ASCT2 regulates glutamine uptake and cell growth in endometrial carcinoma

Glutamine commonly becomes a conditionally essential amino acid in cancer. Glutamine is supplied to the cell by transporters such as ASCT2 (SLC1A5), which is frequently upregulated in multiple cancers. Here we investigated the expression of ASCT2 in endometrial carcinoma, and evaluated the contribution of ASCT2 to glutamine uptake and endometrial cancer cell growth. Analysis of human gene expression data showed that ASCT2 was significantly upregulated in both endometrioid and serous subtypes of endometrial carcinoma, compared to normal, age-matched endometrium. Furthermore, immunohistochemical staining of primary human endometrioid adenocarcinomas showed that tumours stain positive for ASCT2 in either a uniform or mosaic expression pattern, while normal adjacent glands appeared predominantly negative for ASCT2 staining. Chemical inhibition of glutamine transport by benzylserine or GPNA led to a significant decrease in endometrial cancer cell growth and spheroid cross-sectional area. ASCT2 knockdown recapitulated the decrease of cell growth and spheroid cross-sectional area in HEC1A cells, suggesting a reliance on ASCT2-mediated glutamine uptake. ASCT2 knockdown in Ishikawa cells led to lower glutamine uptake and cell growth, but did not affect spheroid area. Ishikawa cells express higher levels of the glutamine transporter SNAT1 compared to HEC1A cells, suggesting these cells may rely on both ASCT2 and SNAT1 for glutamine uptake. Since SNAT1 is also significantly upregulated in the endometrioid and serous subtypes, these data indicate that ASCT2 and SNAT1 could be used as markers of malignancy, and/or potential therapeutic targets in patients with endometrial carcinoma

ASCT2/SLC1A5 controls glutamine uptake and tumour growth in triple-negative basal-like breast cancer

Alanine, serine, cysteine-preferring transporter 2 (ASCT2; SLC1A5) mediates uptake of glutamine, a conditionally essential amino acid in rapidly proliferating tumour cells. Uptake of glutamine and subsequent glutaminolysis is critical for activation of the mTORC1 nutrient-sensing pathway, which regulates cell growth and protein translation in cancer cells. This is of particular interest in breast cancer, as glutamine dependence is increased in high-risk breast cancer subtypes. Pharmacological inhibitors of ASCT2-mediated transport significantly reduced glutamine uptake in human breast cancer cell lines, leading to the suppression of mTORC1 signalling, cell growth and cell cycle progression. Notably, these effects were subtype-dependent, with ASCT2 transport critical only for triple-negative (TN) basal-like breast cancer cell growth compared with minimal effects in luminal breast cancer cells. Both stable and inducible shRNA-mediated ASCT2 knockdown confirmed that inhibiting ASCT2 function was sufficient to prevent cellular proliferation and induce rapid cell death in TN basal-like breast cancer cells, but not in luminal cells. Using a bioluminescent orthotopic xenograft mouse model, ASCT2 expression was then shown to be necessary for both successful engraftment and growth of HCC1806 TN breast cancer cells in vivo. Lower tumoral expression of ASCT2 conferred a significant survival advantage in xenografted mice. These responses remained intact in primary breast cancers, where gene expression analysis showed high expression of ASCT2 and glutamine metabolism-related genes, including GLUL and GLS, in a cohort of 90 TN breast cancer patients, as well as correlations with the transcriptional regulators, MYC and ATF4. This study provides preclinical evidence for the feasibility of novel therapies exploiting ASCT2 transporter activity in breast cancer, particularly in the high-risk basal-like subgroup of TN breast cancer where there is not only high expression of ASCT2, but also a marked reliance on its activity for sustained cellular proliferation

Training simulated patients: evaluation of a training approach using self-assessment and peer/tutor feedback to improve performance

<p>Abstract</p> <p>Background</p> <p>Most medical schools use simulated patients (SPs) for teaching. In this context the authenticity of role play and quality of feedback provided by SPs is of paramount importance. The available literature on SP training mostly addresses instructor led training where the SPs are given direction on their roles. This study focuses on the use of peer and self evaluation as a tool to train SPs.</p> <p>Methods</p> <p>SPs at the medical school participated in a staff development and training programme which included a) self-assessment of their performance while observing video-tapes of their role play using a structured guide and b) peer group assessment of their performance under tutor guidance. The pre and post training performance in relation to authenticity of role play and quality of feedback was blindly assessed by students and tutors using a validated instrument and the scores were compared. A focus group discussion and a questionnaire assessed acceptability of the training programme by the SPs.</p> <p>Results</p> <p>The post-training performance assessment scores were significantly higher (p < 0.05) than the pre-training scores. The degree of improvement in the quality of feedback provided to students was more when compared to the improvement of role play. The acceptability of the training by the SPs was very satisfactory scoring an average of 7.6 out of 10. The majority of the SPs requested the new method of training to be included in their current training programme as a regular feature.</p> <p>Conclusion</p> <p>Use of structured self-reflective and peer-interactive, practice based methods of SP training is recommended to improve SP performance. More studies on these methods of training may further refine SP training and lead to improvement of SP performance which in turn may positively impact medical education.</p

The Fusion of CLEC12A and MIR223HG Arises from a trans-Splicing Event in Normal and Transformed Human Cells

Chimeric RNAs are often associated with chromosomal rearrangements in cancer. In addition, they are also widely detected in normal tissues, contributing to transcriptomic complexity. Despite their prevalence, little is known about the characteristics and functions of chimeric RNAs. Here, we examine the genetic structure and biological roles of CLEC12A-MIR223HG, a novel chimeric transcript produced by the fusion of the cell surface receptor CLEC12A and the miRNA-223 host gene (MIR223HG), first identified in chronic myeloid leukemia (CML) patients. Surprisingly, we observed that CLEC12A-MIR223HG is not just expressed in CML, but also in a variety of normal tissues and cell lines. CLEC12A-MIR223HG expression is elevated in pro-monocytic cells resistant to chemotherapy and during monocyte-to-macrophage differentiation. We observed that CLEC12A-MIR223HG is a product of trans-splicing rather than a chromosomal rearrangement and that transcriptional activation of CLEC12A with the CRISPR/Cas9 Synergistic Activation Mediator (SAM) system increases CLEC12A-MIR223HG expression. CLEC12A-MIR223HG translates into a chimeric protein, which largely resembles CLEC12A but harbours an altered C-type lectin domain altering key disulphide bonds. These alterations result in differences in post-translational modifications, cellular localization, and protein–protein interactions. Taken together, our observations support a possible involvement of CLEC12A-MIR223HG in the regulation of CLEC12A function. Our workflow also serves as a template to study other uncharacterized chimeric RNAs

The progeroid phenotype of Ku80 deficiency Is dominant over DNA-PK CS deficiency

Ku80 and DNA-PKCS are both involved in the repair of double strand DNA breaks via the nonhomologous end joining (NHEJ) pathway. While ku80-/- mice exhibit a severely reduced lifespan and size, this phenotype is less pronounced in dna-pkcs -/- mice. However, these observations are based on independent studies with varying genetic backgrounds. Here, we generated ku80-/-, dna-pkcs -/- and double knock out mice in a C57Bl6/J*FVB F1 hybrid background and compared their lifespan, end of life pathology and mutation frequency in liver a

The Fusion of CLEC12A and MIR223HG Arises from a trans-Splicing Event in Normal and Transformed Human Cells

Chimeric RNAs are often associated with chromosomal rearrangements in cancer. In addition, they are also widely detected in normal tissues, contributing to transcriptomic complexity. Despite their prevalence, little is known about the characteristics and functions of chimeric RNAs. Here, we examine the genetic structure and biological roles of CLEC12A-MIR223HG, a novel chimeric transcript produced by the fusion of the cell surface receptor CLEC12A and the miRNA-223 host gene (MIR223HG), first identified in chronic myeloid leukemia (CML) patients. Surprisingly, we observed that CLEC12A-MIR223HG is not just expressed in CML, but also in a variety of normal tissues and cell lines. CLEC12A-MIR223HG expression is elevated in pro-monocytic cells resistant to chemotherapy and during monocyte-to-macrophage differentiation. We observed that CLEC12A-MIR223HG is a product of trans-splicing rather than a chromosomal rearrangement and that transcriptional activation of CLEC12A with the CRISPR/Cas9 Synergistic Activation Mediator (SAM) system increases CLEC12A-MIR223HG expression. CLEC12A-MIR223HG translates into a chimeric protein, which largely resembles CLEC12A but harbours an altered C-type lectin domain altering key disulphide bonds. These alterations result in differences in post-translational modifications, cellular localization, and protein–protein interactions. Taken together, our observations support a possible involvement of CLEC12A-MIR223HG in the regulation of CLEC12A function. Our workflow also serves as a template to study other uncharacterized chimeric RNAs

PSMA-RLT in Patients with Metastatic Hormone-Sensitive Prostate Cancer : A Retrospective Study

