Pan-inhibition of the three H2S synthesizing enzymes restrains tumor progression and immunosuppression in breast cancer.

BACKGROUND: Hydrogen sulfide (H2S) is a significant endogenous mediator that has been implicated in the progression of various forms of cancer including breast cancer (BC). Cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) are the three principal mammalian enzymes responsible for H2S production. Overexpression of CBS, CSE and 3MST was found to be associated with poor prognosis of BC patients. Moreover, H2S was linked to an immune-suppressive tumor microenvironment in BC. Recently it was observed that BC cells, in response to single or dual inhibition of H2S synthesizing enzymes, develop an escape mechanism by overexpressing alternative sources of H2S generation. Thus, the aim of this work is to escape the H2S compensatory mechanism by pan repressing the three enzymes using microRNAs (miRNAs) and to investigate their impact on the oncogenic and immunogenic profile of BC cells. METHODS: BC female patients (n = 25) were recruited. In-silico analysis was used to identify miRNAs targeting CBS, CSE, and 3MST. MDA-MB-231 cells were cultured and transfected using oligonucleotides. Total RNA was extracted using Biazol, reverse transcribed and quantified using qRT-PCR. H2S levels were measured using AzMc assay. BC hallmarks were assessed using trans-well migration, wound healing, MTT, and colony forming assays. RESULTS: miR-193a and miR-548c were validated by eight different bioinformatics software to simultaneously target CBS, CSE and 3MST. MiR-193a and miR-548c were significantly downregulated in BC tissues compared to their non-cancerous counterparts. Ectopic expression of miR-193a and miR-548c in MDA-MB-231 TNBC cells resulted in a marked repression of CBS, CSE, and 3MST transcript and protein levels, a significant decrease in H2S levels, reduction in cellular viability, inhibition of migration and colony forming ability, repression of immune-suppressor proteins GAL3 GAL9, and CD155 and upregulation of the immunostimulatory MICA and MICB proteins. CONCLUSION: This study sheds the light onto miR-193a and miR-548c as potential pan-repressors of the H2S synthesizing enzymes. and identifies them as novel tumor suppressor and immunomodulatory miRNAs in TNBC.

Antibacterial and in vitro anticancer activities of the antimicrobial peptide NRC-07 encapsulated in chitosan nanoparticles.

Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics and chemotherapy in the treatment of multidrug-resistant pathogens and drug-resistant cancers. Clinical application of AMPs is limited due to low stability and inefficient transport. Encapsulation in nanocarriers may improve their therapeutic potential. Chitosan nanoparticles (CS-NPs) are efficient carriers for proteins and peptides, improving the treatment of microbial infections and targeted drug delivery. We examined toxicity against cancer cell lines and antibacterial activities of the pleurocidin-like AMP NRC-07 upon encapsulation in CS-NPs by ionotropic gelation. The biological activities of various formulations of free and encapsulated NRC-07 and free nanoparticles were evaluated against Pseudomonas aeruginosa and breast cancer cells, using assays for cell viability and lactate dehydrogenase cytolysis with non-cancer cell lines as controls. NRC-07-containing nanoparticles decreased the bacterial and cancer cell viability in a concentration-dependent manner. Activities of encapsulated peptide were >2-fold higher than those of free NRC-07 peptide. Unloaded CS-NPs and free peptide were not cytotoxic against control cells. Encapsulation of NRC-07 into CS-NPs enhanced the antibacterial and selective cytotoxicity of the peptide, possibly enhancing anticancer activities. Encapsulation presents a promising tool for the development of efficient drug delivery systems.

Impact of N6-methyladenosine (m6A) modification on immunity.

N6-methyl-adenosine (m6A) is the most prevalent modification on mRNAs and long noncoding RNAs (lnRNAs) in higher eukaryotes. Modulation of m6A relies on m6A writers, erasers and readers. m6A modification contributes to diverse fundamental biological functions at the molecular, cellular, and physiological levels. The dysregulation of m6A modification has been implicated in various human diseases. Thus, m6A modification has now become a research hotspot for its potential therapeutic applications in the treatment of various cancers and diseases. The immune system is essential to provide defense against infections and cancers. This review summarizes the current knowledge about the roles of m6A in regulating immune cell functions and immune responses. Video abstract.

MALAT-1/p53/miR-155/miR-146a ceRNA circuit tuned by methoxylated quercitin glycoside alters immunogenic and oncogenic profiles of breast cancer.

Triple-Negative Breast Cancer (TNBC) is one of the most aggressive and hot BC subtypes. Our research group has recently shed the light on the utility of natural compounds as effective immunotherapeutic agents. The aim of this study is to investigate the role of a methoxylated quercetin glycoside (MQG) isolated from Cleome droserifolia in harnessing TNBC progression and tuning the tumor microenvironment and natural killer cells cytotoxicity. Results showed that MQG showed the highest potency (IC50 = 12 µM) in repressing cellular proliferation, colony-forming ability, migration, and invasion capacities. Mechanistically, MQG was found to modulate a circuit of competing endogenous RNAs where it was found to reduce the oncogenic MALAT-1 lncRNA and induce TP53 and its downstream miRNAs; miR-155 and miR-146a. Accordingly, this leads to alteration in several downstream signaling pathways such as nitric oxide synthesizing machinery, natural killer cells' cytotoxicity through inducing the expression of its activating ligands such as MICA/B, ULBP2, CD155, and ICAM-1 and trimming of the immune-suppressive cytokines such as TNF-α and IL-10. In conclusion, this study shows that MQG act as a compelling anti-cancer agent repressing TNBC hallmarks, activating immune cell recognition, and alleviating the immune-suppressive tumor microenvironment experienced by TNBC patients.

LncRNA HEIH/miR-939-5p interplay modulates triple-negative breast cancer progression through NOS2-induced nitric oxide production.

This study aimed to unravel the regulatory role of noncoding RNAs (ncRNA) on the nitric oxide (NO) machinery system in triple-negative breast cancer (TNBC) patients and to further assess the influence of NO-modulating ncRNAs on TNBC progression, immunogenic profile, and the tumor microenvironment (TME). The results revealed miR-939-5p and lncRNA HEIH as novel ncRNAs modulating NO machinery in TNBC. MiR-939-5p, an underexpressed microRNA (miRNA) in BC patients, showed an inhibitory effect on NOS2 and NOS3 transcript levels on TNBC cells. In contrast, HEIH was found to be markedly upregulated in TNBC patients and showed a modulatory role on miR-939-5p/NOS2/NO axis. Functionally, miR-939-5p was characterized as a tumor suppressor miRNA while HEIH was categorized as a novel oncogenic lncRNA in TNBC. Finally, knocking down of HEIH resulted in improvement of immunogenic profile of TNBC cells through inducing MICA/B and suppressing the immune checkpoint inhibitor PDL1. In the same context, knockdown of HEIH resulted in the alleviation of the immune-suppressive TME by repressing interleukin-10 and tumor necrosis factor-α levels. In conclusion, this study identifies miR-939-5p as a tumor suppressor miRNA while HEIH as an oncogenic lncRNA exhibiting its effect through miR-939-5p/NOS2/NO axis. Therefore, repressing BC hallmarks, improving TNBC immunogenic profile, and trimming TME.

The long noncoding RNA sONE represses triple-negative breast cancer aggressiveness through inducing the expression of miR-34a, miR-15a, miR-16, and let-7a.

Triple-negative breast cancer (TNBC) represents an aggressive breast cancer subtype. Among young females, TNBC is the leading cause of cancer-related mortalities. Recently, long noncoding RNAs (lncRNAs) are representing a promising pool of regulators for tuning the aggressiveness of several solid malignancies. However, this still needs further investigations in TNBC. The main aim of this study is to unravel the expression pattern of sONE lncRNA and its mechanistic role in TNBC. Results showed that sONE is restrictedly expressed in TNBC patients; its expression level is inversely correlated with the aggressiveness of the disease. sONE acts as a posttranscriptional regulator to endothelial nitric oxide synthase (eNOS) and thus affecting eNOS-induced nitric oxide (NO) production from TNBC cells measured by Greiss reagent. Mechanistically, sONE is a potential tumor suppressor lncRNA in TNBC cells; repressing cellular viability, proliferation, colony-forming ability, migration, and invasion capacities of MDA-MB-231. Furthermore, sONE effects were found to be extended to affect the maestro tumor suppressor TP53 and the oncogenic transcription factor c-Myc. Knocking down of sONE resulted in a marked decrease in TP53 and increase in c-Myc and consequently altering the expression status of their downstream tumor suppressor microRNAs (miRNAs) such as miR-34a, miR-15, miR-16, and let-7a. In conclusion, this study highlights sONE as a downregulated tumor suppressor lncRNA in TNBC cells acting through repressing eNOS-induced NO production, affecting TP53 and c-Myc proteins levels and finally altering the levels of a panel of tumor suppressor miRNAs downstream TP53/c-Myc proteins.

