Krikor Bijian

Serum Proteomic Approach for the Identification of Serum Biomarkers Contributed by Oral Squamous Cell Carcinoma and Host Tissue Microenvironment

The lack of serum biomarkers for head and neck carcinoma limits early diagnosis, monitoring of advanced disease, and prediction of relapses in patients. We conducted a comprehensive proteomics study on serum from mice bearing orthotopic human oral squamous cell carcinomas (OSCC) with distinct invasive phenotypes. Matched established cell lines were transplanted orthotopically into tongues of RAG-2/gamma(c) mice and mouse serum was analyzed by 2-dimensional-differential gel electrophoresis(2D-DIGE)/liquid chromatography (LC)-MS/MS and by online 2D-LC-MS/MS of iTRAQ labeled samples. We identified several serum proteins as being differentially expressed between control and cancer-bearing mice and between noninvasive and invasive cancer (p<0.05). Differentially expressed proteins of human origin included the epidermal growth factor receptor (EGFR), cytokeratins, G-protein coupled receptor 87, Rab11 GTPase, PDZ-domain containing proteins, and PEST-containing nuclear proteins. Identified proteins of mouse origin included clusterin, titin, vitronectin, vitamin D-binding protein, hemopexin, and kininogen I. The levels of serum and cell secreted EGFR were further validated to match proteomic data regarding the inverse correlation with the invasive phenotype. In summary, we report a comprehensive patient-based proteomics approach for the identification of potential serum biomarkers for OSCC using an orthotopic xenograft mouse model.

Fascin regulates prostate cancer cell invasion and is associated with metastasis and biochemical failure in prostate cancer.

Purpose: Prostate cancer metastasis to secondary organs is considered an initial event in the development of hormone refractory disease and remains the major cause of death among prostate cancer patients. In this study, we investigated the role of fascin, a cytoskeleton actin–bundling protein involved in the formation of filopodia and cell migration, in prostate cancer progression. Experimental Design: Fascin protein expression was examined by immunohistochemistry in a cohort of 196 patients with localized prostate cancer and across several stages of disease progression, including hormone refractory disease. Cellular changes were also assessed in vitro and in vivo in DU145 prostate cancer cell line using fascin gene silencing. Results: Fascin epithelial expression was significantly up-regulated in localized and hormone refractory prostate cancer compared with benign prostate tissue (P < 0.05). Furthermore, high fascin expression was associated with an increased rate of prostate-specific antigen recurrence following radical prostatectomy (P = 0.075), signifying more aggressive clinical course, thus supporting a function for fascin in prostate cancer progression. In cellular models, fascin gene silencing using small interfering RNA in the androgen-independent prostate cancer cell line DU145 decreased cell motility and invasiveness while increasing cell adhesive properties. In addition, fascin small interfering RNA–expressing DU145 cells implanted orthotopically in mouse prostate showed significantly decreased growth (P < 0.005) and drastically prevented the formation of lymph node metastases (P < 0.001) compared with their matched controls. Conclusions: Our data show a function of fascin in the regulation of prostate cancer progression and emphasize the importance of fascin as a prognostic marker for aggressive disease and as a potential therapeutic target for advanced androgen independent disease.

PTEN deletion and heme oxygenase-1 overexpression cooperate in prostate cancer progression and are associated with adverse clinical outcome.

Overexpression of the pro‐survival protein heme oxygenase‐1 (HO‐1) and loss of the pro‐apoptotic tumour suppressor PTEN are common events in prostate cancer (PCA). We assessed the occurrence of both HO‐1 expression and PTEN deletion in two cohorts of men with localized and castration‐resistant prostate cancer (CRPC). The phenotypic cooperation of these markers was examined in preclinical and clinical models. Overall, there was a statistically significant difference in HO‐1 epithelial expression between benign, high‐grade prostatic intraepithelial neoplasia (HGPIN), localized PCA, and CRPC (p < 0.0001). The highest epithelial HO‐1 expression was noted in CRPC (2.00 ± 0.89), followed by benign prostate tissue (1.49 ± 1.03) (p = 0.0003), localized PCA (1.20 ± 0.95), and HGPIN (1.07 ± 0.87) (p < 0.0001). However, the difference between HGPIN and PCA was not statistically significant (p = 0.21). PTEN deletions were observed in 35/55 (63.6%) versus 68/183 (37.1%) cases of CRPC and localized PCA, respectively. Although neither HO‐1 overexpression nor PTEN deletions alone in localized PCA showed a statistically significant association with PSA relapse, the combined status of both markers correlated with disease progression (log‐rank test, p = 0.01). In a preclinical model, inhibition of HO‐1 by shRNA in PTEN‐deficient PC3M cell line and their matched cells where PTEN is restored strongly reduced cell growth and invasion in vitro and inhibited tumour growth and lung metastasis formation in mice compared to cells where only HO‐1 is inhibited or PTEN is restored. In summary, we provide clinical and experimental evidence for cooperation between epithelial HO‐1 expression and PTEN deletions in relation to the PCA patients outcome. These findings could potentially lead to the discovery of novel therapeutic modalities for advanced PCA. Copyright