Background: Prostate-specific membrane antigen (PSMA)-directed radioligand therapy (RLT) is a novel treatment for patients with castration-resistant prostate cancer (CRPC). Given the mode of action, patients in an earlier disease stage, such as hormone-sensitive prostate cancer (HSPC), are also likely to benefit from [177Lu]Lu-PSMA- (177Lu-PSMA) or [225Ac]Ac-PSMA-radioligand treatment (225Ac-PSMA). In this retrospective study, we analyzed the safety and efficacy of PSMARLT in early-stage and hormone-sensitive metastatic prostate cancer patients. Methods: A retrospective study was performed in patients who received 177Lu-PSMA and/or 225Ac-PSMA with early-stage metastatic prostate cancer. The primary outcome parameter evaluated in this study was the progression-free survival (PFS) after PSMA-RLT and toxicity according to the Common Terminology Criteria for Adverse Events. Secondary outcome parameters were prostate-specific antigen (PSA) response and the date of onset of CRPC state. Results: In total, 20 patients were included of which 18 patients received 177Lu-PSMA radioligand and two patients received tandem treatment with both 177Lu-PSMA and 225Ac-PSMA radioligands. Patients received a median of 2 treatment cycles (range 1–6) and a median activity of 6.2 GBq 177Lu-PSMA per cycle (interquartile range (IQR) 5.2–7.4 GBq). PSMA-RLT was overall well-tolerated. The most common grade 1–2 side effects were xerostomia (n = 6) and fatigue (n = 8), which were only temporarily reported. One patient that received 225Ac-PSMA developed grade 3–4 bone marrow toxicity. The median PFS was 12 months (95% confidence interval (CI), 4.09–19.9 months). Seventeen (85%) patients had a ≥50% PSA response following PSMA-RLT. One patient developed CRPC 9 months following PSMA-RLT. Conclusions: In this small cohort study, PSMA-RLT appeared safe and showed encouraging efficacy for (metastasized) early-stage and hormone-sensitive prostate cancer patients. Prospective studies are awaited and should include long-term follow-up

Lutetium-177-PSMA-I&amp;T as metastases directed therapy in oligometastatic hormone sensitive prostate cancer, a randomized controlled trial

Background: In recent years, there is increasing evidence showing a beneficial outcome (e.g. progression free survival; PFS) after metastases-directed therapy (MDT) with external beam radiotherapy (EBRT) or targeted surgery for oligometastatic hormone sensitive prostate cancer (oHSPC). However, many patients do not qualify for these treatments due to prior interventions or tumor location. Such oligometastatic patients could benefit from radioligand therapy (RLT) with 177Lu-PSMA; a novel tumor targeting therapy for end-stage metastatic castration-resistant prostate cancer (mCRPC). Especially because RLT could be more effective in low volume disease, such as the oligometastatic status, due to high uptake of radioligands in smaller lesions. To test the hypothesis that 177Lu-PSMA is an effective treatment in oHSPC to prolong PFS and postpone the need for androgen deprivation therapy (ADT), we initiated a multicenter randomized clinical trial. This is globally, the first prospective study using 177Lu-PSMA-I&T in a randomized multicenter setting. Methods & design: This study compares 177Lu-PSMA-I&T MDT to the current standard of care (SOC); deferred ADT. Fifty-eight patients with oHSPC (≤5 metastases on PSMA PET) and high PSMA uptake (SUVmax > 15, partial volume corrected) on 18F-PSMA PET after prior surgery and/or EBRT and a PSA doubling time of < 6 months, will be randomized in a 1:1 ratio. The patients randomized to the interventional arm will be eligible for two cycles of 7.4GBq 177Lu-PSMA-I&T at a 6-week interval. After both cycles, patients are monitored every 3 weeks (including adverse events, QoL- and xerostomia questionnaires and laboratory testing) at the outpatient clinic. Twenty-four weeks after cycle two an end of study evaluation is planned together with another 18F-PSMA PET and (whole body) MRI. Patients in the SOC arm are eligible to receive 177Lu-PSMA-I&T after meeting the primary study objective, which is the fraction of patients who show disease progression during the study follow up. A second primary objective is the time to disease progression. Disease progression is defined as a 100% increase in PSA from baseline or clinical progression. Discussion: This is the first prospective randomized clinical study assessing the therapeutic efficacy and toxicity of 177Lu-PSMA-I&T for patients with oHSPC. Trial registration: Clinicaltrials.gov identifier: NCT04443062

Pretransplant endotrophin predicts delayed graft function after kidney transplantation

Delayed graft function after kidney transplantation is common and increases morbidity and health care costs. There is evidence that endotrophin, a specific fragment of pro-collagen type VI, promotes the inflammatory response in kidney diseases. We tested the hypothesis that pretransplant endotrophin in kidney transplant recipients may be associated with the risk of delayed graft function. Pretransplant plasma endotrophin was assessed using an enzyme-linked immunosorbent assay in three independent cohorts with 806 kidney transplant recipients. The primary outcome was delayed graft function, i.e., the necessity of at least one dialysis session within one-week posttransplant. In the discovery cohort median pretransplant plasma endotrophin was higher in 32 recipients (12%) who showed delayed graft function when compared to 225 recipients without delayed graft function (58.4 ng/mL [IQR 33.4–69.0]; N = 32; vs. 39.5 ng/mL [IQR 30.6–54.5]; N = 225; P = 0.009). Multivariable logistic regression, fully adjusted for confounders showed, that pretransplant plasma endotrophin as a continuous variable was independently associated with delayed graft function in both validation cohorts, odds ratio 2.09 [95% CI 1.30–3.36] and 2.06 [95% CI 1.43–2.97]. Pretransplant plasma endotrophin, a potentially modifiable factor, was independently associated with increased risk of delayed graft function and may be a new avenue for therapeutic interventions