transforming-growth-factor-beta and Papillomavirus-Infections

Bintrafusp alfa (anti-PD-L1/TGFβRII) is a first-in-class bifunctional agent designed to act both as a checkpoint inhibitor and as a "trap" for TGFβ in the tumor microenvironment (TME). This article is designed to review the preclinical studies interrogating the mode of action of bintrafusp alfa and to present a comprehensive overview of recent bintrafusp alfa clinical studies. Preclinical studies have demonstrated that bintrafusp alfa immune-mediating and antitumor activity can be enhanced by combining it with a human papillomavirus (HPV) therapeutic cancer vaccine, a tumor-targeting interleukin 12 (IL-12) immunocytokine and/or an IL-15 superagonist. The importance of TGFβ in HPV-associated malignancies is also reviewed. The clinical studies reviewed span extended phase I cohorts in patients with a spectrum of malignancies, two randomized phase II studies in lung and one in biliary tract cancers in which bintrafusp alfa did not demonstrate superiority over standard-of-care therapies, and provocative results in patients with HPV-associated malignancies, where as a monotherapy, bintrafusp alfa has shown response rates of 35%, compared to overall response rate (ORR) of 12-24% seen with other Food and Drug Administration (FDA)-approved or standard-of-care agents. This article also reviews preliminary phase II study results of patients with HPV

Topics: B7-H1 Antigen; Clinical Trials, Phase II as Topic; Humans; Immunologic Factors; Immunotherapy; Interleukin-12; Neoplasms; Papillomavirus Infections; Randomized Controlled Trials as Topic; Receptor, Transforming Growth Factor-beta Type II; Transforming Growth Factor beta; Tumor Microenvironment

BACKGROUNDHead and neck squamous cell carcinoma not associated with HPV (HPV-unrelated HNSCC) is associated with a high rate of recurrence and poor survival.METHODSWe conducted a clinical trial in 14 patients with newly diagnosed HPV-unrelated HNSCC to evaluate the safety and efficacy of neoadjuvant bintrafusp alfa, a bifunctional fusion protein that blocks programmed death ligand 1 (PD-L1) and neutralizes TGF-β.RESULTSBintrafusp alfa was well tolerated, and no treatment-associated surgical delays or complications occurred. Objective pathologic responses (PRs) were observed, and 12 of the 14 (86%) patients were alive and disease free at 1 year. Alterations in Treg infiltration and spatial distribution relative to proliferating CD8+ T cells indicated a reversal of Treg immunosuppression in the primary tumor. Detection of neoepitope-specific tumor T cell responses, but not virus-specific responses, correlated with the development of a PR. Detection of neoepitope-specific responses and PRs in tumors was not correlated with genomic features or tumor antigenicity but was associated with reduced pretreatment myeloid cell tumor infiltration. These results indicate that dual PD-L1 and TGF-β blockade can safely enhance tumor antigen-specific immunity and highlight the feasibility of multimechanism neoadjuvant immunotherapy for patients with HPV-unrelated HNSCC.CONCLUSIONOur studies provide insight into the ability of neoadjuvant immunotherapy to induce polyclonal neoadjuvant-specific T cell responses in tumors and suggest that features of the tumor microenvironment, such as myeloid cell infiltration, may be a major determinant of enhanced antitumor immunity following such treatment.TRIAL REGISTRATIONClinicalTrials.gov NCT04247282.FUNDINGThis work was funded by the Center for Cancer Research, the NCI, and the Intramural Research Program of the NIDCD, NIH. Bintrafusp alfa was provided by the health care business of Merck KGaA (Darmstadt, Germany), through a Cooperative Research and Development Agreement with the NCI. Additional funding was provided by ImmunityBio through a Cooperative Research and Development Agreement with the NIDCD.

Topics: Antigens, Neoplasm; B7-H1 Antigen; Head and Neck Neoplasms; Humans; Neoadjuvant Therapy; Papillomavirus Infections; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta; Tumor Microenvironment

Recurrent respiratory papillomatosis (RRP) is a human papillomavirus (HPV) driven neoplastic disorder of the upper aerodigestive tract that causes significant morbidity and can lead to fatal airway obstruction. Prior clinical study demonstrated clinical benefit with the programmed death-ligand 1 (PD-L1) monoclonal antibody avelumab. Bintrafusp alpha is a bifunctional inhibitor of PD-L1 and transforming growth factor-beta (TGF-b) that has shown clinical activity in several cancer types.. We conducted a phase II clinical trial evaluating bintrafusp alpha in adults with RRP. Papilloma samples before and after treatment with bintrafusp alpha were assessed for correlates of response with multiplex immunofluorescence as well as immunological and genomic analyses. Post hoc analyses of papilloma samples before and after treatment with avelumab were assessed for comparison.. Dual PD-L1/TGF-b inhibition failed to abrogate papilloma growth in most subjects and increased the frequency of clinically indicated interventions after treatment in four of eight subjects based on each subject's own historical control. TGF-b neutralization consistently decreased pSMAD3 and p21 and increased Ki67 expression within the basal layers of papillomas, indicating that TGF-b restrained proliferation. These alterations were not observed in papillomas treated with PD-L1 blockade alone. Dual PD-L1/TGF-b inhibition did not enhance anti-HPV immunity within papillomas beyond that observed with PD-L1 blockade. Genomic alterations in TGF-b superfamily genes were infrequent in papillomas and normal mucosa but present in a significant fraction of head and neck carcinomas.. Intact TGF-b signaling restrains proliferation within papillomas, and the use of clinical agents that abrogate this pathway should be avoided in patients with RRP.. NCT03707587 and NCT02859454.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Female; Humans; Immune Checkpoint Inhibitors; Immunologic Factors; Immunotherapy; Mice; NIH 3T3 Cells; Papilloma; Papillomavirus Infections; Respiratory Tract Infections; Transforming Growth Factor beta; Tumor Microenvironment

Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of transforming growth factor (TGF)-βRII (a TGF-β 'trap') fused to a human IgG1 mAb blocking programmed cell death ligand 1. This is the largest analysis of patients with advanced, pretreated human papillomavirus (HPV)-associated malignancies treated with bintrafusp alfa.. In these phase 1 (NCT02517398) and phase 2 trials (NCT03427411), 59 patients with advanced, pretreated, checkpoint inhibitor-naive HPV-associated cancers received bintrafusp alfa intravenously every 2 weeks until progressive disease, unacceptable toxicity, or withdrawal. Primary endpoint was best overall response per Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1; other endpoints included safety.. As of April 17, 2019 (phase 1), and October 4, 2019 (phase 2), the confirmed objective response rate per RECIST V.1.1 in the checkpoint inhibitor-naive, full-analysis population was 30.5% (95% CI, 19.2% to 43.9%; five complete responses); eight patients had stable disease (disease control rate, 44.1% (95% CI, 31.2% to 57.6%)). In addition, three patients experienced a delayed partial response after initial disease progression, for a total clinical response rate of 35.6% (95% CI, 23.6% to 49.1%). An additional patient with vulvar cancer had an unconfirmed response. Forty-nine patients (83.1%) experienced treatment-related adverse events, which were grade 3/4 in 16 patients (27.1%). No treatment-related deaths occurred.. Bintrafusp alfa showed clinical activity and manageable safety and is a promising treatment in HPV-associated cancers. These findings support further investigation of bintrafusp alfa in patients with advanced, pretreated HPV-associated cancers.

Topics: B7-H1 Antigen; Female; Humans; Male; Middle Aged; Neoplasms; Papillomaviridae; Papillomavirus Infections; Transforming Growth Factor beta

Chalcones are synthetic and naturally occurring compounds that have been widely investigated as anticancer agents. In this work, the effect of chalcones

Topics: Animals; Cervix Uteri; Chalcone; Chalcones; Female; HeLa Cells; Humans; Interleukin-10; Interleukin-4; Jurkat Cells; Lipopolysaccharides; Macrophages; Male; Mice; Papillomavirus Infections; Prostate; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: B7-H1 Antigen; Head and Neck Neoplasms; Humans; Immunotherapy; Neoadjuvant Therapy; Papillomavirus Infections; Transforming Growth Factor beta

TGFβ1 and TGFβ receptor 1 (TGFβR1) participate in regulation of the host's immune system and inflammatory responses and may serve as prognostic biomarkers for human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC).. This study included 1,013 patients with incident OPSCC, of whom 489 had tumor HPV16 status determined. All patients were genotyped for two functional polymorphisms: TGFβ1 rs1800470 and TGFβR1 rs334348. Univariate and multivariate Cox regression models were performed to evaluate associations between the polymorphisms and overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS).. Patients with TGFβ1 rs1800470 CT or CC genotype had 70%-80% reduced risks of OS, DSS, and DFS compared with patients with TT genotype, and patients with TGFβR1 rs334348 GA or GG genotype had 30%-40% reduced risk of OS, DSS, and DFS compared with patients with AA genotype. Furthermore, among patients with HPV-positive (HPV+) OPSCC, the same patterns were observed but the risk reductions were greater: up to 80%-90% for TGFβ1 rs1800470 CT or CC genotype and 70%-85% for TGFβR1 rs334348 GA or GG genotype. The risk reductions were still greater (up to 17 to 25 times reduced) for patients with both TGFβ1 rs1800470 CT or CC genotype and TGFβR1 rs334348 GA or GG genotype compared with patients with both TGFβ1 rs1800470 TT genotype and TGFβR1 rs334348 AA genotype among patients with HPV+ OPSCC.. Our findings indicate that TGFβ1 rs1800470 and TGFβR1 rs334348 may individually or jointly modify risks of death and recurrence in patients with OPSCC, particularly those with HPV+ OPSCC undergoing definitive radiotherapy, and may serve as prognostic biomarkers, which could lead to better personalized treatment and improved prognosis.

