Targeting Aberrant Expression of STAT3 and AP-1 Oncogenic Transcription Factors and HPV Oncoproteins in Cervical Cancer by Berberis aquifolium

Background: Present study examines phytochemical preparation that uses berberine’s plant source B. aquifolium root for availability of similar anti-cervical cancer (CaCx) and anti-HPV activities to facilitate repurposing of the B. aquifolium based drug in the treatment of CaCx.Purpose: To evaluate therapeutic potential of different concentrations of ethanolic extract of B. aquifolium root mother tincture (BAMT) against HPV-positive (HPV16: SiHa, HPV18: HeLa) and HPV-negative (C33a) CaCx cell lines at molecular oncogenic level.Materials and Methods: BAMT was screened for anti-proliferative activity by MTT assay. Cell cycle progression was analyzed by flowcytometry. Then, the expression level of STAT3, AP-1, HPV E6 and E7 was detected by immunoblotting, whereas nuclear localization was observed by fluorescence microscopy. Phytochemicals reportedly available in BAMT were examined for their inhibitory action on HPV16 E6 by in silico molecular docking.Results: BAMT induced a dose-dependent decline in CaCx cell viability in all cell types tested. Flowcytometric evaluation of BAMT-treated cells showed a small but specific cell growth arrest in G1-phase. BAMT-treatment resulted in reduced protein expression of key transcription factors, STAT3 with a decline of its active form pSTAT3 (Y705); and components of AP-1 complex, JunB and c-Jun. Immunocytochemistry revealed that BAMT did not prevent the entry of remnant active transcription factor to the nucleus, but loss of overall transcription factor activity resulted in reduced availability of transcription factors in the cancer cells. These changes were accompanied by gradual loss of HPV E6 and E7 protein in BAMT-treated HPV-positive cells. Molecular docking of reported active phytochemicals in B. aquifolium root was performed, which indicated a potential interference of HPV16 E6’s interaction with pivotal cellular targets p53, E6AP or both by constituent phytochemicals. Among these, berberine, palmatine and magnoflorine showed highest E6 inhibitory potential.Conclusion: Overall, BAMT showed multi-pronged therapeutic potential against HPV infection and cervical cancer and the study described the underlying molecular mechanism of its action.

Download Full-text

Silencing of hpv16 e6 and e7 oncogenic activities by small interference rna induces autophagy and apoptosis in human cervical cancer cells

Journal of Nucleic Acids Investigation ◽

10.4081/jnai.2011.2583 ◽

2011 ◽

Vol 2 (1) ◽

pp. 10 ◽

Cited By ~ 1

Author(s):

Jonathan Salazar-León ◽

Fabiola Reyes-Román ◽

Angélica Meneses-Acosta ◽

Horacio Merchant ◽

Alfredo Lagunas-Martínez ◽

...

Keyword(s):

Cervical Cancer ◽

Gene Therapy ◽

Cancer Cells ◽

Biological Effects ◽

Cellular Transformation ◽

Hpv16 E6 ◽

Small Interference Rna ◽

Hpv E6 ◽

E6 And E7 ◽

Interference Rna

Cervical cancer is the second most common form of death by cancer in women worldwide and has special attention for the development of new treatment strategies. Human Papilloma Virus (HPV) persistent infection is the main etiological agent of this neoplasia, and the main cellular transformation mechanism is by disruption of p53 and pRb function by interaction with HPV E6 and E7 oncoproteins. This generates alterations in cellular differentiation and cellular death inhibition. Thus, HPV E6 and E7 oncogenes represent suitable targets for the development of gene therapy strategies against cervical cancer. An attractive technology platform is developing for post-transcriptional selective silencing of gene expression, using small interference RNA. Therefore, in the present study, we used SiHa cells (HPV16+) transiently transfected with specific siRNA expression plasmids for HPV16 E6 and E7 oncogenes. In this model we detected repression of E6 and E7 oncogene and oncoprotein expression, an increase in p53 and hypophosphorylated pRb isoform protein expression, and autophagy and apoptosis morphology features. These findings suggest that selective silencing of HPV16 E6 and E7 oncogenes by siRNAs, has significant biological effects on the survival of human cancer cells and is a potential gene therapy strategy against cervical cancer.

