Xue-Ning Shen

Apogossypol derivative BI-97C1 (Sabutoclax) targeting Mcl-1 sensitizes prostate cancer cells to mda-7/IL-24–mediated toxicity

Limited options are available for treating patients with advanced prostate cancer (PC). Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), an IL-10 family cytokine, exhibits pleiotropic anticancer activities without adversely affecting normal cells. We previously demonstrated that suppression of the prosurvival Bcl-2 family member, myeloid cell leukemia-1 (Mcl-1), is required for mda-7/IL-24–mediated apoptosis of prostate carcinomas. Here we demonstrate that pharmacological inhibition of Mcl-1 expression with the unique Apogossypol derivative BI-97C1, also called Sabutoclax, is sufficient to sensitize prostate tumors to mda-7/IL-24–induced apoptosis, whereas ABT-737, which lacks efficacy in inhibiting Mcl-1, does not sensitize mda-7/IL-24–mediated cytotoxicity. A combination regimen of tropism-modified adenovirus delivered mda-7/IL-24 (Ad.5/3-mda-7) and BI-97C1 enhances cytotoxicity in human PC cells, including those resistant to mda-7/IL-24 or BI-97C1 alone. The combination regimen causes autophagy that facilitates NOXA- and Bim-induced and Bak/Bax-mediated mitochondrial apoptosis. Treatment with Ad.5/3-mda-7 and BI-97C1 significantly inhibits the growth of human PC xenografts in nude mice and spontaneously induced PC in Hi-myc transgenic mice. Tumor growth inhibition correlated with increased TUNEL staining and decreased Ki-67 expression in both PC xenografts and prostates of Hi-myc mice. These findings demonstrate that pharmacological inhibition of Mcl-1 with the Apogossypol derivative, BI-97C1, sensitizes human PCs to mda-7/IL-24–mediated cytotoxicity, thus potentially augmenting the therapeutic benefit of this combinatorial approach toward PC.

A novel “patient‐like” model of cholangiocarcinoma progression based on bile duct inoculation of tumorigenic rat cholangiocyte cell lines

Intrahepatic cholangiocarcinoma typically presents in an advanced stage in which treatment options are limited. In an effort to recapitulate key biological and clinical features of the progressive disease, we established a novel rat model based on bile duct inoculation of rat cholangiocyte cell lines in different stages of tumor progression. Our BDEneu cell line, which is highly tumorigenic, originated from an immortalized rat cholangiocyte cell line (BDE1 cells) that was stably transfected to constitutively overexpress mutationally activated rat neu oncogene. Our less aggressive tumorigenic BDEsp cholangiocyte cell line was derived from the spontaneous in vitro neoplastic transformation of the same parent BDE1 cell line. Unlike BDEneu cells, BDEsp cells expressed wild‐type c‐neu and exhibited in vitro growth rates intermediate between those of BDEneu and BDE1 cholangiocytes. Cyclooxygenase‐2 and activated Akt were significantly overexpressed in BDEsp cells over those of BDE1 cells, and at higher levels than those expressed in BDEneu cells. Only BDEneu cells overexpressed activated p185neu, which was associated with a significant increase in phospho‐p44/42 mitogen‐activated protein kinase (MAPK). Mucin 1 (MUC1) messenger RNA (mRNA), an indicator of cholangiocarcinoma cell progression, was also significantly overexpressed in BDEneu cells over that of BDEsp cells. BDEneu cells inoculated into the bile duct of isogenic rats resulted over a 21‐ to 26‐day period in rapid exponential cholangiocarcinoma tumor growth within liver, paralleled by increases in bile duct obstruction and gross peritoneal metastases. Under comparable conditions, BDEsp cells yielded only small nonmetastatic intrahepatic cholangiocarcinomas without bile duct obstruction. Conclusions: A novel model of cholangiocarcinoma progression mimicking progressive development of the advanced human disease has been established, which may serve as a powerful preclinical platform to study cholangiocarcinoma progression and for rapidly testing treatment approaches. (HEPATOLOGY 2008.)

