Laurencia johnstonii extract reverses early lesions in the K14E7HPV16 murine cervical carcinogenesis model.

Persistent infection with high-risk human papillomavirus (HR-HPV) is a well-established risk factor to the development of cervical intraepithelial neoplasia (CIN), a condition that can progress to cervical cancer (CC) a major health problem worldwide. Recently, there has been growing interest in exploring alternative therapies utilizing natural products, among which is the algae species Laurencia johnstonii Setchell & Gardner, 1924 (L. johnstonii), proposed for the management of precancerous lesions. The aim of this work was to determine the effect of an organic extract from L. johnstonii (ELj) in early cervical lesions (CIN 1). These CIN 1 lesions were generated in a murine model expressing the HR-HPV16 E7 oncoprotein (K14E7HPV transgenic mice) with a single exogenous hormonal stimulus using 17ß-estradiol. The histopathological studies, the determination of cell proliferation and of the apoptotic levels in cervical tissue, showed that, seven doses of ELj (30 mg/kg weight per day diluted in a DMSO-saline solution [1:7]) lead to recovery the architecture of cervical epithelium. Accordingly, in the transgenic mice it was observed a statistically significant decrease of the PCNA expression levels, a marker of cell proliferation, and a statistically significant increase in the apoptosis levels using Caspase 3 as a marker. In addition, we determined the expression levels of the tumor suppressor miR-218 and the oncomiRNA miR-21. Interestingly, our results may suggest that ELj treatment tended to restore the normal expression of both miRNAs as compared with controls being more evident in the non-transgenic induced mice. Differences of p < 0.05 were considered statistically significant through the whole study. Based on these results, we propose that the use of ELj could be an alternative for the treatment of cervical early lesions.

Dual regulation of energy metabolism by p53 in human cervix and breast cancer cells.

The role of p53 as modulator of OxPhos and glycolysis was analyzed in HeLa-L (cells containing negligible p53 protein levels) and HeLa-H (p53-overexpressing) human cervix cancer cells under normoxia and hypoxia. In normoxia, functional p53, mitochondrial enzyme contents, mitochondrial electrical potential (ΔΨm) and OxPhos flux increased in HeLa-H vs. HeLa-L cells; whereas their glycolytic enzyme contents and glycolysis flux were unchanged. OxPhos provided more than 70% of the cellular ATP and proliferation was abolished by anti-mitochondrial drugs in HeLa-H cells. In hypoxia, both cell proliferations were suppressed, but HeLa-H cells exhibited a significant decrease in OxPhos protein contents, ΔΨm and OxPhos flux. Although glycolytic function was also diminished vs. HeLa-L cells in hypoxia, glycolysis provided more than 60% of cellular ATP in HeLa-H cells. The energy metabolism phenotype of HeLa-H cells was reverted to that of HeLa-L cells by incubating with pifithrin-α, a p53-inhibitor. In normoxia, the energy metabolism phenotype of breast cancer MCF-7 cells was similar to that of HeLa-H cells, whereas p53shRNAMCF-7 cells resembled the HeLa-L cell phenotype. In hypoxia, autophagy proteins and lysosomes contents increased 2-5 times in HeLa-H cells suggesting mitophagy activation. These results indicated that under normoxia p53 up-regulated OxPhos without affecting glycolysis, whereas under hypoxia, p53 down-regulated both OxPhos (severely) and glycolysis (weakly). These p53 effects appeared mediated by the formation of p53-HIF-1α complexes. Therefore, p53 exerts a dual and contrasting regulatory role on cancer energy metabolism, depending on the O2level.

The HPV16 E6 oncoprotein and UVB irradiation inhibit the tumor suppressor TGFß pathway in the epidermis of the K14E6 transgenic mouse.

High-risk human papillomaviruses (HR-HPVs) are the causative agents of cervical cancer, and they are also associated with a subset of head and neck squamous cell carcinomas. In addition, HPVs have also been postulated in the development of non-melanoma skin cancers (NMSC). In these cancers, the oncogene E6 is best known for its ability to inactivate the tumor suppressor p53 protein. Interestingly, in transgenic mice for HPV16 E6 (K14E6), it was reported that E6 alone induced epithelial hyperplasia and delay in differentiation in skin epidermis independently of p53 inactivation. Transforming growth factor ß (TGFß) is an important regulator of cell growth/differentiation and apoptosis, and this pathway is often lost during tumorigenesis. Ultraviolet radiation B (UVB) exposure activates diverse cellular responses, including DNA damage and apoptosis. In this study, we investigated whether the E6 oncogene alone or in combination with UVB dysregulate some components of the TGFß pathway in the epidermis of K14E6 mice. We used 8-day-old K14E6 and non-transgenic mice irradiated and unirradiated with a single dose of UVB. We found that the E6 oncogene and UVB irradiation impair the TGFß pathway in epidermis of K14E6 mice by downregulation of the TGFß type II receptor (TßRII). This loss of TßRII prevents downstream activation of Smad2 and target genes as p15, an important regulator of cell cycle progression. In summary, the TGFß signalling in cells of the epidermis is downregulated in our mouse model by both the E6 oncoprotein and the UVB irradiation.