Download Full-text

Novel microRNA multivariate biomarkers of response to immunotherapy against HPV E6 oncogene

10.1101/2020.06.26.174441 ◽

2020 ◽

Author(s):

Rebecca M. Fleeman ◽

Gina Deiter ◽

Kristen Lambert ◽

Elizabeth A. Proctor ◽

Rébécca Phaëton

Keyword(s):

Cervical Cancer ◽

Therapeutic Potential ◽

Univariate Analysis ◽

Underlying Mechanism ◽

Experimental Models ◽

Hpv E6 ◽

Novel Interactions ◽

E6 And E7 ◽

And Control ◽

Insight Into

AbstractCervical cancer is caused by the persistent infection high-risk types of human papillomavirus (HPV) in over 99.9% of cases. To favor malignant transformation, HPV E6 and E7 oncogenes disrupt both p53 and retinoblastoma (Rb) respectively and control microRNA (miR) networks. We have previously demonstrated the therapeutic potential of anti-HPV E6 monoclonal antibodies (mAbs) in experimental models of human cervical cancer; yet the underlying mechanism remains unclear. Here, we sought to determine if anti-HPV E6 mAbs modulate the miR signatures of HPV E6 oncogenes. To this end, we performed qRT-PCR to measure the expression of thirty-four miRs and found that univariate analysis is not able to identify novel interactions characteristic of complex biological systems. Thus, we utilized partial least squares discriminant analysis (PLSDA) to identify signatures of co-varying miRs specific to mAb treatment. These miR signatures predictively discriminate between anti-HPV E6 mAb response and control mAb treatment, which may provide mechanistic insight into the action of anti-HPV E6 mAbs.

Download Full-text

Association of cervical carcinogenesis risk with HPV16 E6 and E7 variants in the Taizhou area, China

BMC Cancer ◽

10.1186/s12885-021-08531-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Mei-Zhen Dai ◽

Yi Qiu ◽

Xing-Hong Di ◽

Wei-Wu Shi ◽

Hui-Hui Xu

Keyword(s):

Cervical Cancer ◽

Cervical Carcinogenesis ◽

Chi Square ◽

Major Health Problem ◽

Hpv16 E6 ◽

Exact Test ◽

Chi Square Test ◽

E6 And E7

Abstract Background Human papillomavirus (HPV) type 16 accounts for a larger share of cervical cancer and has been a major health problem worldwide for decades. The progression of initial infection to cervical cancer has been linked to viral sequence properties; however, the role of HPV16 variants in the risk of cervical carcinogenesis, especially with longitudinal follow-up, is not fully understood in China. Methods We aimed to investigate the genetic variability of HPV16 E6 and E7 oncogenes in isolates from cervical exfoliated cells. Between December 2012 and December 2014, a total of 310 single HPV16-positive samples were selected from women living in the Taizhou area, China. Sequences of all E6 and E7 oncogenes were analysed by PCR-sequencing assay. Detailed sequence comparison, genetic heterogeneity analyses and maximum-likelihood phylogenetic tree construction were performed with BioEdit Sequence Alignment Editor and MEGA X software. Data for cytology tests and histological diagnoses were obtained from our Taizhou Area Study with longitudinal follow-up for at least 5 years. The relationship between HPV16 variants and cervical carcinogenesis risk was analysed by the chi-square test or Fisher’s exact test. Results In this study, we obtained 64 distinct variation patterns with the accession GenBank numbers MT681266-MT681329. Phylogenetic analysis revealed that 98.3% of HPV16 variants belong to lineage A, in which the A4 (Asian) sublineage was dominant (64.8%), followed by A2 (12.1%), A1 (11.4%), and A3 (10.0%). The A4 (Asian) sublineage had a higher risk of CIN2+ than the A1–3 (European) sublineages (OR = 2.69, 95% CI = 1.04–6.97, P < 0.05). Furthermore, nucleotide variation in HPV16 E6 T178G is associated with the development of cervical cancer. Conclusion These data could provide novel insights into the role of HPV16 variants in cervical carcinogenesis risk in China.

