Hsin-Tzu Tsai

Enhancement of antitumor immune response by targeted interleukin-12 electrogene transfer through antiHER2 single-chain antibody in a murine bladder tumor model.

Interleukin-12 (IL-12), despite exerting antitumor activity, has limited therapeutic uses due to its systemic toxicity. Since HER2 (also known as ErbB-2, neu, and HER2/neu) is frequently overexpressed on cancer cells, HER2-targeted delivery of IL-12 to tumors may be a promising strategy for enhancing antitumor immunity. Here we showed that intramuscular electrogene transfer of an expression vector encoding a fusion protein antiHER2scFv-IL12, which consists of antiHER2 single-chain variable fragment (scFv) and single-chain IL-12, significantly retarded tumor growth and prolonged the survival in a syngeneic bladder tumor model. Elevated IL-12 and interferon-gamma (IFN-gamma) levels, increased infiltration of CD4(+) and CD8(+) T cells, and reduced vascular endothelial growth factor (VEGF) expression in the tumors, as well as enhanced cytolytic activity of splenocytes were noted in the treated mice. Our results suggest that this approach may be effective for the treatment of HER2-overexpressing tumors.

Prognostic Relevance of Prothymosin-α Expression in Human Upper Urinary Tract Transitional Cell Carcinoma

OBJECTIVES To investigate the prognostic role of prothymosin-alpha (PTMA) expression in human upper urinary tract transitional cell carcinoma (UUT-TCC). METHODS Paraffin-embedded tissues were collected from 91 patients with UUT-TCC and from 15 paired normal renal cortex and 13 paired urothelial walls. The primary antibody for PTMA (2F11) used was validated in 4 human urothelial cancer cell lines before assessing the surgical specimen. Immunohistochemistry was then conducted to determine the expression intensity of PTMA, the calculation of immunostaining density using imaging analysis, and for immunostaining localization. The correlates with clinicopathologic characteristics and patient survival were explored. RESULTS The expression intensity of PTMA demonstrated a significant enhancement of PTMA expression in UUT-TCCs compared with both paired normal tissues (P = .0002 and P = .0004 for UUT-TCC vs the urothelial wall and vs the renal cortex, respectively). As for the localization of PTMA immunoreactivity, of the 91 tumor specimens, 33 (36.3%) were cytoplasmic PTMA-expressing, 51 (56.0%) were nuclear PTMA-expressing, and 7 (7.7%) were PTMA-negative tumors. On univariate and multivariate analyses, PTMA expression localization was the sole independent prognostic indicator for recurrence-free survival (hazard ratio 4.90, 95% confidence interval 1.73-13.9; P = .003), although pathologic staging was an independent prognostic indicator for both progression-free survival (hazard ratio 22.6, 95% confidence interval 2.56-198; P = .005) and disease-specific overall survival (hazard ratio 5.60, 95% confidence interval 1.48-21.2; P = .011). The limitations of our study included small patient numbers and short follow-up. CONCLUSIONS The results of our study have shown that PTMA is overexpressed in UUT-TCCs and that cytoplasmic PTMA expression can provide significant prognostic information for subsequent tumor recurrence in the residual urinary tract after nephroureterectomy.

Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy

The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-γ, IL-1β, and tumor necrosis factor-α proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

Potent antitumor activity of Oct4 and hypoxia dual-regulated oncolytic adenovirus against bladder cancer

Most solid tumors undergo hypoxia, leading to rapid cell division, metastasis and expansion of a cell population with hallmarks of cancer stem cells (CSCs). Tumor-selective replication of oncolytic adenoviruses may be hindered by oxygen deprivation in tumors. It is desirable to develop a potent oncolytic adenovirus, retaining its antitumor activity even in a hypoxic environment. We have previously generated an Oct4-dependent oncolytic adenovirus, namely Ad9OC, driven by nine copies of the Oct4 response element (ORE) for specifically killing Oct4-overexpressing bladder tumors. Here, we developed a novel Oct4 and hypoxia dual-regulated oncolytic adenovirus, designated AdLCY, driven by both hypoxia response element (HRE) and ORE. We showed that hypoxia-inducible factor (HIF)-2α and Oct4 were frequently overexpressed in hypoxic bladder cancer cells, and HIF-2α was involved in HRE-dependent and Oct4 transactivation. AdLCY exhibited higher cytolytic activities than Ad9OC against hypoxic bladder cancer cells, while sparing normal cells. AdLCY exerted potent antitumor effects in mice bearing human bladder tumor xenografts and syngeneic bladder tumors. It could target hypoxic CD44- and CD133-positive bladder tumor cells. Therefore, AdLCY may have therapeutic potential for targeting hypoxic bladder tumors and CSCs. As Oct4 is expressed in various cancers, AdLCY may be further explored as a broad-spectrum anticancer agent.

MP98-15 NUCLEAR PROTHYMOSIN-α CAN ENHANCE PTEN EXPRESSION TRANSCRIPTIONALLY AND NRF2 EXPRESSION THROUGH INHIBITING TRIM-21-MEDIATED UBIQUITINATION IN HUMAN BLADDER CANCER

Many of the genes affected by HOTAIR expression are in the canonical Wnt-pathway. HOTAIR is a known to facilitate EMT through the canonical Wnt-pathway in other tumors. Determining the importance of the Wnt-pathway in UBC may open up new treatment options. Here we show that HOTAIR is necessary for Wnt-responsiveness and its expression increases during Wnt-pathway activation. EMT is also regulated through intercellular communication mediated by ECVs. Given HOTAIR regulates thousands of genes, we hypothesized that ECVs from HOTAIR knockdown cells would have limited ability to facilitate EMT. In fact, HOTAIR knockdown cells produce fewer exosomes with altered protein cargo and do not facilitate migration or invasion, suggesting that targeting of HOTAIR therapeutically would affect EMT through both the Wnt-pathway and ECVs functionality. Objective: To evaluate the role of HOTAIR in WNT-mediated and EVCmediated EMT METHODS: UBCs treated with LiCl or rWNT and gene expression was analyzed by qRTPCR, western blot and immunohistochemistry. We used scratch and 3D spheroid invasion assays to measure in vitro EMT in rWNT treated or untreated UBC cells. shRNA or siRNA against HOTAIR were used and WNT target and antagonist gene expression was measured by qRT-PCR. Migration and invasion were measured using scratch wound assay and 3D spheroid assay. TCF7L2 binding sites were identified in the promoter region of HOTAIR by sequencing. siRNA against TCF7L2 or beta-catenin reduced HOTAIR expression. ECVs isolatedd by ultracentrifugation and sucrose gradient were analyzed using the Nanosight. ECVs protein analysis was performed with LC MS/MS mass spectrometry and western blot. EVC-mediated migration and invasion was evaluated by wound and 3D invasion assay. RESULTS: TCGA data reveals WNT pathway genes are affected in human UBC. LiCl or rWNT treated UBCs have increased EMT related gene expression. rWnt facilitates UBC in vitro migration and invasion in a HOTAIR-dependent fashion. Reduced HOTAIR expression correlates with decreased WNT-target and increased WNTantagonist gene expression. Importantly, HOTAIR is a target of canonical WNT signaling. Reduced HOTAIR expression affects UBC EVC number, content and in vitro migration and invasion. CONCLUSIONS: These data support a role for the canonical WNT-pathway in UBC in a manner dependent on HOTAIR expression. Therefore, therapeutic targeting of the WNT-pathway may affect UBC tumor progression through reduced HOTAIR expression. Importantly, loss of HOTAIR affects the expression of hundreds of genes that results in reduced ECVs number, content, and ability to affect in vitro migration and invasion.