Blechacz, Boris

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of severe immune activation and dysregulation resulting in extreme and often life‐threatening inflammation. HLH has been well recognized in pediatric populations, and most current diagnostic and therapeutic guidelines are based on pediatric HLH. Recently there has been recognition of HLH in adults, especially secondary to immune deregulation by an underlying rheumatologic, infectious, or malignant condition. This review is focused on malignancy‐associated HLH (M‐HLH), in which possible mechanisms of pathogenesis include severe inflammation, persistent antigen stimulation by the tumor cells, and loss of immune homeostasis because of chemotherapy, hematopoietic stem cell transplantation, or infection. Previously considered rare, M‐HLH may occur in up to 1% of patients with hematologic malignancies. M‐HLH is often missed or diagnosed late in most published studies, and it has been associated with a poor median survival of less than 2 months. Identification of the clinical and laboratory features specific to M‐HLH in adults may allow early detection, consultation with HLH experts, and intervention. Improved management of adult M‐HLH with optimal combinations of T‐lympholytic and immunosuppressive agents and the incorporation of novel agents based on the pediatric experience hopefully will improve outcomes in adults with M‐HLH. Cancer 2017;123:3229‐40. © 2017 American Cancer Society.

Abstract Background: The Tumor Growth Factor-β (TGF-β) pathway is a potent tumor suppressor pathway. Its inactivation has been implicated in hepatocarcinogenesis. However, in patients with advanced stage Hepatocellular Carcinoma (HCC), TGF-β1 serum concentrations are elevated and correlated to tumor size, recurrence and shortened survival. The mechanisms mediating TGF-β1 ligand overexpression in HCC are unknown. Material and Methods: Immunohistochemical analysis of human HCC samples was performed using Tissue Micro Array. Established human HCC cell lines were used for in vitro studies. Gene expression was assessed by qRT-PCR, ELISA and immunoblot analysis. shRNA was used for gene silencing. Transcription factor DNA binding was evaluated by chromatin immunoprecipitation. For promoter analysis, transcription factor binding site containing DNA fragments were cloned into the enhancer or promoter position of luciferase expression vectors and subsequent transfected. Results: In up to 68% of advanced stage HCC tumors Stem Cell Factor (SCF) is overexpressed, and SMAD2 and STAT3 are constitutively activated. SCF expression in human HCC tumors is intratumorally and intracellularly correlated to expression of pSer465/467SMAD2 and pTyr705STAT3. In HCC cell lines, SCF stimulation induces TGF-β1 transcription, expression and secretion, all of which are abrogated by genetic silencing of STAT3. TGF-β1 stimulation of human HCC cells induces TGF-β1 transcription, and genetic silencing of SCF prevents TGF-β1 autoregulation. We identified two STAT3 recognition sites within the TGF-β1 gene. In human HCC cells, TGF-β1 stimulation results in STAT3 binding to one of the identified recognition sites. TGF-β1 activates luciferase expression vectors under control of the corresponding STAT3 binding site but not in its absence. TGF-β1-induced STAT3 binding and expression vector activation is abrogated in HCC cells with genetically silenced SCF. TGF-β1 induces SCF expression but SMAD2 knockdown inhibits TGF-β1-induced SCF transcription and expression. We identified 7 putative SMAD2-binding elements (SBE) within the SCF promoter. TGF-β1 stimulation results in SMAD2 binding to the SCF promoter and activation of luciferase expression vectors regulated by the SCF promoter; conversely, genetic silencing of SMAD2 abrogates TGF-β1-induced SCF promoter activation. Importantly, knockdown of SCF restored the antiproliferative functions of TGF-β, and prevented TGF-β-induced tumor cell migration and invasion. Conclusion: In advanced stage HCC, SCF drives TGF-β1 autoregulation through STAT3-mediated regulation of the TGF-β1 promoter. Thereby, SCF and TGF-β1 form a positive feedback loop that functionally switches the TGF-β pathway from a tumor suppressor to a tumor promoter in advanced stage HCC. Citation Format: Pingyu Zhang, Andres Rojas, Ying Wang, Nina M. Munoz, Lianchun Xiao, Jing Wang, Gregory J. Gores, Mien-Chie Hung, Boris R. A. Blechacz. A positive feedback loop between TGF-β and SCF mediates TGF-β1 ligand overexpression in advanced hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3911. doi:10.1158/1538-7445.AM2015-3911

Abstract Background: Current systemic agents for advanced Hepatocellular carcinoma (HCC) achieve minimal survival benefits. Tumor Growth Factor-β (TGF-β) is a potent tumor suppressor via its SMAD2/3 signaling axis; its inactivation has been implicated in hepatocarcinogenesis. Paradoxically, TGF-β serum concentrations are elevated in patients with advanced HCC. The TGF-β receptor is currently explored in clinical trials as a therapeutic target. However, the mechanisms mediating the functional switch of the TGF-β pathway to an oncogenic role in advanced HCC are unknown. Material and Methods: Gene expression profiling of human HCC was performed using the Oncomine database. Immunohistochemical analysis of human HCC samples was performed using Tissue Micro Arrays. Established human HCC cell lines were used for in vitro studies. Gene expression was assessed by qRT-PCR, ELISA and immunoblot analyses. shRNA was used for gene silencing. Cell cycle analysis was performed by FACS. Cell proliferation was assessed by microscopic quantification, MTS- and BrdU-assay. Apoptosis was analyzed by TUNEL assay and DAPI. Migration was evaluated by wound assay, and invasion by Boyden Chamber assay. Results: In human HCC, SMAD2 overexpression and decreased expression of the negative TGF-β/SMAD regulator SKI was correlated to shortened survival. Intranuclear pSer465/467-SMAD2 was identified in up to 65% of human HCC tumors, and correlated with TNM-stage, survival and pTyr705STAT3. In 50% of human HCC cell lines, the TGF-β/SMAD2 axis was functional. Exclusively in HCC cells with intact SMAD-signaling, TGF-β induced STAT3 Tyr705-phosporylation and nuclear translocation. We show that TGF-β1 transcriptionally regulates expression of the c-KIT ligand Stem Cell Factor (SCF). SCF is overexpressed in advanced HCC tumors, and correlated to pSMAD2, metastases and decreased survival. In human HCC cells, SCF activates STAT3 via auto- and paracrine stimulation of its cognate receptor c-KIT. TGF-β1-induced activation of STAT3 is inhibited by SCF-knockdown, inhibition of c-KIT or JAK1. The TGF-β1/STAT3 axis neutralizes the cell cycle inhibitory function of TGF-β in human HCC cell lines, and instead promotes EMT, tumor cell migration and invasion. Disruption of the TGF-β/STAT3 signaling axis inhibits its tumor promoting effects and restores TGF-β antiproliferative effects. Conclusion: In advanced HCC, TGF-β/SMAD2-signaling is constitutively activated and correlated to worse outcomes. The functional switch of TGF-β is mediated through aberrant expression of SCF with subsequent stimulation of the c-KIT/STAT3 signaling axis. Disruption of this axis restores TGF-β tumor suppressor function. Disruption of the molecular network between TGF-β1/SMAD2 and SCF/STAT3 constitutes a novel therapeutic strategy aiming at restoration of a dysregulated tumor suppressor. Citation Format: Andres Rojas, Pingyu Zhang, Ying Wang, Nina M. Munoz, Lianchun Xiao, Jing Wang, Gregory J. Gores, Mien-Chie Hung, Boris R. A. Blechacz. TGF-β/SMAD2 signaling drives tumor-progression via c-KIT/STAT3-signaling in advanced HCC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1962. doi:10.1158/1538-7445.AM2015-1962

Abstract Apoptosis induction by BH3 mimetics is a therapeutic strategy for human cancer. These mimetics exert single-agent activity in cells “primed” for cell death. Primed cells are dependent upon antiapoptotic Bcl-2 proteins for survival and are characterized by the ability of the BH3 mimetic to induce cytochrome c release from their isolated mitochondria. Our aim was to examine the single-agent activity of obatoclax, a BH3 mimetic in cholangiocarcinoma cell lines. In clonogenic assays, inhibition of colony formation was observed by obatoclax treatment. Despite single-agent activity by obatoclax, the mitochondria from these cells did not release cytochrome c after incubation with this BH3 mimetic. However, immunofluorescence and cell fractionation studies identified Bax activation and translocation to mitochondria after treatment with obatoclax. shRNA targeted knockdown of Bax doubled the IC50 for obatoclax but did not abrogate its cytotoxicity, whereas knockdown of Bak did not alter the IC50. In a cell-free system, obatoclax induced an activating conformational change of Bax, which was attenuated by a site-directed mutagenesis of a previously identified protein activation site. Finally, the drug also elicited a significant in vivo response in a rodent model of this disease. In conclusion, single-agent obatoclax treatment results in Bax activation, which contributes, in part, to cell death in cholangiocarcinoma cells. These data indicate that BH3 mimetics may also function as direct activators of Bax and induce cytotoxicity in cells not otherwise primed for cell death. Cancer Res; 70(5); 1960–9