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Molecular Carcinogenesis Mar 2022The FoxQ1 is an oncogenic transcription factor that is overexpressed in basal-like and luminal-type human breast cancers when compared to the normal mammary tissue. The...
The FoxQ1 is an oncogenic transcription factor that is overexpressed in basal-like and luminal-type human breast cancers when compared to the normal mammary tissue. The FoxQ1 is implicated in mammary tumor progression. However, the mechanism by which FoxQ1 promotes mammary tumorigenesis is not fully understood. In this study, we present experimental evidence for a novel function of FoxQ1 in the regulation of complex I activity of the electron transport chain. The RNA-seq data from FoxQ1 overexpressing basal-like SUM159 cells revealed a statistically significant increase in the expression of complex I subunits NDUFS1 and NDUFS2 when compared to the empty vector (EV) transfected control cells. Consistent with these results, the basal and ATP-linked oxygen consumption rates were significantly increased by FoxQ1 overexpression in SUM159 and luminal-type MCF-7 cells. The FoxQ1 overexpression in both cell lines resulted in increased intracellular levels of pyruvate, lactate, and ATP that was associated with overexpression of pyruvate dehydrogenase and pyruvate carboxylase proteins. Activity and assembly of complex I were significantly enhanced by FoxQ1 overexpression in SUM159 and MCF-7 cells that correlated with increased mRNA and/or protein levels of complex I subunits NDUFS1, NDUFS2, NDUFV1, and NDUFV2. The chromatin immunoprecipitation assay revealed the recruitment of FoxQ1 at the promoters of both NDUFS1 and NDUFV1. The cell proliferation of SUM159 and MCF-7 cells was increased significantly by overexpression of NDUFS1 as well as NDUFV1 proteins. In conclusion, we propose that increased complex I-linked oxidative phosphorylation is partly responsible for oncogenic role of FoxQ1 at least in human breast cancer cells.
Topics: Adenosine Triphosphate; Breast Neoplasms; Electron Transport; Electron Transport Complex I; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Pyruvic Acid
PubMed: 34939230
DOI: 10.1002/mc.23381 -
Stem Cell Research & Therapy Jan 2021Mesenchymal stem cell (MSC)-based therapy has the potential for immunomodulation and enhancement of tissue regeneration. Genetically modified MSCs that over-express...
BACKGROUND
Mesenchymal stem cell (MSC)-based therapy has the potential for immunomodulation and enhancement of tissue regeneration. Genetically modified MSCs that over-express specific cytokines, growth factors, or chemokines have shown great promise in pre-clinical studies. In this regard, the anti-inflammatory cytokine interleukin (IL)-4 converts pro-inflammatory M1 macrophages into an anti-inflammatory M2 phenotype; M2 macrophages mitigate chronic inflammation and enhance osteogenesis by MSC lineage cells. However, exposure to IL-4 prematurely inhibits osteogenesis of MSCs in vitro; furthermore, IL-4 overexpressing MSCs inhibit osteogenesis in vivo during the acute inflammatory period. Platelet-derived growth factor (PDGF)-BB has been shown to enhance osteogenesis of MSCs with a dose-dependent effect.
METHODS
In this study, we generated a lentiviral vector that produces PDGF-BB under a weak promoter (phosphoglycerate kinase, PGK) and lentiviral vector producing IL-4 under a strong promoter (cytomegalovirus, CMV). We infected MSCs with PDGF-BB and IL-4-producing lentiviral vectors separately or in combination to investigate cell proliferation and viability, protein expression, and the capability for osteogenesis.
RESULTS
PDGF-BB and IL-4 co-overexpression was observed in the co-infected MSCs and shown to enhance cell proliferation and viability, and osteogenesis compared to IL-4 overexpressing MSCs alone.
CONCLUSIONS
Overexpression of PDGF-BB together with IL-4 mitigates the inhibitory effect of IL-4 on osteogenesis by IL-4 overexpressing MSCS. PDGF-BB and IL-4 overexpressing MSCs may be a potential strategy to facilitate osteogenesis in scenarios of both acute and chronic inflammation.
