FOXD1-ALDH1A3 signaling is a determinant for the self-renewal and tumorigenicity of mesenchymal glioma stem cells

Glioma stem-like cells (GSC) with tumor-initiating activity orchestrate the cellular hierarchy in glioblastoma and engender therapeutic resistance. Recent work has divided GSC into two subtypes with a mesenchymal (MES) GSC population as the more malignant subtype. In this study, we identify the FOXD1-ALDH1A3 signaling axis as a determinant of the MES GSC phenotype. The transcription factor FOXD1 is expressed predominantly in patient-derived cultures enriched with MES, but not with the proneural GSC subtype. shRNA-mediated attenuation of FOXD1 in MES GSC ablates their clonogenicity in vitro and in vivo. Mechanistically, FOXD1 regulates the transcriptional activity of ALDH1A3, an established functional marker for MES GSC. Indeed, the functional roles of FOXD1 and ALDH1A3 are likely evolutionally conserved, insofar as RNAi-mediated attenuation of their orthologous genes in Drosophila blocks formation of brain tumors engineered in that species. In clinical specimens of high-grade glioma, the levels of expression of both FOXD1 and ALDH1A3 are inversely correlated with patient prognosis. Finally, a novel small-molecule inhibitor of ALDH we developed, termed GA11, displays potent in vivo efficacy when administered systemically in a murine GSC-derived xenograft model of glioblastoma. Collectively, our findings define a FOXD1-ALDH1A3 pathway in controling the clonogenic and tumorigenic potential of MES GSC in glioblastoma tumors

New Insights into Therapy-Induced Progression of Cancer

The malignant tumor is a complex heterogeneous set of cells functioning in a no less heterogeneous microenvironment. Like any dynamic system, cancerous tumors evolve and undergo changes in response to external influences, including therapy. Initially, most tumors are susceptible to treatment. However, remaining cancer cells may rapidly reestablish the tumor after a temporary remission. These new populations of malignant cells usually have increased resistance not only to the first-line agent, but also to the second- and third-line drugs, leading to a significant decrease in patient survival. Multiple studies describe the mechanism of acquired therapy resistance. In past decades, it became clear that, in addition to the simple selection of pre-existing resistant clones, therapy induces a highly complicated and tightly regulated molecular response that allows tumors to adapt to current and even subsequent therapeutic interventions. This review summarizes mechanisms of acquired resistance, such as secondary genetic alterations, impaired function of drug transporters, and autophagy. Moreover, we describe less obvious molecular aspects of therapy resistance in cancers, including epithelial-to-mesenchymal transition, cell cycle alterations, and the role of intercellular communication. Understanding these molecular mechanisms will be beneficial in finding novel therapeutic approaches for cancer therapy

Therapy-induced stress response is associated with downregulation of pre-mRNA splicing in cancer cells

Abstract Background Abnormal pre-mRNA splicing regulation is common in cancer, but the effects of chemotherapy on this process remain unclear. Methods To evaluate the effect of chemotherapy on slicing regulation, we performed meta-analyses of previously published transcriptomic, proteomic, phosphoproteomic, and secretome datasets. Our findings were verified by LC-MS/MS, western blotting, immunofluorescence, and FACS analyses of multiple cancer cell lines treated with cisplatin and pladienolide B. Results Our results revealed that different types of chemotherapy lead to similar changes in alternative splicing by inducing intron retention in multiple genes. To determine the mechanism underlying this effect, we analyzed gene expression in 101 cell lines affected by ɣ-irradiation, hypoxia, and 10 various chemotherapeutic drugs. Strikingly, оnly genes involved in the cell cycle and pre-mRNA splicing regulation were changed in a similar manner in all 335 tested samples regardless of stress stimuli. We revealed significant downregulation of gene expression levels in these two pathways, which could be explained by the observed decrease in splicing efficiency and global intron retention. We showed that the levels of active spliceosomal proteins might be further post-translationally decreased by phosphorylation and export into the extracellular space. To further explore these bioinformatics findings, we performed proteomic analysis of cisplatin-treated ovarian cancer cells. Finally, we demonstrated that the splicing inhibitor pladienolide B impairs the cellular response to DNA damage and significantly increases the sensitivity of cancer cells to chemotherapy. Conclusions Decreased splicing efficiency and global intron retention is a novel stress response mechanism that may promote survival of malignant cells following therapy. We found that this mechanism can be inhibited by pladienolide B, which significantly increases the sensitivity of cancer cells to cisplatin which makes it a good candidate drug for improving the efficiency of cancer therapy

Methylation of the PTENP1 pseudogene as potential epigenetic marker of age-related changes in human endometrium.

The processed pseudogene PTENP1 is involved in the regulation of the expression of the PTEN and acts as a tumor suppressor in many types of malignances. In our previous study we showed that PTENP1 methylation is present not only in tumor, but also in normal endometrium tissues of women over 45 years old. Here we used methylation-specific PCR to analyze methylation status of CpG island located near promoter region of PTENP1 in malignant and non-malignant endometrium tissues collected from 236 women of different age groups. To confirm our results, we also analyzed RNA sequencing and microarray data from 431 women with endometrial cancer from TCGA database. We demonstrated that methylation of PTENP1 is significantly increased in older patients. We also found an age-dependent increase in the level of PTENP1 expression in endometrial tissue. According to our data, PTENP1 methylation elevates the level of the pseudogene sense transcript. In turn, a high level of this transcript correlates with a more favorable prognosis in endometrial cancer. The data obtained suggested that PTENP1 methylation is associated with age-related changes in normal and hyperplastic endometrial tissues. We assumed that age-related increase in PTENP1 methylation and subsequent elevation of its expression may serve as a protective mechanism aimed to prevent malignant transformation of endometrial tissue in women during the perimenopause, menopause, and postmenopause periods

The Nitro Group Reshapes the Effects of Pyrido[3,4-<i>g</i>]quinazoline Derivatives on DYRK/CLK Activity and RNA Splicing in Glioblastoma Cells

Serine-threonine protein kinases of the DYRK and CLK families regulate a variety of vital cellular functions. In particular, these enzymes phosphorylate proteins involved in pre-mRNA splicing. Targeting splicing with pharmacological DYRK/CLK inhibitors emerged as a promising anticancer strategy. Investigation of the pyrido[3,4-g]quinazoline scaffold led to the discovery of DYRK/CLK binders with differential potency against individual enzyme isoforms. Exploring the structure–activity relationship within this chemotype, we demonstrated that two structurally close compounds, pyrido[3,4-g]quinazoline-2,10-diamine 1 and 10-nitro pyrido[3,4-g]quinazoline-2-amine 2, differentially inhibited DYRK1-4 and CLK1-3 protein kinases in vitro. Unlike compound 1, compound 2 efficiently inhibited DYRK3 and CLK4 isoenzymes at nanomolar concentrations. Quantum chemical calculations, docking and molecular dynamic simulations of complexes of 1 and 2 with DYRK3 and CLK4 identified a dramatic difference in electron donor-acceptor properties critical for preferential interaction of 2 with these targets. Subsequent transcriptome and proteome analyses of patient-derived glioblastoma (GBM) neurospheres treated with 2 revealed that this compound impaired CLK4 interactions with spliceosomal proteins, thereby altering RNA splicing. Importantly, 2 affected the genes that perform critical functions for cancer cells including DNA damage response, p53 signaling and transcription. Altogether, these results provide a mechanistic basis for the therapeutic efficacy of 2 previously demonstrated in in vivo GBM models

DR5-Selective TRAIL Variant DR5-B Functionalized with Tumor-Penetrating iRGD Peptide for Enhanced Antitumor Activity against Glioblastoma

TRAIL (TNF-related apoptosis-inducing ligand) and its derivatives are potentials for anticancer therapy due to the selective induction of apoptosis in tumor cells upon binding to death receptors DR4 or DR5. Previously, we generated a DR5-selective TRAIL mutant variant DR5-B overcoming receptor-dependent resistance of tumor cells to TRAIL. In the current study, we improved the antitumor activity of DR5-B by fusion with a tumor-homing iRGD peptide, which is known to enhance the drug penetration into tumor tissues. The obtained bispecific fusion protein DR5-B-iRGD exhibited dual affinity for DR5 and integrin αvβ3 receptors. DR5-B-iRGD penetrated into U-87 tumor spheroids faster than DR5-B and demonstrated an enhanced antitumor effect in human glioblastoma cell lines T98G and U-87, as well as in primary patient-derived glioblastoma neurospheres in vitro. Additionally, DR5-B-iRGD was highly effective in a xenograft mouse model of the U-87 human glioblastoma cell line in vivo. We suggest that DR5-B-iRGD may become a promising candidate for targeted therapy for glioblastoma

Targeting NEK2 attenuates glioblastoma growth and radioresistance by destabilizing histone methyltransferase EZH2

Accumulating evidence suggests that glioma stem cells (GSCs) are important therapeutic targets in glioblastoma (GBM). In this study, we identified NIMA-related kinase 2 (NEK2) as a functional binding protein of enhancer of zeste homolog 2 (EZH2) that plays a critical role in the posttranslational regulation of EZH2 protein in GSCs. NEK2 was among the most differentially expressed kinase-encoding genes in GSC-containing cultures (glioma spheres), and it was required for in vitro clonogenicity, in vivo tumor propagation, and radioresistance. Mechanistically, the formation of a protein complex comprising NEK2 and EZH2 in glioma spheres phosphorylated and then protected EZH2 from ubiquitination-dependent protein degradation in a NEK2 kinase activity-dependent manner. Clinically, NEK2 expression in patients with glioma was closely associated with EZH2 expression and correlated with a poor prognosis. NEK2 expression was also substantially elevated in recurrent tumors after therapeutic failure compared with primary untreated tumors in matched GBM patients. We designed a NEK2 kinase inhibitor, compound 3a (CMP3a), which efficiently attenuated GBM growth in a mouse model and exhibited a synergistic effect with radiotherapy. These data demonstrate a key role for NEK2 in maintaining GSCs in GBM by stabilizing the EZH2 protein and introduce the small-molecule inhibitor CMP3a as a potential therapeutic agent for GBM.NIH [P01CA163205, R01NS083767, R21CA175875, R01NS087913, R01CA183991, 3T32GM008804-10S1, 5T32GM008804-10]; First Affiliated Hospital of Xi'an Jiaotong University; China Scholar Council; Russian Foundation for Basic Research [16-04-01209]; Russian Federation; Korea Health Technology R&D Project through Korea Health Industry Development Institute (KHIDI); Ministry of Health & Welfare, Republic of Korea [HI14C3418]Authors retain rights to present the work without prior permission in original, revised, adapted, or derivative form, provided that all such use is for personal or nonprofit (and noncommercial) benefit, is consistent with any employment agreement, and references the original publication citation. Examples: reproduction in nonprofit publications; lecture display (slides, overheads, or digitized media); hosting on personal or curriculum vitae-oriented websites; and inclusion in institutional and/or funding-body repositories.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]