Tumor Suppressor pRb Research Articles

The HPV101 E7 protein shares host cellular targets and biological activities with high-risk HPV16 E7

Human papillomaviruses (HPVs) are a diverse family of viruses with over 450 members that have been identified and fully sequenced. They are classified into five phylogenetic genera: alpha, beta, gamma, mu, and nu. The high-risk alpha HPVs, such as HPV16, have been studied the most extensively due to their medical significance as cancer-causing agents. However, while nearly 70% of all HPVs are members of the gamma genus, they are almost entirely unstudied. This is because gamma HPVs have been considered medically irrelevant commensals as most of them infect the skin and are not known to cause significant clinical lesions in immunocompetent individuals. Members of the gamma 6 HPVs, however, have been detected in the anogenital tract mucosa and HPV101 has been isolated from a premalignant cervical lesion. Moreover, gamma 6 HPVs have a unique genome structure. They lack E6 proteins but in place of E6, they encode unique, tiny hydrophobic proteins without any close viral or cellular homologs that have been termed E10. Here we report that HPV101 E7 shares biochemical activities with the high-risk alpha HPV16 E7, including the ability to target the pRB and PTPN tumor suppressor for degradation. This study underscores the importance of further characterizing HPV101 and other unstudied HPV species.

The anti-tumor efficacy of a recombinant oncolytic herpes simplex virus mediated CRISPR/Cas9 delivery targeting in HPV16-positive cervical cancer

Cervical cancer, often driven by high-risk human papillomavirus (HPV) infections such as HPV16 or HPV18, remains a leading cause of cancer-related deaths. HPV16, found in about 90% of cervical cancer patients, harbors key oncogenic related genes (E6, E7, E2, E5) and an upstream regulatory region (URR) that contribute to cancer progression. This study introduces a novel approach using a recombinant oncolytic herpes simplex virus type 1 (HSV-1) named SONC103, armed with a CRISPR/Cas9 gene editing system. The aim was to target and disrupt integrated HPV16 genes in cervical cancer cells. Results demonstrated SONC103's capability to specifically and effectively knock down HPV16 oncogenes, thereby reducing cell proliferation and promoting apoptosis. Analyses further revealed loss of HPV16 DNA probes in infected cells' chromosomes, significant regulation of cellular processes related to tumor apoptosis, and downregulation of E6/E7 oncoproteins while increasing tumor suppressor proteins P53 and pRB. Notably, SONC103 exhibited substantial inhibition of tumor growth in a murine xenograft cervical cancer model. This study showcases the potential of the recombinant oncolytic HSV-1 virus (SONC103) in combating HPV16-positive cervical cancer by targeting oncogenes and facilitating oncolysis.

The N-Terminal Region of the Transcription Factor E2F1 Contains a Novel Transactivation Domain and Recruits General Transcription Factor GTF2H2.

The transcription factor E2F1 is the principal target of the tumor suppressor pRB. E2F1 promotes cell proliferation by activating growth-promoting genes upon growth stimulation. In contrast, E2F1 contributes to tumor suppression by activating tumor suppressor genes, such as ARF, upon loss of pRB function, a major oncogenic change. The transactivation domain of E2F1 has previously been mapped to the C-terminal region. We show here that the N-terminal region of E2F1 is critical for the activation of tumor suppressor genes. Deletion of the N-terminal region dramatically compromised E2F1 activation of tumor suppressor genes. The N-terminal region showed transactivation ability when fused to the DNA-binding domain of GAL4. A search for novel interacting factors with the N-terminal region, using a yeast two-hybrid system, identified the general transcription factor GTF2H2. Overexpression of GTF2H2 enhanced E2F1 activation of tumor suppressor genes and induction of cell death. Conversely, the knockdown of GTF2H2 compromised both. E2F1 binding enhanced the binding of GTF2H2 to target promoters depending on the integrity of the N-terminal region. Taken together, these results suggest that the N-terminal region of E2F1 contains a novel transactivation domain that mediates the activation of tumor suppressor genes, at least in part, by recruiting GTF2H2.

Comparative Molecular Docking of Apigenin and Luteolin versus Conventional Ligands for TP-53, pRb, APOBEC3H, and HPV-16 E6: Potential Clinical Applications in Preventing Gynecological Malignancies.

