The mitotic spindle has long been known to play a crucial role in mitosis, orchestrating the segregation of chromosomes into two daughter cells during mitosis with high fidelity. Intracellular forces generated by the mitotic spindle are increasingly well understood, and recent work has revealed that the efficiency and the accuracy of mitosis is ensured by the scaling of mitotic spindle size with cell size. However, the role of the spindle in cancer progression has largely been ignored. Two recent studies point toward the role of mitotic spindle evolution in cancer progression through extracellular force generation. Cancer cells with lengthened spindles exhibit highly increased metastatic potential. Further, interpolar spindle elongation drives protrusive extracellular force generation along the mitotic axis to allow mitotic elongation, a morphological change that is required for cell division. Together, these findings open a new research area studying the role of the mitotic spindle evolution in cancer metastasis.
Gain-of-function mutations of SHP2, especially D61Y and E76K, lead to the development of neoplasms in hematopoietic cells. Previously, we found that SHP2-D61Y and -E76K confer HCD-57 cells cytokine-independent survival and proliferation via activation of MAPK pathway. Metabolic reprogramming is likely to be involved in leukemogenesis led by mutant SHP2. However, detailed pathways or key genes of altered metabolisms are unknown in leukemia cells expressing mutant SHP2. In this study, we performed transcriptome analysis to identify dysregulated metabolic pathways and key genes using HCD-57 transformed by mutant SHP2. A total of 2443 and 2273 significant differentially expressed genes (DEGs) were identified in HCD-57 expressing SHP2-D61Y and -E76K compared with parental cells as the control, respectively. Gene ontology (GO) and Reactome enrichment analysis showed that a large proportion of DEGs were involved in the metabolism process. Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis showed that DEGs were the mostly enriched in glutathione metabolism and biosynthesis of amino acids in metabolic pathways. Gene Set Enrichment Analysis (GSEA) revealed that the expression of mutant SHP2 led to a significant activation of biosynthesis of amino acids pathway in HCD-57 expressing mutant SHP2 compared with the control. Particularly, we found that ASNS, PHGDH, PSAT1, and SHMT2 involved in the biosynthesis of asparagine, serine, and glycine were remarkably up-regulated. Together, these transcriptome profiling data provided new insights into the metabolic mechanisms underlying mutant SHP2-driven leukemogenesis.
ABSTRACT During late mitosis and early G 1 , replication origins are licensed for replication by binding to double hexamers of MCM2-7. Here, we investigate how licensing and proliferative commitment are coupled in the small-intestinal epithelium. We developed a method for identifying cells in intact tissue containing DNA-bound MCM2-7. Interphase cells above the transit-amplifying compartment had no DNA-bound MCM2-7, but still expressed MCM2-7 protein, suggesting that licensing is inhibited immediately upon differentiation. Strikingly, we found most proliferative Lgr5(+) stem cells are in an unlicensed state. This suggests that the elongated cell-cycle of intestinal stem-cells is caused by an increased G 1 length, characterised by dormant periods with unlicensed origins. Significantly, the unlicensed state is lost In Apc mutant epithelium, which lacks a functional restriction point, causing licensing immediately upon G 1 entry. We propose that the unlicensed G 1 of intestinal stem cells creates a temporal window when proliferative fate decisions can be made.
Abstract Colorectal cancer (CRC) is a leading cause of death from cancer worldwide. Thus, there is an emerging need for new experimental models that allow identification and validation of biomarkers for CRC-specific progression. In this study, we propose a repeated sphere-forming assay as a strategy to select a malignant subpopulation from a CRC line, HCT116. We validated our assay by confirming that three canonical stemness markers, Nanog, Oct4, and Lgr5, were up-regulated in the sphere state at every generation of the selection assay. The resulting line, after eight rounds of selection, exhibited an increased sphere-forming capacity in vitro and tumorgenicity in vivo . Furthermore, dipeptidase 1 (DPEP1) was identified as the major differentially expressed gene in the selected clone, and depletion of DPEP1 suppressed the elevated sphere-forming capacity in vitro and tumorgenicity in vivo . Overall, we have established an experimental strategy for the isolation of a malignant subpopulation from a CRC cell line. Results from our model also suggested that DPEP1 can serve as a promising prognostic biomarker for CRC.
Colorectal cancer (CRC) is a leading cause of cancer‑associated mortality worldwide; therefore, there is an emerging need for novel experimental models that allow for the identification and validation of biomarkers for CRC‑specific progression. In the present study, a repeated sphere‑forming assay was used as a strategy to select a malignant subpopulation from a CRC cell line, namely HCT116. The assay was validated by confirming that canonical stemness markers were upregulated in the sphere state at every generation of the selection assay. The resulting subpopulation, after eight rounds of selection, exhibited increased sphere‑forming capacity in vitro and increased tumorigenicity in vivo. Furthermore, dipeptidase 1 (DPEP1) was identified as the major differentially expressed gene in the selected clone, and its depletion suppressed the elevated sphere‑forming capacity in vitro and tumorigenicity in vivo. Overall, the present study established an experimental strategy to isolate a malignant subpopulation from a CRC cell line. Additionally, results from the present model revealed that DPEP1 may serve as a promising prognostic biomarker for CRC.
Abstract Cancer cells maintain their telomeres by either re-activating telomerase or adopting the homologous recombination (HR)-based Alternative Lengthening of Telomere (ALT) pathway. Among the many prominent features of ALT cells, C-circles (CC) formation is considered to be the most specific and quantifiable biomarker of ALT. However, the molecular mechanism behind the initiation and maintenance of CC formation in ALT cells is still largely unknown. We reported previously that depletion of the FANCM complex (FANCM-FAAP24-MHF1&2) in ALT cells induced pronounced replication stress, which primarily takes place at their telomeres. Here, we characterized the changes in ALT associated phenotypes in cells deficient of the FANCM complex. We found that depletion of FAAP24 or FANCM, but not MHF1&2, induces a dramatic increase of CC formation. Most importantly, we identified multiple DNA damage response (DDR) and DNA repair pathways that stimulate the dramatic increase of CC formation in FANCM deficient cells, including the dissolvase complex (BLM-TOP3A-RMI1/2, or BTR), DNA damage checkpoint kinases (ATR and Chk1), HR proteins (BRCA2, PALB2, and Rad51), as well as proteins involved in Break-Induced Replication (BIR) (POLD1 and POLD3). In addition, FANCD2, another Fanconi Anemia (FA) protein, is also required for CC formation, likely through promoting the recruitment of BLM to the replication stressed ALT telomeres. Finally, we demonstrated that TERRA R-loops accumulate at telomeres in FANCM deficient ALT cells and downregulation of which attenuates the ALT-associated PML bodies (APBs), replication stress and CC formation. Taken together, our data suggest that FANCM prevents replisomes from stalling/collapsing at ALT telomeres by disrupting TERRA R-loops.
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