Tirbanibulin decreases cell proliferation and downregulates protein expression of oncogenic pathways in human papillomavirus containing HeLa cells.

Tirbanibulin 1% ointment is a synthetic antiproliferative agent approved in 2021 by the European Union for treating actinic keratoses (AK). Topical tirbanibulin has clinically resolved HPV-57 ( +) squamous cell carcinoma (SCC), HPV-16 ( +) vulvar high-grade squamous intraepithelial lesion, epidermodysplasia verruciformis, and condyloma. We examined how tirbanibulin might affect HPV oncoprotein expression and affect other cellular pathways involved in cell proliferation and transformation. We treated the HeLa cell line, containing integrated HPV-18, with increasing doses of tirbanibulin to determine the effects on cell proliferation. Immunoblotting was performed with antibodies against the Src canonical pathway, HPV 18 E6 and E7 transcription regulation, apoptosis, and invasion and metastasis pathways. Cell proliferation assays with tirbanibulin determined the half-maximal inhibitory concentration (IC50) of HeLa cells to be 31.49 nmol/L. Increasing concentrations of tirbanibulin downregulates the protein expression of Src (p < 0.001), phospho-Src (p < 0.001), Ras (p < 0.01), c-Raf (p < 0.001), ERK1 (p < 0.001), phospho-ERK1 (p < 0.001), phospho-ERK2 (p < 0.01), phospho-Mnk1 (p < 0.001), eIF4E (p < 0.01), phospho-eIF4E (p < 0.001), E6 (p < 0.01), E7 (p < 0.01), Rb (p < 0.01), phospho-Rb (p < 0.001), MDM2 (p < 0.01), E2F1 (p < 0.001), phospho-FAK (p < 0.001), phospho-p130 Cas (p < 0.001), Mcl-1 (p < 0.01), and Bcl-2 (p < 0.001), but upregulates cPARP (p < 0.001), and cPARP/fPARP (p < 0.001). These results demonstrate that tirbanibulin may impact expression of HPV oncoproteins via the Src- MEK- pathway. Tirbanibulin significantly downregulates oncogenic proteins related to cell cycle regulation and cell proliferation while upregulating apoptosis pathways.

Tirbanibulin is Promising Novel Therapy for Human Papillomavirus (HPV)-associated Diseases.Tirbanibulin 1% ointment is an approved synthetic topical ointment for treating actinic keratoses (AK), a precancer of skin cancer. Topical tirbanibulin has previously been reported to clinically resolve human papillomavirus (HPV)-( +) diseases.In this study, we examine how tirbanibulin may affect the HPV and pathways associated with cancer.We treated the HeLa cell line to determine the effects on HPV cell proliferation. Increasing the concentration of tirbanibulin statistically significantly affected numerous cellular pathways often associated with cancer.These results demonstrate that tirbanibulin may impact expression of HPV oncoproteins and thereby kill cancer cells.

The effect of selinexor on prostaglandin synthesis in virus-positive Merkel cell carcinoma cell lines.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with high rates of metastasis and mortality. In vitro studies suggest that selinexor (KPT-330), an inhibitor of exportin 1, may be a targeted therapeutic option for MCC. This selective inhibitor prevents the transport of oncogenic mRNA out of the nucleus. Of note, 80% of MCC tumors are integrated with Merkel cell polyomavirus (MCPyV), and virally encoded tumor-antigens, small T (sT) and large T (LT) mRNAs may require an exportin transporter to relocate to the cytoplasm and modulate host tumor-suppressing pathways. To explore selinexor as a targeted therapy for MCC, we examine its ability to inhibit LT and sT antigen expression in vitro and its impact on the prostaglandin synthesis pathway. Protein expression was determined through immunoblotting and quantified by densitometric analysis. Statistical significance was determined with t-test. Treatment of MCPyV-infected cell lines with selinexor resulted in a significant dose-dependent downregulation of key mediators of the prostaglandin synthesis pathway. Given the role of prostaglandin synthesis pathway in MCC, our findings suggest that selinexor, alone or in combination with immunotherapy, could be a promising treatment for MCPyV-infected MCC patients who are resistant to chemotherapy and immunotherapy.

Effect of selinexor on lipogenesis in virus-positive Merkel cell carcinoma cell lines.

BACKGROUND: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine cutaneous carcinoma aetiologically linked to the Merkel cell polyomavirus (MCPyV). Immune checkpoint inhibitors are currently the first-line therapy for metastatic MCC; however, the treatment is effective in only about half of patients, highlighting the need for alternative therapies. Selinexor (KPT-330) is a selective inhibitor of nuclear exportin 1 (XPO1) and has been shown to inhibit MCC cell growth in vitro, but the pathogenesis has not been established. Decades of research have established that cancer cells significantly upregulate lipogenesis to meet an increased demand for fatty acids and cholesterol. Treatments that inhibit lipogenic pathways may halt cancer cell proliferation. AIM: To determine the effect of increasing doses of selinexor on fatty acid and cholesterol synthesis in MCPyV-positive MCC (MCCP) cell lines and aid in elucidating the mechanism by which selinexor prevents and reduces MCC growth. METHODS: MKL-1 and MS-1 cell lines were treated with increasing doses of selinexor for 72 h. Protein expression quantification was determined using chemiluminescent Western immunoblotting and densitometric analysis. Fatty acids and cholesterol were quantified using free fatty acid assay and cholesterol ester detection kits. RESULTS: Selinexor causes statistically significant reductions of the lipogenic transcription factors sterol regulatory element-binding proteins 1 and 2, and lipogenic enzymes acetyl-CoA carboxylase, fatty acid synthase, squalene synthase and 3ß-hydroxysterol Δ-24-reductase in a dose-dependent manner in two MCCP cell lines. Although inhibiting the fatty acid synthesis pathway results in meaningful decreases in fatty acids, the cellular cholesterol levels did not demonstrate such reductions. CONCLUSION: For patients with metastatic MCC refractory to immune checkpoint inhibitors, selinexor may provide clinical benefit through the inhibition of the lipogenesis pathway; however, further research and clinical trials are needed to evaluate these findings.

Selinexor is a novel inhibitor of DNA damage response in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly lethal cutaneous carcinoma, which in ~80% of cases in the USA is aetiologically linked to Merkel cell polyomavirus (MCPyV). Immune checkpoint inhibitors (ICIs) can successfully treat ~50% of patients with metastatic MCC, but some MCCs are refractory to ICIs, possibly due to altered DNA damage response (DDR). Selinexor, an anticancer therapy that is currently approved in combination with chemotherapy for multiple myeloma, downregulates the small T and large T tumour antigens in MCC through selective inhibition of nuclear exportin 1 (XPO1). We examined the effect of varying doses of selinexor on DDR protein expression in MCPyV-positive and MCPyV-negative MCC cells. Selinexor was found to inhibit DDR protein expression in both MCPyV-positive and MCPyV-negative cells. Addition of selinexor alone or combined with ICI may be a promising treatment for MCC, but further in vivo research and clinical trials are required to validate these findings.

Trichodysplasia spinulosa polyomavirus small T antigen synergistically modulates S6 protein translation and DNA damage response pathways to shape host cell environment.

TSPyV is a viral agent linked to Trichodysplasia spinulosa, a disfiguring human skin disease which presents with hyperkeratotic spicule eruption in immunocompromised hosts. This proliferative disease state requires extensive modulation of the host cell environment. While the small T (sT) antigen of TSPyV has been postulated to cause widespread cellular perturbation, its specific substrates and their mechanistic connection are unclear. To identify the cellular substrates and pathways perturbed by TSPyV sT and propose a nuanced model that reconciles the multiple arms of TSPyV pathogenesis, changes in expression of several proteins and phospho-proteins in TSPyV sT expressing and TSPyV sT deletion mutant-expressing cell lysates were interrogated using Western blot assays. TSPyV sT expression exploits the DNA damage response pathway, by inducing hyperphosphorylation of ATM and 53BP1 and upregulation of BMI-1. Concurrently, sT dysregulates the S6 protein translation pathway via hyperphosphorylation of CDC2, p70 S6 kinase, S6, and PP1α. The S6S244/247 and p-PP1αT320 phospho-forms are points of overlap between the DDR and S6 networks. We propose a mechanistic rationale for previous reports positioning sT antigen as the key driver of TSPyV pathogenesis. We illuminate novel targets in the S6 and DDR pathways and recognize a potential synergy between these pathways. TSPyV may sensitize the cell to both unrestricted translation and genomic instability. This multi-pronged infection model may inform future therapeutic modalities against TSPyV and possibly other viruses with overlapping host substrates.

