RESUMO
Human papillomavirus (HPV) 11/16 E6/E7 proteins have been recognized to be pivotal in viral pathogenesis. This study sought to uncover the potential mechanisms of how HPV11/16 E6/E7-transfected keratinocytes inhibit cytokine secretion in peripheral blood mononuclear cells (PBMC). Upon co-culturing HPV11/16 E6/E7-transfected keratinocytes with PBMC in a non-contact manner, we observed a marked decrease in various cytokines secreted by PBMC. To determine if this suppression was mediated by specific common secreted factors, we conducted transcriptomic sequencing on these transfected cells. This analysis identified 53 common differentially secreted genes in all four HPV-transfected cells. Bioinformatics analysis demonstrated these genes were predominantly involved in immune regulation. Results from quantitative PCR (qPCR) and an extensive literature review suggested the downregulation of 12 genes (ACE2, BMP3, BPIFB1, CLU, CST6, CTF1, HMGB2, MMP12, PDGFA, RNASE7, SULF2, TGM2), and upregulation of 7 genes (CCL17, CCL22, FBLN1, PLAU, S100A7, S100A8, S100A9), may be crucial in modulating tumor immunity and combating pathogenic infections, with genes S100A8 and S100A9, and IL-17 signaling pathway being particularly noteworthy. Thus, HPV11/16 E6/E7 proteins may inhibit cytokine secretion of immune cells by altering the expression of host-secreted genes. Further exploration of these genes may yield new insights into the complex dynamics of HPV infection.
Assuntos
Citocinas , Leucócitos Mononucleares , Proteínas Oncogênicas Virais , Humanos , Citocinas/metabolismo , Citocinas/genética , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Leucócitos Mononucleares/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas Oncogênicas Virais/imunologia , Queratinócitos/virologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 11/genética , Papillomavirus Humano 11/imunologia , Perfilação da Expressão Gênica , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/genética , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas E7 de Papillomavirus/imunologia , Técnicas de Cocultura , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/genéticaRESUMO
The Human papillomavirus (HPV) causes tumors in part by hijacking the host cell cycle and forcing uncontrolled cellular division. While there are >200 genotypes of HPV, 15 are classified as high-risk and have been shown to transform infected cells and contribute to tumor formation. The remaining low-risk genotypes are not considered oncogenic and result in benign skin lesions. In high-risk HPV, the oncoprotein E7 contributes to the dysregulation of cell cycle regulatory mechanisms. High-risk E7 is phosphorylated in cells at two conserved serine residues by Casein Kinase 2 (CK2) and this phosphorylation event increases binding affinity for cellular proteins such as the tumor suppressor retinoblastoma (pRb). While low-risk E7 possesses similar serine residues, it is phosphorylated to a lesser degree in cells and has decreased binding capabilities. When E7 binding affinity is decreased, it is less able to facilitate complex interactions between proteins and therefore has less capability to dysregulate the cell cycle. By comparing E7 protein sequences from both low- and high-risk HPV variants and using site-directed mutagenesis combined with NMR spectroscopy and cell-based assays, we demonstrate that the presence of two key nonpolar valine residues within the CK2 recognition sequence, present in low-risk E7, reduces serine phosphorylation efficiency relative to high-risk E7. This results in significant loss of the ability of E7 to degrade the retinoblastoma tumor suppressor protein, thus also reducing the ability of E7 to increase cellular proliferation and reduce senescence. This provides additional insight into the differential E7-mediated outcomes when cells are infected with high-risk verses low-risk HPV. Understanding these oncogenic differences may be important to developing targeted treatment options for HPV-induced cancers.
Assuntos
Proteínas E7 de Papillomavirus , Fosforilação , Proteínas E7 de Papillomavirus/metabolismo , Proteínas E7 de Papillomavirus/genética , Humanos , Caseína Quinase II/metabolismo , Caseína Quinase II/genética , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/genética , Ligação Proteica , Proteína do Retinoblastoma/metabolismo , Proteína do Retinoblastoma/genética , Papillomaviridae/genética , Papillomaviridae/metabolismo , Papillomaviridae/fisiologia , Ciclo Celular , Mutagênese Sítio-DirigidaRESUMO
Tirbanibulin 1% ointment is a synthetic antiproliferative agent approved in 2021 by the European Union for treating actinic keratoses (AK). Topical tirbanibulin has clinically resolved HPV-57 ( +) squamous cell carcinoma (SCC), HPV-16 ( +) vulvar high-grade squamous intraepithelial lesion, epidermodysplasia verruciformis, and condyloma. We examined how tirbanibulin might affect HPV oncoprotein expression and affect other cellular pathways involved in cell proliferation and transformation. We treated the HeLa cell line, containing integrated HPV-18, with increasing doses of tirbanibulin to determine the effects on cell proliferation. Immunoblotting was performed with antibodies against the Src canonical pathway, HPV 18 E6 and E7 transcription regulation, apoptosis, and invasion and metastasis pathways. Cell proliferation assays with tirbanibulin determined the half-maximal inhibitory concentration (IC50) of HeLa cells to be 31.49 nmol/L. Increasing concentrations of tirbanibulin downregulates the protein expression of Src (p < 0.001), phospho-Src (p < 0.001), Ras (p < 0.01), c-Raf (p < 0.001), ERK1 (p < 0.001), phospho-ERK1 (p < 0.001), phospho-ERK2 (p < 0.01), phospho-Mnk1 (p < 0.001), eIF4E (p < 0.01), phospho-eIF4E (p < 0.001), E6 (p < 0.01), E7 (p < 0.01), Rb (p < 0.01), phospho-Rb (p < 0.001), MDM2 (p < 0.01), E2F1 (p < 0.001), phospho-FAK (p < 0.001), phospho-p130 Cas (p < 0.001), Mcl-1 (p < 0.01), and Bcl-2 (p < 0.001), but upregulates cPARP (p < 0.001), and cPARP/fPARP (p < 0.001). These results demonstrate that tirbanibulin may impact expression of HPV oncoproteins via the Src- MEK- pathway. Tirbanibulin significantly downregulates oncogenic proteins related to cell cycle regulation and cell proliferation while upregulating apoptosis pathways.
Tirbanibulin is Promising Novel Therapy for Human Papillomavirus (HPV)-associated Diseases.Tirbanibulin 1% ointment is an approved synthetic topical ointment for treating actinic keratoses (AK), a precancer of skin cancer. Topical tirbanibulin has previously been reported to clinically resolve human papillomavirus (HPV)-( +) diseases.In this study, we examine how tirbanibulin may affect the HPV and pathways associated with cancer.We treated the HeLa cell line to determine the effects on HPV cell proliferation. Increasing the concentration of tirbanibulin statistically significantly affected numerous cellular pathways often associated with cancer.These results demonstrate that tirbanibulin may impact expression of HPV oncoproteins and thereby kill cancer cells.
Assuntos
Proliferação de Células , Regulação para Baixo , Papillomavirus Humano 18 , Proteínas Oncogênicas Virais , Humanos , Células HeLa , Proliferação de Células/efeitos dos fármacos , Proteínas Oncogênicas Virais/metabolismo , Regulação para Baixo/efeitos dos fármacos , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/tratamento farmacológico , Proteínas E7 de Papillomavirus/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/virologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/metabolismo , Quinases da Família src/metabolismo , Quinases da Família src/antagonistas & inibidores , Feminino , Papillomavirus Humano , Proteínas de Ligação a DNARESUMO
Human papillomaviruses (HPV) are a major cause of malignancy, contributing to ~5% of all human cancers worldwide, including most cervical cancer cases and a growing number of anogenital and oral cancers. The major HPV viral oncogenes, E6 and E7, manipulate many host cellular pathways that promote cell proliferation and survival, predisposing infected cells to malignant transformation. Despite the availability of highly effective vaccines, there are still no specific anti-viral therapies targeting HPV or treatments for HPV-associated cancers. As such, a better understanding of viral-host interactions may allow the identification of novel therapeutic targets. Here, we demonstrate that the actin-binding protein LASP1 is upregulated in cervical cancer and significantly correlates with a poorer overall survival. In HPV positive cervical cancer, LASP1 depletion significantly inhibited the oncogenic phenotype in vitro, whilst having minimal effects in HPV negative cervical cancer cells. Furthermore, we demonstrate that the LASP1 SH3 domain is essential for LASP1-mediated oncogenicity in these cells. Mechanistically, we show that HPV E7 regulates LASP1 at the post-transcriptional level by repressing the expression of miR-203, which negatively regulates LASP1 mRNA levels by binding to its 3'UTR. Finally, we demonstrate that LASP1 expression is required for the growth of HPV positive cervical cancer cells in an in vivo tumourigenicity model. Together, these data demonstrate that HPV induces LASP1 expression to promote proliferation and survival in cervical cancer, thus identifying a potential therapeutic target in these cancers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proliferação de Células , Proteínas do Citoesqueleto , Proteínas com Domínio LIM , MicroRNAs , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Neoplasias do Colo do Útero/virologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , MicroRNAs/genética , Humanos , Feminino , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células/genética , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/patologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Animais , Camundongos , Regulação Neoplásica da Expressão Gênica , Linhagem Celular TumoralRESUMO
