Specific Downstream Effectors Research Articles

Subclinical dose irradiation triggers human breast cancer migration via mitochondrial reactive oxygen species

BackgroundDespite technological advances in radiotherapy, cancer cells at the tumor margin and in diffusive infiltrates can receive subcytotoxic doses of photons. Even if only a minority of cancer cells are concerned, phenotypic consequences could be important considering that mitochondrial DNA (mtDNA) is a primary target of radiation and that damage to mtDNA can persist. In turn, mitochondrial dysfunction associated with enhanced mitochondrial ROS (mtROS) production could promote cancer cell migration out of the irradiation field in a natural attempt to escape therapy. In this study, using MCF7 and MDA-MB-231 human breast cancer cells as models, we aimed to elucidate the molecular mechanisms supporting a mitochondrial contribution to cancer cell migration induced by subclinical doses of irradiation (< 2 Gy).MethodsMitochondrial dysfunction was tested using mtDNA multiplex PCR, oximetry, and ROS-sensitive fluorescent reporters. Migration was tested in transwells 48 h after irradiation in the presence or absence of inhibitors targeting specific ROS or downstream effectors. Among tested inhibitors, we designed a mitochondria-targeted version of human catalase (mtCAT) to selectively inactivate mitochondrial H2O2.ResultsPhoton irradiation at subclinical doses (0.5 Gy for MCF7 and 0.125 Gy for MDA-MB-231 cells) sequentially affected mtDNA levels and/or integrity, increased mtROS production, increased MAP2K1/MEK1 gene expression, activated ROS-sensitive transcription factors NF-κB and AP1 and stimulated breast cancer cell migration. Targeting mtROS pharmacologically by MitoQ or genetically by mtCAT expression mitigated migration induced by a subclinical dose of irradiation.ConclusionSubclinical doses of photon irradiation promote human breast cancer migration, which can be countered by selectively targeting mtROS.

Discovery of CDC42 Inhibitors with a Favorable Pharmacokinetic Profile and Anticancer In Vivo Efficacy.

We previously reported trisubstituted pyrimidine lead compounds, namely, ARN22089 and ARN25062, which block the interaction between CDC42 with its specific downstream effector, a PAK protein. This interaction is crucial for the progression of multiple tumor types. Such inhibitors showed anticancer efficacy in vivo. Here, we describe a second class of CDC42 inhibitors with favorable drug-like properties. Out of the 25 compounds here reported, compound 15 (ARN25499) stands out as the best lead compound with an improved pharmacokinetic profile, increased bioavailability, and efficacy in an in vivo PDX tumor mouse model. Our results indicate that these CDC42 inhibitors represent a promising chemical class toward the discovery of anticancer drugs, with ARN25499 as an additional lead candidate for preclinical development.

The role of PI3k/AKT signaling pathway in attenuating liver fibrosis: a comprehensive review.

Excessive accumulation of extracellular matrix (ECM) components within the liver leads to a pathological condition known as liver fibrosis. Alcohol abuse, non-alcoholic fatty liver disease (NAFLD), autoimmune issues, and viral hepatitis cause chronic liver injury. Exploring potential therapeutic targets and understanding the molecular mechanisms involved in liver fibrosis are essential for the development of effective interventions. The goal of this comprehensive review is to explain how the PI3K/AKT signaling pathway contributes to the reduction of liver fibrosis. The potential of this pathway as a therapeutic target is investigated through a summary of results from in vivo and in vitro studies. Studies focusing on PI3K/AKT activation have shown a significant decrease in fibrosis markers and a significant improvement in liver function. The review emphasizes how this pathway may prevent ECM synthesis and hepatic stellate cell (HSC) activation, ultimately reducing the fibrotic response. The specific mechanisms and downstream effectors of the PI3K/AKT pathway in liver fibrosis constitute a rapidly developing field of study. In conclusion, the PI3K/AKT signaling pathway plays a significant role in attenuating liver fibrosis. Its complex role in regulating HSC activation and ECM production, demonstrated both in vitro and in vivo, underscores its potential as a effective therapeutic approach for managing liver fibrosis and slowing disease progression. A comprehensive review of this field provides valuable insights into its future developments and implications for clinical applications.

Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages.

In general, it is known that the paramyxovirus gains access to host cells through direct penetration at the plasma membrane; however, emerging evidence suggests more complex entry mechanisms for paramyxoviruses. The endocytic entry of Newcastle disease virus (NDV), a representative member of the paramyxovirus family, into multiple types of cells has been recently reported. Herein, we demonstrate the binding of NDV to induce ganglioside-activated Src signaling, which is responsible for the endocytic entry of NDV through caveolae-mediated endocytosis. This process involved Src-dependent activation of the caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPase and downstream effectors, thereby orchestrating the endocytic entry process of NDV. Our findings uncover a novel molecular mechanism of endocytic entry of NDV into host cells and provide novel insight into paramyxovirus mechanisms of entry.

Severe Asthmatic Responses: The Impact of TSLP.

Asthma is a chronic inflammatory disease that affects the lower respiratory system and includes several categories of patients with varying features or phenotypes. Patients with severe asthma (SA) represent a group of asthmatics that are poorly responsive to medium-to-high doses of inhaled corticosteroids and additional controllers, thus leading in some cases to life-threatening disease exacerbations. To elaborate on SA heterogeneity, the concept of asthma endotypes has been developed, with the latter being characterized as T2-high or low, depending on the type of inflammation implicated in disease pathogenesis. As SA patients exhibit curtailed responses to standard-of-care treatment, biologic therapies are prescribed as adjunctive treatments. To date, several biologics that target specific downstream effector molecules involved in disease pathophysiology have displayed superior efficacy only in patients with T2-high, eosinophilic inflammation, suggesting that upstream mediators of the inflammatory cascade could constitute an attractive therapeutic approach for difficult-to-treat asthma. One such appealing therapeutic target is thymic stromal lymphopoietin (TSLP), an epithelial-derived cytokine with critical functions in allergic diseases, including asthma. Numerous studies in both humans and mice have provided major insights pertinent to the role of TSLP in the initiation and propagation of asthmatic responses. Undoubtedly, the magnitude of TSLP in asthma pathogenesis is highlighted by the fact that the FDA recently approved tezepelumab (Tezspire), a human monoclonal antibody that targets TSLP, for SA treatment. Nevertheless, further research focusing on the biology and mode of function of TSLP in SA will considerably advance disease management.

Design, Synthesis, In Vitro and In Vivo Characterization of CDC42 GTPase Interaction Inhibitors for the Treatment of Cancer.

CDC42 GTPases (RHOJ, CDC42, and RHOQ) are overexpressed in multiple tumor types and activate pathways critical for tumor growth, angiogenesis, and metastasis. Recently, we reported the discovery of a novel lead compound, ARN22089, which blocks the interaction of CDC42 GTPases with specific downstream effectors. ARN22089 blocks tumor growth in BRAF mutant mouse melanoma models and patient-derived xenografts (PDXs) in vivo. ARN22089 also inhibits tumor angiogenesis in three-dimensional vascularized microtumor models in vitro. Notably, ARN22089 belongs to a novel class of trisubstituted pyrimidines. Based on these results, we describe an extensive structure-activity relationship of ∼30 compounds centered on ARN22089. We discovered and optimized two novel inhibitors (27, ARN25062, and 28, ARN24928), which are optimal back-up/follow-up leads with favorable drug-like properties and in vivo efficacy in PDX tumors. These findings further demonstrate the potential of this class of CDC42/RHOJ inhibitors for cancer treatment, with lead candidates ready for advanced preclinical studies.

Engineered kinases as a tool for phosphorylation of selected targets in vivo.

Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors.

