Human Papillomavirus 16 lineage D is associated with high risk of cervical cancer in the Brazilian Northeast region

BackgroundLike HPV types, different lineages also appear to have different carcinogenic capabilities. Studies have shown that oncogenic HPV specific to the genotype lineage is associated with different risks for the development of cervical intraepithelial neoplasia (CIN2 / CIN3) and cervical cancer. This study aims to analyze the genetic diversity of HPV 16 genotype in cases of cervical intraepithelial neoplasia (CIN2/CIN3) and cervical cancer in women from the Northeast region of Brazil.Methods and ResultsA cross-sectional hospital-based study conducted in the Northeast region of Brazil from 2014 through 2016. In the sample, there were 196 cases of HPV-16 variant (59 cervical intraepithelial neoplasia - CIN2/CIN3 and 137 cases of cervical cancer). Difference of proportion test was used to compare groups of CIN2/CIN3 and cervical cancer by viral lineage (p-value <0.05) in respect to HPV-16 lineage prevalence.The percentage of lineage frequencies by histopathological diagnosis showed a borderline difference of lineage A in the CIN2/CIN3 group compared to the cervical cancer group (p = 0.053). In relation to lineage D, the proportion was higher in cancer cases (32.8%) compared to the CIN2/CIN3 group (16.9%), p-value of 0.023.ConclusionsHPV16 lineage A was the most frequent in both CIN2/CIN3 and cervical cancer samples, while lineage D predominated in cervical cancer, suggesting a possible association of HPV-16 lineage D with cervical cancer.

SIRT1 promotes proliferation, migration, and invasion of cervical cancer cell and is upregulated by human papillomavirus 16 E7 oncoprotein

SIRT1 (silent information regulator 1), a NAD+-dependent III class histone deacetylase, plays crucial roles in cell proliferation, apoptosis, senescence, metabolism, and stress responses. Nevertheless, the role of SIRT1 in tumorigenesis remains unclear. In the present study, we measured expression levels of SIRT1 and HPV16 E7 protein in cervical cancer tissue and calculated their correlations. We measured the effect of silencing SIRT1 on the proliferation, migration, invasion, and apoptosis in human cervical cancer SiHa cells. Immunohistochemistry results revealed that the expression of SIRT1 was upregulated with progression from CINII-III to cervical cancer, but was not expressed in normal cervical tissues and CINI. There was a positive correlation between SIRT1 expression and HPV16 E7 expression in cervical cancer tissues, and silencing of HPV16 E7 downregulated the expression of SIRT1. Depletion of SIRT1 significantly downregulated SIRT1 expression, and inhibited proliferation, migration, and invasion of SiHa cells, inducing apoptosis. Taken together, the data suggest that SIRT1 promotes cervical cancer carcinogenesis. SIRT1 inhibition is a potential treatment strategy for cervical cancer.

The Anaphase Promoting Complex/ cyclosome co-activator, Cdh1, is a novel target of human Papillomavirus 16 E7 oncoprotein in cervical oncogenesis

The transforming properties of the high risk human papillomavirus E7 oncoprotein are indispensable for driving the virus life cycle and pathogenesis. Besides inactivation of retinoblastoma (Rb) family of tumor suppressors as part of its oncogenic endeavors, E7-mediated perturbations of eminent cell cycle regulators, checkpoint proteins and proto-oncogenes are considered to be the tricks of its transformative traits. However, many such critical interactions are still unknown. In the present study, we have identified the anaphase promoting complex/ cyclosome (APC/C) co-activator, Cdh1, as a novel interacting partner and a degradation target of E7. We found that HPV16 E7-induced inactivation of Cdh1 promoted abnormal accumulation of multiple Cdh1 substrates. Such a mode of deregulation possibly contributes to HPV-mediated cervical oncogenesis. Our mapping studies recognized the carboxyl-terminal zinc finger motif of E7 to associate with Cdh1 and interfere with the timely degradation of FoxM1, a bona fide Cdh1 substrate and a potent oncogene. Importantly, the E7 mutant with impaired interaction with Cdh1 exhibited defects in its ability for overriding typical cell cycle transition and oncogenic transformation, thereby validating the functional and pathological significance of the E7-Cdh1 axis during cervical carcinoma progression. Altogether, the findings from our study discover a unique nexus between E7 and APC/C-Cdh1, thereby adding to our understanding of the mechanism of E7-induced carcinogenesis and provide a promising target for the management of cervical carcinoma.

The Retinoblastoma Tumor Suppressor Is Required for the NUP98-HOXA9-Induced Aberrant Nuclear Envelope Phenotype

Chromosomal translocations involving the nucleoporin NUP98 gene are recurrently identified in leukemia; yet, the cellular defects accompanying NUP98 fusion proteins are poorly characterized. NUP98 fusions cause changes in nuclear and nuclear envelope (NE) organization, in particular, in the nuclear lamina and the lamina associated polypeptide 2α (LAP2α), a regulator of the tumor suppressor retinoblastoma protein (RB). We demonstrate that, for NUP98-HOXA9 (NHA9), the best-studied NUP98 fusion protein, its effect(s) on nuclear architecture largely depend(s) on RB. Morphological alterations caused by the expression of NHA9 are largely diminished in the absence of RB, both in human cells expressing the human papillomavirus 16 E7 protein and in mouse embryonic fibroblasts lacking RB. We further show that NHA9 expression associates with distinct histone modification. Moreover, the pattern of trimethylation of histone H3 lysine-27 is affected by NHA9, again in an RB-dependent manner. Our results pinpoint to an unexpected interplay between NUP98 fusion proteins and RB, which may contribute to leukemogenesis.

CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle

Human papillomaviruses are causative agents in around 5% of all cancers, with no specific antiviral therapeutics available for treating infections or resultant cancers. In this report, we demonstrate that phosphorylation of HPV16 E2 by CK2 promotes formation of a complex with the cellular protein TopBP1 in vitro and in vivo .

Combining Photodynamic Therapy with Immunostimulatory Nanoparticles Elicits Effective Anti-Tumor Immune Responses in Preclinical Murine Models

Photodynamic therapy (PDT) has shown encouraging but limited clinical efficacy when used as a standalone treatment against solid tumors. Conversely, a limitation for immunotherapeutic efficacy is related to the immunosuppressive state observed in large, advanced tumors. In the present study, we employ a strategy, in which we use a combination of PDT and immunostimulatory nanoparticles (NPs), consisting of poly(lactic-co-glycolic) acid (PLGA)-polyethylene glycol (PEG) particles, loaded with the Toll-like receptor 3 (TLR3) agonist poly(I:C), the TLR7/8 agonist R848, the lymphocyte-attracting chemokine, and macrophage inflammatory protein 3α (MIP3α). The combination provoked strong anti-tumor responses, including an abscopal effects, in three clinically relevant murine models of cancer: MC38 (colorectal), CT26 (colorectal), and TC-1 (human papillomavirus 16-induced). We show that the local and distal anti-tumor effects depended on the presence of CD8+ T cells. The combination elicited tumor-specific oncoviral- or neoepitope-directed CD8+ T cells immune responses against the respective tumors, providing evidence that PDT can be used as an in situ vaccination strategy against cancer (neo)epitopes. Finally, we show that the treatment alters the tumor microenvironment in tumor-bearing mice, from cold (immunosuppressed) to hot (pro-inflammatory), based on greater neutrophil infiltration and higher levels of inflammatory myeloid and CD8+ T cells, compared to untreated mice. Together, our results provide a rationale for combining PDT with immunostimulatory NPs for the treatment of solid tumors.

Human papillomavirus 16 L1 gene methylation as a potential biomarker for predicting anal intraepithelial neoplasia in men who have sex with men (MSM)

The human papillomavirus (HPV) 16 early promoter and L1 gene methylation were quantitatively measured using pyrosequencing assay in anal cells collected from men who have sex with men (MSM) to determine potential biomarkers for HPV-related anal cancer. The methylation patterns of HPV16 genes, including the early promoter (CpG 31, 37, 43, 52, and 58) and L1 genes (CpG 5600, 5606, 5609, 5615, 7136, and 7145), were analyzed in 178 anal samples. The samples were diagnosed as normal, anal intraepithelial neoplasia (AIN) 1, AIN2, and AIN3. Low methylation levels of the early promoter (< 10%) and L1 genes (< 20%) were found in all detected normal anal cells. In comparison, medium to high methylation (≥ 20–60%) in the early promoter was found in 1.5% (1/67) and 5% (2/40) of AIN1 and AIN2-3 samples, respectively. Interestingly, slightly increased L1 gene methylation levels (≥ 20–60%), especially at the HPV16 5’L1 regions CpGs 5600 and 5609, were demonstrated in AIN2-3 specimen. Moreover, a negative correlation between high HPV16 L1 gene methylation at CpGs 5600, 5609, 5615, and 7145 and a percentual CD4 count was found in AIN3 HIV positive cases. When comparing the methylation status of AIN2-3 to that of normal/AIN1 lesions, the results indicated the potential of using HPV16 L1 gene methylation as a biomarker for HPV-related cancer screening.

Biological Activity Characterization of the Diagnostically Relevant Human Papillomavirus 16 E1C RNA

The spliced human papillomavirus 16 (HPV16) E1C RNA is associated with high-grade precursor lesions and cervical cancer. This qualifies E1C as a biomarker for high-grade lesions in HPV-based cervical cancer precursor screening. Here, we aimed to characterize the biological activity of HPV16 E1C RNA. In HEK-293T cells overexpressing HPV16 E1C RNA, we detected 9 kDa E1C protein in the cytoplasm using immunological assays with a newly generated E1C-specific monoclonal antibody or in mass spectrometry only after proteasome inhibition with MG132, indicating instability of the E1C protein. In HPV16-transformed cervical cancer cell lines in which the level of endogenous E1C RNA is much lower, E1C protein was not detected even after proteasome inhibition. Transient E1C overexpression in HEK-293T cells, co-transfected with a firefly luciferase reporter gene under the control of the HPV16 upstream regulatory region (URR), activated the HPV16 URR by 38%. This activation was also present when E1C translation was abolished by mutation. However, a construct expressing a random RNA sequence with similar GC content and 45% homology to the E1C RNA sequence also stimulated URR activity, indicating that special E1C RNA motifs might be responsible for the activation. In HPV16-transformed cell lines W12-episomal (W12-epi), W12-integrated HPV (W12-int), CaSki and SiHa stably overexpressing E1C RNA from lentiviral transduction, levels of endogenous HPV16 RNAs E6*I and E7 remained unchanged, while E1^E4 levels were significantly reduced by 20–30% in W12-epi, W12-int and CaSki cells. Overall, our study shows that E1C RNA is active and might contribute to transformation independent of the E6*I or E7 pathways. However, E1C overexpression resulted in only subtle changes in HPV16 RNA expression and very low copies of endogenous E1C RNA were detected in cervical cancer cell lines. This could weigh towards a less prominent role of E1C RNA in natural HPV transformation.