Bioinformatics Analysis Reveals E6 and E7 of HPV 16 Regulate Metabolic Reprogramming in Cervical Cancer, Head and Neck Cancer, and Colorectal Cancer through the PHD2-VHL-CUL2-ELOC-HIF-1α Axis.

Human papillomavirus 16 (HPV 16) infection is associated with several types of cancer, such as head and neck, cervical, anal, and penile cancer. Its oncogenic potential is due to the ability of the E6 and E7 oncoproteins to promote alterations associated with cell transformation. HPV 16 E6 and E7 oncoproteins increase metabolic reprogramming, one of the hallmarks of cancer, by increasing the stability of hypoxia-induced factor 1 α (HIF-1α) and consequently increasing the expression levels of their target genes. In this report, by bioinformatic analysis, we show the possible effect of HPV 16 oncoproteins E6 and E7 on metabolic reprogramming in cancer through the E6-E7-PHD2-VHL-CUL2-ELOC-HIF-1α axis. We proposed that E6 and E7 interact with VHL, CUL2, and ELOC in forming the E3 ubiquitin ligase complex that ubiquitinates HIF-1α for degradation via the proteasome. Based on the information found in the databases, it is proposed that E6 interacts with VHL by blocking its interaction with HIF-1α. On the other hand, E7 interacts with CUL2 and ELOC, preventing their binding to VHL and RBX1, respectively. Consequently, HIF-1α is stabilized and binds with HIF-1ß to form the active HIF1 complex that binds to hypoxia response elements (HREs), allowing the expression of genes related to energy metabolism. In addition, we suggest an effect of E6 and E7 at the level of PHD2, VHL, CUL2, and ELOC gene expression. Here, we propose some miRNAs targeting PHD2, VHL, CUL2, and ELOC mRNAs. The effect of E6 and E7 may be the non-hydroxylation and non-ubiquitination of HIF-1α, which may regulate metabolic processes involved in metabolic reprogramming in cancer upon stabilization, non-degradation, and translocation to the nucleus.

miR-218-5p, miR-124-3p and miR-23b-3p act synergistically to modulate the expression of NACC1, proliferation, and apoptosis in C-33A and CaSki cells.

Background: In cervical cancer (CC), miR-218-5p, -124-3p, and -23b-3p act as tumor suppressors. These miRNAs have specific and common target genes that modulate apoptosis, proliferation, invasion, and migration; biological processes involved in cancer. Methods: miR-218-5p, -124-3p, and -23b-3p mimics were transfected into C-33A and CaSki cells, and RT-qPCR was used to quantify the level of each miRNA and NACC1. Proliferation was assessed by BrdU and apoptosis by Annexin V/PI. In the TCGA and The Human Protein Atlas databases, the level of NACC1 mRNA and protein (putative target of the three miRNAs) was analyzed in CC and normal tissue. The relationship of NACC1 with the overall survival in CC was analyzed in GEPIA2. NACC1 mRNA and protein levels were higher in CC tissues compared with cervical tissue without injury. Results: An increased expression of NACC1 was associated with lower overall survival in CC patients. The levels of miR-218-5p, -124-3p, and -23b-3p were lower, and NACC1 was higher in C-33A and CaSki cells compared to HaCaT cells. The increase of miR-218-5p, -124-3p, and -23b-3p induced a significant decrease in NACC1 mRNA. The transfection of the three miRNAs together caused more drastic changes in the level of NACC1, in the proliferation, and in the apoptosis with respect to the individual transfections of each miRNA. Conclusion: The results indicate that miR-218-5p, -124-3p, and -23b-3p act synergistically to decrease NACC1 expression and proliferation while promoting apoptosis in C-33A and CaSki cells. The levels of NACC1, miR-218-5p, -124-3p, and -23b-3p may be a potential prognostic indicator in CC.

