Identification of microRNAs that stabilize p53 in HPV-positive cancer cells

2021 ◽  
Author(s):  
Gustavo Martínez-Noël ◽  
Patricia Szajner ◽  
Rebecca E. Kramer ◽  
Kathleen A. Boyland ◽  
Asma Sheikh ◽  
...  
Keyword(s):  
Cancer Cells ◽  
High Throughput ◽  
Ubiquitin Ligase ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Human Papillomaviruses ◽  
Live Cell ◽  
Viral Oncoprotein ◽  
Protein Levels ◽  
P53 Stability

Etiologically, 5% of all cancers worldwide are caused by the high-risk human papillomaviruses (hrHPVs). These viruses encode two oncoproteins (E6 and E7) whose expression is required for cancer initiation and maintenance. Among their cellular targets are the p53 and the retinoblastoma tumor suppressor proteins. Inhibition of the hrHPV E6-mediated ubiquitylation of p53 through the E6AP ubiquitin ligase results in the stabilization of p53, leading to cellular apoptosis. We utilized a live cell high throughput screen to determine whether exogenous microRNA (miRNA) transfection had the ability to stabilize p53 in hrHPV-positive cervical cancer cells expressing a p53-fluorescent protein as an in vivo reporter of p53 stability. Among the miRNAs whose transfection resulted in the greatest p53 stabilization was 375-3p that has previously been reported to stabilize p53 in HeLa cells, providing validation of the screen. The top 32 miRNAs in addition to 375-3p were further assessed using a second cell-based p53 stability reporter system as well as in non-reporter HeLa cells to examine their effects on endogenous p53 protein levels, resulting in the identification of 23 miRNAs whose transfection increased p53 levels in HeLa cells. While a few miRNAs that stabilized p53 led to decreases in E6AP protein levels, all targeted HPV oncoprotein expression. We further examined subsets of these miRNAs for their abilities to induce apoptosis and determined whether it was p53-mediated. The introduction of specific miRNAs revealed surprisingly heterogeneous responses in different cell lines. Nonetheless, some of the miRNAs described here have potential as therapeutics for treating HPV-positive cancers. Importance Human papillomaviruses cause approximately 5% of all cancers worldwide and encode genes that contribute to both the initiation and maintenance of these cancers. The viral oncoprotein E6 is expressed in all HPV-positive cancers and functions by targeting the degradation of p53 through the engagement of the cellular ubiquitin ligase E6AP. Inhibiting the degradation of p53 leads to apoptosis in HPV-positive cancer cells. Using a high throughput live cell assay we identified several miRNAs whose transfection stabilize p53 in HPV-positive cells. These miRNAs have the potential to be used in the treatment of HPV-positive cancers.

scholarly journals Stabilization of p53 by microRNAs in HPV-positive cervical cancer cells

2020 ◽  
Author(s):  
Gustavo Martínez-Noël ◽  
Patricia Szajner ◽  
Jennifer A. Smith ◽  
Kathleen Boyland ◽  
Rebecca E. Kramer ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
High Throughput ◽  
Ubiquitin Ligase ◽  
Hela Cells ◽  
P53 Protein ◽  
Human Papillomaviruses ◽  
Live Cell ◽  
Protein Levels ◽  
Cervical Cancer Cells

