scholarly journals Oncogenic RAS sensitizes cells to drug-induced replication stress via transcriptional silencing of P53

2021 ◽  
Author(s):  
Hendrika Alida Segeren ◽  
Elsbeth A van Liere ◽  
Frank M Riemers ◽  
Alain de Bruin ◽  
Bart Westendorp
Keyword(s):  
Cancer Cells ◽  
Transcriptional Control ◽  
Target Genes ◽  
Mapk Pathway ◽  
Transcriptional Response ◽  
Replication Stress ◽  
Mapk Signaling ◽  
S Phase ◽  
Drug Induced ◽  
Oncogenic Ras

Cancer cells often experience high basal levels of DNA replication stress (RS), for example due to hyperactivation of oncoproteins like MYC or RAS. Therefore, cancer cells are considered to be sensitive to drugs that exacerbate the level of RS or block the intra S-phase checkpoint. Consequently, RS-inducing drugs including ATR and CHK1 inhibitors are used or evaluated as anti-cancer therapies. However, drug resistance and lack of biomarkers predicting therapeutic efficacy limit efficient use. This raises the question what determines sensitivity of individual cancer cells to RS. Here, we report that oncogenic RAS does not only enhance the sensitivity to ATR/CHK1 inhibitors by directly causing RS. Instead, we observed that HRASG12V dampens the activation of the P53-dependent transcriptional response to drug-induced RS, which in turn confers sensitivity to RS. We demonstrate that inducible expression of HRASG12V sensitized retina pigment epithelial (RPE-hTERT) as well as osteosarcoma (U2OS) cells to ATR and CHK1 inhibitors. Using RNA-sequencing of FACS-sorted cells we discovered that P53 signaling is the sole transcriptional response to RS. However, oncogenic RAS attenuates the transcription of P53 and its target genes. Accordingly, live cell imaging showed that HRASG12V exacerbates RS in S/G2-phase, which could be rescued by stabilization of P53. Thus, our results demonstrate that transcriptional control of P53 is a prime determinant in the response to ATR/CHK1 inhibitors and show that hyperactivation of the MAPK pathway impedes this response. Our findings suggest that the level of oncogenic MAPK signaling could predict sensitivity to intra-S-phase inhibition in cancers with intact P53.

scholarly journals Brassinin Inhibits Proliferation in Human Liver Cancer Cells via Mitochondrial Dysfunction

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 332
Author(s):  
Taeyeon Hong ◽  
Jiyeon Ham ◽  
Jisoo Song ◽  
Gwonhwa Song ◽  
Whasun Lim
Keyword(s):  
Mitochondrial Dysfunction ◽  
Cancer Cells ◽  
Cell Lines ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Nuclear Antigen ◽  
Cruciferous Vegetable ◽  
Antiproliferative Effects ◽  
Hep3b Cells ◽  
Human Liver Cancer Cells

Brassinin is a phytochemical derived from Chinese cabbage, a cruciferous vegetable. Brassinin has shown anticancer effects on prostate and colon cancer cells, among others. However, its mechanisms and effects on hepatocellular carcinoma (HCC) have not been elucidated yet. Our results confirmed that brassinin exerted antiproliferative effects by reducing proliferating cell nuclear antigen (PCNA) activity, a proliferation indicator and inducing cell cycle arrest in human HCC (Huh7 and Hep3B) cells. Brassinin also increased mitochondrial Ca2+ levels and depolarized the mitochondrial membrane in both Huh7 and Hep3B cells. Moreover, brassinin generated high amounts of reactive oxygen species (ROS) in both cell lines. The ROS scavenger N-acetyl-L-cysteine (NAC) inhibited this brassinin-induced ROS production. Brassinin also regulated the AKT and mitogen-activated protein kinases (MAPK) signaling pathways in Huh7 and Hep3B cells. Furthermore, co-administering brassinin and pharmacological inhibitors for JNK, ERK1/2 and P38 decreased cell proliferation in both HCC cell lines more than the pharmacological inhibitors alone. Collectively, our results demonstrated that brassinin exerts antiproliferative effects via mitochondrial dysfunction and MAPK pathway regulation on HCC cells.

scholarly journals A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3317
Author(s):  
Eric Moeglin ◽  
Dominique Desplancq ◽  
Audrey Stoessel ◽  
Christian Massute ◽  
Jeremy Ranniger ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mammalian Cells ◽  
Chromatin Modification ◽  
Replication Stress ◽  
Living Cells ◽  
Single Step ◽  
Dna Double Strand Breaks ◽  
Drug Induced ◽  
Histone H2ax ◽  
C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

scholarly journals p27kip1 controls H-Ras/MAPK activation and cell cycle entry via modulation of MT stability

2015 ◽  
Vol 112 (45) ◽  
pp. 13916-13921 ◽  
Author(s):  
Linda Fabris ◽  
Stefania Berton ◽  
Ilenia Pellizzari ◽  
Ilenia Segatto ◽  
Sara D’Andrea ◽  
...  
Keyword(s):  
Phase Transition ◽  
Cell Cycle ◽  
Signaling Pathway ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
S Phase ◽  
Point Of View ◽  
Cell Cycle Entry ◽  
Pathway Activation ◽  
Direct Binding

The cyclin-dependent kinase (CDK) inhibitor p27kip1 is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27kip1-null mice is reverted by concomitant deletion of stathmin in p27kip1/stathmin double-KO mice, suggesting that a CDK-independent function of p27kip1 contributes to the control of cell proliferation. Whether the regulation of MT stability by p27kip1 impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27kip1-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27kip1 and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27kip1, by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27kip1 and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway.

scholarly journals Dormant replication origin firing links replication stress to whole chromosomal instability in human cancer

2021 ◽  
Author(s):  
Ann-Kathrin Schmidt ◽  
Nicolas Boehly ◽  
Xiaoxiao Zhang ◽  
Benjamin O. Slusarenko ◽  
Magdalena Hennecke ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Chromosomal Instability ◽  
Replication Origin ◽  
Human Cancer ◽  
Chromosome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Origin Firing ◽  
Human Cancer Cells ◽  
Chromosome Missegregation

Chromosomal instability (CIN) is a hallmark of cancer and comprises structural CIN (S-CIN) and whole chromosome instability (W-CIN). Replication stress (RS), a condition of slowed or stalled DNA replication during S phase, has been linked to S-CIN, whereas defects in mitosis leading to chromosome missegregation and aneuploidy can account for W-CIN. It is well established that RS can activate additional replication origin firing that is considered as a rescue mechanism to suppress chromosomal instability in the presence of RS. In contrast, we show here that an increase in replication origin firing during S phase can contribute to W-CIN in human cancer cells. Increased origin firing can be specifically triggered by overexpression of origin firing genes including GINS1 and CDC45, whose elevated expression significantly correlates with W-CIN in human cancer specimens. Moreover, endogenous mild RS present in cancer cells characterized by W-CIN or modulation of the origin firing regulating ATR-CDK1-RIF1 axis induces dormant origin firing, which is sufficient to trigger chromosome missegregation and W-CIN. Importantly, chromosome missegregation upon increased dormant origin firing is mediated by increased microtubule growth rates leading to the generation of lagging chromosomes in mitosis, a condition prevalent in chromosomally unstable cancer cells. Thus, our study identified increased or dormant replication origin firing as a hitherto unrecognized, but cancer-relevant trigger for chromosomal instability.

scholarly journals Post-transcriptional Down-regulation of ROCKI/Rho-kinase through an MEK-dependent Pathway Leads to Cytoskeleton Disruption in Ras-transformed Fibroblasts

2002 ◽  
Vol 13 (1) ◽  
pp. 336-347 ◽  
Author(s):  
Geraldine Pawlak ◽  
David M. Helfman
Keyword(s):  
Mapk Pathway ◽  
Rho Kinase ◽  
Mapk Signaling ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Cell Junctions ◽  
Lim Kinase ◽  
Oncogenic Ras ◽  
Cytoskeleton Disruption ◽  
Kinase Expression

Transformation by oncogenic Ras profoundly alters actin cytoskeleton organization. We investigated Ras-dependent signaling pathways involved in cytoskeleton disruption by transfecting normal rat kidney (NRK) cells with different Ras mutants. RasV12S35, a mutant known to activate specifically the Raf/MAPK pathway, led to stress fiber and focal contact disruption, whereas the adherens junctions remained intact. Next, we found that pharmacological inhibition of MEK was sufficient to restore the cytoskeletal defects of ras-transformed NRK cells, including assembly of stress fibers and focal contacts, but it did not induce reorganization of the cell-cell junctions. Investigating the mechanism underlying this phenotypic reversion, we found that the sustained MAPK signaling resulting from Ras-transformation down-regulated the expression of ROCKI and Rho-kinase, two-Rho effectors required for stress fiber formation, at the post-transcriptional level. On MEK inhibition, ROCKI/Rho-kinase expression and cofilin phosphorylation were increased, demonstrating that the Rho-kinase/LIM-kinase/cofilin pathway was functionally restored. Finally, using dominant negative or constitutively active mutants, we demonstrated that expression of ROCKI/Rho-kinase was both necessary and sufficient to promote cytoskeleton reorganization in NRK/ras cells. These findings further establish the Ras/MAPK pathway as the critical pathway involved in cytoskeleton disruption during Ras-transformation, and they suggest a new mechanism, involving alteration in ROCKI/Rho-kinase expression, by which oncogenic Ras can specifically target the actin-based cytoskeleton and achieve morphological transformation of the cells.

The Transforming Potential of the Homeobox Gene Cdx2 Is Depending on Its N-Terminal Transactivation Domain In Vivo and Can Be Antagonized by the MEK1/2 Inhibitor in a Mouse Model of t(12;13) Positive AML.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 7-7
Author(s):  
Vijay P.S. Rawat ◽  
Natalia Arseni Arseni ◽  
Vegi M. Naidu ◽  
John P. Lynch ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
Microarray Analysis ◽  
Target Genes ◽  
Kinase Inhibitors ◽  
Mapk Pathway ◽  
Ectopic Expression ◽  
Specific Inhibitor ◽  
Homeobox Gene ◽  
Mapk Signaling ◽  
Transactivation Domain

Abstract In AML the translocation t(12;13)(p13;q12) results in the ectopic expression of the homeobox gene Cdx2 and the expression of the ETV6-CDX2 fusion. We have shown that the ectopic expression of the proto-oncogene Cdx2 and not the expression of the ETV6-CDX2 fusion is the key event in initiating myeloid leukemogenesis in a murine model of t(12;13) AML (PNAS, Rawat et. al. 2004). We now analyzed the functional relevance of the different Cdx2 domains and explored the potential of kinase inhibitors to antagonize Cdx2 induced leukemia. For this we generated different mutants, inactivating the PBX1-interacting motif (W167A-Cdx2), or deleting the N-terminal transactivation domain (Ndel-Cdx2). Expression of Cdx2 and the different mutants was induced in primary murine BM cells by retroviral gene transfer. Target genes were identified by cDNA microarray analysis. Mice transplanted with BM cells expressing Cdx2 and its W167A-Cdx2 mutant developed transplantable AML (n=14, avg. latency 90 days). In contrast, mice transplanted with the NDel-Cdx2 mutant did not show any leukemic phenotype in vivo (n=13). In order to identify gene expression signatures associated with Cdx2 induced transformation, we performed microarray analysis on highly purified normal Sca1+/lin− HSC and Sca1+/lin+ progenitor cells transduced with the leukemogenic Cdx2 compared to the non-leukemogenic NDel-Cdx2 mutant and the GFP control vector after 72h of retrovirally induced expression of the different constructs. Compared to the NDel-Cdx2 mutant and the GFP control Cdx2 up regulated genes, which are associated with self-renewal (Wint2, Hoxb3, Etv6, Abcg2,), leukemogenesis (Lmo2, Pim-2, Hoxa9) and in signal transduction pathways (e.g. MAPK family). In addition, Cdx2 transduced BM cells showed an activated Erk1/2 pathway on the protein level. Based on these results we tested whether inhibition of the MAPK pathway would impair the leukemogenic potential of Cdx2. When Cdx2 transduced BM cells were incubated with the MEK1/2 inhibitor PD98059, a 78% reduction of viable cells (n=3, p<0.03) and of the proportion of blast-like Sca1+ positive cells were observed compared to untreated cells (n=3, p<0.005) in liquid culture after 7 days. Furthermore, incubation with the MEK1/2 inhibitor PD98059 decreased the activity of Cdx2 at the level of the short-term repopulating stem cell 8-fold as assessed in the ΔCFU-S after 7 days in vitro culture (n=7, p<0.001). In contrast, incubation with the p38 specific inhibitor SB 28049 did not show any decrease in Cdx2 activity in ΔCFU-S assay, indicating that the transforming potential of Cdx2 depends on the MEK1/2 pathway, but not on the p38 pathway. These data demonstrate that the leukemogenic potential of the homeobox gene Cdx2 depends on the N-terminal activation domain. Furthermore, our data link the oncogenic capacity of the transcription factor Cdx2 to MAPK signaling, opening the possibility to counteract homeobox-associated leukemogenesis by kinase inhibitors.

scholarly journals Replication Stress, Defective S-phase Checkpoint and Increased Death in Plk2-Deficient Human Cancer Cells

Cell Cycle ◽  
2007 ◽  
Vol 6 (20) ◽  
pp. 2571-2578 ◽  
Author(s):  
Elizabeth M. Matthew ◽  
Timothy J. Yen ◽  
David T. Dicker ◽  
Jay F. Dorsey ◽  
Wensheng Yang ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Human Cancer ◽  
Replication Stress ◽  
S Phase ◽  
Human Cancer Cells ◽  
S Phase Checkpoint

scholarly journals PlatyphyllenoneExerts Anti-Metastatic Effects on Human Oral Cancer Cells by Modulating Cathepsin L Expression, MAPK Pathway and Epithelial–Mesenchymal Transition

2021 ◽  
Vol 22 (9) ◽  
pp. 5012
Author(s):  
V. Bharath Kumar ◽  
Jen-Tsun Lin ◽  
B. Mahalakshmi ◽  
Yi-Ching Chuang ◽  
Hsin-Yu Ho ◽  
...  
Keyword(s):  
Oral Cancer ◽  
Cancer Cells ◽  
Epithelial Mesenchymal Transition ◽  
Mapk Pathway ◽  
Cathepsin L ◽  
Mapk Signaling ◽  
Cancer Prognosis ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Oral Cancer Cells

Advanced-stage oral cancers with lymph node metastasis are associated with poor prognosis and a high mortality rate. Although recent advancement in cancer treatment has effectively improved the oral cancer prognosis, the majority of therapeutic interventions are highly expensive and are associated with severe sideeffects. In the present study, we studied the efficacy of a diarylheptanoid derivative, platyphyllenone, in modulating the metastatic potential of human oral cancer cells. Specifically, we treated the human oral cancer cells (FaDu, Ca9-22, and HSC3) with different concentrations of platyphyllenone and measured the cell proliferation, migration, and invasion. The study findings revealed that platyphyllenonesignificantly inhibited the motility, migration, and invasion of human oral cancer cells. Mechanistically, platyphyllenone reduced p38 phosphorylation, decreased β-catenin and Slug, increased E-cadherin expression, and reduced cathepsin L expression, which collectively led to a reduction in cancer cell migration and invasion. Taken together, our study indicates that platyphyllenone exerts significant anti-metastatic effects on oral cancer cells by modulating cathepsin L expression, the MAPK signaling pathway, and the epithelial–mesenchymal transition process.

scholarly journals Akt-mediated Ephexin1–Ras interaction promotes oncogenic Ras signaling and colorectal and lung cancer cell proliferation

2021 ◽  
Vol 12 (11) ◽  
Author(s):  
Jeeho Kim ◽  
Young Jin Jeon ◽  
Sung-Chul Lim ◽  
Joohyun Ryu ◽  
Jung-Hee Lee ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Target Genes ◽  
Critical Role ◽  
Survival Rates ◽  
Tumor Grade ◽  
Mapk Signaling ◽  
Ras Signaling ◽  
Poor Overall Survival ◽  
Downstream Target ◽  
Oncogenic Ras

AbstrctEphexin1 was reported to be highly upregulated by oncogenic Ras, but the functional consequences of this remain poorly understood. Here, we show that Ephexin1 is highly expressed in colorectal cancer (CRC) and lung cancer (LC) patient tissues. Knockdown of Ephexin1 markedly inhibited the cell growth of CRC and LC cells with oncogenic Ras mutations. Ephexin1 contributes to the positive regulation of Ras-mediated downstream target genes and promotes Ras-induced skin tumorigenesis. Mechanically, Akt phosphorylates Ephexin1 at Ser16 and Ser18 (pSer16/18) and pSer16/18 Ephexin1 then interacts with oncogenic K-Ras to promote downstream MAPK signaling, facilitating tumorigenesis. Furthermore, pSer16/18 Ephexin1 is associated with both an increased tumor grade and metastatic cases of CRC and LC, and those that highly express pSer16/18 exhibit poor overall survival rates. These data indicate that Ephexin1 plays a critical role in the Ras-mediated CRC and LC and pSer16/18 Ephexin1 might be an effective therapeutic target for CRC and LC.

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