All-trans-retinoic acid modulates glycolysis via H19 and telomerase: the role of mir-let-7a in estrogen receptor-positive breast cancer cells.

BACKGROUND: Breast cancer (BC) is the most commonly diagnosed cancer in women. Treatment approaches that differ between estrogen-positive (ER+) and triple-negative BC cells (TNBCs) and may subsequently affect cancer biomarkers, such as H19 and telomerase, are an emanating delight in BC research. For instance, all-trans-Retinoic acid (ATRA) could represent a potent regulator of these oncogenes, regulating microRNAs, mostly let-7a microRNA (miR-let-7a), which targets the glycolysis pathway, mainly pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) enzymes. Here, we investigated the potential role of ATRA in H19, telomerase, miR-let-7a, and glycolytic enzymes modulation in ER + and TNBC cells. METHODS: MCF-7 and MDA-MB-231 cells were treated with 5 µM ATRA and/or 100 nM fulvestrant. Then, ATRA-treated or control MCF-7 cells were transfected with either H19 or hTERT siRNA. Afterward, ATRA-treated or untreated MDA-MB-231 cells were transfected with estrogen receptor alpha ER(α) or beta ER(ß) expression plasmids. RNA expression was evaluated by RT‒qPCR, and proteins were assessed by Western blot. PKM2 activity was measured using an NADH/LDH coupled enzymatic assay, and telomerase activity was evaluated with a quantitative telomeric repeat amplification protocol assay. Student's t-test or one-way ANOVA was used to analyze data from replicates. RESULTS: Our results showed that MCF-7 cells were more responsive to ATRA than MDA-MB-231 cells. In MCF-7 cells, ATRA and/or fulvestrant decreased ER(α), H19, telomerase, PKM2, and LDHA, whereas ER(ß) and miR-let-7a increased. H19 or hTERT knockdown with or without ATRA treatment showed similar results to those obtained after ATRA treatment, and a potential interconnection between H19 and hTERT was found. However, in MDA-MB-231 cells, RNA expression of the aforementioned genes was modulated after ATRA and/or fulvestrant, with no significant effect on protein and activity levels. Overexpression of ER(α) or ER(ß) in MDA-MB-231 cells induced telomerase activity, PKM2 and LDHA expression, in which ATRA treatment combined with plasmid transfection decreased glycolytic enzyme expression. CONCLUSIONS: To the best of our knowledge, our study is the first to elucidate a new potential interaction between the estrogen receptor and glycolytic enzymes in ER + BC cells through miR-let-7a.

Evaluating synergistic effects of metformin and simvastatin on ovarian cancer cells.

BACKGROUND: Ovarian Cancer (OC) stands as the most lethal gynecological malignancy, presenting an urgent clinical challenge in the quest to improve response rates. One approach to address this challenge is through drug repurposing, exemplified by the investigation of metabolic-modulating drugs such as Metformin (MTF) and Simvastatin (SIM). This study aims to explore the molecular mechanisms contributing to the potential synergistic anti-cancer effects between MTF and SIM on ovarian cancer cells. METHODS: We assessed the effects of the combination on the proliferation and viability of two cell lines OVCAR-3 and SKOV-3. IC50 concentrations of MTF and SIM were determined using a proliferation assay, followed by subtoxic concentrations to explore the potential synergistic effects on the viability of both cell lines. Transcriptomic analysis was conducted on OVCAR-3 treated cells, and the findings were validated by assessing the expression levels of differentially expressed genes (DEGs) through real-time PCR in both cell lines SK-OV-3 and OVCAR-3. RESULTS: Cytotoxicity analysis guided the selection of treatment concentrations as such MTF 10 mM and SIM 5 µM. The combined treatment of MTF and SIM demonstrated a synergistic inhibition of proliferation and viability in both cell lines. In OVCAR-3, exclusive identification of 507 DEGs was seen in the combination arm. Upregulation of FOXO3, RhoA, and TNFα, along with downregulation of PIK3R1, SKP2, and ATP6V1D levels, was observed in OVCAR-3 treated cells. Real-time PCR validation confirmed the consistency of expression levels for the mentioned DEGs. CONCLUSION: Our data strongly supports the presence of synergy between MTF and SIM in OC cells. The combination's effect is associated with the dysregulation of genes in the key regulators AMPK and mTOR alongside other interconnected pathways.

