Gene regulatory network underlying the immortalization of epithelial cells

Abstract Background: Tumorigenic transformationofhumanepithelialcellsinvitrohasbeendescribedexperimentallyas thepotentialresultofspontaneousimmortalization.Thisprocessischaracterizedbyaseriesofcell–statetransitions,in whichnormalepithelialcellsacquirefirstasenescentstatewhichislatersurpassedtoattainamesenchymalstem–like phenotypewithapotentiallytumorigenicbehavior.Inthispaperweaimtoprovideasystem–levelmechanistic explanationtotheemergenceofthesecelltypes,andtothetime–orderedtransitionpatternsthatarecommonto neoplasiasofepithelialorigin.Tothisend,wefirstintegratepublishedfunctionalandwell–curatedmoleculardataof thecomponentsandinteractionsthathavebeenfoundtobeinvolvedinsuchcellstatesandtransitionsintoa networkof41molecularcomponents.Wethenreducethisinitialnetworkbyremovingsimplemediators(i.e.,linear pathways),andformalizetheresultingregulatorycoreintologicalrulesthatgovernthedynamicsofeachofthe networkcomponentsasafunctionofthestatesofitsregulators. Results: ComputationaldynamicanalysisshowsthatourproposedGeneRegulatoryNetworkmodelrecoversexactly threeattractors,eachofthemdefinedbyaspecificgeneexpressionprofilethatcorrespondstotheepithelial, senescent,andmesenchymalstem–likecellularphenotypes,respectively.Weshowthatalthoughamesenchymal stem–likestatecanbeattainedevenunderunperturbedphysiologicalconditions,thelikelihoodofconvergingtothis stateisincreasedwhenpro–inflammatoryconditionsaresimulated,providingasystems–levelmechanistic explanationforthecarcinogenicroleofchronicinflammatoryconditionsobservedintheclinic.Wealsofoundthat theregulatorycoreyieldsanepigeneticlandscapethatrestrictstemporalpatternsofprogressionbetweenthesteady states,suchthatrecoveredpatternsresemblethetime–orderedtransitionsobservedduringthespontaneous immortalizationofepithelialcells,bothinvivoandinvitro. Conclusion: Ourstudystronglysuggeststhattheinvitrotumorigenictransformationofepithelialcells,which stronglycorrelateswiththepatternsobservedduringthepathologicalprogressionofepithelialcarcinogenesisinvivo, emergesfromunderlyingregulatorynetworksinvolvedinepithelialtrans–differentiationduringdevelopment. Keywords: Carcinomas,Generegulatorynetworks,Epigeneticlandscape,Booleanmodels,Phenotypicattractor

Recurrent patterns of DNA copy number alterations in tumors reflect metabolic selection pressures.

Copy number alteration (CNA) profiling of human tumors has revealed recurrent patterns of DNA amplifications and deletions across diverse cancer types. These patterns are suggestive of conserved selection pressures during tumor evolution but cannot be fully explained by known oncogenes and tumor suppressor genes. Using a pan-cancer analysis of CNA data from patient tumors and experimental systems, here we show that principal component analysis-defined CNA signatures are predictive of glycolytic phenotypes, including 18F-fluorodeoxy-glucose (FDG) avidity of patient tumors, and increased proliferation. The primary CNA signature is enriched for p53 mutations and is associated with glycolysis through coordinate amplification of glycolytic genes and other cancer-linked metabolic enzymes. A pan-cancer and cross-species comparison of CNAs highlighted 26 consistently altered DNA regions, containing 11 enzymes in the glycolysis pathway in addition to known cancer-driving genes. Furthermore, exogenous expression of hexokinase and enolase enzymes in an experimental immortalization system altered the subsequent copy number status of the corresponding endogenous loci, supporting the hypothesis that these metabolic genes act as drivers within the conserved CNA amplification regions. Taken together, these results demonstrate that metabolic stress acts as a selective pressure underlying the recurrent CNAs observed in human tumors, and further cast genomic instability as an enabling event in tumorigenesis and metabolic evolution

Roles of Tumor Suppressor Signaling on Reprogramming and Stemness Transition in Somatic Cells

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Characterizing the C-terminal Region of Human Adenovirus E1A: An Undiscovered Country

Human Adenovirus (HAdV) E1A is the first protein expressed during viral infection. The primary function of E1A is to reprogram the cell for viral replication, but it is additionally capable of transforming primary rodent cells in co-operation with other oncogenes such as HAdV E1B. Despite extensive study, little is known about the function and cellular targets of the C-terminal region of E1A. Importantly, this region is required for the transforming ability of E1A with E1B, but can also suppress transformation with Ras. Previous studies showed that interaction with the C-terminal Binding Protein (CtBP) plays a role in both functions described above. However, other factors must be necessary, as there are mutants of E1A that retain CtBP binding but fail to contribute to either effect. Given the recent identification of new targets of this region of E1A, including FOXK1/2, DYRK1A/1B, and HAN11, I sought to re-evaluate and further characterize the mechanism by which the C-terminus of E1A carries out its functions. I performed an extensive and systematic mutational analysis of the C-terminus of E1A as a means of identifying residues specifically required for binding each cellular target. We then tested our panel of mutants for their ability to transform primary baby rat kidney cells in cooperation with E1B or Ras. Contrary to the current understanding of how the C-terminus of E1A performs its functions, my findings indicate that while CtBP binding is required for transformation with E1B, it is not necessary for the suppression of transformation with Ras. This suggests that other targets in this region play critical roles in this activity. I also discovered that E1A requires a second patch of basic residues upstream of the canonical nuclear localization sequence (NLS) for nuclear localization. Thus, the previously described monopartite NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified. Finally, I also began investigating the global changes in gene expression mediated by the C-terminal targets of E1A during infection using next-generation RNAseq. These studies have expanded on our understanding of the mechanisms by which E1A reprograms the infected cell to induce oncogenic transformation

Human T-Cell Leukemia Virus Type 1 Oncogenesis between Active Expression and Latency: A Possible Source for the Development of Therapeutic Targets

: The human T-cell leukemia virus type 1 (HTLV-1) is the only known human oncogenic retrovirus. HTLV-1 can cause a type of cancer called adult T-cell leukemia/lymphoma (ATL). The virus is transmitted through the body fluids of infected individuals, primarily breast milk, blood, and semen. At least 5-10 million people in the world are infected with HTLV-1. In addition to ATL, HTLV-1 infection can also cause HTLV-I-associated myelopathy (HAM/TSP). ATL is characterized by a low viral expression and poor prognosis. The oncogenic mechanism triggered by HTLV-1 is extremely complex and the molecular pathways are not fully understood. However, viral regulatory proteins Tax and HTLV-1 bZIP factor (HBZ) have been shown to play key roles in the transformation of HTLV-1-infected T cells. Moreover, several studies have shown that the final fate of HTLV-1-infected transformed Tcell clones is the result of a complex interplay of HTLV-1 oncogenic protein expression with cellular transcription factors that subvert the cell cycle and disrupt regulated cell death, thereby exerting their transforming effects. This review provides updated information on the mechanisms underlying the transforming action of HTLV-1 and highlights potential therapeutic targets to combat ATL

Proteomic patterns of cervical cancer cell lines, a network perspective

<p>Abstract</p> <p>Background</p> <p>Cervical cancer is a major mortality factor in the female population. This neoplastic is an excellent model for studying the mechanisms involved in cancer maintenance, because the Human Papilloma Virus (HPV) is the etiology factor in most cases. With the purpose of characterizing the effects of malignant transformation in cellular activity, proteomic studies constitute a reliable way to monitor the biological alterations induced by this disease. In this contextual scheme, a systemic description that enables the identification of the common events between cell lines of different origins, is required to distinguish the essence of carcinogenesis.</p> <p>Results</p> <p>With this study, we sought to achieve a systemic perspective of the common proteomic profile of six cervical cancer cell lines, both positive and negative for HPV, and which differ from the profile corresponding to the non-tumourgenic cell line, HaCaT. Our objectives were to identify common cellular events participating in cancer maintenance, as well as the establishment of a pipeline to work with proteomic-derived results. We analyzed by means of 2D SDS-PAGE and MALDI-TOF mass spectrometry the protein extracts of six cervical cancer cell lines, from which we identified a consensus of 66 proteins. We call this group of proteins, the "central core of cervical cancer". Starting from this core set of proteins, we acquired a PPI network that pointed, through topological analysis, to some proteins that may well be playing a central role in the neoplastic process, such as 14-3-3ζ. <it>In silico </it>overrepresentation analysis of transcription factors pointed to the overexpression of c-Myc, Max and E2F1 as key transcription factors involved in orchestrating the neoplastic phenotype.</p> <p>Conclusions</p> <p>Our findings show that there is a "central core of cervical cancer" protein expression pattern, and suggest that 14-3-3ζ is key to determine if the cell proliferates or dies. In addition, our bioinformatics analysis suggests that the neoplastic phenotype is governed by a non-canonical regulatory pathway.</p

Role of Bovine Ileal Sub-epithelial Myofibroblasts and Epithelial Cells in Innate Immunity

