IKKβ targeting reduces KRAS-induced lung cancer angiogenesis in vitro and in vivo: A potential anti-angiogenic therapeutic target

Objectives: The ability of tumor cells to drive angiogenesis is an important cancer hallmark that positively correlates with metastatic potential and poor prognosis. Therefore, targeting angiogenesis is a rational therapeutic approach and dissecting proangiogenic pathways is important, particularly for malignancies driven by oncogenic KRAS, which are widespread and lack effective targeted therapies. Based on published studies showing that oncogenic RAS promotes angiogenesis by upregulating the proangiogenic NF-κB target genes IL-8 and VEGF, that NF-κB activation by KRAS requires the IKKβ kinase, and that targeting IKKβ reduces KRAS-induced lung tumor growth in vivo, but has limited effects on cell growth in vitro, we hypothesized that IKKβ targeting would reduce lung tumor growth by inhibiting KRAS-induced angiogenesis. Materials and methods: To test this hypothesis, we targeted IKKβ in KRAS-mutant lung cancer cell lines either by siRNA-mediated transfection or by treatment with Compound A (CmpdA), a highly specific IKKβ inhibitor, and used in vitro and in vivo assays to evaluate angiogenesis. Results and conclusions: Both pharmacological and siRNA-mediated IKKβ targeting in lung cells reduced expression and secretion of NF-κB-regulated proangiogenic factors IL-8 and VEGF. Moreover, conditioned media from IKKβ-targeted lung cells reduced human umbilical vein endothelial cell (HUVEC) migration, invasion and tube formation in vitro. Furthermore, siRNA-mediated IKKβ inhibition reduced xenograft tumor growth and vascularity in vivo. Finally, IKKβ inhibition also affects endothelial cell function in a cancer-independent manner, as IKKβ inhibition reduced pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Taken together, these results provide a novel mechanistic understanding of how the IKKβ pathway affects human lung tumorigenesis, indicating that IKKβ promotes KRAS-induced angiogenesis both by cancer cell-intrinsic and cancer cell-independent mechanisms, which strongly suggests IKKβ inhibition as a promising antiangiogenic approach to be explored for KRAS-induced lung cancer therapy

Visualization of fidaxomicin association with the exosporium layer of Clostridioides difficile spores

Indexación ScopusBackground: Fidaxomicin has novel pharmacologic effects on C. difficile spore formation including outgrowth inhibition and persistent spore attachment. However, the mechanism of fidaxomicin attachment on spores has not undergone rigorous microscopic studies. Materials & methods: Fidaxomicin attachment to C. difficile spores of three distinct ribotypes and C. difficile mutant spores with inactivation of exosporium or spore-coat protein-coding genes were visualized using confocal microscopy with a fidaxomicin-bodipy compound (green fluorescence). The pharmacologic effect of the fidaxomicin-bodipy compound was determined. Confocal microscopy experiments included direct effect on C. difficile wild-type and mutant spores, effect of exosporium removal, and direct attachment to a comparator spore forming organism, Bacillus subtilis. Results: The fidaxomicin-bodipy compound MIC was 1 mg/L compared to 0.06 mg/L for unlabeled fidaxomicin, a 16-fold increase. Using confocal microscopy, the intracellular localization of fidaxomicin into vegetative C. difficile cells was observed consistent with its RNA polymerase mechanism of action and inhibited spore outgrowth. The fidaxomicin-bodipy compound was visualized outside of the core of C. difficile spores with no co-localization with the membrane staining dye FM4-64. Exosporium removal reduced fidaxomicin-bodipy association with C. difficile spores. Reduced fidaxomicin-bodipy was observed in C. difficile mutant spores for the spore surface proteins CdeC and CotE. Conclusion: This study visualized a direct attachment of fidaxomicin to C. difficile spores that was diminished with mutants of specific exosporium and spore coat proteins. These data provide advanced insight regarding the anti-spore properties of fidaxomicin. © 2021 The Authorshttps://www-sciencedirect-com.recursosbiblioteca.unab.cl/science/article/pii/S1075996421000354?via%3Dihu

Role of Chaperone Mediated Autophagy (CMA) in the Degradation of Misfolded N-CoR Protein in Non-Small Cell Lung Cancer (NSCLC) Cells

Nuclear receptor co-repressor (N-CoR) plays important role in transcriptional control mediated by several tumor suppressor proteins. Recently, we reported a role of misfolded-conformation dependent loss (MCDL) of N-CoR in the activation of oncogenic survival pathway in acute promyelocytic leukemia (APL). Since N-CoR plays important role in cellular homeostasis in various tissues, therefore, we hypothesized that an APL like MCDL of N-CoR might also be involved in other malignancy. Indeed, our initial screening of N-CoR status in various leukemia and solid tumor cells revealed an APL like MCDL of N-CoR in primary and secondary tumor cells derived from non-small cell lung cancer (NSCLC). The NSCLC cell specific N-CoR loss could be blocked by Kaletra, a clinical grade protease inhibitor and by genistein, an inhibitor of N-CoR misfolding previously characterized by us. The misfolded N-CoR presented in NSCLC cells was linked to the amplification of ER stress and was subjected to degradation by NSCLC cell specific aberrant protease activity. In NSCLC cells, misfolded N-CoR was found to be associated with Hsc70, a molecular chaperone involved in chaperone mediated autophagy (CMA). Genetic and chemical inhibition of Lamp2A, a rate limiting factor of CMA, significantly blocked the loss of N-CoR in NSCLC cells, suggesting a crucial role of CMA in N-CoR degradation. These findings identify an important role of CMA-induced degradation of misfolded N-CoR in the neutralization of ER stress and suggest a possible role of misfolded N-CoR protein in the activation of oncogenic survival pathway in NSCLC cells

