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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 1,099 records · Page 61
All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

RLIP76-dependent suppression of PI3K/AKT/Bcl-2 pathway by miR-101 induces apoptosis in prostate cancer

MicroRNA-101 (miR-101) participates in carcinogenesis and tumor progression in various cancers. However, its biological functions in prostate cancer are still unclear. Here, we demonstrate that miR-101 represents a critical role in regulating cell apoptosis in prostate cancer cells. We first demonstrated that miR-101 treatment promoted apoptosis in DU145 and PC3 cells by using flow cytometric analysis and transmission electron microscopy (TEM). To verify the mechanisms, we identified a novel miR-101 target, Ral binding protein 1 (RLIP76). We found miR-101 transfection significantly suppresses RLIP76 expression, which can transactivate phosphorylation of PI3K-Akt signaling, and resulted in an amplification of Bcl2-induced apoptosis. Furthermore, we demonstrated that RLIP76 overexpression could reverse the anti-tumor effects of miR-101 in DU145 and PC3 cells by using flow cytometry assay and MTT assay. Taken together, our results revealed that the effect of miR-101 on prostate cancer cell apoptosis was due to RLIP76 regulation of the PI3K/Akt/Bcl-2 signaling pathway. - Highlights: • miR-101 inhibited prostate cancer cell proliferation and enhanced apoptosis. • miR-101 directly targeted and regulated RLIP76 expression. • miR-101 suppressed PI3K/Akt/Bcl-2 signaling pathway by targeting RLIP76.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Skeletal stem cell and bone implant interactions are enhanced by LASER titanium modification

Purpose: To evaluate the osteo-regenerative potential of Titanium (Ti) modified by Light Amplification by Stimulated Emission of Radiation (LASER) beam (Yb-YAG) upon culture with human Skeletal Stem Cells (hSSCs{sup 1}). Methods: Human skeletal cell populations were isolated from the bone marrow of haematologically normal patients undergoing primary total hip replacement following appropriate consent. STRO-1{sup +} hSSC{sup 1} function was examined for 10 days across four groups using Ti discs: i) machined Ti surface group in basal media (Mb{sup 2}), ii) machined Ti surface group in osteogenic media (Mo{sup 3}), iii) LASER-modified Ti group in basal media (Lb{sup 4}) and, iv) LASER-modified Ti group in osteogenic media (Lo{sup 5}). Molecular analysis and qRT-PCR as well as functional analysis including biochemistry (DNA, Alkaline Phosphatase (ALP{sup 6}) specific activity), live/dead immunostaining (Cell Tracker Green (CTG{sup 7})/Ethidium Homodimer-1 (EH-1{sup 8})), and fluorescence staining (for vinculin and phalloidin) were undertaken. Inverted, confocal and Scanning Electron Microscopy (SEM) approaches were used to characterise cell adherence, proliferation, and phenotype. Results: Enhanced cell spreading and morphological rearrangement, including focal adhesions were observed following culture of hSSCs{sup 1} on LASER surfaces in both basal and osteogenic conditions. Biochemical analysis demonstrated enhanced ALP{sup 6} specific activity on the hSSCs{sup 1}-seeded on LASER-modified surface in basal culture media. Molecular analysis demonstrated enhanced ALP{sup 6} and osteopontin expression on titanium LASER treated surfaces in basal conditions. SEM, inverted microscopy and confocal laser scanning microscopy confirmed extensive proliferation and migration of human bone marrow stromal cells on all surfaces evaluated. Conclusions: LASER-modified Ti surfaces modify the behaviour of hSSCs.{sup 1} In particular, SSC{sup 1} adhesion, osteogenic gene expression, cell morphology and cytoskeleton structure were affected. The current studies show Ti LASER modification can enhance the osseointegration between Ti and skeletal cells, with important implications for orthopaedic application. - Highlights: • Bone stem cells on LASER Ti surface display enhanced cell growth and viability. • Bone stem cells on LASER Ti surface exhibit marked biocompatibility. • Human bone stem cells on LASER Ti surface exhibit altered morphology. • LASER Ti enhance osteogenic differentiation of human bone skeletal stem cells. • LASER Ti provides a unique approach to enhance osseointegration with the material.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Cloning and expressions of peroxisome proliferator activated receptor alpha1 and alpha2 (PPARα1 and PPARα2) in loach (Misgurnus anguillicaudatus) and in response to different dietary fatty acids