Topics: Binding Sites; Biomarkers; Carcinoma, Squamous Cell; Head and Neck Neoplasms; Humans; MicroRNAs; Oropharyngeal Neoplasms; Papillomavirus Infections; Prognosis; Receptor, Transforming Growth Factor-beta Type I; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta

The role of natural killer (NK) cells in juvenile-onset recurrent respiratory papillomatosis (JORRP) patients remains elusive. In this study, we find increased NK cell percentage, particularly CD11b-CD27- (DN) subsets in peripheral blood of JORRP patients and associated with disease activity. RNA sequencing shows a downregulated "natural killer cell-mediated cytotoxicity" feature in JORRP tumors. We also find impaired cytotoxic capacity and lower expression of NK cell-activating receptors including NKp30 and NKp46. Higher transforming growth factor-beta 1 (TGF-β1) is found both in plasma and tumor tissues of JORRP, and anti-TGF-β1 antibody could restore NK cell cytolytic activity and upregulate NKp30 and NKG2D expression. Also, we find a significantly higher Chemokine receptor type 6 (CXCR6) on NK cells in tumors compared with that in peripheral blood. Finally, RT-PCR analysis show that both HPV6-E6-E7 and HPV11-E6-E7 overexpression leads to higher

Topics: Humans; Killer Cells, Natural; Papilloma; Papillomavirus Infections; Receptors, Natural Killer Cell; Respiratory Tract Infections; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Papillomavirus Infections; RNA; Telomerase; Transforming Growth Factor beta; Uterine Cervical Neoplasms

Patients with HPV-unrelated head and neck squamous cell carcinoma (HPV-unrelated HNSCC) show only modest benefit from treatment with PD-1 inhibitors (PD-1i). Targeting transforming growth factor β (TGF-β) may make PD-1i more effective by inducing T cell responses. In this issue of the JCI, Redman et al. performed a clinical trial in 14 patients with HPV-unrelated HNSCC using bintrafusp alfa, a bifunctional fusion protein that blocks PD-L1 and TGF-β. Primary tumors displayed pathologic responses with 5 of 14 patients having at least a partial response. While no primary tumor or metastatic lymph node demonstrated a complete pathologic response, the findings suggest that concurrent neoadjuvant inhibition of PD-L1 and TGF-β may provide a rational strategy to improve pathologic response and clinical outcome in patients with HPV-unrelated HNSCC.

Topics: B7-H1 Antigen; Clinical Trials as Topic; Head and Neck Neoplasms; Humans; Immune Checkpoint Inhibitors; Immunologic Factors; Immunotherapy; Neoadjuvant Therapy; Papillomavirus Infections; Squamous Cell Carcinoma of Head and Neck; Synapses; T-Lymphocytes; Transforming Growth Factor beta

Topics: Disease Progression; Human papillomavirus 11; Humans; Intercellular Adhesion Molecule-1; Interleukin-10; Interleukin-4; Killer Cells, Natural; Natural Cytotoxicity Triggering Receptor 3; Neoplasm Recurrence, Local; Papilloma; Papillomavirus Infections; Respiratory Tract Infections; Transforming Growth Factor beta

Immune checkpoint blockade (ICB) therapy, while groundbreaking, must be improved to promote enhanced durable responses and to prevent the development of treatment-refractory disease. Cancer therapies that engage, enable, and expand the antitumor immune response will likely require rationally designed combination strategies. Targeting multiple immunosuppressive pathways simultaneously may provide additional therapeutic benefit over singular targeting. We therefore hypothesized that the use of two molecules which inhibit three independent, but overlapping, pathways (TIGIT:CD155, PD-1/PD-L1, and TGFβ) would provide significant antitumor efficacy in the syngeneic ICB resistant colorectal tumor model MC38 expressing human carcinoembryonic antigen (CEA) in CEA transgenic mice. This novel combination treatment strategy has significant antitumor activity and survival benefit in two models of murine carcinomas, MC38-CEA (CRC) and TC1 (HPV

Topics: Animals; B7-H1 Antigen; Carcinoembryonic Antigen; Carcinoma; CD8-Positive T-Lymphocytes; Colonic Neoplasms; Humans; Immune Checkpoint Inhibitors; Mice; Papillomavirus Infections; Programmed Cell Death 1 Receptor; Receptors, Immunologic; Transforming Growth Factor beta