Download Full-text

Complementary Targeting of Rb Phosphorylation and Growth in Cervical Cancer Cell Cultures and a Xenograft Mouse Model by SHetA2 and Palbociclib

Cancers ◽

10.3390/cancers12051269 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1269 ◽

Cited By ~ 1

Author(s):

Amy L. Kennedy ◽

Rajani Rai ◽

Zitha Redempta Isingizwe ◽

Yan Daniel Zhao ◽

Stanley A. Lightfoot ◽

...

Keyword(s):

Cervical Cancer ◽

Cyclin D1 ◽

Cell Lines ◽

Cancer Cell ◽

Xenograft Model ◽

Cervical Cancer Cell ◽

Chi Square ◽

Western Blots ◽

Hpv E6 ◽

E6 And E7

Cervical cancer is caused by high-risk human papillomavirus (HPV) types and treated with conventional chemotherapy with surgery and/or radiation. HPV E6 and E7 proteins increase phosphorylation of retinoblastoma (Rb) by cyclin D1/cyclin dependent kinase (CDK)4/6 complexes. We hypothesized that cyclin D1 degradation by the SHetA2 drug in combination with palbociclib inhibition of CDK4/6 activity synergistically reduces phosphorylated Rb (phospho-Rb) and inhibits cervical cancer growth. The effects of these drugs, alone, and in combination, were evaluated in SiHa and CaSki HPV-positive and C33A HPV-negative cervical cancer cell lines using cell culture, western blots and ELISA, and in a SiHa xenograft model. Endpoints were compared by isobolograms, ANOVA, and Chi-Square. In all cell lines, combination indexes documented synergistic interaction of SHetA2 and palbociclib in association SHetA2 reduction of cyclin D1 and phospho-Rb, palbociclib reduction of phospho-Rb, and enhanced phospho-Rb reduction upon drug combination. Both drugs significantly reduced phospho-Rb and growth of SiHa xenograft tumors as single agents and acted additively when combined, with no evidence of toxicity. Dilated CD31-negative blood vessels adjacent to, or within, areas of necrosis and apoptosis were observed in all drug-treated tumors. These results justify development of the SHetA2 and palbociclib combination for targeting phospho-Rb in cervical cancer treatment.

Download Full-text

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

Cell Death and Differentiation ◽

10.1038/s41418-020-00693-9 ◽

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.

Download Full-text

Flax seed oil reduced tumor growth, modulated immune responses and decreased HPV E6 and E7 oncoprotein expression in a murine model of ectopic cervical cancer

Prostaglandins & Other Lipid Mediators ◽

10.1016/j.prostaglandins.2019.04.002 ◽

2019 ◽

Vol 143 ◽

pp. 106332 ◽

Cited By ~ 2

Author(s):

Rashmi Deshpande ◽

Prerna Raina ◽

Kavita Shinde ◽

Prakash Mansara ◽

Manjiri Karandikar ◽

...

Keyword(s):

Cervical Cancer ◽

Immune Responses ◽

Tumor Growth ◽

Murine Model ◽

Seed Oil ◽

Flax Seed ◽

E7 Oncoprotein ◽

Hpv E6 ◽

E6 And E7

Download Full-text

HPV-targeted tumor-infiltrating lymphocytes for cervical cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2014.32.18_suppl.lba3008 ◽

2014 ◽

Vol 32 (18_suppl) ◽

pp. LBA3008-LBA3008 ◽

Cited By ~ 2

Author(s):

Christian S. Hinrichs ◽

Sanja Stevanovic ◽

Lindsey Draper ◽

Robert Somerville ◽

John Wunderlich ◽

...

Keyword(s):