Human papillomavirus type 16 E7 oncoprotein upregulates the retinoic acid receptor-beta expression in cervical cancer cell lines and K14E7 transgenic mice.

Persistent infection with high-risk human papillomaviruses is the main etiological factor in cervical cancer (CC). The human papillomavirus type 16 (HPV16) E7 oncoprotein alters several cellular processes, regulating the expression of many genes in order to avoid cell cycle control. Retinoic acid receptor beta (RARB) blocks cell growth, inducing differentiation and apoptosis. This tumor suppressor gene is gradually silenced in late passages of foreskin keratinocytes immortalized with HPV16 and in various tumors, including CC, mainly by epigenetic modifications. We investigated the effect of E7 oncoprotein on RARB gene expression. We found that HPV16 E7 increases RARB mRNA and RAR-beta protein expression both in vitro and in the cervix of young K14E7 transgenic mice. In E7-expressing cells, RARB overexpression is further increased in the presence of the tumor suppressor p53 (TP53) R273C mutant. This effect does not change when either C33-A or E7-expressing C33-A cell line is treated with Trichostatin A, suggesting that E7 enhances RARB expression independently of histone deacetylases inhibition. These findings indicate that RARB overexpression is part of the early molecular events induced by the E7 oncoprotein.

Global expression profiling of CD10 + /CD19 + pre-B lymphoblasts from Hispanic B-ALL patients correlates with comparative TARGET database analysis.

Mexico City has one of the highest incidences of acute lymphoblastic leukemia (ALL) globally, with patients showing low survival, and high relapse rates. To gain more insight into the molecular features of B-ALL in Mexican children, we isolated CD10 + /CD19 + precursor B lymphoblasts from four bone marrow and nine peripheral blood samples of B-ALL patients using a fluorescence-activated cell sorting protocol. The global gene expression profile (BM vs PB) revealed 136 differentially expressed genes; 62 were upregulated (45.6%) and 74 were downregulated (54.4%). Pearson's correlation coefficient was calculated to determine the similarity between pre-B lymphoblast populations. We selected 26 highly significant genes and validated 21 by RT-qPCR (CNN3, STON2, CALN1, RUNX2, GADD45A, CDC45, CDC20, PLK1, AIDA, HCK, LY86, GPR65, PIK3CG, LILRB2, IL7R, TCL1A, DOCK1, HIST1H3G, PTPN14, CD72, and NT5E). The gene set enrichment analysis of the total expression matrix and the ingenuity pathway analysis of the 136 differentially expressed genes showed that the cell cycle was altered in the bone marrow with four overexpressed genes (PLK1, CDC20, CDC45, and GADD45A) and a low expression of IL7R and PIK3CG, which are involved in B cell differentiation. A comparative bioinformatics analysis of 15 bone marrow and 10 peripheral blood samples from Hispanic B-ALL patients collected by the TARGET program, corroborated the genes observed, except for PIK3CG. We conclude the Mexican and the Hispanic B-ALL patients studied present common driver alterations and histotype-specific mutations that could facilitate risk stratification and diagnostic accuracy and serve as potential therapeutic targets.

Expression of miR-34a and miR-15b during the progression of cervical cancer in a murine model expressing the HPV16 E7 oncoprotein.