Peroxisome proliferator activated receptor alpha1 and alpha2 (PPARα1 and PPARα2) were investigated in loach (Misgurnus anguillicaudatus) by RACE (rapid amplification of cDNA ends) and qPCR (real-time quantitative PCR) for the first time. The cDNA sequences of PPARα1 and PPARα2 were 2042bp and 2407bp, respectively encoding 467 and 465 amino acids. Sequence alignments of deduced amino acids showed significant homology between the two subtypes of PPARα, indicating 70% identity. The two genes revealed sensible changes in transcriptions during early life stages of the loach, and the highest transcriptions of the two genes both appeared at some day after hatching. PPARα1 predominantly expressed in liver, while PPARα2 markedly expressed in heart. The expression regulation of PPARα1 and PPARα2 in response to dietary fatty acids was determined in livers of loaches fed with diets containing fish oil (FO group) and soybean oil (SO group) for 75 days. The expression level of PPARα1 in FO group was significantly higher than those in SO group (P < 0.01), while the expression level of PPARα2 in FO group was also significantly higher than those in SO group (P < 0.05). There was no significant difference in the expression level between PPARα1 and PPARα2 in SO group, whereas significant difference in FO group. These indicated that lipid resources could regulate the expressions of these two genes in the loach. Our results will provide opportunities to better understand the functional characterization of PPARα1 and PPARα2 in further studies. - Highlights: • The full-length cDNAs of loach PPARα1 and PPARα2 were obtained by a RACE PCR method. • Phylogenetic and protein characterizations of these two genes were predicted. • These two genes differentially expressed at different early life stages and tissues indicating their different functions. • n-3 PUFA may regulate the activation of PPARα in the loach.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Prolyl-tRNA synthetase inhibition promotes cell death in SK-MEL-2 cells through GCN2-ATF4 pathway activation

Protein translation is highly activated in cancer tissues through oncogenic mutations and amplifications, and this can support survival and aberrant proliferation. Therefore, blocking translation could be a promising way to block cancer progression. The process of charging a cognate amino acid to tRNA, a crucial step in protein synthesis, is mediated by tRNA synthetases such as prolyl tRNA synthetase (PRS). Interestingly, unlike pan-translation inhibitors, we demonstrated that a novel small molecule PRS inhibitor (T-3861174) induced cell death in several tumor cell lines including SK-MEL-2 without complete suppression of translation. Additionally, our findings indicated that T-3861174-induced cell death was caused by activation of the GCN2-ATF4 pathway. Furthermore, the PRS inhibitor exhibited significant anti-tumor activity in several xenograft models without severe body weight losses. These results indicate that PRS is a druggable target, and suggest that T-3861174 is a potential therapeutic agent for cancer therapy. - Highlights: • A novel small molecule prolyl tRNA synthetase inhibitor, T-3861174, was tested. • T-3861174 induced cell death in several tumor cell lines. • T-3861174 cytotoxicity was mediated by the GCN2-ATF4 pathway. • Prolyl tRNA synthetase inhibition hindered tumor growth in vivo in xenograft models.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Identification and molecular cloning of novel transcripts of the human kallikrein-related peptidase 10 (KLK10) gene using next-generation sequencing

Tissue kallikrein and kallikrein-related peptidases (KLKs) form the largest group of serine proteases in the human genome, sharing many structural and functional characteristics. Multiple alternative transcripts have been reported for the most human KLK genes, while many of them are aberrantly expressed in various malignancies, thus possessing significant prognostic and/or diagnostic value. Alternative splicing of cancer-related genes is a common cellular mechanism accounting for cancer cell transcriptome complexity, as it affects cell cycle control, proliferation, apoptosis, invasion, and metastasis. In this study, we describe the identification and molecular cloning of eight novel transcripts of the human KLK10 gene using 3′ rapid amplification of cDNA ends (3′ RACE) and next-generation sequencing (NGS), as well as their expression analysis in a wide panel of cell lines, originating from several distinct cancerous and normal tissues. Bioinformatic analysis revealed that the novel KLK10 transcripts contain new alternative splicing events between already annotated exons as well as novel exons. In addition, investigation of their expression profile in a wide panel of cell lines was performed with nested RT-PCR using variant-specific pairs of primers. Since many KLK mRNA transcripts possess clinical value, these newly discovered alternatively spliced KLK10 transcripts appear as new potential biomarkers for diagnostic and/or prognostic purposes or as targets for therapeutic strategies. - Highlights: • NGS was used to identify novel transcripts of the human KLK10 gene. • 8 novel KLK10 transcripts were identified. • A novel 3′UTR was detected and characterized. • The expression profiles of all 8 novel KLK10 transcripts were identified.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Effect of sodium fluoride on the sperm mitochondrial DNA in mice

