Prasanna K. Santhekadur

Increased RNA-Induced Silencing Complex (RISC) Activity Contributes to Hepatocellular Carcinoma

There is virtually no effective treatment for advanced hepatocellular carcinoma (HCC) and novel targets need to be identified to develop effective treatment. We recently documented that the oncogene Astrocyte elevated gene‐1 (AEG‐1) plays a seminal role in hepatocarcinogenesis. Employing yeast two‐hybrid assay and coimmunoprecipitation followed by mass spectrometry, we identified staphylococcal nuclease domain containing 1 (SND1), a nuclease in the RNA‐induced silencing complex (RISC) facilitating RNAi‐mediated gene silencing, as an AEG‐1 interacting protein. Coimmunoprecipitation and colocalization studies confirmed that AEG‐1 is also a component of RISC and both AEG‐1 and SND1 are required for optimum RISC activity facilitating small interfering RNA (siRNA) and micro RNA (miRNA)‐mediated silencing of luciferase reporter gene. In 109 human HCC samples SND1 was overexpressed in ≈74% cases compared to normal liver. Correspondingly, significantly higher RISC activity was observed in human HCC cells compared to immortal normal hepatocytes. Increased RISC activity, conferred by AEG‐1 or SND1, resulted in increased degradation of tumor suppressor messenger RNAs (mRNAs) that are target of oncomiRs. Inhibition of enzymatic activity of SND1 significantly inhibited proliferation of human HCC cells. As a corollary, stable overexpression of SND1 augmented and siRNA‐mediated inhibition of SND1 abrogated growth of human HCC cells in vitro and in vivo, thus revealing a potential role of SND1 in hepatocarcinogenesis. Conclusion: We unravel a novel mechanism that overexpression of AEG‐1 and SND1 leading to increased RISC activity might contribute to hepatocarcinogenesis. Targeted inhibition of SND1 enzymatic activity might be developed as an effective therapy for HCC. (HEPATOLOGY 2011;)

Astrocyte Elevated Gene-1 (AEG-1): a multifunctional regulator of normal and abnormal physiology

Since its initial identification and cloning in 2002, Astrocyte Elevated Gene-1 (AEG-1), also known as metadherin (MTDH), 3D3 and LYsine-RIch CEACAM1 co-isolated (LYRIC), has emerged as an important oncogene that is overexpressed in all cancers analyzed so far. Examination of a large cohort of patient samples representing diverse cancer indications has revealed progressive increase in AEG-1 expression with stages and grades of the disease and an inverse relationship between AEG-1 expression level and patient prognosis. AEG-1 functions as a bona fide oncogene by promoting transformation. In addition, it plays a significant role in invasion, metastasis, angiogenesis and chemoresistance, all important hallmarks of an aggressive cancer. AEG-1 is also implicated in diverse physiological and pathological processes, such as development, inflammation, neurodegeneration, migraine and Huntingtons disease. AEG-1 is a highly basic protein with a transmembrane domain and multiple nuclear localization signals and it is present in the cell membrane, cytoplasm, nucleus, nucleolus and endoplasmic reticulum. In each location, AEG-1 interacts with specific proteins thereby modulating diverse intracellular processes the combination of which contributes to its pleiotrophic properties. The present review provides a snapshot of the current literature along with future perspectives on this unique molecule.

Targeted Apoptotic Effects of Thymoquinone and Tamoxifen on XIAP Mediated Akt Regulation in Breast Cancer

X-linked inhibitor of apoptosis protein (XIAP) is constitutively expressed endogenous inhibitor of apoptosis, exhibit its antiapoptotic effect by inactivating key caspases such as caspase-3, caspase-7 and caspase-9 and also play pivotal role in rendering cancer chemoresistance. Our studies showed the coadministration of TQ and TAM resulting in a substantial increase in breast cancer cell apoptosis and marked inhibition of cell growth both in vitro and in vivo. Anti-angiogenic and anti-invasive potential of TQ and TAM was assessed through in vitro studies. This novel combinatorial regimen leads to regulation of multiple cell signaling targets including inactivation of Akt and XIAP degradation. At molecular level, TQ and TAM synergistically lowers XIAP expression resulting in binding and activation of caspase-9 in apoptotic cascade, and interfere with cell survival through PI3-K/Akt pathway by inhibiting Akt phosphorylation. Cleaved caspase-9 further processes other intracellular death substrates such as PARP thereby shifting the balance from survival to apoptosis, indicated by rise in the sub-G1 cell population. This combination also downregulates the expression of Akt-regulated downstream effectors such as Bcl-xL, Bcl-2 and induce expression of Bax, AIF, cytochrome C and p-27. Consistent with these results, overexpression studies further confirmed the involvement of XIAP and its regulatory action on Akt phosphorylation along with procaspase-9 and PARP cleavage in TQ-TAM coadministrated induced apoptosis. The ability of TQ and TAM in inhibiting XIAP was confirmed through siRNA-XIAP cotransfection studies. This novel modality may be a promising tool in breast cancer treatment.

Multifunction protein staphylococcal nuclease domain containing 1 (SND1) promotes tumor angiogenesis in human hepatocellular carcinoma through novel pathway that involves nuclear factor κB and miR-221.

Background: Staphylococcal nuclease domain-containing 1 (SND1) is overexpressed in human hepatocellular carcinoma (HCC). Results: SND1 augments tumor angiogenesis by activating NF-κB, resulting in the induction of miR-221, which subsequently induces angiogenin and CXCL16. Conclusion: A novel pathway activated by SND1 is identified as contributing to tumor angiogenesis. Significance: SND1 promotes hepatocarcinogenesis by multiple ways indicating that small molecule inhibitors of SND1 might be effective therapies for HCC. Staphylococcal nuclease domain-containing 1 (SND1) is a multifunctional protein that is overexpressed in multiple cancers, including hepatocellular carcinoma (HCC). Stable overexpression of SND1 in Hep3B cells expressing a low level of SND1 augments, whereas stable knockdown of SND1 in QGY-7703 cells expressing a high level of SND1 inhibits establishment of xenografts in nude mice, indicating that SND1 promotes an aggressive tumorigenic phenotype. In this study we analyzed the role of SND1 in regulating tumor angiogenesis, a hallmark of cancer. Conditioned medium from Hep3B-SND1 cells stably overexpressing SND1 augmented, whereas that from QGY-SND1si cells stably overexpressing SND1 siRNA significantly inhibited angiogenesis, as analyzed by a chicken chorioallantoic membrane assay and a human umbilical vein endothelial cell differentiation assay. We unraveled a linear pathway in which SND1-induced activation of NF-κB resulted in induction of miR-221 and subsequent induction of angiogenic factors Angiogenin and CXCL16. Inhibition of either of these components resulted in significant inhibition of SND1-induced angiogenesis, thus highlighting the importance of this molecular cascade in regulating SND1 function. Because SND1 regulates NF-κB and miR-221, two important determinants of HCC controlling the aggressive phenotype, SND1 inhibition might be an effective strategy to counteract this fatal malady.

Insulin-like Growth Factor–Binding Protein-7 Functions as a Potential Tumor Suppressor in Hepatocellular Carcinoma

Purpose: Hepatocellular carcinoma (HCC) is a highly virulent malignancy with no effective treatment, thus requiring innovative and effective targeted therapies. The oncogene astrocyte-elevated gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis and profoundly downregulates insulin-like growth factor–binding protein-7 (IGFBP7). The present study focuses on analyzing potential tumor suppressor functions of IGFBP7 in HCC and the relevance of IGFBP7 downregulation in mediating AEG-1 function. Experimental Design: IGFBP7 expression was detected by immunohistochemistry in HCC tissue microarray and real-time PCR and ELISA in human HCC cell lines. Dual FISH was done to detect LOH at IGFBP7 locus. Stable IGFBP7-overexpressing clones were established in the background of AEG-1–overexpressing human HCC cells and were analyzed for in vitro proliferation and senescence and in vivo tumorigenesis and angiogenesis. Results: IGFBP7 expression is significantly downregulated in human HCC samples and cell lines compared with normal liver and hepatocytes, respectively, and inversely correlates with the stages and grades of HCC. Genomic deletion of IGFBP7 was identified in 26% of patients with HCC. Forced overexpression of IGFBP7 in AEG-1–overexpressing HCC cells inhibited in vitro growth and induced senescence, and profoundly suppressed in vivo growth in nude mice that might be an end result of inhibition of angiogenesis by IGFBP7. Conclusion: The present findings provide evidence that IGFBP7 functions as a novel putative tumor suppressor for HCC and establish the corollary that IGFBP7 downregulation can effectively modify AEG-1 function. Accordingly, targeted overexpression of IGFBP7 might be a potential novel therapy for HCC. Clin Cancer Res; 17(21); 6693–701. ©2011 AACR.