Topics: Becaplermin; Bone Regeneration; Interleukin-4; Mesenchymal Stem Cells; Osteogenesis
PubMed: 33413614
DOI: 10.1186/s13287-020-02086-8 -
Biological & Pharmaceutical Bulletin 2023Phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) is a highly conserved enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI(4,5)P) by phosphorylating...
Phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) is a highly conserved enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI(4,5)P) by phosphorylating phosphatidylinositol 4-phosphate (PI(4)P). Schizosaccharomyces pombe (S. pombe) its3-1 is a loss-of-function mutation in the essential its3 gene that encodes a PI4P5K. Its3 regulates cell proliferation, cytokinesis, cell integrity, and membrane trafficking, but little is known about the regulatory mechanisms of Its3. To identify regulators of Its3, we performed a genetic screening utilizing the high-temperature sensitivity (TS) of its3-1 and identified puf3 and puf4, encoding Pumilio/PUF family RNA-binding proteins as multicopy suppressors of its3-1 cells. The deletions of the PUF domains in the puf3 and puf4 genes resulted in the reduced ability to suppress its3-1, suggesting that the suppression by Puf3 and Puf4 may involve their RNA-binding activities. The gene knockout of Puf4, but not that of Puf3, exacerbated the TS of its3-1. Interestingly, mutant Its3 expression levels both at mRNA and protein levels were lower than those of the wild-type (WT) Its3. Consistently, the overexpression of the mutant its3-1 gene suppressed the its3-1 phenotypes. Notably, Puf3 and Puf4 overexpression increased the mRNA and protein expression levels of both Its3 and Its3-1. Collectively, our genetic screening revealed a functional relationship between the Pumilio/PUF family RNA-binding proteins and PI4P5K.
Topics: RNA, Messenger; RNA-Binding Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 36724944
DOI: 10.1248/bpb.b22-00569 -
Journal of the... 2022A novel collagen called type XXVIII collagen (COL28) is involved in cancer and lung fibrosis. Preliminary data showed that renal tubular epithelial cells could...
BACKGROUND
A novel collagen called type XXVIII collagen (COL28) is involved in cancer and lung fibrosis. Preliminary data showed that renal tubular epithelial cells could proliferate, migrate, and undergo an epithelial-mesenchymal transition (EMT) when COL28 was overexpressed; however, it is still unknown how this occurs and what the underlying mechanism is.
METHODS
We analyzed the differential expression of genes (DEGs) in the stable COL28 overexpression HK-2 cell lines by RNA-sequencing analysis, before which Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analyses were performed. Genes related to COL28 promoting HK-2 cell proliferation and EMT were screened and verified. By using western blot and immunofluorescence, the effects of COL28 on the expression of -SMA, E-cadherin, Snail, HKDC1, and SREBP1 were detected. The effect of COL28 overexpression on renal fibrosis in unilateral ureteral obstruction (UUO) mice was detected by H&E and Masson staining. HKDC1 interference agent was synthesized and transfected into the HK-2 cell line stably overexpressing COL28. In HK-2 cells, the effects of HKDC1 interference on the expression of -SMA, E-cadherin, and Snail were detected.
RESULTS
We screened and verified that HKDC1 was related to COL28 and promoted HK-2 cell proliferation and EMT. WB showed that in HK-2 cells, COL28 overexpression increased -SMA, Snail, HKDC1, and SREBP1 expressions and decreased E-cadherin expression. Overexpression of COL28 aggravated renal interstitial fibrosis in UUO mice; upregulated -SMA, Snail, HKDC1, and SREBP1 expressions; and decreased the E-cadherin protein expression in UUO mice. Interference of HKDC1 expression promoted the E-cadherin protein expression while inhibiting -SMA, Snail, HKDC1, and SREBP1 protein expressions.