AGXT2 and DDAH-1 genetic variants are highly correlated with serum ADMA and SDMA levels and with incidence of coronary artery disease in Egyptians.

Dimethylarginine aminodehydrolase (DDAH1) and alanine glyoxylate aminotransferase2 (AGXT2) are two enzymes that contribute in asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) metabolism. Hence they affect production and bioavailability of eNOS-derived nitric oxide (NO) and consequently healthy blood vessels. The major aims of the current study were to investigate the association of genetic variants of AGXT2 rs37369, AGXT2 rs16899974 and DDAH1 rs997251 SNPs with incidence of coronary artery disease (CAD) in Egyptians and to correlate these variants with the serum levels of ADMA and SDMA. The study included 150 subjects; 100 CAD patients and 50 healthy controls. Genotyping was performed by qPCR while the ADMA and SDMA concentrations were assayed by ELISA. Both serum ADMA and SDMA concentrations were significantly higher in CAD patients compared to controls (both p < 0.0001). Genotype distributions for all studied SNPs were significantly different between CAD patients and controls. Carriers of AGXT2 rs37369-T allele (CT + TT genotypes) and AGXT2 rs16899974-A allele (CA + AA genotypes) had 2.4- and 2.08-fold higher risk of having CAD than CC genotype in both SNPs (p = 0.0050 and 0.0192, respectively). DDAH1 rs997251 TC + CC genotypes were associated with 2.3-fold higher risk of CAD than TT genotype (p = 0.0063). Moreover, the AGXT2 rs37369 TT and AGXT2 rs16899974 AA genotypes were associated with the highest serum ADMA and SDMA while DDAH1 rs997251 CC genotype was associated with the highest ADMA. AGXT2 rs37369-T, AGXT2 rs16899974-A, and DDAH1 rs997251-C alleles represent independent risk factors for CAD in the Egyptians.

Investigation of brain-derived neurotrophic factor (BDNF) gene expression in hypothalamus of obese rats: Modulation by omega-3 fatty acids.

PURPOSE: The aim of this study was investigating the effect of omega-3 fatty acids (ω-3 FAs) on brain-derived neurotrophic factor (BDNF) gene expression, using in vivo and in vitro models, to unravel the potential mechanisms of polyunsaturated fatty acids use in obesity. MATERIALS AND METHODS: Twenty-nine Sprague-Dawley rats were divided into three groups; lean controls fed normal chow diet for 14 weeks, obese controls fed 60% of their diet as saturated fats for 14 weeks, and ω-3 FAs-treated rats fed 60% saturated fat diet for 14 weeks with concomitant oral administration of 400 mg/kg/day ω-3 FAs, mainly docosahexaenoic acid and EPA, from week 12 to week 14. For the in vitro experiment, hypothalamic cells from six obese rats were cultured in the presence of different concentrations of ω-3 FAs to determine its direct effect on BDNF expression. RESULTS: In vivo results showed that obesity has negative effect on BDNF gene expression in rat hypothalamus that was reversed by administration of ω-3 FAs. Obese rats showed hypercholesterolemia, hypertriglyceridemia, normoinsulinemia, hyperglycemia and hyperleptinemia. Treatment with ω-3 FAs showed significant decrease in serum total cholesterol and TAG. Also serum glucose level and HOMA index were decreased significantly. In vitro results demonstrated the increase in BDNF expression by ω-3 FAs in a dose-dependent manner. CONCLUSIONS: Obesity causes down-regulation of BDNF gene expression that can be reversed by ω-3 FAs treatment, making them an interesting treatment approach for obesity and metabolic disease.