Previous studies demonstrated that fluoride, as a widespread environmental pollutant, induced the reproductive toxicity at high dose. Besides the decrease of sperm characteristics like concentration, survival, and sperm motility, it was found that high fluoride induced the destructive mitochondrial ultrastructure and decreased ATP production from mitochondrial respiration. However, whether fluoride exposure can damage the mitochondrial DNA (mtDNA) of sperm is still unknown. In this study, 100 male mice were randomly divided into four groups 25 each, which were administrated with the distilled water containing 0, 25, 50 and 100 mg L{sup −1} NaF, respectively for 60 days. After exposure, sperm mtDNA copy number was measured by real-time PCR, mtDNA integrity by acridine orange (AO) staining, and nuclear DNA (nDNA) integrity through long PCR amplification, as well as MTCYB and MTATP6 mutations by PCR and direct sequencing. The results showed that 100 mg L{sup −1} NaF significantly increased sperm mtDNA copy number, and reduced nDNA integrity. There were no changes observed in mtDNA integrity, and mutations of MTCYB and MTATP6 between fluoride groups and the control group. These results indicated that along with low sperm quality, sperm mtDNA copy number is also a sensitive biomarker to reflect the sperm toxicity of fluoride. - Highlights: • NaF significantly increased sperm mtDNA copy number, and reduced nDNA integrity. • NaF did not change sperm mtDNA integrity, and mutations of MTCYB and MTATP6. • Sperm mtDNA copy number can be a potential biomarker of fluoride-induced toxicity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Endothelial Robo4 regulates IL-6 production by endothelial cells and monocytes via a crosstalk mechanism in inflammation

Highlights: • Robo4 knockout in mouse endotoxemia models reduced circulating IL-6 levels. • Robo4 knockdown suppressed IL-6 and GM-CSF production by endothelial cells (ECs). • Robo4 enhanced IL-6 production by monocytes via EC-derived GM-CSF. • Robo4 regulates cytokine production by both ECs and immune cells in inflammation. Roundabout4 (Robo4) is an endothelial cell-specific receptor that stabilizes vasculature in pathological angiogenesis. Previous studies have shown that Robo4 is a potential therapeutic target for inflammatory diseases, but its precise roles in inflammation remain unclear. To investigate physiological Robo4 functions in inflammation, we performed a loss-of-function study in vitro and in vivo using lipopolysaccharide (LPS)-induced endotoxemia models. Subcutaneous injection of LPS into Robo4-knockout mice reduced circulating IL-6 levels. siRNA-mediated Robo4 knockdown suppressed IL-6 production induced by LPS, IL-1β, and TNFα, in human umbilical vein endothelial cells (HUVECs). Coculture experiments with HUVECs and a monocytic cell line, U937 cells, demonstrated that Robo4 knockdown suppresses IL-6 production by both endothelial cells and U937 cells. Further coculture experiments demonstrated that Robo4 knockdown inhibited a novel IL-6 amplification mechanism mediated by crosstalk between endothelial cells and U937 cells via direct interactions and two mediators, GM-CSF and IL-1β. Taken together, we demonstrated novel Robo4 functions in inflammation, i.e., it promotes IL-6 production by endothelial cells and immune cells via crosstalk.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Exosomes derived from human umbilical vein endothelial cells promote neural stem cell expansion while maintain their stemness in culture