CONCLUSION
Overexpression of COL28 can aggravate renal interstitial fibrosis by encouraging renal tubular epithelial cells to undergo EMT, and interference with HKDC1 expression can alleviate fibrosis by reversing EMT induced by COL28 overexpression.
Topics: Animals; Mice; Cadherins; Collagen; Epithelial-Mesenchymal Transition; Fibrosis; Hexokinase; Kidney Diseases; Sterol Regulatory Element Binding Protein 1; Transforming Growth Factor beta1; Ureteral Obstruction
PubMed: 36518326
DOI: 10.1155/2022/9582559 -
Scientific Reports Oct 2022Transgenic mice over-expressing human PRNP or murine Prnp transgenes on a mouse prion protein knockout background have made key contributions to the understanding of...
Transgenic mice over-expressing human PRNP or murine Prnp transgenes on a mouse prion protein knockout background have made key contributions to the understanding of human prion diseases and have provided the basis for many of the fundamental advances in prion biology, including the first report of synthetic mammalian prions. In this regard, the prion paradigm is increasingly guiding the exploration of seeded protein misfolding in the pathogenesis of other neurodegenerative diseases. Here we report that a well-established and widely used line of such mice (Tg20 or tga20), which overexpress wild-type mouse prion protein, exhibit spontaneous aggregation and accumulation of misfolded prion protein in a strongly age-dependent manner, which is accompanied by focal spongiosis and occasional neuronal loss. In some cases a clinical syndrome developed with phenotypic features that closely resemble those seen in prion disease. However, passage of brain homogenate from affected, aged mice failed to transmit this syndrome when inoculated intracerebrally into further recipient animals. We conclude that overexpression of the wild-type mouse prion protein can cause an age-dependent protein misfolding disorder or proteinopathy that is not associated with the production of an infectious agent but can produce a phenotype closely similar to authentic prion disease.
Topics: Animals; Brain Diseases; Humans; Mammals; Mice; Mice, Transgenic; Prion Diseases; Prion Proteins; Prions
PubMed: 36229637
DOI: 10.1038/s41598-022-21608-3 -
Biochimica Et Biophysica Acta.... Dec 2022In the endoplasmic reticulum (ER) membrane, transmembrane (TM) domain insertion occurs through the Sec61 channel with its auxiliary components, including Sec62. Sec62...
In the endoplasmic reticulum (ER) membrane, transmembrane (TM) domain insertion occurs through the Sec61 channel with its auxiliary components, including Sec62. Sec62 interacts with the Sec61 channel and is located on the front side of the Sec61 lateral gate, an entry site for TM domains to the lipid bilayer. Overexpression of Sec62 led to a growth defect in yeast, and we investigated its effects on protein translocation and membrane insertion by pulse labeling of Sec62 client proteins. Our data show that the insertion efficiency of marginally hydrophobic TM segments is reduced upon Sec62 overexpression. This result suggests a potential regulatory role of Sec62 as a gatekeeper of the lateral gate, thereby modulating the insertion threshold of TM segments.
Topics: Endoplasmic Reticulum; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Membrane Transport Proteins; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 36122759
DOI: 10.1016/j.bbamem.2022.184051 -
Virchows Archiv : An International... Oct 2019Overexpression of ETS genes is involved in prostate cancer (PrCa), but there is little information on the non-ERG components of this family. We have investigated ETV1,...