AbstractEtiologically 5% of cancers worldwide are caused by the high-risk human papillomaviruses (hrHPVs). These viruses encode two oncoproteins (E6 and E7) whose expression is required for cancer initiation and maintenance. Among the cellular targets of these viral oncoproteins are the p53 and the retinoblastoma tumor suppressor proteins. Inhibition of E6-mediated ubiquitylation of p53 through the E6AP ubiquitin ligase results in the stabilization of p53, leading to cellular apoptosis. We utilized a live cell high-throughput screen to assess the ability of 885 microRNAs (miRNAs) to stabilize p53 in human papillomavirus (HPV)-positive cervical cancer cells expressing a p53-fluorescent protein as an in vivo reporter of p53 stability. The 32 miRNAs whose expression resulted in the greatest p53 stabilization were further assessed in validation experiments using a second cell-based p53 stability reporter system as well as in HeLa cells to examine their effects on endogenous p53 protein levels. The positive miRNAs identified included 375-3p that has previously been reported as stabilizing p53 in HeLa cells, providing validation of the screen. Additional miRNAs that stabilized p53 led to decreases in E6AP protein levels, while others, including members of the 302/519 family of miRNAs, targeted HPV oncoprotein expression. We further examined a subset of these miRNAs for their abilities to induce apoptosis and determined whether the apoptosis was p53-mediated. The miRNAs described here have potential as therapeutics for treating HPV-positive cancers.Author summaryHuman papillomaviruses cause approximately 5% of cancers worldwide and encode genes that contribute to both the initiation and maintenance of these cancers. The viral gene E6 is expressed in all HPV-positive cancers and functions by targeting the degradation of the p53 protein through its engagement of the cellular ubiquitin ligase E6AP. Inhibiting the degradation of p53 results in apoptosis in HPV-positive cancer cells. We have developed a high-throughput live cell assay to identify molecules that stabilize p53 in HPV-positive cells and we present the results of a screen we have carried out examining miRNAs for their abilities to stabilize p53 and induce apoptosis in HPV-positive cervical cancer cells. These miRNAs have the potential to be used in the treatment of HPV-positive cancers.

Live cell, image-based high-throughput screen to quantitate p53 stabilization and viability in human papillomavirus positive cancer cells

Virology ◽  
2021 ◽  
Author(s):  
Gustavo Martínez-Noël ◽  
Valdimara Corrêa Vieira ◽  
Patricia Szajner ◽  
Erin M. Lilienthal ◽  
Rebecca E. Kramer ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Cancer Cells ◽  
High Throughput ◽  
Live Cell ◽  
High Throughput Screen ◽  
Cell Image ◽  
Live Cell Image

scholarly journals A novel role for BRCA1 in regulating breast cancer cell spreading and motility

2011 ◽  
Vol 192 (3) ◽  
pp. 497-512 ◽  
Author(s):  
Elisabeth D. Coene ◽  
Catarina Gadelha ◽  
Nicholas White ◽  
Ashraf Malhas ◽  
Benjamin Thomas ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cancer Cells ◽  
Ubiquitin Ligase ◽  
Fluorescent Protein ◽  
Cell Spreading ◽  
Dominant Negative ◽  
Full Length ◽  
Suppressor Activity ◽  
Ubiquitin Ligase Activity ◽  
Mcf 7

BRCA1 C-terminal (BRCT) domains in BRCA1 are essential for tumor suppressor function, though the underlying mechanisms remain unclear. We identified ezrin, radixin, and moesin as BRCA1 BRCT domain–interacting proteins. Ezrin–radixin–moesin (ERM) and F-actin colocalized with BRCA1 at the plasma membrane (PM) of cancer cells, especially at leading edges and focal adhesion sites. In stably expressing cancer cells, high levels of enhanced green fluorescent protein (EGFP)-BRCA11634–1863 acted as a dominant-negative factor, displacing endogenous BRCA1 from the PM. This led to delayed cell spreading, increased spontaneous motility, and irregular monolayer wound healing. MCF-7 cells (intact BRCA1) showed lower motility than HCC1937 cells (truncated BRCA1), but expression of EGFP-BRCA11634–1863 in MCF-7 increased motility. Conversely, full-length BRCA1 expression in HCC1937 decreased motility but only if the protein retained ubiquitin ligase activity. We conclude that full-length BRCA1 is important for complete tumor suppressor activity via interaction of its BRCT domains with ERM at the PM, controlling spreading and motility of cancer cells via ubiquitin ligase activity.

