Upregulation of dihydropyrimidinase-like 3 (DPYSL3) protein predicts poor prognosis in urothelial carcinoma.

BACKGROUND: Dihydropyrimidinase-like 3 (DPYSL3) is a cytosolic phosphoprotein expressed in the nervous system and is crucial for neurogenesis. A previous study showed that increased DPYSL3 expression promotes tumour aggressiveness in pancreatic ductal adenocarcinoma, gastric cancer, and colon cancer. However, the role of DPYSL3 in affecting the biological behaviour of urothelial carcinoma (UC) is not yet understood. METHODS: A UC transcriptomic dataset from the Gene Expression Omnibus and the Urothelial Bladder Cancer (BLCA) dataset from The Cancer Genome Atlas were used for the in silico study. We collected 340 upper urinary tract urothelial carcinoma (UTUC) and 295 urinary bladder urothelial carcinoma (UBUC) samples for the immunohistochemical study. Fresh tumour tissue from 50 patients was used to examine the DPYSL3 mRNA level. In addition, urothelial cell lines with and without DPYSL3 knockdown were used for the functional study. RESULTS: The in silico study revealed that DPYSL3 correlated with advanced tumour stage and metastasis development while functioning primarily in the nucleobase-containing compound metabolic process (GO:0006139). DPYSL3 mRNA expression is significantly upregulated in advanced UC. Furthermore, overexpression of the DPYSL3 protein is significantly associated with the aggressive behaviour of UTUC and UBUC. DPYSL3 expression independently predicts disease-specific survival (DSS) and metastatic-free survival (MFS) in patients with UC. In non-muscle-invasive UBUC, DPYSL3 expression predicts local recurrence-free survival. UC cell lines with DPYSL3 knockdown exhibited decreased proliferation, migration, invasion, and human umbilical vein endothelial cells (HUVECs) tube formation but increased apoptosis and G1 arrest. Gene ontology enrichment analysis revealed that the enriched processes related to DPYSL3 overexpression in UC were tissue morphogenesis, cell mesenchyme migration, smooth muscle regulation, metabolic processes, and RNA processing. In vivo study revealed DPYSL3 knockdown in UC tumours significantly suppressed the growth of tumours and decreased MYC and GLUT1 protein expression. CONCLUSIONS: DPYSL3 promotes the aggressiveness of UC cells by changing their biological behaviours and is likely associated with cytoskeletal and metabolic process modifications. Furthermore, DPYSL3 protein overexpression in UC was associated with aggressive clinicopathological characteristics and independently predicted poor clinical outcomes. Therefore, DPYSL3 can be used as a novel therapeutic target for UC.

Regulation of Dihydropyrimidinase-like 3 Gene Expression by MicroRNAs in PC12 Cells with Induced Ischaemia and Hypothermia.

Although hypothermic treatment has been reported to have some beneficial effects on ischaemia at the clinical level, the mechanism of ischaemia suppression by hypothermia remains unclear due to a lack of mechanism understanding and insufficient data. The aim of this study was to isolate and characterize microRNAs specifically expressed in ischaemia-hypothermia for the dihydropyrimidinase-like 3 (Dpysl3) gene. PC12 cells were induced with CoCl2 for chemical ischaemia and incubated at 32 ℃ for hypothermia. In ischaemia-hypothermia, four types of microRNAs (miR-106b-5p, miR-194-5p, miR-326-5p, and miR-497-5p) were highly related to the Dpysl3 gene based on exosomal microRNA analysis. Dpysl3 gene expression was up-regulated by miR-497-5p but down-regulated by miR-106b-5p, miR-194-5p and miR-326-5p. Our results suggest that these four microRNAs are involved in the regulation of Dpysl3 gene expression. These findings provide valuable clues that exosomal microRNAs could be used as therapeutic targets for effective treatment of ischaemia.

DPYSL3 modulates mitosis, migration, and epithelial-to-mesenchymal transition in claudin-low breast cancer.

A Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomic analysis prioritized dihydropyrimidinase-like-3 (DPYSL3) as a multilevel (RNA/protein/phosphoprotein) expression outlier specific to the claudin-low (CLOW) subset of triple-negative breast cancers. A PubMed informatics tool indicated a paucity of data in the context of breast cancer, which further prioritized DPYSL3 for study. DPYSL3 knockdown in DPYSL3-positive ([Formula: see text]) CLOW cell lines demonstrated reduced proliferation, yet enhanced motility and increased expression of epithelial-to-mesenchymal transition (EMT) markers, suggesting that DPYSL3 is a multifunctional signaling modulator. Slower proliferation in DPYSL3-negative ([Formula: see text]) CLOW cells was associated with accumulation of multinucleated cells, indicating a mitotic defect that was associated with a collapse of the vimentin microfilament network and increased vimentin phosphorylation. DPYSL3 also suppressed the expression of EMT regulators SNAIL and TWIST and opposed p21 activated kinase 2 (PAK2)-dependent migration. However, these EMT regulators in turn induce DPYSL3 expression, suggesting that DPYSL3 participates in negative feedback on EMT. In conclusion, DPYSL3 expression identifies CLOW tumors that will be sensitive to approaches that promote vimentin phosphorylation during mitosis and inhibitors of PAK signaling during migration and EMT.

Inhibition of cell-adhesion protein DPYSL3 promotes metastasis of lung cancer.

BACKGROUND: Our previous screening study suggested that the cell-adhesions protein Dihydropyrimidinase-like 3 (DPYSL3) was a candidate metastatic lung cancer related molecule. This study aimed to analyze the correlation between DPYSL3 and metastatic lung cancer. METHODS: Stable DPYSL3 knockdown Lewis lung carcinoma (LLC) cells were constructed with a retroviral system. Cell migration and invasion assays were performed to determine the role of DPYSL3 in LLC cells' migration and invasion changes. A metastatic lung tumor model in which the stable DPYSL3 knockdown LLC cells were injected through tail vein was used to analyze the role of DPYSL3 in tumor metastasis in vivo. The correlation between DPYSL3 expression and the survival time of lung cancer patients were analyzed in KMPLOT database. RESULTS: Knockdown of DPYSL3 promoted the migratory and invasive of LLC cells compared to the control group. Meanwhile, the motility of LLC cells was also increased with the inhibition of DPYSL3. The TGFß-induced EMT increased when DPYSL3 was inhibited. The expression of EMT markers, TWIST1 and N-cadherin, significantly increased to almost two times with the knockdown of DPYSL3. Furthermore, inhibition of DPYSL3 promoted the progression of metastatic xenograft in C57BL/6 mice. The expression level of DPYSL3 decreased in lung cancer patients with distant metastasis. CONCLUSIONS: Knockdown of DPYSL3 promoted the metastatic ability of LLC cells in vitro and in vivo.

Suppression of Prostate Cancer Metastasis by DPYSL3-Targeted saRNA.

Metastasis is the sole cause of cancer death and there is no curable means in clinic. Cellular protein CRMP4 (DPYSL3 gene) was previously defined as a metastasis suppressor in human prostate cancers since its expression is dramatically reduced in lymphatic metastatic diseases and DPYSL3 overexpression in prostate cancer cells significantly suppressed cancer cell migration and invasion. To develop a CRMP4-based antimetastasis therapeutic approach, the small activating RNA (saRNA) technique was utilized to enhance CRMP4 expression in prostate cancer cells. A total of 14 saRNAs were synthesized and screened in multiple prostate cancer cell lines. Two saRNAs targeting the isoform-2 promoter region were determined to have significant activating effect on DPYSL3 gene expression at the mRNA and protein levels. These saRNA also largely reduced prostate cancer cell migration and invasion in vitro and in vivo. Most significantly, PSMA aptamer-mediated prostate cancer cell homing of these saRNAs blocked distal metastasis in an orthotopic nude mouse model. In conclusion, our data demonstrated that saRNA-based DPYSL3 gene enhancement is capable of suppressing tumor metastasis in prostate cancer, which provides a potential therapeutic approach for cancer management.

CRMP4 Up-regulates M2 Macrophages and Myeloid-derived Suppressor Cells to Promote Pancreatic Cancer in Mice.

BACKGROUND/AIM: We previously observed higher prevalence of high-grade pancreatic intraepithelial neoplasia (PanIN) in LSL-KrasG12D/+; Pdx1Cre/+ (KC-Crmp4wild) mice than LSL-KrasG12D/+; Pdx1Cre/+; Crmp4-/- (KC-Crmp4-/-) mice. This study investigated the relationship between collapsin response mediator protein 4 (CRMP4) and immune cell infiltration in pancreatic cancer. MATERIALS AND METHODS: PanIN was induced by intraperitoneal injection of caerulein into KC-Crmp4wild and KC-Crmp4-/- mice, and immune cells in PanIN lesions were compared. Subcutaneous tumors were created by injecting Pan02 cells, and tumor diameter was compared between Crmp4wild and Crmp4-/- mice every 7 days. Peritumoral immune cells were examined immunohisto chemically. RESULTS: High-grade PanIN in KC mice showed statistically significantly high expression of CD163 (p=0.031) and CD11b (p=0.027). Following subcutaneous injection of Pan02 cells, tumor diameter was greater in Crmp4wild mice than Crmp4-/- mice. Crmp4wild mice exhibited higher CD163 and CD11b expression than Crmp4-/- mice in tumors (p<0.001). CONCLUSION: CRMP4 might promote pancreatic cancer by up-regulating M2 macrophages and myeloid-derived suppressor cells.

CRMP4 Inhibits Bone Formation by Negatively Regulating BMP and RhoA Signaling.

We identified the neuroprotein collapsing response mediator protein-4 (CRMP4) as a noncanonical osteogenic factor that regulates the differentiation of mouse bone marrow skeletal stem cells (bone marrow stromal stem cells [mBMSCs]) into osteoblastic cells. CRMP4 is the only member of the CRMP1-CRMP5 family to be expressed by mBMSCs and in osteoprogenitors of both adult mouse and human bones. In vitro gain-of-function and loss-of-function of CRMP4 in murine stromal cells revealed its inhibitory effect on osteoblast differentiation. In addition, Crmp4-deficient mice (Crmp4-/- ) displayed a 40% increase in bone mass, increased mineral apposition rate, and bone formation rate, compared to wild-type controls. Increased bone mass in Crmp4-/- mice was associated with enhanced BMP2 signaling and BMP2-induced osteoblast differentiation in Crmp4-/- osteoblasts (OBs). Furthermore, Crmp4-/- OBs exhibited enhanced activation of RhoA/focal adhesion kinase (FAK) signaling that led to cytoskeletal changes with increased cell spreading. In addition, Crmp4-/- OBs exhibited increased cell proliferation that was mediated via inhibiting cyclin-dependent kinase inhibitor 1B, p27Kip1 and upregulating cyclin D1 expression which are targets of RhoA signaling pathway. Our findings identify CRMP4 as a novel negative regulator of osteoblast differentiation. © 2016 American Society for Bone and Mineral Research.

Enhancing DPYSL3 gene expression via a promoter-targeted small activating RNA approach suppresses cancer cell motility and metastasis.

To explore a novel strategy in suppressing tumor metastasis, we took the advantage of a recent RNA activation (RNAa) theory and used small double-strand RNA molecules, termed as small activating RNAs (saRNA) that are complimentary to target gene promoter, to enhance transcription of metastasis suppressor gene. The target gene in this study is Dihydro-pyrimidinase-like 3 (DPYSL3, protein name CRMP4), which was identified as a metastatic suppressor in prostate cancers. There are two transcriptional variants of DPYSL3 gene in human genome, of which the variant 2 is the dominant transcript (DPYSL3v2, CRMP4a) but is also significantly down-regulated in primary prostate cancers. A total of 8 saRNAs for DPYSL3v1 and 14 saRNAs for DPYSL3v2 were tested in multiple prostate cancer cell lines. While none of the saRNAs significantly altered DPYSL3v1 expression, 4 saRNAs showed a strong enhancing effect on DPYSL3v2 expression, resulting in reduced cell mobility in vitro. To achieve a prostate cancer-specific delivery for in vivo testing, we conjugated the most potent saV2-9 RNA molecule with the prostate-specific membrane antigen (PSMA)-targeting aptamer A10-3.2. The conjugates successful increased DPYSL3v2 gene expression in PSMA-positive but not PSMA-negative prostate cancer cells. In nude mice bearing orthotopic xenograft of prostate cancer, a 10-day consecutive treatment with the saV2-9 conjugates significantly suppress distal metastasis compared to the control saRNAs. Analysis of xenograft tissues revealed that DPYSL3v2 expression was largely increased in saV2-9 conjugate-treated group compared to the control group. In conclusion, DPYSL3v2 promoter-targeted saRNA molecules might be used as an adjunctive therapy to suppress prostate cancer metastasis.