Protein Tyrosine Phosphatase SHP2 Controls Interleukin-8 Expression in Breast Cancer Cells

Treatment of metastasis remains a clinical challenge and the majority of breast cancer-related deaths are the result of drug-resistant metastases. The protein tyrosine phosphatase SHP2 encoded by the proto-oncogene PTPN11 promotes breast cancer progression. Inhibition of SHP2 has been shown to decrease metastases formation in various breast cancer models, but specific downstream effectors of SHP2 remain poorly characterized. Certain cytokines in the metastatic cascade facilitate local invasion and promote metastatic colonization. In this study, we investigated cytokines affected by SHP2 that could be relevant for its pro-tumorigenic properties. We used a cytokine array to investigate differentially released cytokines in the supernatant of SHP2 inhibitor-treated breast cancer cells. Expression of CXCL8 transcripts and protein abundance were assessed in human breast cancer cell lines in which we blocked SHP2 using shRNA constructs or an allosteric inhibitor. The impact of SHP2 inhibition on the phospho-tyrosine-proteome and signaling was determined using mass spectrometry. From previously published RNAseq data (Aceto et al. in Nat. Med. 18:529–37, 2012), we computed transcription factor activities using an integrated system for motif activity response analysis (ISMARA) (Balwierz et al. in Genome Res. 24:869–84, 2014). Finally, using siRNA against ETS1, we investigated whether ETS1 directly influences CXCL8 expression levels. We found that IL-8 is one of the most downregulated cytokines in cell supernatants upon SHP2 blockade, with a twofold decrease in CXCL8 transcripts and a fourfold decrease in IL-8 protein. These effects were also observed in preclinical tumor models. Analysis of the phospho-tyrosine-proteome revealed that several effectors of the mitogen-activated protein kinase (MAPK) pathway are downregulated upon SHP2 inhibition in vitro. MEK1/2 inhibition consistently reduced IL-8 levels in breast cancer cell supernatants. Computational analysis of RNAseq data from SHP2-depleted tumors revealed reduced activity of the transcription factor ETS1, a direct target of ERK and a transcription factor reported to regulate IL-8 expression. Our work reveals that SHP2 mediates breast cancer progression by enhancing the production and secretion of the pro-metastatic cytokine IL-8. We also provide mechanistic insights into the effects of SHP2 inhibition and its downstream repercussions. Overall, these results support a rationale for targeting SHP2 in breast cancer.

Mutation of the Polyproline Sequence in CD3ε Evidences TCR Signaling Requirements for Differentiation and Function of Pro-Inflammatory Tγδ17 Cells.

Tγδ17 cells have emerged as a key population in the development of inflammatory and autoimmune conditions such as psoriasis. Thus, the therapeutic intervention of Tγδ17 cells can exert protective effects in this type of pathologies. Tγδ cells commit to IL-17 production during thymus development, and upon immune challenge, additional extrathymic signals induce the differentiation of uncommitted Tγδ cells into Tγδ17 effector cells. Despite the interest in Tγδ17 cells during the past 20 years, the role of TCR signaling in the generation and function of Tγδ17 cells has not been completely elucidated. While some studies point to the notion that Tγδ17 differentiation requires weak or no TCR signaling, other works suggest that Tγδ17 require the participation of specific kinases and adaptor molecules downstream of the TCR. Here we have examined the differentiation and pathogenic function of Tγδ17 cells in “knockin” mice bearing conservative mutations in the CD3ε polyproline rich sequence (KI-PRS) with attenuated TCR signaling due to lack of binding of the essential adaptor Nck. KI-PRS mice presented decreased frequency and numbers of Tγδ17 cells in adult thymus and lymph nodes. In the Imiquimod model of skin inflammation, KI-PRS presented attenuated skin inflammation parameters compared to wild-type littermates. Moreover, the generation, expansion and effector function Tγδ17 cells were impaired in KI-PRS mice upon Imiquimod challenge. Thus, we conclude that an intact CD3ε-PRS sequence is required for optimal differentiation and pathogenic function of Tγδ17 cells. These data open new opportunities for therapeutic targeting of specific TCR downstream effectors for treatment of Tγδ17-mediated diseases.

Passive coupling of membrane tension and cell volume during active response of cells to osmosis

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.

