scholarly journals PIK3R3, part of the regulatory domain of PI3K, is upregulated in sarcoma stem-like cells and promotes invasion, migration, and chemotherapy resistance

2021 ◽  
Vol 12 (8) ◽  
Author(s):  
Changhwan Yoon ◽  
Jun Lu ◽  
Sandra W. Ryeom ◽  
M. Celeste Simon ◽  
Sam S. Yoon

AbstractTo identify drivers of sarcoma cancer stem-like cells (CSCs), we compared gene expression using RNA sequencing between HT1080 fibrosarcoma and SK-LMS-1 leiomyosarcoma spheroids (which are enriched for CSCs) compared with the parent populations. The most overexpressed survival signaling-related gene in spheroids was phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3), a regulatory subunit of PI3K, which functions in tumorigenesis and metastasis. In a human sarcoma microarray, PIK3R3 was also overexpressed by 4.1-fold compared with normal tissues. PIK3R3 inhibition using shRNA in the HT1080, SK-LMS-1, and DDLS8817 dedifferentiated liposarcoma in spheroids and in CD133+ cells (a CSC marker) reduced expression of CD133 and the stem cell factor Nanog and blocked spheroid formation by 61–71%. Mechanistic studies showed that in spheroid cells, PIK3R3 activated AKT and ERK signaling. Inhibition of PIK3R3, AKT, or ERK using shRNA or inhibitors decreased expression of Nanog, spheroid formation by 68–73%, and anchorage-independent growth by 76–91%. PIK3R3 or ERK1/2 inhibition similarly blocked sarcoma spheroid cell migration, invasion, secretion of MMP-2, xenograft invasion into adjacent normal tissue, and chemotherapy resistance. Together, these results show that signaling through the PIK3R3/ERK/Nanog axis promotes sarcoma CSC phenotypes such as migration, invasion, and chemotherapy resistance, and identify PIK3R3 as a potential therapeutic target in sarcoma.

Oncogenesis ◽  
2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Changhwan Yoon ◽  
Jun Lu ◽  
Brendan C. Yi ◽  
Kevin K. Chang ◽  
M. Celeste Simon ◽  
...  

AbstractThe self-renewal transcription factor Nanog and the phosphoinositide 3-kinase (PI3K)–Akt pathway are known to be essential for maintenance of mesenchymal stem cells. We evaluated their contribution to the maintenance of CD133(+) cancer stem-like cells (CSCs) and spheroid-forming cells in patient-derived cell lines from three human sarcoma subtypes: HT1080 fibrosarcoma, SK-LMS-1 leiomyosarcoma, and DDLS8817 dedifferentiated liposarcoma. Levels of Nanog and activated Akt were significantly higher in sarcoma cells grown as spheroids or sorted for CD133 expression to enrich for CSCs. shRNA knockdown of Nanog decreased spheroid formation 10- to 14-fold, and reversed resistance to both doxorubicin and radiation in vitro and in H1080 flank xenografts. In the HT1080 xenograft model, doxorubicin and Nanog knockdown reduced tumor growth by 34% and 45%, respectively, and the combination reduced tumor growth by 74%. Using a human phospho-kinase antibody array, Akt1/2 signaling, known to regulate Nanog, was found to be highly activated in sarcoma spheroid cells compared with monolayer cells. Pharmacologic inhibition of Akt using LY294002 and Akt1/2 knockdown using shRNA in sarcoma CSCs decreased Nanog expression and spheroid formation and reversed chemotherapy resistance. Akt1/2 inhibition combined with doxorubicin treatment of HT1080 flank xenografts reduced tumor growth by 73%. Finally, in a human sarcoma tumor microarray, expression of CD133, Nanog, and phospho-Akt were 1.8- to 6.8-fold higher in tumor tissue compared with normal tissue. Together, these results indicate that the Akt1/2–Nanog pathway is critical for maintenance of sarcoma CSCs and spheroid-forming cells, supporting further exploration of this pathway as a therapeutic target in sarcoma.


2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
Jie Zhang ◽  
Qianqian Song ◽  
Jinxia Liu ◽  
Lina Lu ◽  
Yuqing Xu ◽  
...  

Cyclin-dependent kinase regulatory subunit 2 (CKS2) is a member of the cell cycle-dependent protein kinase subunit family, which is implicated as an oncogene in various malignancies. However, the clinical significance, oncogenic functions, and related mechanisms of CKS2 in hepatocellular carcinoma (HCC) remain largely unclear. In the present study, expression features and prognostic value of CKS2 were evaluated in the bioinformatic databases and HCC tissues. The effects of CKS2 on the malignant phenotypes of HCC cells were explored in vitro. According to the analyses of three bioinformatic databases, mRNA levels of CKS2 were elevated in HCC tissues compared with the normal tissues. Immunohistochemical assays found that high CKS2 expression was closely associated with liver cirrhosis (P=0.019), poor differentiation (P=0.02), portal vein invasion (P<0.001), TNM stage (P=0.019), tumor metastasis (P=0.008), and recurrence (P=0.003). The multivariate regression analyses suggested that CKS2 was an independent prognostic factor for overall survival (HR=2.088, P=0.014) and disease-free survival (HR=2.511, P=0.002) of HCC patients. Moreover, the bioinformatic analyses indicated that CKS2 might be associated with the malignant phenotypes in HCC progression. In addition, in vitro assays showed that CKS2 expression was higher in HCC cell lines than in normal liver cells. Knockdown of CKS2 remarkably repressed the proliferation, colony formation (P=0.0003), chemoresistance, migration (P=0.0047), and invasion (P=0.0012) of HCC cells. Taken together, overexpression of CKS2 was significantly correlated with poor prognosis of HCC patients and the malignant phenotypes of HCC cells, suggesting that it was a novel prognostic biomarker and potential target of HCC.


