Autophagy Regulation on Cancer Stem Cell Maintenance, Metastasis, and Therapy Resistance

Cancer stem cells (CSCs) are a subset of the tumor population that play critical roles in tumorigenicity, metastasis, and relapse. A key feature of CSCs is their resistance to numerous therapeutic strategies which include chemotherapy, radiation, and immune checkpoint inhibitors. In recent years, there is a growing body of literature that suggests a link between CSC maintenance and autophagy, a mechanism to recycle intracellular components during moments of environmental stress, especially since CSCs thrive in a tumor microenvironment that is plagued with hypoxia, acidosis, and lack of nutrients. Autophagy activation has been shown to aid in the upkeep of a stemness state along with bolstering resistance to cancer treatment. However, recent studies have also suggested that autophagy is a double-edged sword with anti-tumorigenic properties under certain circumstances. This review summarizes and integrates what has been published in the literature in terms of what role autophagy plays in stemness maintenance of CSCs and suggests that there is a more complex interplay between autophagy and apoptosis which involves multiple pathways of regulation. Future cancer therapy strategies are needed to eradicate this resistant subset of the cell population through autophagy regulation.

Mouse CHD4-NURD is Required for Neonatal Spermatogonia Survival and Normal Gonad Development.

Testis development and sustained germ cell production in adults rely on the establishment and maintenance of spermatogonia stem cells and their proper differentiation into spermatocytes. Chromatin remodeling complexes regulate critical processes during gamete development by restricting or promoting accessibility of DNA repair and gene expression machineries to the chromatin. Here, we investigated the role of CHD4 and CHD3 catalytic subunits of the NURD complex during spermatogenesis. Germ cell-specific deletion of Chd4 early in gametogenesis, but not Chd3, resulted in arrested early gamete development due to failed cell survival of neonate undifferentiated spermatogonia stem cell population. Candidate assessment revealed that CHD4 controls expression of Dmrt1 and its downstream target Plzf, both described as prominent regulators of spermatogonia stem cell maintenance. Our results show the requirement of CHD4 in mammalian gametogenesis pointing to functions in gene expression early in the process.

Longitudinal single-cell transcriptomics reveals distinct patterns of recurrence in acute myeloid leukemia

The heterogeneity and evolution of AML blasts can render therapeutic interventions ineffective in a yet poorly understood patient-specific manner. To gain insight into the clonal heterogeneity of diagnosis (Dx) and relapse (Re) pairs, we employed whole-exome sequencing and single-cell RNA-seq to longitudinally profile two t(8;21) (AML1-ETO = RUNX1-RUNX1T1), and four FLT3-ITD AML cases. The single cell RNA data underpinned the tumor heterogeneity amongst patient blasts. The Dx-Re transcriptomes of high risk FLT3-ITD pairs formed a continuum from extensively changed in the absence of significantly mutational changes in AML-associated genes to rather similar Dx-Re pair of an intermediate risk FLT3-ITD. In one high risk FLT3-ITD pair, a pathway switched from an AP-1 regulated network in Dx to mTOR signaling in Re. The distinct AML1-ETO pairs comprise clusters that share genes related to hematopoietic stem cell maintenance and cell migration suggesting that the Re leukemic stem cell-like (LSC-like) cells probably evolved from the Dx LSC-like cells. In summary, our study revealed a continuum from drastic transcriptional changes to extensive similarities between respective Dx-Re pairs that are poorly explained by the well-established model of clonal evolution. Our results suggest alternative and currently unappreciated and unexplored mechanisms leading to therapeutic resistance and AML recurrence.

