Progressive, multi-organ, and multi-layered nature of cancer cachexia.

Cancer cachexia is a complex, multifaceted condition that negatively impacts the health, treatment efficacy, and economic status of cancer patients. The management of cancer cachexia is an essential clinical need. Cancer cachexia is currently defined mainly according to the severity of weight loss and sarcopenia (i.e., macrosymptoms). However, such macrosymptoms may be insufficient to give clinicians clues on how to manage this condition as these symptoms appear at the late stage of cancer. We need to understand earlier events during the progression of cancer cachexia so as not to miss a clinical opportunity to control this complex syndrome. Recent research indicates that cancer-induced changes in the host are much wider than previously recognized, including disruption of liver function and the immune system. Furthermore, such changes are observed before the occurrence of visible distant metastases (i.e., in early, localized cancers). In light of these findings, we propose to expand the definition of cancer cachexia to include all cancer-induced changes to host physiology, including changes caused by early, localized cancers. This new definition of cancer cachexia can provide a new perspective on this topic, which can stimulate the research and development of novel cancer cachexia therapies.

Time-series blood cytokine profiles correlate with treatment responses in triple-negative breast cancer patients.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most heterogeneous breast cancer subtype. Partly due to its heterogeneity, it is currently challenging to stratify TNBC patients and predict treatment outcomes. METHODS: In this study, we examined blood cytokine profiles of TNBC patients throughout treatments (pre-treatment, during chemotherapy, pre-surgery, and 1 year after the surgery in a total of 294 samples). We analyzed the obtained cytokine datasets using weighted correlation network analyses, protein-protein interaction analyses, and logistic regression analyses. RESULTS: We identified five cytokines that correlate with good clinical outcomes: interleukin (IL)-1α, TNF-related apoptosis-inducing ligand (TRAIL), Stem Cell Factor (SCF), Chemokine ligand 5 (CCL5 also known as RANTES), and IL-16. The expression of these cytokines was decreased during chemotherapy and then restored after the treatment. Importantly, patients with good clinical outcomes had constitutively high expression of these cytokines during treatments. Protein-protein interaction analyses implicated that these five cytokines promote an immune response. Logistic regression analyses revealed that IL-1α and TRAIL expression levels at pre-treatment could predict treatment outcomes in our cohort. CONCLUSION: We concluded that time-series cytokine profiles in breast cancer patients may be useful for understanding immune cell activity during treatment and for predicting treatment outcomes, supporting precision medicine. TRIAL REGISTRATION: The study has been registered with the University Hospital Medical Information Network Clinical Trials Registry ( http://www.umin.ac.jp/ctr/index-j.htm ) with the unique trial number UMIN000023162. The association Japan Breast Cancer Research Group trial number is JBCRG-22. The clinical outcome of the JBCRG-22 study was published in Breast Cancer Research and Treatment on 25 March 2021. https://doi.org/10.1007/s10549-021-06184-w .

Tracing the evolutionary history of blood cells to the unicellular ancestor of animals.

Blood cells are thought to have emerged as phagocytes in the common ancestor of animals followed by the appearance of novel blood cell lineages such as thrombocytes, erythrocytes, and lymphocytes, during evolution. However, this speculation is not based on genetic evidence and it is still possible to argue that phagocytes in different species have different origins. It also remains to be clarified how the initial blood cells evolved; whether ancient animals have solely developed de novo programs for phagocytes or they have inherited a key program from ancestral unicellular organisms. Here, we traced the evolutionary history of blood cells, and cross-species comparison of gene expression profiles revealed that phagocytes in various animal species and Capsaspora (C.) owczarzaki, a unicellular organism, are transcriptionally similar to each other. We also found that both phagocytes and C. owczarzaki share a common phagocytic program, and that CEBPα is the sole transcription factor highly expressed in both phagocytes and C. owczarzaki. We further showed that the function of CEBPα to drive phagocyte program in nonphagocytic blood cells has been conserved in tunicate, sponge, and C. owczarzaki. We finally showed that, in murine hematopoiesis, repression of CEBPα to maintain nonphagocytic lineages is commonly achieved by polycomb complexes. These findings indicate that the initial blood cells emerged inheriting a unicellular organism program driven by CEBPα and that the program has also been seamlessly inherited in phagocytes of various animal species throughout evolution.

Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation.

Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in ∼3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs.

A single female-specific piRNA is the primary determiner of sex in the silkworm.

The silkworm Bombyx mori uses a WZ sex determination system that is analogous to the one found in birds and some reptiles. In this system, males have two Z sex chromosomes, whereas females have Z and W sex chromosomes. The silkworm W chromosome has a dominant role in female determination, suggesting the existence of a dominant feminizing gene in this chromosome. However, the W chromosome is almost fully occupied by transposable element sequences, and no functional protein-coding gene has been identified so far. Female-enriched PIWI-interacting RNAs (piRNAs) are the only known transcripts that are produced from the sex-determining region of the W chromosome, but the function(s) of these piRNAs are unknown. Here we show that a W-chromosome-derived, female-specific piRNA is the feminizing factor of B. mori. This piRNA is produced from a piRNA precursor which we named Fem. Fem sequences were arranged in tandem in the sex-determining region of the W chromosome. Inhibition of Fem-derived piRNA-mediated signalling in female embryos led to the production of the male-specific splice variants of B. mori doublesex (Bmdsx), a gene which acts at the downstream end of the sex differentiation cascade. A target gene of Fem-derived piRNA was identified on the Z chromosome of B. mori. This gene, which we named Masc, encoded a CCCH-type zinc finger protein. We show that the silencing of Masc messenger RNA by Fem piRNA is required for the production of female-specific isoforms of Bmdsx in female embryos, and that Masc protein controls both dosage compensation and masculinization in male embryos. Our study characterizes a single small RNA that is responsible for primary sex determination in the WZ sex determination system.