2002 ◽  
Vol 13 (2) ◽  
pp. 480-492 ◽  
Author(s):  
Tom D. Wolkow ◽  
Tamar Enoch

Fission yeast Rad3 is a member of a family of phosphoinositide 3-kinase -related kinases required for the maintenance of genomic stability in all eukaryotic cells. In fission yeast, Rad3 regulates the cell cycle arrest and recovery activities associated with the G2/M checkpoint. We have developed an assay that directly measures Rad3 kinase activity in cells expressing physiological levels of the protein. Using the assay, we demonstrate directly that Rad3 kinase activity is stimulated by checkpoint signals. Of the five other G2/M checkpoint proteins (Hus1, Rad1, Rad9, Rad17, and Rad26), only Rad26 was required for Rad3 kinase activity. Because Rad26 has previously been shown to interact constitutively with Rad3, our results demonstrate that Rad26 is a regulatory subunit, and Rad3 is the catalytic subunit, of the Rad3/Rad26 kinase complex. Analysis of Rad26/Rad3 kinase activation in rad26.T12, a mutant that is proficient for cell cycle arrest, but defective in recovery, suggests that these two responses to checkpoint signals require quantitatively different levels of kinase activity from the Rad3/Rad26 complex.


1996 ◽  
Vol 109 (2) ◽  
pp. 289-300 ◽  
Author(s):  
T.R. Jackson ◽  
I.J. Blader ◽  
L.P. Hammonds-Odie ◽  
C.R. Burga ◽  
F. Cooke ◽  
...  

Application of nerve growth factor (NGF) to PC12 cells stimulates a programme of physiological changes leading to the development of a sympathetic neuron like phenotype, one aspect of which is the development of a neuronal morphology characterised by the outgrowth of neuritic processes. We have investigated the role of phosphoinositide 3-kinase in NGF-stimulated morphological differentiation through two approaches: firstly, preincubation with wortmannin, a reputedly specific inhibitor of phosphoinositide kinases, completely inhibited initial morphological responses to NGF, the formation of actin filament rich microspikes and subsequent neurite outgrowth. This correlated with wortmannin inhibition of NGF-stimulated phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) and phosphatidylinositol(3,4)bisphosphate (PtdIns(3,4)P2) production and with inhibition of NGF-stimulated phosphoinositide 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Secondly, the overexpression of a mutant p85 regulatory subunit of the phosphoinositide 3-kinase, which cannot interact with the catalytic p110 subunit, also substantially inhibited the initiation of NGF-stimulated neurite outgrowth. In addition, we found that wortmannin caused a rapid collapse of more mature neurites formed following several days exposure of PC12 cells to NGF. These results indicate that NGF-stimulated neurite outgrowth requires the activity of a tyrosine kinase regulated PI3-kinase and suggest that the primary product of this enzyme, PtdInsP3, is a necessary second messenger for the cytoskeletal and membrane reorganization events which occur during neuronal differentiation.


2019 ◽  
Vol 20 (5) ◽  
pp. 1036 ◽  
Author(s):  
Kang Zhang ◽  
Yun Han ◽  
Yabo Zhao ◽  
Yingfei Sun ◽  
Mengyun Zou ◽  
...  

Mycoplasma gallisepticum (MG) mainly infects chickens to initiate chronic respiratory disease (CRD). microRNAs (miRNAs) play vital roles according to previously reported studies. Our previous study showed that gga-miR-16-5p, in MG-infected lungs of chicken embryo, was upregulated by Illumina sequencing. The study aimed to reveal what role gga-miR-16-5p plays in CRD progression. gga-miR-16-5p was upregulated in MG-infected fibroblast cells (DF-1). Phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) was demonstrated as the target gene of gga-miR-16-5p. Furthermore, PIK3R1 expression was lower in MG-infected groups than it in noninfected controls measured by qPCR. Additionally, overexpressed gga-miR-16-5p could downregulate PIK3R1 and phosphorylated serine/threonine kinase (p-Akt) to express protein, whereas there is an opposite effect on inhibition. Overexpressed gga-miR-16-5p resulted in decreased activity of tumor necrosis factor alpha (TNF-α) and the nuclear factor-kappaB (NF-κB) by qPCR. Furthermore, overexpressed gga-miR-16-5p restricted cell multiplication, cycle progression, and increased apoptosis of MG-infected DF-1 cells, whereas inhibited gga-miR-16-5p led to the opposite effect. Collectively, upregulated gga-miR-16-5p could decrease multiplication, cycle progression, and increase apoptosis of MG-infected DF-1 cells, at least partly through directly targeting PIK3R1 and inhibiting PI3K/Akt/NF-κB pathway to exert an anti-inflammatory effect. Our results will provide more experimental evidence to bring pathogenesis of MG infection to light.


2000 ◽  
Vol 20 (21) ◽  
pp. 8069-8083 ◽  
Author(s):  
Randall D. York ◽  
Derek C. Molliver ◽  
Savraj S. Grewal ◽  
Paula E. Stenberg ◽  
Edwin W. McCleskey ◽  
...  

ABSTRACT Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.


2007 ◽  
Vol 104 (9) ◽  
pp. 3466-3471 ◽  
Author(s):  
L. Soroceanu ◽  
S. Kharbanda ◽  
R. Chen ◽  
R. H. Soriano ◽  
K. Aldape ◽  
...  

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