Beatriz Hernández-Suárez

G-quadruplexes have been identified as a promising anti-cancer target because of their ability to modulate the stability of mRNAs encoding oncogenes, tumor suppressor genes, and other potential therapeutic targets. Deregulation of DNA damage and Unfolded Protein Response pathways in cancer cells may create vulnerabilities that can be exploited therapeutically. Previous studies have shown variations in the relative expression of DDR and UPR components in canine lymphoma and leukemia cell lines CLBL-1, CLB70, and GL-1. In the present study, we report the presence of G-quadruplex structures in these canine cell lines. Downregulation of the expression of DDR and UPR components at the mRNA level was observed in the CLBL-1 and CLB70 cell lines after stabilization of G4 structures using the ligand PhenDC3. In contrast, in GL-1 cells, important components of the DDR pathway, such as PARP1, GADD45A, and PIK3CB were upregulated in response to PhenDC3 treatment. Downregulation of DDIT4 mRNA expression, which encodes an important UPR component, was detected in the CLBL-1 and GL-1 cell lines after PhenDC3 exposure. These results suggest that G4 structures can be used to manipulate the expression of potential targets to treat lymphoma in dogs. A substantial enrichment of DNA replication and pyrimidine metabolism pathways was found in the GL-1 cell line after G4 stabilization. This finding suggests that PhenDC3 may induce DNA replication stress in this cell line. Collectively, these results support the feasibility of employing canine cancer cells as a model system to investigate the role of G-quadruplex structures in cancer.

Cancer cells rely on mitochondria for their bioenergetic supply and macromolecule synthesis. Central to mitochondrial function is the regulation of mitochondrial protein synthesis, which primarily depends on the cytoplasmic translation of nuclear-encoded mitochondrial mRNAs whose protein products are imported into mitochondria. Despite the growing evidence that mitochondrial protein synthesis contributes to the onset and progression of cancer, and can thus offer new opportunities for cancer therapy, knowledge of the underlying molecular mechanisms remains limited. Here, we show that RNA G-quadruplexes (RG4s) regulate mitochondrial function by modulating cytoplasmic mRNA translation of nuclear-encoded mitochondrial proteins. Our data support a model whereby the RG4 folding dynamics, under the control of oncogenic signaling and modulated by small molecule ligands or RG4-binding proteins, modifies mitochondria-localized cytoplasmic protein synthesis. Ultimately, this impairs mitochondrial functions, affecting energy metabolism and consequently cancer cell proliferation.

Introduction: New and more effective therapies for canine cancer patients are urgently required and this necessitates advanced experimental research. Dogs are good models for studies in comparative oncology; however, canine cancer cell biology research is currently limited by low availability of validated antibody reagents and techniques. This study characterises the expression of key components of the unfolded protein response (UPR) in a panel of haematopoietic canine cancer cell lines using commercially available antibodies, and validates the methods used to study this pathway. Material and Methods: The CLBL-1 canine lymphoma cell line and the GL-1 canine leukaemia cell line sourced externally and two counterparts established in house (CNK-89 and CLB70) were used as models of different lymphoma and leukaemia canine cell lines for the study. The human U2OS cell line served as the control. Antibodies were selected for identifying UPR proteins according to known canine cell reactivity and canine–murine and canine–human homology. Endoplasmic reticulum stress was induced with thapsigargin and MG132 in the cell lines. Etoposide was used to induce DNA damage in the cells. The techniques used for this validation analysis were RNA sequencing to observe the expression of UPR components in canine cell lines, Western blot to observe changes of protein expression levels after inducing ER stress in the cells, and flow cytometry in order to study cell death. Results: Substantial variations in both the basic expression and agonist-induced activation of the UPR pathway were observed in canine cancer cell lines, although the biological significance of these differences requires further investigation. Conclusion: These findings will be a starting point for future studies on cancer biology in dogs. They will also contribute to developing novel anticancer therapies for canine patients and may provide new insights into human oncology.

