Welcome to the IPVC 2023 Conference Program Scheduling
The meeting will officially run on Washington DC, USA Time (EDT)
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The sessions can be viewed through the IPVC Virtual Platform. You can scroll through the program to the right using the arrows on the left side of the calendar.
NON-CANONICAL HPV CARCINOGENESIS DRIVES RADIO-SENSITIZATION OF HEAD AND NECK TUMORS
Abstract
Introduction
Human papillomavirus-associated (HPV+) head and neck squamous cell carcinoma (HNSCC) has surpassed cervical cancer and is now the most common HPV associated cancer and its incidence continues to increase. HPV mediated oncogenesis is generally thought to rely on integration of the viral DNA into the host genome, loss of HPV E2 expression, activating alterations of PIK3CA, and APOBEC-mediated mutagenesis. We report the identification of a novel subclass of HPV+ carcinomas comprising approximately 45% of HPV+ HNSCC that is not associated with any of these classic features of HPV oncogenesis. Patients in this subgroup have improved clinical outcomes, and that cell culture models of this class have increased sensitivity to radiation.
Methods
We analyzed novel transcriptional profiling data from 104 HPV+ HNSCC tumors in tandem with two publicly available sources. Transcriptional data was correlated with viral integration, tumor genomic features and clinical outcomes. Derived tumor subclasses were modeled with HPV+ cells in culture.
Results
We found a single transcriptional module that naturally subclassifies HPV+ HNSCC tumors based on a bimodal pattern of gene expression. The subclass-defining gene set was correlated with NF-κB target expression. Tumors with high expression of this NF-κB module, were rarely associated with activating PIK3CA alterations or viral integration, expressed higher levels of E2 and had decreased APOBEC mutagenesis. Alternatively, they harbored alterations of NF-κB regulators, TRAF3 and CYLD. HPV+ HNSCC cells in culture with experimental depletion of TRAF3 or CYLD displayed increased expression of the subclass-defining genes and robust radio-sensitization, thus recapitulating both the tumor transcriptional state and improved treatment response observed in patient data.
Conclusions
Identification of a cohesive class of HPV-driven tumors without canonical features of HPV oncogenesis challenges the accepted models of HPV carcinogenesis. Recognition of this highly identifiable, biologically distinct tumor subclass with improved response to radio-therapy may fundamentally alter how HPV+ HNSCC patients are treated.
FUNCTIONAL SCREENS FOR MICRORNAS AND THEIR MRNA TARGETS REGULATING ANCHORAGE-INDEPENDENT GROWTH IN HUMAN CERVICAL CANCER CELLS
Abstract
Introduction
The progression of anchorage-dependent epithelial cells to anchorage-independent (AI) cells is a critical hallmark and an early event of malignant transformation. Hence, alterations in genes involved in AI growth can predict the progression of precancerous lesions to invasive cancer. Using an in vitro model of HPV-transformed cells representing cervical precancerous lesions, we previously showed that acquisition of AI growth is associated with (epi)genetic changes, including altered expression of miRNAs. Here we developed a novel high-throughput functional screening method using Ultra-Low-Attachment (ULA) plates to investigate miRNAs driving AI growth.
Methods
A proof-of-concept functional screen on cervical cancer cells SiHa was developed using 96-well ULA plates (Huseinovic, 2022). Cells were transfected with a miRNA mimic library (n=2019) and cell viability was measured to identify miRNAs affecting AI growth. The screen was further optimized on 384-well adherent and ULA plates, and performed with a comprehensive library of 2574 miRNA mimics in HPV16/18-transformed keratinocytes (FK16A/FK18B). Additionally, we performed a complementary CRISPR/Cas9 screen with a genome-wide knock-out CRISPR library with anchorage-independent FK18B cells cultured on adherent and ULA plates to search for genes affecting AI growth.