BACKGROUND: It has been demonstrated that exosomes derived from HPV-16 E7-over-expressiong non-small cell lung cancer (NSCLC) cells (E7 Exo) trigger increased levels of epidermal growth factor receptor (EGFR) and miR-381-3p. The purpose of this investigation was to examine the role of E7 Exo in NSCLC angiogenesis, and to analyze the contribution of exosomal EGFR and miR-381-3p to it. METHODS: The influence of E7 Exo on the proliferation and migration of human umbilical vein endothelial cells (HUVECs) was assessed using colony formation and transwell migration assays. Experiments on both cells and animal models were conducted to evaluate the angiogenic effect of E7 Exo treatment. The involvement of exosomal EGFR and miR-381-3p in NSCLC angiogenesis was further investigated through suppressing exosome release or EGFR activation, or by over-expressing miR-381-3p. RESULTS: Treatment with E7 Exo increased the proliferation, migration, and tube formation capacities of HUVECs, as well as angiogenesis in animal models. The suppression of exosome release or EGFR activation in NSCLC cells decreased the E7-induced enhancements in HUVEC migration and tube formation, and notably reduced vascular endothelial growth factor A (VEGFA) and Ang-1 levels. HUVECs that combined miR-381-3p mimic transfection and E7 Exo treatment exhibited a more significant tube-forming capacity than E7 Exo-treated HUVECs alone, but were reversed by the miR-381-3p inhibitor. CONCLUSION: The angiogenesis induced by HPV-16 E7 in NSCLC is mediated through exosomal EGFR and miR-381-3p.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Movimento Celular , Proliferação de Células , Receptores ErbB , Exossomos , Células Endoteliais da Veia Umbilical Humana , Neoplasias Pulmonares , MicroRNAs , Neovascularização Patológica , Proteínas E7 de Papillomavirus , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Exossomos/metabolismo , Exossomos/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/irrigação sanguínea , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Nus , Papillomavirus Humano 16/genética , AngiogêneseRESUMO
BACKGROUND: Cervical cancer is among the highest-ranking types of cancer worldwide, with human papillomavirus (HPV) as the agent driving the malignant process. One aspect of the infection's evolution is given by epigenetic modifications, mainly DNA methylation and chromatin alteration. These processes are guided by several chromatin remodeling complexes, including NuRD. The purpose of this study was to evaluate the genome-wide binding patterns of the NuRD complex components (MBD2 and MBD3) in the presence of active HPV16 E6 and E7 oncogenes and to determine the potential of identified genes through an experimental model to differentiate between cervical precursor lesions, with the aim of establishing their utility as biomarkers. METHODS: The experimental model was built using the CaSki cell line and shRNA for E6 and E7 HPV16 silencing, ChIP-seq, qRT-PCR, and Western blot analyses. Selected genes' expression was also assessed in patients. RESULTS: Several genes have been identified to exhibit altered transcriptional activity due to the influence of HPV16 E6/E7 viral oncogenes acting through the MBD2/MBD3 NuRD complex, linking them to viral infection and cervical oncogenesis. CONCLUSIONS: The impacted genes primarily play roles in governing gene transcription, mRNA processing, and regulation of translation. Understanding these mechanisms offers valuable insights into the process of HPV-induced oncogenesis.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA , Papillomavirus Humano 16 , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Proteínas Oncogênicas Virais , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Carcinogênese/genética , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/patogenicidade , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Neoplasias do Colo do Útero/virologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Recurrent respiratory papillomatosis (RRP) is a rare benign tumor caused mainly by the infection of the respiratory tract epithelial cells by the human papillomavirus (HPV) type 6/11. However, the specific mechanisms underlying the inhibition of the host's innate immune response by HPV remain unclear. For this purpose, we employed single-cell RNA sequencing to analyze the states of various immune cells in RRP samples post-HPV infection and utilized a cellular model of HPV infection to elucidate the mechanisms by which HPV evades the innate immune system in RRP. The results revealed distinct immune cell heterogeneity in RRP and demonstrated that HPV11 E7 can inhibit the phosphorylation of the stimulator of interferon genes protein, thereby circumventing the body's antiviral response. In vitro co-culture experiments demonstrated that stimulation of macrophages to produce interferon-beta induced the death of HPV-infected epithelial cells, also reducing HPV viral levels. In summary, our study preliminarily identifies the potential mechanisms by which HPV evades the host's antiviral immune response, as well as the latent antiviral functions exhibited by activated macrophages. This research serves as an initial exploration of antiviral immune evasion in RRP, laying a solid foundation for investigating immunotherapeutic approaches for the disease.IMPORTANCESurgical tumor reduction is the most common treatment for recurrent respiratory papillomatosis (RRP). One of the characteristics of RRP is its persistent recurrence, and multiple surgeries are usually required to control the symptoms. Recently, some adjuvant therapies have shown effectiveness, but none of them can completely clear human papillomavirus (HPV) infection, and thus, a localized antiviral immune response is significant for disease control; after all, HPV infection is limited to the epithelium. Inhibition of interferon-beta (IFN-ß) secretion by HPV11 E7 viral proteins in epithelial cells by affecting stimulator of interferon genes phosphorylation may account for the persistence of low-risk HPV replication in the RRP. Moreover, suppression of the IFN-I pathway in RRP cell types might provide clues regarding the hyporeactive function of local immune cells. However, activation of macrophage groups to produce IFN-ß can still destroy HPV-infected cells.
