DDRE-24. ARF4-MEDIATED RETROGRADE TRAFFICKING PROMOTES CHEMORESISTANCE IN GBM

Glioblastoma (GBM) is the most common type of adult malignant brain tumor, with a median survival of only 21 months. This is partly due to the high rate of resistance to conventional therapy, including temozolomide (TMZ), leading to recurrence rates close to 100%. To identify the unknown genes driving the development of this resistance, we performed a genome-wide CRISPR knockout screen comparing a DMSO-treated population with a TMZ-treated population over 14 days. Results showed significant enrichment of ~200 novel genes and pathways. From this list, we identified 4 previously unstudied genes showing significant elevations in RNA expression (p< 0.05) when comparing recurrent and primary tumors in patient datasets, along with significant survival benefits corresponding to low gene expression (p< 0.05). Validation experiments in vitro showed significant elevations in RNA and protein expression in multiple patient-derived xenografts (PDX) lines during TMZ-treated conditions, while knocking out these genes also resulted in significantly heightened sensitivity to TMZ (p< 0.01). We investigated one particularly enriched gene, ARF4, known to be involved in retrograde trafficking. With previous studies showing that ARF4 is upregulated under ER stress, we first confirmed the increased expression of ER stress markers during TMZ treatment to explain the increased expression of ARF4 during treatment. Further investigation via live-cell imaging also showed a consequent increase in retrograde trafficking in TMZ-treated cells, as evidenced by significantly increased trafficking of transferrin receptors, a retrograde transport marker, as well as EGFR, known to play a role in promoting chemoresistance through strengthened DNA repair response. ARF4-overexpressed GBM cells similarly showed increased trafficking of transferrin receptors and EGFR to the nucleus, while ARF4-knockdowns showed decreased trafficking and nuclear EGFR expression. Ultimately, our CRISPR-Cas9 screen has identified a promising therapeutic target, ARF4, which may drive GBM’s robust resistance to chemotherapy through increased retrograde trafficking of chemoresistance-promoting nuclear EGFR.

Reference Ranges of Iron Profiles in Apparently Healthy Elderly in Sokoto, Nigeria

Background: Iron is an important micronutrient in the body, lead to anaemia, frailty and cognitive disorders in the elderly when deficient. Aim: This study was aimed to determine reference values of iron profile in apparently healthy elderly persons in Sokoto and compared with the local reference values. Study Design: This was a comparative study Duration of Study: The study lasted for a period of one year between January to December, 2020. Methodology: This was a comparative study involving 105 apparently healthy elderly persons aged 60 years and above in Sokoto metropolis. Serum iron and total-iron binding capacity (TIBC) were determined using Iron Ferrozine method. Serum ferritin, Serum transferrin (Tf) and Serum Transferrin Receptors (sTfR) were assayed using enzyme-linked immunosorbent assay (ELISA). Transferrin Saturation (TS) and Serum Transferrin Receptors ferritin log (sTfR/FL) was calculated. Data were expressed as percentiles, mean and standard deviation and analysed using t-test and one way ANOVA. Results: The study established reference ranges of Serum iron,TIBC, Serum ferritin, Tf, sTfR, TS and sTfR/FL was calculated. in Sokoto. The study showed that iron and ferritin have high reference ranges than the local values in Sokoto. The local values for TIBC, ferritin, sTfR, TS and sTfR/FL were not available. Mean Ferritin (µg/L), sTfR (ng/L) and sTfR/Fl the test subjects were significantly higher in males than females in Sokoto (p=0.026), (p= 0.001), (p=0.044) and (p= 0.003) respectively. Iron, ferritin and TS increased as the BMI was increasing (p=<0.001). Conclusion: In conclusion, normal reference values obtained in this study notably vary with the local reference ranges used in the Sokoto metropolis. There is a need for each locality to have separate reference ranges for the elderly for their proper diagnosis and management of iron related disorders.

DIAGNOSTICS AND TREATMENT OF ANEMIC SYNDROME IN PATIENTS WITH BREAST CANCER ON THE BACKGROUND OF NEOADJUVANT CHEMOTHERAPY

Introduction. In recent years, a separate publications have appeared indicating that interleukin 6 (IL-6) and the protein hepcidin 25 (GP25) play a significant role for the development of functional iron deficiency (FID) in oncological patients with a widespread tumor process. It is important to differentiate between FID and iron deficiency anemia (IDA), since they have the same morphological characteristics, but their treatment is fundamentally different.The aim of this study was to study the main metabolites of ferrokinetics, IL-6 and C-reactive protein (CRP) expression parameters in patients with breast cancer on the background of neoadjuvant chemotherapy to develop individual approaches to the diagnosis and treatment of anemic syndrome (AS), prediction, early detection of anemia and its adequate correction.Materials and methods. The study was conducted in 31 breast cancer patients, during of 6 cycles of chemotherapy. The main metabolites of ferrokinetics were studied: GP25, ferritin, soluble transferrin receptors, transferrin, iron, erythropoietin, IL-6 and CRP indices. The control group consisted of 29 apparently healthy women.Results. AS was detected in 14 (45.1 %) of breast cancer patients. IDA prevailed with microcytic, hypochromic characteristics of erythrocytes, a low concentration of ferritin, iron, GP25, IL-6, CRP, and a high level of transferrin and soluble transferrin receptors. A some patients were diagnosed with FID, mainly with the III and IV stages of the disease. Unlike IDA, they had a high concentration of ferritin, CRP and significant production of GP25, IL-6. Erythropoietin level was not optimal for the majority of patients with AS. A few patients on the background of treatment with recombinant erythropoietins revealed a deficiency of vitamins B12 (cyanocobalamin) and folic acid.Conclusion. Early diagnosis, a personalized approach to the prescription of iron preparations, recombinant erythropoietins, vitamins B12 and folic acid in patients with AS allowed for 6 cycles neoadjuvant chemotherapy without a significant decrease in erythrocytes, hemoglobin and hematocrit in most of them. The data obtained on IL-6, GP25, and CRP indicate relationship between them in the development of FID in breast cancer patients with a widespread tumor process and require further study.

