Evolution and heterogeneity of non-hereditary colorectal cancer revealed by single-cell exome sequencing.

Recently single-cell whole-exome sequencing (scWES) has deeply expanded and sharpened our knowledge of cancer evolution and subclonality. Herein, with scWES and matched bulk whole-exome sequencing (bulk WES) on two colorectal cancer (CRC) patients with normal or adenomatous polyps, we found that both the adenoma and cancer were of monoclonal origin, and both shared partial mutations in the same signaling pathways, but each showed a specific spectrum of heterogeneous somatic mutations. In addition, the adenoma and cancer further developed intratumor heterogeneity with the accumulation of nonrandom somatic mutations specifically in GPCR, PI3K-Akt and FGFR signaling pathways. We identified novel driver mutations that developed during adenoma and cancer evolution, particularly in OR1B1 (GPCR signaling pathway) for adenoma evolution, and LAMA1 (PI3K-Akt signaling pathway) and ADCY3 (FGFR signaling pathway) for CRC evolution. In summary, we demonstrated that both colorectal adenoma and CRC are monoclonal in origin, and the CRCs further diversified into different subclones with heterogeneous mutation profiles accumulating in GPCR, PI3K-Akt and FGFR signaling pathways. ScWES provides evidence for the importance of mutations in certain pathways that would not be as apparent from bulk sequencing of tumors, and can potentially establish whether specific mutations are mutually exclusive or occur sequentially in the same subclone of cells.

Cloning of a cDNA encoding cathepsin D from salamander, Hynobius leechii, and its expression in the limb regenerates.

Cathepsin D is a major lysosomal aspartic proteinase participating in the degradation or modification of intra- and extracellular matrix molecules, and its activity is known to increase in the process of tissue reorganization during the early phase of salamander limb regeneration. Here, we report the cloning of a salamander cathepsin D cDNA from Hynobius leechii and its expression profile in normal and retinoic acid (RA) treated limb regenerates. The gene expression of cathepsin D increased notably during the dedifferentiation stage and decreased gradually thereafter. Furthermore, RA that enhances dedifferentiation of regenerating salamander limb caused the elevation of cathepsin D expression both in terms of level and duration. These results suggest that cathepsin D plays important role(s) in the dedifferentiation process, and enhancement of cathepsin D expression might be closely related to RA-evoked pattern duplication.

Fringe forms a complex with Notch.

The Fringe protein of Drosophila and its vertebrate homologues function in boundary determination during pattern formation. Fringe has been proposed to inhibit Serrate-Notch signalling but to potentiate Delta-Notch signalling. Here we show that Fringe and Notch form a complex through both the Lin-Notch repeats and the epidermal growth factor repeats 22-36 (EGF22-36) of Notch when they are co-expressed. The Abruptex59b (Ax59b) and AxM1 mutations, which are caused by missense mutations in EGF repeats 24 and 25, respectively, abolish the Fringe-Notch interaction through EGF22-36, whereas the l(1)N(B) mutation in the third Lin-Notch repeat of Notch abolishes the interaction through Lin-Notch repeats. Ax mutations also greatly affect the Notch response to ectopic Fringe in vivo. Results from in vitro protein mixing experiments and subcellular colocalization experiments indicate that the Fringe-Notch complex may form before their secretion. These findings explain how Fringe acts cell-autonomously to modulate the ligand preference of Notch and why the Fringe-Notch relationship is conserved between phyla and in the development of very diverse structures.

Upregulation of cathepsin D expression in the dedifferentiating salamander limb regenerates and enhancement of its expression by retinoic acid.

In the regenerating urodele limb, catheptic activity has been known to be present in the stump tissue undergoing histolysis, and it has been implicated in the modification of some intracellular and extracellular matrix molecules. In this study, we compared spatial and temporal gene expression profiles of cathepsin D in normal, retinoic acid-treated, or denervated larval limb regenerates of Hynobius leechii and compared cathepsin D activities between normal and retinoic acid-treated limb regenerates. The results showed that the expression of cathepsin D increased markedly in the vicinity of an amputation site under the wound epidermis at dedifferentiation stage, whereas the expression level of cathepsin D was low in the denervated limb. With retinoic acid treatment, the expression of cathepsin D was elevated in terms of both level and duration. In addition, the profile of cathepsin D activity coincided well with the expression profile of cathepsin D in normal and retinoic acid-treated limb regenerates. These results suggest that the increase of cathepsin D activity during the dedifferentiation period is due to the upregulation of cathepsin D transcription, and nerve factors are involved in this process. Furthermore, retinoic acid appears to upregulate cathepsin D expression, which might be linked to the enhanced dedifferentiation in the retinoic acid-treated limb regenerates.

Pattern duplication by retinoic acid treatment in the regenerating limbs of Korean salamander larvae, Hynobius leechii, correlates well with the extent of dedifferentiation.

In the regenerating limbs of Korean salamanders, Hynobius leechii, retinoic acid (RA) induces duplication of skeletal structures in the proximodistal (PD) axis and often in the transverse axes. In the present study, the stage-dependent effects of RA for the duplication of limb skeletal structures at two amputation levels, the distal stylopodium and the distal zeugopodium, were studied using larval limbs of Korean salamanders. The results showed that the mean level of proximalization (MLP) by RA treatment increased during the stages of dedifferentiation and early bud formation while the MLP declined thereafter in both amputation levels. The decline of the MLP at the later stages of regeneration was due to the high frequency of hypomorphic regeneration or blocked regeneration. When the effects of RA treatment at two amputation levels were compared, the overall trends were similar but the actual timing was delayed for 2-4 days in the proximal level of amputation. Furthermore, the peak level of proximalization was achieved earlier and the peak level remained longer in the distal stylopodial level of amputation compared to the distal zeugopodial level of amputation. Since the histological observations revealed that the dedifferentiation period was also extended up to 2-4 days in the proximal level of amputation, the acid phosphatase activity during the course of regeneration was measured to look for a quantitative relationship between the enzyme activity and the states of dedifferentiation. The results show that the level and the duration of acid phosphatase activity in the upper arm regenerates are both higher and longer than those in the lower arm regenerates. Furthermore, RA treatment caused an increase in acid phosphatase activity. Thus our results suggest that the state of dedifferentiation might be closely linked to the extent of proximalization of regenerating limbs by RA treatment.

Stage-dependent effects of retinoic acid on the regenerating limbs of larval Korean salamander, Hynobius leechii, at two amputation levels.

The morphological and biochemical data obtained in this series of experiments clearly confirm the previous finding about the relationship between the stage of limb regeneration and the effects of RA on duplication, i.e., the most sensitive stage for RA-induced duplication is the stage of dedifferentiation, regardless of the level of amputation. However, when the RA effects were expressed as a function of time after amputation, the upper arm regenerates clearly showed a prolonged and delayed response to RA treatment compared to the lower arm regenerates. These findings were further supported by the data on acid phosphatase activity, which may reflect the level of dedifferentiation. Furthermore, the slow increase of this enzyme activity above the control level until 8 days after RA-injection corresponds to the late appearance of limb regenerates after RA treatment. At present, we do not have any clear explanation why upper arm regenerates and lower arm regenerates show differences in the MLP's and different temporal profiles for the RA-evoked limb pattern duplications. However, the difference in the level of dedifferentiation after simple amputation and after RA-treatment suggests that RA-induced changes in the positional identity of blastema cells may require a prolonged and augmented state of dedifferentiation. In this scheme, the level and type of CRABP may play a critical role as suggested in the case of regenerating axolotl limbs (McCormick et al., 1988).