The SWIB/MDM2 motif of UBE4B activates the p53 pathway.

The tumor suppressor p53 plays a critical role in cancer pathogenesis, and regulation of p53 expression is essential for maintaining normal cell growth. UBE4B is an E3/E4 ubiquitin ligase involved in a negative-feedback loop with p53. UBE4B is required for Hdm2-mediated p53 polyubiquitination and degradation. Thus, targeting the p53-UBE4B interactions is a promising anticancer strategy for cancer therapy. In this study, we confirm that while the UBE4B U box does not bind to p53, it is essential for the degradation of p53 and acts in a dominant-negative manner, thereby stabilizing p53. C-terminal UBE4B mutants lose their ability to degrade p53. Notably, we identified one SWIB/Hdm2 motif of UBE4B that is vital for p53 binding. Furthermore, the novel UBE4B peptide activates p53 functions, including p53-dependent transactivation and growth inhibition, by blocking the p53-UBE4B interactions. Our findings indicate that targeting the p53-UBE4B interaction presents a novel approach for p53 activation therapy in cancer.

DNA damage response revisited: the p53 family and its regulators provide endless cancer therapy opportunities.

Antitumor therapeutic strategies that fundamentally rely on the induction of DNA damage to eradicate and inhibit the growth of cancer cells are integral approaches to cancer therapy. Although DNA-damaging therapies advance the battle with cancer, resistance, and recurrence following treatment are common. Thus, searching for vulnerabilities that facilitate the action of DNA-damaging agents by sensitizing cancer cells is an active research area. Therefore, it is crucial to decipher the detailed molecular events involved in DNA damage responses (DDRs) to DNA-damaging agents in cancer. The tumor suppressor p53 is active at the hub of the DDR. Researchers have identified an increasing number of genes regulated by p53 transcriptional functions that have been shown to be critical direct or indirect mediators of cell fate, cell cycle regulation, and DNA repair. Posttranslational modifications (PTMs) primarily orchestrate and direct the activity of p53 in response to DNA damage. Many molecules mediating PTMs on p53 have been identified. The anticancer potential realized by targeting these molecules has been shown through experiments and clinical trials to sensitize cancer cells to DNA-damaging agents. This review briefly acknowledges the complexity of DDR pathways/networks. We specifically focus on p53 regulators, protein kinases, and E3/E4 ubiquitin ligases and their anticancer potential.

p63, a key regulator of Ago2, links to the microRNA-144 cluster.

As a key component of the RNA-induced silencing complex (RISC), Argonaute2 (Ago2) exhibits a dual function regulatory role in tumor progression. However, the mechanistic basis of differential regulation remains elusive. p63 is a homolog of the tumor suppressor p53. p63 isoforms play a critical role in tumorigenesis and metastasis. Herein, we show that p63 isoforms physically interact with and stabilize Ago2. Expression of p63 isoforms increases the levels of Ago2 protein, while depletion of p63 isoforms by shRNA decreases Ago2 protein levels. p63 strongly guides Ago2 dual functions in vitro and in vivo. Ectopic expression of the miR-144/451 cluster increases p63 protein levels; TAp63 transactivates the miR-144/451 cluster, forming a positive feedback loop. Notably, miR-144 activates p63 by directly targeting Itch, an E3 ligase of p63. Ectopic expression of miR-144 induces apoptosis in H1299 cells. miR-144 enhances TAp63 tumor suppressor function and inhibits cell invasion. Our findings uncover a novel function of p63 linking the miRNA-144 cluster and the Ago2 pathway. FACTS AND QUESTIONS: Identification of Ago2 as a p63 target. Ago2 exhibits a dual function regulatory role in tumor progression; however, the molecular mechanism of Ago2 regulation remains unknown. p63 strongly guides Ago2 dual functions in vitro and in vivo. Unraveling a novel function of p63 links the miRNA-144 cluster and the Ago2 pathway.