Results
We successfully performed miRNA mimic screens and, based on the validation in three cell lines, were able to identify 40 miRNAs affecting AI growth during HPV-induced transformation. CRISPR/Cas9 screen and subsequent gene ontology analysis revealed the enrichment of several relevant pathways involved in AI growth. Further functional validation and integrated data analysis of miRNA mimic and CRISPR/Cas9 screens are ongoing to identify miRNAs and their target mRNAs involved in the acquisition of anchorage independence.
Conclusions
We successfully developed and completed miRNA and CRISPR/Cas9 functional screens on ULA plates. We identified miRNAs, genes, and pathways that regulate AI growth. These findings can be used for risk stratification and treatment of precancerous lesions that are at the edge of malignant transformation.
HUMAN PAPILLOMAVIRUS ONCOGENES MAY PROMOTE CERVICAL DYSPLASIA PROGRESSION THROUGH DYSREGULATION OF MIR-4488 AND WNT SIGNALING
Abstract
Introduction
Many women are diagnosed with human papillomavirus (HPV)-associated low-grade cervical dysplasia (CIN) each year. Most women will exhibit restoration to a healthy cervix; however, a few will progress to high-grade CIN, which increases their risk of developing cancer. We have demonstrated that miRNAs, including miR-4488, are dysregulated in women with progressive CIN. Pathway analysis using miRPathDB2.0 revealed enrichment of mRNA involved in Wnt (Wingless and Int-1) signaling as targets of the miRNAs downregulated in progressive CIN. Wnt signaling, previously found to be activated in cervical cancer, promotes migration in part through the action of matrix metalloproteinases (MMPs), also previously found upregulated in cervical cancer. We hypothesize that HPV onco-proteins E6/E7 promote cervical dysplasia progression by downregulating miRNAs and promoting migratory phenotypes through Wnt signaling activation and induction of MMP expression.
Methods
MiR-4488 expression was analyzed in primary cervical cells expressing HPV-16 E6/E7 (Primary-E6/E7) compared to vector controls. Synthetic miR-4488 mimics and inhibitors were used to confirm putative mRNA target MMP15 via 3’UTR reporter assays and for increased cell migration using a wound healing assay. Expression of MMP15 was examined in cells expressing HPV-16 E6/E7 compared to vector controls by qPCR. Wnt signaling activation was measured in HPV-16 E6/E7 expressing C-33A cells (C-33A-E6/E7) compared to vector controls using a Wnt responsive reporter assay.
Results
Expression of HPV oncogenes E6/E7 led to downregulation of miR-4488 (2-fold), increased Wnt signaling activation (p<0.01), and increased MMP15 gene expression (p<0.05). Increased migration was observed in cells transfected with an inhibitor of miR-4488 (p<0.05).
Conclusions
Downregulation of miR-4488, expression of MMP15, and activation of Wnt signaling may be early indicators of oncogenic HPV E6/E7 overexpression and CIN progression.
DISRUPTION OF MUS MUSCULUS PAPILLOMAVIRUS 1 (MMUPV1) E8^E2 ACTIVITY INDUCES E4 PROTEIN EXPRESSION AND PREVENTS WART FORMATION
Abstract
Introduction
The E8^E2 protein is a conserved repressor of papillomavirus replication and transcription. We have recently reported that the inactivation of the E8 start codon in the MmuPV1 genome (E8-) increases viral gene expression in cultured keratinocytes, but surprisingly fails to form warts in immune deficient Foxn1nu/nu mice.
