Biology subjects

Johnson, P. F.

Publications and source records attributed to Johnson, P. F..

5 recordsLinked to original sources

bioRxiv2024

The long noncoding RNA lnc-FANCI-2 restricts RAS signaling and phosphorylation of Akt and Erk in HPV16-infected cervical cancer cells

We recently discovered the expression of a long noncoding RNA, lnc-FANCI-2, coinciding with cervical lesion progression from CIN1, CIN2-3 to cervical cancer. Viral E7 of high-risk HPVs and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression. To explore possible roles of lnc-FANCI-2 in HPV-induced cervical carcinogenesis, we ablated the expression of lnc-FANCI-2 in the HPV16-positive cervical cancer cell line, CaSki. Knock-out (KO) single cell clones expressed HPV16 oncogenes normally but displayed altered cell morphology and proliferation when compared with the parental cells. Proteomic profiling of cytosolic and secreted proteins from the parental and KO cells showed that lnc-FANCI-2 regulates expression of a subset of cell soluble receptors responsible for cell signaling, epithelial mesenchymal transition, and IFN responses. RNA-seq analyses revealed that, relative to the parental cells, lnc-FANCI-2 KO cells exhibited significantly increased RAS signaling but decreased IFN pathways. In the KO cells, phosphorylated Akt and Erk1/2, two important RAS pathway effectors, were increased more than 3-fold, accompanied by increase of IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decrease of RAC3 expression. High levels of lnc-FANCI-2 and lower levels of MCAM in cervical cancer patients are associated with improved survival. We found that lnc-FANCI-2 interacts specifically with 32 host proteins, including H13, HNRH1, K1H1, MAP4K4, and RNPS1, and knockdown of MAP4K4 in CaSki cells led to an increase of phosphorylation of Erk1/2 and somewhat Akt. In summary, a key function of lnc-FANCI-2 is to intrinsically regulate RAS signaling to impact cervical lesion progression and affect cervical cancer prognosis. SignificanceExpression of lnc-FANCI-2 is related to cervical lesion progression. Knock-out (KO) or knock-down (KD) of lnc-FANCI-2 expression in HPV16-positive cervical cancer cells CaSki significantly increases RAS signaling, phosphorylation of Akt and Erk1/2, and the epithelial mesenchymal transition factors. lnc-FANCI-2 KO also regulates the expression of a subset of cell soluble receptor proteins IGFBP3 and MCAM. A high level of lnc-FANCI-2 and lower level of MCAM in cervical cancer patients are associated with improved survival. lnc-FANCI-2 in CaSki cells interacts specifically with 32 host proteins, including MAP4K4. KD of MAP4K4 expression in CaSki cells led to an increase in phosphorylation of Erk1/2 and Akt. Thus, one lnc-FANCI-2 function is to intrinsically regulate RAS signaling to impact cervical lesion progression.

cancer biology↗

bioRxiv2023

Single-cell mRNA-regulation analysis reveals cell type-specific mechanisms of type 2 diabetes

Perturbed secretion of insulin and other pancreatic islet hormones is the main cause of type 2 diabetes (T2D). The islets harbor five cell types that are potentially altered differently by T2D. Whole-islet transcriptomics and single-cell RNA-sequencing (scRNAseq) studies have revealed differentially expressed genes without reaching consensus. Here, we demonstrate that unprecedented insights into disease mechanisms can be obtained by network-based analysis of scRNAseq data. We developed differential gene coordination network analysis (dGCNA) and analyzed islet scRNAseq data from 16 T2D and 16 non-T2D individuals. dGCNA revealed T2D-induced cell type-specific networks of dysregulated genes with remarkable ontological specificity, thus allowing for a comprehensive and unbiased functional classification of genes involved in T2D. In beta cells eleven networks of genes were detected, revealing that mitochondrial electron transport chain, glycolysis, cytoskeleton organization, cell proliferation, unfolded protein response and three networks of beta cell transcription factors are perturbed, whereas exocytosis, lysosomal regulation and insulin translation programs are instead enhanced in T2D. Furthermore, we validated the ability of dGCNA to reveal disease mechanisms and predict the functional context of genes by showing that TMEM176A/B regulates the beta cell cytoskeleton and that CEPBG is a key regulator of the unfolded protein response. In addition, comparing beta- and alpha and cells, we found substantial differences, reproduced across independent datasets, confirming cell type-specific alterations in T2D. We conclude that analysis of networks of differentially coordinated genes provides outstanding insight into cell type-specific gene function and T2D pathophysiology.

cell biology↗

bioRxiv2022

CK2 signaling from TOLLIP-dependent perinuclear endosomes is an essential feature of KRAS mutant cancers