3' end formation of PIWI-interacting RNAs in vitro.

PIWI-interacting RNAs (piRNAs) are 23-30 nucleotides small RNAs that act with PIWI proteins to silence transposon activity in animal gonads. In contrast to microRNAs and small interfering RNAs, the biogenesis of piRNAs, including how 3' ends are formed, remains largely unknown. Here, by using lysate from BmN4, a silkworm ovary-derived cell line, we have developed a cell-free system that recapitulates key steps of piRNA biogenesis: loading of long single-stranded precursor RNAs into PIWI proteins with 5'-nucleotide bias, followed by Mg(2+)-dependent 3' to 5' exonucleolytic trimming and 2'-O-methylation at 3' ends. Importantly, 3' end methylation is tightly coupled with trimming and yet is not a prerequisite for determining the mature piRNA length. Our system provides a biochemical framework for dissecting piRNA biogenesis.

Silkworm HP1a transcriptionally enhances highly expressed euchromatic genes via association with their transcription start sites.

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein across different eukaryotic species and is crucial for heterochromatin establishment and maintenance. The silkworm, Bombyx mori, encodes two HP1 proteins, BmHP1a and BmHP1b. In order to investigate the role of BmHP1a in transcriptional regulation, we performed genome-wide analyses of the transcriptome, transcription start sites (TSSs), chromatin modification states and BmHP1a-binding sites of the silkworm ovary-derived BmN4 cell line. We identified a number of BmHP1a-binding loci throughout the silkworm genome and found that these loci included TSSs and frequently co-occurred with neighboring euchromatic histone modifications. In addition, we observed that genes with BmHP1a-associated TSSs were relatively highly expressed in BmN4 cells. RNA interference-mediated BmHP1a depletion resulted in the transcriptional repression of highly expressed genes with BmHP1a-associated TSSs, whereas genes not coupled with BmHP1a-binding regions were less affected by the treatment. These results demonstrate that BmHP1a binds near TSSs of highly expressed euchromatic genes and positively regulates their expression. Our study revealed a novel mode of transcriptional regulation mediated by HP1 proteins.

Histone H2B ubiquitin ligase RNF20 is required for MLL-rearranged leukemia.

Mixed-lineage leukemia (MLL) fusions are potent oncogenes that initiate aggressive forms of acute leukemia. As aberrant transcriptional regulators, MLL-fusion proteins alter gene expression in hematopoietic cells through interactions with the histone H3 lysine 79 (H3K79) methyltransferase DOT1L. Notably, interference with MLL-fusion cofactors like DOT1L is an emerging therapeutic strategy in this disease. Here, we identify the histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20) as an additional chromatin regulator that is necessary for MLL-fusion-mediated leukemogenesis. Suppressing the expression of Rnf20 in diverse models of MLL-rearranged leukemia leads to inhibition of cell proliferation, under tissue culture conditions as well as in vivo. Rnf20 knockdown leads to reduced expression of MLL-fusion target genes, effects resembling Dot1l inhibition. Using ChIP-seq, we found that H2B ubiquitination is enriched in the body of MLL-fusion target genes, correlating with sites of H3K79 methylation and transcription elongation. Furthermore, Rnf20 is required to maintain local levels of H3K79 methylation by Dot1l at Hoxa9 and Meis1. These findings support a model whereby cotranscriptional recruitment of Rnf20 at MLL-fusion target genes leads to amplification of Dot1l-mediated H3K79 methylation, thereby rendering leukemia cells dependent on Rnf20 to maintain their oncogenic transcriptional program.

Hsp90 facilitates accurate loading of precursor piRNAs into PIWI proteins.

PIWI-interacting RNAs (piRNAs) defend the genome against transposon activity in animal gonads. The Hsp90 chaperone machinery has been implicated in the piRNA pathway, but its exact role remains obscure. Here, we examined the effect of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90-specific inhibitor, on the piRNA pathway. In the silkworm ovary-derived BmN4 cells, 17-AAG treatment reduced the level of piRNAs and PIWI proteins. In vitro, the 5'-nucleotide preference upon precursor piRNA loading was compromised by 17-AAG, whereas 3'-end trimming and 2'-O-methylation were unaffected. Our data highlight a role of Hsp90 in accurate loading of precursor piRNAs into PIWI proteins.

The comprehensive epigenome map of piRNA clusters.

PIWI-interacting RNA (piRNA) clusters act as anti-transposon/retrovirus centers. Integration of selfish jumping elements into piRNA clusters generates de novo piRNAs, which in turn exert trans-silencing activity against these elements in animal gonads. To date, neither genome-wide chromatin modification states of piRNA clusters nor a mode for piRNA precursor transcription have been well understood. Here, to understand the chromatin landscape of piRNA clusters and how piRNA precursors are generated, we analyzed the transcriptome, transcription start sites (TSSs) and the chromatin landscape of the BmN4 cell line, which harbors the germ-line piRNA pathway. Notably, our epigenomic map demonstrated the highly euchromatic nature of unique piRNA clusters. RNA polymerase II was enriched for TSSs that transcribe piRNA precursors. piRNA precursors possessed 5'-cap structures as well as 3'-poly A-tails. Collectively, we envision that the euchromatic, opened nature of unique piRNA clusters or piRNA cluster-associated TSSs allows piRNA clusters to capture new insertions efficiently.