Deregulations of RNA Pol II Subunits in Cancer

Deregulated transcription is a well-known characteristic of cancer cells, with differentially expressed genes being a common feature of several cancers. Often, deregulated transcription is a consequence of alterations in transcription factors (TFs), which play a crucial role in gene expression and can act as tumour suppressors or proto-oncogenes. In eukaryotic organisms, transcription is carried out by three distinct RNA polymerase complexes: Pol I, Pol II, and Pol III. Pol II, specifically, is responsible for transcribing messenger RNA (mRNA), the protein coding part of the genome, as well as long non-coding RNAs (lncRNAs). While there is considerable research on the impact of specific deregulated transcription factors in cancer development, there is a lack of studies focusing on defects within the RNA polymerase complexes and their subunits. This review aims to shed light in particular on the Pol II complex and highlight the deregulation of its subunits that have a significant impact on tumour development, prognosis, and survival. By providing a comprehensive overview of our current understanding of Pol II subunits in cancer, this review emphasizes the importance of further research in this area. It suggests that exploring these subunits’ deregulations could lead to the identification of valuable biomarkers and potential therapeutic targets, making it a topic of collective interest

Parathyroid Hormone (PTH)-Related Peptides Family: An Intriguing Role in the Central Nervous System

: Parathyroid Hormone (PTH) plays a crucial role in the maintenance of calcium homeostasis directly acting on bone and kidneys and indirectly on the intestine. However, a large family of PTH-related peptides exists that exerts other physiological effects on different tissues and organs, such as the Central Nervous System (CNS). In humans, PTH-related peptides are Parathyroid Hormone (PTH), PTH-like hormones (PTHrP and PTHLH), and tuberoinfundibular peptide of 39 (TIP39 or PTH2). With different affinities, these ligands can bind parathyroid receptor type 1 (PTH1R) and type 2 (PTH2R), which are part of the type II G-protein-coupled-receptors (GPCRs) family. The PTH/PTHrP/PTH1R system has been found to be expressed in many areas of the brain (hippocampus, amygdala, hypothalamus, caudate nucleus, corpus callosum, subthalamic nucleus, thalamus, substantia nigra, cerebellum), and literature data suggest the system exercises a protective action against neuroinflammation and neurodegeneration, with positive effects on memory and hyperalgesia. TIP39 is a small peptide belonging to the PTH-related family with a high affinity for PTH2R in the CNS. The TIP39/PTH2R system has been proposed to mediate many regulatory and functional roles in the brain and to modulate auditory, nociceptive, and sexual maturation functions. This review aims to summarize the knowledge of PTH-related peptides distribution and functions in the CNS and to highlight the gaps that still need to be filled

An integrated human health risk assessment framework for alkylphenols due to drinking water and edible crop consumption

INTRODUCTION The scarcity of clean freshwater is becoming a major issue for present and future generations, especially in densely urbanised areas. This situation promotes the potential cross-contamination of different environmental compartments by contaminants of emerging concern (CECs) which, in fact, have already been detected worldwide in surface water, groundwater and soils. In particular, the CECs released by wastewater treatment plants (WWTPs) can end up both in the recipient surface water and groundwater, both of which are used as drinking water (DW) sources. Furthermore, if those water sources and reclaimed wastewater are used for irrigation, CECs can be directly absorbed by crops intended for human consumption or accumulate in soil and translocate to crops over time. Hence, both DW and edible crops are critical CEC exposure pathways for humans, the combined effect of which requires further investigation. This work is aimed at developing an integrated framework for a quantitative chemical risk assessment due to CECs in complex multiple-use scenarios, combining DW and edible crop consumption, as a decision-making support tool for optimising solutions to minimise risks and social costs. METHODOLOGY The developed procedure includes several steps. Firstly, the analysed system boundaries are defined, to evaluate all the phenomena affecting the fate of CECs from source to end user. Then, CEC migration (e.g. diffusion in surface water, infiltration in soil, uptake by food crops) and human exposure (via water and edible crop consumption) are modelled in an integrated framework as a function of boundary conditions, CECs and by-products characteristics, and proposed interventions. Exposure models are calibrated through literature data, field monitoring and lab tests where, for instance, the CECs’ fate and uptake by vegetables from contaminated soils have been investigated. In the hazard assessment step, a toxicological characterisation was performed to obtain single CEC adverse effect potencies, aimed at applying the Relative Potency Factors methodology for combining CECs that affect the same endpoint. Lastly, exposure and hazard assessment steps are combined to quantitatively estimate the risk to human health from a mixture of CECs, which includes uncertainty analyses to account for knowledge gaps and to provide decision-makers with the confidence level of the risk estimation. RESULTS The developed quantitative risk assessment procedure has been applied to a case study on the mixture of two alkylphenols, i.e. bisphenol-A (BPA) and nonylphenol (NP), used as reference CECs. Literature and field-monitoring data were used to feed the model, with an estimate of BPA and NP concentration in DW up to 0.1 and 0.35 μg/L, respectively, as a function of different system boundary conditions. As for their uptake in edible crops, lab tests with contaminated soil (BPA=75 μg/kg and NP=10 mg/kg, according to the range reported in literature for soil irrigated with reclaimed wastewater or amended with biosolids) demonstrated a significant transfer of NP from soil to vegetables, with concentrations of up to 230 μg/kg fresh weight (f.w.) in the edible parts. No BPA (<8 μg/kg f.w.) was found in vegetables, unlike its metabolite para-hydroxybenzoic acid (up to 56 μg/kg f.w). Those results highlight that both DW and edible crop consumption exposure pathways are critical for the risk to human health due to BPA, NP and their by-products. Several interventions in WWTPs or in DW treatment plants and distribution networks were simulated, demonstrating promising cumulative risk reduction. DISCUSSION Integrated modelling of the fate of CEC mixtures in complex multiple-use water systems, combined with quantitative risk assessment, has proven to be an effective tool to identify the main causes of risk for humans and to assign the various CEC source contributions. Lab tests proved to be useful to investigate the fate of CECs, including metabolites, in the soil system and potential transfer to food crops, corroborating the information from literature and monitoring data for model calibration. Integrated modelling also made it possible to explore several intervention strategies to be adopted at different points of the water system, identifying those that achieve the minimum overall mixture risk. Moreover, in addition to CEC toxicological characterisation, this procedure allows decision-makers to prioritise CECs to be regulated not only based on their exposure levels but looking at their contribution to the overall mixture risk. Lastly, uncertainty analysis made it possible to properly consider the availability and quality of CEC data, especially as regards their physical-chemical behaviour and toxicity, thereby providing the degree of confidence for the estimated risk, which is a key factor for taking informed decisions concerning CEC