Background: Dogs present a significant opportunity for studies in comparative oncology. However, the study of cancer biology phenomena in canine cells is currently limited by restricted availability of validated antibody reagents and techniques. Here, we provide an initial characterization of the expression and activity of key components of the DNA Damage Response (DDR) in a panel of hematopoietic canine cancer cell lines, with the use of commercially available antibody reagents.Materials and methods: The techniques used for this validation analysis were western blot, qPCR, and DNA combing assay.Results: Substantial variations in both the basal expression (ATR, Claspin, Chk1, and Rad51) and agonist-induced activation (p-Chk1) of DDR components were observed in canine cancer cell lines. The expression was stronger in the CLBL-1 (B-cell lymphoma) and CLB70 (B-cell chronic lymphocytic leukemia) cell lines than in the GL-1 (B-cell leukemia) cell line, but the biological significance of these differences requires further investigation. We also validated methodologies for quantifying DNA replication dynamics in hematopoietic canine cancer cell lines, and found that the GL-1 cell line presented a higher replication fork speed than the CLBL-1 cell line, but that both showed a tendency to replication fork asymmetry.Conclusion: These findings will inform future studies on cancer biology, which will facilitate progress in developing novel anticancer therapies for canine patients. They can also provide new knowledge in human oncology.

The DNA damage response (DDR) is a complex signal transduction network that is activated when endogenous or exogenous genotoxins damage or interfere with the replication of genomic DNA. Under such conditions, the DDR promotes DNA repair and ensures accurate replication and division of the genome. High levels of genomic instability are frequently observed in cancers and can stem from germline loss‐of‐function mutations in certain DDR genes, such as BRCA1, BRCA2, and p53, that form the basis of human cancer predisposition syndromes. In addition, mutation and/or aberrant expression of multiple DDR genes are frequently observed in sporadic human cancers. As a result, the DDR is considered to represent a viable target for cancer therapy in humans and a variety of strategies are under investigation. Cancer is also a significant cause of mortality in dogs, a species that offers certain advantages for experimental oncology. Domestic dogs present numerous inbred lines, many of which display predisposition to specific forms of cancer and the study of which may provide insight into the biological basis of this susceptibility. In addition, clinical trials are possible in dogs and may lead to therapeutic insights that could ultimately be extended to humans. Here we review what is known specifically about the DDR in dogs and discuss how this knowledge could be extended and exploited to advance experimental oncology in this species.

Background: The study investigated four flavanone-derived γ-oxa-ε-lactones: a parent unsubstituted compound and its three derivatives with the methoxy group in positions 2′, 4′ and 8. Our objective was to find out if the introduction of the methoxy group into the aromatic ring affects in vitro anti-tumor potency of the investigated lactones. Methods: Cytotoxic and pro-apoptotic effects were assessed with cytometric tests with propidium iodide, annexin V, and Western blot techniques. We also investigated potential synergistic potency of the tested lactones and glucocorticoids in canine lymphoma/leukemia cell lines. Results: The tested flavanone-derived lactones showed anti-cancer activity in vitro. Depending on its location, the methoxy group either increased or decreased cytotoxicity of the derivatives as compared with the parent compound. The most potent lactone was the one with the methoxy group at position 4′ of the B ring (compound 3), and the weakest activity was observed when the group was located at C-8 in the A ring. A combination of the lactones with glucocorticoids confirmed their synergy in anti-tumor activity in vitro. Conclusions: Methoxy-substituted flavanone-derived lactones effectively kill canine lymphoma/leukemia cells in vitro and, thanks to their synergistic action with glucocorticoids, may potentially be applied in the treatment of hematopoietic cancers.

Background: Ubiquitin-specific protease 7 (USP7) belongs to the group of deubiquitinating enzymes (DUBs), which remove ubiquitin which controls various cellular processes such as chromosome segregation, DNA repair, gene expression, protein localization, kinase activity, protein degradation, cell cycle progression, and apoptosis. It is critical for several important functions in the cell, and therefore dysregulation of USP7 can contribute to tumorigenesis. Objectives: Alterations in the USP7 protein have been identified in various malignancies of humans. Our aim was to examine whether USP7 could be a potential therapeutic target in hematopoietic cancers of dogs. Methods: The expression level of USP7 in lymphocytes from healthy dogs and canine lymphoma cells was determined, and the effect of USP7 inhibition on the vital functions of canine cancer cells was examined. Results: We showed that USP7 was overexpressed in lymphomas in dogs. The USP7 inhibitor P5091 has selective cytotoxic activity in canine lymphoma and leukemia cell lines. Our results indicate that inhibition of USP7 leads to a disruption of cell cycle progression, and triggers DNA damage and apoptosis. The observed proapoptotic effect of the USP7 inhibitor most likely is not dependent on the p53 pathway. Conclusions and Clinical Importance: Our results suggest that USP7 could be explored as a potential therapeutic target in dogs with lymphoma. The effectiveness of USP7 inhibition in malignant cells is predicted to be independent of their p53 status.