Overexpression of ETS genes is involved in prostate cancer (PrCa), but there is little information on the non-ERG components of this family. We have investigated ETV1, ETV4, and ETV5 overexpression, with or without PTEN loss, and their association with grade group (GG), pathological stage, focality, and PSA recurrence in PrCa. ETS gene expression was analyzed by qPCR in 104 cases. ETV1 and PTEN immunohistochemistry was assessed in TMA sections from 194 additional cases (PSMAR-Biobank, Barcelona, Spain). ETS mRNA overexpression was found in 23.1%, being ETV1 the most frequently overexpressed (18.3%). ETV1 protein overexpression was detected in 30.4% cases (moderate in 19.6%, strong in 10.8%). PTEN protein expression loss was detected in 36.1% cases and was not associated with ETV1. Strong-moderate ETV1 protein overexpression reaches its highest values in GG3-4, whereas its negativity was significantly more common in GG1 tumors (p = 0.034). ETV1-overexpressing tumors were more often unifocal (p = 0.0007) and high stage (p = 0.032). PTEN loss was less common in GG1 (p = 0.012) and showed a trend to be less frequent in pT2 (p = 0.062) tumors. Strong ETV1 immunostaining (histoscore > 177) was associated with shorter time to PSA recurrence in the univariate (p = 0.002) and in the multivariate analysis (p = 0.018). Moreover, when strong ETV1 overexpression was not combined with PTEN loss, its association with PSA recurrence was even stronger (p = 0.0004). In conclusion, non-ERG ETS overexpression, particularly ETV1 overexpression, has a non-negligible role in PrCa. Strong ETV1 protein expression has a negative impact on prostate cancer outcome that seems to be independent of PTEN status.
Topics: Aged; Biomarkers, Tumor; Cytoplasm; DNA-Binding Proteins; Humans; Male; Middle Aged; Prostatic Neoplasms; Transcription Factors
PubMed: 31016435
DOI: 10.1007/s00428-019-02573-1 -
Cell Reports Apr 2024Overexpression of Cyclin E1 perturbs DNA replication, resulting in DNA lesions and genomic instability. Consequently, Cyclin E1-overexpressing cancer cells increasingly...
Overexpression of Cyclin E1 perturbs DNA replication, resulting in DNA lesions and genomic instability. Consequently, Cyclin E1-overexpressing cancer cells increasingly rely on DNA repair, including RAD52-mediated break-induced replication during interphase. We show that not all DNA lesions induced by Cyclin E1 overexpression are resolved during interphase. While DNA lesions upon Cyclin E1 overexpression are induced in S phase, a significant fraction of these lesions is transmitted into mitosis. Cyclin E1 overexpression triggers mitotic DNA synthesis (MiDAS) in a RAD52-dependent fashion. Chemical or genetic inactivation of MiDAS enhances mitotic aberrations and persistent DNA damage. Mitosis-specific degradation of RAD52 prevents Cyclin E1-induced MiDAS and reduces the viability of Cyclin E1-overexpressing cells, underscoring the relevance of RAD52 during mitosis to maintain genomic integrity. Finally, analysis of breast cancer samples reveals a positive correlation between Cyclin E1 amplification and RAD52 expression. These findings demonstrate the importance of suppressing mitotic defects in Cyclin E1-overexpressing cells through RAD52.
Topics: Humans; Cyclin E; Genomic Instability; Rad52 DNA Repair and Recombination Protein; Mitosis; Oncogene Proteins; DNA Replication; Cell Line, Tumor; DNA Damage; DNA; Breast Neoplasms
PubMed: 38625790
DOI: 10.1016/j.celrep.2024.114116 -
The Journal of Physiology Sep 2021The maintenance of mitochondrial integrity is critical for skeletal muscle health. Mitochondrial dynamics play key roles in mitochondrial quality control; however, the...
KEY POINTS
The maintenance of mitochondrial integrity is critical for skeletal muscle health. Mitochondrial dynamics play key roles in mitochondrial quality control; however, the exact role that mitochondrial fission plays in the muscle ageing process remains unclear. Here we report that both Drp1 knockdown and Drp1 overexpression late in life in mice is detrimental to skeletal muscle function and mitochondrial health. Drp1 knockdown in 18-month-old mice resulted in severe skeletal muscle atrophy, mitochondrial dysfunction, muscle degeneration/regeneration, oxidative stress and impaired autophagy. Overexpressing Drp1 in 18-month-old mice resulted in mild skeletal muscle atrophy and decreased mitochondrial quality. Our data indicate that silencing or overexpressing Drp1 late in life is detrimental to skeletal muscle integrity. We conclude that modulating Drp1 expression is unlikely to be a viable approach to counter the muscle ageing process.