This study presents a comparative analysis of molecular docking data, focusing on the binding interactions of the natural compounds apigenin and luteolin with the proteins TP-53, pRb, and APOBEC, in comparison to conventional pharmacological ligands. Advanced bioinformatics techniques were employed to evaluate and contrast binding energies, showing that apigenin and luteolin demonstrate significantly higher affinities for TP-53, pRb, and APOBEC, with binding energies of -6.9 kcal/mol and -6.6 kcal/mol, respectively. These values suggest strong potential for therapeutic intervention against HPV-16. Conventional ligands, by comparison, exhibited lower affinities, with energies ranging from -4.5 to -5.5 kcal/mol. Additionally, protein-protein docking simulations were performed to assess the interaction between HPV-16 E6 oncoprotein and tumor suppressors TP-53 and pRb, which revealed high binding energies around -976.7 kcal/mol, indicative of their complex interaction. A conversion formula was applied to translate these protein-protein interaction energies to a comparable scale for non-protein interactions, further underscoring the superior binding potential of apigenin and luteolin. These findings highlight the therapeutic promise of these natural compounds in preventing HPV-16-induced oncogenesis, warranting further experimental validation for clinical applications.

Insulin and IGF-1 extend the lifespan of Caenorhabditis elegans by inhibiting insulin/insulin-like signaling and mTOR signaling pathways: C. elegans - Focused cancer research

The mutations in Caenorhabditis elegans (C. elegans) that extend lifespan slow down aging by interfering with several signaling pathways, including the insulin/IGF-1 signaling (IIS) pathway, AMP-activated protein kinase (AMPK), and mechanistic target of rapamycin (mTOR). The tumor suppressor pRb (retinoblastoma protein) is believed to be involved in almost all human cancers. Lin-35, the C. elegans orthologue of the tumor suppressor pRb, was included in the study to explore the effects of insulin and IGF-1 because it has been linked to cancer-related pRb function in mammals and exhibits a tumor suppressor effect by inhibiting mTOR or IIS signaling. According to our results, IGF-1 or insulin increased the lifespan of lin-35 worms compared to N2 worms by increasing fertilization efficiency, also causing a significant increase in body size. It was concluded that the expression of daf-2 and rsks-1 decreased after insulin or IGF-1 administration, thus extending the lifespan of C. elegans lin-35 worms through both IIS and mTOR-dependent mechanisms. This suggests that it was mediated by the combined effect of the TOR and IIS pathways. These results, especially obtained in cancer-associated mutant lin-35 worms, will be useful in elucidating the C. elegans cancer model in the future.

Interaction of pRb and beta-catenin in cancer and normal tissue in the human prostate

Prostate cancer (PCa) is one of the most common oncological diseases, which goes through two stages in its development. The first stage, localized prostate cancer, can proceed indefinitely in a dormant form that does not require active medical intervention, or suddenly turn into an aggressive metastatic form with lethal outcome. The pathogenesis of the transition of the dormant form of PCa to the metastatic form remains not fully understood. The signaling pathways of the tumor suppressor pRb and the proto-oncogene β-catenin are probably the most involved in the pathogenesis of PCa but the role of their interaction in the pathogenesis of prostate cancer has not been studied. The publication on the pathogenesis of tumors in other tissues suggests that pRb may lose some properties of a tumor suppressor at the initial stage of PCa development due to its interaction with β-catenin that enables tumor cells to gain competitive advantages for reproduction. In this work, we have shown that the RB and β-catenin (CTNNB1) genes are well expressed in tumor and normal prostate tissue. Unlike β-catenin, pRb is not detected by immunoblotting in tumor and normal prostate tissue, but is easily determined in this way in extracts of control T98G cells. Co-immunoprecipitation with antibodies to pRb from extracts of tumor and normal prostate tissue makes it possible to detect this protein and β-catenin by subsequent immunoblotting, which indicates the physical interaction of these proteins in prostate tissue. On the other hand, immunoprecipitation of β-catenin with antibodies to its C-terminal fragment does not detect this protein in prostate extracts by subsequent immunoblotting using the same antibody. In contrast to prostate tissue, β-catenin is readily detected by immunoprecipitation combined with immunoblotting in T98G control cell extracts. The obtained data suggest that pRb and β-catenin physically interact with each other in cells of different tissue specificity. In T98G cells, this interaction probably occurs through the C-terminal fragment of β-catenin, but in prostate cells it occurs in a different way, since the C-fragment of β-catenin is shielded from such interaction, possibly due to its physical association with pRb.