Identification of a targetable KRAS-mutant epithelial population in non-small cell lung cancer

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencingin aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas

Primary staging and follow-up in melanoma patients – monocenter evaluation of methods, costs and patient survival

In a German cohort of 661 melanoma patients the performance, costs and survival benefits of staging methods (history and physical examination; chest X-ray; ultrasonography of the abdomen; high resolution sonography of the peripheral lymph nodes) were assessed at initial staging and during follow-up of stage I/II+III disease. At initial staging, 74% (23 out of 31) of synchronous metastases were first detected by physical examination followed by sonography of the lymph nodes revealing 16% (5 out of 31). Other imaging methods were less efficient (Chest X-ray: one out of 31; sonography of abdomen: two out of 31). Nearly 24% of all 127 first recurrences and 18% of 73 second recurrences developed in patients not participating in the follow-up programme. In follow-up patients detection of first or second recurrence were attributed to history and physical examination on a routine visit in 47 and 52% recurrences, respectively, and to routine imaging procedures in 21 and 17% of cases, respectively. Lymph node sonography was the most successful technical staging procedure indicating 13% of first relapses, but comprised 24% of total costs of follow-up in stage I/II. Routine imaging comprised nearly 50% of total costs for follow-up in stage I/II and in stage III. The mode of detecting a relapse (‘patient vs. doctor-diagnosed’ or ‘symptomatic vs asymptomatic’) did not significantly influence patients overall survival. Taken together, imaging procedures for routine follow-up in stage I/II and stage III melanoma patients were inefficient and not cost-efficient

RelB-Dependent Stromal Cells Promote T-Cell Leukemogenesis

BACKGROUND: The Rel/NF-kappaB transcription factors are often activated in solid or hematological malignancies. In most cases, NF-kappaB activation is found in malignant cells and results from activation of the canonical NF-kappaB pathway, leading to RelA and/or c-Rel activation. Recently, NF-kappaB activity in inflammatory cells infiltrating solid tumors has been shown to contribute to solid tumor initiation and progression. Noncanonical NF-kappaB activation, which leads to RelB activation, has also been reported in breast carcinoma, prostate cancer, and lymphoid leukemia. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a novel role for RelB in stromal cells that promote T-cell leukemogenesis. RelB deficiency delayed leukemia onset in the TEL-JAK2 transgenic mouse model of human T acute lymphoblastic leukemia. Bone marrow chimeric mouse experiments showed that RelB is not required in the hematopoietic compartment. In contrast, RelB plays a role in radio-resistant stromal cells to accelerate leukemia onset and increase disease severity. CONCLUSIONS/SIGNIFICANCE: The present results are the first to uncover a role for RelB in the crosstalk between non-hematopoietic stromal cells and leukemic cells. Thus, besides its previously reported role intrinsic to specific cancer cells, the noncanonical NF-kappaB pathway may also play a pro-oncogenic role in cancer microenvironmental cells

In vitro activity of eravacycline against common ribotypes of Clostridioides difficile

Background Eravacycline is a novel synthetic fluorocycline antibacterial approved for complicated intra-abdominal infections. Objectives The purpose of this study was to assess the in vitro activities of eravacycline and comparator antibiotics against contemporary clinical isolates of Clostridioides difficile representing common ribotypes, including isolates with decreased susceptibility to metronidazole and vancomycin. Methods Clinical C. difficile strains from six common or emerging ribotypes were used to test the in vitro activities of eravacycline and comparator antibiotics (fidaxomicin, vancomycin and metronidazole) by broth microdilution. In addition, MBC experiments, time–kill kinetic studies and WGS experiments were performed. Results A total of 234 isolates were tested, including ribotypes RT001 (n = 37), RT002 (n = 41), RT014-020 (n = 39), RT027 (n = 42), RT106 (n = 38) and RT255 (n = 37). MIC50/90 values were lowest for eravacycline (≤0.0078/0.016 mg/L), followed by fidaxomicin (0.016/0.063 mg/L), metronidazole (0.25/1.0 mg/L) and vancomycin (2.0/4.0 mg/L). MBCs were lower for eravacycline compared with vancomycin for all ribotypes tested. Both vancomycin and eravacycline demonstrated bactericidal killing, including for epidemic RT027. The presence of the tetM or tetW resistance genes did not affect the MIC of eravacycline. Conclusions This study demonstrated potent in vitro activity of eravacycline against a large collection of clinical C. difficile strains that was not affected by ribotype, susceptibility to vancomycin or the presence of certain tet resistance genes. Further development of eravacycline as an antibiotic to be used in patients with Clostridioides difficile infection is warranted.</p