scholarly journals CRISPR/Cas9-based inactivation of human papillomavirus oncogenes E6 and E7 induces senescence in cervical cancer cells

2020 ◽  
Author(s):  
Raviteja Inturi ◽  
Per Jemth
Keyword(s):  
Cancer Cells ◽  
Cellular Senescence ◽  
Hela Cells ◽  
Human Papillomaviruses ◽  
Alternative Outcome ◽  
Cervical Cancer Cells ◽  
Nucleus Surface ◽  
Hpv18 E6 ◽  
E6 And E7 ◽  
E7 Proteins

ABSTRACTHuman papillomaviruses (HPVs) such as HPV16 and HPV18 can cause cancers of the cervix, vagina, vulva, penis, anus and oropharynx. Continuous expression of the HPV viral oncoproteins E6 and E7 are essential for transformation and maintenance of cancer cells. Therefore, therapeutic targeting of E6 and E7 genes can potentially be used to treat HPV-related cancers. Previous CRISPR/Cas9 studies on inactivation of E6 and E7 genes confirmed cell cycle arrest and apoptosis. Here we report that CRISPR/Cas9-based knockout of E6 and E7 can also trigger cellular senescence in HPV18 immortalized HeLa cells. Specifically, HeLa cells in which E6 and E7 were inactivated exhibited characteristic senescence markers like enlarged cell and nucleus surface area, increased β-galactosidase expression, and loss of lamin B1 with detection of cytoplasmic chromatin fragments. Furthermore, the knockout of HPV18 E6 and E7 proteins resulted in upregulation of p53/p21 and pRb/p21 levels in senescent cells. These senescent cells were devoid of characteristic apoptotic markers and re-introduction of codon-modified HPV18 E6 decreased p53 levels. Taken together, our study demonstrates that cellular senescence is as an alternative outcome of HPV oncogene inactivation by the CRISPR/Cas9 methodology.

scholarly journals E6-mediated activation of JNK drives EGFR signalling to promote proliferation and viral oncoprotein expression in cervical cancer

2020 ◽  
Author(s):  
Ethan L. Morgan ◽  
James A. Scarth ◽  
Molly R. Patterson ◽  
Christopher W. Wasson ◽  
Georgia C. Hemingway ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Human Papillomaviruses ◽  
Signalling Pathway ◽  
Viral Oncoprotein ◽  
Hpv E6 ◽  
Cervical Cancer Cells ◽  
E6 And E7 ◽  
Novel Therapeutic

AbstractHuman papillomaviruses (HPV) are a major cause of malignancy worldwide, contributing to ~5% of all human cancers including almost all cases of cervical cancer and a growing number of ano-genital and oral cancers. HPV-induced malignancy is primarily driven by the viral oncogenes, E6 and E7, which manipulate host cellular pathways to increase cell proliferation and enhance cell survival, ultimately predisposing infected cells to malignant transformation. Consequently, a more detailed understanding of viral-host interactions in HPV-associated disease offers the potential to identify novel therapeutic targets. Here, we identify that the c-Jun N-terminal kinase (JNK) signalling pathway is activated in cervical disease and in cervical cancer. The HPV E6 oncogene induces JNK1/2 phosphorylation in a manner that requires the E6 PDZ binding motif. We show that blockade of JNK1/2 signalling using small molecule inhibitors, or knockdown of the canonical JNK substrate c-Jun, reduces cell proliferation and induces apoptosis in cervical cancer cells. We further demonstrate that this phenotype is at least partially driven by JNK-dependent activation of EGFR signalling via increased expression of EGFR and the EGFR ligands EGF and HB-EGF. JNK/c-Jun signalling promoted the invasive potential of cervical cancer cells and was required for the expression of the epithelial to mesenchymal transition (EMT)-associated transcription factor Slug and the mesenchymal marker Vimentin. Furthermore, JNK/c-Jun signalling is required for the constitutive expression of HPV E6 and E7, which are essential for cervical cancer cell growth and survival. Together, these data demonstrate a positive feedback loop between the EGFR signalling pathway and HPV E6/E7 expression, identifying a regulatory mechanism in which HPV drives EGFR signalling to promote proliferation, survival and EMT. Thus, our study has identified a novel therapeutic target that may be beneficial for the treatment of cervical cancer.