HSP27 modulates tumoural immune evasion by enhancing STAT3-mediated upregulation of PD-L1 and NLRC5 in ovarian cancer.

Multiple preclinical studies have demonstrated that the addition of hyperthermia (HT) to immunotherapy could enhance tumour immunogenicity and stimulate an antitumour immune response, primarily via heat shock proteins (HSPs). However, antitumour immune responses are often impeded by immune evasion mechanisms, such as the overexpression of programmed death-ligand1 (PD-L1) and the loss of major histocompatibility complex class 1 (MHC-1) expression. In this context, we sought to investigate the effect of HT on PD-L1 and NOD-like receptor family CARD domain containing 5 (NLRC5) identified as the key transcriptional activator of MHC-1 genes, and their interaction in ovarian cancer. A coculture of ovarian cancer cell lines (IGROV1 and SKOV3) with peripheral blood mononuclear cells was set up. Then, culture media conditioned with IGROV1 or SKOV3 subjected to HT was tested on untreated cell cultures. Knocking down heat shock protein B1 (HSPB1 or HSP27), heat shock protein A1 (HSPA1 or HSP70), and pharmacological inhibition of STAT3 phosphorylation were performed. Subsequently, we measured expression levels of PD-L1, NLRC5, and proinflammatory cytokines. The correlation between PD-L1 and NLRC5 expression in ovarian cancer was evaluated using the Cancer Genome Atlas database. We found that HT produces a concomitant decrease in PD-L1 and NLRC5 expression in coculture. Notably, however, the conditioned media by heat-shocked cells increases their expression. HSP27 knockdown can reverse this increase. Adding STAT3 phosphorylation inhibitor significantly enhanced the expression inhibition of PD-L1 and NLRC5 induced by HSP27 silencing. Correlation analysis showed a positive correlation in ovarian cancer between NLRC5 and PD-L1. These findings demonstrate that HSP27 modulates PD-L1 and NLRC5 expression through the activation of a common regulator 'STAT3'. Moreover, the positive correlation between PD-L1 and NLRC5 led us to conclude that the upregulation of PD-L1 and the downregulation of MHC class I are two mutually exclusive mechanisms of immune evasion in ovarian cancer.

Microarray analysis of breast cancer gene expression profiling in response to 2-deoxyglucose, metformin, and glucose starvation.

BACKGROUND: Breast cancer (BC) is the most frequently diagnosed cancer in women. Altering glucose metabolism and its effects on cancer progression and treatment resistance is an emerging interest in BC research. For instance, combining chemotherapy with glucose-lowering drugs (2-deoxyglucose (2-DG), metformin (MET)) or glucose starvation (GS) has shown better outcomes than with chemotherapy alone. However, the genes and molecular mechanisms that govern the action of these glucose deprivation conditions have not been fully elucidated. Here, we investigated the differentially expressed genes in MCF-7 and MDA-MB-231 BC cell lines upon treatment with glucose-lowering drugs (2-DG, MET) and GS using microarray analysis to study the difference in biological functions between the glucose challenges and their effect on the vulnerability of BC cells. METHODS: MDA-MB-231 and MCF-7 cells were treated with 20 mM MET or 4 mM 2-DG for 48 h. GS was performed by gradually decreasing the glucose concentration in the culture medium to 0 g/L, in which the cells remained with fetal bovine serum for one week. Expression profiling was carried out using Affymetrix Human Clariom S microarrays. Differentially expressed genes were obtained from the Transcriptome Analysis Console and enriched using DAVID and R packages. RESULTS: Our results showed that MDA-MB-231 cells were more responsive to glucose deprivation than MCF-7 cells. Endoplasmic reticulum stress response and cell cycle inhibition were detected after all three glucose deprivations in MDA-MB-231 cells and only under the metformin and GS conditions in MCF-7 cells. Induction of apoptosis and inhibition of DNA replication were observed with all three treatments in MDA-MB-231 cells and metformin-treated MCF-7 cells. Upregulation of cellular response to reactive oxygen species and inhibition of DNA repair mechanisms resulted after metformin and GS administration in MDA-MB-231 cell lines and metformin-treated MCF-7 cells. Autophagy was induced after 2-DG treatment in MDA-MB-231 cells and after metformin in MCF-7 cells. Finally, inhibition of DNA methylation were observed only with GS in MDA-MB-231 cells. CONCLUSION: The procedure used to process cancer cells and analyze their expression data distinguishes our study from others. GS had the greatest effect on breast cancer cells compared to 2-DG and MET. Combining MET and GS could restrain both cell lines, making them more vulnerable to conventional chemotherapy.

Poor Biointegration of Porcine Acellular Dermal Matrix Associated with Unfavorable Gingival Healing: A Report of Three Cases.

AIM: The aim of the present work was to explain the poor biointegration of acellular dermal xenogeneic matrix, leading to an unfavorable gingival healing following a grafting procedure for the treatment of soft tissue deficiencies. BACKGROUND: Numerous works have demonstrated the successful use of acellular dermal matrix (ADM) in soft tissue augmentation procedures. However, spare human investigations reported adverse healing outcomes at microscopic level. CASE DESCRIPTION: Three patients showing various soft tissue deficiencies (recession, gingival thickening) requiring a gingival augmentation were grafted using an ADM porcine acellular dermal matrices (pADM) as a soft tissue substitute. For this purpose, appropriate soft tissue augmentation surgeries were performed and the grafted pADM was left for proper healing. Biopsies were harvested from two out of the three patients, respectively, at 11 and 27 weeks in order to conduct a histological evaluation of the pADM's doubtful biointegration. Moreover, the ultrastructural analysis of pADM was performed using scanning electron microscopy, and additional histological procedures were used to assess its ability to support human gingival fibroblast cultures. Signs of gingival inflammation persisted several months postoperatively. Histologically, numerous inflammatory cells characterized the grafted site. Indeed, the high number of foreign body giant cell granulomas and the very densified newly formed collagen fibers highlighted a fibrotic process within gingival connective tissue. The ultrastructural and histological analysis showed that pADM was characterized by very thick and dense collagen bundles demonstrating a nonphysiological collagen network organization. Cell culture experiments showed fibroblasts proliferating on the matrix surface, sparing its deeper part, even though the collagen matrix degradation seemed to occur following a gradient from the pADM surface inward. CONCLUSION: The unfavorable clinical results may be caused by the poor colonization of matrix cells and poor angiogenesis leading to the inadequate biointegration of pADM. Hence, the pADM structure in terms of porosity and degradability should be further investigated. CLINICAL SIGNIFICANCE: The present cases highlighted a poor integration of pADM following soft tissue grafting procedures, which was caused by the inadequate ultrastructure of the used pADM. Therefore, despite the utility of such tissue substitutes, their manufacturing improvement could be required to obtain a better biointegration.

Glucose restriction combined with chemotherapy decreases telomere length and cancer antigen-125 secretion in ovarian carcinoma.

Although chemotherapy is the standard treatment for ovarian cancer (OC), recent studies have focused on its coupling with hypoglycemic drugs to decrease glucose availability. Similarly to cancer antigen 125 (Ca-125), telomerase, the key protein for telomere lengthening, is overexpressed in 90% of OC cases. The aim of the present study was to investigate the effect of the combination of glucose restriction and chemotherapy on telomere length and Ca-125 secretion in OC cells. SKOV-3, OVCAR-3 and Igrov-1 cells were treated with 20 µM cisplatin and 100 nM paclitaxel for 48 h in three different glucose concentrations: i) 4.5 g/l, ii) 1 g/l and iii) 0.5 g/l. The same treatment was repeated once per week for 6 consecutive weeks. The surviving cells were considered platinum-taxane escape (PTES) cells. The expression levels of telomerase and Ca-125 in treated and PTES cells were quantified by qPCR, and Ca-125 secretion by ELISA. Telomere length was evaluated by qPCR according to the Cawthon method. The modulation of Ca-125 by telomerase was assessed using inhibitors, small interfering RNA and transfection with human telomerase reverse transcriptase (hTERT) vectors. The implication of phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/Akt/mTOR) in Ca-125 modulation was investigated using specific inhibitors. An increase in hTERT and Ca-125 expression levels (range, 1.5-3 fold) was observed in short-term treated cells. However, an opposite effect was detected in PTES cells, where the rate of decrease in the expression levels of hTERT and Ca-125 reached 60% after treatment in 0.5 g/l glucose. Moreover, telomere length was decreased by 30% in cells treated with 0.5 g/l glucose. Inhibition of hTERT expression significantly decreased Ca-125 secretion, suggesting a potential modulation of Ca-125 by hTERT. The inhibition of the PI3K/Akt/mTOR pathway also decreased Ca-125 secretion; however, the effect of this treatment was not enhanced when coupled with telomerase inhibitors. In conclusion, the combination of chemotherapy and glucose restriction was observed to decrease Ca-125 secretion and telomerase expression leading to shortening in telomere length. Thus, decreasing glucose availability for OC cells during treatment may lead to a better clinical outcome and potentially improve the prognosis of patients with OC.