Expanding Roles of the E2F-RB-p53 Pathway in Tumor Suppression.

The transcription factor E2F links the RB pathway to the p53 pathway upon loss of function of pRB, thereby playing a pivotal role in the suppression of tumorigenesis. E2F fulfills a major role in cell proliferation by controlling a variety of growth-associated genes. The activity of E2F is controlled by the tumor suppressor pRB, which binds to E2F and actively suppresses target gene expression, thereby restraining cell proliferation. Signaling pathways originating from growth stimulative and growth suppressive signals converge on pRB (the RB pathway) to regulate E2F activity. In most cancers, the function of pRB is compromised by oncogenic mutations, and E2F activity is enhanced, thereby facilitating cell proliferation to promote tumorigenesis. Upon such events, E2F activates the Arf tumor suppressor gene, leading to activation of the tumor suppressor p53 to protect cells from tumorigenesis. ARF inactivates MDM2, which facilitates degradation of p53 through proteasome by ubiquitination (the p53 pathway). P53 suppresses tumorigenesis by inducing cellular senescence or apoptosis. Hence, in almost all cancers, the p53 pathway is also disabled. Here we will introduce the canonical functions of the RB-E2F-p53 pathway first and then the non-classical functions of each component, which may be relevant to cancer biology.

Silencing E6/E7 Oncoproteins in SiHa Cells Treated with siRNAs and Oroxylum indicum Extracts Induced Apoptosis by Upregulating p53/pRb Pathways.

E6 and E7 human papillomavirus (HPV) oncoproteins play a significant role in the malignant transformation of infected cervical cancer cells via suppression of tumour suppressor pathways by targeting p53 and pRb, respectively. This study aimed to investigate the anticancer effects of Oroxylum indicum (OI) leaves' methanol extract on SiHa cervical cancer cells. Expression of apoptosis-related proteins (Bcl-2, caspase (cas)-3, and cas-9), viral oncoproteins (E6 and E7), and tumour suppressor proteins (p53 and pRb) were evaluated using western blot analysis before and after E6/E7 small interfering RNAs (siRNAs) transfection. In addition, the E6/E7 mRNA expression levels were assessed with real-time (RT)-PCR. The present study showed that the OI extract effectively hindered the proliferation of SiHa cells and instigated increments of cas-3 and cas-9 expressions but decreased the Bcl-2 expressions. The OI extract inhibited E6/E7 viral oncoproteins, leading to upregulation of p53 and pRb tumour suppressor genes in SiHa cells. Additionally, combinatorial treatment of OI extract and gossypin flavonoid induced restorations of p53 and pRb. Treatment with OI extract in siRNA-transfected cells also further suppressed E6/E7 expression levels and further upregulations of p53 and pRb proteins. In conclusion, OI extract treatment on siRNAs-transfected SiHa cells can additively and effectively block E6- and E7-dependent p53 and pRb degradations. All these data suggest that OI could be explored for its chemotherapeutic potential in cervical cancer cells with HPV-integrated genomes.

