Human papillomavirus E6/E7 oncoproteins promote radiotherapy-mediated tumor suppression by globally hijacking host DNA damage repair.

Rationale: Whatever the mucosa primary infected, HPV-positive cancers are traditionally associated with a favorable outcome, attributable to a high sensitivity to radiation therapy. However, the direct impact of viral E6/E7 oncoproteins on the intrinsic cellular radiosensitivity (and, globally, on host DNA repair) remains mostly speculative. Methods: Using several isogenic cell models expressing HPV16 E6 and/or E7, the effect of viral oncoproteins on global DNA damage response was first investigated by in vitro/in vivo approaches. The binary interactome of each individual HPV oncoprotein with factors involved in the various host DNA damage/repair mechanisms was then precisely mapped by Gaussia princeps luciferase complementation assay (and validated by co-immunoprecipitation). The stability/half-life of protein targets for HPV E6 and/or E7 as well as their subcellular localizations were determined. At last, the host genome integrity following E6/E7 expression and the synergy between radiotherapy and compounds targeting DNA repair were analyzed. Results: We first showed that the sole expression of one viral oncoprotein from HPV16 was able to significantly increase the sensitivity to irradiation of cells without affecting their basal viability parameters. In total, 10 novel targets (CHEK2, CLK2, CLK2/3, ERCC3, MNAT1, PER1, RMI1, RPA1, UVSSA and XRCC6) for E6 and 11 (ALKBH2, CHEK2, DNA2, DUT, ENDOV, ERCC3, PARP3, PMS1, PNKP, POLDIP2 and RBBP8) for E7 were identified. Importantly, not degraded following their interaction with E6 or E7, these proteins have been shown to be less linked to host DNA and to colocalize with HPV replication foci, denoting their crucial implication in viral life cycle. Finally, we found that E6/E7 oncoproteins globally jeopardize host genome integrity, increase the cellular sensitivity to DNA repair inhibitors and enhance their synergy with radiotherapy. Conclusion: Taken together, our findings provide a molecular insight into the direct hijacking of host DNA damage/repair responses by HPV oncoproteins, demonstrate the significant impact of this phenomenon on both intrinsic cellular radiosensitivity and host DNA integrity and suggest novel connected therapeutic vulnerabilities.

HPV infection alters vaginal microbiome through down-regulating host mucosal innate peptides used by Lactobacilli as amino acid sources.

Despite the high prevalence of both cervico-vaginal human papillomavirus (HPV) infection and bacterial vaginosis (BV) worldwide, their causal relationship remains unclear. While BV has been presumed to be a risk factor for HPV acquisition and related carcinogenesis for a long time, here, supported by both a large retrospective follow-up study (n = 6,085) and extensive in vivo data using the K14-HPV16 transgenic mouse model, we report a novel blueprint in which the opposite association also exists. Mechanistically, by interacting with several core members (NEMO, CK1 and ß-TrCP) of both NF-κB and Wnt/ß-catenin signaling pathways, we show that HPV E7 oncoprotein greatly inhibits host defense peptide expression. Physiologically secreted by the squamous mucosa lining the lower female genital tract, we demonstrate that some of these latter are fundamental factors governing host-microbial interactions. More specifically, several innate molecules down-regulated in case of HPV infection are hydrolyzed, internalized and used by the predominant Lactobacillus species as amino acid source sustaining their growth/survival. Collectively, this study reveals a new viral immune evasion strategy which, by its persistent/negative impact on lactic acid bacteria, ultimately causes the dysbiosis of vaginal microbiota.

Extracellular HMGB1 blockade inhibits tumor growth through profoundly remodeling immune microenvironment and enhances checkpoint inhibitor-based immunotherapy.

BACKGROUND: High-mobility group box 1 (HMGB1) is a multifunctional redox-sensitive protein involved in various intracellular (eg, chromatin remodeling, transcription, autophagy) and extracellular (inflammation, autoimmunity) processes. Regarding its role in cancer development/progression, paradoxical results exist in the literature and it is still unclear whether HMGB1 mainly acts as an oncogene or a tumor suppressor. METHODS: HMGB1 expression was first assessed in tissue specimens (n=359) of invasive breast, lung and cervical cancer and the two distinct staining patterns detected (nuclear vs cytoplasmic) were correlated to the secretion profile of malignant cells, patient outcomes and the presence of infiltrating immune cells within tumor microenvironment. Using several orthotopic, syngeneic mouse models of basal-like breast (4T1, 67NR and EpRas) or non-small cell lung (TC-1) cancer, the efficacy of several HMGB1 inhibitors alone and in combination with immune checkpoint blockade antibodies (anti-PD-1/PD-L1) was then investigated. Isolated from retrieved tumors, 14 immune cell (sub)populations as well as the activation status of antigen-presenting cells were extensively analyzed in each condition. Finally, the redox state of HMGB1 in tumor-extruded fluids and the influence of different forms (oxidized, reduced or disulfide) on both dendritic cell (DC) and plasmacytoid DC (pDC) activation were determined. RESULTS: Associated with an unfavorable prognosis in human patients, we clearly demonstrated that targeting extracellular HMGB1 elicits a profound remodeling of tumor immune microenvironment for efficient cancer therapy. Indeed, without affecting the global number of (CD45+) immune cells, drastic reductions of monocytic/granulocytic myeloid-derived suppressor cells (MDSC) and regulatory T lymphocytes, a higher M1/M2 ratio of macrophages as well as an increased activation of both DC and pDC were continually observed following HMGB1 inhibition. Moreover, blocking HMGB1 improved the efficacy of anti-PD-1 cancer monoimmunotherapy. We also reported that a significant fraction of HMGB1 encountered within cancer microenvironment (interstitial fluids) is oxidized and, in opposite to its reduced isoform, oxidized HMGB1 acts as a tolerogenic signal in a receptor for advanced glycation endproducts-dependent manner. CONCLUSION: Collectively, we present evidence that extracellular HMGB1 blockade may complement first-generation cancer immunotherapies by remobilizing antitumor immune response.