The neural stem cell (NSC) niche in subventricular zone (SVZ) of adult mammalian brain contains dense vascular plexus, where endothelial cells (ECs) regulate NSCs by releasing plenty of angiocrine factors. However, the role of ECs-derived exosomes, a novel type of mediators of intercellular communications, in the regulation of NSCs remains unclear. In the current study, primary NSCs isolated from embryonic mouse brains form more neurospheres when cultured in the presence of human umbilical vein endothelial cells (HUVECs). The supportive role of ECs in the coculture was significantly attenuated when GW4869, a blocker of exosome formation, was included, suggesting that HUVECs-derived exosomes played a significant role in supporting NSCs. In order to investigate the role of ECs-derived exosomes on NSCs, we collected exosomes from HUVECs. We found that HUVECs-derived exosomes could significantly promote the formation of neurospheres by primary murine NSCs. EdU incorporation and TUNEL assays indicated that the proliferation of NSCs increased while apoptosis decreased when cultured in the presence of HUVECs-derived exosomes. NSCs incubated with the HUVECs-derived exosomes maintained their potential of multi-lineage differentiation potentials. The expression of stemness-related genes was up-regulated. These data suggested that ECs-derived exosomes could play an importantly role in NSC niche, and they might be used as a reagent for ex vivo NSC amplification for medical application.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Deficiency of Atg6 impairs beneficial effect of metformin on intestinal stem cell aging in Drosophila

Highlights: • Knockdown of Atg6 in ISC induces age-related phenotypes of ISC. • Metformin inhibits age-related phenotypes of ISC in Atg6-dependent manner. • This study provides an intervention mechanism of metformin on adult stem cell aging. Age-related changes of adult stem cell are crucial for tissue aging and age-related diseases. Thus, clarifying mechanisms to prevent adult stem cell aging is indispensable for healthy aging. Metformin, a drug for type 2 diabetes, has been highlighted for its anti-aging and anti-cancer effect. In Drosophila intestinal stem cell (ISC), we previously reported the inhibitory effect of metformin on age-related phenotypes of ISC. Here, we showed that knockdown of Atg6, a crucial autophagy-related factor, in ISC induces age-related phenotypes of ISC such as hyperproliferation, centrosome amplification, and DNA damage accumulation. Then, we revealed that metformin inhibits ISC aging phenotypes in Atg6-dependent manner. Taken together, our study suggests that Atg6 is required for the inhibitory effect of metformin on ISC aging, providing an intervention mechanism of metformin on adult stem cell aging.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

SURF4 has oncogenic potential in NIH3T3 cells

Highlights: • SURF4 was found to be amplified and highly expressed in the tumor tissues of several human cancer patients. • Patients with tumors exhibiting high SURF4 expression had significantly shorter overall survival than low SURF4 expression. • SURF4 can induce cellular transformation and cell migration in vitro and promotes oncogenic transformation in vivo. SURF4, which is located in the Surfeit gene cluster, encodes for a conserved integral membrane protein containing multiple putative transmembrane regions. However, the physiological role of SURF4 has not been determined. We found that SURF4 demonstrated aberrant amplification and increased expression in the tumor tissues of several human cancer patients. Overexpression of SURF4 led to increased cell proliferation, migration, and maintenance of anchorage-independent growth. In addition, NIH3T3 cells overexpressing SURF4 induced tumor growth in the mice. Collectively, our findings demonstrate that SURF4 has the potential for inducing cellular transformation and cell migration in vitro and has oncogenic transformation ability in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

MiR-455-3p suppresses renal fibrosis through repression of ROCK2 expression in diabetic nephropathy

Highlights: • Downregulation of miR-455-3p expression in DN. • ROCK2 as a target of miR-455-3p. • Re-introduction of ROCK2 attenuated the effect of miR-455-3p on cell proliferation, ECM accumulation and EMT in vitro. • Reductions in proinflammatory levels and renal fibrosis by miR-455-3p usages, in vivo. Emerging evidence has shown that microRNAs (miRNAs) play a mediatory role in the pathogenesis of diabetic nephropathy (DN), but the function of the involved miRNAs is still incomplete. Here, we found that miR-455-3p was down-regulated in the human mesangial cells (HMC) and human proximal tubule epithelial cells (HK-2) stimulated with high glucose (HG) or transforming growth factor beta 1 (TGF-β1). Rho-associated coiled coil-containing protein kinase 2 (ROCK2) was identified as a directed target of miR-455-3p. Overexpression of ROCK2 significantly attenuated the inhibitory effects of miR-455-3p on cell proliferation, extracellular matrix (ECM) synthesis and epithelial-mesenchymal transition (EMT) in HG-treated cells. Furthermore, the DN model was prepared by using high-fat feeding combined with Streptozotocin (STZ) induced rats, and the DN group was treated by injecting miR-455-3p agomir. The results of periodic acid-Schiff (PAS) and Masson staining showed that miR-455-3p overexpression improved the pathological changes of glomerular hypertrophy, mesangial amplification, and renal fibrosis. Additionally, miR-455-3p overexpression decreased ROCK2, proliferating cell nuclear antigen (PCNA) and Collagen I levels, and also reduced inflammatory cytokines TNF-α, MCP-1 and IL-1β levels in vivo. Altogether, these results suggest that miR-455-3p plays an essential role in the treatment of renal fibrosis through repressing ROCK2 expression; and miR-455-3p might be an effective therapy for DN.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