BACKGROUND This study aimed to investigate the expression of long noncoding RNA (lncRNA) loc285194 in cervical squamous cell carcinoma (CSCC) biopsies that were positive and negative for human papillomavirus (HPV) and in human CSCC cell lines SiHa and C33A and to investigate the overexpression of lncRNA loc285194. MATERIAL AND METHODS Cervical biopsy tissue and plasma samples from 66 patients with histologically confirmed CSCC, that were HPV16-positive (N=22), HPV18-positive (N=27), and HPV-negative (N=17), and healthy controls (N=20) and human CSCC cell lines SiHa (HPV16-positive) and C33A (HPV-negative) were studied. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to measure the expression of lncRNA loc285194 in cervical biopsies and plasma. Enzyme-linked immunosorbent assay (ELISA) and Western blot were used to measure levels of transforming growth factor-ß1 (TGF-ß1). A lncRNA loc285194 expression vector was constructed and transfected into SiHa and C33A cells that underwent a transwell assay for cell migration. RESULTS Expression of lncRNA loc285194 was downregulated in HPV-positive and HPV-negative tissue samples and plasma from patients with CSCC and distinguished between patients and healthy controls. Plasma levels of loc285194 and TGF-ß1 were significantly correlated with the presence of CSCC. In SiHa and C33A cells, TGF-ß1 expression was downregulated, and cell migration was inhibited following lncRNA loc285194 overexpression. Although lncRNA loc285194 expression was not affected by TGF-ß1 treatment, its effects on cell migration were reduced by TGF-ß1. CONCLUSIONS The expression of lncRNA loc285194 inhibited the migration of CSCC cells in vitro through the inactivation of TGF-ß1.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; China; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Papillomaviridae; Papillomavirus Infections; RNA, Long Noncoding; Transforming Growth Factor beta; Transforming Growth Factors; Uterine Cervical Neoplasms

The development of cervical cancer (CeCa) is associated with high-risk human papilloma virus (HR-HPV) infections, mainly HPV-16, which is present in more than 50% of cases. The presence of immunosuppressive factors in the early stages of the disease is also strongly linked to CeCa progression. In this context, it is unknown whether ectonucleotidases CD39 and CD73, which are involved in the production of adenosine (Ado) that suppresses the specific antitumor immune response, are present in precursor lesions of CeCa. In this pilot study, we analyzed the presence of CD39 and CD73 and their capacity to generate Ado in 25 cervical samples from patients with grade 1 cervical intraepithelial neoplasms (CIN-1) and 25 samples from normal donors (NDs) free of HPV infection. Cells obtained from cervical samples of CIN-1 patients positive for HPV-16 showed higher CD39 and CD73 contents compared to samples obtained from CIN-1 patients negative for HPV-16 and NDs. Interestingly, solubilized cervical mucus from these patients also showed higher contents of soluble CD39 and CD73, which were associated with a greater capacity to produce Ado from the hydrolysis of adenosine triphosphate (ATP) and adenosine monophosphate (AMP). In addition, serum samples of these patients showed higher levels of TGF-

Topics: 5'-Nucleotidase; Adenosine Monophosphate; Adenosine Triphosphate; Adolescent; Adult; Antigens, CD; Apyrase; Cross-Sectional Studies; Female; Human papillomavirus 16; Humans; Papillomavirus Infections; Transforming Growth Factor beta; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms; Young Adult

Persistent high-risk human papillomavirus (HPV) infection is the major causal factor in cervical and other anogenital cancers. Because there are currently no therapeutics capable of preventing neoplastic progression of HPV infections, understanding the mechanisms of HPV-mediated persistence, including immune evasion, is a major research priority. The multifunctional growth factor transforming growth factor beta (TGFβ) has been shown to inhibit expression of early viral transcripts from cells harboring integrated HPV genomes or cells infected with retroviruses expressing HPV oncoproteins. However, the mechanism of TGFβ-induced inhibition has not been fully defined. In this study, we have observed a previously uncharacterized ability of TGFβ to repress the differentiation-induced upregulation of late HPV16 gene expression. In addition, interferon kappa (IFN-κ), a keratinocyte-specific, constitutively expressed cytokine suppressed by differentiation, can be transcriptionally induced by TGFβ1. TGFβ-mediated IFN-κ transcription only occurs in cells containing HPV16, and this is due to TGFβ1-mediated reversal of HPV-induced methylation of the IFN-κ promoter through active DNA demethylation mediated by thymine DNA glycosylase (TDG). This novel interaction between growth factor and innate immune signaling may shed light on the mechanisms of HPV persistence and how the virus manipulates both immune and growth factor signaling to promote its life cycle.

Topics: DNA Methylation; Gene Expression Regulation, Viral; Human papillomavirus 16; Humans; Interferon Type I; Keratinocytes; Papillomavirus Infections; Promoter Regions, Genetic; Transcriptional Activation; Transforming Growth Factor beta

Following cytotoxic therapy, 70% of patients with human papillomavirus (HPV)-positive oropharyngeal head and neck squamous cell carcinoma (HNSCC) are alive at 5 years compared with 30% of those with similar HPV-negative cancer. Loss of TGFβ signaling is a poorly studied consequence of HPV that could contribute to patient outcome by compromising DNA repair.. Human HNSCC cell lines (. Analysis of HNSCC specimens. HPV-positive HNSCC cells are unresponsive to TGFβ. Abrogated TGFβ signaling compromises repair by HRR and increases reliance on alt-EJ, which provides a mechanistic basis for sensitivity to PARP inhibitors. The effect of HPV in HNSCC provides critical validation of TGFβ's role in DNA repair proficiency and further raises the translational potential of TGFβ inhibitors in cancer therapy.