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)

MDA-9/Syntenin and IGFBP-2 Promote Angiogenesis in Human Melanoma

Melanoma differentiation-associated gene-9 (mda-9/syntenin) encodes an adapter scaffold protein whose expression correlates with and mediates melanoma progression and metastasis. Tumor angiogenesis represents an integral component of cancer metastasis prompting us to investigate a possible role of mda-9/syntenin in inducing angiogenesis. Genetic (gain-of-function and loss-of-function) and pharmacologic approaches were used to modify mda-9/syntenin expression in normal immortal melanocytes, early radial growth phase melanoma, and metastatic melanoma cells. The consequence of modifying mda-9/syntenin expression on angiogenesis was evaluated using both in vitro and in vivo assays, including tube formation assays using human vascular endothelial cells, chorioallantoic membrane (CAM) assays and xenograft tumor animal models. Gain-of-function and loss-of-function experiments confirm that MDA-9/syntenin induces angiogenesis by augmenting expression of several proangiogenic factors/genes. Experimental evidence is provided for a model of angiogenesis induction by MDA-9/syntenin in which MDA-9/syntenin interacts with the extracellular matrix (ECM), activating Src and FAK resulting in activation by phosphorylation of Akt, which induces hypoxia inducible factor 1-α (HIF-1α). The HIF-1α activates transcription of insulin growth factor-binding protein-2 (IGFBP-2), which is secreted thereby promoting angiogenesis and further induces endothelial cells to produce and secrete VEGF-A augmenting tumor angiogenesis. Our studies delineate an unanticipated cell nonautonomous function of MDA-9/syntenin in the context of angiogenesis, which may directly contribute to its metastasis-promoting properties. As a result, targeting MDA-9/syntenin or its downstream-regulated molecules may provide a means of simultaneously impeding metastasis by both directly inhibiting tumor cell transformed properties (autonomous) and indirectly by blocking angiogenesis (nonautonomous).

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.

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.

The presence and severity of nonalcoholic steatohepatitis is associated with specific changes in circulating bile acids

The histologic spectrum of nonalcoholic fatty liver disease (NAFLD) includes fatty liver (NAFL) and steatohepatitis (NASH), which can progress to cirrhosis in up to 20% of NASH patients. Bile acids (BA) are linked to the pathogenesis and therapy of NASH. We (1) characterized the plasma BA profile in biopsy‐proven NAFL and NASH and compared to controls and (2) related the plasma BA profile to liver histologic features, disease activity, and fibrosis. Liquid chromatography/mass spectrometry quantified BAs. Descriptive statistics, paired and multiple group comparisons, and regression analyses were performed. Of 86 patients (24 controls, 25 NAFL, and 37 NASH; mean age 51.8 years and body mass index 31.9 kg/m2), 66% were women. Increased total primary BAs and decreased secondary BAs (both P < 0.05) characterized NASH. Total conjugated primary BAs were significantly higher in NASH versus NAFL (P = 0.047) and versus controls (P < 0.0001). NASH had higher conjugated to unconjugated chenodeoxycholate (P = 0.04), cholate (P = 0.0004), and total primary BAs (P < 0.0001). The total cholate to chenodeoxycholate ratio was significantly higher in NAFLD without (P = 0.005) and with (P = 0.02) diabetes. Increased key BAs were associated with higher grades of steatosis (taurocholate), lobular (glycocholate) and portal inflammation (taurolithocholate), and hepatocyte ballooning (taurocholate). Conjugated cholate and taurocholate directly and secondary to primary BA ratio inversely correlated to NAFLD activity score. A higher ratio of total secondary to primary BA decreased (odds ratio, 0.57; P = 0.004) and higher conjugated cholate increased the likelihood of significant fibrosis (F≥2) (P = 0.007). Conclusion: NAFLD is associated with significantly altered circulating BA composition, likely unaffected by type 2 diabetes, and correlated with histological features of NASH; these observations provide the foundation for future hypothesis‐driven studies of specific effects of BAs on specific aspects of NASH. (Hepatology 2018;67:534‐548).