Bio-O1-08 - PLCγ1/PKCθ and its downstream effectors in cutaneous T-cell lymphoma development and progression

Developing mechanistic rationales can improve the clinical management of cutaneous T-cell lymphomas (CTCL). In the last few years, the use of Next Generation Sequencing (NGS) approaches has revealed a rich landscape of genomic alterations potentially influencing the biology of these entities. In this regard, multiple genetic and biological evidences point at a malignant network of signaling mechanisms, highly influenced by deregulated TCR/PLCγ1 activity, to control the biology of CTCLs. In addition, activated STAT3 is associated with clinical progression, although the specific mechanisms responsible for this have not been fully elucidated. Here we studied PLCγ1-dependent mechanisms that can activate JAK/STAT and control tumor growth and progression. Downstream of PLCγ1, we identified PKCθ (a specific T cell PKC isoform) to mediate JAK/STAT3 activation and control CTCL cell proliferation and viability. In vivo, PKCθ blockage in CTCL cells, impaired tumor development and cells spreading to the liver and the lungs. In light of these results, we sought to identify specific PKCθ downstream effectors, that in addition to JAK/STATs, could help understanding its biological role in CTCL. To this end, we studied PKCθ-dependent transcriptome as well as its protein interactome in CTCL cells. This approach helped detecting a number of hits that can potentially explain STAT3 phosphorylation in CTCL cells (i.e. RACK1), and highlight a number of oncogenic activities controlled by PKCq, like mitochondrial activity (i.e. ASK1, HSPA9) and cytoskeletal regulation (i.e. ARHGAP-19 or CCT4, 5). As a result, this dual approach helped identifying a number of protein effectors and target genes that can participate in important CTCL activities like for example cytokine signaling, mitochondrial activity, TP53, and actin cytoskeleton dynamics. Finally, we took advantage of Nanostring to study the expression of a selection of PKCθ target genes, in a cohort of 81 human MF samples and control inflammatory dermatoses. In this context, PRKCQ (PKCθ) gene expression was significantly upregulated in MF cases vs. controls. Moreover, the expression of target genes like PRKACB (encoding for PKA isoform B) or the lymphocyte specific actin binding LCP1 and LCP1, positively correlated with that of PRKCQ expression in MF cases. On the other hand, genes like FGFR3 and RHOB showed a significant yet inverse correlation with PRKCQ in robust agreement with the data generated in CTCL cells. Overall, the results show a PKCθ gene signature that is significant and differentially expressed amongst different MF cases, at different stages of the disease. Moreover, this signature highlights novel CTCL disease mechanisms with translational potential in diagnosis and targeted therapy. In summary, we have uncover a series of PKCθ-dependent effectors/routes that can play a central role in the biology of CTCLs and help to develop novel approaches to diagnose and treat these malignancies.

Cardiac cAMP-PKA Signaling Compartmentalization in Myocardial Infarction.

Under physiological conditions, cAMP signaling plays a key role in the regulation of cardiac function. Activation of this intracellular signaling pathway mirrors cardiomyocyte adaptation to various extracellular stimuli. Extracellular ligand binding to seven-transmembrane receptors (also known as GPCRs) with G proteins and adenylyl cyclases (ACs) modulate the intracellular cAMP content. Subsequently, this second messenger triggers activation of specific intracellular downstream effectors that ensure a proper cellular response. Therefore, it is essential for the cell to keep the cAMP signaling highly regulated in space and time. The temporal regulation depends on the activity of ACs and phosphodiesterases. By scaffolding key components of the cAMP signaling machinery, A-kinase anchoring proteins (AKAPs) coordinate both the spatial and temporal regulation. Myocardial infarction is one of the major causes of death in industrialized countries and is characterized by a prolonged cardiac ischemia. This leads to irreversible cardiomyocyte death and impairs cardiac function. Regardless of its causes, a chronic activation of cardiac cAMP signaling is established to compensate this loss. While this adaptation is primarily beneficial for contractile function, it turns out, in the long run, to be deleterious. This review compiles current knowledge about cardiac cAMP compartmentalization under physiological conditions and post-myocardial infarction when it appears to be profoundly impaired.