Oncogenic RAS induces perinuclear translocation of the effector kinases ERK and CK2 and their scaffold, KSR1, forming endosomal signaling hubs termed perinuclear signaling centers (PSCs). PSCs are present in all cancer cell lines and tissues examined, suggesting that subcellular compartmentalization of oncogenic kinases drives tumorigenesis. However, the mechanism of perinuclear targeting, whether this location affects kinase substrate specificity, and the importance of PSCs in cancer are unclear. Here we show that the endosomal adaptor, TOLLIP, specifically tethers RAB11A+ signaling endosomes containing CK2 and KSR1 to the perinuclear ER. A predicted {beta}-hairpin fold in TOLLIP mediates binding to the KSR1 CA5 pseudo-kinase domain, recruiting CK2/KSR1 complexes to perinuclear endosomes. TOLLIP is essential for proliferation/survival of tumor cells carrying KRAS and NRAS mutations but not HRAS, BRAF, ERBB or PTEN lesions, or non-transformed cells. KRasG12D-induced lung lesions in Tollip-/- mice displayed reduced numbers of carcinomatous lesions, implicating TOLLIP in malignant progression. TOLLIP-dependent perinuclear CK2 was shown to phosphorylate discrete substrates, including proteins involved in translation and ribosome biogenesis such as RIOK1. Thus, TOLLIP is a key RAS pathway signaling adaptor in K/NRAS tumors whose inhibition is a specific vulnerability of these cancers.

cancer biology↗

bioRxiv2022

3'UTR-dependent dynamic changes in TP53 mRNA localization regulate p53 tumor suppressor activity

The tumor suppressor p53 triggers senescence in response to oncogenic stress in primary cells. However, the mechanisms by which tumor cells retaining p53 bypass senescence are not fully understood. Here we report that p53 cytostatic activity is inhibited in tumor cells by the 3 untranslated region (3UTR) of its mRNA, without altering p53 levels. 3UTR inhibition requires a long U-rich element (URE) and its binding protein, HuR. The 3UTR excluded TP53 mRNAs from a perinuclear compartment containing the CK2 kinase, suppressing p53 phosphorylation on an activating CK2 site, Ser392. In primary cells undergoing oncogene-induced senescence and tumor cells treated with genotoxic agents, TP53 mRNAs became concentrated in the perinuclear cytoplasm, coinciding with p53 phosphorylation and activation by CK2. In both cases, perinuclear re-localization of TP53 transcripts required AMPK2-dependent HuR nuclear translocation. ATM kinase activity was essential for DNA damage-induced spatial reprogramming of TP53 mRNAs, likely through phosphorylation and inactivation of MDM2. MDM2 was required for peripheral localization of TP53 transcripts and negatively regulated levels of the AMPK2 activating kinase, CaMKK{beta}. Our findings reveal a critical role for 3UTR sequences in suppressing p53 protein activity and provide a new mechanistic framework for p53 activation by DNA damaging agents.

cancer biology↗

bioRxiv2022

3'UTR-directed, kinase proximal mRNA decay inhibits C/EBPβ phosphorylation/activation to suppress senescence in tumor cells

C/EBP{beta} is a potent regulator of oncogene-induced senescence (OIS) and the SASP. C/EBP{beta} is post-translationally activated in OIS cells by the effector kinases ERK1/2 and CK2. However, in tumor cells C/EBP{beta} activation is suppressed by its 3UTR. 3'UTR regulation of protein activity (UPA) requires a G/U-rich element (GRE) and its cognate binding protein, HuR. These components segregate CEBPB transcripts away from a perinuclear compartment harboring ERK1/2 and CK2, restricting C/EBP{beta} from its activating kinases. We report here that the mRNA decay proteins UPF1 and Staufen1/2 are essential UPA factors enriched within the perinuclear cytoplasm. STAU1/2 and UPF1 overlap with CK2 on perinuclear signaling endosomes where they promote localized CEBPB mRNA decay. UPF1 or STAU1/2 depletion in tumor cells increased CEBPB transcripts adjacent to CK2 foci, coinciding with C/EBP{beta} activation and senescence. The GRE and an adjacent STAU binding site independently suppress C/EBP{beta}-mediated senescence, while a distinct 3UTR region inhibits its SASP-inducing activity. KrasG12D-driven lung tumors in mice carrying a Cebpb GRE deletion rarely progressed to malignant adenocarcinomas, demonstrating the importance of UPA to enable tumor progression in vivo. Thus, kinase-proximal mRNA decay is a novel mechanism that inhibits C/EBP{beta} activation in tumor cells to facilitate senescence bypass.

cancer biology↗