Identification of Mir-182-3p/FLI-1 Axis as a Key Signaling in Immune Response in Cervical Cancer: A Comprehensive Bioinformatic Analysis.

miRNAs modulate gene expression and play critical functions as oncomiRs or tumor suppressors. The miR-182-3p is important in chemoresistance and cancer progression in breast, lung, osteosarcoma, and ovarian cancer. However, the role of miR-182-3p in cervical cancer (CC) has not been elucidated. AIM: To analyze the role of miR-182-3p in CC through a comprehensive bioinformatic analysis. METHODS: Gene Expression Omnibus (GEO) databases were used for the expression analysis. The mRNA targets of miR-182-3p were identified using miRDB, TargetScanHuman, and miRPathDB. The prediction of island CpG was performed using the MethPrimer program. The transcription factor binding sites in the FLI-1 promoter were identified using ConSite+, Alibaba2, and ALGGEN-PROMO. The protein-protein interaction (PPI) analysis was performed in STRING 11.5. RESULTS: miR-182-3p was significantly overexpressed in CC patients and has potential as a diagnostic. We identified 330 targets of miR-182-3p including FLI-1, which downregulates its expression in CC. Additionally, the aberrant methylation of the FLI-1 promoter and Ap2a transcription factor could be involved in downregulating FLI1 expression. Finally, we found that FLI-1 is a possible key gene in the immune response in CC. CONCLUSIONS: The miR-182-3p/FLI-1 axis plays a critical role in immune response in CC.

REST/NRSF Silencing Modifies Neuronal Gene Expression in siRNA-Treated HeLa Cells: A Preliminary Exploration in the Search for Neuronal Biomarkers of Cervical Cancer.

Background and Objectives: REST (RE1-silencing transcription factor) diminution is associated with transcriptional relaxation, neuropeptide overexpression, and phenotype redefinition in neuroendocrine cancers, but this effect has barely been studied in cervical cancer (CC). We previously reported reduced expressions of REST in samples with premalignant lesions and CC; however, the transcriptional consequences for neural genes associated with reduced REST expression in CC are unknown. Therefore, the objective of this work was to evaluate the expression of neuronal genes in cancerous cells with reduced expression levels of REST. Materials and Methods: Here, we monitored levels of REST by immunostaining along the premalignant lesions and in invasive cervical squamous cell carcinoma (SCC) and endocervical adenocarcinoma (ADC) in tissue samples from female patients from southern Mexico and the derivative cell lines SiHa and HeLa, respectively. Next, we selected REST target genes in silico and explored the effect of REST silencing by RT-PCR in siRNA-treated HeLa cells. Results: The results show a REST diminution in premalignant lesions, SCC, ADC, and cancerous cell lines. Further REST silencing in HeLa cells altered the expression of genes containing the RE1 (Restrictive Element 1) sequence, including CgA (chromogranin A), CHRNß2 (cholinergic receptor nicotinic ß 2 subunit), BDNF (brain-derived neurotrophic factor), CRF (corticotropin-releasing factor), and RASSF1A (Ras association domain family 1). Conclusions: This work provides preliminary evidence of the role of REST loss in the transcriptional regulation of its target genes in HeLa cells, which could have positive implications for the search for new biomarkers of cervical cancer.

Prevalence and Distribution of Human Papillomavirus Genotypes (1997-2019) and Their Association With Cervical Cancer and Precursor Lesions in Women From Southern Mexico.

BACKGROUND: Cervical cancer (CC) is the fourth most common malignancy of the female genital tract. Human Papillomavirus (HPV) is the main cause of precancerous lesions and CC cases worldwide. OBJECTIVE: We assessed the prevalence and distribution of HPV types and their association with precancerous lesions and CC. METHODS: HPV genotypes were detected by 3 methods depending on the year of in which the sample was analyzed: MY09/11 RFLPs (1997 to 2010), GP5+/6+ primer systems (2005 to 2010) and INNO-LiPA HPV Genotyping Extra (2010 to 2019) in cervical samples (No-IL: 4445; LSIL: 2464; HSILs: 151 and CC: 253) from women from southern Mexico. RESULTS: The overall HPV prevalence was 54.17%, and hpv-16 was the most common genotype. In single infection, the high-risk HPV genotypes (group 1) were associated with squamous intraepitelial lesions (LSIL: HPV-39 (OR = 10.58, 95% CI 4.09-27.36, P < .001); HSIL: HPV-31 (OR = 14.76, 95% CI 6.56-33.20, P < .001); and CC: HPV-16 (OR = 25.01, 95% CI 18.83-33.21, P < .001). In multiple infections, the HPV genotypes (HPV-16 and HPV-18) were also associated with a high risk of lesions [LSIL: HPV-18 (OR = 3.45; 95% CI 1.36-8.91; P = .009); HSIL: HPV-18 (OR = 5.12; 95% CI 1.21-21.68; P = .026); and CC: HPV-16 (OR = 3.03; 95% CI 1.72-5.32; P < .001)] compared to single infection. In the analysis adjusted for age, giving birth, and cigarette smoking, a significant increase in the risk of LSIL, HSIL, and CC was maintained. CONCLUSIONS: This study provides current data on the prevalence and distribution of HPV genotypes in women from southern Mexico, which could serve as a valuable reference to guide nationwide CC screening programs and provide scientific evidence that could be useful for vaccine development efforts. Likewise, it was identified that infection with carcinogenic HPV genotypes is an independent risk factor for LSIL, HSIL, and CC.

Bioactivity of a Linoleic Acid-Rich Fraction of Ricinus communis L. (Euphorbiaceae) Leaves against the Yellow Sugarcane Aphid, Sipha flava (Hemiptera: Aphididae).

ABSTRACT: The insecticidal activity of a Ricinus communis leaf hexane extract and its fractions against adult yellow sugarcane aphids (Sipha flava) was evaluated using a contact bioassay after fumigation. The n-hexane extract at 10,000 ppm achieved the highest mortality (80%); the positive control had 100% mortality and the negative control had only 4% mortality over the 72-h experiment time. Chemical fractionation of the hexane extract of R. communis leaves produced multiple fractions, and 10,000 ppm of the F4 fraction resulted in 92% aphid mortality at 72 h. Gas chromatography-mass spectrometry of the F4 fraction revealed linoleic acid as the major compound (84.5%). The R. communis n-hexane extract and linoleic acid could be used for integrated pest control as an ecologically friendly alternative to synthetic chemical insecticides.

Effect of HPV 16 E6 Oncoprotein Variants on the Alterations of the Proteome of C33A Cells.

BACKGROUND/AIM: The E6 genotypic variants of HPV 16 identified in lesions of women with cervical cancer (CC) in Southern of Mexico include the E-G350, AAa, AAc, E-C188/G350, and E-A176/G350, transcriptomic analysis cells transfected with those variants showed to induce differential expression of the host genes involved in the development of CC, the aim of this work was to understand how the over-expression of the E6 oncoprotein and its variants can induce molecular mechanisms that lead to more aggressive HPV 16 phenotypes in cervical cancer and which proteins could be associated with the process. MATERIALS AND METHODS: Total extracts from C33A, C33A mock, C33A AAa, C33A E-C188/G350, C33A E-A176/G350, and C33A E-prototype cells were analyzed using 2D electrophoresis, PDQuest software and mass spectrometry, validation of results was performed through qPCR. RESULTS: Statistically significant differential expression of 122 spots was detected, 12 of the identified proteins were associated with metabolism and metabolic programming. Out of these CCT8, ENO and ALDH1A were further validated. CONCLUSION: CCT8 and ALDH1A were found to be over-expressed in C33A AAa and C33A E-A176/G350, compared to the E prototype. Both proteins could be associated with a most aggressive phenotype due to their relationship with metabolism, protein folding and stemness, mechanisms associated to E6 that could be useful in the design of new therapies.

TOP2A/MCM2, p16INK4a, and cyclin E1 expression in liquid-based cytology: a biomarkers panel for progression risk of cervical premalignant lesions.

BACKGROUND: To improve the efficiency of early diagnosis systems for cervical cancer, the use of cellular and viral markers for identifying precancerous lesions with a greater probability to progress to cancer has been proposed. Several cellular proteins and markers of oxidative DNA damage have been suggested as possible biomarkers of cervical carcinogenesis; however, they have not been evaluated together. In this study, we analyzed the expression of the cellular markers p16INK4a, Ki-67, CyclinE1, TOP2A/MCM2, and telomerase, as well as the DNA oxidative damage markers ROS and 8-OHdG. The analyses were performed in liquid-based cervical cytology samples or biopsies with premalignant lesions or cervical cancer diagnosis, with the purpose of selecting a panel of biomarkers that allow the identification of precursor lesions with greater risk of progression to cervical cancer. METHODS: We analyzed 1485 liquid-based cytology samples, including 239 non-squamous intraepithelial lesions (NSIL), 901 low-grade squamous intraepithelial lesions (LSIL), 54 high-grade squamous intraepithelial lesions (HSIL), and 291 cervical cancers (CC). The biomarkers were analyzed by immunocytochemistry and Human Papilloma Virus (HPV) genotyping with the INNO-LiPA genotyping Extra kit. RESULTS: We found that all tested cellular biomarkers were overexpressed in samples with high risk-HPV infection, and the expression levels increased with the severity of the lesion. TOP2A/MCM2 was the best biomarker for discriminating between LSIL and HSIL, followed by p16INK4a and cyclinE1. Statistical analysis showed that TOP2A/MCM2 provided the largest explanation of HSIL and CC cases (93.8%), followed by p16INK4a (91%), cyclin E1 (91%), Ki-67 (89.3%), and telomerase (88.9%). CONCLUSIONS: We propose that the detection of TOP2A/MCM2, p16INK4a and cyclin E1 expression levels is useful as a panel of biomarkers that allow identification of cervical lesions with a higher risk for progression to CC with high sensitivity and precision; this can be done inexpensively, in a single and non-invasive liquid-based cytology sample.