Growing Patient Education in Dermatology: An Upside-Down Tree Analogy for Immunomodulatory Therapies

As patient autonomy gains momentum in the era of readily accessible health information, dermatologists bear a growing responsibility in patient education. With the rapid evolution and development of targeted therapies for dermatologic conditions, clinicians are faced with questions from patients regarding the mechanisms of action and side effects of novel therapeutic agents. We present an educational aid to be used in patient counseling to describe and compare the mechanisms of traditional immunosuppressive medications and targeted agents such as biologics and small molecule inhibitors. Using an upside-down tree as an analogy for the immune system, traditional immunosuppressives can be represented by an axe, chopping at large branches (ie, upstream immune pathways), while targeted immunomodulators can be represented by pruning shears, trimming select small branches and leaves (ie, specific downstream effectors). This approachable visual aid can guide practitioners in explaining the complexities of the immune system and immunomodulatory therapies with the goal of augmenting patient understanding and addressing patient concerns regarding new medications. J Drugs Dermatol. 2020;19(1):28-29.

Sema3A inhibits axonal regeneration of retinal ganglion cells via ROCK2

Successful regeneration of injured axons in the adult mammalian central nervous system (CNS) is mainly limited by lesion-induced neuronal apoptosis and the inhibitory environment consisting of numerous extrinsic and intrinsic factors. Semaphorin 3A (Sema3A), a classic axonal guidance cue, contributes to the failure of axonal regeneration and can be neutralized to enhance axonal regeneration. Previous studies have suggested that blockage of rho-associated protein kinase 2 (ROCK2) also exerts a protective effect on the survival and axonal regeneration of retinal ganglion cells (RGC, RGCs) after injury. Yet unresolved question is the interaction between the two factors. We thus evaluated the role of Sema3A and ROCK2 in RGC axonal regeneration. In this study, we first examined the expression of Sema3A and ROCK2 against optic nerve crush in vivo and oxygen-glucose deprivation insult to RGCs in vitro at different time points. Then Sema3A, ROCK2 inhibitor Y-27632, combination of both and phosphate-buffered saline (PBS) only were injected into the vitreous cavity after optic nerve crush at various times in different experiments. In order to assess axonal regeneration, we detected the mRNA levels of small proline-rich protein 1A (Sprr1A) and growth-associated protein 43 (GAP43) by quantitative real time-polymerase chain reaction (RT-qPCR), evaluated visual function by Flash Visual Evoked Potentials (F-VEPs), and checked the protein level of GAP43 by immunofluorescent staining. Our results demonstrated that Sema3A significantly suppressed optic nerve regeneration and this effect can be attenuated via blocking ROCK2. Moreover, Sema3A promoted the phosphorylation of myosin light chain 2 (MLC2) (specific downstream effector of ROCK2 concerning neurite growth). Collectively, Sema3A may negatively regulate axonal regeneration through ROCK2 in RGCs.

Regulatory role of sphingosine kinase and sphingosine-1-phosphate receptor signaling in progenitor/stem cells.

Balanced sphingolipid signaling is important for the maintenance of homeostasis. Sphingolipids were demonstrated to function as structural components, second messengers, and regulators of cell growth and survival in normal and disease-affected tissues. Particularly, sphingosine kinase 1 (SphK1) and its product sphingosine-1-phosphate (S1P) operate as mediators and facilitators of proliferation-linked signaling. Unlimited proliferation (self-renewal) within the regulated environment is a hallmark of progenitor/stem cells that was recently associated with the S1P signaling network in vasculature, nervous, muscular, and immune systems. S1P was shown to regulate progenitor-related characteristics in normal and cancer stem cells (CSCs) via G-protein coupled receptors S1Pn (n = 1 to 5). The SphK/S1P axis is crucially involved in the regulation of embryonic development of vasculature and the nervous system, hematopoietic stem cell migration, regeneration of skeletal muscle, and development of multiple sclerosis. The ratio of the S1P receptor expression, localization, and specific S1P receptor-activated downstream effectors influenced the rate of self-renewal and should be further explored as regeneration-related targets. Considering malignant transformation, it is essential to control the level of self-renewal capacity. Proliferation of the progenitor cell should be synchronized with differentiation to provide healthy lifelong function of blood, immune systems, and replacement of damaged or dead cells. The differentiation-related role of SphK/S1P remains poorly assessed. A few pioneering investigations explored pharmacological tools that target sphingolipid signaling and can potentially confine and direct self-renewal towards normal differentiation. Further investigation is required to test the role of the SphK/S1P axis in regulation of self-renewal and differentiation.

