The roles of ubiquitin modifying enzymes in neoplastic disease.

The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses. In this review, we highlight the known oncogenic and tumour suppressive effects of ubiquitin modifying enzymes in cancer relevant pathways with specific focus on PI3K, MAPK, TGFß, WNT, and YAP pathways. Moreover, we discuss the capacity of targeting DUBs as a novel anticancer therapeutic strategy.

Identification of proteins derived from Listeria monocytogenes inducing human dendritic cell maturation.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that can promote antitumor immunity when pulsed with tumor antigens and then matured by stimulatory agents. Despite apparent progress in DC-based cancer immunotherapy, some discrepancies were reported in generating potent DCs. Listeria monocytogenes as an intracellular microorganism is able to effectively activate DCs through engaging pattern-recognition receptors (PRRs). This study aimed to find the most potent components derived from L. monocytogenes inducing DC maturation. The preliminary results demonstrated that the ability of protein components is higher than DNA components to promote DC maturation and activation. Protein lysate fractionation demonstrated that fraction 2 HIC (obtained by hydrophobic interaction chromatography) was able to efficiently mature DCs. F2HIC-matured DCs are able to induce allogeneic CD8(+) T cells proliferation better than LPS-matured DCs and induce IFN-γ producing CD8(+) T cells. Mass spectrometry results showed that F2HIC contains 109 proteins. Based on the bioinformatics analysis for these 109 proteins, elongation factor Tu (EF-Tu) could be considered as a PRR ligand for stimulating DC maturation.

Tolerogenic dendritic cells: key regulators of peripheral tolerance in health and disease.

Dendritic cells (DCs) as professional antigen-presenting cells are able to induce immunity and tolerance in different conditions, which leads to strong immune responses against hostile agents or tolerance to self-antigens. Although for subsets of DCs, a regulatory function has been shown, the role of the microenvironment is momentous to generate tolerogenic DCs (tDCs). Different microorganisms and tumor cells escape from immune responses by producing mediators and components that influence DCs to show tolerogenic characteristics. In this review, tDC induction was explained in steady state and disease conditions. Moreover, an overview was presented on the efforts to generate in vitro tDCs, their clinical applications and the problems which remain to be solved in this field.

Adaptive Responses as Mechanisms of Resistance to BRAF Inhibitors in Melanoma.

: The introduction of v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitors in melanoma patients with BRAF (V600E) mutations has demonstrated significant clinical benefits. However, rarely do tumours regress completely. Frequently, the reason for this is that therapies targeting specific oncogenic mutations induce a number of intrinsic compensatory mechanisms, also known as adaptive responses or feedback loops, that enhance the pro-survival and pro-proliferative capacity of a proportion of the original tumour population, thereby resulting in tumour progression. In this review we will summarize the known adaptive responses that limit BRAF mutant therapy and discuss potential novel combinatorial therapies to overcome resistance.

c-Met activation leads to the establishment of a TGFß-receptor regulatory network in bladder cancer progression.

Treatment of muscle-invasive bladder cancer remains a major clinical challenge. Aberrant HGF/c-MET upregulation and activation is frequently observed in bladder cancer correlating with cancer progression and invasion. However, the mechanisms underlying HGF/c-MET-mediated invasion in bladder cancer remains unknown. As part of a negative feedback loop SMAD7 binds to SMURF2 targeting the TGFß receptor for degradation. Under these conditions, SMAD7 acts as a SMURF2 agonist by disrupting the intramolecular interactions within SMURF2. We demonstrate that HGF stimulates TGFß signalling through c-SRC-mediated phosphorylation of SMURF2 resulting in loss of SMAD7 binding and enhanced SMURF2 C2-HECT interaction, inhibiting SMURF2 and enhancing TGFß receptor stabilisation. This upregulation of the TGFß pathway by HGF leads to TGFß-mediated EMT and invasion. In vivo we show that TGFß receptor inhibition prevents bladder cancer invasion. Furthermore, we make a rationale for the use of combinatorial TGFß and MEK inhibitors for treatment of high-grade non-muscle-invasive bladder cancers.

Author Correction: c-Met activation leads to the establishment of a TGFß-receptor regulatory network in bladder cancer progression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ubiquitination and adaptive responses to BRAF inhibitors in Melanoma.

Response to targeted therapies is limited by the activation or inhibition of feedback loops. Here we report the ubiquitin specific peptidase 28/F-box WD repeat-containing protein 7 (USP28/FBW7) complex functions as a negative regulator of mitogen-activated protein kinase (MAPK) pathway by targeting v-raf murine sarcoma viral oncogene homolog B (BRAF) for degradation, a process which is lost in a large proportion of BRAF mutant melanoma patients, resulting in resistance to BRAF inhibitor therapies.

Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies.