Assessment of the link between endothelin K198n Snp, endothelin concentration and acute myocardial infarction in Egyptians.

The aim of the current study was to assess the link between EDN K198N SNP, ET-1 serum concentration and acute myocardial infarction (AMI) in Egyptians. The study cohort consisted of 84 patients at AMI onset and 84 age-matched healthy controls. Endothelin genotypes and concentrations were determined by sequencing and ELISA, respectively. Genotype distribution was not significantly different between AMI patients and controls (P=.8341). The mean serum ET-1 concentration of patients (13.83±0.7 pg/mL) was significantly higher than controls (7.26±0.2 pg/mL) (P<.0001). ET-1 serum concentrations did not vary significantly among various EDN genotypes in patients (P=.378) and controls (P=.6164). Hence, we conclude that EDN K198N genotypes were not related to either ET-1 concentration or incidence of early-onset AMI in Egyptians. But, AMI patients had higher ET-1 concentrations than controls.

The Role of Nitric Oxide from Neurological Disease to Cancer.

Until the beginning of the 1980s, nitric oxide (NO) was just a toxic molecule of a lengthy list of environmental pollutants such as cigarette smoke and smog. In fact, NO had a very bad reputation of being destroyer of ozone, suspected carcinogen and precursor of acid rain. However, by the early 1990s it was well recognized by the medical research community. Over the last two decades, the picture has been totally changed. Diverse lines of evidence have converged to show that this sometime poison is a fundamental player in the everyday business of the human body. NO activity was probed in the brain, arteries, immune system, liver, pancreas, uterus, peripheral nerves, lungs, and almost every system in the human body. NO is a major player in the cardiovascular system as it is involved in regulating blood pressure. In the CNS, it is involved in memory formation and the regulation of cerebral blood flow to ensure adequate supply of blood to the brain. Because NO is involved in many pathways, it has a role in several diseases related to modern life as hypertension, coronary heart diseases, Alzheimer's Disease, stroke and cancer. This chapter focuses on the discussion of the role of NO in neurological diseases and cancer and how can this Janus-faced molecule play a role in the pathology and personalized treatment of these diseases.

Genetic variation in vitamin D receptor gene (Fok1:rs2228570) is associated with risk of coronary artery disease.

OBJECTIVE: The Fok1 polymorphism (rs2228570) in vitamin D receptor gene appears to be the only polymorphism influencing size of translated protein. Investigations into its association with coronary artery disease (CAD) are sparse. METHODS: Male patients (n = 98) with verified CAD were recruited alongside age- and sex-matched controls (n = 55). Genotyping was performed by PCR-RFLP and plasma 25-Hydroxyvitamin D levels were assessed by HPLC-UV. RESULTS: The C-variant (mutant) was predominantly expressed in patients compared to controls (68.9% versus 55.5%; p = 0.025). The observed genotypes were not associated with 25-Hydroxyvitamin D levels. CONCLUSION: This study presents Fok1 polymorphism as a potential genetic marker for CAD.

Vitamin D receptor gene polymorphisms (TaqI and ApaI) in relation to 25-hydroxyvitamin D levels and coronary artery disease incidence.

CONTEXT/OBJECTIVE: Previous studies have illustrated the association of the ApaI and TaqI polymorphisms of the vitamin D receptor gene, located in non-coding and coding regions, respectively, with diseases such as cancer and cardiovascular disease; however, investigating such association in Egyptian patients with coronary artery disease (CAD) has never been formerly attempted. MATERIALS AND METHODS: Male patients (n = 137), 35-50 years of age, with verified CAD, were recruited alongside age- and sex-matched controls (n = 58). Genotyping and 25-hydroxyvitamin D [25(OH)D] measurement were performed by polymerase chain reaction RFLP and HPLC, respectively. RESULTS: Comparison of the genotypic distribution of both the TaqI and ApaI polymorphisms between patients and controls yielded insignificant results (p = 0.55 and 0.7, respectively). Comparison of the allelic distribution of both polymorphisms also yielded insignificant results. The TaqI polymorphism was not found to predict 25(OH)D levels, whereas the wild-type genotype of the ApaI polymorphism was associated with greater levels of 25(OH)D (p = 0.02), taking all subjects into consideration. DISCUSSION/CONCLUSION: This study presents the ApaI and TaqI polymorphisms as non-influencing players in the pathogenesis of CAD in Egyptian males and the ability of only the ApaI polymorphism to predict 25(OH)D levels, thus warranting further investigations of the triangular relationship between the polymorphisms, 25(OH)D and CAD incidence.