Region-specific proteolysis differentially modulates type 2 and type 3 inositol 1,4,5-trisphosphate receptor activity in models of acute pancreatitis

Fine-tuning of the activity of inositol 1,4,5-trisphosphate receptors (IP3R) by a diverse array of regulatory inputs results in intracellular Ca2+ signals with distinct characteristics. These events allow the activation of specific downstream effectors. We reported previously that region-specific proteolysis represents a novel regulatory event for type 1 IP3R (R1). Specifically, caspase-fragmented R1 display a marked increase in single-channel open probability. More importantly, the distinct characteristics of the Ca2+ signals elicited via fragmented R1 can activate alternate downstream effectors. In this report, we expand these studies to investigate whether all IP3R subtypes are regulated by proteolysis. We now show that type 2 and type 3 IP3R (R2 and R3, respectively) are proteolytically cleaved in rodent models of acute pancreatitis. Surprisingly, fragmented IP3R retained tetrameric architecture, remained embedded in endoplasmic reticulum membranes and were not functionally disabled. Proteolysis was associated with a marked attenuation of the frequency of Ca2+ signals in pancreatic lobules. Consistent with these data, expression of DNAs encoding complementary R2 and R3 peptides mimicking fragmented receptors at particular sites, resulted in a significant decrease in the frequency of agonist-stimulated Ca2+ oscillations. Further, proteolysis of R2 resulted in a marked decrease in single-channel open probability. Taken together, proteolytic fragmentation modulates R2 and R3 activity in a region-specific manner, and this event may contribute to the altered Ca2+ signals in pancreatic acinar cells during acute pancreatitis.

Combination of melatonin and rapamycin for head and neck cancer therapy: Suppression of AKT/mTOR pathway activation, and activation of mitophagy and apoptosis via mitochondrial function regulation.

Head and neck squamous cell carcinoma (HNSCC) clearly involves activation of the Akt mammalian target of rapamycin (mTOR) signalling pathway. However, the effectiveness of treatment with the mTOR inhibitor rapamycin is often limited by chemoresistance. Melatonin suppresses neoplastic growth via different mechanisms in a variety of tumours. In this study, we aimed to elucidate the effects of melatonin on rapamycin-induced HNSCC cell death and to identify potential cross-talk pathways. We analysed the dose-dependent effects of melatonin in rapamycin-treated HNSCC cell lines (Cal-27 and SCC-9). These cells were treated with 0.1, 0.5 or 1mmol/L melatonin combined with 20 nM rapamycin. We further examined the potential synergistic effects of melatonin with rapamycin in Cal-27 xenograft mice. Relationships between inhibition of the mTOR pathway, reactive oxygen species (ROS), and apoptosis and mitophagy reportedly increased the cytotoxic effects of rapamycin in HNSCC. Our results demonstrated that combined treatment with rapamycin and melatonin blocked the negative feedback loop from the specific downstream effector of mTOR activation S6K1 to Akt signalling, which decreased cell viability, proliferation and clonogenic capacity. Interestingly, combined treatment with rapamycin and melatonin-induced changes in mitochondrial function, which were associated with increased ROS production, increasing apoptosis and mitophagy. This led to increase cell death and cellular differentiation. Our data further indicated that melatonin administration reduced rapamycin-associated toxicity to healthy cells. Overall, our findings suggested that melatonin could be used as an adjuvant agent with rapamycin, improving effectiveness while minimizing its side effects.

Detection of Smo, Gli2 and Gli3 Among Basal Cell Carcinoma Patients in Sulaimani Province.

Basal-cell carcinoma (BCC hedgehog (Shh) of BCC. The specific downstream effector in the Shh pathway leading to cancer development is unclear. However in vertebrates, specific downstream effectors in the Shh signaling pathway including zinc-finger transcription factors Gli2 and Gli3 play the Shh pathway. S and controlled cell proliferation. The expression of transducers had not been reported yet in BCC skin of local patients. The aim of this study was to investigate the expression of S BCC biopsies taken from sun Five BCC skin biopsy specimens were taken from the sun which served as material for the study. RNA extracted from the samples, cDNA synthesised carried out and specific primers for each of the S genes were designed. Reverse transcription polymerase chain reaction (RT samples expressed genes for S patients in Sulaimani Province. These findings support the role of S the Shh–receptor complex. These results establish S oncogenes in skin and suggest a pivotal role for these transcription factors in the development of BCC. This method can be used in the diagnosis of BCC and fur study of downstream effectors in the Shh pathway may lead to an effective anti cancer therapy. Key Words: Basal cell carcinoma, Smo, Gli2, Gli3, RT-PCR. JZS (2017) 19 – 2 (Part-A) 1 Detection of Smo, Gli2 and Gli3 Among Basal Cell Carcinoma Patients in Sulaimani Province Department of Biology, College of Science, University of Sulaimani, Sulaimani, Kurdistan Region, Iraq. Basal cell carcinoma is the most common malignancy in humans. Although rarely metasta capable of significant local destruction and disfigurement. Skin cancer is accounting for about half of all cancers occur. BCC constitute approximately 80% of all nonmelanoma skin cancers [ in the world has been reported in Australia [2]. Exposure to ult radiation is generally accepted as the major cause of BCC and the risk of this disease is significantly increased by recreational exposure to the sun during childhood and adolescence cluding fair complexion, red or blond hair, and light eye color, influence responsiveness to but are also independent risk factors [4]; exposures to ionizing radiation, arsenic, and oral methoxsalen ave also been linked to the development of BCC[5,6] Journal homepage www.jzs.univsul.edu.iq Journal of Zankoy Sulaimani Part-A- (Pure and Applied Sciences) Abstract cell carcinoma (BCC) is the most common type of cancer in human. Sonic hedgehog (Shh) signaling pathway impairment plays a key role in the pathogenesis of BCC. The specific downstream effector in the Shh pathway leading to cancer development is unclear. However in vertebrates, specific downstream effectors in e Shh signaling pathway including smoothened, frizzled class receptor (S finger transcription factors Gli2 and Gli3 play an important role in regulating the Shh pathway. SMO, Gli2, and Gli3 family proteins are necessary for adequate and controlled cell proliferation. The expression of SMO, Gli2 and Gli3 signal transducers had not been reported yet in BCC skin of local patients. The aim of this study was to investigate the expression of SMO BCC biopsies taken from sun-exposed skin areas of patients in Sulaimani Provinc Five BCC skin biopsy specimens were taken from the sun which served as material for the study. RNA extracted from the samples, cDNA synthesised carried out and specific primers for each of the S genes were designed. Reverse transcription polymerase chain reaction (RT-PCR) analyses samples expressed genes for SMO, Gli2 and Gli3 in BCC skin biopsies taken from patients in Sulaimani Province. These findings support the role of SMO, Gli2 and Gli3 as a signa receptor complex. These results establish SMO, Gli2 and Gli3 as potent oncogenes in skin and suggest a pivotal role for these transcription factors in the development of BCC. This method can be used in the diagnosis of BCC and fur study of downstream effectors in the Shh pathway may lead to an effective anti cancer therapy.

C-Myc Alteration Determines the Therapeutic Response to FGFR Inhibitors.

