Mechano-Sensing Channel PIEZO2 Enhances Invasive Phenotype in Triple-Negative Breast Cancer.

BACKGROUND: Mechanically gated PIEZO channels lead to an influx of cations, activation of additional Ca2+ channels, and cell depolarization. This study aimed to investigate PIEZO2's role in breast cancer. METHODS: The clinical relevance of PIEZO2 expression in breast cancer patient was analyzed in a publicly available dataset. Utilizing PIEZO2 overexpressed breast cancer cells, and in vitro and in vivo experiments were conducted. RESULTS: High expression of PIEZO2 was correlated with a worse survival in triple-negative breast cancer (TNBC) but not in other subtypes. Increased PEIZO2 channel function was confirmed in PIEZO2 overexpressed cells after mechanical stimulation. PIEZO2 overexpressed cells showed increased motility and invasive phenotypes as well as higher expression of SNAIL and Vimentin and lower expression of E-cadherin in TNBC cells. Correspondingly, high expression of PIEZO2 was correlated with the increased expression of epithelial-mesenchymal transition (EMT)-related genes in a TNBC patient. Activated Akt signaling was observed in PIEZO2 overexpressed TNBC cells. PIEZO2 overexpressed MDA-MB-231 cells formed a significantly higher number of lung metastases after orthotopic implantation. CONCLUSION: PIEZO2 activation led to enhanced SNAIL stabilization through Akt activation. It enhanced Vimentin and repressed E-cadherin transcription, resulting in increased metastatic potential and poor clinical outcomes in TNBC patients.

Ion Current-Based Proteomic Profiling for Understanding the Inhibitory Effect of Tumor Necrosis Factor Alpha on Myogenic Differentiation.

Despite a demonstrated role for TNF-α in promoting muscle wasting and cachexia, the associated molecular mechanisms and signaling pathways of myoblast differentiation dysregulated by TNF-α remain poorly understood. This study presents well-controlled proteomic profiling as a means to investigate the mechanisms of TNF-α-regulated myogenic differentiation. Primary human muscle precursor cells (MPCs) cultured in growth medium (GM), differentiation medium (DM) to induce myogenic differentiation, and DM with 20 ng/mL of TNF-α (n = 5/group) were comparatively analyzed by an ion current-based quantitative platform consisting of reproducible sample preparation/on-pellet digestion, a long-column nano-LC separation, and ion current-based differential analysis. The inhibition of myogenic differentiation by TNF-α was confirmed by reduced formation of multinucleated myotubes and the recovered expression of altered myogenic proteins such as MYOD and myogenin during myogenic differentiation. Functional analysis and validation by immunoassay analysis suggested that the cooperation of NF-κB and STAT proteins is responsible for dysregulated differentiation in MPCs by TNF-α treatment. Increased MHC class I components such as HLA-A, HLA-B, HLA-C, and beta-2-microglobulin were also observed in cultures in DM treated with TNF-α. Interestingly, inhibition of the cholesterol biosynthesis pathway during myogenic differentiation induced by serum starvation was not recovered by TNF-α treatment, which combined with previous reports, implies that this process may be an early event of myogenesis. This finding could lay the foundation for the potential use of statins in modulating myogenesis through cholesterol, for example, in stem cell-based myocardial infarction treatment, where differentiation of myoblasts and stem cells into force-generating mature muscle cells is a key step to the therapeutic capacity. In conclusion, the landscapes of altered transcription regulators, metabolic processes, and signaling pathways in MPCs are revealed in the regulation of myogenic differentiation by TNF-α, which is valuable for myogenic cellular therapeutics.

Mutations in mitochondrial DNA polymerase-gamma promote breast tumorigenesis.

Decreased mitochondrial oxidative phosphorylation (OXPHOS) is one of the hallmarks of cancer. To date, the identity of nuclear gene(s) responsible for decreased OXPHOS in tumors remains unknown. It is also unclear whether mutations in nuclear gene(s) responsible for decreased OXPHOS affect tumorigenesis. Polymerase-gamma (POLG) is the only DNA polymerase known to function in human mitochondria. Mutations in POLG are known to cause mitochondrial DNA (mtDNA) depletion and decreased OXPHOS, resulting in mtDNA depletion syndrome in humans. We therefore sequenced all coding exons (2-23) and flanking intron/splice junctions of POLG in breast tumors. We found that the POLG gene was mutated in 63% of breast tumors. We identified a total of 17 mutations across the POLG gene. Mutations were found in all three domains of the POLG protein, including T251I (the exonuclease domain), P587L (the linker region) and E1143G (the polymerase domain). We identified two novel mutations that include one silent (A703A) and one missense (R628Q) mutation in the evolutionarily conserved POLG linker region. In addition, we identified three novel mutations in the intronic region. Our study also revealed that mtDNA was depleted in breast tumors. Consistently, mutant POLG, when expressed in breast cancer cells, induced a depletion of mtDNA, decreased mitochondrial activity, decreased mitochondrial membrane potential, increased levels of reactive oxygen species and increased Matrigel invasion. Together, our study provides the first comprehensive analysis of the POLG gene mutation in human cancer and suggests a function for POLG (1) in decreased OXPHOS in cancers and (2) in promoting tumorigenicity.

[Total knee arthroplasty in the cardiac transplant patient--a case report]. / Aloartroplastika koljena u bolesnika s transplantiranim srcem--prikaz bolesnika.

We present a 66 year-old diabetic patient who 4 years ago underwent cardiac transplantation and 1 year ago was implanted a total knee arthroplasty due to arthrosis. In the literature and reference books the arthroplasty in such patients is mainly related to aseptic necrosis of the joints as a result of taking immunosuppressive therapy (corticosteroids). Our patient didn't have an aseptic necrosis but a classical arthrosis of the knee. Although our patient didn't have so strong pain as patients with aseptic necrosis, we decided to do the knee arthroplasty. The procedure went properly and postoperative time, recovery and knee function were excellent. Implantation of endoprosthesis in a cardiac transplant patient shouldn't be retained only for cases of aseptic necrosis of a joint, which occurs due to immunosuppressive therapy, but also for the cases of classical arthrosis. Good preoperative, perioperative and postoperative care and good surgery technic make this procedure safe in cardiac transplant patients as in nontransplanted patients. To the best of our knowledge this is the first case of implantation of knee endoprosthesis in a cardiac transplant patient in Croatia.

Genetic determinants of mitochondrial response to arsenic in yeast Saccharomyces cerevisiae.

We have used yeast Saccharomyces cerevisiae as a tool to identify the importance of mitochondrial processes involved in arsenic-induced carcinogenicity in humans. We screened 466 single-gene knockout strains of yeast S. cerevisiae known to be involved in biogenesis of mitochondria for sodium arsenite (AsIII) and sodium arsenate (AsV) sensitivity. We identified 72 arsenite-sensitive and 81 arsenate-sensitive mutants. We categorized the identified mutants based on the various mitochondrial processes, including nucleic acid metabolism, oxidative phosphorylation, protein synthesis, and vacuolar acidification. We have identified 65 human orthologues to proteins involved in arsenite sensitivity and 3 human orthologues to arsenite resistance. Furthermore, 23 human orthologues to arsenate sensitivity and 20 human orthologues to arsenate-resistant proteins, including MSH3, COX10, GCSH, PPOX, and MTHFD1, were also identified. Using PathwayAssist software, we did cellular network analysis between identified mitochondrial proteins. Three types of interactions, (a) protein-protein interactions, (b) common transcriptional regulators, and (c) common target genes, were identified. We found that RTG (retrograde) genes involved in mitochondria-to-nucleus signaling regulate both arsenite sensitivity and resistance. Furthermore, our study revealed that ABF1, a multifunctional transcriptional factor, regulates genes involved in both arsenite and arsenate sensitivity and resistance. However, REB1 and RAP1 transcriptional regulators were common to only arsenate- and arsenite-sensitive genes, respectively. These studies indicate that multiple pathways involved in mitochondrial biogenesis protect yeast S. cerevisiae from arsenic-induced toxicity. Together, our studies suggest that evolutionary conserved mitochondrial networks identified in yeast S. cerevisiae must play an important role in arsenic-induced carcinogenesis in humans.

DNA replication stress is a determinant of chronological lifespan in budding yeast.

The chronological lifespan of eukaryotic organisms is extended by the mutational inactivation of conserved growth-signaling pathways that regulate progression into and through the cell cycle. Here we show that in the budding yeast S. cerevisiae, these and other lifespan-extending conditions, including caloric restriction and osmotic stress, increase the efficiency with which nutrient-depleted cells establish or maintain a cell cycle arrest in G1. Proteins required for efficient G1 arrest and longevity when nutrients are limiting include the DNA replication stress response proteins Mec1 and Rad53. Ectopic expression of CLN3 encoding a G1 cyclin downregulated during nutrient depletion increases the frequency with which nutrient depleted cells arrest growth in S phase instead of G1. Ectopic expression of CLN3 also shortens chronological lifespan in concert with age-dependent increases in genome instability and apoptosis. These findings indicate that replication stress is an important determinant of chronological lifespan in budding yeast. Protection from replication stress by growth-inhibitory effects of caloric restriction, osmotic and other stresses may contribute to hormesis effects on lifespan. Replication stress also likely impacts the longevity of higher eukaryotes, including humans.

Genome-wide screen identifies genes whose inactivation confer resistance to cisplatin in Saccharomyces cerevisiae.

To identify novel genes that mediate cellular resistance to cisplatin, we have screened the collection of Saccharomyces cerevisiae deletion strains. We have found reproducibly 22 genes/open reading frames (ORF), which when deleted, confer resistance to cisplatin at a concentration that is lethal to wild-type cells. Complementation of individual deletion strains with the corresponding wild-type gene abolished cisplatin resistance, confirming that specific gene deletions caused the resistance. Twenty of the genes/ORFs identified have not been previously linked to cisplatin resistance and belong to several distinct functional groups. Major functional groups encode proteins involved in nucleotide metabolism, mRNA catabolism, RNA-polymerase-II-dependent gene regulation and vacuolar transport systems. In addition, proteins that function in ubiquitination, sphingolipid biogenesis, cyclic AMP-dependent signaling, DNA repair, and genome stability are also associated with cisplatin resistance. More than half of the identified genes are known to have sequences or functional homology to mammalian counterparts. Some deletion strains are cross-resistant to selected cytotoxic agents whereas hypersensitive to others. The sensitivity of certain resistant strains to other cytotoxic agents suggests that our findings may point to particular drug combinations that can overcome resistance caused by inactivation of specific genes.