TRAIL Receptor Signaling Regulation of Chemosensitivity In Vivo but Not In Vitro

Background: Signaling by Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) and Fas ligand (FasL) has been proposed to contribute to the chemosensitivity of tumor cells treated with various other anti-cancer agents. However, the importance of these effects and whether there are differences in vitro and in vivo is unclear. Methodology/Principal Findings: To assess the relative contribution of death receptor pathways to this sensitivity and to determine whether these effects are intrinsic to the tumor cells, we compared the chemosensitivity of isogenic BJAB human lymphoma cells where Fas and TRAIL receptors or just TRAIL receptors were inhibited using mutants of the adaptor protein FADD or by altering the expression of the homeobox transcription factor Six1. Inhibition of TRAIL receptors did not affect in vitro tumor cell killing by various anti-cancer agents indicating that chemosensitivity is not significantly affected by the tumor cell-intrinsic activation of death receptor signaling. However, selective inhibition of TRAIL receptor signaling caused reduced tumor regression and clearance in vivo when tested in a NOD/SCID mouse model. Conclusions: These data show that TRAIL receptor signaling in tumor cells can determine chemosensitivity in vivo but not in vitro and thus imply that TRAIL resistance makes tumors less susceptible to conventional cytotoxic anti-cancer drugs a

Bone Structure and Turnover in Postmenopausal Women With Long‐Standing Type 1 Diabetes

ABSTRACT Compromised bone structural and mechanical properties are implicated in the increased fracture risk in type 1 diabetes (T1D). We investigated bone structure and turnover by histomorphometry in postmenopausal women with T1D and controls without diabetes using tetracycline double‐labeled transiliac bone biopsy. After in vivo tetracycline double labeling, postmenopausal women with T1D of at least 10 years and without diabetes underwent transiliac bone biopsy. An expert blinded to the study group performed histomorphometry. Static and dynamic histomorphometry measurements were performed and compared between the two groups. The analysis included 9 postmenopausal women with T1D (mean age 58.4 ± 7.1 years with 37.9 ± 10.9 years of diabetes and HbA1c 7.1% ± 0.4%) and 7 postmenopausal women without diabetes (mean age 60.9 ± 3.3 years and HbA1c 5.4% ± 0.2%). There were no significant differences in serum PTH (38.6 ± 8.1 versus 51.9 ± 23.9 pg/mL), CTX (0.4 ± 0.2 versus 0.51 ± 0.34 ng/mL), or P1NP (64.5 ± 26.2 versus 87.3 ± 45.3 ng/mL). Serum 25‐hydroxyvitamin D levels were higher in T1D than in controls (53.1 ± 20.8 versus 30.9 ± 8.2 ng/mL, p < 0.05). Bone structure metrics (bone volume, trabecular thickness, trabecular number, and cortical thickness) were similar between the groups. Indices of bone formation (osteoid volume, osteoid surface, and bone formation rate) were 40% lower in T1D and associated with lower activation frequency. However, the differences in bone formation were not statistically significant. Long‐standing T1D may affect bone turnover, mainly bone formation, without significantly affecting bone structure. Further research is needed to understand bone turnover and factors affecting bone turnover in people with T1D. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research

FADD-DD and FADD-DD V108E do not inhibit chemotherapy-induced death in HCT-116 cells.

<p>HCT-116 cells transfected with GFP, FADD-DD or FADD-DDV108E expression constructs were treated with increasing doses of TRAIL, FasL, etoposide, 5-FU, or doxorubicin as indicated and cell viability was assessed. FADD-DD and FADD-DDV108E inhibited FasL and TRAIL as in BJAB cells, but had no effect on tumor cell killing by the chemotherapy drugs.</p

Six1-mediated TRAIL resistance reduces the effectiveness of etoposide in vivo, but not in vitro.

<p>Panel A, parental BJAB cells and BJAB cells expressing Six1 were treated in vitro with increasing doses of TRAIL, FasL and etoposide, and cell viability was assessed by the MTT assay. Six1 caused TRAIL resistance but had little effect on FasL or etoposide-induced cell death. Panel B illustrates how etoposide treatment of subcutaneous tumors from BJAB or BJAB-Six1 cells reduces the growth of both tumors relative to untreated controls, but is less effective in the Six1-expressing cells (* p<0.001 by t-test at day 14 for the control versus Six1 expressing cells).</p

FADD-DD and FADD-DDV108E reduce the effectiveness of tumor eradication by etoposide in vivo.