Assuntos
Papillomavirus Humano 11 , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus , Infecções Respiratórias , Adulto , Feminino , Humanos , Masculino , Células Epiteliais/virologia , Células Epiteliais/imunologia , Papillomavirus Humano 11/genética , Papillomavirus Humano 11/imunologia , Evasão da Resposta Imune , Imunidade Inata , Interferon beta/metabolismo , Interferon beta/imunologia , Interferon beta/genética , Macrófagos/imunologia , Macrófagos/virologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Infecções Respiratórias/virologia , Infecções Respiratórias/imunologiaRESUMO
Human papillomavirus (HPV) infection has been reported to be associated with N6-methyladenosine (m6A) modification in cancers. However, the underlying mechanism by which m6A methylation participates in HPV-related cervical squamous cell carcinoma (CSCC) remains largely unclear. In this study, we observed that m6A regulators methyltransferase like protein (METTL14) and insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) were upregulated in HPV-positive CSCC tissues and cell lines, and their high expression predicted poor prognosis for HPV-infected CSCC patients. Cellular functional experiments verified that HPV16 oncogenes E6/E7 upregulated the expression of METTL14 and IGF2BP3 to promote cell proliferation and epithelial mesenchymal transition of CSCC cells. Next, we found that E6/E7 stabilized fascin actin-bundling protein 1 (FSCN1) mRNA and elevated FSCN1 expression in CSCC cells through upregulating METTL14/IGF2BP3-mediated m6A modification, and FSCN1 expression was also validated to be positively associated with worse outcomes of HPV-positive CSCC patients. Finally, HPV16-positive CSCC cell lines SiHa and CaSki were transfected with knockdown vector for E6/E7 or METTL14/IGF2BP3 and overexpressing vector for FSCN1, and functional verification experiments were performed through using MTT assay, flow cytometry, wound healing assay and tumour formation assay. Results indicated that knockdown of E6/E7 or METTL14/IGF2BP3 suppressed cell proliferation, migration and tumorigenesis, and accelerated cell apoptosis of HPV-positive CSCC cells. Their tumour-suppressive effects were abolished through overexpressing FSCN1. Overall, HPV E6/E7 advanced CSCC development through upregulating METTL14/IGF2BP3-mediated FSCN1 m6A modification.
Assuntos
Carcinoma de Células Escamosas , Papillomavirus Humano 16 , Metiltransferases , Proteínas dos Microfilamentos , Infecções por Papillomavirus , Proteínas de Ligação a RNA , Neoplasias do Colo do Útero , Feminino , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Carcinoma de Células Escamosas/virologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Metilação , Metiltransferases/metabolismo , Metiltransferases/genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Proteínas Repressoras , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias do Colo do Útero/virologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/metabolismoRESUMO
Multiple cellular pathways are affected by HPV E6 and E7 oncoproteins, including endocytic and cellular trafficking. HPV-16 E7 can target the adaptor protein (AP) complex, which contains proteins important during endocytosis transport. To further investigate the role of HPV E7 during this process, we analysed the expression of cell surface proteins in NIKS cells expressing HPV-16 E7. We show that different cell surface proteins are regulated by HPV-16 E7 via interaction with AP2. We observed that the expression of MET and CD109 membrane protein seems to be upregulated in cells expressing E7. Moreover, the interaction of MET and CD109 with AP2 proteins is disrupted by HPV-16 E7. In addition, in the absence of HPV-16 E7, there is a downregulation of the cell membrane expression of MET and CD109 in HPV-positive cell lines. These results expand our knowledge of the functions of E7 and open new potential cellular pathways affected by this oncoprotein.
Assuntos
Antígenos CD , Papillomavirus Humano 16 , Proteínas E7 de Papillomavirus , Proteínas Proto-Oncogênicas c-met , Humanos , Proteínas E7 de Papillomavirus/metabolismo , Proteínas E7 de Papillomavirus/genética , Antígenos CD/metabolismo , Antígenos CD/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 16/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Membrana Celular/metabolismo , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Endocitose , Proteínas Ligadas por GPIRESUMO
The altered protein expression of inverted CCAAT box-binding protein of 90 kDa/ubiquitin-like with PHD and RING finger domains 1 (ICBP90/UHRF1), and Np95-like ring finger protein (NIRF)/UHRF2, which belong to the ubiquitin-like with PHD and RING finger domains (UHRF) family, is linked to tumor malignancy and the progression of various cancers. In this study, we analyzed the UHRF family expression in cervical cancers, and it's regulation by human papillomavirus (HPV). Western blotting was performed to analyze protein expression in cervical cancer cell lines. Immunohistochemical analysis were used to investigate the expression of UHRF family and MIB-1 in cervical cancer tissues. Transfection were done for analyze the relationship between UHRF family and HPVs. We showed that NIRF expression was decreased and ICBP90 expression was increased in cervical cancers compared to normal counterparts. Western blotting also showed that NIRF expression was quite low levels, but ICBP90 was high in human cervical cancer cell lines. Interestingly, ICBP90 was up regulated by high risk type HPV16 E6 and E7, but not low-risk type HPV11. On the other hand, NIRF was down regulated by high risk type HPV16 E6 but not by E7. Low risk type HPV11 E6 did not affect the NIRF expression at all. We propose that ICBP90 overexpression, and reduced NIRF expression, found in cervical cancers, is an important event of a cervical carcinogenesis, and especially ICBP90 may offer a proliferating marker and therapeutic target for treating uterine cervical cancers.
Assuntos
Proteínas Oncogênicas Virais , Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/patologia , Papillomavirus Humano 16/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Ubiquitinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismoRESUMO
BACKGROUND INFORMATION: The precise etiology of breast cancer is not completely understood, although women with BRCA1 gene mutations have a significantly increased risk of developing the disease. In addition, sporadic breast cancer is frequently associated with decreased BRCA1 gene expression. Growing evidence of Human papillomaviruses (HPVs) infections in breast tumors has raised the possibility of the involvement of HPVs in the pathogenesis of breast cancer. We investigated whether the effects of HPV oncoproteins E6 and E7 were influenced by the expression levels of BRCA1. HPV16E6E7 (prototype or E6D25E/E7N29S Asian variant type) were stably expressed in MDA-MB231 breast cancer cells, wild type for BRCA1, or with BRCA1 knocked down. RESULTS: Expression of HPV16E6E7 oncogenes did not affect BRCA1 levels and the abundance of HPV16E6E7 was not altered by BRCA1 knockdown. BRCA1 levels did not alter HPV16E6E7-dependent degradation of G1-S cell cycle proteins p53 and pRb. However, we found that the expression of G2-M cell cycle protein cyclin B1 enhanced by HPV16E6E7 was impacted by BRCA1 levels. Especially, we found the correlation between BRCA1 and cyclin B1 expression and this was also confirmed in breast cancer samples from a Thai cohort. We further demonstrated that the combination of HPV oncoproteins and low levels of BRCA1 protein appears to enhance proliferation and invasion. Transactivation activities of HPV16E6E7 on genes regulating cell proliferation and invasion (TGF-ß and vimentin) were significantly increased in BRCA1-deficient cells. CONCLUSIONS: Our results indicate that a deficiency of BRCA1 promotes the transactivation activity of HPV16E6E7 leading to increase of cell proliferation and invasion. SIGNIFICANCE: HPV infection appears to have the potential to enhance the aggressiveness of breast cancers, especially those deficient in BRCA1.
Assuntos
Neoplasias da Mama , Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Feminino , Humanos , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Ciclina B1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias da Mama/genética , Infecções por Papillomavirus/genética , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismoRESUMO
Human papillomavirus (HPV) is predominantly associated with HPV-related cancers, however, the precise mechanisms underlying the HPV-host epigenetic architectures in HPV carcinogenesis remain elusive. Here, we employed high-throughput chromosome conformation capture (Hi-C) to comprehensively map HPV16/18-host chromatin interactions. Our study identified the transcription factor Sp1 as a pivotal mediator in programming HPV-host interactions. By targeting Sp1, the active histone modifications (H3K27ac, H3K4me1, and H3K4me3) and the HPV-host chromatin interactions are reprogrammed, which leads to the downregulation of oncogenes located near the integration sites in both HPV (E6/E7) and the host genome (KLF5/MYC). Additionally, Sp1 inhibition led to the upregulation of immune checkpoint genes by reprogramming histone modifications in host cells. Notably, humanized patient-derived xenograft (PDX-HuHSC-NSG) models demonstrated that Sp1 inhibition promoted anti-PD-1 immunotherapy via remodeling the tumor immune microenvironment in cervical cancer. Moreover, single-cell transcriptomic analysis validated the enrichment of transcription factor Sp1 in epithelial cells of cervical cancer. In summary, our findings elucidate Sp1 as a key mediator involved in the programming and reprogramming of HPV-host epigenetic architecture. Inhibiting Sp1 with plicamycin may represent a promising therapeutic option for HPV-related carcinoma.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Cromatina/genética , Epigênese Genética , Papillomavirus Humano 16/metabolismo , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/metabolismo , Papillomavirus Humano , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/terapia , Fatores de Transcrição/genética , Microambiente Tumoral , Neoplasias do Colo do Útero/patologiaRESUMO
BACKGROUND: MAL (T-lymphocyte maturation-associated protein) is highly downregulated in most cancers, including cervical cancer (CaCx), attributable to promoter hypermethylation. Long noncoding RNA genes (lncGs) play pivotal roles in CaCx pathogenesis, by interacting with human papillomavirus (HPV)-encoded oncoproteins, and epigenetically regulating coding gene expression. Hence, we attempted to decipher the impact and underlying mechanisms of MAL downregulation in HPV16-related CaCx pathogenesis, by interrogating the interactive roles of MAL antisense lncRNA AC103563.8, E7 oncoprotein and PRC2 complex protein, EZH2. RESULTS: Employing strand-specific RNA-sequencing, we confirmed the downregulated expression of MAL in association with poor overall survival of CaCx patients bearing HPV16, along with its antisense long noncoding RNA (lncRNA) AC103563.8. The strength of positive correlation between MAL and AC103563.8 was significantly high among patients compared to normal individuals. While downregulated expression of MAL was significantly associated with poor overall survival of CaCx patients bearing HPV16, AC103563.8 did not reveal any such association. We confirmed the enrichment of chromatin suppressive mark, H3K27me3 at MAL promoter, using ChIP-qPCR in HPV16-positive SiHa cells. Subsequent E7 knockdown in such cells significantly increased MAL expression, concomitant with decreased EZH2 expression and H3K27me3 marks at MAL promoter. In silico analysis revealed that both E7 and EZH2 bear the potential of interacting with AC103563.8, at the same binding domain. RNA immunoprecipitation with anti-EZH2 and anti-E7 antibodies, respectively, and subsequent quantitative PCR analysis in E7-silenced and unperturbed SiHa cells confirmed the interaction of AC103563.8 with EZH2 and E7, respectively. Apparently, AC103563.8 seems to preclude EZH2 and bind with E7, failing to block EZH2 function in patients. Thereby, enhanced EZH2 expression in the presence of E7 could potentially inactivate the MAL promoter through H3K27me3 marks, corroborating our previous results of MAL expression downregulation in patients. CONCLUSION: AC103563.8-E7-EZH2 axis, therefore, appears to crucially regulate the expression of MAL, through chromatin inactivation in HPV16-CaCx pathogenesis, warranting therapeutic strategy development.
Assuntos
Proteínas Proteolipídicas Associadas a Linfócitos e Mielina , Proteínas Oncogênicas Virais , RNA Longo não Codificante , Neoplasias do Colo do Útero , Feminino , Humanos , Cromatina/metabolismo , Metilação de DNA , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histonas/metabolismo , Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias do Colo do Útero/patologia , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismoRESUMO
Human papillomaviruses (HPVs) are a major cause of cancer. While surgical intervention remains effective for a majority of HPV-caused cancers, the urgent need for medical treatments targeting HPV-infected cells persists. The pivotal early genes E6 and E7, which are under the control of the viral genome's long control region (LCR), play a crucial role in infection and HPV-induced oncogenesis, as well as immune evasion. In this study, proteomic analysis of endosomes uncovered the co-internalization of ErbB2 receptor tyrosine kinase, also called HER2/neu, with HPV16 particles from the plasma membrane. Although ErbB2 overexpression has been associated with cervical cancer, its influence on HPV infection stages was previously unknown. Therefore, we investigated the role of ErbB2 in HPV infection, focusing on HPV16. Through siRNA-mediated knockdown and pharmacological inhibition studies, we found that HPV16 entry is independent of ErbB2. Instead, our signal transduction and promoter assays unveiled a concentration- and activation-dependent regulatory role of ErbB2 on the HPV16 LCR by supporting viral promoter activity. We also found that ErbB2's nuclear localization signal was not essential for LCR activity, but rather the cellular ErbB2 protein level and activation status that were inhibited by tucatinib and CP-724714. These ErbB2-specific tyrosine kinase inhibitors as well as ErbB2 depletion significantly influenced the downstream Akt and ERK signaling pathways and LCR activity. Experiments encompassing low-risk HPV11 and high-risk HPV18 LCRs uncovered, beyond HPV16, the importance of ErbB2 in the general regulation of the HPV early promoter. Expanding our investigation to directly assess the impact of ErbB2 on viral gene expression, quantitative analysis of E6 and E7 transcript levels in HPV16 and HPV18 transformed cell lines unveiled a noteworthy decrease in oncogene expression following ErbB2 depletion, concomitant with the downregulation of Akt and ERK signaling pathways. In light of these findings, we propose that ErbB2 holds promise as potential target for treating HPV infections and HPV-associated malignancies by silencing viral gene expression.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Humanos , Linhagem Celular Tumoral , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Repressoras/metabolismoRESUMO