Soluble transferrin receptors and ferritin index in the diagnosis of iron deficiency in patients with spondyloarthritis and anemia

Aim. To assess the diagnostic value of the detection of soluble transferrin receptors (sTfR) and ferritin index (sTfR/log Fer) in patients with spondyloarthritis (SpA) and anemia for the revealing absolute iron deficiency (ID). Materials and methods. The study included 68 patients with SpA: median age 39 [34; 47] years, men: 38 (55.9%). Hemogram, C-reactive protein levels and ferrokinetics parameters were assessed, including sTfR testing by the method of quantitative enzyme-linked immunosorbent assay (Monobind Inc., USA). We also calculated sTfR/log Fer. Based on ferrokinetics parameters and C-reactive protein levels, chronic disease anemia (CDA), iron deficiency anemia (IDA), or their combination (CDA/IDA) were diagnosed. Results. CDA was diagnosed in 16 patients, CDA/IDA in 32 patients, and 20 patients had no anemia. An increase in sTfR concentration in patients with CDA/IDA (1.7 [1.4; 2.2] mg/L) compared with patients with CDA (1.5 [1.1; 1.7] mg/L, p0.05) was revealed. sTfR/log Fer in patients with CDA/IDA (0.93 [0.82; 1.24]) was higher than in patients with CDA (0.64 [0.48; 0.75], p0.0001). When evaluating the ROC curves, it was found that sTfR levels 1.39 mg/L and sTfR/log Fer levels 0.83 indicate the presence of absolute ID. The area under the ROC curve for sTfR was 0.72 (95% confidence interval 0.600.82, p0.001), for sTfR/log Fer 0.85 (95% confidence interval 0.740.92, p0.001). The sensitivity and specificity of sTfR/log Fer (75 and 83%, respectively) were higher compared with sTfR (53 and 81%, respectively). Conclusion. In patients with SpA having CDA/IDA, sTfR and sTfR/log Fer are statistically significantly increased. The results obtained indicate the possibility of diagnosing ID by using these parameters.

Vitamin A deficiency regulates the expression of ferritin in young male Wistar rats

ABSTRACT Objective Iron deficiency and vitamin A deficiency are two of the main micronutrient deficiencies. Both micronutrients are essential for human life and children's development. This study aimed to investigate the effects of vitamin A deficiency on ferritin and transferrin receptors' expression and its relationship with iron deficiency. Methods Five diets with different vitamin A-to-iron ratios were given to thirty five 21-day-old male Wistar rats (separated in groups of seven animals each). The animals received the diet for six weeks before being euthanized. Serum iron and retinol levels were measured as biochemical parameters. Their duodenums, spleens, and livers were analyzed for the expression of ferritin and transferrin receptors by Western Blotting. Results Regarding biochemical parameters, the results show that when both vitamin A and iron are insufficient, the serum iron content (74.74µg/dL) is significantly lower than the control group (255.86µg/dL). The results also show that vitamin A deficiency does not influence the expression of the transferrin receptor, but only of the ferritin one. Conclusion Vitamin A deficiency regulates the expression of ferritin in young male Wistar rats.

Transferrin Receptors in Erythropoiesis

Erythropoiesis is a highly dynamic process giving rise to red blood cells from hematopoietic stem cells present in the bone marrow. Red blood cells transport oxygen to tissues thanks to the hemoglobin comprised of α- and β-globin chains and of iron-containing hemes. Erythropoiesis is the most iron-consuming process to support hemoglobin production. Iron delivery is mediated via transferrin internalization by the endocytosis of transferrin receptor type 1 (TFR1), one of the most abundant membrane proteins of erythroblasts. A second transferrin receptor—TFR2—associates with the erythropoietin receptor and has been implicated in the regulation of erythropoiesis. In erythroblasts, both transferrin receptors adopt peculiarities such as an erythroid-specific regulation of TFR1 and a trafficking pathway reliant on TFR2 for iron. This review reports both trafficking and signaling functions of these receptors and reassesses the debated role of TFR2 in erythropoiesis in the light of recent findings. Potential therapeutic uses targeting the transferrin-TFR1 axis or TFR2 in hematological disorders are also discussed.