Significant decrease of a master regulator of genes (REST/NRSF) in high-grade squamous intraepithelial lesion and cervical cancer.

BACKGROUND: The repressor element 1-silencing transcription factor (REST) is a regulator of gene expression, and the Ras association domain family member 1 A (RASSF1A) is an important tumor suppressor gene involved in cancer development. Although extensive characterization of the roles of REST and RASSF1A in cancer development have been reported in cellular models, the link between them and their possible role in the development of squamous intraepithelial lesions (SIL) and squamous cell carcinoma (SCC) of the cervix have not been explored. The aim of this study was to evaluate the expression of REST and RASSF1A in cervical cytological and histological samples from patients diagnosed with SIL or SCC and in CC-derived cell lines. METHODS: We analyzed the expression of REST and RASSF1A by immunocyto/histochemistry in cervical samples from patients (n = 271) and in cancer cell lines. Data analyses were performed using the Kruskal-Wallis test and generalized linear models. RESULTS: We identified binding sites for REST in RASSF1A and observed a significant reduction in REST and RASSF1A nuclear expression in samples from patients with high-grade SIL (HSIL) and SCC. For REST, we observed an average decrease of 334 and 423 r.u.d. for HSIL (n = 21) and SCC (n = 18) compared with non-LSIL (n = 72), whereas for RASSF1A, this decrease was 126 and 217 r.u.d., respectively (p < 0.001). CONCLUSION: Our results provide evidence of the altered expression of REST and RASSF1A in SIL and SCC, with a significant decrease in HSIL, SCC, and SCC-derived cell lines; findings that can contribute to the diagnosis, prognosis, and post-treatment follow-up of patients diagnosed with SIL or SCC.

Women with chronic follicular gastritis positive for Helicobacter pylori express lower levels of GKN1.

In women, serum levels of CTSB, GKN2, LIPF, LIPFG, AZGP1, TOP2A and PGA4 are proposed as predictive markers of gastric cancer. It is unknown whether GKN1 expression varies with the sex of patients with chronic gastritis or gastric cancer. We studied 36 patients with histopathological diagnosis of chronic gastritis from the state of Guerrero, Mexico. PCR was performed for H. pylori detection and GKN1 expression was determined by RT-qPCR and western blot. GKN1 mRNA expression was significantly lower in patients with chronic follicular gastritis than in those with chronic chemical gastritis (p = 0.00071). The mRNA and protein level of expression of GKN1 were significantly lower in women with chronic follicular gastritis than in men with the same condition (p = 0.0279 and p = 0.0014, respectively); the lowest levels of GKN1 were detected in women with H. pylori-positive follicular gastritis (p = 0.0175 and p = 0.0111, respectively). Through a bioinformatic analysis, estrogen response elements were identified in the GKN1 promoter.

Leptin induces cell migration and invasion in a FAK-Src-dependent manner in breast cancer cells.

Breast cancer is the most common invasive neoplasia, and the second leading cause of the cancer deaths in women worldwide. Mammary tumorigenesis is severely linked to obesity, one potential connection is leptin. Leptin is a hormone secreted by adipocytes, which contributes to the progression of breast cancer. Cell migration, metalloproteases secretion, and invasion are cellular processes associated with various stages of metastasis. These processes are regulated by the kinases FAK and Src. In this study, we utilized the breast cancer cell lines MCF7 and MDA-MB-231 to determine the effect of leptin on FAK and Src kinases activation, cell migration, metalloprotease secretion, and invasion. We found that leptin activates FAK and Src and induces the localization of FAK to the focal adhesions. Interestingly, leptin promotes the activation of FAK through a Src- and STAT3-dependent canonical pathway. Specific inhibitors of FAK, Src and STAT3 showed that the effect exerted by leptin in cell migration in breast cancer cells is dependent on these proteins. Moreover, we established that leptin promotes the secretion of the extracellular matrix remodelers, MMP-2 and MMP-9 and invasion in a FAK and Src-dependent manner. Our findings strongly suggest that leptin promotes the development of a more aggressive invasive phenotype in mammary cancer cells.

Extracellular-Signal Regulated Kinase: A Central Molecule Driving Epithelial-Mesenchymal Transition in Cancer.

Epithelial-mesenchymal transition (EMT) is a reversible cellular process, characterized by changes in gene expression and activation of proteins, favoring the trans-differentiation of the epithelial phenotype to a mesenchymal phenotype. This process increases cell migration and invasion of tumor cells, progression of the cell cycle, and resistance to apoptosis and chemotherapy, all of which support tumor progression. One of the signaling pathways involved in tumor progression is the MAPK pathway. Within this family, the ERK subfamily of proteins is known for its contributions to EMT. The ERK subfamily is divided into typical (ERK 1/2/5), and atypical (ERK 3/4/7/8) members. These kinases are overexpressed and hyperactive in various types of cancer. They regulate diverse cellular processes such as proliferation, migration, metastasis, resistance to chemotherapy, and EMT. In this context, in vitro and in vivo assays, as well as studies in human patients, have shown that ERK favors the expression, function, and subcellular relocalization of various proteins that regulate EMT, thus promoting tumor progression. In this review, we discuss the mechanistic roles of the ERK subfamily members in EMT and tumor progression in diverse biological systems.

Methylation of the L1 gene and integration of human papillomavirus 16 and 18 in cervical carcinoma and premalignant lesions.

High-risk human papillomavirus (HPV) is the primary cause of cervical carcinoma (CC). Viral integration into the host chromosomes is associated with neoplastic progression, and epigenetic changes may occur as a result. The objective of the present study was to analyze HPV L1 gene methylation and to compare the use of quantitative polymerase chain reaction (qPCR), in situ hybridization (ISH) and L1 methylation analysis as methods for detecting HPV integration. Cervical scrapes or biopsy samples positive for HPV 16 or 18, from 187 female patients with CC, squamous intraepithelial lesions (SILs) or no intraepithelial lesion (non-IL) were analyzed. Methylation of the L1 gene was determined using bisulfite modification followed by PCR, and HPV integration was subsequently analyzed. HPV 16 L1 gene methylation was revealed to increase with histological grade, with statistically significant differences observed as follows: Low-grade SIL vs. CC, P<0.0001 and non-IL vs. CC, P<0.0001. HPV 18 L1 gene methylation also increased according to histological grade, however, no statistically significant differences were observed. Methylation at CpG site 5608 of the HPV 16 L1 gene was associated with all grades of cervical lesions, whereas methylation at CpG site 5617 demonstrated the strongest association with CC (odds ratio, 42.5; 95% confidence interval, 4.7-1861; P<0.0001). The concordance rates between the various methods for the detection of the physical status of HPV 16 and HPV 18 were 96.1% for qPCR and ISH, 76.7% for qPCR and L1 gene methylation, and 84.8% for ISH and L1 gene methylation. In conclusion, methylation of the HPV 16 L1 gene increases significantly according to the grade of the cervical lesion, and methylation at CpG sites 5608 and 5617 of this gene may be used as prognostic biomarkers. ISH and L1 gene methylation have good concordance with qPCR with regards to the detection of HPV integration. Therefore, these are useful methods in determining the physical state of HPV.

Leptin induces partial epithelial-mesenchymal transition in a FAK-ERK dependent pathway in MCF10A mammary non-tumorigenic cells.

Epithelial-mesenchymal transition (EMT) is a biological process involved in different steps of tumor progression and metastasis of breast cancer cells. Epidemiological studies suggest a link between obesity and the progression of breast cancer. Leptin is an adipocyte-secreted hormone which can promote cell migration and invasion as part of EMT in breast cancer cells. We investigated the effect of leptin on expression of EMT markers in MCF10A cells, as well as, the role of FAK and ERK in this process. We found that leptin induces morphological changes from an epithelial phenotype towards a mesenchymal phenotype and promotes cell migration in MCF10A cells. Moreover, leptin induces an increase in vimentin expression, changes in the cellular localization of E-cadherin and increase in FAK and ERK phosphorylation. Furthermore, using FAK and ERK chemical inhibitors we show that leptin regulates EMT markers in a FAK and ERK dependent manner. In conclusion, leptin promotes vimentin expression and cell migration in a FAK and ERK dependent pathway in the non-tumorigenic epithelial cell line MCF10A.