The RNA binding protein Swm is critical for Drosophila melanogaster intestinal progenitor cell maintenance

The regulation of stem cell survival, self-renewal, and differentiation is critical for the maintenance of tissue homeostasis. Although the involvement of signaling pathways and transcriptional control mechanisms in stem cell regulation have been extensively investigated, the role of post-transcriptional control is still poorly understood. Here we show that the nuclear activity of the RNA-binding protein Second Mitotic Wave Missing (Swm) is critical for Drosophila intestinal stem cells (ISCs) and their daughter cells, enteroblasts (EBs), to maintain their identity and function. Loss of swm in these intestinal progenitor cells leads ISCs and EBs to lose defined cell identities, fail to proliferate, and detach from the basement membrane, resulting in severe progenitor cell loss. swm loss further causes nuclear accumulation of poly(A)+ RNA in progenitor cells. Swm associates with transcripts involved in epithelial cell maintenance and adhesion, and the loss of swm, while not generally affecting the levels of these Swm-bound mRNAs, leads to elevated expression of proteins encoded by some of them, including the fly orthologs of Filamin and Talin. Taken together, this study indicates a role for Swm in adult stem cell maintenance, and raises the possibility that nuclear post-transcriptional gene regulation plays vital roles in controlling adult stem cell maintenance and function.

Transcriptome Analysis Reveals Genes Respond to Chlorophyll Deficiency in Green and Yellow Leaves of Chrysanthemum morifolium Ramat

Chlorophyll is vital for photosynthesis to produce sugars and other useful biochemical products in green plants. However, the molecular effects of chlorophyll deficiency in Chrysanthemum are largely unknown. In this study, we identified a bud sport mutant chrysanthemum belonging to the variety ‘Nannong Binyun’, which has yellow branches. Plant physiological studies have shown that the yellow color is revealed due to chlorophyll loss. RNA extracts of yellow and green tissues were analyzed using high-throughput RNA-sequencing, and a total of 11,649 tissue enriched unigenes that respond to chlorophyll deficiency were identified, including 4803 unigenes upregulated in yellow tissues and 6846 unigenes in green tissues. GO analysis revealed that these tissue-enriched genes may involve in the physiological processes of chlorophyll accumulation and photosynthesis. In addition, many DEGs from the families of AP2-EREBP, bHLH, MYB, and FAR1 that are associated with plant development and stress response were detected. Our study found that most of the genes from the GRAS family were downregulated in yellow leaves, indicating their putative roles in stem cell maintenance and possible contribution to leaf size determination.

A phosphorylation code regulates the multi-functional protein RETINOBLASTOMA-RELATED1 in Arabidopsis thaliana

The RETINOBLASTOMA-RELATED (RBR) proteins play a central role coordinating cell division, cell differentiation and cell survival within an environmental and developmental context. These roles reflect RBR ability to engage in multiple protein-protein interactions (PPIs), which are regulated by multi-site phosphorylation. However the functional outcomes of RBR phosphorylation in multicellular organisms remain largely unexplored. Here we test the hypothesis that phosphorylation allows diversification of RBR functions in multicellular context. Using a representative collection of transgenic loss- and gain of function point mutations in RBR phospho-sites, we analysed their complementation capacity in Arabidopsis thaliana root meristems. While the number of mutated residues often correlated to the phenotypic strength of RBR phospho-variants, phospho-sites contributed differentially to distinct phenotypes. For example, the pocket-domain has a greater influence on meristematic cell proliferation, whereas the C-terminal region associates to stem cell maintenance. We found combinatorial effects between the T406 phopspho-site with others in different protein domains. Moreover, a phospho-mimetic and a phospho-defective variant, both promoting cell death, indicate that RBR controls similar cell fate choices by distinct mechanisms. Thus, additivity and specificity of RBR phospho-sites fine tune RBR activity across its multiple roles. Interestingly, a mutation disrupting RBR interactions with the LXCXE motif suppresses dominant phospho-defective RBR phenotypes. By probing protein-protein interactions of RBR variants, we found that LXCXE-containing members of the DREAM complex constitute an important component of phosphorylation-regulated RBR function, but also that RBR participates in stress or environmental responses independently of its phosphorylation state. We conclude that developmental-related, but not stress- or environmental-related functions of RBR are defined and separable by a combinatorial phosphorylation code.