Merkel Cell Polyomavirus Small T Antigen Induces DNA Damage Response.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine cancer of the skin with high rates of metastasis and mortality. Besides well-established factors including genetic mutations and UV-induced DNA damage in Merkel cell carcinogenesis, the recent discovery of the Merkel cell polyomavirus (MCPyV) has shed light on the viral etiology of MCC. In the current study, we provide novel evidence that MCPyV small T (sT) antigen induces the DNA damage response (DDR) pathway. Our data show that in human MCC cells, the presence of MCPyV is associated with hyperphosphorylation of histone H2AX, a marker for DNA damage. We observed that overexpression of MCPyV sT antigen induced the phosphorylation of histone H2AX as well as the activation of ataxia telangiectasia mutant (ATM), an upstream kinase important for H2AX phosphorylation. Moreover, we observed that MCPyV sT expression also induced the hyperphosphorylation of other ATM downstream molecules (including 53BP1 and CHK2) as well as the hypermethylation of histone 3 and histone 4. These findings disclose a novel link between MCPyV sT and the DDR pathway in MCC. Given that measurement of DDR is clinically useful for evaluating treatment response to radio- and chemotherapy, our findings warrant further investigation to evaluate the potential implications of this pathway for MCC management.

Human polyomavirus 7 (HPyV7)-associated dermatoses: novel molecular mechanism driven by viral activation of 4E-BP1 and MEK-ERK-cJun.

A number of pruritic skin conditions arising in immunocompromised patients are associated with viral infection. Recently, human polyomavirus 7 (HPyV7) has been implicated in the pathogenesis of eruptive pruritic parakeratotic and dyskeratotic dermatoses with distinct "peacock plumage" histology. While expression of HPyV7 viral protein, namely small tumor (sT) antigen, is prominent within lesional tissue, the functional role of HPyV7 in cutaneous pathobiology is not yet known. In this study, we demonstrate a novel role for HPyV7 sT antigen in pathways important for the maintenance of keratinocyte structure and function. In particular, HPyV7 sT was found to dysregulate protein phosphatase 2A through physical interactions that led to activation of MEK/ERK/c-Jun and 4E-BP1 (proteins that contribute to disorganized keratinocyte growth as well as hyperproliferative and inflammatory states). Given that HPyV7 actively infects keratinocytes and sT antigen is highly expressed in pruritic dyskeratotic/parakeratotic dermatoses, our data provide important mechanistic evidence supporting a pathogenic role for HPyV7 in cutaneous disease.

Beta Human Papillomavirus Infection Is Prevalent in Elephantiasis and Exhibits a Productive Phenotype: A Case-Control Study.

Elephantiasis is considered a cutaneous region of immune deficiency with cobblestone-like surface caused by a wart-like eruption. Verrucosis is a diffuse human papillomavirus (HPV) infection linked to immunodeficiency disorders. The objective of this study was to examine the prevalence of HPV infection in lymphedema and its pathogenic role in elephantiasis. A retrospective case-control study was performed examining lymphedematous skin and controls of peritumoral normal skin. HPV infection was evaluated at the DNA, protein, and histopathologic levels by polymerase chain reaction, immunohistochemistry, and light microscopy, respectively. Overall, 540 HPV DNAs were detected in 120 of 122 cutaneous samples (median 4 HPV DNAs per sample, range 0-9). Compared with controls, no differences existed in type or number of HPVs identified. Instead, a diverse spectrum of HPV-related histopathologies were evident, likely reflecting the multiplicity of HPV genotypes detected. Most notably, increasing histopathologic lymphedema stage significantly correlated with markers of productive HPV infection such as altered keratohyaline granules and HPV L1 capsid expression. Limitations of this study are the absence of normal skin controls not associated with neoplasia or subclinical lymphedema, and lack of assessment of HPV copy number per keratinocyte infected. In conclusion, productive HPV infection, not HPV type or numbers detected, distinguished lymphedematous skin from controls. These findings support the theory that lymphedema creates a region of depressed immunity that permits productive HPV infection, manifested clinically by diffuse papillomatosis, characteristic of elephantiasis.

Molecular mechanisms supporting a pathogenic role for human polyomavirus 6 small T antigen: Protein phosphatase 2A targeting and MAPK cascade activation.