Methods
keratinocyte culture, mouse model
Results
To confirm that the loss of E8^E2 is responsible for the failure to form warts in vivo, we mutated the E8 splice donor (E8 SD mt) used to generate the E8^E2 RNA. This resulted in increased viral transcription in mouse keratinocytes comparable to E8-. HPV E8^E2 proteins recruit NCoR/SMRT complexes to repress transcription and replication. Co-immunoprecipitation experiments revealed that NCoR/SMRT complex components HDAC3, GPS2, and TBLR1 interact with MmuPV1 E8^E2 and that these interactions are reduced by mutation of conserved E8 residues K2R/L3P/K4R. E8 RPR mt genomes displayed increased viral transcription in mouse keratinocytes, and siRNA knock-downs confirmed that NCoR/SMRT components contribute to the inhibition of transcription and replication by E8^E2. This suggests that the E8^E2 protein functionally interacts with NCoR/SMRT corepressor complexes similar to HPV. Consistent with E8- genomes, E8 SD mt and E8 RPR mt genomes do not induce warts in Foxn1nu/numice. Interestingly, MmuPV1 genomes with impaired E8^E2 activity lead to a greatly enhanced number of mouse keratinocytes expressing the late viral E4 protein despite being maintained in undifferentiated conditions.
Conclusions
The increased gene expression from MmuPV1 E8-, E8 SD mt and E8 RPR mt genomes is due to the loss of E8^E2 or preventing its interaction with NCoR/SMRT complexes, and the data confirm that E8^E2 is required for wart formation in vivo. The induction of E4 protein expression in cultured murine keratinocytes from E8^E2 mt genomes suggests that the viral late phase is aberrantly induced in undifferentiated cells which might be incompatible with tumour formation in vivo.
DOWNREGULATION OF HSA-MIR-193A/B-3P CONTRIBUTES TO ANCHORAGE-INDEPENDENT GROWTH IN HPV-TRANSFORMED KERATINOCYTES THROUGH THE PI3K/AKT PATHWAY
Abstract
Introduction
Cervical cancer is caused by a persistent infection with high-risk types of HPV and accumulation of (epi)genetic alterations in the host cell. Acquisition of anchorage-independent growth represents a critical hallmark during HPV-induced carcinogenesis, thereby yielding most valuable biomarkers for early diagnosis and therapeutic targets. In a previous study, we found that miR-193a-3p and miR-193b-3p were involved in anchorage-independent growth. This study aimed to delineate the role of miR-193a/b-3p in HPV-induced carcinogenesis and to identify their target genes related anchorage-independent growth.
Methods
Cell viability and colony formation were assessed in SiHa cancer cells and HPV-16 and -18 immortalized keratinocytes (FK16A/FK18B) upon miR-193a/b-3p overexpression to validate their functional involvement. Online target predicting programs (StarBase and miRDB) were used to find candidate mRNAs targets of miR-193a/b-3p. mRNA targets were further selected based on publicly available expression data. Targets were validated by RT-qPCR and luciferase assays.
Results
miR-193a-3p and miR-193b-3p largely reduced cell growth of SiHa, FK16A and FK18B in anchorage-independent conditions and showed minor effect in anchorage-dependent conditions. Fifteen genes were identified as potential targets related to anchorage-independence. Seven genes showed reduced mRNA expression upon miR-193a-3p and miR-193b-3p overexpression. A direct interaction was confirmed using luciferase assays for 6 genes: LAMC1, KRAS, PPP2R5C, PTK2, SOS2 and STMN1. Both miR-193a/b-3p are downregulated in high-grade CIN lesions and miR-193b-3p also downregulated in cervical cancers. All 6 targets were overexpressed in cervical cancers and/or high-grade CIN lesions.
Conclusions
miR-193a-3p and miR-193b-3p reduce anchorage-independent growth of HPV-transformed cells through targeting LAMC1, KRAS, PPP2R5C, PTK2, SOS2 and STMN1. Target upregulation in cervical cancer underlines the biological relevance of miR-193a-3p and miR193b-3p downregulation during HPV-induced carcinogenesis.
MASTOMYS COUCHA: A UNIQUE ANIMAL MODEL FOR PAPILLOMAVIRUS-INDUCED SKIN CARCINOGENESIS
Abstract
Introduction
Animals are indispensable to provide an empirical bona fide equivalent for pathophysiological processes in humans. To act as a preclinical model, an animal has to fulfill certain criteria by reflecting face validity, complexity and predictability of a disease. Here, I will describe some properties of the rodent Mastomys coucha. Although still not yet very common in the scientific community, the model is well known in various research fields and was successfully used to study the role of cutaneous papillomaviruses in non-melanoma skin cancer (NMSC) development, in testing vaccines and in dissecting the mechanisms of viral immune escape.