Topics: Animals; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mice; Papillomaviridae; Papillomavirus Infections; Recombinational DNA Repair; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV-), and HCK1T (normal). Proteins were analyzed by 2D gel electrophoresis coupled to MALDI-TOF-MS. Enrichment of secreted proteins with characteristic profiles for each cell line was followed by the identification of differentially expressed proteins. Particularly, transforming growth factor-beta-induced protein ig-h3 (Beta ig-h3) and peroxiredoxin-2 (PRDX2) overexpression in the secretome of cancer cell lines was detected and confirmed by Western blot. Bioinformatics analysis identified the transcription factor NRF2 as a regulator of differentially expressed proteins in the cervical cancer secretome. NRF2 levels were measured by both Western blot and Multiple Reaction Monitoring (MRM) in the total cell extract of the four cell lines. NRF2 was upregulated in SiHa and C33A compared to HCK1T. In conclusion, the secreted proteins identified in cervical cancer cell lines indicate that aberrant NRF2-mediated oxidative stress response (OSR) is a prominent feature of cervical carcinogenesis.

Topics: Algorithms; Carcinogenesis; Cell Line, Tumor; Computational Biology; Electrophoresis, Gel, Two-Dimensional; Extracellular Matrix Proteins; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Human papillomavirus 16; Humans; NF-E2-Related Factor 2; Papillomavirus Infections; Peptides; Peroxiredoxins; Proteomics; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry; Transforming Growth Factor beta; Uterine Cervical Neoplasms

Only a minority of those exposed to human papillomavirus (HPV) develop HPV-related cervical and oropharyngeal cancer. Because host immunity affects infection and progression to cancer, we tested the hypothesis that genetic variation in immune-related genes is a determinant of susceptibility to oropharyngeal cancer and other HPV-associated cancers by performing a multitier integrative computational analysis with oropharyngeal cancer data from a head and neck cancer genome-wide association study (GWAS). Independent analyses, including single-gene, gene-interconnectivity, protein-protein interaction, gene expression, and pathway analysis, identified immune genes and pathways significantly associated with oropharyngeal cancer. TGFβR1, which intersected all tiers of analysis and thus selected for validation, replicated significantly in the head and neck cancer GWAS limited to HPV-seropositive cases and an independent cervical cancer GWAS. The TGFβR1 containing p38-MAPK pathway was significantly associated with oropharyngeal cancer and cervical cancer, and TGFβR1 was overexpressed in oropharyngeal cancer, cervical cancer, and HPV(+) head and neck cancer tumors. These concordant analyses implicate TGFβR1 signaling as a process dysregulated across HPV-related cancers. This study demonstrates that genetic variation in immune-related genes is associated with susceptibility to oropharyngeal cancer and implicates TGFβR1/TGFβ signaling in the development of both oropharyngeal cancer and cervical cancer. Better understanding of the immunogenetic basis of susceptibility to HPV-associated cancers may provide insight into host/virus interactions and immune processes dysregulated in the minority of HPV-exposed individuals who progress to cancer.

Topics: Female; Genome-Wide Association Study; Host-Pathogen Interactions; Humans; MAP Kinase Signaling System; Neoplasms; p38 Mitogen-Activated Protein Kinases; Papillomaviridae; Papillomavirus Infections; Polymorphism, Single Nucleotide; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

The recent elucidation of the genomic landscape of head and neck squamous cell carcinoma (HNSCC) has provided a unique opportunity to develop selective cancer treatment options. These efforts will require the establishment of relevant HNSCC models for preclinical testing. Here, we performed full exome and transcriptome sequencing of a large panel of HNSCC-derived cells from different anatomical locations and human papillomavirus (HPV) infection status. These cells exhibit typical mutations in TP53, FAT1, CDK2NA, CASP8, and NOTCH1, and copy number variations (CNVs) and mutations in PIK3CA, HRAS, and PTEN that reflect the widespread activation of the PI3K-mTOR pathway. SMAD4 alterations were observed that may explain the decreased tumor suppressive effect of TGF-β in HNSCC. Surprisingly, we identified HPV+ HNSCC cells harboring TP53 mutations, and documented aberrant TP53 expression in a subset of HPV+ HNSCC cases. This analysis also revealed that most HNSCC cells harbor multiple mutations and CNVs in epigenetic modifiers (e.g., EP300, CREBP, MLL1, MLL2, MLL3, KDM6A, and KDM6B) that may contribute to HNSCC initiation and progression. These genetically-defined experimental HNSCC cellular systems, together with the identification of novel actionable molecular targets, may now facilitate the pre-clinical evaluation of emerging therapeutic agents in tumors exhibiting each precise genomic alteration.