High-Throughput Screening and Triage Assays Identify Small Molecules Targeting c-MYC in Cancer Cells

2021 ◽  
Vol 26 (2) ◽  
pp. 216-229
Author(s):  
Lorena A. Kallal ◽  
Anna Waszkiewicz ◽  
Jon-Paul Jaworski ◽  
Anthony Della Pietra ◽  
Tom Berrodin ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cancer Cells ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Detection System ◽  
Antibody Detection ◽  
Protein Levels ◽  
Cellular Assays

While c-MYC is well established as a proto-oncogene, its structure and function as a transcription factor have made c-MYC a difficult therapeutic target. To identify small-molecule inhibitors targeting c-MYC for anticancer therapy, we designed a high-throughput screening (HTS) strategy utilizing cellular assays. The novel approach for the HTS was based on the detection of cellular c-MYC protein, with active molecules defined as those that specifically decreased c-MYC protein levels in cancer cells. The assay was based on a dual antibody detection system using Förster/fluorescence resonance energy transfer (FRET) and was utilized to detect endogenous c-MYC protein in the MYC amplified cancer cell lines DMS273 and Colo320 HSR. The assays were miniaturized to 1536-well plate format and utilized to screen the GlaxoSmithKline small-molecule collection of approximately 2 million compounds. In addition to the HTS assay, follow-up assays were developed and used to triage and qualify compounds. Two cellular assays used to eliminate false-positive compounds from the initially selected HTS hits were (1) a cellular toxicity assay and (2) an unstable protein reporter assay. Three positive selection assays were subsequently used to qualify compounds: (1) 384-well cell cycle flow cytometry, (2) 384-well cell growth, and (3) c-MYC gene signature reverse transcription quantitative PCR (RT-qPCR). The HTS and follow-up assays successfully identified three compounds that specifically decreased c-MYC protein levels in cancer cells and phenocopied c-MYC siRNA in terms of cell growth inhibition and gene signatures. The HTS, triage, and three compounds identified are described.

scholarly journals The Steroidogenic Enzyme AKR1C3 Regulates Stability of the Ubiquitin Ligase Siah2 in Prostate Cancer Cells

2015 ◽  
Vol 290 (34) ◽  
pp. 20865-20879 ◽  
Author(s):  
Lingling Fan ◽  
Guihong Peng ◽  
Arif Hussain ◽  
Ladan Fazli ◽  
Emma Guns ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cell Growth ◽  
Cancer Cells ◽  
Ubiquitin Ligase ◽  
Castration Resistant Prostate Cancer ◽  
Prostate Cancer Cells ◽  
Cancer Cell Growth ◽  
Protein Levels ◽  
Castration Resistant

Re-activation of androgen receptor (AR) activity is the main driver for development of castration-resistant prostate cancer. We previously reported that the ubiquitin ligase Siah2 enhanced AR transcriptional activity and prostate cancer cell growth. Among the genes we found to be regulated by Siah2 was AKR1C3, which encodes a key androgen biosynthetic enzyme implicated in castration-resistant prostate cancer development. Here, we found that Siah2 inhibition in CWR22Rv1 prostate cancer cells decreased AKR1C3 expression as well as intracellular androgen levels, concomitant with inhibition of cell growth in vitro and in orthotopic prostate tumors. Re-expression of either wild-type or catalytically inactive forms of AKR1C3 partially rescued AR activity and growth defects in Siah2 knockdown cells, suggesting a nonenzymatic role for AKR1C3 in these outcomes. Unexpectedly, AKR1C3 re-expression in Siah2 knockdown cells elevated Siah2 protein levels, whereas AKR1C3 knockdown had the opposite effect. We further found that AKR1C3 can bind Siah2 and inhibit its self-ubiquitination and degradation, thereby increasing Siah2 protein levels. We observed parallel expression of Siah2 and AKR1C3 in human prostate cancer tissues. Collectively, our findings identify a new role for AKR1C3 in regulating Siah2 stability and thus enhancing Siah2-dependent regulation of AR activity in prostate cancer cells.