<p>Panel A, isogenic BJAB cells expressing GFP control, GFP-FADD-DD and GFP-FADD-DDV108E were implanted subcutaneously and tumors grown for 10 days prior to treatment with etoposide. Untreated tumors continued to grow. In control BJAB cells, etoposide caused tumor eradication; whereas, in tumors expressing either FADD-DD or FADD-DDV108E, etoposide treatment led to stabilization of tumor mass but no eradication (p<0.05 by t-test at 18 for the control versus FADD-DD and FADD-DDV108E expressing cells). Panel B, Western blot of tumor tissue from GFP control, GFP-FADD-DD and GFP-VFADD-DD V108E demonstrating similar expression of the GFP-tagged protein in all tumors.</p

FADD-DD blocks TRAIL-induced but not etoposide-induced death in long-term assays.

<p>Isogenic control or FADD-DD expressing BJAB cells were treated with TRAIL or etoposide as indicated for 24 hours, then washed and replaced into growth media. Long term growth of surviving cells was determined by counting viable cells. Control BJAB cells died rapidly and were unable to recover any long term growth. Etoposide treated cells were completely unable to recover growth capacity whether or not FADD-DD was expressed. However FADD-DD expression protected the TRAIL-treated cells as demonstrated by overlapping growth curves with the untreated controls.</p

FADD-DD and FADD-DD V108E do not inhibit killing by other apoptotic stimuli.

<p>Isogenic BJAB cell lines were treated with increasing doses of etoposide, MS-275, oxamflatin, doxorubicin, MG132, UV, temozolomide, 5-FU, staurosporine or sorbitol as indicated followed by MTT assay to assess cell viability. All dose response curves overlap for each stimulus.</p

ATF6-mediated signaling contributes to PARP Inhibitor Resistance in Ovarian Cancer.

High grade serous ovarian cancer (HGSOC) is the deadliest ovarian cancer histotype due in-part to the lack of therapeutic options for chemotherapy resistant disease. Poly(ADP)-ribose polymerase inhibitors (PARPi) represent a targeted treatment. However, PARPi resistance is becoming a significant clinical challenge. There is an urgent need to overcome resistance mechanisms to extend disease-free intervals. We established isogeneic PARPi-sensitive and -resistant HGSOC cell lines. In three PARPi-resistant models there is a significant increase in AP-1 transcriptional activity and DNA repair capacity. Using RNA-seq and an shRNA screen, we identified Activating Transcription Factor 6 (ATF6) as a mediator of AP-1 activity, DNA damage response, and PARPi resistance. In publicly available datasets, ATF6 expression is elevated in HGSOC and portends a poorer recurrence free survival. In a cohort of primary HGSOC tumors, higher ATF6 expression significantly correlated to PARPi resistance. In PARPi-resistant cell lines and a PDX model, inhibition of a known ATF6 regulator, p38, attenuated AP-1 activity and RAD51 foci formation, enhanced DNA damage, significantly inhibited tumor burden, and reduced accumulation of nuclear ATF6. Implications: This study highlights that a novel p38-ATF6 mediated AP-1 signaling axis contributes to PARPi resistance and provides a clinical rationale for combining PARPi and AP-1 signaling inhibitors

Multi-omic approaches identify metabolic and autophagy regulators important in ovarian cancer dissemination.

International audienceHigh-grade serous ovarian cancers (HGSOCs) arise from exfoliation of transformed cells from the fallopian tube, indicating that survival in suspension, and potentially escape from anoikis, is required for dissemination. We report here the results of a multi-omic study to identify drivers of anoikis escape, including transcriptomic analysis, global non-targeted metabolomics, and a genome-wide CRISPR/Cas9 knockout (GeCKO) screen of HGSOC cells cultured in adherent and suspension settings. Our combined approach identified known pathways, including NOTCH signaling, as well as novel regulators of anoikis escape. Newly identified genes include effectors of fatty acid metabolism, ACADVL and ECHDC2, and an autophagy regulator, ULK1. Knockdown of these genes significantly inhibited suspension growth of HGSOC cells, and the metabolic profile confirmed the role of fatty acid metabolism in survival in suspension. Integration of our datasets identified an anoikis-escape gene signature that predicts overall survival in many carcinomas