Topics: Base Sequence; Cadherins; Carcinoma, Squamous Cell; Caspase 8; Cell Line, Tumor; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Cyclin-Dependent Kinase 2; DNA Copy Number Variations; Gene Dosage; Gene Expression Profiling; Head and Neck Neoplasms; Humans; Papillomaviridae; Papillomavirus Infections; Phosphatidylinositol 3-Kinases; Precision Medicine; Proto-Oncogene Proteins p21(ras); PTEN Phosphohydrolase; Receptor, Notch1; Sequence Analysis, DNA; Smad4 Protein; Squamous Cell Carcinoma of Head and Neck; TOR Serine-Threonine Kinases; Transcriptome; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Tumor cells are known to secrete cytokines that modify their microenvironment in order to favor their survival and continuous proliferation. In this work, we evaluated if TGF-β secreted in vitro by cervical cancer cells could interfere with the proliferation and survival of lymphocytes.. Lymphocytes were obtained from peripheral blood of healthy human volunteers, and isolated by density gradient centrifugation and cultured in 96-well plates. Lymphocyte proliferation was induced with phytohemaglutinin and co-cultured with conditioned media (CM) from cervical cancer cell lines, and the inhibition of proliferation was evaluated after 72 h by the incorporated radioactivity and a CFSE-labeling assay. TGF-β quantification on these CM was evaluated by ELISA. Non-apoptotic cellular death was evaluated through disruption of cell membrane integrity by measuring the liberation of lactate dehydrogenase. The apoptosis process was evaluated by annexin-V and active caspase-3. The presence of CD4+ or CD8+ lymphocytes was evaluated by flow cytometry using specific antibodies.. It was found that the conditioned media from these cells significantly inhibited the proliferation of lymphocytes and induced them to go into apoptosis. Antibodies against TGF-β almost completely blocked this activity, suggesting that this cytokine is responsible for the inhibitory activity. When the induced apoptosis on subpopulations of lymphocytes was evaluated, it was detected that the CD4+ cells were specifically targeted.. Cervical cancer cells secrete TGF-β that inhibits lymphocyte proliferation and induces apoptosis in CD4+, but not in CD8+ lymphocytes.

Topics: Alphapapillomavirus; Apoptosis; Carcinoma; CD4-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Culture Media, Conditioned; Female; Humans; Papillomavirus Infections; Transforming Growth Factor beta; Uterine Cervical Neoplasms

Thriving of tumors amidst rich immune infiltrates is an unexplained paradox.. Immune markers on lymphocytic infiltrates in HPV-positive cervicitis, cervical intraepithelial neoplasia III (CIN III), squamous cell carcinoma (SCC) and normal cervices were characterized immunohistochemically. Regulatory T cells were enumerated and phenotypically characterized using antibodies to FOXP3.. SCCs had higher numbers of CD4 and CD8 cells; infiltrates expressed more CD25, TGFbeta, and IL10 but had significantly lower IL2 compared with cervicitis and CIN III. Expression of CD25 and IL2 correlated well in cervicitis and CIN III but not in SCC. FOXP3 expression was also higher and ratios of CD4/FOXP3 and CD8/FOXP3 were lower in SCC. A fraction of cervicitis, CIN I, CIN II and CIN III had natural (n) regulatory T cells (Tregs); their lesional distribution was predominantly intraepithelial in cervicitis, while in CIN they were also present in the stroma. The proportion of FOXP3(+) CD25(+); FOXP3(+) CD25(-) and TGFbeta(+) CD25(+) in invasive tumors was 17; 19 and 22 respectively.. Cervical tumors are marked by the presence of an immunoregulatory environment, and harbor equal proportions of 'inactive' n Tregs; activated n Tregs; and Tregs operating via TGFbeta. nTregs in cervicitis and CIN may be a potential marker of persistence.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Female; Forkhead Transcription Factors; Humans; Immunohistochemistry; Lymphocytes, Tumor-Infiltrating; Middle Aged; Papillomavirus Infections; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms; Uterine Cervicitis

E7 is regarded as one of the main oncoproteins of high-risk human papillomaviruses (HPVs). It may affect the transforming growth factor beta 1 (TGF-beta1) signaling pathway. In this study, the relationship between HPV-16 infection and the functions of three critical factors of the TGF-beta1/Smads pathway was explored to assess the possible role of E7 in the development of cervical cancer.. The expression of E7, TGF-beta1, TbetaR-II and Smad4 was detected by immunohistochemistry in paraffin-embedded cervical samples, and by RT-PCR and Western blotting in cervical cancer cell lines. The effect of TGF-beta1 on the growth of cervical cancer cells were tested by methyl thiazolyl tetrazolium (MTT), and the effects of HPV-16 E7 protein on normal and malignant cervical cells were investigated by flow cytometry.. During the progression from benign to malignant lesions, the expression levels of TGF-beta1 and Smad4 increased significantly in cervical carcinoma tissues. The expression of TGF-beta1 was positively correlated with E7 expression. In vitro experiments showed that TGF-beta1 could not inhibit the proliferation of several cervical carcinoma cell lines in long-term regulation, but could inhibit immunologic reactions of peripheral blood mononuclear cells (PBMCs). Blocking E7 expression could lower the expression level of TGF-beta1 and induce cells to enter apoptosis.. Our data indicate that HPV-16 E7 protein plays an important role during the development of cervical cancer by immuno-inhibition and stimulation of tumor cell proliferation through the TGF-beta1/Smads signaling pathway.