Regulation of the tumor suppressor PTEN in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer (BCa) in which estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) are not expressed. Although TNBC cases account for approximately 15% of all BCa cases, TNBC patients' prognosis is poor compared with that of other BCa subtypes. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) plays an important role in cell proliferation and migration by negatively regulating the PI3K/Akt pathway. PTEN is one of the most commonly inactivated tumor suppressors in BCa. PTEN inactivity is associated with larger tumor sizes, multiple lymph node metastases, and an aggressive triple-negative phenotype. This review primarily focuses on two key points: (1) PTEN and its function. (2) The regulation of tumor suppressor PTEN in TNBC. We provide a summary of genomic alterations of PTEN in BCa. We further discuss the transcriptional regulation of PTEN and how PTEN is regulated by posttranscription and posttranslational modification, as well as by protein interactions. Finally, we discuss the perspectives of the PTEN protein in TNBC.

Hsp70 acts as a fine-switch that controls E3 ligase CHIP-mediated TAp63 and ΔNp63 ubiquitination and degradation.

The major clinical problem in human cancer is metastasis. Metastases are the cause of 90% of human cancer deaths. TAp63 is a critical suppressor of tumorigenesis and metastasis. ΔNp63 acts as a dominant-negative inhibitor to block the function of p53 and TAp63. Although several ubiquitin E3 ligases have been reported to regulate p63 stability, the mechanism of p63 regulation remains partially understood. Herein, we show that CHIP, an E3 ligase with a U-box domain, physically interacts with p63 and promotes p63 degradation. Notably, Hsp70 depletion by siRNA stabilizes TAp63 in H1299 cells and destabilizes ΔNp63 in SCC9 cells. Loss of Hsp70 results in a reduction in the TAp63-CHIP interaction in H1299 cells and an increase in the interaction between ΔNp63 and CHIP in SCC9 cells. Our results reveal that Hsp70 acts as a molecular switch to control CHIP-mediated ubiquitination and degradation of p63 isoforms. Furthermore, regulation of p63 by the Hsp70-CHIP axis contributes to the migration and invasion of tumor cells. Hence, our findings demonstrate that Hsp70 is a crucial regulator of CHIP-mediated ubiquitination and degradation of p63 isoforms and identify a new pathway for maintaining TAp63 or ΔNp63 stability in cancers.

Pirh2, an E3 ligase, regulates the AIP4-p73 regulatory pathway by modulating AIP4 expression and ubiquitination.

Pirh2 is an E3 ligase belonging to the RING-H2 family and shown to bind, ubiquitinate and downregulate p73 tumor suppressor function without altering p73 protein levels. AIP4, an E3 ligase belonging to the HECT domain family, has been reported to be a negative regulatory protein that promotes p73 ubiquitination and degradation. Herein, we found that Pirh2 is a key regulator of AIP4 that inhibits p73 function. Pirh2 physically interacts with AIP4 and significantly downregulates AIP4 expression. This downregulation is shown to involve the ubiquitination of AIP4 by Pirh2. Importantly, we demonstrated that the ectopic expression of Pirh2 inhibits the AIP4-p73 negative regulatory pathway, which was restored when depleting endogenous Pirh2 utilizing Pirh2-siRNAs. We further observed that Pirh2 decreases AIP4-mediated p73 ubiquitination. At the translational level and specifically regarding p73 cell cycle arrest function, Pirh2 still ensures the arrest of p73-mediated G1 despite AIP4 expression. Our study reveals a novel link between two E3 ligases previously thought to be unrelated in regulating the same effector substrate, p73. These findings open a gateway to explain how E3 ligases differentiate between regulating multiple substrates that may belong to the same family of proteins, as it is the case for the p53 and p73 proteins.

Graphene Oxide Nanoparticles Induce Apoptosis in wild-type and CRISPR/Cas9-IGF/IGFBP3 knocked-out Osteosarcoma Cells.

