Epigenetic and Transcriptomic Regulation Landscape in HPV+ Cancers: Biological and Clinical Implications.

Human Papillomavirus (HPV) is an oncogenic virus that causes the highest number of viral-associated cancer cases and deaths worldwide, with more than 690,000 new cases per year and 342,000 deaths only for cervical cancer (CC). Although the incidence and mortality rates for CC are declining in countries where screening and vaccination programs have been implemented, other types of cancer in which HPV is involved, such as oropharyngeal cancer, are increasing, particularly in men. Mutational and transcriptional profiles of various HPV-associated neoplasms have been described, and accumulated evidence has shown the oncogenic capacity of E6, E7, and E5 genes of high-risk HPV. Interestingly, transcriptomic analysis has revealed that although a vast majority of the human genome is transcribed into RNAs, only 2% of transcripts are translated into proteins. The remaining transcripts lacking protein-coding potential are called non-coding RNAs. In addition to the transfer and ribosomal RNAs, there are regulatory non-coding RNAs classified according to size and structure in long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and small RNAs; such as microRNAs (miRNAs), piwi-associated RNAs (piRNAs), small nucleolar RNAs (snoRNAs) and endogenous short-interfering RNAs. Recent evidence has shown that lncRNAs, miRNAs, and circRNAs are aberrantly expressed under pathological conditions such as cancer. In addition, those transcripts are dysregulated in HPV-related neoplasms, and their expression correlates with tumor progression, metastasis, poor prognosis, and recurrence. Nuclear lncRNAs are epigenetic regulators involved in controlling gene expression at the transcriptional level through chromatin modification and remodeling. Moreover, disruption of the expression profiles of those lncRNAs affects multiple biological processes such as cell proliferation, apoptosis, and migration. This review highlights the epigenetic alterations induced by HPV, from infection to neoplastic transformation. We condense the epigenetic role of non-coding RNA alterations and their potential as biomarkers in transformation's early stages and clinical applications. We also summarize the molecular mechanisms of action of nuclear lncRNAs to understand better their role in the epigenetic control of gene expression and how they can drive the malignant phenotype of HPV-related neoplasia. Finally, we review several chemical and epigenetic therapy options to prevent and treat HPV-associated neoplasms.

Gene expression profiles and cytokine environments determine the in vitro proliferation and expansion capacities of human hematopoietic stem and progenitor cells.

OBJECTIVE: The interplay between intrinsic and extrinsic elements involved in the physiology of hematopoietic cells is not completely understood. In the present study, we analyzed the transcriptional profiles of human cord blood-derived hematopoietic stem cells (HSCs), as well as myeloid (MPCs) and erythroid (EPCs) progenitors, and assessed their proliferation and expansion kinetics in vitro. METHODS: All cell populations were obtained by cell-sorting, and were cultured in liquid cultures supplemented with different cytokine combinations. Their gene expression profiles were determined by RNA microarrays right after cell-sorting, before culture. RESULTS: HSCs showed the highest proliferation and expansion capacities in culture, and were found to be more closely related, in transcriptional terms, to MPCs than to EPCs. This correlated with the fact that after 30 days, only cultures initiated with HSCs and MPCs were sustained. Expression of cell cycle and cell division-related genes was enriched in EPCs. Such cells showed significantly higher proliferation than MPCs, however, their expansion potential was reduced, so that cultures initiated with EPCs declined after 15 days and became exhausted by day 30. Proliferation and expansion of HSCs and EPCs were higher in the presence of a cytokine combination that favors erythropoiesis, whereas the growth of MPCs was higher under a cytokine combination that favors myelopoiesis. CONCLUSION: This study shows a correlation between the transcriptional profiles of HSCs, MPCs, and EPCs, and their respective in vitro growth under particular culture conditions. These results may be relevant in the development of ex vivo systems for the expansion of hematopoietic cells for clinical application.

Prevalence of HPV in Mexican Patients with Head and Neck Squamous Carcinoma and Identification of Potential Prognostic Biomarkers.

Head and neck squamous cell carcinomas (HNSCC) show a variety of biological and clinical characteristics that could depend on the association with the human papillomavirus (HPV). Biological and clinical characterization is essential to stratify patients based on prognostic and predictive factors. Reports on HNSCC are scarce in Mexico. Herein, we analyzed 414 Mexican patients with HNSCC, including oropharynx (OPSCC), larynx (LASCC), and oral cavity (OCSCC), and identified HPV DNA and p16 expression. Global gene expression profiles were analyzed in 25 HPV+/p16+ vs. HPV-/p16- cases. We found 32.3% p16+ and 22.3% HPV+ samples, HPV 16, 18, 39, 52, and 31 being the most frequent genotypes. For OPSCC, LASCC and OCSCC, 39.2, 14.7, and 9.6% were HPV+/p16+, respectively. High expression of SLIRP, KLF10, AREG, and LIMA was associated with poor survival; in contrast, high expression of MYB and SYCP2 correlated with better survival. In HPV+ cases, high expression of SLC25A39 and GJB2 was associated with poor survival. Likewise, EGFR, IL-1, IL-6, JAK-STAT, WNT, NOTCH, and ESR1 signaling pathways were downregulated in HPV+ cases. CSF1R, MYC, and SRC genes were identified as key hubs and therapeutic targets. Our study offers information regarding the molecular and clinical characteristics of HNSCC in Mexican patients.

Adipose-derived mesenchymal stem cells promote the malignant phenotype of cervical cancer.

Epidemiological studies indicate that obesity negatively affects the progression and treatment of cervical-uterine cancer. Recent evidence shows that a subpopulation of adipose-derived stem cells can alter cancer properties. In the present project, we described for the first time the impact of adipose-derived stem cells over the malignant behavior of cervical cancer cells. The transcriptome of cancer cells cultured in the presence of stem cells was analyzed using RNA-seq. Changes in gene expression were validated using digital-PCR. Bioinformatics tools were used to identify the main transduction pathways disrupted in cancer cells due to the presence of stem cells. In vitro and in vivo assays were conducted to validate cellular and molecular processes altered in cervical cancer cells owing to stem cells. Our results show that the expression of 95 RNAs was altered in cancer cells as a result of adipose-derived stem cells. Experimental assays indicate that stem cells provoke an increment in migration, invasion, angiogenesis, and tumorigenesis of cancer cells; however, no alterations were found in proliferation. Bioinformatics and experimental analyses demonstrated that the NF-kappa B signaling pathway is enriched in cancer cells due to the influence of adipose-derived stem cells. Interestingly, the tumor cells shift their epithelial to a mesenchymal morphology, which was reflected by the increased expression of specific mesenchymal markers. In addition, stem cells also promote a stemness phenotype in the cervical cancer cells. In conclusion, our results suggest that adipose-derived stem cells induce cervical cancer cells to acquire malignant features where NF-kappa B plays a key role.

Changes in the transcriptome profile of breast cancer cells grown as spheroids.

BACKGROUND: Multicellular tumor spheroids mimic the functional organization of tumors in vivo, providing biological readouts that predict the behavior of cancer cells more accurately. The current study aimed to evaluate the transcriptome (mRNAs and long non-coding RNAs) of multicellular tumor spheroids from breast cancer cells. METHODS: MCF-7 cell spheroids were used; the transcriptome was analyzed using RNAseq and RNA microarrays; the secretion of macrophage migration inhibitor (MIF), a cytokine exported by the cholesterol efflux regulatory protein, was measured by ELISA. Linc00052 was inhibited using short-hairpin RNAs (shRNAs). RESULTS: We found several differentially regulated mRNAs and lncRNAs in MCF-7 cell spheroids. We also found significant enrichment of the Wnt/B-catenin death receptor and the cholesterol metabolic processes. Interestingly, we also found an increased concentration of MIF. Further, at 12 and 20 days of 3D culture we found 221 and 1146 dysregulated lncRNAs, respectively; including linc00052 (long intergenic non-protein coding RNA 52), which has been involved in breast cancer. Linc00052 knock-down experiments suggest that it could be a key regulator of cholesterol pathways in breast cancer. CONCLUSIONS: Our data shows that tumor spheroids can induce changes in the transcriptome of the cultured cells, including both mRNAs and ncRNA. One of the major changes included the deregulation of cholesterol pathways, of which linc00052 is apparently a key regulator.

IKKε regulates the breast cancer stem cell phenotype.

The Inhibitor of Nuclear Factor Kappa B Kinase Subunit Epsilon (IKKε) is an oncogenic protein that is up-regulated in various types of human cancers, including breast tumors. This kinase regulates diverse processes associated with malignant progression including proliferation, invasion, and metastasis. To delve into the molecular mechanisms regulated by this kinase we performed RNA-seq and network analysis of breast cancer cells overexpressing IKKε. We found that the TNF/NF-κB cascade was clearly enriched, and in accordance, NF-κB pathway inhibition in these cells resulted in a decreased expression of IKKε target genes. Interestingly, we also found an enrichment of a mammary stemness functional pathway. Upregulation of IKKε led to an increase of a stem CD44+/CD24-/low population accompanied by a high expression of stem markers such as ALDH1A3, NANOG, and KLF4 and with an increased clonogenic ability and mammosphere formation capacity. These results were corroborated with in vivo dilution assays in zebrafish embryos which showed a significant increase in the number of Cancer Stem Cells (CSCs). Finally, we found that Triple-Negative breast tumors, which are enriched in CSCs, display higher levels of IKKε than other breast tumors, supporting the association of this kinase with the stem phenotype. In conclusion, our results highlight the role of IKKε kinase in the regulation of the stem cell phenotype in breast cancer cells, as assessed by expression, functional and in vivo assays. These results add to the potential use of this kinase as a therapeutic target in this neoplasia.

The emerging role of lncRNAs in the regulation of cancer stem cells.

BACKGROUND: Tumors contain a functional subpopulation of cells that exhibit stem cell properties. These cells, named cancer stem cells (CSCs), play significant roles in the initiation and progression of cancer. Long non-coding RNAs (lncRNAs) can act at the transcriptional, posttranscriptional and translational level. As such, they may be involved in various biological processes such as DNA damage repair, inflammation, metabolism, cell survival, cell signaling, cell growth and differentiation. Accumulating evidence indicates that lncRNAs are key regulators of the CSC subpopulation, thereby contributing to cancer progression. The aim of this review is to overview current knowledge about the functional role and the mechanisms of action of lncRNAs in the initiation, maintenance and regulation of CSCs derived from different neoplasms. These lncRNAs include CTCF7, ROR, DILC, HOTAIR, H19, HOTTIP, ATB, HIF2PUT, SOX2OT, MALAT-1, CUDR, Lnc34a, Linc00617, DYNC2H1-4, PVT1, SOX4 and ARSR Uc.283-plus. Furthermore, we will illustrate how lncRNAs may regulate asymmetric CSC division and contribute to self-renewal, drug resistance and EMT, thus affecting the metastasis and recurrence of different cancers. In addition, we will highlight the implications of targeting lncRNAs to improve the efficacy of conventional drug therapies and to hamper CSC survival and proliferation. CONCLUSIONS: lncRNAs are valuable tools in the search for new targets to selectively eliminate CSCs and improve clinical outcomes. LncRNAs may serve as excellent therapeutic targets because they are stable, easily detectable and expressed in tissue-specific contexts.

NRP1-positive lung cancer cells possess tumor-initiating properties.

Tumor-initiating cells possess the capacity for self-renewal and to create heterogeneous cell lineages within a tumor. Therefore, the identification and isolation of cancer stem cells is an essential step in the analysis of their biology. The aim of the present study was to determine whether the cell surface protein neuropilin 1 (NRP1) can be used as a biomarker of stem-like cells in lung cancer tumors. For this purpose, NRP1-negative (NRP1-) and NRP1-positive (NRP1+) cell subpopulations from two lung cancer cell lines were sorted by flow cytometry. The NRP1+ cell subpopulation showed an increased expression of pluripotency markers OCT-4, Bmi-1 and NANOG, as well as higher cell migration, clonogenic and self-renewal capacities. NRP1 gene knockdown resulted not only in a decreased expression of stemness markers but also in a decrease in the clonogenic, cell migration and self-renewal potential. In addition, the NRP1+ cell subpopulation exhibited dysregulated expression of epithelial-to-mesenchymal transition-associated genes, including the ΔNp63 isoform protein, a previously reported characteristic of cancer stem cells. Notably, a genome-wide expression analysis of NRP1-knockdown cells revealed a potential new NRP1 pathway involving OLFML3 and genes associated with mitochondrial function. In conclusion, we demonstrated that NRP1+ lung cancer cells have tumor-initiating properties. NRP1 could be a useful biomarker for tumor-initiating cells in lung cancer tumors.