Wednesday, January 29, 2020

Natural Killers Responsive Through Transposable Elements

Mechanisms of immunity are generally referred through MHC, HLA, antigens and other molecules that describe the presentation and interaction of cell surface receptors with cells of the adaptive immune system. Innate immune interactions occur through inhibitory and activating receptors including on Natural Killer (NK) Cells, but rarely does research elucidate the genomic activity of the target cell and its relationship to upstream activities on the cell surface. 

Transposable Elements (TE's) are DNA sections of a gene that can change their location by insertion. They were a focus of our early work that included the study of repeats in intron's (regulatory sections of genes) and their potential relationship to protein expression. Interestingly the most recurrent TE's are among the youngest in the genomic evolutionary chain and are predominantly expressed from intergenic loci associated with antiviral or DNA damage responses. In Drosophila melanogaster, the genomic regions surrounding 84 TE's located near genes involved in stress response, behavior and development indicated an adaptive effect. Recently it has become more widely accepted that TE expression in tumors is associated with immune infiltration and increased antigenicity.

In plant immunity a TE has been domesticated for service through histone marks and generation of alternative mRNA isoforms that were both directly linked to response to a particular pathogen. In vivo, an intron1 site co-opted the TE-associated histone mark to facilitate epigenetic control of pre-mRNA processing, which established a unique mechanism for regulation of immune gene expression in plants. Although in vivo proof in animal or human cells is more complex to obtain, increasing amounts of research has been directed to determine whether TE's are a widely deployed, histone associated, epigenetic mechanism for gene expression.

p53 transcription sites evolved through epigenetic methylation, deamination and histone regulation that constituted a universal mechanism found to generate various transcription-factor binding sites in short TE's or Alu repeats. A study into the evolution of immune antigen receptor's (AgR's) proposed their origin from NK-like receptors that recognized MHC-like molecules. The team went on to provide evidence of such. They found that all AgR rearrangements are likely derived from the huMHCpara-19 precursor by invasion of a TE on the RAG gene that was split, by double-stranded DNA breaks (DSB) at variable (V), diversity (D), and joining (J) segments that could also be recombined. In mature NK cells recombination of the V and J element does not frequently occur, but in immature NK precursor populations RAG altered heterogeneity, cytotoxic capacity, cellular fitness and differentiation.

To persist RAG DSBs must escape efficient repair, avoid the activation of p53 cell death pathways, dissociate from their post-cleavage complex, associate with other DSBs to which they will ultimately join and successfully navigate end joining pathways. In lymphocyte precursors of scid's patients (severe combined immunodeficiency) RAG V J recombination activates a p53-dependent DNA damage checkpoint.

Mutant NK cells lacking RAG activity or Wild Type NK cells lacking a history of RAG expression are more terminally differentiated and highly cytolytic, but characterized by greater apoptosis following DNA damage. In contrast, WT NK cells with a history of RAG expression are less terminally differentiated and cytotoxic but can generate long-life memory cells following antigen-specific proliferation, characterized by increased survival and ability to repair DSBs. Therefore, an unexpected functional RAG dichotomy exists between NK cell populations to effectively combat pathogens.

Natural killer cells do not rearrange DNA to generate antigen receptors and are thus innate immune cells. However NK cells do have reciprocal relationship with cells of the adaptive immune system. The integrated dynamics of TE's in RAG DSB's and p53 binding sites implicate innate and adaptive immunity mediated through diverse NK cell population, education and antigen production possibly dating back to MHC evolution and reproductive allorecognition in which p53 plays a central role.

189 gastrointestinal cancer patients across three cancer types: 95 stomach, colorectal esophageal were examined for any aberration in DNA repair pathways that could be associated with L1 retro-transposition. Out of 15 DNA repair pathways, only the TP53 repair pathway showed a significant association. L1 retro-transposition is inversely correlated with expression of immunologic response genes. Frequent TP53 mutations in tumors with a higher load of L1 insertions suggest the critical role of TP53 in restricting retrotransposons as a guardian of L1 expression and cancer immunity.

Monday, January 13, 2020

Impotent Natural Killers by Cancer Stem Cells and Ageing

Cancer stem cells have been found, through various mechanisms to alter the sentinel function and innate, immune surveillance of Natural Killer cells (NK). In senescent cells that have stopped cell division, including in cancer stem cell niches and NK induced vascular remodeling (as found in the developing placenta) NK's sentinel vigilance is also reduced.

Senescence-associated mitochondrial dysfunction, a significant trigger of multiple dimensions of the senescent phenotype is caused by disruption of normal mitochondrial autophagy (mitophagy). Mitophagy increases with aging and this age-dependent rise is abrogated by PINK1 or parkin deficiency. Deletion of a p53 response element on PINK1 promoter impacts p53-mediated PINK1 transcriptional repression. This p53-mediated negative regulation of autophagy has been found to be PINK1-dependent and constitutes a p53-PINK1 loop in nucleus and cytoplasm.

Further, mitophagy controls the activities of tumor suppressor p53 to regulate, at least hepatic cancer stem cells via Nanog. Prostate cancer cells escape NK attack by Nanog down-regulating ICAM1 (LFA1), to which NK would normally bind its target. In lung cancer NK have been found to limit the efficient clearance of senescent tumor cells from the mouse lung after p53 restoration. This indicated p53 may promote conditions for cellular survival and NK induced vascular remodeling or angiogenesis, necessary for the growth of tumors.

When under stress and inner mitochondrial membrane pressure gradient moves toward depolarization, Pink1 slots into the membrane, binds and phosphorylates p53 at Serine 392 (p53s392) and aids phagophore formation to enhance mitophagy. Mitophagy traps cytoplasmic p53s392, which reduces its transport to the nucleus where it would otherwise disrupt transcription of Nanog. (As illustrated below). 
Activated p53s392 nucleoside concentrations are effected by mitophagy
On the other hand, the sentinel function of NK may be subject to this PINK1 mediated mitochondrial switch. In prostate cancer cells Nanog promoted ICAM1 transcription required for NK binding target and cell killing. In prostate cancer cells Nanog over-expression restricts ICAM1, which promotes tumor formation. (As illustrated below). Investigating further, the direct functional link between p53 and ICAM-1 (CD54) in senescence and age-related disorders appears to be deeply integrated in mitophagy, senescence and immunity.

Nanog over-expression appears to be deterministic 
In stem cells where normal expression of Nanog transcribes ICAM1 and cancer stem cells where over-expression of Nanog restricts ICAM1, the variable PINK1-p53 switch may represent a "canary" that signals the state of  mitochondrial health to sentinel NK. However in some cancer cells where normal mitophagy is impaired and Nanog expression is restricted by p53s392, other p53 isoforms may directly promote the transcription of ICAM1.

In  two manipulation experiments using five different fibroblast cell lines that accelerated development of senescent associated secretory phenotypes a striking result was observed: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated pro-malignant paracrine signaling activities. Experiments show that PINK1 and Parkin, which are regulated by p53 specifically regulate mitochondrial antigen presentation of both MHC classes.

So, the question is whether the p53-PINK1 mitochondrial switch acts as cell-health "canary" for sentinel NK, where its inherent variables and regulatory loop may be fertile ground for the challenges of developing cancers?