Osteosarcoma affects both adolescents and adults, and some improvement in the survival rate for affected patients has been reached in the last decade. Still, non-specificity and systemic toxicity may limit traditional therapeutic approaches to some extent. The insulin growth factor 1 (IGF1) and its binding protein (IGFBP3) have been implicated in the tumorigenesis. Nanoparticles, such as graphene oxide (GO), can provide an effective treatment for cancer as they can specifically target cancer cells while reducing undesired side effects. This study aimed to evaluate the toxicity of GO on osteosarcoma in vitro using tumor cell lines with and without knocking out the IGF and IGFBP3 genes. Human osteosarcoma cell lines, U2OS and SAOS2, and the normal osteoblast cell line hFOB1.19 were used. The IGF1 and IGFBP3 genes were eliminated using CRISPR/Cas9. Tumor cells were cultured and treated with GO. Apoptosis and reactive oxygen species (ROS) were analyzed by Annexin V-FITC and ROS assays. The nuclear factor erythroid 2-related factor 2 (NRF2), which is a crucial regulator of cellular resistance to oxidants, was investigated by Western blotting. We found a significantly higher rate of apoptosis in the OS than hFOB1.19, especially in U2OS cells in which IGF1 and IGFBP3 were knocked out. ROS increase due to GO exposure was remarkably time and concentration-dependent. Based on the rate of apoptosis, ROS, Nrf-2 decrease, and cytomorphological changes, GO has a significant cytotoxic effect against OS. Targeting the IGF1 and IGFBP3 signaling pathway may strengthen GO-related cytotoxicity with the potential to increase the survival of patients affected by this tumor.

Cancer Stem Cells and their Management in Cancer Therapy.

BACKGROUND: In the last decade, the proposed Cancer Stem Cell (CSC) hypothesis has steadily changed the way cancer treatment is approached. CSCs may be the source of the heterogeneous non-tumorigenic cell population included in a neoplasm. Intratumor and intertumoral heterogeneity is a well-known phenomenon that massively entangles the diagnosis and treatment of cancer. The literature seems to suggest that heterogeneity develops progressively within tumor-initiating stem cells. CSCs harbor genetic and/or epigenetic alterations that allow them to differentiate into multiple tumor cell types sequentially. OBJECTIVE: The CSC hypothesis, cellular therapy, and the most recent patents on CSCs were reviewed. METHODS: PubMed, Scopus, and Google Scholar were screened for this information. Also, an analysis of the most recent data targeting CSCs in pediatric cancer developed at two Canadian institutions is provided. The genes involved with the activation of CSCs and the drugs used to antagonize them are also highlighted. RESULTS: It is underlined that (1) CSCs possess stem cell-like properties, including the ability for self-renewal; (2) CSCs can start carcinogenesis and are responsible for tumor recurrence after treatment; (3) Although some limitations have been raised, which may oppose the CSC hypothesis, cancer progression and metastasis have been recognized to be caused by CSCs. CONCLUSION: The significant roles of cell therapy may include an auto-transplant with high-dose treatment, an improvement of the immune function, creation of chimeric antigen receptor T cells, and the recruitment of NK cell-based immunotherapy.

Osteosarcoma progression is associated with increased nuclear levels and transcriptional activity of activated ß-Catenin.

Osteosarcoma (OS) is an aggressive primary bone malignancy that has peak incidence in children and young adults <25 years of age. Despite current multimodal treatments, no significant change in patient outcome has been observed in two decades. Presently, there is a lack of established, reliable baseline prognostic markers for aggressive OS, other than extent and site of disease involvement. The canonical Wnt/ß-catenin pathway controls multiple cellular processes, and is known to be a critical pathway in OS progression. This pathway regulates cellular levels of ß-catenin, which is a significant player in the oncogenesis and progression of many cancers. We investigated the relationship between ß-catenin, more specifically, the transcriptionally active form of ß-catenin, Activated ß-Catenin (ABC), and OS progression. Using an in vitro model, we observed that cellular/nuclear ABC levels, but not cellular/nuclear ß-catenin levels, increase with the degree of aggressiveness in OS. Our results demonstrate a strong association between nuclear-ABC levels and aggressive OS in vitro. Furthermore, we observed significant correlation between positive nuclear-ABC and patient age and tumor stage. Our results support the potential use of ABC as a predictive marker for risk stratification in OS.

The Regulation of Tumor Suppressor p63 by the Ubiquitin-Proteasome System.

The protein p63 has been identified as a homolog of the tumor suppressor protein p53 and is capable of inducing apoptosis, cell cycle arrest, or senescence. p63 has at least six isoforms, which can be divided into two major groups: the TAp63 variants that contain the N-terminal transactivation domain and the ΔNp63 variants that lack the N-terminal transactivation domain. The TAp63 variants are generally considered to be tumor suppressors involved in activating apoptosis and suppressing metastasis. ΔNp63 variants cannot induce apoptosis but can act as dominant negative inhibitors to block the function of TAp53, TAp73, and TAp63. p63 is rarely mutated in human tumors and is predominately regulated at the post-translational level by phosphorylation and ubiquitination. This review focuses primarily on regulation of p63 by the ubiquitin E-3 ligase family of enzymes via ubiquitination and proteasome-mediated degradation, and introduces a new key regulator of the p63 protein.

