Role of Syk in the regulation of cytoskeleton and stress granules in breast cancer

The Syk protein-tyrosine kinase, a well-characterized modulator of immune recognition receptor signaling, also plays important, but poorly characterized, roles in tumor progression, acting as an inhibitor of cellular motility and metastasis in highly invasive cancer cells. Multiharmonic atomic force microscopy (AFM) was used to map nanomechanical properties of live MDA-MB-231 breast cancer cells either lacking or expressing Syk. The expression of Syk dramatically altered the cellular topography, reduced cell height, increased elasticity, increased viscosity, and allowed visualization of a more substantial microtubule network. The microtubules of Syk-expressing cells were more stable to nocodazole-induced depolymerization and were more highly acetylated than those of Syk-deficient cells. Silencing of MAP1B, a major substrate for Syk in MDA-MB-231 cells, attenuated Syk-dependent microtubule stability and reversed much of the effect of Syk on cellular topography, stiffness, and viscosity. This study illustrates the use of multiharmonic AFM both to quantitatively map the local nanomechanical properties of living cells and to identify the underlying mechanisms by which these properties are modulated by signal transduction machinery. Proteomic analyses of Syk-binding proteins identified several interacting partners also found to be recruited to stress granules. Treatment of cells with inducers of stress granule formation leads to the recruitment of Syk to these protein-RNA complexes. This recruitment requires the phosphorylation of Syk on tyrosine and results in the phosphorylation of proteins at or near the stress granule. Grb7 is identified as a Syk-binding protein involved in the recruitment of Syk tothe stress granule. This recruitment promotes the formation of autophagosomes and the clearance of stress granules from the cell once the stress is relieved, enhancing the ability of cells to survive the stress stimulus

Microbial and epigenetic factors in the pathogenesis of nasopharyngeal carcinoma

While uncommon in most of the world, nasopharyngeal carcinoma (NPC) shows an unusual geographic and ethnic distribution, being highly prevalent in Southern China and Southeast Asia. Genetic susceptibility, Epstein-Barr virus (EBV) infection and additional environmental exposures are well established risk factors for NPC in endemic areas. However, the detailed molecular mechanisms of NPC pathogenesis remain largely unknown. In this thesis, several novel pathogenic mechanisms of NPC development and progression are presented. The interaction of EBV encoded latent membrane protein 2A (LMP2A) with cellular proteins promoting invasiveness of NPC cells is described in paper I. Spleen tyrosine kinase (Syk) interacts with integrin β4 subunit (ITGβ4) in epithelial cells through an ITAM-like motif, and concurrent LMP2A expression interferes with this interaction by competitive binding to Syk. Both Syk and LMP2A affect cell surface expression of ITGβ4. Particularly, ITGβ4 concentrates at cellular protrusions in LMP2A expressing cells, which may contribute to the migration property of NPC-cells. Paper II and paper III focus on the epigenetic alteration of candidate tumor suppressor genes (TSGs) and their possible role in NPC tumorigenesis. Cadherin 4 (CDH4) and ubiquitin-conjugating enzyme 2L6 (UBE2L6) are downregulated due to promoter hypermehtylation in NPC. Both genes suppress the proliferation and colony formation of NPC-cells. CDH4 impedes cell migration and elicits cell communication; UBE2L6 induce apoptosis of NPC cells and counteracts degradation of adipocyte triglyceride lipase (ATGL) through ISG15-conjugation of valosin-containing protein (VCP). CDH4 and UBE2L6 could be involved in both initiation and progression of NPC. Suppression of UBE2L6 encoded protein UbcH8 correlated with poor outcome in NPC patients. In paper IV, we compared the response of NPC and normal nasopharyngeal epithelial (NNE) cell lines to bacteria and bacterial cell wall components. Strong nuclear translocation of NF-κB and significant induction of proinflammatory factors IL6, IL8, IL1α and CXCL2 were observed in NNE cells, but not in NPC cells upon exposure to Gram-positive bacteria streptococci and peptidoglycan (PGN). We identified three different mechanisms by which the activation of NF-κB in NPC cells could be hampered. It could be trapped by an enhanced accumulation of cytoplasmic lipids. I-κB degradation could be impaired due to downregulation of UBE2L6. We also showed that overexpression of lysine-specific demethylase-1 (LSD1) blocked the transcriptional activation of proinflammatory genes. Together these mechanisms might contribute to decreased immune reactivity in NPC and thus affect tumor progression

The SYK tyrosine kinase suppresses autolysosome biogenesis via activation of mTORC1 in pancreatic cancer cell lines

Spleen tyrosine kinase (SYK) regulates mitogenic signaling, inflammatory responses and cell fate in a number of diverse cell types. KRAS is a proto-oncogene that controls cell growth and proliferation through several mitogenic pathways. In pancreatic cancer, KRAS is frequently mutated, resulting in constitutive activation in 90% of pancreatic cancer cell lines. We previously showed that SYK is highly expressed in a subset of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines. We demonstrated that SYK kinase inhibition with PRT062607 (SYKi) causes decreased cell proliferation of PDAC cell lines. Furthermore, combined SYKi and MEK inhibitor (MEKi) treatment promotes additive effects on suppression of PDAC cell proliferation and clonogenic growth. Mechanistically, SYK activates the mTORC1 kinase complex as shown by reduced phosphorylation of ribosomal S6 protein and its upstream kinase p70 S6 kinase (p70S6K) following SYKi treatment in PDAC cell lines. SYK-mediated mTORC1 activation occurs independently of MEK/ERK and PI3K/AKT effector signaling pathways. The mTORC1 complex suppresses lysosome biogenesis and macroautophagy (autophagy). Consequently, mTORC1 suppression via SYK inhibition or shRNA-mediated depletion causes accumulation of autolysosomes. These effects are mediated by the enhanced nuclear localization of MITF, a key transcriptional regulator of genes involved in lysosome biogenesis and autophagy pathway activation. In summary, SYK positively regulates mTORC1 activation in a subset of PDAC cell lines to suppress hyperactivation of autophagy. These findings open new avenues for further exploration of SYK as a critical regulator of the autophagy pathway in KRAS/mTORC1-dependent PDAC, and how this may be exploited for therapeutic benefit

High Throughput Screening For Drug Discovery In Head And Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and despite advancements in traditional therapies, the survival rate of ~40% remains unchanged, thus highlighting the need for novel treatments in HNSCC. High throughput robotic screening of a panel of HNSCC cell lines was carried out against 1,505 compounds, where drug activity was measured using metabolic agent alamarBlue and quantified by percent activity. Initial screening found that the majority of active compounds could be grouped based upon their cellular target(s) and/or function including: cell cycle regulation, cytoskeleton disruption, and DNA topoisomerase function. Potency was confirmed with dose response curves for 23 hit compounds and ER27319 maleate and NSC146109 hydrochloride were selected for further investigation. ER27319 maleate was observed to control tumour growth in vivo. This systematic high throughput screen of large panels of drugs identified a multitude of potentially effective agents for the treatment of HNSCC

Expression of Syk and MAP4 proteins in ovarian cancer

PurposeWe have previously reported on the prognostic importance of the calpain family of proteins in ovarian cancer, especially calpain-2. Spleen tyrosine kinase (Syk) phosphorylates a variety of cytoskeletal proteins with studies suggesting potential interactions between Syk and conventional calpains. Microtubule-associated protein 4 (MAP4) has been reported to be regulated by Syk.MethodsThe current study assessed Syk and MAP4 protein expression, by immunohistochemistry on a tissue microarray comprised of cores from primary ovarian carcinomas (n = 575), to evaluate associations with patient clinical outcomes and other clinicopathological factors and sought to determine whether there were any correlations between the expression of Syk, MAP4 and the calpain system.ResultsMAP4 expression was significantly associated with ovarian cancer histological subtype (P [less than] 0.001), stage (P = 0.001), grade (P [less than] 0.001) and residual tumour (P = 0.005). Despite this finding, we found no significant association existing between MAP4 expression and overall survival. Syk expression was also found significantly associated with histological subtype (P [less than] 0.001). Syk seems to play a contradictory role with respect to tumour progression: low cytoplasmic Syk expression was significantly associated with low stage (P = 0.013), and low nuclear Syk expression with chemo-resistance in patients treated with taxane-containing therapy (P = 0.006). Interestingly, despite the lack of association in the whole cohort, high nuclear Syk expression was significantly associated with better overall survival in certain subgroups (P = 0.001).ConclusionsThe current study indicates a lack of correlation between calpain-2 expression and Syk and MAP4. Syk, MAP4 and calpain-1 appeared to significantly correlate with each other in the whole cohort, with calpain-1 being more highly associated with MAP4 and Syk in mucinous carcinomas. Overall, the current results suggest that Syk, MAP4, and calpain-1 expression are correlated with each other and these proteins may be involved in early stages of tumour spread

SPROUTY 2: A Novel Attenuator of B Cell Receptor and MAPK Signaling in Chronic Lymphocytic Leukemia

Clinical heterogeneity is a major barrier to effective treatment of Chronic Lymphocytic Leukemia (CLL). Emerging evidence suggests that constitutive activation of various signaling pathways plays a role in the heterogeneous clinical outcome of CLL patients. MAPK-Erk signaling represents one such pathway with a demonstrated role in CLL pathogenesis. In this study, we have investigated the role of Sprouty2 (SPRY2) as a negative regulator of receptor and non-receptor tyrosine kinase signaling in the pathogenesis of CLL. We show that SPRY2 expression is significantly decreased in CLL cells, particularly from poor prognosis patients compared to those from good prognosis patients. Over-expression of SPRY2 in CLL cells from poor prognosis patients decreased their proliferation while increasing their apoptosis. Conversely, down-regulation of SPRY2 in CLL cells from good prognosis patients resulted in increased proliferation. Furthermore, CLL cells with low SPRY2 expression grew more rapidly in a xenograft model of CLL. Strikingly, B-cell specific transgenic over-expression of spry2 in mice led to a decrease in the frequency of B1 cells, the precursors of CLL cells in rodents. Mechanistically, we show that SPRY2 attenuates the BCR and MAPK-Erk signaling by binding to and antagonizing the activities of RAF1, BRAF and SYK in normal B cells and CLL cells. We identified that SPRY2 is targeted by miR-21 which in turn leads to increased activity of Syk and Erk in CLL cells. We also show that the activation of miR-21 is mediated by IL-10 induced STAT3 signaling in CLL cells. Taken together, these results establish SPRY2 as a critical negative regulator of BCR-mediated MAPK-Erk signaling in CLL, thereby providing one of the molecular mechanisms to explain the clinical heterogeneity of CLL

NOVEL ROLE OF PROSTATE APOPTOSIS RESPONSE-4 TUMOR SUPPRESSOR IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic Lymphocytic Leukemia (CLL) is defined by the accumulation of clonally expanded CD5+ and CD19+ B lymphocytes in blood and secondary lymphoid organs with impaired apoptotic mechanisms. CLL represents one third of all leukemia cases with an average age of 72 years at diagnosis making it the most common adult leukemia. The Eµ-Tcl1 mouse serves as an excellent model to study the development of CLL as they progress to a CLL like disease by 9-14 months of age, due to overexpression of an oncogene, T cell Leukemia 1(Tcl1), specifically in B cells through the Ig VH promoter and Eµ enhancer (Bichi et al. PNAS. 2002). In an adoptive transfer model, intravenous or intraperitoneal injection of primary CD5+CD19+ CLL cells from the Eµ-Tcl1 CLL mouse into recipient syngeneic mice leads to the development of a CLL like disease within 3-8 weeks of transfer. We have characterized the growth of CLL cells in these mice by periodic submandibular bleeding, spleen ultrasonography and flow cytometry. We find that Eµ-Tcl1 CLL cells express more Prostate apoptosis response-4 protein (Par-4), a known pro-apoptotic tumor suppressor protein, than normal B-1 or B-2 cells in mice. Par-4 is silenced by promoter methylation in more than 30% of all cancers and has been shown to be secreted and to induce apoptosis selectively in various types of cancer cells but not in normal cells. We found that CLL cells have constitutively active B-cell receptor signaling (BCR) and that inhibition of BCR signaling with FDA approved drugs causes a decrease in Par-4 protein, mRNA levels, and an increase in apoptosis. In particular, activities of Src family kinases, spleen tyrosine kinase and Bruton’s tyrosine kinase are required for Par-4 expression in CLL cells, suggesting a novel regulation of Par-4 through BCR signaling in both Eµ-Tcl1 CLL cells and primary human CLL samples. Consistent with this, lenti-viral shRNA mediated knockdown of Lyn kinase leads to a decrease in Par-4 expression in MEC-1 cells, a human CLL derived cell line. Igα (CD79a) silencing in primary human CLL cells also results in down regulation of Par-4 expression. Additionally, we knocked down expression of Par-4 in MEC-1 cells which resulted in a decrease in cell growth that could be attributed to an increase in p21 expression and a reduction in the G1/S cell cycle transition. We have also observed this phenomenon by crossing mice deficient in Par-4 with the Eµ-Tcl1 mouse where lack of Par-4 delays CLL growth in the mouse significantly (time to euthanization due to poor body condition - Eµ-Tcl1: 8.9mo vs Par4-/-EµTcl1: 11.97 mo, p = 0.0472) and splenic B-CLL cells from these mice also have increased expression of p21. Since mice in this cohort are whole body knockout for Par-4, the difference in survival times between the Par-4 +ve and Par-4 –ve EµTcl1 mice could be due to the influence of Par-4 on CLL cells as well as the effect of Par-4 secreted by the CLL cells on the microenvironment. There could be other potential roles for Par-4 in the context of CLL which are under further investigation. We have also investigated the site of CLL growth in mouse models to determine that the spleen is the primary organ to accumulate the CLL tumor burden. We have found that splenectomy significantly delays the development of CLL in the primary Eμ-Tcl1 mouse model and prevents growth and development in the adoptive transfer model. Interestingly, splenectomy did not delay CLL development as significantly in animals deficient for Par-4 compared to C57BL/6 wild type mice. Par-4 appears to regulate a specific microenvironment required for CLL growth. Current studies are investigating the role of Par-4 in the microenvironment and the cell types that are critical for CLL growth within the splenic niche

SIMULTANEOUS INHIBITION OF DRIVER AND EFFECTOR KINASES PROMOTES POTENT GROWTH ARREST OF AML CELLS IN VITRO AND IN VIVO

Acute myelogenous leukemia (AML) is caused by successive mutations acquired in hematopoietic progenitor cells that lead to the overpopulation of the bone marrow and peripheral blood by immature myeloid cells. The overall survival rate with current therapy is 25%, which decreases steadily with patient age. While numerous genetic alterations occur in AML, mutations to the FMS-like tyrosine kinase 3 (FLT3) are the most common, occurring in 30% of patients. Two types of FLT3 mutations occur in AML: internal tandem duplications (ITDs) and tyrosine kinase domain point mutations. FLT3 mutations drive AML pathogenesis and numerous small molecule kinase inhibitors have been designed as therapeutics for FLT3-mutated AML. However, these FLT3 inhibitors have had limited clinical success owing to lack of potency in vivo, toxicity, or short duration of response due to the development of resistance. Constitutively active FLT3 links to multiple downstream tyrosine kinases that are critical for AML cell survival and proliferation. Gene silencing studies have shown that several of these FLT3-associated kinases are individually crucial for FLT3 oncogenic potential, including SYK, FES, and the myeloid Src-family kinase, HCK. These previous observations raised the hypothesis that a small molecule inhibitor with a selectivity profile targeting FLT3 plus these associated kinases may be a potent AML drug lead with reduced propensity for acquired resistance. To test this hypothesis, we screened a library of N-phenylbenzamide compounds and identified a compound with three-digit nanomolar activity against each of these AML-associated kinases in vitro. Remarkably, this compound (TL02-59) inhibited FLT3-ITD+ cell growth in the picomolar range. Furthermore, TL02-59 demonstrated efficacy against primary AML bone marrow samples and a mouse xenograft model of AML. To explore the full range of targets for TL02-59, we performed a KINOMEscan assay and determined the expression of the TL02-59 target kinases in primary AML bone marrow samples. We discovered that while myeloid Src-family kinases HCK, LYN and FGR are critical to TL02-59 efficacy, inhibition of SYK, FES and the Ser/Thr kinases p38α and TAOK3 also plays a role. Future work will explore the advantages of this potent, multi-targeted kinase inhibitor in combating acquired resistance in AML