Investigating the Potential Role of IKZF3 in HPV-Associated Carcinogenesis

HPVs cause approximately 5% of all human cancers. They encode two viral transforming proteins, E6 and E7, that are consistently expressed in these cancers. The HPV E6 and E7 proteins contribute to carcinogenesis by binding and functionally compromising multiple cellular regulatory proteins, most prominently the p53 and retinoblastoma (pRB) tumor suppressors, respectively. To discover additional cellular regulatory circuits that are dysregulated by E6 and E7 expression, we performed RNA sequencing analyses of HPV16 E6 and E7 expressing primary human genital epithelial cells. These experiments revealed that the Ikaros Zinc Finger 3 (IKZF3) transcription factor is highly upregulated in HPV16 E6/E7 expressing keratinocytes as compared to control keratinocytes. IKZF3 plays a key role in lymphocyte development, and when over-expressed or mutated, contributes to hematopoietic malignancies. IKZF3 is expressed at a very low level in normal epithelial cells, but the expression is upregulated in solid tumors, including invasive breast, cervical, and lung cancers. IKZF3 expression in these tumors causes upregulation of lymphocyte-specific genes, conferring lymphocyte-like behavior referred to as "lymphocyte mimicry," which presumably enables cancer cell survival in the bloodstream, thus potentially contributing to cancer metastasis. There is an FDA-approved drug, lenalidomide, that targets IKZF3 for degradation. Hence, we set out to determine whether IKZF3 might contribute to cervical carcinogenesis. We treated HPV16-positive SiHa and CaSki cervical cancer cells with 10 uM of lenalidomide for 12 hours. Cells were treated with equal amounts of DMSO as a negative control. Then, IKZF3 and E7 mRNA expression were determined using qRT-PCR after 12 hours of treatment. Gene expression was analyzed relative to GAPDH, and the results were analyzed using 2-way ANOVAs with Dunnett's correction for multiple comparisons. Western Blot experiments were performed to examine IKZF3, pRB, E7, and p53 (to indirectly assess E6) protein expression levels after 12 hours of lenalidomide treatment. Actin protein was used as a loading control. Each of these experiments were performed 3 times, qRT-PCR experiments were each performed in triplicate, and the results were graphed using a scientific 2-D graphing and statistics software. Although Western Blot showed that IKZF3 protein levels were significantly decreased by lenalidomide treatment, there was no change in pRB, E7, and p53 protein expression upon IKZF3 depletion. Similarly, there was no decrease in E7 mRNA expression. We showed by western blotting that targeting IKZF3 for proteasome-mediated degradation with lenalidomide effectively decreased the steady-state levels of IKZF3. However, we did not consistently observe an associated decrease in HPV gene expression. We are currently analyzing the transcriptome changes that IKZF3 depletion causes in cervical carcinoma cells to determine whether this may affect anoikis resistance.

Effects of a Sertoli cell-specific knockout of Connexin43 on maturation and proliferation of postnatal Sertoli cells

Adult male Sertoli cell-specific Connexin43 knockout mice (SCCx43KO) exhibit higher Sertoli cell (SC) numbers per seminiferous tubule compared to their wild type (WT) littermates. Thus, deletion of this testicular gap junction protein seems to affect the proliferative potential and differentiation of “younger” SC. Although SC have so far mostly been characterised as postmitotic cells that cease to divide and become an adult, terminally differentiated cell population at around puberty, there is rising evidence that there exist exceptions from this for a very long time accepted paradigm. Aim of this study was to investigate postnatal SC development and to figure out underlying causes for observed higher SC numbers in adult KO mice. Therefore, the amount of SC mitotic figures was compared, resulting in slightly more and prolonged detection of SC mitotic figures in KO mice compared to WT. SC counting per tubular cross section revealed significantly different time curves, and comparing proliferation rates using Bromodesoxyuridine and Sox9 showed higher proliferation rates in 8-day old KO mice. SC proliferation was further investigated by Ki67 immunohistochemistry. SC in KO mice displayed a delayed initiation of cell-cycle-inhibitor p27Kip1 synthesis and prolonged synthesis of the phosphorylated tumour suppressor pRb and proliferation marker Ki67. Thus, the higher SC numbers in adult male SCCx43KO mice may arise due to two different reasons: Firstly, in prepubertal KO mice, the proliferation rate of SC was higher. Secondly, there were differences in their ability to cease proliferation as shown by the delayed initiation of p27Kip1 synthesis and the prolonged production of phosphorylated pRb and Ki67. Immunohistochemical results indicating a prolonged period of SC proliferation in SCCx43KO were confirmed by detection of proliferating SC in 17-days-old KO mice. In conclusion, deletion of the testicular gap junction protein Cx43 might prevent normal SC maturation and might even alter also the proliferation potential of adult SC.