Glucose restriction reverses the Warburg effect and modulates PKM2 and mTOR expression in breast cancer cell lines.

Aerobic glycolysis, known as the "Warburg effect", is one of several hallmarks of cancer cells. The conversion of phosphoenolpyruvate (PEP) to pyruvate can be down regulated by the re-expression of the embryonic isoform 2 of pyruvate kinase (PKM2). This mechanism allows the accumulation of glycolytic intermediates for the biosynthesis of macromolecules, such as proteins, lipids and nucleic acids. PKM2 is favored by the well-known PI3K/Akt/mTOR proliferative pathway. This pathway is induced by high glucose levels, and the mTOR kinase is the central activator of the Warburg effect. In this study, we investigated the role of glucose restriction (GR) and mTOR inhibition  in reversing the Warburg effect in MDA-MB 231 and MCF-7 breast cancer cell lines. PKM2 expression was measured by western blot. Lactate production by cells was determined by a colorimetric assay. The concentration of glucose in the supernatant of cells was measured using the Trinder method. ATP level  was evaluated by using a Colorimetric/Fluorometric ATP Assay Kit. Our results showed that MDA-MB 231 cells increased glucose consumption when the glucose concentration was 0 g/L (P <0.01). In MCF-7 cells, glucose deprivation reduced lactate secretion by 80% (P =0.0001) but tripled glucose consumption (P = 0.0041). ATP concentration increased approximately when MCF-7 cells were deprived of glucose (P = 0.02). GSK1059615 does not significantly modulate lactate secretion and glucose uptake in both cell lines. Glucose restriction contribute to the reduction of the Warburg effect through mTOR inhibition and regulation of PKM2 kinases.

Different TP53 mutants in p53 overexpressed epithelial ovarian carcinoma can be associated both with altered and unaltered glycolytic and apoptotic profiles.

BACKGROUND: p53 is a tumor suppressor and key regulator of glycolysis in cancer cells, however highly mutated in tumors. In ovarian cancer, studies concerning p53 mutations focus on the DNA binding domain since the majority of hotspot mutations affects this region. Yet, mutations in other regions such as the proline rich domain may also affect the protein's expression and activity. The aim of this study is to investigate the effect of various positions of mutations in TP53 gene on glycolysis, apoptosis and transcription of p53 target genes. METHODS: Mutations frequency and their effect on p53 expression were assessed by PCR-SSCP, sequencing and immunohistochemistry on 30 ovarian cancer biopsies. Six tumors were cultured, as well as SK-OV-3, OVCAR-3 and Igrov-1. SK-OV-3 cells were transfected with 2 TP53 mutants. p53 transcriptional activity was assayed by qPCR, apoptosis by flow cytometry and glycolysis by glucose and lactate measurements, with quantification of glycolytic enzymes expression. RESULTS: Our results showed a high frequency of the P72R mutant, associated with p53 overexpression in the ovarian biopsies. However, P72R mutant cells showed similar apoptosis and glycolysis as WT cells. DNA binding domain mutations decreased the transcriptional activity of the protein and increased glucose consumption and lactate production. CONCLUSION: Despite the overexpression of the P72R mutated protein in the biopsies, it showed a similar apoptotic activity and glucose regulation ability as WT p53. Knowing that p53 expression status is used for chemotherapeutic approaches and prognosis in ovarian cancer, the results obtained highlight the importance of locating TP53 mutations.

Gastrointestinal cancer cells treatment with bevacizumab activates a VEGF autoregulatory mechanism involving telomerase catalytic subunit hTERT via PI3K-AKT, HIF-1α and VEGF receptors.

BACKGROUND: Targeting angiogenesis has been considered a promising treatment of choice for a large number of malignancies, including gastrointestinal cancers. Bevacizumab is an anti-vascular endothelial growth factor (anti-VEGF) being used for this purpose. However, treatment efficacy is largely questioned. Telomerase activity, responsible for cancer cell immortality, is detected in 85-95% of human cancers and is considered a potential regulator of VEGF. The aim of our study was to investigate the interrelationship between VEGF and hTERT in gastrointestinal cancers and to explore cell response to a combined inhibition of telomerase and VEGF. METHODS: AGS (gastric cancer), Caco-2 (colorectal cancer) and HepG2/C3A (hepatocellular carcinoma), were treated with telomerase inhibitors BIBR-1232 (10µM) and costunolide (10µM), with bevacizumab (Avastin® at 5 ng/ml or 100µg/ml) or with a combination of both types of inhibitors. VEGF and hTERT mRNA levels, and telomerase activity were detected by RT-PCR. VEGF levels were quantified by ELISA. Telomerase was knocked down using hTERT siRNA and hTERT was overexpressed in the telomerase negative cell line, Saos-2 (osteosarcoma), using constructs expressing either wild type hTERT (hTERT-WT) or dominant negative hTERT (hTERT-DN). Tube formation by HUVECs was assessed using ECMatrix™ (EMD Millipore). RESULTS: Our results showed that telomerase regulates VEGF expression and secretion through its catalytic subunit hTERT in AGS, Caco2, and HepG2/C3A, independent of its catalytic activity. Interestingly, VEGF inhibition with bevacizumab (100µg/ml) increased hTERT expression 42.3% in AGS, 94.1% in Caco2, and 52.5% in HepG2/C3A, and increased telomerase activity 30-fold in AGS, 10.3-fold in Caco2 and 8-fold in HepG2/C3A. A further investigation showed that VEGF upregulates hTERT expression in a mechanism that implicates the PI3K/AKT/mTOR pathway and HIF-1α. Moreover, bevacizumab treatment increased VEGFR1 and VEGFR2 expression in cancer cells and human umbilical vein endothelial cells (HUVECs) through hTERT. Thus, the combination of bevacizumab with telomerase inhibitors decreased VEGF expression and secretion by cancer cells, inhibited VEGFR1 and VEGFR2 upregulation, and reduced tube formation by HUVECs. CONCLUSIONS: Taken together, our results suggest that bevacizumab treatment activates a VEGF autoregulatory mechanism involving hTERT and VEGF receptors and that an inhibition of this pathway could improve tumor cell response to anti-VEGF treatment.

Telomerase inhibition decreases alpha-fetoprotein expression and secretion by hepatocellular carcinoma cell lines: in vitro and in vivo study.

Alpha-fetoprotein (AFP) is a diagnostic marker for hepatocellular carcinoma (HCC). A direct relationship between poor prognosis and the concentration of serum AFP has been observed. Telomerase, an enzyme that stabilizes the telomere length, is expressed by 90% of HCC. The aim of this study was to investigate the effect of telomerase inhibition on AFP secretion and the involvement of the PI3K/Akt/mTOR signaling pathway. Proliferation and viability tests were performed using tetrazolium salt. Apoptosis was determined through the Annexin V assay using flow cytometry. The concentrations of AFP were measured using ELISA kits. The AFP mRNA expression was evaluated using RT-PCR, and cell migration was evaluated using a Boyden chamber assay. The in vivo effect of costunolide on AFP production was tested in NSG mice. Telomerase inhibition by costunolide and BIBR 1532 at 5 and 10 µM decreased AFP mRNA expression and protein secretion by HepG2/C3A cells. The same pattern was obtained with cells treated with hTERT siRNA. This treatment exhibited no apoptotic effect. The AFP mRNA expression and protein secretion by PLC/PRF/5 was decreased after treatment with BIBR1532 at 10 µM. In contrast, no effect was obtained for PLC/PRF/5 cells treated with costunolide at 5 or 10 µM. Inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP concentration. In contrast, the MAPK/ERK pathway appeared to not be involved in HepG2/C3A cells, whereas ERK inhibition decreased the AFP concentration in PLC/PRF/5 cells. Modulation of the AFP concentration was also obtained after the inhibition or activation of PKC. Costunolide (30 mg/kg) significantly decreased the AFP serum concentration of NSG mice bearing HepG2/C3A cells. Both the inhibition of telomerase and the inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP production of HepG2/C3A and PLC/PRF/5 cells, suggesting a relationship between telomerase and AFP expression through the PI3K/Akt/mTOR pathway.