Gastro-intestinal (GI) tract harbors largest number of microbiota as well as the largest number of immune cells for a given tissue. The host needs to mount an effective immune response against invading pathogens and tolerance against commensals. Thus, regulatory mechanism and barrier function of the GI tract are of utmost importance for appropriate host microbe interaction and gut homeostasis. Intestinal epithelial cells (IECs) act as the first line of defense against invading pathogens. IECs recognize pathogens and commensals and mount an effective innate immune response. Such recognition of pathogens is mediated through germ line encoded pattern recognition receptors (PRRs). Intestinal sub-epithelial myofibroblasts (ISEMFs) reside just beneath the surface epithelium and are involved in maturation and differentiation of epithelium. ISEMFs protect from pathogens that breach surface epithelium by expressing PRRs. Lack of stable intestinal epithelial and sub-epithelial myofibroblast cell lines has slowed down scientific studies on these cells. In this study, we established and characterized ISEMF cells from the ileum of a 2-day old calf. We also had generated stable bovine ileal epithelial cell (BIEC-c4) cultures in our lab. On real time-quantitative polymerase chain reaction (RT-qPCR) analysis both these cell types expressed Toll-like receptors (TLRs) 1-9. To investigate their responses to various pathogen-associated molecular patterns (PAMPs), we stimulated both cell types for 3 hours and 24 hours with various PAMPs. The RT-qPCR assay was used to investigate changes in TLR gene expression and in cytokine genes following stimulation. Lipopolysaccharide, peptidoglycan, and flagellin were used as bacterial ligands of surface PRRs. Similarly, γ-D-Glu-mDAP, muramyl dipeptide, polyinosonic:polycytidylic acid, poly I:C complexed with lyovec, and imiquimod were used as ligands of cytosolic and endosomal PRRs. Bovine ileal ISEMFs responded to bacterial PAMPs and to ligands of cytosolic and endosomal PRRs by significantly altering TLR gene expression. Unlike bovine ISEMFs, BIEC-c4 cells responded only to bacterial ligands. Thus, we conclude that bovine ileal ISEMF can be a good model to study innate immune responses and signaling pathways occurring at subepithelial compartment. However, BIEC-c4 cells may serve as a good in-vitro model to study enteric infectious disease pathogenesis and innate immune responses associated with them

N6-methyladenosine contributes to cellular phenotype in a genetically-defined model of breast cancer progression

The mRNA modification N6-methyladenosine (m6A) is involved in many post- transcriptional regulatory processes including mRNA stability and translational efficiency. However, it is also imperative to correlate these processes with phenotypic outputs during cancer progression. Here we report that m6A levels are significantly decreased in genetically-defined immortalized and oncogenically-transformed human mammary epithelial cells (HMECs), as compared with their primary cell predecessor. Furthermore, the m6A methyltransferase (METTL3) is decreased and the demethylase (ALKBH5) is increased in the immortalized and transformed cell lines, providing a possible mechanism for this basal change in m6A levels. Although the immortalized and transformed cells showed lower m6A levels than their primary parental cell line, overexpression of METTL3 and METTL14, or ALKBH5 knockdown to increase m6A levels in transformed cells increased proliferation and migration. Remarkably, these treatments had little effect on the immortalized cells. Together, these results suggest that m6A modification may be downregulated in immortalized cells as a brake against malignant progression. Finally, we found that m6A levels in the immortalized and transformed cells increased in response to hypoxia without corresponding changes in METTL3, METTL14 or ALKBH5 expression, suggesting a novel pathway for regulation of m6A levels under stress

Human papillomavirus 16 E6 induces FoxM1B in oral keratinocytes through GRHL2

High-risk human papillomavirus (HPV) is a major risk factor for oral and pharyngeal cancers (OPCs), yet the detailed mechanisms by which HPV promotes OPCs are not understood. Forkhead box M1B (FoxM1B) is an oncogene essential for cell cycle progression and tumorigenesis, and it is aberrantly overexpressed in many tumors. We previously showed that FoxM1B was the putative target of an epithelial-specific transcription factor, Grainyhead-like 2 (GRHL2). In the current study, we demonstrate that HPV type 16 (HPV-16) E6 induces FoxM1B in human oral keratinocytes (HOKs) and tonsillar epithelial cells (TECs) in part through GRHL2. FoxM1B was barely detectable in cultured normal human oral keratinocytes (NHOKs) and progressively increased in immortalized HOKs harboring HPV-16 genome (HOK-16B) and tumorigenic HOK-16B/BaP-T cells. Retroviral expression of HPV-16 E6 and/or E7 in NHOKs, TECs, and hypopharyngeal carcinoma cells (FaDu) revealed induction of FoxM1B and GRHL2 by the E6 protein but not E7. Both GRHL2 and FoxM1B were strongly induced in the epidermis of HPV-16 E6 transgenic mice and HPVoral squamous cell carcinomas. Ectopic expression of FoxM1B led to acquisition of transformed phenotype in HOK-16B cells. Loss of FoxM1B by lentiviral short hairpin RNA vector or chemical inhibitor led to elimination of tumorigenic characteristics of HOK-16B/BaP-T cells. Luciferase reporter assay revealed that GRHL2 directly bound and regulated the FoxM1B gene promoter activity. Using epithelial-specific Grhl2 conditional knockout mice, we exposed wild-type (WT) and Grhl2 KO mice to 4-nitroquinolin 1-oxide (4-NQO), which led to induction of FoxM1B in the tongue tissues and rampant oral tumor development in the WT mice. However, 4-NQO exposure failed to induce tongue tumors or induction of FoxM1B expression in Grhl2 KO mice. Collectively, these results indicate that HPV-16 induces FoxM1B in part through GRHL2 transcriptional activity and that elevated FoxM1B level is required for oropharyngeal cancer development