The β-aryl-δ-halo-γ-lactones are known for their antiproliferative activity towards numerous cancer cell lines. The aim of this study was to obtain in the biotransformation process new β-aryl-δ-hydroxy-γ-lactones and compare their activity with the antiproliferative activity of parent compounds. The racemic cis-5-(1-iodoethyl)-4-phenyldihydrofuran-2-one as well as separate enantiomers were transformed in fungal cultures. Among ten tested biocatalysts, three (Absidia cylindrospora AM336, Absidia glauca AM254, and Fusarium culmorum AM10) were able to catalyze the hydrolytic dehalogenation process. The biotransformations processes were highly stereoselective and enantiomerically pure hydroxylactones were obtained (ee ≥ 99%). The iodo- and hydroxylactone enantiomers were subjected to cytotoxic activity evaluation on canine leukemia and lymphoma cell lines. The iodolactones exhibited higher biological potential towards tested cell lines than hydroxylactones. Higher cytotoxic potential was also characteristic for (+)-(4S,5S,6R)-enantiomer of iodolactone compared to its antipode.

Chalcones are interesting candidates for anti-cancer drugs due to the ease of their synthesis and their extensive biological activity. The study presents antitumor activity of newly synthesized chalcone analogues with a methoxy group on a panel of canine lymphoma and leukemia cell lines. The antiproliferative effect of the 2′-hydroxychalcone and its methoxylated derivatives was evaluated in MTT assay after 48 h of treatment in different concentrations. The proapoptotic activity was studied by cytometric analysis of cells stained with Annexin V/FITC and propidium iodide and by measure caspases 3/7 and 8 activation. The DNA damage was evaluated by Western blot analysis of phosphorylated histone H2AX. The new compounds had selective antiproliferative activity against the studied cell lines, the most effective were the 2′-hydroxy-2″,5″-dimethoxychalcone and 2′-hydroxy-4′,6′-dimethoxychalcone. 2′-Hydroxychalcone and the two most active derivatives induced apoptosis and caspases participation, but some percentage of necrotic cells was also observed. Comparing phosphatidylserine externalization after treatment with the different compounds it was noted that the addition of two methoxy groups increased the proapoptotic potential. The most active compounds triggered DNA damage even in the cell lines resistant to chalcone-induced apoptosis. The results confirmed that the analogues could have anticancer potential in the treatment of canine lymphoma or leukemia.

The DNA damage-activated protein kinase Chk1 is known to undergo auto-phosphorylation, however the sites and functional significance of this modification remain poorly understood. We have identified two novel Chk1 auto-phosphorylation sites, threonines 378 and 382 (T378/382), located in a highly conserved motif within the C-terminal Kinase Associated 1 (KA1) domain. T378/382 occur within optimal consensus Chk1 phosphorylation motifs and substitution with phospho-mimetic aspartic acid residues results in a constitutively active mutant Chk1 kinase (Chk1-DD) that arrests cell cycle progression in G2 phase of the cell cycle in the absence of DNA damage. Remarkably, the mutant Chk1-DD protein is also subject to very rapid proteasomal degradation, with a half-life approximately one tenth that of wild-type Chk1. Consistent with this, T378/T382 auto-phosphorylation also accelerates the proteasomal degradation of constitutively active Chk1 KA1 domain structural mutants. T378/382 auto-phosphorylation and accelerated degradation of wild-type Chk1 occurs at low levels during unperturbed growth, but surprisingly, is not augmented in response to genotoxic stress. Taken together, these observations demonstrate that Chk1 T378/T382 auto-phosphorylation within the KA1 domain is linked to kinase activation and rapid proteasomal degradation, and suggest a non-canonical mechanism of regulation.