BRAF inhibitors are highly effective therapies in treating a subset of melanomas but are associated with induction of secondary cutaneous squamous cell carcinoma (cSCC). Recently, Human Polyomavirus 6 (HPyV6) was found to actively express viral proteins in BRAF inhibitor-induced cSCCs; however, the specific cellular mechanisms by which HPyV6 may facilitate neoplastic cell growth require further investigation. The current study describes a novel pathogenic mechanism of action for HPyV6 small tumor (sT) antigen which involves binding to protein phosphatase 2A (PP2A) via its WFG motif and zinc binding sites. Our findings demonstrate an important role of HPyV6 sT for activation of PP2A's downstream oncogenic pathways (MEK/ERK/c-Jun), which may underlie the pathogenesis of BRAF inhibitor-induced neoplasms. J. Med. Virol. 89:742-747, 2017. © 2016 Wiley Periodicals, Inc.

Merkel cell polyomavirus in Merkel cell carcinogenesis: small T antigen-mediates c-Jun phosphorylation.

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer associated with the Merkel cell polyomavirus (MCPyV). The MCPyV genome, which is clonally integrated in the majority of MCCs, encodes the regulatory small T (sT) antigen. Previously, reports have established MCPyV sT antigen as a potent oncogene capable of inducing cell transformation. In the current study, we demonstrate a distinct role for c-Jun hyperactivation in MCPyV sT antigen pathogenesis. As MCPyV sT antigen's association with aggressive cancer growth has been previously established, this finding may represent a potential therapeutic target for the treatment of MCCs.

Emerging differential roles of the pRb tumor suppressor in trichodysplasia spinulosa-associated polyomavirus and Merkel cell polyomavirus pathogeneses.

BACKGROUND: Merkel cell carcinoma (MCC) and trichodysplasia spinulosa (TS) are two proliferative cutaneous diseases caused by the Merkel cell polyomavirus (MCPyV) and trichodysplasia spinulosa-associated polyomavirus (TSPyV) respectively. Recently, studies have elucidated a key role of the small tumor (sT) antigen in the proliferative pathogenic mechanisms of MCPyV and likely TSPyV. While both sT antigens have demonstrated a capacity in regulating cellular pathways, it remains unknown whether MCPyV and TSPyV sT antigens contribute similarly or differentially to cell proliferation. OBJECTIVES: The present study aims to explore the proliferative potential of MCPyV and TSPyV sT antigens by investigating their regulatory effects on the retinoblastoma protein (pRb) tumor suppressor. STUDY DESIGN: Inducible cell lines expressing MCPyV sT or TSPyV sT were created using a lentiviral packaging system. Cellular proteins were extracted and subjected to SDS-PAGE followed by Western blot detection and densitometric analysis. RESULTS: Expression of TSPyV sT markedly enhanced the phosphorylation of pRb in Western blot experiments. In contrast, expression of MCPyV sT did not alter pRb phosphorylation under the same experimental conditions. Densitometric analysis revealed that TSPyV sT antigen expression nearly doubled the ratio of phosphorylated to total pRb (P<0.001, Student's T-test), while MCPyV sT antigen expression did not cause significant change in pRb phosphorylation status. CONCLUSION: Given that hyperphosphorylation of pRb is associated with dysregulation of the cell cycle, S-phase induction, and increased cell proliferation, our findings support an important role of TSPyV-mediated pRb deactivation in the development of TS. The observation that the pRb tumor suppressor is inactivated by TSPyV sT but not MCPyV sT provides further insights into the distinct pathobiological mechanisms of MCC and TS.

Cutaneous Human Polyomavirus Small T Antigens and 4E-BP1 Targeting.

Merkel cell polyomavirus (MCPyV), trichodysplasia spinulosa-associated polyomavirus (TSPyV), human polyomavirus 6 (HPyV6), and human polyomavirus 7 (HPyV7) are implicated in the pathogeneses of distinct hyperproliferative cutaneous growths and encode small tumor (sT) antigens. The current study demonstrates that the four sT antigens differentially regulate 4E-binding protein 1 (4E-BP1) serine 65 hyperphosphorylation. MCPyV and HPyV7 sT antigens were found to promote the presence of the hyperphosphorylated 4E-BP1-δ isoform, while TSPyV and HPyV6 sT antigens had no significant effects. Given that hyperphosphorylated 4E-BP1 is associated with an aggressive cancer phenotype, our findings confirm the previously reported pathogenicity of MCPyV sT and highlight a novel mechanism by which HPyV7 sT may mediate oncogenesis.