DNA copy number gain-mediated lncRNA LINC01061 upregulation predicts poor prognosis and promotes papillary thyroid cancer progression

Several DNA copy number amplifications (CNAs) have been reported in papillary thyroid cancer (PTC). However, the functional role of CNAs in PTC remains very unclear. And whether there is a correlation between long noncoding RNA (lncRNA) and CNA requires to be explored. Here, we identified a novel lncRNA LINC01061. The genomic copy number of LINC01061 is amplified, which leads to its elevated expression level in PTC tissues. Moreover, increased level of LINC01061 was correlated with aggressive clinicopathological characteristics. Functional study indicated that LINC01061 silence significantly inhibited the proliferation, cell-cycle and invasion of PTC cells in vitro and tumor growth in vivo. Mechanistically, we showed that LINC01061 interacted with miR-4316 to promote E2F6 expression. The expression of miR-4316 was downregulated in PTC tissues while that of E2F6 was upregulated. Through rescue assay, we demonstrated that LINC01061 promoted PTC cell proliferation, cell-cycle progression and invasion by regulating miR-4316/E2F6 signaling pathway. In conclusion, our research indicated that LINC01061 might be a target for PTC therapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Anti-EGFR antibody cetuximab is secreted by oral squamous cell carcinoma and alters EGF-driven mesenchymal transition

Genetic amplification, overexpression, and increased signaling from the epidermal growth factor receptor (EGFR) are often found in oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed effects of cetuximab in control of EGF-driven malignant traits of OSCC cells. EGF stimulation promoted progression level of mesenchymal traits in OSCC cells, which were attenuated by cetuximab but incompletely. We pursued a potential mechanism underlying such incomplete attenuation of OSCC malignant traits. Cetuximab promoted secretion of EGFR-EVs by OSCC cells and failed to inhibit EGF-driven secretion of EGFR-EVs. Cetuximab was also found to be robustly secreted with the EGFR-EVs by the OSCC cells. Thus, EGF promotes the level of mesenchymal traits of OSCC cells and secretion of EGFR-EVs, which involve cetuximab resistance.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Tissue-specific variation in 5-terminal exons of mouse Anoctamin 1 transcript induces N-terminal variation of its protein via alternative translational start sites

Anoctamin 1 (encoded by the Ano1 gene) is a Ca{sup 2+}-activated Cl{sup −} channel critical to many physiological functions. It has been speculated that Ano1 expression is regulated in a tissue-dependent manner via alternative promoters. However, variation in the 5-end sequence of mouse Ano1 (mAno1) and its tissue-dependent regulation are poorly understood. We identified a novel 5-terminal exon (designated exon 1a) of mAno1 instead of the known 5-terminal exon (exon 0) using 5-rapid amplification of cDNA ends (RACE) analysis. Unexpectedly, the novel 5-end variant mAno1{sub Ex1a} was abundantly expressed in many tissues including the salivary and mammary glands, rectum, lung, trachea and prostate. In contrast, the known variant mAno1{sub Ex0} predominated only in male reproductive tissues such as the epididymis and testis. In a heterologous expression system, mAno1{sub Ex0} encoded a longer protein than mAno1{sub Ex1a}, and this long isoform was abolished by a mutation in the exon 0 start codon. Moreover, the mAno1{sub Ex0}-specific N-terminal sequence was immunohistochemically detected in epididymis but not in salivary gland. Our data suggest that mAno1 expression is regulated via alternative promoters, and its transcriptional variation results in variation of the N-terminal sequence of the Ano1 protein due to the alternative translation initiation sites. These tissue-specific variations might contribute to the regulation of mAno1 expression and activity according to the physiological function of each tissue.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Sterol regulatory element binding protein-1: Molecular cloning, tissue distribution and gene expression level in response to nutritional regulation in mud crab, Scylla paramamosain