Withania somnifera extract reduces gastric cancerous properties through inhibition of gankyrin in cellular milieu produced by Helicobacter pylori and Epstein Barr virus

Helicobacter pylori and Epstein Barr virus (EBV) are group1 carcinogens and their role in Gastric cancer (GC) is well established. Previously we have shown that H. pylori and EBV appears to support aggressive gastric oncogenesis through the upregulation of oncoprotein Gankyrin. Natural plant active molecules have the potential to interrupt oncogenesis. Herein, we investigated the potential of Withania somnifera root extract (WSE) as a possible chemotherapeutic agent against host oncoprotein Gankyrin whose expression was altered by H. pylori and EBV-associated modified cellular milieu. The results show that WSE does not have any inhibitory effect on H. pylori and EBV-associated gene transcripts except for the lmps (lmp1, lmp2a, and lmp2B). Moreover, the WSE exert their anticancer activity via host cellular response and decreased the expression of cell-migratory (mmp3 and mmp7); cell-cycle regulator (pcna); antiapoptotic gene (bcl2); increased the expression of the proapoptotic gene (apaf1 and bax); and tumor suppressor (p53, prb, and pten). Knockdown of Gankyrin followed by the treatment of WSE also decreases the expression of TNF-ɑ, Akt, and elevated the expression of NFkB, PARP, Casp3, and Casp9. WSE also reduces cell migration, and genomic instability and forced the cells to commit programmed cell death. Moreover, molecular simulation studies revealed that out of eight active compounds of WSE, only four compounds such as withaferin A (WFA), withanoside IV (WA4), withanolide B (WNB), and withanolide D (WND) showed direct stable interaction with Gankyrin. This article reports for the first time that treatment of WSE decreased the cancerous properties through host cellular response modulation in gastric epithelial cells coinfected with H. pylori and EBV. Communicated by Ramaswamy H. Sarma

PI3K/AKT/mTOR Signaling Pathway in HPV-Driven Head and Neck Carcinogenesis: Therapeutic Implications.

High-risk human papillomaviruses (HR-HPVs) are the causal agents of cervical, anogenital and a subset of head and neck carcinomas (HNCs). Indeed, oropharyngeal cancers are a type of HNC highly associated with HR-HPV infections and constitute a specific clinical entity. The oncogenic mechanism of HR-HPV involves E6/E7 oncoprotein overexpression for promoting cell immortalization and transformation, through the downregulation of p53 and pRB tumor suppressor proteins, among other cellular targets. Additionally, E6/E7 proteins are involved in promoting PI3K/AKT/mTOR signaling pathway alterations. In this review, we address the relationship between HR-HPV and PI3K/AKT/mTOR signaling pathway activation in HNC with an emphasis on its therapeutic importance.

The TFDP1 gene coding for DP1, the heterodimeric partner of the transcription factor E2F, is a target of deregulated E2F

The TFDP1 gene codes for the heterodimeric partner DP1 of the transcription factor E2F. E2F, principal target of the tumor suppressor pRB, plays central roles in cell proliferation by activating a group of growth-related genes. E2F also mediates tumor suppression by activating tumor suppressor genes such as ARF, an upstream activator of the tumor suppressor p53, when deregulated from pRB upon oncogenic changes. Among 8 E2F family members (E2F1∼E2F8), expression of activator E2Fs (E2F1∼E2F3a) is induced at the G1/S boundary of the cell cycle after growth stimulation by E2F itself. However, mechanisms regulating DP1 expression are not known.We show here that over-expression of E2F1 and forced inactivation of pRB, by adenovirus E1a, induced TFDP1 gene expression in human normal fibroblast HFFs, suggesting that the TFDP1 gene is a target of E2F. Serum stimulation of HFFs also induced TFDP1 gene expression, but with different kinetics from that of the CDC6 gene, a typical growth-related E2F target. Both over-expression of E2F1 and serum stimulation activated the TFDP1 promoter. We searched for E2F1-responsive regions by 5′ and 3’ deletion of the TFDP1 promoter and by introducing point mutations in putative E2F1-responsive elements. Promoter analysis identified several GC-rich elements, mutation of which reduced E2F1-responsiveness but not serum-responsiveness. ChIP assays showed that the GC-rich elements bound deregulated E2F1 but not physiological E2F1 induced by serum stimulation. These results suggest that the TFDP1 gene is a target of deregulated E2F. In addition, knockdown of DP1 expression by shRNA enhanced ARF gene expression, which is specifically induced by deregulated E2F activity, suggesting that activation of the TFDP1 gene by deregulated E2F may function as a failsafe feedback mechanism to suppress deregulated E2F and maintain normal cell growth in the event that DP1 expression is insufficient relative to that of its partner activator E2Fs.a maximum of 6 keywords: E2F, DP1, TFDP1 gene, pRB, gene expression.

Integrated approaches for the separation and purification of recombinant HPV16 E6 protein from Escherichia coli crude extracts

Human papillomavirus (HPV) is a sexually transmissible virus responsible for 5% of global human cancers and associated with 99% of cervical cancer cases. The oncogenic potential of high-risk HPVs is mainly related to the E6 and E7 oncoproteins, which are responsible, at least in part, for inactivating the p53 and pRb tumor suppressor proteins. Due to the critical role of the E6 protein in malignant tumorigenesis, it is widely recognized as a therapeutic target for anti-HPV drug development. Nevertheless, it is required to obtain large amounts of protein with high purity to perform biointeraction studies with the potential inhibitor drugs. In this work, recombinant dual-tagged E6 protein (His6-MBP-E6) was expressed from Escherichia coli (E. coli) cultures and successfully extracted by sonication/ice cycles. Affinity chromatography using MBPtrap columns allowed 85 ± 5% protein recovery with the elimination of major host heterologous proteins in a single fraction. Subsequently, a polishing step was studied by applying anionic exchange (QSepharose), size exclusion (Superdex), or immobilized-metal affinity chromatography (HisTrap). The combination of affinity chromatography with size exclusion or two affinity chromatography techniques allowed us to obtain 82 ± 2% and 94 ± 3%, of highly pure His6-MBP-E6, respectively. Also, the secondary structure of His6-MBP-E6 is preserved in both purification strategies, as appraised by circular dichroism and western-blot studies. Thermal shift assay confirmed the CD results and suggested potential additives for protein stabilization. Altogether, the reproducible strategies established for the purification of His6-MBP-E6 protein could be successfully applied to later perform biointeraction studies and structural characterization of protein–ligand complexes.

Deregulated E2F Activity as a Cancer-Cell Specific Therapeutic Tool.

The transcription factor E2F, the principal target of the tumor suppressor pRB, plays crucial roles in cell proliferation and tumor suppression. In almost all cancers, pRB function is disabled, and E2F activity is enhanced. To specifically target cancer cells, trials have been undertaken to suppress enhanced E2F activity to restrain cell proliferation or selectively kill cancer cells, utilizing enhanced E2F activity. However, these approaches may also impact normal growing cells, since growth stimulation also inactivates pRB and enhances E2F activity. E2F activated upon the loss of pRB control (deregulated E2F) activates tumor suppressor genes, which are not activated by E2F induced by growth stimulation, inducing cellular senescence or apoptosis to protect cells from tumorigenesis. Deregulated E2F activity is tolerated in cancer cells due to inactivation of the ARF-p53 pathway, thus representing a feature unique to cancer cells. Deregulated E2F activity, which activates tumor suppressor genes, is distinct from enhanced E2F activity, which activates growth-related genes, in that deregulated E2F activity does not depend on the heterodimeric partner DP. Indeed, the ARF promoter, which is specifically activated by deregulated E2F, showed higher cancer-cell specific activity, compared to the E2F1 promoter, which is also activated by E2F induced by growth stimulation. Thus, deregulated E2F activity is an attractive potential therapeutic tool to specifically target cancer cells.

Cytosolic DNA sensor IFI16 proteins: Potential molecular integrators of interactions among the aging hallmarks.

Cellular changes that are linked to aging in humans include genomic instability, telomere attrition, epigenetic alterations, mitochondrial dysfunction, cellular senescence, and altered intercellular communications. The extent of the changes in these aging hallmarks and their interactions with each other are part of the human aging. However, the molecular mechanisms through which the aging hallmarks interact with each other remain unclear. Studies have indicated a potential role for the type I interferon (IFN) and p53-inducible IFI16 proteins in interactions with the aging hallmarks. The IFI16 proteins are members of the PYHIN protein family. Proteins in the family share a DNA-binding domain (the HIN domain) and a protein-protein interaction pyrin domain (PYD). IFI16 proteins are needed for cytosolic DNA-induced activation of the cGAS-STING pathway for type I IFN (IFN-β) expression. The pathway plays an important role in aging-related inflammation (inflammaging). Further, increased levels of the IFI16 proteins potentiate the cell growth inhibitory functions of the p53 and pRb tumor suppressors proteins. Moreover, IFI16 proteins are needed for most aging hallmarks. Therefore, here we discuss how an improved understanding of the role of the IFI16 proteins in integration of the aging hallmarks has potential to improve the human health and lifespan.

CIGB-300 Peptide Targets the CK2 Phospho-Acceptor Domain on Human Papillomavirus E7 and Disrupts the Retinoblastoma (RB) Complex in Cervical Cancer Cells.

CIGB-300 is a clinical-grade anti-Protein Kinase CK2 peptide, binding both its substrate’s phospho-acceptor site and the CK2α catalytic subunit. The cyclic p15 inhibitory domain of CIGB-300 was initially selected in a phage display library screen for its ability to bind the CK2 phospho-acceptor domain ofHPV-16 E7. However, the actual role of this targeting in CIGB-300 antitumoral mechanism remains unexplored. Here, we investigated the physical interaction of CIGB-300 with HPV-E7 and its impact on CK2-mediated phosphorylation. Hence, we studied the relevance of targeting E7 phosphorylation for the cytotoxic effect induced by CIGB-300. Finally, co-immunoprecipitation experiments followed by western blotting were performed to study the impact of the peptide on the E7–pRB interaction. Interestingly, we found a clear binding of CIGB-300 to the N terminal region of E7 proteins of the HPV-16 type. Accordingly, the in vivo physical interaction of the peptide with HPV-16 E7 reduced CK2-mediated phosphorylation of E7, as well as its binding to the tumor suppressor pRB. However, the targeting of E7 phosphorylation by CIGB-300 seemed to be dispensable for the induction of cell death in HPV-18 cervical cancer-derived C4-1 cells. These findings unveil novel molecular clues to the means by which CIGB-300 triggers cell death in cervical cancer cells.

Role of Epstein-Barr Virus and Human Papilloma Virus in the Development of Oropharyngeal Cancer: A Literature Review.

The aim of this review was to describe the association and related mechanisms between HPV, EBV, and the development of oral and oropharyngeal cancer. A search for scientific evidence was carried out in electronic databases (MEDLINE/PubMed, SciELO). It was found that, among the carcinogenic mechanisms of HPV, E6 and E7 proteins are responsible for the malignization process, inhibiting tumor suppressors p53 and pRb. As to EBV, it was noted that its “hit and run” phenomenon manipulates the host epigenetic mechanism, triggering the tumor process without the virus being currently present; a “cellular reprogramming” is essentially generated, causing heritable changes in gene expression without DNA mutation. In conclusion, there is an association between oropharyngeal carcinogenesis and HPV and also between the former and EBV. Further studies are required to clarify the causal mechanisms and impact of both viruses on cancer development and to obtain biomarkers of greater specificity in the case of EBV.

Abstract 49: Integrative genomic analysis of human papillomavirus associated head and neck squamous cell carcinoma reveals selective pressure for somatic alteration of E6 and E7 targets