The Drosophila BTB domain protein Jim Lovell has roles in multiple larval and adult behaviors.

Innate behaviors have their origins in the specification of neural fates during development. Within Drosophila, BTB (Bric-a-brac,Tramtrack, Broad) domain proteins such as Fruitless are known to play key roles in the neural differentiation underlying such responses. We previously identified a gene, which we have termed jim lovell (lov), encoding a BTB protein with a role in gravity responses. To understand more fully the behavioral roles of this gene we have investigated its function through several approaches. Transcript and protein expression patterns have been examined and behavioral phenotypes of new lov mutations have been characterized. Lov is a nuclear protein, suggesting a role as a transcriptional regulator, as for other BTB proteins. In late embryogenesis, Lov is expressed in many CNS and PNS neurons. An examination of the PNS expression indicates that lov functions in the late specification of several classes of sensory neurons. In particular, only two of the five abdominal lateral chordotonal neurons express Lov, predicting functional variation within this highly similar group. Surprisingly, Lov is also expressed very early in embryogenesis in ways that suggests roles in morphogenetic movements, amnioserosa function and head neurogenesis. The phenotypes of two new lov mutations that delete adjacent non-coding DNA regions are strikingly different suggesting removal of different regulatory elements. In lov(47) , Lov expression is lost in many embryonic neurons including the two lateral chordotonal neurons. lov(47) mutant larvae show feeding and locomotor defects including spontaneous backward movement. Adult lov(47) males perform aberrant courtship behavior distinguished by courtship displays that are not directed at the female. lov(47) adults also show more defective negative gravitaxis than the previously isolated lov(91Y) mutant. In contrast, lov(66) produces largely normal behavior but severe female sterility associated with ectopic lov expression in the ovary. We propose a negative regulatory role for the DNA deleted in lov(66) .

Tropomyosin is an interaction partner of the Drosophila coiled coil protein yuri gagarin.

The Drosophila gene yuri gagarin is a complex locus encoding three protein isoform classes that are ubiquitously expressed in the organism. Mutations to the gene affect processes as diverse as gravitactic behavior and spermatogenesis. The larger Yuri isoforms contain extensive coiled-coil regions. Our previous studies indicate that one of the large isoform classes (Yuri-65) is required for formation of specialized F-actin-containing structures generated during spermatogenesis, including the so-called actin "cones" that mediate spermatid individualization. We used the tandem affinity purification of a tagged version of Yuri-65 (the TAP-tagging technique) to identify proteins associated with Yuri-65 in the intact organism. Tropomyosin, primarily as the 284-residue isoform derived from the ubiquitously expressed Tropomyosin 1 gene was thus identified as a major Yuri interaction partner. Co-immunoprecipitation experiments confirmed this interaction. We have established that the stable F-actin cones of spermatogenesis contain Tropomyosin 1 (Tm1) and that in mutant yuri(F64), failure of F-actin cone formation is associated with failure of Tm1 to accumulate at the cone initiation sites. In investigating possible interactions of Tm1 and Yuri in other tissues, we discovered that Tm1 and Yuri frequently colocalize with the endoplasmic reticulum. Tropomyosin has been implicated in actin-mediated membrane trafficking activity in other systems. Our findings suggest that Yuri-Tm1 complexes participate in related functions.

Oxygen doping modifies near-infrared band gaps in fluorescent single-walled carbon nanotubes.

Controlled chemical modifications of single-walled carbon nanotubes (SWCNTs) that tune their useful properties have been sought for multiple applications. We found that beneficial optical changes in SWCNTs resulted from introducing low concentrations of oxygen atoms. Stable covalently oxygen-doped nanotubes were prepared by exposure to ozone and then light. Treated samples showed distinct, structure-specific near-infrared fluorescence at wavelengths 10 to 15% longer than displayed by pristine semiconducting SWCNTs. Dopant sites harvest light energy absorbed in undoped nanotube regions by trapping mobile excitons. The oxygen-doped SWCNTs are much easier to detect and image than pristine SWCNTs because they give stronger near-infrared emission and do not absorb at the shifted emission wavelength.

The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus.

Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface.