A glycolytic metabolite bypasses "two-hit" tumor suppression by BRCA2.

Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.

A transcriptional response to replication stress selectively expands a subset of Brca2-mutant mammary epithelial cells.

Germline BRCA2 mutation carriers frequently develop luminal-like breast cancers, but it remains unclear how BRCA2 mutations affect mammary epithelial subpopulations. Here, we report that monoallelic Brca2mut/WT mammary organoids subjected to replication stress activate a transcriptional response that selectively expands Brca2mut/WT luminal cells lacking hormone receptor expression (HR-). While CyTOF analyses reveal comparable epithelial compositions among wildtype and Brca2mut/WT mammary glands, Brca2mut/WT HR- luminal cells exhibit greater organoid formation and preferentially survive and expand under replication stress. ScRNA-seq analysis corroborates the expansion of HR- luminal cells which express elevated transcript levels of Tetraspanin-8 (Tspan8) and Thrsp, plus pathways implicated in replication stress survival including Type I interferon responses. Notably, CRISPR/Cas9-mediated deletion of Tspan8 or Thrsp prevents Brca2mut/WT HR- luminal cell expansion. Our findings indicate that Brca2mut/WT cells activate a transcriptional response after replication stress that preferentially favours outgrowth of HR- luminal cells through the expression of interferon-responsive and mammary alveolar genes.

Adult appendicitis score versus Alvarado score: A comparative study in the diagnosis of acute appendicitis.

Background: Acute Appendicitis (AA) is the most common abdominal surgical emergency. It requires proper management to decrease mortality and morbidity. Clinical scoring systems for diagnosing AA aimed to decrease the use of radiological scans and the rate of negative appendectomies (NA). We aim to assess the adult appendicitis score (AAS) in the diagnosis prediction of AA. Method: A retrospective study with 1303 cases of AA is performed. We compared the correlation of AAS and Alvarado scores to postoperative histopathology. Specificity, sensitivity, Positive Predictive Value (PPV), and Negative Predictive Value (NPV) were assessed. ROC was used. Results: AAS risk stratification was applied to the study population. Group I for a low probability, and groups II and III for an intermediate and high probability of AA. We found that 159 patients were matched in group I, 505, and 639 were in groups II and III of AAS, respectively. The correlation between Alvarado and AAS with HP was significant. AAS ≥ 16 presented sensitivity and specificity of 50 % and 75.47 %, respectively, with PPV of 97.96 % and NPV of 6.02 %, with an accuracy of 51.04 %. Regarding AAS ≥ 11, the sensitivity was 88.96 %, specificity was 39.62 %, PPV was 97.2 %, NPV was 13.21 %, and accuracy was 86.95 %. Conclusion: AAS is relatively more accurate than Alvarado's score, especially in selecting a safe candidate for discharge from an emergency. In addition, AAS is found to decrease the need for radiological images and NA rate more than Alvarado.

Uncommon presentation of complicated internal hernia through the appendices epiploicae ring of adhesion: a clinical case study.

Background: Internal hernias are infrequent, yet serious, medical conditions with potentially severe consequences. An internal hernia resulting from an Appendices epiploicae (AE) ring is an especially rare cause, with a mere nine documented instances worldwide. Case report: This report presents the case of a 58-year-old male who suffered from an internal hernia originating from an AE ring. The condition led to a gangrenous small bowel, which was treated by laparotomy and resection of the affected segment, followed by primary anastomosis. The post-operative recovery was favorable. Conclusion: Internal hernias, although rare, warrant immediate attention and early intervention to preclude detrimental outcomes, as illustrated in this particular case.

BRCA2 deficiency reveals that oxidative stress impairs RNaseH1 function to cripple mitochondrial DNA maintenance.