Topics: Cell Growth Processes; Cell Line, Tumor; Cell Transformation, Neoplastic; Enzyme-Linked Immunosorbent Assay; Female; HeLa Cells; Human papillomavirus 16; Humans; Interferon-gamma; Interleukin-2; Leukocytes, Mononuclear; Oligonucleotides, Antisense; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Papillomavirus Infections; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Smad4 Protein; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

Topics: Carcinoma, Squamous Cell; DNA-Binding Proteins; Head and Neck Neoplasms; Humans; Models, Biological; Papillomaviridae; Papillomavirus Infections; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

The SMADs are a group of interrelated proteins that mediate transforming growth factor beta (TGF-beta) signaling. Upon TGF-beta binding the TGF-beta type I receptor phosphorylates Smad2 and Smad3, which then complex with Smad4 and translocate to the nucleus, with subsequent activation of target genes. Disruption of TGF-beta signaling is thought to contribute to the development of head and neck squamous cell carcinomas (HNSCC). Alterations in the function of the DPC4/Smad4 tumor suppressor gene have been found to inactivate TGF-beta signaling in several tumor types. For example, DPC4/Smad4 is lost or mutated in colorectal, pancreatic, and esophageal cancers. In addition, DPC4/Smad4 transcriptional activity and TGF-beta ability to inhibit DNA synthesis is blocked by the E7 protein of the human papillomavirus type 16 (HPV16) in cervical carcinoma cell lines. HPV16 infection is a risk factor for the development of a subset of HNSCC. This study was undertaken to investigate a potential correlation between expression of components of the TGF-beta signaling pathway and HPV16 status in HNSCC tumors. We examined the expression of TGF-beta signaling proteins Smad2, Smad2-P, and Smad4 by immunohistochemistry in 27 HPV16-negative and 16 HPV16-positive HNSCCs. We compared the expression patterns and assessed their relationship to HPV16 status. No significant differences were detected between HPV16-positive and HPV16-negative tumors in the expression of Smad2 and Smad2-P. Smad4 expression, however, was decreased in 56% of the HPV16-positive tumors and in 39% of HPV16-negative tumors. This difference was statistically significant (P = 0.01) suggesting that loss of Smad4 expression may be involved in HPV16-induced carcinogenesis of HNSCC.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; DNA-Binding Proteins; DNA, Viral; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Staging; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Papillomavirus Infections; Polymerase Chain Reaction; Repressor Proteins; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Human papillomavirus (HPV) is thought to be one of the possible causative factors in cervical carcinogenesis, and cervical carcinoma cells are refractory to tumor transforming growth factor (TGF)-beta1. The purpose of this study is to investigate the possible cause-effect association between HPV and TGF-beta1 during cervical tumorigenesis.. We assessed the expression of HPV capsid proteins, HPV-16 E7, HPV-16 E2 (C and N terminals), TGF-beta1, and their receptors TGF-beta RI and RII by immunohistochemistry in 48 paraffin-embedded blocks of tumor tissue derived from patients of cervical neoplasia.. Expression of TGF-beta1 decreased as tumor cells progressed from cervical intraepithelial neoplasia (CIN)1, CIN2, CIN3, to microinvasive carcinoma (P < 0.05). Levels of TGF-betaRI and TGFbeta-RII stayed the same in all cases. HPV was found in 89.6% of the studied sections, and cervical lesions without HPV infection expressed significantly less TGF-beta1 (P < 0.05). By comparing the expression pattern of TGF-beta1 and HPV in the neoplastic cells with that of normal cervical epithelium in each section, we found loss of HPV-16 E2 higher in CIN3 (15/24) than in CIN1 or CIN2 (3/7), and there is a significant trend that loss of HPV-16 E2 expression correlated with a >50% loss of TGF-beta1 at the lesion site (P < 0.05).. Our result showed co-suppression of HPV and TGF-beta1 expression during progression of cervical squamous cell cancer. Using antibody against HPV-16 E2 may be an auxiliary tool for the investigation of cervical tumor progression.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; DNA-Binding Proteins; Female; Humans; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus Infections; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Virus Infections; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

The symposium on Virus Infections and Tumors of the Oral Mucosae was organized as a joint meeting of the Arbeitskreis Oralpathologie and Oralmedizin and the Arbeitsgemeinschaft Dermatologische Infektiologie (ADI) der Deutschen Dermatologischen Gesellschaft. The main topics of the meeting were herpes virus infections, human papillomavirus (HPV) infections and human immunodeficiency virus (HIV) infections of the oral mucosae. Clinically both diagnostic, differential diagnostic and therapeutic aspects of the virus-associated diseases were discussed in several presentations. Another important issue was the role of these viruses, particularly of HPVs, in the origin and development of oral cancer. Apparently besides smoking and alcohol other risk factors comprise high risk HPVs, immunodeficiency and possibly also genetic factors. Whether neonatal early infections may predispose children to a specific cancer risk in their future life is still at a level of discussion. Some arguments, however were shown that tonsillar carcinoma, which shows the highest prevalence of the high-risk HPV 16- DNA sequences between all oral cancer, is possibly an epidemiologically and etiologically distinct tumor. It is argued that this tumor is probably less dependent on classical carcinogens than other oral malignant tumors.