Methods
The Mastomys coucha colony is naturally infected by the cutaneous MnPV. Serology, ELISAs, VLP, pseudovirion productions and neutralization assays were performed as described.
Results
MnPV acts in combination with UV via a “hit-and-run” mechanism in the development of NMSC. Moreover, monitoring seroconversion during the permissive cycle, a novel humoral escape mechanism was identified. The initial synthesis of a longer L1 isoform unable to form mature capsids only induces non-neutralizing antibodies. This strategy allows viral amplification and spread. Only after a delay of a couple of months neutralizing antibodies appear, now directed against the shorter form of L1 that forms mature viral particles necessary to encapsulate progeny viral DNA. The implication of these findings were discussed, especially in the light of our vaccination experiments where we showed that – in contrast to L1 short – immunization with the only 34 amino acid longer L1 isoform completely failed to prevent skin tumor formation.
Conclusions
Mastomys coucha is a valuable preclinical model to answer a variety of questions in the context of papillomavirus-induced carcinogenesis. In future experiments, the generation of germ-free animals and their subsequent recolonization with defined microorganisms should show the impact of the microbiome on skin cancer formation after infection with cutaneous PVs.
MMUPV1 CAN INDUCE AN INVASIVE CANCER PHENOTYPE IN ADULT IMMUNE-COMPETENT MICE WITHIN TWO WEEKS OF OROPHARYNGEAL INFECTION
Abstract
Introduction
Development of cancer in adult mammals is thought to take months or years from the time of initiation, be it by chemical carcinogens, cancer driver mutations, or cancer-causing pathogens such as oncogenic viruses. The mouse papillomavirus MmuPV1 is a useful preclinical model for human papillomavirus (HPV)-induced cancers, causing cancers in both immune-deficient and certain immune-competent mouse strains (e.g. FVB/NTac). Prior studies on MmuPV1 have focused on cancer development 3-6 months post infection.
Methods
We have assessed lesions in inbred, immune-competent FVB/NTac mice, 1-4 weeks post infection at the base of the tongue (where in humans HPVs cause oropharyngeal cancer). We pursued parallel studies in FVB-KRT14-HPV16E5-transgenic (FVB-E5) mice that express the HPV16 E5 oncogene in stratified epithelia; these mice are more susceptible to MmuPV1-induced disease (Torres et al., Virology, 2020). MmuPV1 does not encode an E5 oncogene.
Results
We were surprised to find that invasive cancers (independently scored by two pathologists: J.P.S & R.H.) developed in FVB and FVB-E5 mice within two weeks of infection, more frequently in E5-transgenic mice than in non-transgenic mice (p = 0.023). Dysplasia developed within one week. Cancers arose in both sexes (p = 0.79). Cancers and dysplastic lesions, positive for MmuPV1 by RNAscope, mostly disappeared by four weeks post infection with ~109 genome equivalents of virus, but cancers persisted for at least four weeks post infection with 1010 genome equivalents of virus. By contrast, infection of the rostro-dorsal (anterior) tongue with 1010 genome equivalents resulted in mild to moderate dysplasia but no cancers (p = 0.0040). No neoplastic lesions arose in mock-infected mice.
Conclusions
These findings indicate MmuPV1 can induce rapid onset of neoplastic lesions including cancers, depending on the site of infection. We are currently investigating the tissue specificity and transience of these lesions and the role they play in longer-term development of MmuPV1-induced disease.
HPV8 E1 AND E2 SUPPRESS THE ACTIVATION OF THE RIG-I-LIKE RECEPTOR MDA5
Abstract
Introduction
Persistent infections of the skin with HPV of genus beta (beta-HPV) in immunocompetent individuals are asymptomatic, but in immunosuppressed patients, beta-HPV infections exhibit much higher viral loads on the skin and are associated with an increased risk of skin cancer. Unlike with HPV16, the impact of beta-HPV early genes on the innate immune sensing of viral nucleic acids has not been studied.