scholarly journals Novel Ubiquitinated Proteins Downstream of the Fanconi Anemia Core Complex

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1116-1116
Author(s):  
Abeer Najjar
Keyword(s):  
Dna Repair ◽  
High Throughput ◽  
Ubiquitin Ligase ◽  
Fanconi Anemia ◽  
Hela Cells ◽  
Repair Pathway ◽  
Core Complex ◽  
Target Proteins ◽  
Ubiquitinated Proteins ◽  
Ubiquitin Ligase Complex

Abstract The Fanconi anemia (FA) pathway is a major player in the control of DNA replication integrity in response to replication stress. Germline defect in the pathway results in the FA syndrome characterized by developmental abnormalities, bone marrow (BM) failure, and genome instability which greatly elevates the incidence of cancers. A pivotal step in the activation of the FA DNA repair pathway is the monoubiquitination of the FANCD2 and FANCI proteins (ID2) by the FA core complex, a unique ubiquitin ligase complex which includes eight proteins (FANCA-FANCG, FANCL, and FAAP100) and UBE2T/FANCT. This monoubiquitination event enables the recruitment of the ID2 complex to chromatin and nuclear foci at sites of DNA damage. Cells with mutations in any of the FA core complex proteins lack the ability to monoubiquitinated ID2, making ID2 ubiquitination a convergence point in the pathway, with an estimation of>90% FA patients defective in this step. Additionally, somatic mutations In FA genes render tumor cells sensitive to DNA crosslinking agents, so identification of FA pathway defects provides an opportunity for therapeutic targeting. In search for additional potential target/substrate of this unique FA core ubiquitin ligase complex, we performed a high throughput genome-wide ubiquitin-specific proteomics (UbiScan) screen and found, in addition to the ID2 complex, many ubiquitinated proteins are dysregulated (mostly downregulated) in FA deficient cells compared with that of FA proficient cells. We used a Ubiquitin Remnant Motif (K- ∑-GG) Antibody Bead Conjugate (Cell Signaling Technology), a proprietary ubiquitin branch ("K- ∑-GG") antibody with specificity for a di-glycine tag that is the remnant of ubiquitin left on protein substrates after trypsin digestion, to enrich ubiquitinated peptides from trypsin digested cell samples (shNT vs shFANCA). This enrichment is followed by LC-MS/MS analysis for quantitative profiles of hundreds to over a thousand nonredundant ubiquitinated sequences. We were successful in demonstrating that under steady-state conditions (without proteasome inhibitor treatment), the ubiquitinated forms of both FANCD2 and FANCI proteins are much higher in control (shNT) HeLa cells compared with that of the cells depleted of FANCA (shFANCA). We then collaborated with the Cell signaling technology to perform a high throughput UbiScan® analysis of total ubiquitinated proteins both in total nuclei and chromatin fractions under replicative stress conditions. UbiScan® enables researchers to isolate, identify and quantitate large numbers of ubiquitin-modified cellular peptides with a high degree of specificity and sensitivity, providing a global overview of the ubiquitination sites in cellular proteins in cell and tissue samples without preconceived biases about where these modified sites occur. A total of 16,249 redundant modified peptide assignments to 7,856 modified sites for the Ubiquitin K-GG Remnant Motif Antibody were obtained. As expected, the amount of monoubiquitinated FANCD2 (at K651) and FANCI (at K523) were highly reduced in both the nuclear and chromatin fractions of Hela cells depleted of FANCA (shA). Consistent with the earlier findings, the amount of ubiquitinated ID2 proteins were extremely low in the chromatin fraction of the Hela cells depleted of FANCA. Since there are numerous ubiquitinated proteins found to be dysregulated in our UbiScan analyses, we used the following criteria to select the target proteins based on; a) -fold changes, and b) proteins that are known to participate in the DNA repair signaling pathways. We validated our UbiScan results by using an assay system to detect endogenous protein ubiquitination. We also found a significant reduction in the ubiquitination of several DNA repair-related proteins (found in our UbiScan analysis) in FANCA deficient cells. To assess FA pathway functions, we generated HAP1 and appropriate cells knock out of these select ubiquitinated target proteins by using CRISPR-Cas9 system. Then, the KO cells were examined for FA pathway functions. These results will be discussed. In conclusion, our findings reveal that the FA core ubiquitin ligase complex regulates (directly or indirectly) the ubiquitinated levels of many novel proteins outside of the ID2 complex, and these novel target proteins may provide important additional mechanistic insights into the FA DNA repair pathway. Disclosures No relevant conflicts of interest to declare.