Oxidative stress is a ubiquitous cellular challenge implicated in aging, neurodegeneration, and cancer. By studying pathogenic mutations in the tumor suppressor BRCA2, we identify a general mechanism by which oxidative stress restricts mitochondrial (mt)DNA replication. BRCA2 inactivation induces R-loop accumulation in the mtDNA regulatory region and diminishes mtDNA replication initiation. In BRCA2-deficient cells, intracellular reactive oxygen species (ROS) are elevated, and ROS scavengers suppress the mtDNA defects. Conversely, wild-type cells exposed to oxidative stress by pharmacologic or genetic manipulation phenocopy these defects. Mechanistically, we find that 8-oxoguanine accumulation in mtDNA caused by oxidative stress suffices to impair recruitment of the mitochondrial enzyme RNaseH1 to sites of R-loop accrual, restricting mtDNA replication initiation. Thus, oxidative stress impairs RNaseH1 function to cripple mtDNA maintenance. Our findings highlight a molecular mechanism that links oxidative stress to mitochondrial dysfunction and is elicited by the inactivation of genes implicated in neurodegeneration and cancer.

High-throughput surface marker screen on primary human breast tissues reveals further cellular heterogeneity.

BACKGROUND: Normal human breast tissues are a heterogeneous mix of epithelial and stromal subtypes in different cell states. Delineating the spectrum of cellular heterogeneity will provide new insights into normal cellular properties within the breast tissue that might become dysregulated in the initial stages of cancer. Investigation of surface marker expression provides a valuable approach to resolve complex cell populations. However, the majority of cell surface maker expression of primary breast cells have not been investigated. METHODS: To determine the differences in expression of a range of uninvestigated cell surface markers between the normal breast cell subpopulations, primary human breast cells were analysed using high-throughput flow cytometry for the expression of 242 cell surface proteins in conjunction with EpCAM/CD49f staining. RESULTS: We identified 35 surface marker proteins expressed on normal breast epithelial and/or stromal subpopulations that were previously unreported. We also show multiple markers were equally expressed in all cell populations (e.g. CD9, CD59, CD164) while other surface markers were confirmed to be enriched in different cell lineages: CD24, CD227 and CD340 in the luminal compartment, CD10 and CD90 in the basal population, and CD34 and CD140b on stromal cells. CONCLUSIONS: Our dataset of CD marker expression in the normal breast provides better definition for breast cellular heterogeneity.

Time-resolved single-cell analysis of Brca1 associated mammary tumourigenesis reveals aberrant differentiation of luminal progenitors.

It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the deterministic role of the cell-of-origin and offering a potential explanation for the tissue specificity of BRCA1 tumours.

Study of Toll-like Receptor 3 Gene Polymorphism as a Novel Risk Factor for HCV-related Hepatocellular Carcinoma in Egypt.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a highly aggressive cancer with few treatment options. Toll-like receptor 3 (TLR3) plays a key role in innate immunity and may affect the development of cancers. This study aimed to investigate the association between TLR3 gene polymorphism and HCV-related hepatocellular carcinoma in Egypt. METHODS: This work was conducted on 70 individuals; fifty HCV cirrhotic patients were included in two groups; with HCC (30 patients) and without HCC (20 patients) compared with a group of 20 apparently healthy controls. All of the studied individuals underwent clinical-laboratory evaluation. TLR3 gene single-nucleotide polymorphism (SNP) (+1234C/T) was tested by polymerase chain reaction- restriction fragment length polymorphism. RESULTS: This study reported that the prevalence of TLR3 +1234TT genotype was significantly increased in cirrhotic patients with HCC than without HCC, while it was not detected at all among the controls. When analyzing the TLR3 SNP +1234C/T with different clinical parameters in HCC patients, there was a significant association between+1234C/T SNP; namely TT genotype and each of the hepatic focal lesionsá¾½ number, size and the patientsá¾½ higher Okuda and BCLC stages. No association could be detected between TLR3 SNP and the age, sex, Child-Pugh grades, MELD score or AFP of the studied HCC cases. CONCLUSION: TLR3 gene SN P +1234C/T could be a novel risk factor for the HCV-related HCC among the Egyptian population.

Hypervascular Nodules and Stiffer Liver are Associated with Recurrence after Microwave Ablation in Patients with Hepatocellular Carcinoma: A Double-Center Analysis.