BET inhibition induces HEXIM1- and RAD51-dependent conflicts between transcription and replication

Summary: BET bromodomain proteins are required for oncogenic transcription activities, and BET inhibitors have been rapidly advanced into clinical trials. Understanding the effects of BET inhibition on processes such as DNA replication will be important for future clinical applications. Here, we show that BET inhibition, and specifically inhibition of BRD4, causes replication stress through a rapid overall increase in RNA synthesis. We provide evidence that BET inhibition acts by releasing P-TEFb from its inhibitor HEXIM1, promoting interference between transcription and replication. Unusually, these transcription-replication conflicts do not activate the ATM/ATR-dependent DNA damage response but recruit the homologous recombination factor RAD51. Both HEXIM1 and RAD51 promote BET inhibitor-induced fork slowing but also prevent a DNA damage response. Our data suggest that BET inhibitors slow replication through concerted action of transcription and recombination machineries and shed light on the importance of replication stress in the action of this class of experimental cancer drugs. : Bowry et al. show that BET inhibitors, emerging cancer therapeutics that target transcription programs, cause conflicts between transcription and replication by deregulating P-TEFb. These conflicts recruit the homologous recombination factor RAD51, which slows down replication and suppresses DNA damage. This highlights the importance of replication stress for BET inhibitor treatment. Keywords: JQ1, I-BET151, BRD4, P-TEFb, homologous recombination, replication fork, replication stress, DNA damage, cance

Functional characterization of a CDKN1B mutation in a Sardinian kindred with multiple endocrine neoplasia type 4 (MEN4)

Inactivating germline mutations of the CDKN1B gene, encoding for the nuclear cyclin-dependent kinase inhibitor p27kip1 protein, have been reported in patients with multiple endocrine neoplasia type 4 (MEN4), a MEN1-like phenotype without MEN1 mutations. The aim of this study was to in vitro characterize the germline CDKN1B mutation c.374_375delCT (S125X) we detected in a patient with MEN4. The proband was affected by multiglandular primary hyperparathyroidism and gastro-entero-pancreatic tumors. We carried out subcellular localization experiments transfecting into eukaryotic HeLa and GH3 cell lines plasmid vectors expressing the CDKN1B wild type (wt) or mutant cDNA. Western blot studies showed that fusion proteins were expressed at equal levels. The mutated protein was shorter compared to the wt protein and lacked the highly conserved C-terminal domain, which includes the bipartite nuclear localization signal at amino acids 152/153 and 166/168. In HeLa and GH3 cells wt p27 localized in the nucleus whereas the p27_S125X protein was retained in the cytoplasm predicting the loss of tumor suppressive function. The proband's tumoral parathyroid tissue did not show allelic loss, since wt and mutant alleles were both present by sequencing the somatic DNA. Immunohistochemistry showed a complete loss of nuclear p27 expression in the parathyroid adenoma removed by the patient at the second surgery. In conclusion, our study confirms the pathogenic role of the c.374_375delCT CDKN1B germline mutation in a patient with MEN4