Astrocyte elevated gene-1 promotes hepatocarcinogenesis: Novel insights from a mouse model†‡

Astrocyte elevated gene‐1 (AEG‐1) is a key contributor to hepatocellular carcinoma (HCC) development and progression. To enhance our understanding of the role of AEG‐1 in hepatocarcinogenesis, a transgenic mouse with hepatocyte‐specific expression of AEG‐1 (Alb/AEG1) was developed. Treating Alb/AEG‐1, but not wild‐type (WT) mice, with N‐nitrosodiethylamine resulted in multinodular HCC with steatotic features and associated modulation of expression of genes regulating invasion, metastasis, angiogenesis, and fatty acid synthesis. Hepatocytes isolated from Alb/AEG‐1 mice displayed profound resistance to chemotherapeutics and growth factor deprivation with activation of prosurvival signaling pathways. Alb/AEG‐1 hepatocytes also exhibited marked resistance toward senescence, which correlated with abrogation of activation of a DNA damage response. Conditioned media from Alb/AEG‐1 hepatocytes induced marked angiogenesis with elevation in several coagulation factors. Among these factors, AEG‐1 facilitated the association of factor XII (FXII) messenger RNA with polysomes, resulting in increased translation. Short interfering RNA–mediated knockdown of FXII resulted in profound inhibition of AEG‐1‐induced angiogenesis. Conclusion: We uncovered novel aspects of AEG‐1 functions, including induction of steatosis, inhibition of senescence, and activation of the coagulation pathway to augment aggressive hepatocarcinogenesis. The Alb/AEG‐1 mouse provides an appropriate model to scrutinize the molecular mechanism of hepatocarcinogenesis and to evaluate the efficacy of novel therapeutic strategies targeting HCC. (HEPATOLOGY 2012;56:1782–1791)

Enhanced delivery of mda‐7/IL‐24 using a serotype chimeric adenovirus (Ad.5/3) in combination with the apogossypol derivative BI‐97C1 (Sabutoclax) improves therapeutic efficacy in low CAR colorectal cancer cells

Adenovirus (Ad)‐based gene therapy represents a potentially viable strategy for treating colorectal cancer. The infectivity of serotype 5 adenovirus (Ad.5), routinely used as a transgene delivery vector, is dependent on Coxsackie‐adenovirus receptors (CAR). CAR expression is downregulated in many cancers thus preventing optimum therapeutic efficiency of Ad.5‐based therapies. To overcome the low CAR problem, a serotype chimerism approach was used to generate a recombinant Ad (Ad.5/3) that is capable of infecting cancer cells via Ad.3 receptors in a CAR‐independent manner. We evaluated the improved transgene delivery and efficacy of Ad.5/3 recombinant virus expressing melanoma differentiation associated gene‐7/interleukin‐24 (mda‐7/IL‐24), an effective wide‐spectrum cancer‐selective therapeutic. In low CAR human colorectal cancer cells RKO, wild‐type Ad.5 virus expressing mda‐7/IL‐24 (Ad.5‐mda‐7) failed to infect efficiently resulting in lack of expression of MDA‐7/IL‐24 or induction of apoptosis. However, a recombinant Ad.5/3 virus expressing mda‐7/IL‐24 (Ad.5/3‐mda‐7) efficiently infected RKO cells resulting in higher MDA‐7/IL‐24 expression and inhibition of cell growth both in vitro and in nude mice xenograft models. Addition of the novel Bcl‐2 family pharmacological inhibitor Apogossypol derivative BI‐97C1 (Sabutoclax) significantly augmented the efficacy of Ad.5/3‐mda‐7. A combination regimen of suboptimal doses of Ad.5/3‐mda‐7 and BI‐97C1 profoundly enhanced cytotoxicity in RKO cells both in vitro and in vivo. Considering the fact that Ad.5‐mda‐7 has demonstrated significant objective responses in a Phase I clinical trial for advanced solid tumors, Ad.5/3‐mda‐7 alone or in combination with BI‐97C1 would be predicted to exert significantly improved therapeutic efficacy in colorectal cancer patients. J. Cell. Physiol. 227: 2145–2153, 2012.

Insulin-like growth factor-binding protein-7 (IGFBP7): a promising gene therapeutic for hepatocellular carcinoma (HCC).