Treatment algorithm and prognostic factors for patients with stage I-III carcinoma of the anal canal: a 20-year multicenter study.

Despite a growing incidence in developed countries and a recent improved understanding of its pathogenesis, anal cancer management has not evolved over the past decades and drug combination used as first-line regimen still largely depends on clinician preferences. Aiming at paving the way for precision medicine, a large cohort of 372 HIV-negative patients diagnosed over a 20-year time period with locally advanced anal carcinoma was collected and carefully characterized at the clinical, demographic, histopathologic, immunologic, and virologic levels. Both the prognostic relevance of each clinicopathological parameter and the efficacy of different concurrent chemoradiation strategies were determined. Overall, the incidence of anal cancer peaked during the sixth decade (mean: 63.4) and females outnumbered males (ratio: 2.51). After completion of treatment, 95 (25.5%) patients experienced progression of persistent disease or local/distant recurrence and 102 (27.4%) died during the follow-up period (median: 53.8 months). Importantly, uni-multivariate analyses indicated that both negative HPV/p16ink4a status and aberrant p53 expression were far better predictors for reduced progression-free survival than traditional risk factors such as tumor size and nodal status. As for overall survival, the significant influences of age at diagnosis, p16ink4a status, cTNM classification as well as both CD3+ and CD4+ T-cell infiltrations within tumor microenvironment were highlighted. Cisplatin-based chemoradiotherapy was superior to both radiotherapy alone and other concurrent chemoradiation therapies in the treatment of HPV-positive tumors. Regarding their HPV-uninfected counterparts, frequent relapses were observed, whatever the treatment regimen administered. Taken together, our findings reveal that current anal cancer management and treatment have reached their limits. A dualistic classification according to HPV/p53 status should be considered with implications for therapy personalization and optimization.

EZH2 and KDM6B Expressions Are Associated with Specific Epigenetic Signatures during EMT in Non Small Cell Lung Carcinomas.

The role of Epigenetics in Epithelial Mesenchymal Transition (EMT) has recently emerged. Two epigenetic enzymes with paradoxical roles have previously been associated to EMT, EZH2 (Enhancer of Zeste 2 Polycomb Repressive Complex 2 (PRC2) Subunit), a lysine methyltranserase able to add the H3K27me3 mark, and the histone demethylase KDM6B (Lysine Demethylase 6B), which can remove the H3K27me3 mark. Nevertheless, it still remains unclear how these enzymes, with apparent opposite activities, could both promote EMT. In this study, we evaluated the function of these two enzymes using an EMT-inducible model, the lung cancer A549 cell line. ChIP-seq coupled with transcriptomic analysis showed that EZH2 and KDM6B were able to target and modulate the expression of different genes during EMT. Based on this analysis, we described INHBB, WTN5B, and ADAMTS6 as new EMT markers regulated by epigenetic modifications and directly implicated in EMT induction.

EMT is associated with an epigenetic signature of ECM remodeling genes.

Type III epithelial-mesenchymal transition (EMT) has been previously associated with increased cell migration, invasion, metastasis, and therefore cancer aggressiveness. This reversible process is associated with an important gene expression reprogramming mainly due to epigenetic plasticity. Nevertheless, most of the studies describing the central role of epigenetic modifications during EMT were performed in a single-cell model and using only one mode of EMT induction. In our study, we studied the overall modulations of gene expression and epigenetic modifications in four different EMT-induced cell models issued from different tissues and using different inducers of EMT. Pangenomic analysis (transcriptome and ChIP-sequencing) validated our hypothesis that gene expression reprogramming during EMT is largely regulated by epigenetic modifications of a wide range of genes. Indeed, our results confirmed that each EMT model is unique and can be associated with a specific transcriptome profile and epigenetic program. However, we could select some genes or pathways that are similarly regulated in the different models and that could therefore be used as a common signature of all EMT models and become new biomarkers of the EMT phenotype. As an example, we can cite the regulation of gene-coding proteins involved in the degradation of the extracellular matrix (ECM), which are highly induced in all EMT models. Based on our investigations and results, we identified ADAM19 as a new biomarker of in vitro and in vivo EMT and we validated this biological new marker in a cohort of non-small lung carcinomas.