Differential SIRT1 expression in hepatocellular carcinomas and cholangiocarcinoma of the liver.

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are two major liver malignancies. Although some phenotypic overlap is known, HCC and CCA are usually different with regard to etiology, histology, and prognosis. Gene expression and deacetylase activity of the class III histone deacetylase SIRT1 are up-regulated in cancer cells due to oncogene overexpression or loss of function of tumor suppressor genes. SIRT1 may play a critical role in tumor initiation, progression, and drug resistance by blocking senescence and apoptosis, and promoting cell growth and angiogenesis, but pleiotropic effects (synchronous or metachronous anti-proliferation and anti-apoptotic mechanisms) have been suggested in some cancers. Our aim was to investigate the expression of SIRT1 in liver epithelial malignancies. Thirty carcinomas of the liver, including 16 HCC and 14 CCA cases, were investigated by immunohistochemistry using monoclonal antibodies against SIRT1 and p53. Western blot analysis (WBA) was carried out for expression of SIRT1 in three CCA cell lines, one HCC cell line, and one cell line of Papova-immortalized normal hepatocytes. An expression of SIRT1 was found in 11 of 16 (68.75%) HCC and in 5 of 14 (35.71%) CCA. Moreover, we found an expression of p53 in 8 out of 16 (50%) HCC and 13 out of 14 (92.86%) CCA. WBA showed expression of SIRT1 in all cell lines studied, although a stronger signal was seen in the HCC cell line. Immunohistochemical data did not correlate to clinical stage or other clinical or histopathological parameters. Sirtuin 1 is a phylogenetically-conserved family of deacetylases and our data seem to indicate that (1) pleiotropic effects may be present in hepatic epithelial malignancies, and (2) there is no specificity of SIRT1 for either HCC or CCA.

Cholangiocarcinoma: risk factors, environmental influences and oncogenesis.

Cholangiocarcinoma (CCA) is one of the most frequent malignant epithelial liver tumors after hepatocellular carcinoma (HCC). Its incidence seems to be increasing worldwide, although risk factors are heterogeneous and differ globally. Although diagnostic and therapeutic medicine have advanced in several countries, tackling this tumor remains a challenge. The causes of CCA's increasing incidence are likely a differential increment of some factors according to the geographical area, which will be considered in this review. Environment-linked risk factors may play a critical role in the carcinogenesis. Liver flukes may play a major role in East Asia, while exposure to chemical compounds, such as naphthenic acids, has been postulated as a source of the rate increase in Western countries. Carcinogenesis is variable and confounding factors also need to be taken into account. Carcinogenesis depends on a sequential process and most probably involves both cholestasis and chronic inflammation as promoting steps after induction. The release and interaction of interleukin-6 (IL-6), transforming growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha), and platelet-derived growth factor (PDGF) are at the basis of the proliferation of biliary epithelial cells or cholangiocytes. Additional steps for the final development of CCA may also involve an increase of the mutation rate of tumor suppressor genes, such as TP53, and the evasion of apoptosis.

Twisted gastrulation expression in cholangiocellular and hepatocellular carcinoma.

AIMS: To assess the expression of Twisted gastrulation (TWSG1) protein, which regulates the activity of bone morphogenetic proteins (BMPs) in the extracellular space in malignant epithelial tumours of the liver. METHODS: Thirteen hepatocellular carcinoma (HCC) samples and 12 intrahepatic cholangiocellular carcinoma (CCA) samples were compiled into diagnosis-specific tissue microarrays. Sections were immunostained with a monoclonal antibody against TWSG1 and a polyclonal antibody against BMP4. Human cell lines were also used, including one HCC cell line (HepG2), three CCA cell lines (OZ, Huh-28, HuCCT-1) and a Papova-immortalised normal hepatocyte cell line (THLE-3) for western blot analysis (WBA). RESULTS: Immunostaining and WBA showed a stronger TWSG1 expression in CCA than in HCC. The difference in expression was significant (p<0.05), and the immunohistochemical signal was particularly evident in the malignant epithelial areas close to desmoplastic stroma in CCA and in the areas of glandular differentiation in HCC. No expression was seen in normal hepatocytes. Interestingly, BMP4 was fully expressed in CCA and only partly in HCC. WBA showed a band for BMP4 in both CCA and HCC cell lines. CONCLUSIONS: TWSG1 is expressed in both malignant epithelial carcinomas, although the level of expression is higher in CCA than in HCC and seems to correlate at least partially with BMP4 expression.