The high-risk human papillomavirus (HR-HPV) E7 oncoprotein appears to be a major determinant for cell immortalization and transformation altering critical processes such as cell proliferation, apoptosis, and immune response. This oncoprotein plays an essential role in cervical carcinogenesis, but other cofactors such as long-term use of hormonal contraceptives are necessary to modulate the risk of cervical cancer (CC). The role of HR-HPVs in the alteration of microRNA (miRNA) levels in persistent viral infections currently remains unclear. The aim of this study was to evaluate the miR-34a and miR-15b expression levels in the murine HPV16K14E7 (K14E7) transgenic model after chronic estrogen (E2) treatment and their involvement in CC. Interestingly, results showed that, although miR-34a expression is elevated by the HPVE7 oncogene, this expression was downregulated in the presence of both the E7 oncoprotein and chronic E2 in cervical carcinoma. On the other hand, miR-15b expression was upregulated along cervical carcinogenesis mainly by the effect of E2. These different changes in the expression levels of miR-34a and miR-15b along cervical carcinogenesis conduced to low apoptosis levels, high cell proliferation and finally, to cancerous cervical tissue development. In this work, we also determined the relative mRNA expression of Cyclin E2 (Ccne2), Cyclin A2 (Ccna2), and B cell lymphoma 2 (Bcl-2) (target genes of miR-34a and miR-15b); Sirtuin 1 (Sirt1), Cmyc, and Bax (miR-34a target genes); and p21/WAF1 (mir15b target gene) and the H-ras oncogene. Given the modifications in the expression levels of miR-34a and miR-15b during the development of cervical cancer, it will be useful to carry out further investigation to confirm them as molecular biomarkers of cancer.

Vitamin A deficiency in K14E7HPV expressing transgenic mice facilitates the formation of malignant cervical lesions.

Infection with high-risk human papillomavirus (HR-HPV) is the main cause of cervical cancer (CC), but viral infection alone does not guarantee the development of this malignancy. Indeed, deficiencies of dietary micronutrients could favor cervical cancer development in individuals that harbor HR-HPV infections. The status of retinoid levels, natural and synthetic derivatives of vitamin A, is important in maintaining cellular differentiation of the cervical epithelium. Moreover, many studies show a link between deficient intake of retinoids or alteration of the retinoid receptors and CC development. In spite of this, the effect of vitamin A deficiency (VAD) in presence of HR-HPV oncoproteins on cervical carcinogenesis in vivo has not been reported. Transgenic mice expressing E6 or E7 oncoproteins (K14E6 or K14E7 mice, respectively) were used to evaluate the possible role of VAD in the development of malignant cervical lesions. The survival of the mice in VAD condition was studied, and histopathological analysis and immunohistochemical detection of molecular cancer markers such as the tumor suppressor retinoic acid receptor beta (RARß), proliferating cell nuclear antigen (PCNA), cleaved caspase 3, and the tumor suppressor protein p16INK4A (inhibitor of CDK4) were performed. Our results show that K14E6/VAD mice showed moderate cervical dysplasia; notably, K14E7/VAD mice developed severe cervical dysplasia and cervical in situ carcinoma at an early age. VAD synergizes with HPV16E7 oncoprotein expression favoring cervical carcinogenesis in vivo.

Circulating miR-15b, miR-34a and miR-218 as promising novel early low-invasive biomarkers of cervical carcinogenesis.

Circulating biological markers, such as miRNAs, hold the greatest possibilities to complement tissue biopsy and clinical diagnostic tests. The objective of this study was to evaluate the relative abundance of three circulating miRNAs in serum from 17 HPV16-positive patients with early cervical lesions known as Low-Grade Squamous Intraepithelial Lesions (LSILs). The expression of circulating microRNAs miR-15b, miR-34a and miR-218 in patients with LSILs was compared to 23 HPV-negative individuals showing normal cervical epithelium (healthy women) and 23 Squamous Cell Carcinoma (SCC) samples. The expression levels of miR-15b remained unchanged while those of miRNAs 34a and 218 were relatively high in serum obtained from LSIL patients in comparison with healthy women (results were statistically significant with a p of < 0.01 or < 0.001). According to previous findings, miR-15b was overexpressed and miRNAs 34a and 218 were underexpressed in serum from SCC patients. Additionally, the mRNA expression levels of some selected gene targets were determined [Cyclin D1 (CCND1), Cyclin E1 (CCNE1), B-cell lymphoma 2 (Bcl-2) and MutS homolog 2 (MSH-2)]. All serum results correlated with tissue samples from the same patients. We propose that circulating microRNAs can be valuable as molecular markers for the early follow-up of cervical carcinogenesis risk.

Early synergistic interactions between the HPV16­E7 oncoprotein and 17ß-oestradiol for repressing the expression of Granzyme B in a cervical cancer model.

Although high-risk human papillomavirus (HR­HPV) infection has a prominent role in the aetiology of cervical cancer (CC), sex steroid hormones may also be involved in this process; however, the cooperation between oestrogen and HR­HPV in the early stages of cervical carcinogenesis is poorly understood. Since 17ß-oestradiol (E2) and the HPV type 16­E7 oncoprotein induce CC in transgenic mice, a microarray analysis was performed in the present study to generate global gene expression profiles from 2­month­old FVB (non­transgenic) and K14E7 (transgenic) mice who were left untreated or were treated for 1 month with E2. Upregulation of cancer-related genes that have not been previously reported in the context of CC, including glycerophosphodiester phosphodiesterase domain containing 3, interleukin 1 receptor type II, natriuretic peptide type C, MGAT4 family member C, lecithin-retinol acyltransferase (phosphatidylcholine-retinol-O-acyltransferase) and glucoside xylosyltransferase 2, was observed. Notably, upregulation of the serine (or cysteine) peptidase inhibitor clade B member 9 gene and downregulation of the Granzyme gene family were observed; the repression of the Granzyme B pathway may be a novel mechanism of immune evasion by cancer cells. The present results provide the basis for further studies on early biomarkers of CC risk and synergistic interactions between HR­HPV and oestrogen.

HPV16-E6 Oncoprotein Activates TGF-ß and Wnt/ß-Catenin Pathways in the Epithelium-Mesenchymal Transition of Cataracts in a Transgenic Mouse Model.

OBJECTIVE: This work aimed to determine if cataractous changes associated with EMT occurring in the K14E6 mice lenses are associated with TGF-ß and Wnt/ß-catenin signaling activation. MATERIALS AND METHODS: Cataracts of K14E6 mice were analysed histologically; and components of TGF-ß and Wnt/ß-catenin signaling were evaluated by Western blot, RT-qPCR, in situ RT-PCR, IHC, or IF technics. Metalloproteinases involved in EMT were also assayed using zymography. The endogenous stabilisation of Smad7 protein was also assessed using an HDAC inhibitor. RESULTS: The K14E6 mice, which displayed binocular cataracts in 100% of the animals, exhibited loss of tissue organisation, cortical liquefaction, and an increase in the number of hyperproliferative-nucleated cells with mesenchymal-like characteristics in the lenses. Changes in lenses' cell morphology were due to actin filaments reorganisation, activation of TGF-ß and Wnt/ß-catenin pathways, and the accumulation of MTA1 protein. Finally, the stabilisation of Smad7 protein diminishes cell proliferation, as well as MTA1 protein levels. CONCLUSION: The HPV16-E6 oncoprotein induces EMT in transgenic mice cataracts. The molecular mechanism may involve TGF-ß and Wnt/ß-catenin pathways, suggesting that the K14E6 transgenic mouse could be a useful model for the study or treatment of EMT-induced cataracts.

Resveratrol induces downregulation of DNA repair genes in MCF-7 human breast cancer cells.

To gain insights into the antitumor mechanisms of resveratrol (RES), we carried out a DNA microarray analysis in the breast cancer cell line MCF-7 to study the global gene expression profile induced by RES treatment. The mRNA expression level of 19 734 well-characterized human genes from MCF-7 cells was determined using Affymetrix microarrays under two different RES treatments: 150 µmol/l (IC(50)) and 250 µmol/l during 48 h. A total of 1211 genes were found to have altered mRNA expression levels of two-fold or more in the 150 µmol/l RES-treated group (518 upregulated and 693 downregulated genes). However, 2412 genes were found to have altered expression levels of two-fold or more in the 250 µmol/l RES-treated group (651 genes upregulated and 1761 downregulated). Under both conditions of RES treatment, several genes of mismatch repair, DNA replication, homologous recombination (HR), and cell cycle were strongly inhibited. Consistently, we found decreased protein levels of the MRN complex (MRE11-NBS1-RAD50), an important complex of the HR DNA repair pathway. The ability to inhibit the expression of DNA repair genes by RES could help to overcome drug resistance commonly shown by transformed cells and to provide a solid basis for carrying out clinical trials with RES, alone or in combination with other agents, to enhance treatment efficacy, reduce toxicity, and overcome chemoresistance. Remarkably, after RES treatment, we found a decrease in NBS1 and MRE11 protein levels, two major proteins involved in HR, which suggests that RES could be used to sensitize cancer cells to cell death in combination with anticancer drugs.

Estrogens and human papilloma virus oncogenes regulate human ether-à-go-go-1 potassium channel expression.

Ether-à-go-go-1 (Eag1) potassium channels are potential tools for detection and therapy of numerous cancers. Here, we show human Eag1 (hEag1) regulation by cancer-associated factors. We studied hEag1 gene expression and its regulation by estradiol, antiestrogens, and human papillomavirus (HPV) oncogenes (E6/E7). Primary cultures from normal placentas and cervical cancer tissues; tumor cell lines from cervix, choriocarcinoma, keratinocytes, and lung; and normal cell lines from vascular endothelium, keratinocytes, and lung were used. Reverse transcription-PCR (RT-PCR) experiments and Southern blot analysis showed Eag1 expression in all of the cancer cell types, normal trophoblasts, and vascular endothelium, in contrast to normal keratinocytes and lung cells. Estradiol and antiestrogens regulated Eag1 in a cell type-dependent manner. Real-time RT-PCR experiments in HeLa cells showed that Eag1 estrogenic regulation was strongly associated with the expression of estrogen receptor-alpha. Eag1 protein was detected by monoclonal antibodies in normal placenta and placental blood vessels. Patch-clamp recordings in normal trophoblasts treated with estradiol exhibited potassium currents resembling Eag1 channel activity. Eag1 gene expression in keratinocytes depended either on cellular immortalization or the presence of HPV oncogenes. Eag1 protein was found in keratinocytes transfected with E6/E7 HPV oncogenes. Cell proliferation of E6/E7 keratinocytes was decreased by Eag1 antibodies inhibiting channel activity and by the nonspecific Eag1 inhibitors imipramine and astemizole; the latter also increased apoptosis. Our results propose novel oncogenic mechanisms of estrogen/antiestrogen use and HPV infection. We also suggest Eag1 as an early indicator of cell proliferation leading to malignancies and a therapeutic target at early stages of cellular hyperproliferation.

Deficiencia de retinoides y consumo de estrógenos como cofactores de riesgo de cáncer cervical / Retinoid deficiency and estrogens as risk cofactors in cervical cancer

La infección persistente por el virus del papiloma humano de alto riesgo (HR-HPV) está relacionada con la aparición de cáncer cervical (CC), una de las principales causas de mortalidad por cáncer en todo el mundo. La infección se produce en la zona de transformación, la región más sensible del cérvix a estrógenos y retinoides. El CC afecta a un bajo porcentaje de mujeres infectadas por HR-HPV y tarda en desarrollarse hasta décadas después de la infección, lo que sugiere que el HR-HPV es necesario pero no suficiente para causar CC. Otros factores son necesarios para la progresión desde la infección por HR-HPV hasta el cáncer, como por ejemplo: uso de anticonceptivos orales por largos períodos, fumar, partos múltiples, falta de micronutrientes, particularmente una dieta baja en retinoides, los cuales alteran la diferenciación epitelial, el crecimiento celular y la apoptosis de las células malignas. La detección precoz del HR-HPV y el manejo de lesiones precancerosas, aunado a un conocimiento detallado de factores de riesgo adicionales, puede ser una estrategia para prevenir esta enfermedad. La presente revisión se enfoca en explicar el efecto de los estrógenos, la deficiencia de retinoides y el HR-HPV en la aparición del CC. Dichos cofactores pueden actuar en conjunto para inducir transformación neoplásica en el epitelio escamoso del cérvix, promoviendo un segundo evento genético o epigenético que lleve a la aparición del CC.

ersistent infection with high-risk human papillomaviruses (HR-HPVs) is involved in cervical cancer (CC),a major cause of cancer mortality worldwide. Infection occurs primarily at the transformation zone (TZ),the most estrogen- and retinoid-sensitive region of the cervix. Development of CC affects a small per-centage of HR-HPV-infected women and often takes decades after infection, suggesting that HR-HPVis a necessary but not sufficient cause of CC. Thus, other cofactors are necessary for progression fromcervical HR-HPV infection to cancer such as long-term use of hormonal contraceptives, multiparity, smo-king, as well as micronutrient depletion and in particular retinoid deficiency, which alters epithelial diffe-rentiation, cellular growth and apoptosis of malignant cells. Therefore, early detection of HR-HPV andmanagement of precancerous lesions together with a profound understanding of additional risk factorscould be a strategy to avoid this disease. In this review we focus on the synergic effect of estrogens,retinoid deficiency and HR-HPVs in the development of CC. These risk factors may act in concert to indu-ce neoplastic transformation in the squamous epithelium of the cervix, setting the stage for secondarygenetic or epigenetic events leading to cervical cancer.