Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Purdue University

Aneuploidy is one of the most common genetic alterations in cancer cell genomes. It greatly contributes to the heterogeneity of cancer cell genomes, and its roles in tumorigenesis are attracting more and more attentions. Zebrafish is emerging as a new genetic model for many human diseases including cancer. The zebrafish cancer model has shown an equivalent degree of aneuploidy as found in corresponding human cancers, thus it provides a great tool for us to study cancer aneuploidy and, in general, cancer biology. Here, we discuss some new advances of aneuploidy and the potential usages of this cancer model system. 

New insight into cancer aneuploidy in zebrafish.

The Hedgehog (Hh) pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue maturity must be coordinated, and how an energy sensor regulates the Hh pathway is not clear. AMP-activated protein kinase (AMPK) is an important sensor of energy stores and controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic-hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency.

AMP-Activated Protein Kinase Directly Phosphorylates and Destabilizes Hedgehog Pathway Transcription Factor GLI1 in Medulloblastoma.

Gene co-option, usually after gene duplication, in the evolution of development is found to contribute to vertebrate morphological innovations, including the endothelium-based vascular system. Recently, a zebrafish kank gene was found expressed in the vascular vessel primordium, suggesting KANK genes are a component of the developmental tool kit for the vertebrate vascular system. However, how the KANK gene family is involved in vascular vessel development during evolution remains largely unknown. First, we analyzed the molecular evolution of the KANK genes in metazoan, and found that KANK1, KANK2, KANK3 and KANK4 emerged in the lineage of vertebrate, consistent with the two rounds of vertebrate whole-genome duplications (WGD). Moreover, KANK genes were further duplicated in teleosts through the bony-fish specific WGD, while only kank1 and kank4 duplicates were retained in some of the examined fish species. We also found all zebrafish kank genes, except kank1b, are primarily expressed during embryonic vascular development. Compared to invertebrate KANK gene expression in the central nervous system, the vascular expression of zebrafish kank genes suggested KANK genes were co-opted for vertebrate vascular development. Given the cellular roles of KANK genes, our results suggest that this co-option may facilitate the evolutionary origin of vertebrate vascular vessels.

Evolutionary and developmental analysis reveals KANK genes were co-opted for vertebrate vascular development.

The mouse double minute 1 (Mdm1) gene was first reported and cloned in mouse tumor cell lines as an oncogene candidate. Later, it was found that mutation of Mdm1 might cause age-related retinal degeneration 2 in mice by genetic linkage analysis. Additionally, the MDM1 protein was found to be expressed in the centrosomes, cilia, and the nucleus of multiciliated tracheal epithelial cells in mice. These observations suggest that MDM1 may have some basal functions in cell physiology. However, the evolutionary history of this gene and its expression during embryonic development remain largely unexplored.

Molecular Evolution of MDM1, a "Duplication-Resistant" Gene in Vertebrates.

Currently, human cancer genomics is making great progress, and many mutations of new cancer driver genes have been detected at an unprecedented rate in a variety of human cancers. Many details of the genetic alterations in cancer cell genomes have been revealed by the massively parallel sequencing. Long-lasting aneuploidy caused large-scale somatic copy number alterations remains a difficulty as there are too many genes located on such big chromosomal fragments, and this cannot simply be solved by increasing sequencing depth and tumor sample numbers. Comparative oncogenomics may provide us with a solution to this problem. Here, we review some of the common animal cancer models and propose to analyze cancer cell genomics in vertebrate phylogenetic backgrounds. Thus phylooncogenomics may provide us with a unique perspective on he nature of cancer biology unattainable by single species studies.

Phylooncogenomics: Examining the cancer genome in the context of vertebrate evolution.

Malignant peripheral nerve sheath tumors (MPNSTs) are a type of rare sarcomas with a poor prognosis due to its highly invasive nature and limited treatment options. Currently there is no targeted-cancer therapy for this type of malignancy. Thus, it is important to identify more cancer driver genes that may serve as targets of cancer therapy. Through comparative oncogenomics, we have found that KANK1 was a candidate tumor suppressor gene (TSG) for human MPNSTs. Although KANK1 is known as a cytoskeleton regulator, its tumorigenic function in MPNSTs remains largely unknown. In this study, we report that restoration of KANK1 in human MPNST cells inhibits cell growth both in human cell culture and xenograft mice by increasing apoptosis. Consistently, knockdown of KANK1 in neurofibroma cells promoted cell growth. Using RNA-seq analysis, we identified CXXC5 and other apoptosis-related genes, and demonstrated that CXXC5 is regulated by KANK1. Knockdown of CXXC5 was found to diminish KANK1-induced apoptosis in MPNST cells. Thus, KANK1 inhibits MPNST cell growth though CXXC5 mediated apoptosis. Our results suggest that KANK1 may function as a tumor suppressor in human MPNSTs, and thus it may be useful for targeted therapy.

KANK1 inhibits cell growth by inducing apoptosis though regulating CXXC5 in human malignant peripheral nerve sheath tumors.

IFN regulatory factor 4 (IRF4) is a key transcription factor that promotes effector CD8 T cell differentiation and expansion. The roles of IRF4 in regulating the CD8 T cell response to cytokines have not been explored. In this article, we show that IL-2 and IL-15 signaling and STAT5 activation regulate IRF4 expression in CD8 T cells. Gene-expression profile analysis has also revealed that IRF4 is required for expression of the receptors of IL-2 family cytokines CD122 and CD127. We found that IRF4 binds directly to CD122 and CD127 gene loci, indicating that it may directly promote CD122 and CD127 gene transcription. As a consequence, IRF4-deficient CD8 T cells show diminished sensitivity to IL-2, IL-15, and IL-7 treatment in vitro. Furthermore, we found that IRF4-deficient CD8 T cells had lower expression of CD122 and CD127 in vivo during influenza virus infection. These data suggest that IRF4 regulates the sensitivity of CD8 T cells to IL-2 family cytokines, which correlates with the diminished effector and memory CD8 T cell responses in IRF4-deficient CD8 T cells.

GuangJun Zhang

Purdue University

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors