Sutao Zhu

Malignant mesothelioma growth inhibition by agents that target the VEGF and VEGF-C autocrine loops.

Malignant mesothelioma (MM) is a locally aggressive tumor that originates from the mesothelial cells of the pleural and sometimes peritoneal surface. Conventional treatments for MM, consisting of chemotherapy or surgery give little survival benefit to patients, who generally die within 1 year of diagnosis. Hence, there is an urgent need for the development of alternative therapies. Vascular endothelial growth factor (VEGF) is an autocrine growth factor for MM. The closely related molecule, VEGF‐C, is also implicated in malignant mesothelioma growth. VEGF‐C and its cognate receptor VEGFR‐3 are co‐expressed in mesothelioma cell lines. A functional VEGF‐C autocrine growth loop was demonstrated in mesothelioma cells by targeting VEGF‐C expression and binding to VEGFR‐3. The ability of novel agents that reduce the levels of VEGF and VEGF‐C to inhibit mesothelioma cell growth in vitro was assessed. Antisense oligonucleotide (ODN) complementary to VEGF that inhibited VEGF and VEGF‐C expression simultaneously specifically inhibited mesothelioma cell growth. Similarly, antibodies to VEGF receptor (VEGFR‐2) and VEGF‐C receptor (VEGFR‐3) were synergistic in inhibiting mesothelioma cell growth. In addition, a diphtheria toxin‐VEGF fusion protein (DT‐VEGF), which is toxic to cells that express VEGF receptors was very effective in inhibiting mesothelioma cell growth in vitro. These results indicate that targeting VEGF and VEGF‐C simultaneously may be an effective therapeutic approach for malignant mesothelioma.

EphB4 Expression and Biological Significance in Prostate Cancer

Prostate cancer is the most common cancer in men. Advanced prostate cancer spreading beyond the gland is incurable. Identifying factors that regulate the spread of tumor into the regional nodes and distant sites would guide the development of novel diagnostic, prognostic, and therapeutic targets. The aim of our study was to examine the expression and biological role of EphB4 in prostate cancer. EphB4 mRNA is expressed in 64 of 72 (89%) prostate tumor tissues assessed. EphB4 protein expression is found in the majority (41 of 62, 66%) of tumors, and 3 of 20 (15%) normal prostate tissues. Little or no expression was observed in benign prostate epithelial cell line, but EphB4 was expressed in all prostate cancer cell lines to varying degrees. EphB4 protein levels are high in the PC3 prostate cancer cell line and several folds higher in a metastatic clone of PC3 (PC3M) where overexpression was accompanied by EphB4 gene amplification. EphB4 expression is induced by loss of PTEN, p53, and induced by epidermal growth factor/epidermal growth factor receptor and insulin-like growth factor-I/insulin-like growth factor-IR. Knockdown of the EphB4 protein using EphB4 short interfering RNA or antisense oligodeoxynucleotide significantly inhibits cell growth/viability, migration, and invasion, and induces apoptosis in prostate cancer cell lines. Antisense oligodeoxynucleotide targeting EphB4 in vivo showed antitumor activity in murine human tumor xenograft model. These data show a role for EphB4 in prostate cancer and provide a rationale to study EphB4 for diagnostic, prognostic, and therapeutic applications.

Expression and Significance of Vascular Endothelial Growth Factor Receptor 2 in Bladder Cancer

PURPOSE Vascular endothelial growth factor has a critical role in maintaining tumor microvasculature and, as such, is an attractive target for anti-angiogenic therapy. Aberrant expression of VEGF receptors, especially VEGFR2, on epithelial tumor cells allows VEGF to stimulate growth and migration of tumor cells in an autocrine and/or paracrine manner. Therefore, we studied the expression of VEGF and VEGFR2 in bladder cancer, and the relationship to disease characteristics. MATERIALS AND METHODS Expression of VEGF and VEGFR2 was studied in a cohort of 72 patients with transitional cell cancer of the bladder. Tumor tissues from all patients were analyzed by immunohistochemistry and examined by a pathologist blinded to patient outcome. Patient demographics and disease outcome were correlated with expression of these markers. Bladder cancer cell lines that express VEGFR2 were studied in vitro and in vivo to establish the significance of VEGF/VEGFR2 signaling. RESULTS Expression of VEGF and VEGFR2 was observed in 58% and 50% of urothelial tumor cells, respectively. VEGF expression failed to correlate with clinical variables. However, VEGFR2 expression correlated with disease stage (coefficient 0.23, p = 0.05). In addition, VEGFR2 expression increased with tumor invasion into the muscle (p <0.01). Experiments with VEGFR2 positive bladder cancer cell lines in vitro demonstrated increased invasion in response to VEGF. In addition, VEGF inhibition augmented the effect of docetaxel in a murine xenograft model of bladder cancer with a significant inhibition in proliferative index and microvascular density, and induction of apoptosis. CONCLUSIONS Increased VEGFR2 expression correlates with several features that predict progression of urothelial cancer, including disease stage and invasive phenotype. VEGF targeted therapy may enhance the efficacy of standard therapy for bladder cancer.

EphB4 receptor tyrosine kinase is expressed in bladder cancer and provides signals for cell survival.

We sought to evaluate the biological function of the receptor tyrosine kinase EphB4 in bladder cancer. All of the nine bladder cancer cell lines examined express EphB4 and the receptor could be phosphorylated following stimulation with its cognate ligand, EphrinB2. Out of the 15 fresh bladder cancer specimens examined, 14 expressed EphB4 with a mean sevenfold higher level of expression compared to adjacent normal urothelium. EphB4 expression was regulated by several mechanisms: EPHB4 gene locus was amplified in 27% tumor specimens and 33% cell lines studied; inhibition of EGFR signaling downregulated EphB4 levels; and forced expression of wild-type p53 reduced EphB4 expression. EphB4 knockdown using specific siRNA and antisense oligodeoxynucleotides molecules led to a profound inhibition in cell viability associated with apoptosis via activation of caspase-8 pathway and downregulation of antiapoptotic factor, bcl-xl. Furthermore, EphB4 knockdown significantly inhibited tumor cell migration and invasion. EphB4 knockdown in an in vivo murine tumor xenograft model led to a nearly 80% reduction in tumor volume associated with reduced tumor proliferation, increased apoptosis and reduced tumor microvasculature. EphB4 is thus a potential candidate as a predictor of disease outcome in bladder cancer and as target for novel therapy.

Increased radiation sensitivity of head and neck squamous cell carcinoma with sphingosine kinase 1 inhibition

Sphingosine kinase 1 (SphK1) is an important regulator of apoptosis, survival, and proliferation in cancer cells. SphK1 expression in head and neck squamous cell cancer (HNSCC) cell lines and tumor tissue was assessed, and the efficacy of SphK1 knockdown in increasing tumor radiosensitivity was evaluated in vitro and in vivo.

Up-regulation of EphB4 in mesothelioma and its biological significance.

Purpose: Mesothelioma is a rare malignancy that is incurable and carries a short survival despite surgery, radiation, or chemotherapy. This study was designed to identify novel targets for diagnostic, prognostic, and therapeutic approaches. Experimental Design: The expression and functional significance of the receptor tyrosine kinase EphB4 was studied in vitro and in a murine model of mesothelioma. Results: EphB4 was highly expressed in mesothelioma cell lines and primary tumor tissues but not in normal mesothelium. Knockdown of EphB4 using small interfering RNA and antisense oligodeoxynucleotide showed reduction in cell survival, migration, and invasion. EphB4 knockdown initiated a caspase-8-mediated apoptosis and down-regulation of the antiapoptotic protein bcl-xl. EphB4 knockdown also resulted in reduced phosphorylation of Akt and down-regulation of matrix metalloproteinase-2 transcription. In addition, murine tumor xenograft studies using EphB4 oligodeoxynucleotides showed a marked reduction in tumor growth accompanied by a specific decline in EphB4 protein levels, reduced cell division, apoptosis in tumor tissue, and decreased microvascular density. Conclusions: EphB4 is expressed in mesothelioma, provides a survival advantage to tumor cells, and is therefore a potential novel therapeutic target.

Relationship of eosinophils and plasma cells to biofilm in chronic rhinosinusitis.

Background This study investigates the relationship of eosinophils and plasma cells to biofilm in chronic rhinosinusitis (CRS). A prospective observational study was performed at the Keck Hospital, University of Southern California, Department of Otolaryngology, Los Angeles, CA. Methods A total of 29 patients, 20 undergoing endoscopic sinus surgery for CRS and 9 control patients undergoing septoplasty for nasal obstruction without history or evidence of CRS, were included in this study. Contiguous sinonasal mucosa sample sections were examined by hematoxylin and eosin (H&E), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC) for biofilm, microbes, eosinophil major basic protein (EMBP), and cluster designation 27 (CD27). EMBP and CD27 were used as eosinophil and plasma cell markers, respectively. Results Biofilm was visualized in 15 of 20 patients with CRS on H&E sections, confirmed by microbial presence using FISH. Biofilm was not identified in tissue samples of the nine control patients. On IHC analysis, CD27 and EMBP expression were significantly higher in patients with CRS compared with control (p < 0.05) and had greater expression in biofilm-positive patients compared with biofilm-negative patients. Nasal polyps correlated with higher expression of CD27 and EMBP, but in CRS patients without polyps CD27 and EMBP was also significantly greater in biofilm-positive specimens compared with biofilm-negative specimens. Conclusion Biofilm presence in CRS appears to correlate to host inflammatory response involving plasma cell and eosinophil recruitment.

Natural killer cell cytolytic activity is necessary for in vivo antitumor activity of the dipeptide L-glutamyl-L-tryptophan

A dipeptide, L‐glutamyl L‐tryptophan (L‐glu‐L‐trp), was identified in a screen for immunomodulators in the soluble fraction of the thymus. L‐glu‐L‐trp inhibits tumor growth in mice without showing direct cellular toxicity in a variety of human tumor cell lines. L‐glu‐L‐trp antitumor activity in vivo requires the presence of natural killer (NK) cells. Defective trafficking of cytoplasmic granules caused by the Lyst mutation also resulted in loss of antitumor activity of the dipeptide. The effect of L‐glu‐L‐trp on tumor growth in mice with targeted gene mutations demonstrated the absolute requirement for perforin for antitumor activity. The requirement of 2 major modulators of NK cell activity, gamma interferon (IFNγ) and interleukin (IL)‐12, were also tested. L‐glu‐L‐trp had full antitumor activity in IFNγ knockout mice, but had significantly diminished activity in IL‐12 knockout mice. These data show that L‐glu‐L‐trp antitumor activity in mice is dependent on cytolytic cell activity of NK or NKT cells. L‐glu‐L‐trp in vivo regulates NK cell function independent of IFNγ but partly dependent on IL‐12.

Lower Recurrence in HPV-Positive Head and Neck Squamous Cell Carcinoma Mediated by Down-Regulation of Sphingosine Kinase 1

Background: Human papilloma viruses (HPVs) are implicated in a subgroup of head and neck squamous cell carcinoma (HNSCC) with a more favorable prognosis and lower recurrences. The mechanism of the HPV ‘s protective role remains unclear. We observed that HPV+ tumors express lower levels of sphingosine kinase 1 (Sphk1), an important regulator of apoptosis and proliferation in cancer cells. Previously, we showed that both in vitro and in vivo knockdown of Sphk1 increased radiosensitivity in HNSCC cell lines. Our goal is to determine the potential of Sphk1 as a biomarker for recurrence. Methods: DNA polymerase chain reaction classified HPV status for 16 tumors and 6 primary cell cultures developed from HNSCC. Immunohistochemistry assessed p53, Rb, p16, Sphk1, and TGF-β expression in tumor samples. Western Blot examined Sphk1 expression in primary cell cultures. We gathered clinical information through review of medical records. Results: All HNSCC patients, both HPV+ and HPV-, presented at advanced stage (III/IV). HPV+ samples had lower p53 and Rb and higher p16 expression. On immunohistochemistry Sphk1 expression was lower in HPV+ (46%) than HPV- (70%) tumors (p=0.014). Primary cell cultures from HNSCC patients displayed lower Sphk1 expression in HPV+ cells on Western Blot. TGF-β showed no difference among these groups (p=0.99). HPV+ patients had fewer recurrences (p=0.001) and lower 5-year local recurrence (p=0.03). Conclusion: The study showed that Sphk1 expression was lower in HPV+ than HPV- HNSCC. Primary cell cultures showed robust Sphk1 expression in HPV- cells and diminished expression in HPV+ cells. Sphk1 expression was associated with recurrence, suggesting its utility as an early biomarker for detecting recurrence. Despite the small size of the cohort, we observed significant differences between HPV+ and HPV- groups. We identified a novel pathway through which HPV may confer a favorable prognosis in HNSCC. This pathway may have value for further testing in Sphk1-targeted therapies.