Clinical Trial ◽

Cervical Cancer ◽

T Cells ◽

Tumor Infiltrating Lymphocytes ◽

The Other ◽

High Dose ◽

Complete Regression ◽

Hpv E6 ◽

E6 And E7 ◽

Infiltrating Lymphocytes

LBA3008 Background: Adoptive T-cell therapy (ACT) is a promising cancer treatment modality with potentially broad application. It is not known if ACT can mediate regression of carcinomas, the most common solid tumors in humans. We studied carcinoma of the uterine cervix, a virally induced malignancy that constitutively expresses the HPV E6 and E7 oncoproteins, as a model cancer to test if ACT can mediate regression of an epithelial malignancy. Methods: We initiated a clinical trial to treat metastatic HPV+ cancers with tumor-infiltrating lymphocytes (TIL) selected for HPV E6- and E7-reactivity (HPV-TIL). Patients from the cervical cancer cohort are reported here. HPV-TIL infusion was preceded b y non-myeloablative conditioning and followed by high-dose bolus aldesleukin. HPV-reactivity was assessed by ELISPOT, IFN-gamma production, and CD137 expression assays. Results: Nine patients were treated on the study. They received a median of 81 x 109 T cells (range 33 to 159 x 109) as a single infusion. The infused cells possessed reactivity against high-risk HPV E6 and/or E7 in 6/8 patients. The two patients with no HPV reactivity did not respond to treatment. 3/6 patients with HPV reactivity demonstrated objective tumor responses by RECIST (1 PR and 2 CR). One patient had a 39% best response. Two patients with widespread metastases had complete tumor responses that are ongoing 18 and 11 months after treatment. One patient with a complete response had a chemotherapy-refractory HPV-16+ squamous cell carcinoma and the other a chemoradiation-refractory HPV-18+ adenocarcinoma. Both patients demonstrated prolonged repopulation with HPV-reactive T cells following treatment. Increased frequencies of HPV-specific T cells were detectable after 13 months in one patient and 6 months in the other. Two patients with HPV-reactive TIL that did not respond to treatment did not display repopulation with HPV-reactive T cells. Conclusions: HPV-TIL can mediate durable, complete regression of metastatic cervical cancer. Continued investigation of HPV-TIL for cervical cancer, and possibly other HPV+ malignancies, is warranted. Cellular therapy can mediate complete regression of an epithelial malignancy. Clinical trial information: NCT01585428.

Download Full-text

Disruption of repressive p130–DREAM complexes by human papillomavirus 16 E6/E7 oncoproteins is required for cell-cycle progression in cervical cancer cells

Journal of General Virology ◽

10.1099/vir.0.035352-0 ◽

2011 ◽

Vol 92 (11) ◽

pp. 2620-2627 ◽

Cited By ~ 28

Author(s):

Nurshamimi Nor Rashid ◽

Rohana Yusof ◽

Roger J. Watson

Keyword(s):

Cell Cycle ◽

Cervical Cancer ◽

Cancer Cells ◽

Cell Cycle Progression ◽

Human Papillomaviruses ◽

G1 Arrest ◽

Cycle Progression ◽

Hpv16 E6 ◽

Cervical Cancer Cells ◽

E6 And E7

Human papillomaviruses (HPVs) with tropism for mucosal epithelia are the major aetiological factors in cervical cancer. Most cancers are associated with so-called high-risk HPV types, in particular HPV16, and constitutive expression of the HPV16 E6 and E7 oncoproteins is critical for malignant transformation in infected keratinocytes. E6 and E7 bind to and inactivate the cellular tumour suppressors p53 and Rb, respectively, thus delaying differentiation and inducing proliferation in suprabasal keratinocytes to enable HPV replication. One member of the Rb family, p130, appears to be a particularly important target for E7 in promoting S-phase entry. Recent evidence indicates that p130 regulates cell-cycle progression as part of a large protein complex termed DREAM. The composition of DREAM is cell cycle-regulated, associating with E2F4 and p130 in G0/G1 and with the B-myb transcription factor in S/G2. In this study, we addressed whether p130–DREAM is disrupted in HPV16-transformed cervical cancer cells and whether this is a critical function for E6/E7. We found that p130–DREAM was greatly diminished in HPV16-transformed cervical carcinoma cells (CaSki and SiHa) compared with control cell lines; however, when E6/E7 expression was targeted by specific small hairpin RNAs, p130–DREAM was reformed and the cell cycle was arrested. We further demonstrated that the profound G1 arrest in E7-depleted CaSki cells was dependent on p130–DREAM reformation by also targeting the expression of the DREAM component Lin-54 and p130. The results show that continued HPV16 E6/E7 expression is necessary in cervical cancer cells to prevent cell-cycle arrest by a repressive p130–DREAM complex.

Download Full-text