Purpose: Lately, emerging evidence has suggested that oncogenic kinases are associated with specific downstream effectors to govern tumor growth, suggesting potential translational values in kinase-targeted cancer therapy. Tyrosine kinase FGFR, which is aberrant in various cancer types, is one of the most investigated kinases in molecularly targeted cancer therapy. Herein, we investigated whether there exists key downstream effector(s) that converges FGFR signaling and determines the therapeutic response of FGFR-targeted therapy.Experimental Design: A range of assays was used to assess the role of c-Myc in FGFR aberrant cancers and its translational relevance in FGFR-targeted therapy, including assessment of drug sensitivity using cell viability assay, signaling transduction profiling using immunoblotting, and in vivo antitumor efficacy using cancer cell line-based xenografts and patient-derived xenografts models.Results: We discovered that c-Myc functioned as the key downstream effector that preceded FGFR-MEK/ERK signaling in FGFR aberrant cancer. Disruption of c-Myc overrode the cell proliferation driven by constitutively active FGFR. FGFR inhibition in FGFR-addicted cancer facilitated c-Myc degradation via phosphorylating c-Myc at threonine 58. Ectopic expression of undegradable c-Myc mutant conferred resistance to FGFR inhibition both in vitro and in vivo c-Myc level alteration stringently determined the response to FGFR inhibitors, as demonstrated in FGFR-responsive cancer subset, as well as cancers bearing acquired or de novo resistance to FGFR inhibition.Conclusions: This study reveals a stringent association between FGFR and the downstream effector c-Myc in FGFR-dependent cancers, and suggests the potential therapeutic value of c-Myc in FGFR-targeted cancer therapy. Clin Cancer Res; 23(4); 974-84. ©2016 AACR.

Abnormal Akt signalling in bladder epithelial cell explants from patients with interstitial cystitis/bladder pain syndrome can be induced by antiproliferative factor treatment of normal bladder cells

To determine whether protein kinase B (Akt) signalling and secretion of specific downstream effector proteins are abnormal in specific cell fractions of bladder epithelial cells from patients with interstitial cystitis/bladder pain syndrome (IC/BPS), as explanted bladder epithelial cells from patients with IC/BPS produce a frizzled 8-related glycopeptide antiproliferative factor (APF) that inhibits normal bladder epithelial cell proliferation and expression of several proteins known to be regulated by Akt signalling. A related secondary objective was to determine whether treatment of normal bladder epithelial cells with active synthetic asialo-antiproliferative factor (as-APF) induces similar changes in Akt signalling and specific downstream effector proteins/mRNAs. Cell proteins were extracted into four subcellular fractions from primary bladder epithelial explants of six patients who fulfilled modified National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) criteria for IC/BPS and six age- and gender-matched controls. Total and/or phosphorylated cellular Akt, glycogen synthase kinase 3β (GSK3β), and β-catenin; total cellular JunB; and secreted matrix metalloproteinase 2 (MMP2) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) levels were determined by Western blot. MMP2, JunB, p53, uroplakin 3 (UPK3), and β-actin mRNAs were quantified by quantitative reverse transcriptase-polymerase chain reaction. Akt activity was determined by nonradioactive assay. IC/BPS cells had lower Akt activity, along with lower Akt ser473- and GSK3β ser9-phosphorylation and higher β-catenin ser33,37/thr41-phosphorylation in specific fractions as compared with matched control cells. IC/BPS explants also had evidence of additional downstream abnormalities compared with control cells, including lower nuclear JunB; lower secreted MMP2 and HB-EGF; plus lower MMP2, JunB, and UPK3 mRNAs but higher p53 mRNA relative to β-actin. Each of these IC/BPS cell abnormalities was also induced in normal cells by as-APF. These findings indicate that IC/BPS cells have abnormal Akt activity with downstream protein expression abnormalities including decreased MMP2 and HB-EGF secretion. They also support the hypothesis that APF plays a role in the pathogenesis of IC/BPS via its effects on cell Akt signalling and HB-EGF production.