PM interior’ regions from 16 PMs were performed. Clinical follow-up (median 24 months) was available from resection.Results IHC revealed a statistically significantly higher concentration of TILs expressing immune checkpoint and immunoregulatory molecules in PMs compared with primary bone tumors (including programmed cell death 1 (PD-1), programmed death ligand 1 (PD-L1), lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and indoleamine 2,3-dioxygenase (IDO1). Remarkably, these lymphocytes are excluded at the PM interface compared with PM interior. TILs from PMs exhibited significantly higher amounts of PD-1 and LAG-3 and functional cytokines including interferon-γ (IFNγ) by flow cytometry. Gene expression profiling further confirmed the presence of CD8 and CD4 lymphocytes concentrated at the PM interface, along with upregulation of immunoregulatory molecules and IFNγ-driven genes in the same region. We further discovered a strong alternatively activated macrophage signature throughout the entire PMs along with a polymorphonuclear myeloid-derived suppressor cell signature focused at the PM interface. Expression of PD-L1, LAG-3, and colony-stimulating factor 1 receptor (CSF1R) at the PM interface was associated with significantly worse progression-free survival (PFS), while gene sets indicative of productive T cell immune responses (CD8 T cells, T cell survival, and major histocompatibility complex class 1 expression) were associated with significantly improved PFS.Conclusions Osteosarcoma PMs exhibit immune exclusion characterized by the accumulation of TILs at the PM interface. These TILs produce effector cytokines, suggesting their capability of activation and recognition of tumor antigens. Our findings suggest cooperative immunosuppressive mechanisms in osteosarcoma PMs including immune checkpoint molecule expression and the presence of immunosuppressive myeloid cells. We identify cellular and molecular signatures that are associated with patient outcomes, which could be exploited for successful immunotherapy

Effects of CGEN-856S and captopril administration on (A) systolic tension, (B) diastolic tension, (C) +dT/dt, (D) -dT/dt, (E) coronary flow, and (F) heart rate of rat hearts with myocardial infarction (MI).

<p>Values are expressed as mean ± SEM, n = 7−8 animals. *<i>P</i><0.05 vs. sham; ^#<i>P</i><0.05 vs. MI+vehicle; ^α<i>P</i><0.05 vs. MI+captopril.</p

Effects of CGEN-856S and losartan administration on the deposition of type I collagen (CO I), type III collagen (CO III), and fibronectin (FN) in the left ventricles of isoproterenol (ISO)-treated rats.

<p>(A) Representative confocal photomicrographs and (B) quantification of CO I, CO III, and FN in the left ventricles of animals treated with CGEN-856S. (C) Representative confocal photomicrographs and (D) quantification of CO I, CO III, and FN in the left ventricles of animals treated with losartan. Values are expressed as arbitrary units (mean gray value ± SEM, n = 4−5 animals). *<i>P</i><0.05 vs. ISO+vehicle.</p

Effects of CGEN-856S and captopril administration on left ventricular infarct area.

<p>(A) Representative photomicrographs and (B) quantification of the infarct area of animals treated with CGEN-856S or captopril. Values are expressed as mean ± SEM, n = 7−8 animals. MI: myocardial infarction. *<i>P</i><0.05 vs. sham; ^#<i>P</i><0.05 vs. MI+vehicle.</p

Effects of CGEN-856S and losartan administration on the cardiomyocyte diameters of isoproterenol-treated rats.

<p>Animals were treated with isoproterenol (ISO) for 7 days to induce heart hypertrophy or with olive oil as a control. The effects of CGEN-856S were compared to those of saline as a negative control (Veh) or losartan (LOS) as a positive control. Values are expressed as mean ± standard error of the mean (SEM), n = 4−5 animals. *<i>P</i><0.05 vs. oil +Veh; ^#<i>P</i><0.05 vs. ISO+Veh; ^α<i>P</i><0.05 vs. ISO+LOS.</p