Abstract Purpose: Previous studies implicated viral oncoproteins E6 and E7, of which targets include but are not limited to tumor suppressors p53 and pRb, in human papillomavirus (HPV) associated tumorigenesis. However, HPV-associated tumors must accumulate additional genomic alterations as HPV oncoproteins alone are insufficient for tumor formation in vivo. Experimental Design: Targeted sequencing was conducted with a panel of over 800 cancer related genomic regions and genes in a novel cohort of 525 head and neck squamous cell carcinoma (HNSCC) patients, including 252 HPV(+) and 273 HPV(-) tumors. Variant calling and filtering, followed by analysis with computational tools such as MutSig for somatic mutations and DiNAMIC.Duo for recurrent copy number alterations (CNAs), defined the catalog of driver alterations that are specific to HPV(+) and HPV(-) tumors. Pathway analysis was then used to elucidate the functional role of somatic alterations in the context of previously described HNSCC perturbations and HPV pathophysiology. Results: Integrative genomic analysis in one of the largest HPV(+) HNSCC cohorts to date revealed differential patterns of somatic mutations and CNAs in E6/E7 targets between HPV(+) and HPV(-) tumors. Specifically, EP300, an activator of p53 and target of E6, and retinoblastoma (RB) family members RB1 (pRb) and RBL2 (p130), which are known targets of E7 mediated degradation, were selected as significantly mutated by MutSig in HPV(+) samples only (EP300 & RB1 P<0.001, RBL2 P=0.01), while RBL1 (p107) was significant for both HPV(+) and HPV(-) samples (P<0.001, P=0.02 respectively). Higher fractions of mutations in HPV(+) vs. HPV(-) tumors were observed for EP300 (13% vs 6%, Fisher’s exact test P=0.006), RB1 (7% vs 3%, P=0.07), RBL2 (3% vs 1%, P=0.09), and FOXM1 (4% vs 1%, P=0.02), another cell cycle pathway target of E7, but not for RBL1 (4% vs. 3%, P=1). Lower copy numbers were detected in HPV(+) tumors compared to HPV(-) for chromosome regions containing RB1 (P=0.03), RBL1 (P=0.08), RBL2 (P<0.001), and for another inhibitory target of E6, FADD (P<0.001), which is an activator of the proapoptotic protein caspase-8. Additionally, significant copy number gains were detected in HPV(+) vs. HPV(-) tumors within the genomic region containing E2F1 (P<0.001), the transcription factor whose inhibition by RB family proteins is abrogated in the presence of E7, thus leading to abnormal cell cycle progression and proliferation. Conclusions: Previous studies suggest HPV-associated tumors lack significant levels of somatic alterations in canonical tumor suppressor genes that are known targets of viral oncoproteins. Here, we show additional selective pressure exists for somatic alteration of E6/E7 target genes in at least a subset of HPV(+) HNSCCs, which further deregulate cell cycle and apoptosis pathways in these tumors. Citation Format: Jeremiah Holt, Heejoon Jo, Xiaobei Zhao, Hyo Young Choi, Vonn Walter, Paul Little, Benjamin Wahle, Angela Mazul, Jose P. Zevallos, David Neil Hayes. Integrative genomic analysis of human papillomavirus associated head and neck squamous cell carcinoma reveals selective pressure for somatic alteration of E6 and E7 targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 49.

AIB1 is a novel target of the high-risk HPV E6 protein and a biomarker of cervical cancer progression.

The high‐risk human papillomaviruses (HPV‐16, ‐18) are critical etiologic agents in human malignancy, most importantly in cervical cancer. These oncogenic viruses encode the E6 and E7 proteins that are uniformly retained and expressed in cervical cancers and required for maintenance of the tumorigenic phenotype. The E6 and E7 proteins were first identified as targeting the p53 and pRB tumor suppressor pathways, respectively, in host cells, thereby leading to disruption of cell cycle controls. In addition to p53 degradation, a number of other functions and critical targets for E6 have been described, including telomerase, Myc, PDZ‐containing proteins, Akt, Wnt, mTORC1, as well as others. In this study, we identified Amplified in Breast Cancer 1 (AIB1) as a new E6 target. We first found that E6 and hTERT altered similar profiling of gene expression in human foreskin keratinocytes (HFK), independent of telomerase activity. Importantly, AIB1 was a common transcriptional target of both E6 and hTERT. We then verified that high‐risk E6 but not low‐risk E6 expression led to increases in AIB1 transcript levels by real‐time RT‐PCR, suggesting that AIB1 upregulation may play an important role in cancer development. Western blots demonstrated that AIB1 expression increased in HPV‐16 E6 and E7 expressing (E6E7) immortalized foreskin and cervical keratinocytes, and in three of four common cervical cancer cell lines as well. Then, we evaluated the expression of AIB1 in human cervical lesions and invasive carcinoma using immunohistochemical staining. Strikingly, AIB1 showed positivity in the nucleus of cells in the immediate suprabasal epithelium, while nuclei of the basal epithelium were negative, as evident in the Cervical Intraepithelial Neoplasia 1 (CIN1) samples. As the pathological grading of cervical lesions increased from CIN1, CIN2, CIN3 carcinoma in situ and invasive carcinoma, AIB1 staining increased progressively, suggesting that AIB1 may serve as a novel histological biomarker for cervical cancer development. For cases of invasive cervical carcinoma, AIB1 staining was specific to cancerous lesions. Increased expression of AIB1 was also observed in transgenic mouse cervical neoplasia and cancer models induced by E6E7 and estrogen. Knockdown of AIB1 expression in E6E7 immortalized human cervical cells significantly abolished cell proliferation. Taken together, these data support AIB1 as a novel target of HPV E6 and a biomarker of cervical cancer progression.