Highlights: • The complete SREBP-1 cDNA sequence was cloned from the hepatopancreas of mud crab based on the RACE technology. • Tissue distribution analysis revealed that SREBP-1 transcripts were widely distributed in various organs, with higher mRNA levels in the eyestalk and cranial ganglia. • The expression level of SREBP-1 mRNA were up-regulated in proportion to the replacement of dietary fish oil with soybean oil. In the present study, SREBP-1 cDNA was cloned from the hepatopancreas of mud crab (Scylla paramamosain) and characterized by performing rapid-amplification of cDNA ends. The 3361bp long full-length cDNA encodes a polypeptide with 1039 amino acids. Tissue distribution analysis revealed that SREBP-1 transcripts were widely distributed in various organs, with higher mRNA levels in the eyestalk and cranial ganglia. Further, expression level of SREBP-1 mRNA were up-regulated in proportion to the replacement of dietary fish oil (FO) with soybean oil (SO). These results may contribute to better understanding of the long-chain polyunsaturated fatty acids (LC-PUFA) biosynthetic pathway and regulation mechanism in mud crab.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Effects of molecular crowding on a bimolecular group I ribozyme and its derivative that self-assembles to form ribozyme oligomers

Highlights: • PEG rescued mutations in the bimolecular group I ribozyme more effectively than those in the oligomeric form. In the RNA world, enrichment of self-replicating RNAs would have been beneficial to their survival, amplification, and evolution. Self-assembly of RNAs may be a strategy by which they enrich themselves. We examined the effects of molecular crowding on the activity of a bimolecular group I ribozyme and its derivative that self-assembles to form ribozyme oligomers. In a comparative activity assay using PEG as a molecular crowder, PEG rescued mutations in the parent bimolecular ribozyme more effectively than those in the oligomeric form.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Prolonged MEK inhibition leads to acquired resistance and increased invasiveness in KRAS mutant gastric cancer

Highlights: • MAPK pathway is important for the survival of KRAS mutant gastric cancer cells. • Prolonged MEK inhibition leads to acquired resistance. • c-MET activity is upregulated in MEK inhibitor resistant gastric cancer cells. • MEK inhibitor resistant gastric cancer cells harbor more invasive phenotype. • The increased invasiveness is abrogated by c-MET TKI or PI3K/mTOR dual inhibitor. Gastric cancer (GC) is one of the most common causes of cancer-associated death. However, traditional therapeutic strategies have failed to significantly improve the survival of patient with advanced GC. While KRAS mutations have been found in some patients with gastric cancer, an effective therapy to treat KRAS-driven gastric cancer has not been established yet. To provide a rationale for clinical application of kinase inhibitors targeting RAS pathways, we first determined the sensitivity of GC cell lines harboring KRAS mutations or amplification to RAS pathway inhibitors. We found that MAPK pathway inhibitors (MEKi and ERKi) were more effective than AKT inhibitor, suggesting that KRAS-driven gastric cancer cells are dependent on MAPK pathway for survival. Further, we established a KRAS mutant GC cell line with acquired resistance to MEK inhibitors in order to mimic clinical situation of kinase inhibitor resistance. A comprehensive analysis of tyrosine phosphorylation in receptor tyrosine kinases in combination with small molecule chemical library screening revealed upregulated c-MET phosphorylation in this resistance cell line with elevated sensitivity to c-MET TKI (crizotinib) and PI3K/mTOR dual inhibitor (BEZ235). We also showed that migration and invasion of resistant cells were promoted, and crizotinib and BEZ235 could inhibit this malignant phenotype. Overall, our results indicate that prolonged MAPK pathway inhibition could result in acquired resistance which is associated with increased malignant phenotype in KRAS mutant GC and pharmacological targeting c-MET and PI3K/mTOR could overcome this problem.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Engineering and evolution of Yarrowia lipolytica for producing lipids from lignocellulosic hydrolysates

Yarrowia lipolytica, an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.

60 APPLIED LIFE SCIENCES↗