RAF kinase inhibitors are clinically active in patients with BRAF (V600E) mutant melanoma. However, rarely do tumors regress completely, with the majority of responses being short-lived. This is partially mediated through the loss of negative feedback loops after MAPK inhibition and reactivation of upstream signaling. Here, we demonstrate that the deubiquitinating enzyme USP28 functions through a feedback loop to destabilize RAF family members. Loss of USP28 stabilizes BRAF enhancing downstream MAPK activation and promotes resistance to RAF inhibitor therapy in culture and in vivo models. Importantly, we demonstrate that USP28 is deleted in a proportion of melanoma patients and may act as a biomarker for response to BRAF inhibitor therapy in patients. Furthermore, we identify Rigosertib as a possible therapeutic strategy for USP28-depleted tumors. Our results show that loss of USP28 enhances MAPK activity through the stabilization of RAF family members and is a key factor in BRAF inhibitor resistance.

Identification of Toxoplasma gondii protein fractions induce immune response against melanoma in mice.

Dendritic cells (DCs) play a crucial role in the initiation of adaptive immune responses against tumor cells. We recently found that protein components of Toxoplasma gondii (T. gondii) could mature DCs efficiently. Therefore, in this study, we aimed to find the most effective protein components of T. gondii which are able to mature DCs and consequently instruct immune responses in tumor-bearing mice. Soluble tachyzoite antigens (STAgs) were fractionated by ammonium sulfate precipitation and subsequently by anion-exchange HPLC. Immature DCs (iDCs) were treated by these protein fractions and were monitored for IL-12p70 and IL-10 production. Moreover, the capacity of mature DCs (mDCs) to induce lymphocyte proliferation was investigated. Ultimately, we analyzed the ability of mDCs in instructing immune responses in tumor-bearing mice. We found that ammonium sulfate fraction one (A1) matured-DCs produced higher IL-12 level and IL-12/IL-10 ratio; therefore, this fraction was selected for further fractionation by anion-exchange HPLC. The results showed that anion-exchange HPLC fraction 14 (C14) matured-DCs secrete higher levels of IL-12p70 and IL-12p70/IL-10 ratio. Survival of the mice matured by A1 fraction increased significantly compared to other groups. Moreover, SDS-PAGE electrophoresis showed that different obtained fractions have distinct proteins based on their size. These results demonstrate that two protein fractions of T. gondii are able to mature DCs more efficient.

Listeria monocytogenes protein fraction induces dendritic cells maturation and T helper 1 immune responses.

Fully mature dendritic cells (DCs) play pivotal role in inducing immune responses and converting naïve T lymphocytes into functional Th1 cells. We aimed to evaluate Listeria Monocytogenes-derived protein fractions to induce DC maturation and stimulating T helper (Th)1 immune responses.In the present study, we fractionated Listeria Monocytogenes-derived proteins by adding of ammonium sulfate in a stepwise manner. DCs were also generated from C57BL/6 mice bone marrow precursor cells. Then, the effects of protein fractions on bone marrow derived DC (BMDC) maturation were evaluated. In addition, we assessed the capacity of activated DCs to induce cytokine production and proliferation of lymphocytes.Listeria-derived protein fractions induced fully mature DCs expressing high costimulatory molecules such as CD80, CD86 and CD40. DCs that were activated by selected F3 fraction had low capacity to uptake exogenous antigens while secreted high levels of Interleukine (IL)-12. Moreover, lymphocytes cultured with activated BMDCs produced high amounts of IFN-γ and showed higher proliferation than control. Listeria derived protein fractions differently influenced DC maturation.In conclusion, Listeria protein activated-BMDCs can be used as a cell based vaccine to induce anti-tumor immune responses.

Effect of HLA-B*27 and its subtypes on clinical manifestations and severity of ankylosing spondylitis in Iranian patients.

The aim of this study was to assess the role of HLA-B*27 and it's subtypes in determining severity and clinical manifestations of ankylosing spondylitis (AS).A total of 163 AS patients were assessed for clinical manifestations and severity using structured questionnaires. HLA-B*27 screening and B*27 sub-typing were performed by PCR.One hundred twenty two patients (74.8%) were B*27 positive. The male to female ratio, peripheral arthritis, steroid use, intense dorsal kyphosis and decrease of cervical slope had a significantly higher frequency in B*27 positive patients compared to B*27 negative ones (p=0.01, 0.001, 0.01, 0.04 and 0.04, respectively). However, the age of diagnosis was significantly lower in B*27 positive patients (p=0.005). Trend in uveitis and some severity markers including: BASMI and ASQoL were toward higher values in B*27 positive group with no significant difference. After controlling confounding variables, significant relationship was found only between B*27 and BASMI (p=0.01). B*27 subtypes in patients were included B*2705: 48.4%, B*2702: 42.6%, B*2704: 5.7% and B*2707: 3.3%. No significant differences were seen for severity markers and clinical manifestations between subtypes; although trend toward lower values of severity markers, less intense dorsal kyphosis and less decrease of cervical slope were observed in B*2704 and B*2707 versus other polymorphisms.Clinical features and severity of AS is influenced by HLA-B*27. Trend toward higher severity markers in B*2705 and B*2702 versus other polymorphisms might be subject of interest for evaluation in other ethnicities with concentration to other novel susceptibility genes co-inherited in each B*27 subtype.