Topics: DNA, Viral; Genes, p53; Herpesviridae Infections; HIV Infections; Humans; Leukoplakia, Oral; Lichen Planus, Oral; Mouth Neoplasms; Papilloma; Papillomaviridae; Papillomavirus Infections; Transforming Growth Factor beta; Tumor Virus Infections

Transforming growth factor beta (TGF-beta) suppresses proliferation and potentiates apoptosis of HPV16-immortalized human cervical epithelial cells (ECE16-1). Exposure of ECE16-1 to TGF-beta1 increased expression of p53 and induced cell cycle arrest. We examined, by Western blotting, expression of p53 and related cell cycle regulatory proteins after treatment. p53 levels increased as a function of time and dose. Increased p53 appeared to be active, since TGF-beta1 treatment increased the activity of a p53 transcriptional response element in a luciferase reporter plasmid. Additionally, the proteins of the p53-regulated genes, p21(WAF1), mdm2, and Bax, were increased with similar time and dose responses. We did not observe consistent changes in protein levels of cyclin D, cyclin E, CDK4, CDK6, CDK2, p27(Kip1), p16(INK4a), or RNA levels of p15(INK4b). Activity of CDK4 or 6, measured by phosphorylation of an Rb fragment, remained constant during the response period; however, activity of CDK2 (phosphorylation of histone H1) decreased. Concordantly, increased levels of p21(WAF1) were immunoprecipitated with anti-CDK2 antibodies. During treatment, the phosphorylation state of Rb shifted to a hypophosphorylated form. mRNA for the HPV E6/E7 genes decreased; however, significant changes in the E7 protein were not observed, while increased levels of Rb immunoprecipitated with anti-E7 antibodies were observed. These data are consistent with the following model. In ECE16-1 cells, there exists a fine balance between inhibitory levels of p53 and Rb and the antagonists, E6 and E7. TGF-beta1 treatment decreases steady-state levels of E6/E7 mRNA, which results in a shifted balance (lowered activity of E6) in favor of increased p53 expression, resulting in activation of the cell cycle inhibitory gene, p21(WAF1). This protein binds the cyclin E/CDK2 complex that maintains Rb in a phosphorylated state. Rb shifts to a hypophosphorylated state, resulting in G1 arrest, presumably by binding E2F transcription factors.

Topics: Carrier Proteins; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cell Line, Transformed; Cell Transformation, Viral; Cervix Uteri; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Dose-Response Relationship, Drug; Epithelial Cells; Female; G1 Phase; Gene Expression Regulation, Viral; Humans; Microtubule-Associated Proteins; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus Infections; Phosphorylation; Protein Serine-Threonine Kinases; Retinoblastoma Protein; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Tumor Virus Infections

Human papillomaviruses (HPV) are implicated in the multistep process of cervical carcinogenesis. Transforming growth factor beta(TGF-beta) inhibits the proliferation of epithelial cells, and it has also been found to inhibit HPV gene expression in nontumorigenic epithelial cell lines. In the present study, we examined the expression of TGF-beta 1 and TGF-beta 2 protein immunohistochemically (IHC) in a series of 95 HPV-positive and HPV-negative lesions of the uterine cervix, with special emphasis on HPV type, grade of cervical intraepithelial neoplasia (CIN), and the clinical course of the disease. Expression of TGF-beta 1 was found in 56/95 (59%) and that of TGF-beta 2 in 87/95 (92%) of the specimens. Cytoplasmic TGF-beta 2 staining was localized in the epithelial layers higher than that of TGF-beta 1, which showed also some nuclear staining and was located in the basal cells of the epithelium as well. TGF-beta 1 was expressed in 36/68 (53%) of HPV-positive samples and in 16/21 (76%) of HPV-negative samples; TGF-beta 2 expression was detectable in 63/68 (93%) and 18/21 (86%), respectively. TGF-beta 1 was present slightly more frequently in HPV-CIN lesions (23/41, 56%) than in HPV-NCIN (HPV without CIN) specimens (13/27, 48%). TGF-beta 2 expression was detected in 39/41 (95%) of HPV-CIN and in 24/27 (89%) of HPV-NCIN specimens. TGF-beta 2 expression was not related to the clinical course of the disease. TGF-beta 1 expression was most frequent in regressed and persistent lesions (> 60%), compared to 45% in progressed and 33% in the recurred lesions. The results suggest that TGF-beta (especially TGF-beta 2) expression is common in CIN lesions, but the pattern and intensity of TGF-beta expression examined by IHC are not clearly related to the grade of the lesions or their clinical course. Assessment of the biological activity of TGF-beta s and their influence on HPV genes may shed more light on HPV-associated carcinogenesis.

Topics: Female; Humans; Immunohistochemistry; In Situ Hybridization; Papillomaviridae; Papillomavirus Infections; Prospective Studies; Transforming Growth Factor beta; Tumor Virus Infections; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms