Inhibition of T-antigen expression promoting glycogen synthase kinase 3 impairs merkel cell carcinoma cell growth

2022 ◽  
Vol 524 ◽  
pp. 259-267
Author(s):  
Roland Houben ◽  
Sonja Hesbacher ◽  
Bhavishya Sarma ◽  
Carolin Schulte ◽  
Eva-Maria Sarosi ◽  
...  
Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 919 ◽  
Author(s):  
Bhavishya Sarma ◽  
Christoph Willmes ◽  
Laura Angerer ◽  
Christian Adam ◽  
Jürgen C. Becker ◽  
...  

Merkel cell carcinoma (MCC) is a rare and highly aggressive skin cancer with frequent viral etiology. Indeed, in about 80% of cases, there is an association with Merkel cell polyomavirus (MCPyV); the expression of viral T antigens is crucial for growth of virus-positive tumor cells. Since artesunate—a drug used to treat malaria—has been reported to possess additional anti-tumor as well as anti-viral activity, we sought to evaluate pre-clinically the effect of artesunate on MCC. We found that artesunate repressed growth and survival of MCPyV-positive MCC cells in vitro. This effect was accompanied by reduced large T antigen (LT) expression. Notably, however, it was even more efficient than shRNA-mediated downregulation of LT expression. Interestingly, in one MCC cell line (WaGa), T antigen knockdown rendered cells less sensitive to artesunate, while for two other MCC cell lines, we could not substantiate such a relation. Mechanistically, artesunate predominantly induces ferroptosis in MCPyV-positive MCC cells since known ferroptosis-inhibitors like DFO, BAF-A1, Fer-1 and β-mercaptoethanol reduced artesunate-induced death. Finally, application of artesunate in xenotransplanted mice demonstrated that growth of established MCC tumors can be significantly suppressed in vivo. In conclusion, our results revealed a highly anti-proliferative effect of the approved and generally well-tolerated anti-malaria compound artesunate on MCPyV-positive MCC cells, suggesting its potential usage for MCC therapy.


2019 ◽  
Author(s):  
Reety Arora ◽  
Komal Gupta ◽  
Anjali Vijaykumar ◽  
Sudhir Krishna

AbstractMerkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCV) T antigen expression, post integration (∼80% cases), or by UV mediated DNA damage. Interestingly, overall survival of patients suffering from MCV positive Merkel cell carcinoma is better, making this differential information of significant diagnostic and prognostic value. Also, MCV as a causative agent also provides a direct target for therapy in virus positive MCC patients. Currently, the methods used for diagnosis of MCV in tumours are often tedious, discordant and unreliable. In this study we used a guided molecular scissors based - DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) technique to develop an in vitro molecular diagnostic tool for MCV positive MCC. DETECTR couples recombinase polymerase based amplification of target MCV DNA with Cas12a mediated detection. CRISPR diagnostics couple specific detection followed by cutting of the pathogenic DNA by the Cas enzyme – gRNA complex, with non-specific cutting of ssDNA that provides a measurable visual cue. To detect MCV DNA in MCC tumours, we designed Cas12a gRNAs targeting the MCV DNA and tested their targeting efficiency, and sensitivity using a fluorophore quencher labeled reporter assay. We show that this sophisticated MCV DETECTR system can detect MCV integrated in Merkel tumour rapidly, specifically and with femto-molar sensitivity. This new MCV DNA detecting system is promising and we hope it can be coupled with histopathological and immunohistochemical studies to diagnose the viral status of MCC in clinics in the near future.


2009 ◽  
Vol 125 (6) ◽  
pp. 1243-1249 ◽  
Author(s):  
Masahiro Shuda ◽  
Reety Arora ◽  
Hyun Jin Kwun ◽  
Huichen Feng ◽  
Ronit Sarid ◽  
...  

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Kashif Rasheed ◽  
Baldur Sveinbjørnsson ◽  
Ugo Moens

Abstract Background Approximately 15% of human cancers are attributed to viruses. Numerous studies have shown that high-risk human polyomaviruses (HR-HPV) and Merkel cell polyomavirus (MCPyV) are two human tumor viruses associated with anogenetal and oropharyngeal cancers, and with Merkel cell carcinoma, respectively. MCPyV has been found in HR-HPV positive anogenetal and oropharyngeal tumors, suggesting that MCPyV can act as a co-factor in HR-HPV induced oncogenesis. This prompted us to investigate whether the oncoproteins large T-antigen (LT) and small antigen (sT) of MCPyV could affect the transcriptional activity HPV16 and HPV18 and vice versa whether HPV16 and HPV18 E6 and E7 oncoproteins affected the expression of MCPyV LT and sT. Reciprocal stimulation of these viral oncoproteinscould enhance the oncogenic processes triggered by these tumor viruses. Methods Transient co-transfection studies using a luciferase reporter plasmid with the long control region of HPV16 or HPV18, or the early or late promoter of MCPyV and expression plasmids for LT and sT, or E6 and E7, respectively were performed in the HPV-negative cervical cancer cell line C33A, in the keratinocyte cell line HaCaT, and in the oral squamous cell carcinoma cell line HSC-3. Transfections were also performed with deletion mutants of all these promoters and with mutants of all four oncoproteins. Finally, the effect of E6 and E7 on LT and sT expression in the MCPyV-positive Merkel cell carcinoma cell line WaGa and the effect of LT and sT on the expression of E6 and E7 was monitored by Western blotting. Results LT and sT stimulated the transcriptional activity of the HPV16 and HPV18 LCR and v.v. E6 and E7 potentiated the MCPyV early and late promoter in all cell lines. Induction by E6 and E7 was p53- and pRb-independent, and transactivation by LT did not require DNA binding, nuclear localization and HSC70/pRb interaction, whereas sT stimulated the HPV16/18 LCR activity in a PP2A- and DnaJ-independent manner. Conclusions These results indicate that the co-infection of MCPyV may act as a co-factor in the initiation and/or progression of HPV-induced cancers.


2009 ◽  
Vol 27 (24) ◽  
pp. 4021-4026 ◽  
Author(s):  

Purpose To expedite improved understanding, diagnosis, treatment, and prevention of Merkel cell carcinoma (MCC), a rare malignancy of cutaneous neuroendocrine cells that has a 28% 2-year mortality rate. Methods This article summarizes a workshop that discussed the state-of-the-art research and priorities for research on MCC and on a new human polyomavirus (ie, MCPyV) recently discovered in 80% of MCC tumors. Results Normal Merkel cells are widely distributed in the epidermis near the end of nerve axons and may function as mechanoreceptors or chemoreceptors. Malignant MCC cells typically stain for cytokeratin 20 as well as for other epithelial and neuroendocrine markers. MCC subtypes, which are based on histology, on cell line growth properties, and on gene expression profiles, have been reported but have not been linked to prognosis. Clinical management has been empiric. MCPyV is clonally integrated at various sites in the human genome of MCC tumors, with truncating mutations in the viral, large T antigen gene that interrupt viral replication. MCPyV seroprevalence may be high, as with previously known human polyomaviruses. MCC risk is increased 11-fold with AIDS and with other cell-mediated immune deficiencies, B-cell neoplasms, and ultraviolet radiation exposure. Conclusion Development and validation of a range quantitative polymerase chain reaction and serologic assays for detection of MCPyV, as well as an infectious clone of the virus, would clarify the fundamental biology, natural history, and epidemiology of the virus, of MCC, and of other diseases. Contingent on standardized histologic diagnosis and staging of MCC, consortia are needed to clarify the risks and benefits of sentinel lymph node biopsy, adjuvant radiation therapy, and salvage therapies; consortia are needed also for epidemiologic studies of MCC etiology.


2015 ◽  
Vol 89 (18) ◽  
pp. 9232-9241 ◽  
Author(s):  
Crystal Woodard ◽  
Gangling Liao ◽  
C. Rory Goodwin ◽  
Jianfei Hu ◽  
Zhi Xie ◽  
...  

ABSTRACTThe Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence forin vivophosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death.IMPORTANCEKSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human protein microarray for ERK-primed GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.


Sign in / Sign up

Export Citation Format

Share Document