Background and Aims The aim of this study was to detect the most important risk factors for recurrence after microwave ablation (MWA) of hepatocellular carcinoma (HCC). Methods A total of 92 patients with 110 HCC focal lesions (FLs) underwent MWA therapy. All the patients underwent triphasic CT before and after 1 and 3 months of MWA therapy. Complete ablation and recurrence rates were recorded, and the risk factors associated with recurrence were analyzed. Results Regarding the 110 HCC FLs that were detected pre-MWA, adequate ablation was recorded post-MWA procedure in 88 FLs (80%) and incomplete ablation in 22 FLs (showed residual contrast enhancement). However, there were newly detected lesions (17 FLs). The rate of recurrence was significantly higher in patients with multiple larger (> 4 cm) sized and hypervascular nodules. Diabetics were significantly associated with a higher recurrence rate of HCC. The rate of recurrence was significantly higher in patients with baseline level of serum alfa-fetoprotein (AFP) ≥200 ng/mL. Stiffer liver> 25 kPa had higher incidence for recurrence after ablation. Conclusion Meticulous follow-up is mandatory in diabetic patients, patients with AFP > 200 ng/dL starting value, hypervascular large hepatic FL, and in stiffer liver> 25 kPa, as these patients have higher incidence for recurrence after ablation.

Effects of gamma-irradiation on antibiotic resistance and diagnostic molecular markers of methicillin-resistant Staphylococcus aureus in Egyptian cancer patients.

Purpose: This in-vitro study aimed to assess in 120 [40 community-acquired (CA-MRSA) & 80 hospital-acquired (HA-MRSA)] isolates from cancer patients whether the transmissible staphylococcal cassette chromosome mec (SCCmec) typing, and the Panton-Valentine leukocidin (PVL) virulence genes detection could be employed as tools for molecular diagnostic purposes to distinguish both methicillin-resistant Staphylococcus aureus (MRSA) categories in radiotherapy treated cancer patients.Materials and methods: SCCmec typing was determined by the combination of the type of the cassette chromosome recombinase genes (ccr) gene complex and the class of the methicillin resistance (mec) gene complex. Besides, a rapid slide latex agglutination test (LAT) and antibiotic resistance spectrum determination before and after irradiation were performed.Results: In the strict sense, with the effect of irradiation; the presence of SCCmec subtypes IVa (22.5% vs. 10.0%), b (47.5% vs. 25.0%), & d (7.5 vs. 2.5%) or type V (15.0% vs. 7.5%) genetic elements and PVL genes (p < .001) were not proved as a signature for CA-MRSA. While, the larger SCCmec types II, and III elements were not detected in 14, and 19 from the 38, and 36 typed HA-MRSA isolates (p < .001), respectively. Remarkable effects on class A & class B mec gene complex and type2, type 3 & type 5 ccr gene complex and an increase in agglutination reaction strength in response to gamma irradiation external stimulus were observed.Conclusions: Different heterogeneous genetic composition with upregulation mecA gene expression was detected after irradiation in the HA- MRSA studied population. CA-MRSA showed remarkable ability to acquire multi-antibiotic resistance after irradiation and propose a novel paradigm for future chemotherapy against the multi-resistant pathogens whose proliferation especially among immunocompromised cancer patients is on the increase.

Identifying the murine mammary cell target of metformin exposure.

The heterogeneity of breast cancer makes current therapies challenging. Metformin, the anti-diabetic drug, has shown promising anti-cancer activities in epidemiological studies and breast cancer models. Yet, how metformin alters the normal adult breast tissue remains elusive. We demonstrate metformin intake at a clinically relevant dose impacts the hormone receptor positive (HR+) luminal cells in the normal murine mammary gland. Metformin decreases total cell number, progenitor capacity and specifically reduces DNA damage in normal HR+ luminal cells, decreases oxygen consumption rate and increases cell cycle length of luminal cells. HR+ luminal cells demonstrate the lowest levels of mitochondrial respiration and capacity to handle oxidative stress compared to the other fractions, suggesting their intrinsic susceptibility to long-term metformin exposure. Uncovering HR+ luminal cells in the normal mammary gland as the major cell target of metformin exposure could identify patients that would most benefit from repurposing this anti-diabetic drug for cancer prevention/therapy purposes.