Complement Activates the c-Jun N-Terminal Kinase/Stress-Activated Protein Kinase in Glomerular Epithelial Cells

In the rat passive Heymann nephritis model of membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 activates cytosolic phospholipase A2 (cPLA2), and products of cPLA2-mediated phospholipid hydrolysis modulate GEC injury and proteinuria. In the present study, we demonstrate that C5b-9 activates c-Jun N-terminal kinase (JNK) in cultured rat GECs and that JNK activity is increased in glomeruli isolated from proteinuric rats with passive Heymann nephritis, as compared with control rats. Stable overexpression of cPLA2 in GECs amplified complement-induced release of arachidonic acid (AA) and JNK activity, as compared with neo (control) GECs. Activation of JNK was not affected by indomethacin. Incubation of GECs with complement stimulated production of superoxide, and pretreatment with the antioxidants, N-acetylcysteine, glutathione, and α-tocopherol as well as with diphenylene iodonium, an inhibitor of the NADPH oxidase, inhibited complement-induced JNK activation. Conversely, H2O2 activated JNK, whereas exogenously added AA stimulated both superoxide production and JNK activity. Overexpression of a dominant-inhibitory JNK mutant or treatment with diphenylene iodonium exacerbated complement-dependent GEC injury. Thus, activation of cPLA2 and release of AA facilitate complement-induced JNK activation. AA may activate the NADPH oxidase, leading to production of reactive oxygen species, which in turn mediate the activation of JNK. The functional role of JNK activation is to limit or protect GECs from complement attack.

Focal Adhesion Kinase-Related Proline-Rich Tyrosine Kinase 2 and Focal Adhesion Kinase Are Co-Overexpressed in Early-Stage and Invasive ErbB-2-Positive Breast Cancer and Cooperate for Breast Cancer Cell Tumorigenesis and Invasiveness

Early cancer cell migration and invasion of neighboring tissues are mediated by multiple events, including activation of focal adhesion signaling. Key regulators include the focal adhesion kinase (FAK) and FAK-related proline-rich tyrosine kinase 2 (Pyk2), whose distinct functions in cancer progression remain unclear. Here, we compared Pyk2 and FAK expression in breast cancer and their effects on ErbB-2-induced tumorigenesis and the potential therapeutic utility of targeting Pyk2 compared with FAK in preclinical models of breast cancer. Pyk2 is overexpressed in tissues from early and advanced breast cancers and overexpressed with both FAK and epidermal growth factor receptor-2 (ErbB-2) in a subset of breast cancer cases. Down-regulation of Pyk2 in ErbB-2-positive, FAK-proficient, and FAK-deficient cells reduced cell proliferation, which correlated with reduced mitogen-activated protein kinase (MAPK) activity. In contrast, Pyk2 silencing had little impact on cell migration and invasion. In vivo, Pyk2 down-regulation reduced primary tumor growth induced by a metastatic variant of ErbB-2-positive MDA 231 breast cancer cells but had little effect on lung metastases in contrast to FAK down-regulation. Dual reduction of Pyk2 and FAK expression resulted in strong inhibition of both primary tumor growth and lung metastases. Together, these data support the cooperative function of Pyk2 and FAK in breast cancer progression and suggest that dual inhibition of FAK and Pyk2 is an efficient therapeutic approach for targeting invasive breast cancer.

Glomerular epithelial cell injury associated with mutant α-actinin-4

Focal segmental glomerulosclerosis (FSGS) may be associated with glomerular epithelial cell (GEC; podocyte) apoptosis due to acquired injury or mutations in alpha-actinin-4. This study addresses how FSGS-associated mutant alpha-actinin-4 may induce GEC injury, focusing on endoplasmic reticulum (ER) stress and metabolism of mutant alpha-actinin-4 via the ubiquitin-proteasome system. In a model of experimental FSGS induced by expression of an alpha-actinin-4 K256E transgene in podocytes, we show induction of ER stress, including upregulation of ER chaperones (bip, grp94), phosphorylation of the eukaryotic translation initiation factor-2alpha subunit, and induction of the proapoptotic gene C/EBP homologous protein-10 (CHOP). To address mechanisms of ER stress, we studied signaling in cultured GEC and COS cells expressing alpha-actinin-4 K256E. Previously, we showed that expression of this alpha-actinin-4 mutant in GEC increased apoptosis. In the present study, we show that alpha-actinin-4 K256E upregulates grp94 and CHOP expression in COS cells and significantly exacerbates induction of bip and CHOP in GEC in the presence of tunicamycin. ER stress was associated with aggregation and ubiquitination of alpha-actinin-4 K256E and impairment of the ubiquitin-proteasome system. In addition, alpha-actinin-4 K256E exacerbated apoptosis in the context of mild proteasome inhibition. Thus alpha-actinin-4 K256E triggers several metabolic abnormalities, which may lead to GEC injury and glomerulosclerosis.

Discovery of indoline-based, natural-product-like compounds as probes of focal adhesion kinase signaling pathways.

With the goal of identifying small molecule modulators of protein-protein interactions, we developed a solid-phase synthesis method, which was then successfully utilized in a library generation of 164 aminoindoline-derived, natural-product-like compounds. This library and several other related intermediates synthesized during this project were then subjected to different biological assays in search of small molecule modulators of focal adhesion kinase (FAK)-mediated signaling pathways. This study included (i) an in vitro, full length FAK inhibition assay, (ii) a cell proliferation assay, and (iii) a wound healing assay. In FAK inhibition assay, eight library members (5-12) and three aminoindoline derivatives (13, 14, and 2) were identified as promising candidates. Compounds 13 and 2 inhibited the FAK activity by 25-45% at 10 microM. These two lead compounds also showed activity in a wound healing assay. To our knowledge, these aminoindoline-derived small molecules belong to a new family of FAK inhibitors.

Total Synthesis and Biological Activity of Marine Alkaloid Eudistomins Y1–Y7 and Their Analogues

Eudistomin Y class compounds are a series of β-carbolines which was originally isolated from a marine turnicate or ascidian near the South Korea Sea. These compounds contain bromo-substituted groups, which is one of the typical characters of marine natural products. We report herein the chemical synthesis and biological evaluation of seven new β-carboline-based metabolites, Eudistomins Y1–Y7, and their hydroxyl-methylated phenyl derivatives. Using bromo-substituted tryptamines and bromo-substituted phenylglyoxals as the key intermediates, Eudistomins Y1–Y7 and their derivatives were synthesized via the acid-catalyzed Pictet-Spengler reaction and fully characterized by 1H- and 13C-NMR and mass spectroscopy. Biological studies revealed that all of the compounds showed moderate growth inhibitory activity against breast carcinoma cell line MDA-231 with IC50 of 15–63 μM and the inhibitory activities of hydroxyl-methylated phenyl products were higher than that of the corresponding natural products Eudistomins Y1–Y7.

Total synthesis and bioactivity of the marine alkaloid pityriacitrin and some of its derivatives

We report herein the chemical synthesis and biological evaluation of β-carboline alkaloid pityriacitrin and some of its new derivatives. Using tryptophan or 5-hydroxytryptophan and 5-substituted indole-3-glyoxals as the starting materials, pityriacitrin and some of its derivatives were synthesized via the acid-catalyzed Pictet-Spengler reaction and fully characterized by (1)H and (13)C NMR, mass spectroscopy and IR determinations. Biological studies revealed that pityriacitrin has a weak antiproliferative activity against a panel of breast and prostate cancer cell lines, whereas some of its derivatives exhibited stronger and potent activity, which was associated with induction of both cell apoptosis and necrosis.

Building skeletally diverse architectures on the Indoline Scaffold: the discovery of a chemical probe of focal adhesion kinase signaling networks.

Inspired by bioactive indoline alkaloid natural products, here, we report a divergent synthesis approach that led to skeletally diverse indoline alkaloid-inspired compounds. The natural product-inspired compounds obtained were then subjected to a series of in vitro and cellular assays to examine their properties as modulators of focal adhesion kinase (FAK) activity. This study resulted in the identification of a promising lead inhibitor of FAK (42), which also showed activity in a wound healing and cell invasion assay. The in silico study of the lead compound (42) was also undertaken.