Functional characterization of a CDKN1B mutation in a Sardinian kindred with multiple endocrine neoplasia type 4 (MEN4)

Inactivating germline mutations of the CDKN1B gene, encoding for the nuclear cyclin-dependent kinase inhibitor p27kip1 protein, have been reported in patients with multiple endocrine neoplasia type 4 (MEN4), a MEN1-like phenotype without MEN1 mutations. The aim of this study was to in vitro characterize the germline CDKN1B mutation c.374_375delCT (S125X) we detected in a patient with MEN4. The proband was affected by multiglandular primary hyperparathyroidism and gastro-entero-pancreatic tumors. We carried out subcellular localization experiments transfecting into eukaryotic HeLa and GH3 cell lines plasmid vectors expressing the CDKN1B wild type (wt) or mutant cDNA. Western blot studies showed that fusion proteins were expressed at equal levels. The mutated protein was shorter compared to the wt protein and lacked the highly conserved C-terminal domain, which includes the bipartite nuclear localization signal at amino acids 152/153 and 166/168. In HeLa and GH3 cells wt p27 localized in the nucleus whereas the p27_S125X protein was retained in the cytoplasm predicting the loss of tumor suppressive function. The proband's tumoral parathyroid tissue did not show allelic loss, since wt and mutant alleles were both present by sequencing the somatic DNA. Immunohistochemistry showed a complete loss of nuclear p27 expression in the parathyroid adenoma removed by the patient at the second surgery. In conclusion, our study confirms the pathogenic role of the c.374_375delCT CDKN1B germline mutation in a patient with MEN4

Is There a Crucial Link Between Vitamin D Status and Inflammatory Response in Patients With COVID-19?

Background: Hypovitaminosis D has been suggested to play a possible role in coronavirus disease 2019 (COVID-19) infection. Methods: The aim of this study is to analyze the relationship between vitamin D status and a biochemical panel of inflammatory markers in a cohort of patients with COVID-19. A secondary endpoint was to evaluate the correlation between 25OHD levels and the severity of the disease. Ninety-three consecutive patients with COVID-19-related pneumonia were evaluated from March to May 2020 in two hospital units in Pisa, in whom biochemical inflammatory markers, 25OHD levels, P/F ratio at nadir during hospitalization, and complete clinical data were available. Results: Sixty-five percent of patients presented hypovitaminosis D (25OHD ≤ 20 ng/ml) and showed significantly higher IL-6 [20.8 (10.9–45.6) vs. 12.9 (8.7–21.1) pg/ml, p = 0.02], CRP [10.7 (4.2–19.2) vs. 5.9 (1.6–8.1) mg/dl, p = 0.003], TNF-a [8.9 (6.0–14.8) vs. 4.4 (1.5–10.6) pg/ml, p = 0.01], D-dimer [0.53 (0.25–0.72) vs. 0.22 (0.17–0.35) mg/l, p = 0.002], and IL-10 [3.7 (1.8–6.9) vs. 2.3 (0.5–5.8) pg/ml, p = 0.03]. A significant inverse correlation was found between 25OHD and all these markers, even adjusted for age and sex. Hypovitaminosis D was prevalent in patients with severe ARDS, compared with the other groups (75% vs. 68% vs. 55%, p < 0.001), and 25OHD levels were lower in nonsurvivor patients. Conclusions: The relationship between 25OHD levels and inflammatory markers suggests that vitamin D status needs to be taken into account in the management of these patients. If vitamin D is a marker of poor prognosis or a possible risk factor with beneficial effects from supplementation, this still needs to be elucidated

Mutation of cancer driver MLL2 results in transcription stress and genome instability

Genome instability is a recurring feature of tumorigenesis. Mutation in MLL2, encoding a histone methyltransferase, is a driver in numerous different cancer types, but the mechanism is unclear. Here, we present evidence that MLL2 mutation results in genome instability. Mouse cells in which MLL2 gene deletion can be induced display elevated levels of sister chromatid exchange, gross chromosomal aberrations, 53BP1 foci, and micronuclei. Human MLL2 knockout cells are characterized by genome instability as well. Interestingly, MLL2 interacts with RNA polymerase II (RNAPII) and RECQL5, and, although MLL2 mutated cells have normal overall H3K4me levels in genes, nucleosomes in the immediate vicinity of RNAPII are hypomethylated. Importantly,MLL2 mutated cells display signs of substantial transcription stress, and the most affected genes overlap with early replicating fragile sites, show elevated levels ofγH2AX, and suffer frequent mutation. The requirement for MLL2 in the maintenance of genome stability in genes helps explain its widespread role in cancer and points to transcription stress as a strong driver in tumorigenesis