Wang, Jing

Suppression of ZEB1 through miR-200 expression or HDAC inhibition with mocetinostat sensitizes resistant KRAS mutant lung cancers to MEK inhibition.

Abstract Background: We recently have reported that co-mutations within KRAS-mutated lung adenocarcinoma (LUAD) define a distinct biological phenotype and may offer insights into therapeutic targeting using this classification system. The analysis revealed striking differences in checkpoint ligand expression and T cell infiltration between TP53 and STK11 (LKB1) co-mutated KRAS LUAD. We, therefore, hypothesized that loss of LKB1promotes an immune-suppressed phenotype in KRAS LUAD. To test this, we analyzed 100 immune-related genes from The Cancer Genome Atlas datasets, as well as three other sets of resected tumors from NSLC patients from MD Anderson Cancer Center. Results: Our results revealed that tumors lacking LKB1 (LKB1-loss), as defined by mutation or gene-expression signature, resulted in a down regulation of: tumor associated immunosuppressive ligands, PD-L1, PD-L2, B7-H3, B7-H4, HVEM; T cell associated immunosuppressive ligands PD-1, CTLA-4, BTLA; co-stimulation ligands such as CD80, -86, -40L, and -40. We further examined if this loss of immunosuppression and co-stimulation resulted in changes in markers of T cell phenotypes. Indeed, tumors lacking LKB1-loss tumors demonstrated a distinct lack absence of several T cell markers including CD3, -4, & -8, but also RORC, T-bet, GATA-3, and FoxP3. To determine if myeloid cell populations, known mediators of immune suppression, were also diminished in LKB1-loss tumors, we analyzed expression of CD11c, -103, -11b, -68, and CD33. Consistent with T cell makers and suppressive ligand markers, myeloid markers were down regulated in LKB1-loss tumors. Because common immune suppression markers were lost, we then examined the expression of antigen presentation (HLA-I, HLA-II) and amino acid transporters as possible alternative methods for immune suppression. In LKB1-loss tumors, both HLA-I and HLA-II markers were down regulated, compared to tumors with intact LKB1 expression. Furthermore, genes associated with the antigen presentation complex, LMP2, TAP1&2, and B2M were also down regulated. In contrast to the immune markers described above, cationic transporters (SLC7A2), anionic transporters (SLC7A11), and neutral transporters (SLC3A2) were universally upregulated in LKB1-loss tumors. Conclusions: LKB1 loss is associated with an inert immune phenotype. Our data suggests that reduced antigen presentation and amino acid depletion, but not checkpoint factors or MDSCs, are likely mediator of this phenotype. Citation Format: Warren L. Denning, Ferdinandos Skoulidis, Li Shen, Vassiliki Papadimitrakopoulou, Lixia Diao, Yanyan Lou, Lauren A. Byers, Jing Wang, Jaime R. Canales, Ignacio I. Wistuba, John Weinstein, Don L. Gibbons, John V. Heymach. Loss of LKB1 mediates an immune inert phenotype in human lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4137.

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