scholarly journals Investigation of the interplay between SKP2, CDT1 and Geminin in cancer cells

2015 ◽  
Author(s):  
Andrea Ballabeni ◽  
Raffaella Zamponi
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cancer Cells ◽  
Ubiquitin Ligase ◽  
S Phase ◽  
Physical Interaction ◽  
Protein Levels ◽  
Ubiquitin Ligase Complex ◽  
G2 Phase ◽  
Scf Ubiquitin Ligase

Geminin has a dual role in the regulation of DNA replication: it inhibits replication factor CDT1 activity during the G2 phase of the cell cycle and promotes its accumulation at the G2/M transition. In this way Geminin prevents DNA re-replication during G2 phase and ensures that DNA replication is efficiently executed in the next S phase. CDT1 was shown to associate with SKP2, the substrate recognition factor of the SCF ubiquitin ligase complex. We investigated the interplay between these three proteins in cancer cell lines. We show that Geminin, CDT1 and SKP2 could possibly form a complex and propose the putative regions of CDT1 and Geminin involved in the binding. We also show that, despite the physical interaction, SKP2 depletion does not substantially affect CDT1 and Geminin protein levels. Moreover, we show that while Geminin and CDT1 levels fluctuate, SKP2 levels, differently than in normal cells, are almost steady during the cell cycle of the tested cancer cells.

scholarly journals Electrodelivery of Drugs into Cancer Cells in the Presence of Poloxamer 188

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Iana Tsoneva ◽  
Iordan Iordanov ◽  
Annette J. Berger ◽  
Toma Tomov ◽  
Biliana Nikolova ◽  
...  
Keyword(s):  
Small Molecules ◽  
Cancer Cells ◽  
Nude Mice ◽  
Propidium Iodide ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Electrical Pulse ◽  
Red Fluorescent Protein ◽  
Poloxamer 188 ◽  
Pore Structures

In the present study it is shown that poloxamer 188, added before or immediately after an electrical pulse used for electroporation, decreases the number of dead cells and at the same time does not reduce the number of reversible electropores through which small molecules (cisplatin, bleomycin, or propidium iodide) can pass/diffuse. It was suggested that hydrophobic sections of poloxamer 188 molecules are incorporated into the edges of pores and that their hydrophilic parts act as brushy pore structures. The formation of brushy pores may reduce the expansion of pores and delay the irreversible electropermeability. Tumors were implanted subcutaneously in both flanks of nude mice using HeLa cells, transfected with genes for red fluorescent protein and luciferase. The volume of tumors stopped to grow after electrochemotherapy and the use of poloxamer 188 reduced the edema near the electrode and around the subcutaneously growing tumors.

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