OBJECTIVE: To investigate the anti-cancer efficacy of ENB101-LNP, an ionizable lipid nanoparticles (LNPs) encapsulating siRNA against E6/E7 of HPV 16, in combination therapy with cisplatin in cervical cancer in vitro and in vivo. METHODS: CaSki cells were treated with ENB101-LNP, cisplatin, or combination. Cell viability assessed the cytotoxicity of the treatment. HPV16 E6/E7 gene knockdown was verified with RT-PCR both in vitro and in vivo. HLA class I and PD-L1 were checked by flow cytometry. A xenograft model was made using CaSki cells in BALB/c nude mice. To evaluate anticancer efficacy, mice were grouped. ENB101-LNP was given three times weekly for 3 weeks intravenously, and cisplatin was given once weekly intraperitoneally. Tumor growth was monitored. On day 25, mice were euthanized; tumors were collected, weighed, and imaged. Tumor samples were analyzed through histopathology, immunostaining, and western blot. RESULTS: ENB101-LNP and cisplatin synergistically inhibit CaSki cell growth. The combination reduces HPV 16 E6/E7 mRNA and boosts p21 mRNA, p53, p21, and HLA class I proteins. In mice, the treatment significantly blocked tumor growth and promoted apoptosis. Tumor inhibition rates were 29.7% (1 mpk ENB101-LNP), 29.6% (3 mpk), 34.0% (cisplatin), 47.0% (1 mpk ENB101-LNP-cisplatin), and 68.8% (3 mpk ENB101-LNP-cisplatin). RT-PCR confirmed up to 80% knockdown of HPV16 E6/E7 in the ENB101-LNP groups. Immunohistochemistry revealed increased p53, p21, and HLA-A expression with ENB101-LNP treatments, alone or combined. CONCLUSION: The combination of ENB101-LNP, which inhibits E6/E7 of HPV 16, with cisplatin, demonstrated significant anticancer activity in the xenograft mouse model of cervical cancer.
Assuntos
Lipossomos , Nanopartículas , Proteínas Oncogênicas Virais , Neoplasias do Colo do Útero , Feminino , Humanos , Animais , Camundongos , RNA Interferente Pequeno/genética , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Proteína Supressora de Tumor p53/genética , Camundongos Nus , Xenoenxertos , Linhagem Celular Tumoral , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , RNA Mensageiro/genéticaRESUMO
Human papillomavirus (HPV) E6 and E7 genes are biomarkers and drivers of the progression of cervical cancer (CxCa). The aim of this study was to investigate the relationship between HPV16 E6, E7 gene mutations and the occurrence and development of CxCa. Cervical exfoliated cells and clinical data of patients with cervical diseases were collected. Sample DNA was extracted, the E6 and E7 gene fragments were amplified by PCR, and the mutations were detected by Sanger sequencing and compared with standard sequences. Microarray was used to sequence the transcriptome of cells. Data of transcriptome analyzed and visualized using R software and its packages. Analysis of clinical characteristics demonstrated the association of HPV16 infection with CxCa (Pâ <â .05). Sanger sequencing results showed that the mutation sites of E6 gene included T178G/A, T350G, A131C, and T241G; among these, A131C and T241G were synonymous mutations. The mutation sites of E7 gene included A647G, T846C, G666A, T843C, and T760C, and all of them were synonymous mutations except A647G. There was no significant difference in the distribution of HPV16 E6, E7 mutations among CxCa, cervical intraepithelial neoplasia, and infection groups (Pâ >â .05). Compared with the non- CxCa group, gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes (DEGs) showed more significant enrichment of DEGs in the biological processes, pathways, and diseases closely related to cancer. Compared with the non-mutation group, the DEGs in the E6, E7 gene mutation group were significantly enriched in the events related to infection and immunity. To summarize, HPV16 may be associated with the occurrence and development of CxCa, but HPV16 E6 and E7 gene mutations have little effect on the occurrence and development of CxCa. Individual differences may have a greater effect on the progression of CxCa.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/complicações , Papillomavirus Humano 16/genética , Papillomavirus Humano , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , Transcriptoma , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , MutaçãoRESUMO
Worldwide prevalence of cervical cancer decreased significantly with the use of human papilloma virus (HPV)-targeted prophylactic vaccines. However, these multivalent antiviral vaccines are inert against established tumors, which leave patients with surgical ablative options possibly resulting in long-term reproductive complications and morbidity. In an attempt to bypass this unmet medical need, we designed a new E7 protein-based vaccine formulation using Accum™, a technology platform designed to promote endosome-to-cytosol escape as a means to enhance protein accumulation in target cells. Prophylactic vaccination of immunocompetent mice using the Accum-E7 vaccine (aE7) leads to complete protection from cervical cancer despite multiple challenges conducted with ascending C3.43 cellular doses (0.5-, 1.0-, and 2.0 × 106 cells). Moreover, the humoral response induced by aE7 was higher in magnitude compared with naked E7 protein vaccination and displayed potent inhibitory effects on C3.43 proliferation in vitro. When administered therapeutically to animals with pre-established C3.43 or Tal3 tumors, the vaccine-induced response synergized with multiple immune checkpoint blockers (anti-PD-1, anti-CTLA4, and anti-CD47) to effectively control tumor growth. Mechanistically, the observed therapeutic effect requires cross-presenting dendritic cells as well as CD8 T cells predominantly, with a non-negligible role played by both CD4+ and CD19+ lymphocytes. good laboratory practice (GLP) studies revealed that aE7 is immunogenic and well tolerated by immunocompetent mice with no observed adverse effects despite the use of a fourfold exceeding dose. In a nutshell, aE7 represents an ideal vaccine candidate for further clinical development as it uses a single engineered protein capable of exhibiting both prophylactic and therapeutic activity.
Assuntos
Vacinas Anticâncer , Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Vacinas contra Papillomavirus , Neoplasias do Colo do Útero , Feminino , Humanos , Animais , Camundongos , Neoplasias do Colo do Útero/patologia , Proteínas E7 de Papillomavirus/metabolismo , Linfócitos T CD8-Positivos , Vacinação , Camundongos Endogâmicos C57BL , Infecções por Papillomavirus/prevenção & controle , Proteínas Oncogênicas Virais/genéticaRESUMO
The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV16 upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the HPV16 E7 protein by degrading the components of the S-phase kinase-associated protein 1-CUL1-F-box ubiquitin ligase complex in HPV+ HNC cells. We found that MARCHF8 knockdown in HPV+ HNC cells drastically decreases the HPV16 E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the HPV16 E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases HPV16 E7 protein levels and suppresses tumor growth in vivo. Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the HPV16 E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.IMPORTANCESince human papillomavirus (HPV) oncoprotein E7 is essential for virus replication; HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the E3 ubiquitin ligase complex cullin 1 (CUL1) and ubiquitin-conjugating enzyme E2 L3 (UBE2L3) components play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8) induced by HPV16 E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV16 maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis.
Assuntos
Proteínas Culina , Neoplasias de Cabeça e Pescoço , Proteínas Oncogênicas Virais , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus , Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Humanos , Proteínas Culina/genética , Proteínas Culina/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Papillomavirus Humano , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
There are over 220 identified genotypes of Human papillomavirus (HPV), and the HPV genome encodes 3 major oncogenes, E5, E6, and E7. Conservation and divergence in protein sequence and function between low-risk versus high-risk oncogenic HPV genotypes has not been fully characterized. Here, we used modern computational and structural folding algorithms to perform a comparative analysis of HPV E5, E6, and E7 between multiple low risk and high risk genotypes. We first identified significantly greater sequence divergence in E5 between low- and high-risk genotypes compared to E6 and E7. Next, we used AlphaFold to model the structure of papillomavirus proteins and complexes with high confidence, including some with no established consensus structure. We observed that HPV E5, but not E6 or E7, had a dramatically different 3D structure between low-risk and high-risk genotypes. To our knowledge, this is the first comparative analysis of HPV proteins using Alphafold artificial intelligence (AI) system. The marked differences in E5 sequence and structure in high-risk HPVs may contribute in important and underappreciated ways to the development of HPV-associated cancers.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Papillomavirus Humano , Inteligência Artificial , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Papillomaviridae/genética , GenótipoRESUMO
Cervical cancer (CaCx) is the deadliest malignancy among women which is caused by human papillomavirus (HPV) and anthro-demographical/clinicopathological factors. HPV oncoproteins E6 and E7 target p53 and RB (retinoblastoma) protein degradation, Ataxia telangiectasia mutated (ATM), ATM-RAD3-related (ATR) inactivation and subsequent impairment of non-homologous end joining (NHEJ), homologous recombination, and base excision repair pathways. There is also an accumulation of genetic and epigenetic alterations in Tumor Growth Suppressors (TGS), oncogenes, and DNA repair genes leading to increased genome instability and CaCx development. These alterations might be responsible for differential clinical response to Cisplatin-based chemoradiotherapy (CRT) in patients. This review explores HPV-mediated DNA damage as a risk factor in CaCx development, the mechanistic role of genetic and epigenetic alterations in DNA repair genes and their association with CRT and outcome, It also explores new possibilities for the development of genetic and epigenetic-based biomarkers for diagnostic, prognostic, and molecular therapeutic interventions.