Canine liver transplantation model and the intermediate filaments of the cytoskeleton of the hepatocytes.

Liver transplantation has been a successful therapy for liver failure. However, a significant number of recipients suffer from graft dysfunction. Considerably, ischemia and reperfusion (I/R) injury is the most important factor leading to organ dysfunction, although the pathogenesis has not been fully described. I/R injury have several established features that are accompanied by and/or linked to bile duct loss or ductopenia, cholestasis, and biliary ductular proliferations in the posttransplant liver biopsy. However, biliary marker levels increase usually only 5-7 days after transplantation. Intermediate filaments are one of the three cytoskeletal proteins that have a major role in liver protection and maintaining both cellular structure and integrity of eukaryotic cells. We reviewed the canine liver transplantation model as I/R injury model to delineate the intermediate filaments of the cytoskeleton that are probably the determinants in changing the phenotype of hepatocytes to cholangiocytes. Remarkably, this interesting feature seems to occur earlier than frank cholestasis. We speculate that I/R liver injury through a phenotypical switch of the hepatocytes may contribute to the poor outcome of the liver graft.

Expression of E-cadherin and ß-catenin in two cholangiocarcinoma cell lines (OZ and HuCCT1) with different degree of invasiveness of the primary tumor.

BACKGROUND: Cholangiocarcinoma (CC) is the most frequent malignant epithelial tumor of the biliary system. CC has received increasing interest due to its different etiologic factors, invasiveness, and the difficulty of diagnosis at an early stage. The pathogenesis of CC has not been clearly defined, but cohesiveness of tumor cells seems to be a critical factor. Calcium-dependent adherence proteins or cadherins are a family of proteins essential for connecting the plasma membrane of adjacent cells. Linkage of cadherins with the cytoskeleton occurs through another class of proteins, called catenins. E-cadherin forms a mutually exclusive complex or unit with ß-catenin. Loss of E-cadherin -ß-catenin adhesion represents an important step in the progression of many epithelial malignancies. Cell lines arising from CC are not often investigated and may show a differential expression of cell adhesion molecules, particularly E-cadherin - ß-catenin. We hypothesized that a moderately invasive cell line of CC may co-localize both molecules in cytoplasm and cytoplasmic membrane, indicating a greater "tightness" of the tumor cells, while a metastasizing cell line may show isolated cytoplasmic membrane localization, indicating tumor cells probably more keen to reach the blood stream and give metastases. Thus, our aim was to investigate the expression and localization of E-cadherin and ß-catenin in two CC cell lines, including a rapidly metastasizing cell line and a moderately invasive cell line, correlating to a different degree of invasiveness of the primary tumor. MATERIALS AND METHODS: OZ and HuCCT1 cells represent homogeneous, functional human biliary epithelial tumor cell lines that were originally isolated in Japan. Following cell line growth we extracted total proteins. Western blot analysis, immunofluorescence and confocal laser microscopy were used to identify the protein expression and their cyto-localization and co-localization. RESULTS: Both CC cell lines expressed E-cadherin and ß-catenin, but they showed remarkably different localization patterns. In HuCCT1, both E-cadherin and ß-catenin were localized in the cytoplasm, while in OZ these proteins were localized in the cytoplasmic membrane only. This was attributed to a different degree of invasiveness of the primitive CC from which the cell lines were characterized, OZ being a metastasizing cell line, HuCCT1 being a moderately invasive cell line. CONCLUSION: To the best of our knowledge, this is the first time that E-cadherin and ß-catenin have been studied in detail in these two cell lines. These data seem to be very promising in terms of adding insight into the cell biology of CC and initiating investigations that aim to identify cytoskeletal dynamics and ultimately provide guidelines for developing new therapeutic strategies.