MicroRNA therapy inhibits hepatoblastoma growth in vivo by targeting ß-catenin and Wnt signaling.

Hepatoblastoma (HBL) is the most common pediatric liver cancer. In this malignant neoplasm, beta-catenin protein accumulates and increases Wnt signaling due to recurrent activating mutations in the catenin-beta 1 (CTNNB1) gene. Therefore, beta-catenin is a key therapeutic target in HBL. However, controlling beta-catenin production with therapeutic molecules has been challenging. New biological studies could provide alternative therapeutic solutions for the treatment of HBL, especially for advanced tumors and metastatic disease. In this study, we identified microRNAs (miRNAs) that target beta-catenin and block HBL cell proliferation in vitro and tumor growth in vivo. Using our dual-fluorescence-FunREG system, we screened a library of 1,712 miRNA mimics and selected candidates inhibiting CTNNB1 expression through interaction with its untranslated regions. After validating the regulatory effect of nine miRNAs on beta-catenin in HBL cells, we measured their expression in patient samples. Let-7i-3p, miR-449b-3p, miR-624-5p, and miR-885-5p were decreased in tumors compared to normal livers. Moreover, they inhibited HBL cell growth and Wnt signaling activity in vitro partly through beta-catenin down-regulation. Additionally, miR-624-5p induced cell senescence in vitro, blocked experimental HBL growth in vivo, and directly targeted the beta-catenin 3'-untranslated region. Conclusion: Our results shed light on how beta-catenin-regulating miRNAs control HBL progression through Wnt signaling inactivation. In particular, miR-624-5p may constitute a promising candidate for miRNA replacement therapy for HBL patients. (Hepatology Communications 2017;1:168-183).

Matrix metalloproteinase-9 is a diagnostic marker of heterotopic ossification in a murine model.

Heterotopic ossification (HO) is a serious disorder that occurs when there is aberrant bone morphogenic protein (BMP) signaling in soft tissues. Currently, there are no methods to detect HO before mineralization occurs. Yet once mineralization occurs, there are no effective treatments, short of surgery, to reverse HO. Herein, we used in vivo molecular imaging and confirmatory ex vivo tissue analyses of an established murine animal model of BMP-induced HO to show that matrix metalloproteinase-9 (MMP-9) can be detected as an early-stage biomarker before mineralization. Ex vivo analyses show that active MMP-9 protein is significantly elevated within tissues undergoing HO as early as 48 h after BMP induction, with its expression co-localizing to nerves and vessels. In vivo molecular imaging with a dual-labeled near-infrared fluorescence and micro-positron emission tomography (µPET) agent specific to MMP-2/-9 expression paralleled the ex vivo observations and reflected the site of HO formation as detected from microcomputed tomography 7 days later. The results suggest that the MMP-9 is a biomarker of the early extracellular matrix (ECM) re-organization and could be used as an in vivo diagnostic with confirmatory ex vivo tissue analysis for detecting HO or conversely for monitoring the success of tissue-engineered bone implants that employ ECM biology for engraftment.

An unexpected role for keratin 10 end domains in susceptibility to skin cancer.

Keratin 10 (K10) is a type I keratin that is expressed in post-mitotic suprabasal keratinocytes of the skin. Based on cell culture experiments and transgenic mouse studies, it has been proposed that K10 suppresses cell proliferation and tumor formation in the skin. Furthermore, the ability of K10 to suppress cell proliferation was mapped to its unique N- and C-terminal protein domains. In the present study, we modified the endogenous keratin 14 (K14) gene of mice using a knock-in approach to encode a chimeric keratin that consists of the K14 rod domain fused to the K10 head and tail domains (K1014chim). This transgene was expressed in the basal layer of the epidermis and the outer root sheath of hair follicles. Unexpectedly, we found that the K10 end domains had no effect on basal keratinocyte proliferation in vivo. Moreover, when subjected to a chemical skin carcinogenesis protocol, papilloma formation in mutant mice was accelerated instead of being inhibited. Our data suggest that the increased tumor susceptibility of K1014chim mice is in part due to a suppression of apoptosis in mutant keratinocytes. Our results support the notion that intermediate filaments, in addition to their function as cytoskeletal components, affect tumor susceptibility of epithelial cells.

Desmocollin 3 is required for pre-implantation development of the mouse embryo.

Desmocollin 3 (Dsc3) is a transmembrane glycoprotein that belongs to the cadherin family of cell adhesion receptors. Together with desmoglein(s), it forms the transmembrane core of desmosomes, a multiprotein complex involved in cell adhesion, organization of the cytoskeleton, cell sorting and cell signaling. Previous reports have suggested that Dsc3 synthesis is largely restricted to stratified epithelia, and that it plays a role in the proper differentiation of these tissues during mammalian embryonic development. To test these hypotheses, we generated Dsc3-null mice. Unexpectedly, homozygous mutants show a pre-implantation lethal phenotype. In fact, most mutants die even before mature desmosomes are formed in the embryo, suggesting a new and unexpected role of Dsc3 during early development.

Tumor necrosis factor-alpha and CD80 modulate CD28 expression through a similar mechanism of T-cell receptor-independent inhibition of transcription.

Replicative senescence of human T cells is characterized by the loss of CD28 expression, exemplified by the clonal expansion of CD28(null) T cells during repeated stimulation in vitro as well as in chronic inflammatory and infectious diseases and in the normal course of aging. Because CD28 is the major costimulatory receptor for the induction of T cell-mediated immunity, the mechanism(s) underlying CD28 loss is of paramount interest. Current models of replicative senescence involve protracted procedures to generate CD28(null) cells from CD28(+) precursors; hence, a T-cell line model was used to examine the dynamics of CD28 expression. Here, we show the versatility of the JT and Jtag cell lines in tracking CD28(null) <--> CD28(hi) phenotypic transitions. JT and Jtag cells were CD28(null) and CD28(lo), respectively, but expressed high levels of CD28 when exposed to phorbol 12-myristate 13-acetate. This was a result of the reconstitution of the CD28 gene transcriptional initiator (INR). Tumor necrosis factor-alpha reduced CD28 expression because of the inhibition of INR-driven transcription. Ligation of CD28 by an antibody or by CD80 also down-regulated CD28 transcription through the same mechanism, providing evidence that CD28 can generate a T cell receptor-independent signal with a unique biological outcome. Collectively, these data unequivocally demonstrate the critical role of the INR in the regulation of CD28 expression. T cell lines with transient expression of CD28 are invaluable in the dissection of the biochemical processes involved in the transactivation of the CD28 INR, the silencing of which is a key event in the ontogenesis of senescent T cells.