A dualistic model of primary anal canal adenocarcinoma with distinct cellular origins, etiologies, inflammatory microenvironments and mutational signatures: implications for personalised medicine.

BACKGROUND: Primary adenocarcinoma of the anal canal is a rare and aggressive gastrointestinal disease with unclear pathogenesis. Because of its rarity, no clear clinical practice guideline has been defined and a targeted therapeutic armamentarium has yet to be developed. The present article aimed at addressing this information gap by in-depth characterising the anal glandular neoplasms at the histologic, immunologic, genomic and epidemiologic levels. METHODS: In this multi-institutional study, we first examined the histological features displayed by each collected tumour (n = 74) and analysed their etiological relationship with human papillomavirus (HPV) infection. The intratumoural immune cell subsets (CD4, CD8, Foxp3), the expression of immune checkpoints (PD-1, PD-L1), the defect in mismatch repair proteins and the mutation analysis of multiple clinically relevant genes in the gastrointestinal cancer setting were also determined. Finally, the prognostic significance of each clinicopathological variable was assessed. RESULTS: Phenotypic analysis revealed two region-specific subtypes of anal canal adenocarcinoma. The significant differences in the HPV status, density of tumour-infiltrating lymphocytes, expression of immune checkpoints and mutational profile of several targetable genes further supported the separation of these latter neoplasms into two distinct entities. Importantly, anal gland/transitional-type cancers, which poorly respond to standard treatments, displayed less mutations in downstream effectors of the EGFR signalling pathway (i.e., KRAS and NRAS) and demonstrated a significantly higher expression of the immune inhibitory ligand-receptor pair PD-1/PD-L1 compared to their counterparts arising from the colorectal mucosa. CONCLUSIONS: Taken together, the findings reported in the present article reveal, for the first time, that glandular neoplasms of the anal canal arise by HPV-dependent or independent pathways. These etiological differences leads to both individual immune profiles and mutational landscapes that can be targeted for therapeutic benefits.

PEGylated and Functionalized Aliphatic Polycarbonate Polyplex Nanoparticles for Intravenous Administration of HDAC5 siRNA in Cancer Therapy.

Guanidine and morpholine functionalized aliphatic polycarbonate polymers are able to deliver efficiently histone deacetylase 5 (HDAC5) siRNA into the cytoplasm of cancer cells in vitro leading to a decrease of cell proliferation were previously developed. To allow these biodegradable and biocompatible polyplex nanoparticles to overcome the extracellular barriers and be effective in vivo after an intravenous injection, polyethylene glycol chains (PEG750 or PEG2000) were grafted on the polymer structure. These nanoparticles showed an average size of about 150 nm and a slightly positive ζ-potential with complete siRNA complexation. Behavior of PEGylated and non-PEGylated polyplexes were investigated in the presence of serum, in terms of siRNA complexation (fluorescence correlation spectroscopy), size (dynamic light scattering and single-particle tracking), interaction with proteins (isothermal titration calorimetry) and cellular uptake. Surprisingly, both PEGylated and non-PEGylated formulations presented relatively good behavior in the presence of fetal bovine serum (FBS). Hemocompatibility tests showed no effect of these polyplexes on hemolysis and coagulation. In vivo biodistribution in mice was performed and showed a better siRNA accumulation at the tumor site for PEGylated polyplexes. However, cellular uptake in protein-rich conditions showed that PEGylated polyplex lost their ability to interact with biological membranes and enter into cells, showing the importance to perform in vitro investigations in physiological conditions closed to in vivo situation. In vitro, the efficiency of PEGylated nanoparticles decreases compared to non-PEGylated particles, leading to the loss of the antiproliferative effect on cancer cells.

Impact of the structure of biocompatible aliphatic polycarbonates on siRNA transfection ability.

RNAi therapeutics are promising therapeutic tools that have sparked the interest of many researchers. In an effort to provide a safe alternative to PEI, we have designed a series of new guanidinium- and morpholino-functionalized biocompatible and biodegradable polycarbonate vectors. The impact of different functions (morpholino-, guanidinium-, hydrophobic groups) of the architecture (linear homopolymer to dumbbell-shape) and of the molecular weight of these copolymers on their capacity to form polyplexes and to decrease the expression of two epigenetic regulators of gene expression, HDAC7 and HDAC5, was evaluated. The use of one of these polymers combining morpholine and guanidine functions at the ratio >1 and hydrophobic trimethylene carbonate groups showed a significant decrease of mRNA and protein level in HeLa cells, similar to PEI. These results highlight the potential of polycarbonate vectors for future in vivo application as an anticancer therapy.