Triangular relationship between single nucleotide polymorphisms in the CYP2R1 gene (rs10741657 and rs12794714), 25-hydroxyvitamin d levels, and coronary artery disease incidence.

OBJECTIVE: To investigate the relationship between the rs10741657 and rs12794714 polymorphisms in the CYP2R1 gene, 25(OH)D levels, and coronary artery disease (CAD) incidence. METHODS: In total, 134 male patients with verified CAD were recruited, alongside 109 age- and sex-matched controls. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism, using the corresponding restriction enzyme for each polymorphism, whereas 25(OH)D levels were analyzed by HPLC-UV. RESULTS: 25(OH)D levels were significantly lower in patients. The genotypic and allelic distributions of the rs10741657 polymorphism were significantly different between patients and controls, whereas insignificant results were obtained for the rs12794714 polymorphism. Furthermore, rs10741657, but not rs12794714, predicted 25(OH)D levels. CONCLUSION: The rs10741657 polymorphism is a novel genetic marker for CAD.

C242T polymorphism of NADPH oxidase p22phox gene reduces the risk of coronary artery disease in a random sample of Egyptian population.

The p22phox protein subunit is essential for NADPH oxidase activity. The prevalence of C242T variants of p22phox gene was studied in 101 healthy Egyptian controls and 104 acute myocardial infarction (AMI) Egyptian patients. Contribution of oxidative stress, represented by serum oxidized-LDL (ox-LDL), in development of AMI was also examined and correlated with C242T gene variants. Genotyping and ox-LDL were assessed by PCR-RFLP and ELISA. Results showed that wild type CC genotype is prevalent in 27 % of controls; CT and TT are in 72 and 1 %. In patients, the distribution was 40.2, 59.8 and 0 % for CC, CT and TT; respectively, showing a significant difference (p = 0.0259). Serum ox-LDL levels were higher in patients than controls (p ≤ 0.0001). Subjects having CT genotype had lower levels of ox-LDL than CC genotype (p ≤ 0.005). C242T polymorphism of p22phox gene of NADPH oxidase is a novel genetic marker associated with reduced susceptibility to AMI.

Interplay of vitamin D and nitric oxide in post-menopausal knee osteoarthritis.

AIM: Mounting evidence has presented nitric oxide (NO) and vitamin D (vitD) as having independently complex roles in osteoarthritis (OA). However, a mechanistic or an observational connection between them has never been investigated in the disease. This study investigates the correlation between circulating 25-hydroxyvitamin D [25(OH)D] and total NO as nitrate/nitrite (NO x ) in patients with knee OA. METHODS: The recruited subjects comprised 36 post-menopausal women with knee OA, ages 50-60 years, as well as 10 healthy males, 20-30 years of age. 25(OH)D and NO x levels were determined using high-performance liquid chromatography and spectrophotometrically using Griess reaction, respectively. RESULTS: The mean (SEM) 25(OH)D and NO x concentrations of OA patients were 25.0 (1.6) ng/mL and 32.45 (2.18) µM, respectively, and 35.4 (2.1) ng/mL and 25.49 (2.23) µM, respectively, for controls. Comparison of mean 25(OH)D and NO x concentrations of OA patients and controls yielded significant results (P = 0.001 and 0.034, respectively). NO notably decreased with decreasing 25(OH)D concentration in patients. However, significant results in terms of mean NO x concentration were observed in the comparison of normal and deficient vitD OA groups (P = 0.048). CONCLUSION: Results suggest that vitD increases NO production and inducible NO synthase expression in osteoarthritic chondrocytes possibly leading to a protective effect.

Role of Hydrogen Sulfide in Oncological and Non-Oncological Disorders and Its Regulation by Non-Coding RNAs: A Comprehensive Review.

Recently, myriad studies have defined the versatile abilities of gasotransmitters and their synthesizing enzymes to play a "Maestro" role in orchestrating several oncological and non-oncological circuits and, thus, nominated them as possible therapeutic targets. Although a significant amount of work has been conducted on the role of nitric oxide (NO) and carbon monoxide (CO) and their inter-relationship in the field of oncology, research about hydrogen sulfide (H2S) remains in its infancy. Recently, non-coding RNAs (ncRNAs) have been reported to play a dominating role in the regulation of the endogenous machinery system of H2S in several pathological contexts. A growing list of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are leading the way as upstream regulators for H2S biosynthesis in different mammalian cells during the development and progression of human diseases; therefore, their targeting can be of great therapeutic benefit. In the current review, the authors shed the light onto the biosynthetic pathways of H2S and their regulation by miRNAs and lncRNAs in various oncological and non-oncological disorders.

Association of suboptimal 25-hydroxyvitamin D levels with knee osteoarthritis incidence in post-menopausal Egyptian women.

The main aim of this study was to assess the vitamin D status of newly diagnosed knee osteoarthritis (OA) patients. Thirty-six post-menopausal Egyptian females of mean age 54.7 years with knee OA were recruited alongside ten healthy males of mean age 25.8 years. The body mass index of all knee OA patients was calculated, and full patient history was gathered to screen for vitamin D status altering conditions or medication. Total 25-hydroxyvitamin D [25(OH)D] was assessed using HPLC which permitted an individualized assessment of both forms of the vitamin's metabolite, 25(OH)D2 and 25(OH)D3. Results showed that mean 25(OH)D ± SEM concentrations were 25.0 ± 1.6 ng/mL and 35.4 ± 2.1 ng/mL for female patients and healthy male participants, respectively. Student's t test statistical comparison yielded a significant result (P = 0.001) when comparing healthy and osteoarthritic participants, and insignificant results when comparing patients of different BMI class, and the different forms of the vitamin's metabolite (P = 0.184 and 0.335, respectively). The 95 % confidence interval associated with knee OA incidence is 21.9-28.1 ng/mL, which is in the vitamin D insufficiency zone. In Conclusion, suboptimal 25(OH)D levels are associated with knee OA incidence in post-menopausal Egyptian females which further fortifies accumulating evidence.

Let-7a/cMyc/CCAT1/miR-17-5p Circuit Re-sensitizes Atezolizumab Resistance in Triple Negative Breast Cancer through Modulating PD-L1.

BACKGROUND: Triple negative breast cancer (TNBC) is an immunogenically hot tumor. The immune checkpoint blockades (ICBs) have been recently emerged as promising therapeutic candidates for several malignancies including TNBC. Yet, the development of innate and/or adaptive resistance by TNBC patients towards ICBs such as programmed death-ligand 1 (PD-L1) inhibitors (e.g. Atezolizumab) shed the light on importance of identifying the underlying mechanisms regulating PD-L1 in TNBC. Recently, it was reported that non-coding RNAs (ncRNAs) perform a fundamental role in regulating PD-L1 expression in TNBC. Hence, this study aims to explore a novel ncRNA axis tuning PD-L1 in TNBC patients and investigate its possible involvement in fighting Atezolizumab resistance. METHODS: In-silico screening was executed to identify ncRNAs that could potentially target PD-L1. Screening of PD-L1 and the nominated ncRNAs (miR-17-5p, let-7a and CCAT1 lncRNA) was performed in BC patients and cell lines. Ectopic expression and/or knockdown of respective ncRNAs were performed in MDA-MB-231. Cellular viability, migration and clonogenic capacities were evaluated using MTT, scratch assay and colony-forming assay, respectively. RESULTS: PD-L1 was upregulated in BC patients, especially in TNBC patients. PD-L1 is positively associated with lymph node metastasis and high Ki-67 in recruited BC patients. Let-7a and miR-17-5p were nominated as potential regulators of PD-L1. Ectopic expression of let-7a and miR-17-5p caused a noticeable reduction in PD-L1 levels in TNBC cells. In order to investigate the whole ceRNA circuit regulating PD-L1 in TNBC, intensive bioinformatic studies were performed. The lncRNA, Colon Cancer-associated transcript 1 (CCAT1), was reported to target PD-L1 regulating miRNAs. Results showed that CCAT1 is an upregulated oncogenic lncRNA in TNBC patients and cell lines. CCAT1 siRNAs induced a noticeable reduction in PD-L1 levels and a marked increase in miR-17-5p level, building up a novel regulatory axis CCAT1/miR-17-5p/PD-L1 in TNBC cells that was tuned by the let-7a/c-Myc engine. On the functional level, co-treatment of CCAT-1 siRNAs and let-7a mimics efficiently relieved Atezolizumab resistance in MDA-MB-231 cells. CONCLUSION: The present study revealed a novel PD-L1 regulatory axis via targeting let-7a/c-Myc/CCAT/miR-17-5p. Additionally, it sheds the light on the potential combinational role of CCAT-1 siRNAs and Let-7a mimics in relieving Atezolizumab resistance in TNBC patients.

High Glucose Increases DNA Damage and Elevates the Expression of Multiple DDR Genes.

The DNA Damage Response (DDR) pathways sense DNA damage and coordinate robust DNA repair and bypass mechanisms. A series of repair proteins are recruited depending on the type of breaks and lesions to ensure overall survival. An increase in glucose levels was shown to induce genome instability, yet the links between DDR and glucose are still not well investigated. In this study, we aimed to identify dysregulation in the transcriptome of normal and cancerous breast cell lines upon changing glucose levels. We first performed bioinformatics analysis using a microarray dataset containing the triple-negative breast cancer (TNBC) MDA-MB-231 and the normal human mammary epithelium MCF10A cell lines grown in high glucose (HG) or in the presence of the glycolysis inhibitor 2-deoxyglucose (2DG). Interestingly, multiple DDR genes were significantly upregulated in both cell lines grown in HG. In the wet lab, we remarkably found that HG results in severe DNA damage to TNBC cells as observed using the comet assay. In addition, several DDR genes were confirmed to be upregulated using qPCR analysis in the same cell line. Our results propose a strong need for DDR pathways in the presence of HG to oppose the severe DNA damage induced in cells.

Dual targeting of H2S synthesizing enzymes; cystathionine ß-synthase and cystathionine γ-lyase by miR-939-5p effectively curbs triple negative breast cancer.

Introduction: Hydrogen sulfide (H2S) has been recently scrutinized for its critical role in aggravating breast cancer (BC) tumorigenicity. Several cancers aberrantly express H2S synthesizing enzymes; Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE). However, their levels and interdependence in BC require further studies. Objectives: Firstly, this study aimed to demonstrate a comparative expression profile of H2S synthesizing enzymes in BC vs normal tissue. Moreover, to investigate the reciprocal relationship between CBS and CSE and highlight the importance of dual targeting. Finally, to search for a valid dual repressor of the H2S synthesizing enzymes that could cease H2S production and reduce TNBC pathogenicity. Methods: Pairwise analysis of tumor vs. normal tissues of 40 BC patients was carried out. The TNBC cell line MDA-MB-231 was transfected with oligonucleotides to study the H2S mediated molecular mechanisms. In silico screening was performed to identify dual regulator(s) for CBS and CSE. Gene expression analysis was performed using qRT-PCR and was confirmed on protein level using Western blot. TNBC hallmarks were evaluated using MTT, migration, and clonogenicity assays. H2S levels were detected using a AzMc fluorescent probe. Results: BC tissues exhibited elevated levels of both CBS and CSE. Interestingly, upon CBS knockdown, CSE levels increased compensating for H2S production in TNBC cells, underlining the importance of dually targeting both enzymes in TNBC. In silico screening suggested miR-939-5p as a regulator of both CBS and CSE with high binding scores. Low expression levels of miR-939-5p were found in BC tissues, especially the aggressive subtypes. Ectopic expression of miR-939-5p significantly repressed CBS and CSE transcript and protein levels, diminished H2S production and attenuated TNBC hallmarks. Moreover, it improved the immune surveillance potency of TNBC cells through up regulating the NKG2D ligands, MICB and ULBP2 and reducing the immune suppressive cytokine IL-10. Conclusion: This study sheds light on the reciprocal relationship between CBS and CSE and on the importance of their dual targeting, particularly in TNBC. It also postulates miR-939-5p as a potent dual repressor for CBS and CSE overcoming their redundancy in H2S production, a mechanism that can potentially attenuate TNBC oncogenicity and improves the immunogenic response.