ABSTRACT
Sarcopenia, the ageing-related loss of skeletal muscle mass and function, is a debilitating process negatively impacting the quality of life of afflicted individuals. Although the mechanisms underlying sarcopenia are still only partly understood, impairments in mitochondrial dynamics, and specifically mitochondrial fission, have been proposed as an underlying mechanism. Importantly, conflicting data exist in the field and both excessive and insufficient mitochondrial fission were proposed to contribute to sarcopenia. In Drosophila melanogaster, enhancing mitochondrial fission in midlife through overexpression of dynamin-1-like protein (Drp1) extended lifespan and attenuated several key hallmarks of muscle ageing. Whether a similar outcome of Drp1 overexpression is observed in mammalian muscles remains unknown. In this study, we investigated the impact of knocking down and overexpressing Drp1 protein for 4 months in skeletal muscles of late middle-aged (18 months) mice using intra-muscular injections of adeno-associated viruses expressing shRNA targeting Drp1 or full Drp1 cDNA. We report that knocking down Drp1 expression late in life triggers severe muscle atrophy, mitochondrial dysfunctions, degeneration/regeneration, oxidative stress and impaired autophagy. Drp1 overexpression late in life triggered mild muscle atrophy and decreased mitochondrial quality. Taken altogether, our results indicate that both overexpression and silencing of Drp1 in late middle-aged mice negatively impact skeletal muscle mass and mitochondrial health. These data suggest that Drp1 content must remain within a narrow physiological range to preserve muscle and mitochondrial integrity during ageing. Altering Drp1 expression is therefore unlikely to be a viable target to counter sarcopenia.
Topics: Animals; Cytoskeletal Proteins; Drosophila melanogaster; Dynamins; GTP-Binding Proteins; Mice; Mitochondria; Mitochondrial Dynamics; Muscle, Skeletal; Quality of Life
PubMed: 34269418
DOI: 10.1113/JP281752 -
Genes May 2021Colorectal cancer (CRC) remains one of the leading causes of cancer-related death worldwide. The high mortality of CRC is related to its ability to metastasize to...
Colorectal cancer (CRC) remains one of the leading causes of cancer-related death worldwide. The high mortality of CRC is related to its ability to metastasize to distant organs. The kallikrein-related peptidase Kallikrein 6 (KLK6) is overexpressed in CRC and contributes to cancer cell invasion and metastasis. The goal of this study was to identify KLK6-associated markers for the CRC prognosis and treatment. Tumor Samples from the CRC patients with significantly elevated transcript levels were identified in the RNA-Seq data from Cancer Genome Atlas (TCGA) and their expression profiles were evaluated using Gene Ontology (GO), Phenotype and Reactome enrichment, and protein interaction methods. KLK6-high cases had a distinct spectrum of mutations in titin (), , , and genes. Differentially expressed genes (DEGs) found in the KLK6-overexpressing CRCs were associated with cell signaling, extracellular matrix organization, and cell communication regulatory pathways. The top KLK6-interaction partners were found to be the members of kallikrein family (KLK7, KLK8, KLK10), extracellular matrix associated proteins (keratins, integrins, small proline rich repeat, S100A families) and TGF-β, FOS, and Ser/Thr protein kinase signaling pathways. Expression of selected KLK6-associated genes was validated in a subset of paired normal and tumor CRC patient-derived organoid cultures. The performed analyses identified KLK6 itself and a set of genes, which are co-expressed with KLK6, as potential clinical biomarkers for the management of the CRC disease.
Topics: Adenomatous Polyposis Coli Protein; CA-125 Antigen; Colorectal Neoplasms; Connectin; Extracellular Matrix Proteins; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Kallikreins; Male; Membrane Proteins; Proto-Oncogene Proteins p21(ras); Signal Transduction; Transcriptome; Tumor Cells, Cultured; Up-Regulation
PubMed: 34065672
DOI: 10.3390/genes12050749