Utility of RGNEF in the Prediction of Clinical Prognosis in Patients with Rectal Cancer Receiving Preoperative Concurrent Chemoradiotherapy

Rectal cancer is a heterogeneous malignancy with different clinical responses to preoperative concurrent chemoradiotherapy (CCRT). To discover the significant genes associated with CCRT response, we performed data mining of a transcriptomic dataset (GSE35452), including 46 rectal cancer patients who received preoperative CCRT and underwent standardized curative resection. We identified ARHGEF28 as the most significantly upregulated gene correlated with resistance to CCRT among the genes related to Rho guanyl-nucleotide exchange factor activity (GO:0005085). We enrolled 172 patients with rectal cancer receiving CCRT with radical surgery. The expression of ARHGEF28 encoded protein, Rho guanine nucleotide exchange factor (RGNEF), was assessed using immunohistochemistry. The results showed that upregulated RGNEF immunoexpression was considerably correlated with poor response to CCRT (p = 0.018), pre-CCRT positive nodal status (p = 0.004), and vascular invasion (p < 0.001). Furthermore, high RGNEF expression was significantly associated with worse local recurrence-free survival (p < 0.0001), metastasis-free survival (MeFS) (p = 0.0029), and disease-specific survival (DSS) (p < 0.0001). The multivariate analysis demonstrated that RGNEF immunoexpression status was an independent predictor of DSS (p < 0.001) and MeFS (p < 0.001). Using Gene Ontology enrichment analysis, we discovered that ARHGEF28 overexpression might be linked to Wnt/β-catenin signaling in rectal cancer progression. In conclusion, high RGNEF expression was related to unfavorable pathological characteristics and independently predicted worse clinical prognosis in patients with rectal cancer undergoing CCRT, suggesting its role in risk stratification and clinical decision making.

A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation

In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Translational control is primarily exerted through a conformational switch in eIF2’s nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2. Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B’s β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed (Schoof et al. 2021) A/I-State model of allosteric ISR regulation.

GDP/GTP exchange factor MADD drives activation and recruitment of secretory Rab GTPases to Weibel-Palade bodies

Von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized and secreted by endothelial cells and stored in Weibel-Palade bodies (WPBs). The secretory Rab GTPases Rab27A, Rab3B and Rab3D have been linked with WPB trafficking and secretion. How these Rabs are activated and recruited to WPBs remains elusive. In this study, we identified MAP kinase-activating death domain (MADD) as the guanine nucleotide exchange factor (GEF) for Rab27A and both Rab3 isoforms in primary human endothelial cells. Rab activity assays revealed a reduction in Rab27A, Rab3D, and Rab3B activation upon MADD silencing. Rab activation, but not binding, was dependent on the DENN domain of MADD, indicating potential existence of two Rab interaction modules. Furthermore, immunofluorescent analysis showed that Rab27A, Rab3B, and Rab3D recruitment to WPBs was dramatically decreased upon MADD knockdown, revealing that MADD drives Rab membrane targeting. Artificial mistargeting of MADD using a TOMM70-tag abolished Rab27A localization to WPB membranes in a DENN domain-dependent manner, indicating that normal MADD localization in the cytosol is crucial. Activation of Rab3B and Rab3D was reduced upon Rab27A silencing, suggesting that activation of these Rabs is enhanced through prior activation of Rab27A by MADD. MADD silencing did not affect WPB morphology, but reduced VWF intracellular content. Furthermore, MADD-depleted cells exhibited decreased histamine-evoked VWF release, similar to Rab27A-depleted cells. In conclusion, MADD acts as a master regulator in VWF secretion by coordinating the activation and membrane targeting of secretory Rabs to WPBs.

Abstract P470: Disrupted HRAS/GRB2 Signaling In Induced-cardiomyocytes Derived From Patients With Noonan Syndrome Carrying SOS1 Gene Mutation