Methods
We used primary skin keratinocytes (PHK) expressing individual HPV8 early genes and U2OS cells expressing all HPV8 genes as well as well-defined ligands of the nucleic-acid-sensing receptors RIG-I, MDA5, TLR3, and STING to analyze a potential functional interaction using cell culture, qRT-PCR and ELISA.
Results
We found that PHK and U2OS cells express RIG-I, MDA5, TLR3, and STING, but not TLR7, TLR8, or TLR9. While HPV16-E6 downregulated the expression of RIG-I, MDA5, TLR3, and STING and, in conjunction with HPV16-E7, effectively suppressed type I IFN in response to MDA5 activation, the presence of HPV8 early genes showed little effect on the expression of these immune receptors, except for HPV8-E2, which was associated with an elevated expression of TLR3. Nevertheless, whole HPV8 genome expression, as well as the selective expression of HPV8-E1 or HPV8-E2, was found to suppress MDA5-induced type I IFN and the proinflammatory cytokine IL-6. Furthermore, RNA isolated from HPV8-E2 expressing PHK, but not control cells, stimulated a type I IFN response in peripheral blood mononuclear cells, indicating that the expression of HPV8-E2 in keratinocytes leads to the formation of stimulatory RNA ligands that require the active suppression of immune recognition.
Conclusions
These results identify E1 and E2 as viral proteins that are responsible for the immune escape of HPV8 from the innate recognition of viral nucleic acids, a mechanism that may be necessary for establishing persistent beta-HPV infections.
POLY(I:C) TREATMENT PREVENTS SKIN TUMOR FORMATION IN THE PRECLINICAL HPV8 TRANSGENIC MOUSE MODEL
Abstract
Introduction
The development of actinic keratoses and squamous cell carcinoma of the skin is associated with infections with HPV of genus beta (betaHPV), especially in immunosuppressed patients. To date, targeted therapy against betaHPV-associated skin cancer is not available due to the large number of betaHPV without defined high-risk types. We therefore tested whether an in-situ autovaccination strategy through activation of innate antiviral immunity may represent a suitable approach to develop a personalized immunotherapy preventing the formation of betaHPV-associated skin cancer.
Methods
K14-HPV8 transgenic mice were used in which skin tumor development can be induced within 3 weeks after physical skin damage such as tattooing. We administered the following nucleic acid receptor ligands locally by tattooing into the murine skin: Y-DNA (cGAS/STING ligand), CpG1826 (TLR9 ligand), R848 (TLR7/8 ligand) and poly(I:C) (MDA5/TLR3 ligand). Mice were examined for tumor development and biopsies from tattooed skin were analysed by FACS, qRT-PCR and immunohistochemical stainings.
Results
While skin tumors were induced in control animals, tattooing TLR7/8 (R848) and TLR9 (CpG1826) ligands reduced tumor formation by 50% and the cGAS/STING ligand (Y-DNA) by 25%. Strikingly, poly(I:C) treatment completely prevented tumor growth in K14-HPV8 mice. Induction of the interferon-induced genes CXCL10 and IFIT1 by poly(I:C) depended on the activation of MDA5. FACS analysis revealed increased numbers of total as well as activated CD4+and CD8+ T-cells in poly(I:C) treated skin. By using immunohistochemistry, T-cells were found in the skin of poly(I:C)-treated animals but not in skin tumors of untreated control mice. T-cell depletion demonstrated a predominant role of CD4 T cells in HPV8 induced tumor development.
Conclusions
Taken together, our findings identify the MDA5 ligand poly(I:C) as a promising candidate for in situ autovaccination approaches, which might very well serve as a benchmark for the development of treatment strategies against betaHPV-related diseases.