Androgen Receptor Signalling Promotes a Luminal Phenotype in Mammary Epithelial Cells.

Androgens influence mammary gland development but the specific role of the androgen receptor (AR) in mammary function is largely unknown. We identified cell subsets that express AR in vivo and determined the effect of AR activation and transgenic AR inhibition on sub-populations of the normal mouse mammary epithelium by flow cytometry and immunohistochemistry. Immunolocalisation of AR with markers of lineage identity was also performed in human breast tissues. AR activation in vivo significantly decreased the proportion of basal cells, and caused an accumulation of cells that expressed a basal cell marker but exhibited morphological features of luminal identity. Conversely, in AR null mice the proportion of basal mammary epithelial cells was significantly increased. Inhibition of AR increased basal but not luminal progenitor cell activity in vitro. A small population of AR-positive cells in a basal-to-luminal phenotype transition was also evident in human breast lobules. Collectively, these data support a role for AR in promoting a luminal phenotype in mammary epithelial cells.

Proliferative heterogeneity of murine epithelial cells in the adult mammary gland.

Breast cancer is the most common cancer in females. The number of years menstruating and length of an individual menstrual cycle have been implicated in increased breast cancer risk. At present, the proliferative changes within an individual reproductive cycle or variations in the estrous cycle in the normal mammary gland are poorly understood. Here we use Fucci2 reporter mice to demonstrate actively proliferating mammary epithelial cells have shorter G1 lengths, whereas more differentiated/non-proliferating cells have extended G1 lengths. We find that cells enter into the cell cycle mainly during diestrus, yet the expansion is erratic and does not take place every reproductive cycle. Single cell expression analyses feature expected proliferation markers (Birc5, Top2a), while HR+ luminal cells exhibit fluctuations of key differentiation genes (ER, Gata3) during the cell cycle. We highlight the proliferative heterogeneity occurring within the normal mammary gland during a single-estrous cycle, indicating that the mammary gland undergoes continual dynamic proliferative changes.

Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities.

The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology.

Purification of Distinct Subsets of Epithelial Cells from Normal Human Breast Tissue.

The mammary epithelium is composed of a variety of specialized cell types that function in a coordinated fashion to produce and eject milk through multiple cycles of pregnancy. The ability to identify and purify these subsets of cells in order to interrogate their growth and differentiation capacities, as well as to characterize the molecular mechanisms that regulate their behavior, is essential in identifying the processes associated with breast cancer initiation and progression. This methods chapter outlines the step-by-step methods for dissociating human breast reduction specimens to a single cell suspension of viable cells. As well, strategies for purifying four distinct subsets of epithelial cells by using fluorescence-activated cell sorting and protocols for interrogating the growth and differentiation properties of these purified cells at clonal densities in adherent culture are also described.

Stem and progenitor cell division kinetics during postnatal mouse mammary gland development.

The cycling properties of mammary stem and progenitor cells is not well understood. To determine the division properties of these cells, we administered synthetic nucleosides for varying periods of time to mice at different stages of postnatal development and monitored the rate of uptake of these nucleosides in the different mammary cell compartments. Here we show that most cell division in the adult virgin gland is restricted to the oestrogen receptor-expressing luminal cell lineage. Our data also demonstrate that the oestrogen receptor-expressing, milk and basal cell subpopulations have telomere lengths and cell division kinetics that are not compatible with these cells being hierarchically organized; instead, our data indicate that in the adult homeostatic gland, each cell type is largely maintained by its own restricted progenitors. We also observe that transplantable stem cells are largely quiescent during oestrus, but are cycling during dioestrus when progesterone levels are high.

Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations.

Heterogeneity within the self-renewal durability of adult hematopoietic stem cells (HSCs) challenges our understanding of the molecular framework underlying HSC function. Gene expression studies have been hampered by the presence of multiple HSC subtypes and contaminating non-HSCs in bulk HSC populations. To gain deeper insight into the gene expression program of murine HSCs, we combined single-cell functional assays with flow cytometric index sorting and single-cell gene expression assays. Through bioinformatic integration of these datasets, we designed an unbiased sorting strategy that separates non-HSCs away from HSCs, and single-cell transplantation experiments using the enriched population were combined with RNA-seq data to identify key molecules that associate with long-term durable self-renewal, producing a single-cell molecular dataset that is linked to functional stem cell activity. Finally, we demonstrated the broader applicability of this approach for linking key molecules with defined cellular functions in another stem cell system.

Mammary stem cells have myoepithelial cell properties.

Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt actin-myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using two independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage-tracing approach we follow the progeny of myoepithelial cells that express α-smooth muscle actin and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy.

The influence of tamoxifen on normal mouse mammary gland homeostasis.

INTRODUCTION: Lineage tracing using inducible genetic labeling has emerged to be a powerful method for interrogating the developmental fate of cells in intact tissues. A common induction mechanism is the use of tamoxifen-dependent Cre recombinase (CreER and CreERT2), but the effects of tamoxifen at doses normally used in lineage-tracing studies on normal adult mammary gland homeostasis are not known. METHODS: We used flow cytometry and immunostaining of intact glands to determine whether varying doses of tamoxifen skew the distribution and the apoptosis and proliferation status of different types of mammary epithelial cells in vivo. We also examined how tamoxifen influences the number of progenitor and mammary repopulating units (MRUs). RESULTS: Our results indicate that ≥5 mg/25 g body weight of tamoxifen induces a transient increase in cell proliferation and in the number of basal cells in the adult mammary epithelium up to 7 days after tamoxifen administration. However, in the medium term (3 weeks), all doses of tamoxifen≥1 mg/25 g body weight result in a decrease in the number of basal and EpCAM+CD49b- luminal cells and a decrease in progenitor cell function. Tamoxifen at doses≥5 mg/25 g body weight induced a transient increase in caspase-3-mediated apoptotic cell death within the mammary epithelium. However, mammary epithelial cell numbers in all subpopulations were restored to their original levels by 8 weeks. No long-lasting effects of tamoxifen on MRU numbers or on pubertal ductal development were observed. CONCLUSION: Tamoxifen can skew the distribution of mammary cell types in a dose-dependent manner, and thus caution must be taken when interpreting lineage-tracing studies using high doses of tamoxifen, particularly when short-duration analyses of a quantitative nature are being performed.

FAS and FAS ligand gene polymorphisms in Egyptian females with preeclampsia.

We aimed to evaluate the association of Fas polymorphism and the Fas ligand with preeclampsia, investigating whether the G 670 Fas gene variant and the Fas Ligand INV2nt 124 G variant had a differential distribution in patients with preeclampsia. The preeclamptic group consisted of 50 pregnant women who developed preeclampsia, while the control group consisted of 50 age-matched pregnant women with uncomplicated pregnancies. Fas and Fas ligand gene polymorphisms were tested using polymerase chain reaction-restriction fragment length polymorphism. Regarding the Fas 670 A>G polymorphism, statistically significant differences were found between the two groups regarding the AA and GG/AG genotypes as well as the A, G allele frequency, while no statistically significant differences were found regarding AG or GG genotypes. Regarding the FasLG IVS2nt 124 A>G polymorphism, no statistically significant differences were found between the two groups studied. Concerning the Fas 670 A>G gene, no statistically significant differences between the severe and mild preeclampsia groups regarding the A allele frequency were found. Concerning the FasLG IVS2nt 124 A>G gene, there were no statistically significant differences between the severe and mild preeclampsia groups regarding the A allele frequency or the G allele frequency. The presence of the Fas gene polymorphism Fas A670G is associated with an increased risk of preeclampsia, while the presence of FasLG IVS2nt 124 A>G gene may be protective against preeclampsia.