Glycidol induces axonopathy and aberrations of hippocampal neurogenesis affecting late-stage differentiation by exposure to rats in a framework of 28-day toxicity study.

Developmental exposure to glycidol induces aberrations of late-stage neurogenesis in the hippocampal dentate gyrus of rat offspring, whereas maternal animals develop axonopathy. To investigate the possibility whether similar effects on adult neurogenesis could be induced by exposure in a framework of 28-day toxicity study, glycidol was orally administered to 5-week-old male Sprague-Dawley rats by gavage at 0, 30 or 200 mg/kg for 28 days. At 200 mg/kg, animals revealed progressively worsening gait abnormalities as well as histopathological and immunohistochemical changes suggestive of axonal injury as evidenced by generation of neurofilament-L(+) spheroids in the cerebellar granule layer and dorsal funiculus of the medulla oblongata, central chromatolysis in the trigeminal nerve ganglion cells and axonal degeneration in the sciatic nerves. At the same dose, animals revealed aberrations in neurogenesis at late-stage differentiation as evidenced by decreases of both doublecortin(+) and dihydropyrimidinase-like 3(+) cells in the subgranular zone (SGZ) and increased reelin(+) or calbindin-2(+) γ-aminobutyric acid-ergic interneurons and neuron-specific nuclear protein(+) mature neurons in the dentate hilus. These effects were essentially similar to that observed in offspring after maternal exposure to glycidol. These results suggest that glycidol causes aberrations in adult neurogenesis in the SGZ at the late stage involving the process of neurite extension similar to the developmental exposure study in a standard 28-day toxicity study.

Dihydropyrimidinase-like 3 regulates the inflammatory response of activated microglia.

Microglia, the resident immune cells of the CNS, are known to respond to injuries, infection and inflammation in the CNS by producing proinflammatory cytokines and phagocytosing cell debris and pathogens. In this study, we investigated the expression pattern and role of dihydropyrimidinase-like 3 (Dpysl3), a member of collapsin response mediator protein family, on the inflammatory reaction of microglia. Microarray analysis comparing the global gene expression profile of ameboid and ramified microglia has shown that Dpysl3 is mainly expressed in ameboid microglia in the 5-day postnatal rat brain. Immunohistochemical analysis revealed that Dpysl3 was intensely expressed in ameboid microglial cells in the rat brain till postnatal 7th day and then gradually diminished in ramified microglia of 2 weeks postnatal rat brain. Further, in vitro analysis confirmed that Dpysl3 expression was induced in activated BV-2 microglia treated with lipopolysaccharide (LPS). It is well documented that microglial activation by LPS increased the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines through the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity in BV-2 microglia. However, siRNA-mediated knockdown of Dpysl3 prevented the LPS-induced expression of iNOS and cytokines including interleukin-1 beta, and tumor necrosis factor-alpha as well as nuclear translocation of NF-κB in microglia. Remarkably, knockdown of Dpysl3 inhibited the migration of activated microglia coupled with deranged actin filament configuration (as revealed by F-actin cytoskeleton expression) in lamellipodia projecting from the cells. Knockdown of Dpysl3 also inhibited the phagocytic ability of activated microglia. These findings suggest that knockdown of Dpysl3 can inhibit activation, migration and phagocytic capability of microglia and consequently reduce neuroinflammation.