Unraveling the roles of Mast4 in amelogenesis via regulating DLX3 and stem cell maintenance of mouse incisors

Asymmetric division of stem cells allows for maintenance of the cell population and differentiation for harmonious progress. Developing mouse incisors allows for examin ation of how the stem cell niche employs specific insights into essential phases. Microtubule associated serine/threonine kinase family member 4 (Mast4) knockout (KO) mice showed abnormal incisor development with weak hardness as the apical bud was reduced and preameloblasts were shifted to the apical side, resulting in Amelogenesis Imperfecta. In addition, Mast4) KO incisors showed abnormal enamel maturation, and stem cell maintenance was inhibited as amelogenesis accelerated. Distal-Less Homeobox 3 (DLX3), known to be a critical factor Tricho Dento Osseous (TDO) syndrome, is considered to be responsible for A melogenesis Imperfecta in humans. MAST4 directly binds to DLX3 and induces phosphorylation at three residues within the nuclear localization sites (NLS) that promote the nuclear translocation of DLX3. MAST4-mediated phosphorylation of DLX3 ultimately controls the transcription of DLX3 target genes, which are carbonic anhydrase and ion transporter genes involved in the pH regulation process during ameloblast maturation. Taken together, our data reveal a novel role of MAST4 as a critical regulator of ameloblast maturation, which controls DLX3 transcriptional activity.

Diverse tumorigenic consequences of human papillomavirus integration in primary oropharyngeal cancers

Human papillomavirus (HPV) causes 5% of all cancers and frequently integrates into host chromosomes. The HPV oncoproteins E6 and E7 are necessary but insufficient for cancer formation, indicating that additional secondary genetic events are required. Here, we investigate potential oncogenic impacts of virus integration. Analysis of 105 HPV-positive oropharyngeal cancers by whole-genome sequencing detects virus integration in 77%, revealing five statistically significant sites of recurrent integration near genes that regulate epithelial stem cell maintenance (i.e., SOX2, TP63, FGFR, MYC) and immune evasion (i.e., CD274). Genomic copy number hyperamplification is enriched 16-fold near HPV integrants, and the extent of focal host genomic instability increases with their local density. The frequency of genes expressed at extreme outlier levels is increased 86-fold within ±150 kb of integrants. Across 95% of tumors with integration, host gene transcription is disrupted via intragenic integrants, chimeric transcription, outlier expression, gene breaking, and/or de novo expression of noncoding or imprinted genes. We conclude that virus integration can contribute to carcinogenesis in a large majority of HPV-positive oropharyngeal cancers by inducing extensive disruption of host genome structure and gene expression.

TB-3 miR-33a depletion accelerate medulloblastoma generation and invasion

Background and purposes: Lipid metabolism have been shown to be associated with tumorigenicity in various malignancies. The purpose of this study was to investigate the association of miR-33, a key regulator of lipid metabolism, in tumorigenicity and progression of medulloblastoma. Methods: Incidence of medulloblastoma and histopathological findings were compared between ptch1+/- mice and ptch1+/- miR-33a-/- mice. Tumors extracted from these mice were transplanted subcutaneously in nude mice (n=14 for ptch1+/-, n=19 for ptch1+/- miR-33a-/-) and in C57BL/6 mice (n=12 for each). Gene expression profile was compared between tumors from ptch1+/- mice and those from ptch1+/- miR-33a-/- mice. Results: Knockout of miR-33a in ptch1+/- transgenic mouse model increased the incidence of spontaneous generation of medulloblastoma from 34.5% to 84.0% (p< 0.001) at 12 months. Histopathological analysis showed infiltrative tumor borders in ptch1+/- miR-33a-/- tumors as compared with ptch1+/- ones. Tumor formation was observed in 21.4% for ptch1+/- tumors and 68.4% for ptch1+/- miR-33a-/- tumors in nude mice (p= 0.008). It was observed in 0% and 16.7% in immune competent mice. RNA sequencing detected that SCD1 and SREBF1 was upregulated in tumors from miR-33a knockout mice. Discussion: Our results demonstrated that depletion of miR-33a accelerated medulloblastoma generation and invasion. miR-33a may also be important for immune evasion. SCD1, which is reported to play a role in tumor stem cell maintenance and metastasis, can be a potential therapeutic target for medulloblastoma.