Background: Noonan syndrome (NS), a dominant autosomal genetic disorder that prevents normal development, and exhibits cardiac defects, which is estimated to appear in 50% to 90% of patients. Son of sevenless homolog 1 (SOS1) gene mutation has been identified as a major gene causing NS and attributes to the development of cardiomyopathy and congenital heart defects. SOS1 is a guanine nucleotide exchange factor for RAS and is known to interact with growth factor receptor-bound protein 2 (GRB2). Recently, we have generated induced pluripotent stem cells (iPSCs)-derived cardiomyocytes (iCMCs) from cardiac fibroblasts obtained from a NS patient carrying SOS1 gene variant 1654A>G. Hypothesis: Since NS is known to have aberrant RAS-MAPK signaling, we hypothesize that iCMCs derived from NS patient (NS-iCMCs) may have atypical RAS signaling leading to the development of cardiomyopathy. Methods and Results: We have compared the normal skin fibroblast-derived iPSCs (N-iPSCs) and N-iCMCs with NS patient-derived induced NS-iPSCs and NS-iCMCs. Our qRT-PCR results showed that the mRNA expressions of signaling molecules HRAS, GRB2 and SOS1 were significantly decreased in NS-iCMCs compared with N-iCMCs (Figure A), and further confirmed through the protein expression by Western immunoblotting (Figure B). These results were in association with a significantly decreased mRNA and protein expressions of cardiac transcription factor GATA4, and structural proteins alpha sarcomeric actinin-2 (ACTN2), cardiac troponin T (TNNT2) and tropomyosin alpha-1 (TPM1) in NS-iCMCs compared with N-iCMCs. Further studies are underway to explore the difference in the guanine nucleotide exchange factor (GEF) activity and ERK activation between NS-iCMCs and N-iCMCs. Conclusion: Our current findings clearly indicate that the SOS1-associated signaling molecules HRAS and GRB2 were disrupted in NS-iCMCs, which may result in the development of cardiomyopathy in NS patients.

Determination of the Radiation Exchange Factor in the Bundle of Steel Round Bars

A method to obtain a radiation exchange factor FR in the bundle of steel round bars is presented. This parameter is required for determination of the radiative thermal conductivity krd, which is one of the basic thermal properties of the bar bundles. In the presented approach, the analyzed parameter is calculated indirectly. The initial point for calculations is the geometric model of the medium defined as a unit cell. Then, for the elements present in this cell, the thermal resistance of both conduction and radiation is determined. The radiation resistance is calculated from the radiosity balance of the surfaces enclosing the analyzed system. On this basis, the radiation thermal conductivity krd is calculated. Next, taking into account the bar diameter, the value of parameter FR is also determined. The analysis is performed at the process temperature range of 200 to 800 °C for three bar diameters: 10, 20 and 30 mm, and for the three porosities of the bundle. Different emissivity of bars in the range of 0.5 to 0.9 was also taken into account. Finally, a relationship that allows calculating the FR factor correlated with the emissivity of the bars and the bundle porosity was established. The krd obtained from the methodology presented and compared with the values calculated directly do not exceed 9%; however, after averaging over the entire temperature range of the process, the difference does not exceed 0.2%.

Mechanistic Insights on Cytotoxicity of KOLR by targeting Signaling Complexes of phosphodiesterase 3B and Rap guanine nucleotide exchange factor 3

Background Protein signaling complexes play important roles in prevention of several cancer types and can be used for development of targeted therapy. The roles of signaling complexes of phosphodiesterase 3B (PDE3B) and Rap guanine nucleotide exchange factor 3 (RAPGEF3), which are two important enzymes of cyclic adenosine monophosphate (cAMP) metabolism, in cancer have not been fully explored. Methods The natural product Kaempferol-3-O-(3′′,4′′-di-E-p-coumaroyl)-α-L-rhamnopyranoside designated as KOLR was extracted from Cinnamomum pauciflorum Nees leaves using reverse phase chromatography, high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy. The antitumor effect of KOLR was analyzed by multiple cell proliferation and metastasis experiments. The PDE3B/RAPGEF3 complex was found to be the target of KOLR through mRNA sequencing, Co-Immunoprecipitation assay, gene knock-down, gene mutation of drug-resistance cell line, and molecular docking. In vivo studies have shown that KOLR has the same antitumor mechanism. Results KOLR exhibited cytotoxic effects against selected cancer cells, except for AsPC-1 pancreatic cancer cell line. KOLR stabilized PDE3B/RAPGEF3 signaling complex thus inhibiting AKT phosphorylation and Rap-1 activation. Notably, mutation of RAPGEF3 G557A inhibited effect of KOLR on stabilizing PDE3B/RAPGEF3 complex in AsPC-1 cells. Furthermore, downregulation of PDE3B expression inhibited cytotoxic effect of KOLR on tumor cells. Downregulation of RAPGEF3 and Rap-1 expression promoted apoptosis of tumor cells and inhibited tumor metastasis. PDE3B inhibits activity of RAPGEF3 and activation of downstream signaling pathway. Conclusion The findings of this study show that KOLR could stabilize PDE3B/RAPGEF3 signaling complex to play an anti-tumor role and the PDE3B/RAPGEF3 complex is a potential therapeutic cancer target.