Hepatocellular carcinoma (HCC) is a highly fatal disease mandating development of novel, targeted therapies to elicit prolonged survival benefit to the patients. Insulin-like growth factor-binding protein-7 (IGFBP7), a secreted protein belonging to the IGFBP family, functions as a potential tumor suppressor for HCC. In the present study, we evaluated the therapeutic efficacy of a replication-incompetent adenovirus expressing IGFBP7 (Ad.IGFBP7) in human HCC. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing reactive oxygen species (ROS) and activating a DNA damage response (DDR) and p38 MAPK. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intrahepatic metastasis. In a nude mice subcutaneous model, xenografts from human HCC cells were established in both flanks and only left-sided tumors received intratumoral injection of Ad.IGFBP7. Growth of both left-sided injected tumors and right-sided uninjected tumors were markedly inhibited by Ad.IGFBP7 with profound suppression of angiogenesis. These findings indicate that Ad.IGFBP7 might be a potent therapeutic eradicating both primary HCC and distant metastasis and might be an effective treatment option for terminal HCC patients.Hepatocellular carcinoma (HCC) is a highly fatal disease mandating development of novel, targeted therapies to elicit prolonged survival benefit to the patients. Insulin-like growth factor-binding protein-7 (IGFBP7), a secreted protein belonging to the IGFBP family, functions as a potential tumor suppressor for HCC. In the present study, we evaluated the therapeutic efficacy of a replication-incompetent adenovirus expressing IGFBP7 (Ad.IGFBP7) in human HCC. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing reactive oxygen species (ROS) and activating a DNA damage response (DDR) and p38 MAPK. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intrahepatic metastasis. In a nude mice subcutaneous model, xenografts from human HCC cells were established in both flanks and only left-sided tumors received intratumoral injection of Ad.IGFBP7. Growth of both left-sided injected tumors and right-sided uninjected tumors were markedly inhibited by Ad.IGFBP7 with profound suppression of angiogenesis. These findings indicate that Ad.IGFBP7 might be a potent therapeutic eradicating both primary HCC and distant metastasis and might be an effective treatment option for terminal HCC patients.

Astrocyte Elevated Gene-1 Interacts with Akt Isoform 2 to Control Glioma Growth, Survival, and Pathogenesis

The oncogene astrocyte elevated gene-1 (AEG-1; MTDH) is highly expressed in glioblastoma multiforme (GBM) and many other types of cancer, where it activates multiple signaling pathways that drive proliferation, invasion, angiogenesis, chemoresistance, radioresistance, and metastasis. AEG-1 activates the Akt signaling pathway and Akt and c-Myc are positive regulators of AEG-1 transcription, generating a positive feedback loop between AEG-1 and Akt in regulating tumorigenesis. Here, we describe in GBM cells a direct interaction between an internal domain of AEG-1 and the PH domain of Akt2, a major driver in GBM. Expression and interaction of AEG-1 and Akt2 are elevated in GBM and contribute to tumor cell survival, proliferation, and invasion. Clinically, in silico gene expression and immunohistochemical analyses of patient specimens showed that AEG-1 and Akt2 expression correlated with GBM progression and reduced patient survival. AEG-1-Akt2 interaction prolonged stabilization of Akt2 phosphorylation at S474, regulating downstream signaling cascades that enable cell proliferation and survival. Disrupting AEG-1-Akt2 interaction by competitive binding of the Akt2-PH domain led to reduced cell viability and invasion. When combined with AEG-1 silencing, conditional expression of Akt2-PH markedly increased survival in an orthotopic mouse model of human GBM. Our study uncovers a novel molecular mechanism by which AEG-1 augments glioma progression and offers a rationale to block AEG-1-Akt2 signaling function as a novel GBM treatment.

Suppression of miR-184 in malignant gliomas upregulates SND1 and promotes tumor aggressiveness

BACKGROUND Malignant glioma is an aggressive cancer requiring new therapeutic targets. MicroRNAs (miRNAs) regulate gene expression post transcriptionally and are implicated in cancer development and progression. Deregulated expressions of several miRNAs, specifically hsa-miR-184, correlate with glioma development. METHODS Bioinformatic approaches were used to identify potential miR-184-regulated target genes involved in malignant glioma progression. This strategy identified a multifunctional nuclease, SND1, known to be overexpressed in multiple cancers, including breast, colon, and hepatocellular carcinoma, as a putative direct miR-184 target gene. SND1 levels were evaluated in patient tumor samples and human-derived cell lines. We analyzed invasion and signaling in vitro through SND1 gain-of-function and loss-of-function. An orthotopic xenograft model with primary glioma cells demonstrated a role of miR-184/SND1 in glioma pathogenesis in vivo. RESULTS SND1 is highly expressed in human glioma tissue and inversely correlated with miR-184 expression. Transfection of glioma cells with a miR-184 mimic inhibited invasion, suppressed colony formation, and reduced anchorage-independent growth in soft agar. Similar phenotypes were evident when SND1 was knocked down with siRNA. Additionally, knockdown (KD) of SND1 induced senescence and improved the chemoresistant properties of malignant glioma cells. In an orthotopic xenograft model, KD of SND1 or transfection with a miR-184 mimic induced a less invasive tumor phenotype and significantly improved survival of tumor bearing mice. CONCLUSIONS Our study is the first to show a novel regulatory role of SND1, a direct target of miR-184, in glioma progression, suggesting that the miR-184/SND1 axis may be a useful diagnostic and therapeutic tool for malignant glioma.

Genetic deletion of AEG-1 prevents hepatocarcinogenesis.

Activation of the oncogene AEG-1 (MTDH, LYRIC) has been implicated recently in the development of hepatocellular carcinoma (HCC). In mice, HCC can be initiated by exposure to the carcinogen DEN, which has been shown to rely upon activation of NF-κB in liver macrophages. Because AEG-1 is an essential component of NF-κB activation, we interrogated the susceptibility of mice lacking the AEG-1 gene to DEN-induced hepatocarcinogenesis. AEG-1-deficient mice displayed resistance to DEN-induced HCC and lung metastasis. No difference was observed in the response to growth factor signaling or activation of AKT, ERK, and β-catenin, compared with wild-type control animals. However, AEG-1-deficient hepatocytes and macrophages exhibited a relative defect in NF-κB activation. Mechanistic investigations showed that IL6 production and STAT3 activation, two key mediators of HCC development, were also deficient along with other biologic and epigenetics findings in the tumor microenvironment, confirming that AEG-1 supports an NF-κB-mediated inflammatory state that drives HCC development. Overall, our findings offer in vivo proofs that AEG-1 is essential for NF-κB activation and hepatocarcinogenesis, and they reveal new roles for AEG-1 in shaping the tumor microenvironment for HCC development.

Late SV40 factor (LSF) enhances angiogenesis by transcriptionally up-regulating matrix metalloproteinase-9 (MMP-9).

Background: The transcription factor Late SV40 Factor (LSF) is overexpressed in human hepatocellular carcinoma (HCC). Results: LSF augments tumor angiogenesis by transcriptionally up-regulating matrix metalloproteinase-9 (MMP-9). Conclusion: A novel target of LSF is identified contributing to its oncogenic properties. Significance: LSF regulates a network of proteins, including osteopontin, MMP-9, and c-Met, thereby establishing the rationale for LSF inhibition as a potential therapeutic strategy for HCC. The transcription factor late SV40 factor (LSF) is overexpressed in human hepatocellular carcinoma (HCC) fostering a highly aggressive and metastatic phenotype. Angiogenesis is an essential component of cancer aggression and metastasis and HCC is a highly aggressive and angiogenic cancer. In the present studies, we analyzed the molecular mechanism of LSF-induced angiogenesis in HCC. Employing human umbilical vein endothelial cells (HUVEC) differentiation assay and chicken chorioallantoic membrane (CAM) assay we document that stable LSF overexpression augments and stable dominant negative inhibition of LSF (LSFdn) abrogates angiogenesis by human HCC cells. A quest for LSF-regulated factors contributing to angiogenesis, by chromatin immunoprecipitation-on-chip (ChIP-on-chip) assay, identified matrix metalloproteinase-9 (MMP-9) as a direct target of LSF. MMP-9 expression and enzymatic activity were higher in LSF-overexpressing cells and lower in LSFdn-expressing cells. Deletion mutation analysis identified the LSF-responsive regions in the MMP-9 promoter and ChIP assay confirmed LSF binding to the MMP-9 promoter. Inhibition of MMP-9 significantly abrogated LSF-induced angiogenesis as well as in vivo tumorigenesis, thus reinforcing the role of MMP-9 in facilitating LSF function. The present findings identify a novel target of LSF contributing to its oncogenic properties.

Pancreatic cancer-specific cell death induced in vivo by cytoplasmic-delivered polyinosine-polycytidylic acid.

Polyinosine-polycytidylic acid [pIC] is a synthetic dsRNA that acts as an immune agonist of TLR3 and RLR to activate dendritic and natural killer cells that can kill tumor cells. pIC can also trigger apoptosis in pancreatic ductal adenocarcinoma cells (PDAC) but its mechanism of action is obscure. In this study, we investigated the potential therapeutic activity of a formulation of pIC with polyethylenimine ([pIC](PEI)) in PDAC and investigated its mechanism of action. [pIC](PEI) stimulated apoptosis in PDAC cells without affecting normal pancreatic epithelial cells. Mechanistically, [pIC](PEI) repressed XIAP and survivin expression and activated an immune response by inducing MDA-5, RIG-I, and NOXA. Phosphorylation of AKT was inhibited by [pIC](PEI) in PDAC, and this event was critical for stimulating apoptosis through XIAP and survivin degradation. In vivo administration of [pIC](PEI) inhibited tumor growth via AKT-mediated XIAP degradation in both subcutaneous and quasi-orthotopic models of PDAC. Taken together, these results offer a preclinical proof-of-concept for the evaluation of [pIC](PEI) as an immunochemotherapy to treat pancreatic cancer.