Neale T. Hanke

Doxorubicin eliminates myeloid-derived suppressor cells and enhances the efficacy of adoptive T-cell transfer in breast cancer.

Myeloid-derived suppressor cells (MDSC) expand in tumor-bearing hosts and play a central role in cancer immune evasion by inhibiting adaptive and innate immunity. They therefore represent a major obstacle for successful cancer immunotherapy. Different strategies have thus been explored to deplete and/or inactivate MDSC in vivo. Using a murine mammary cancer model, we demonstrated that doxorubicin selectively eliminates MDSC in the spleen, blood, and tumor beds. Furthermore, residual MDSC from doxorubicin-treated mice exhibited impaired suppressive function. Importantly, the frequency of CD4(+) and CD8(+) T lymphocytes and consequently the effector lymphocytes or natural killer (NK) to suppressive MDSC ratios were significantly increased following doxorubicin treatment of tumor-bearing mice. In addition, the proportion of NK and cytotoxic T cell (CTL) expressing perforin and granzyme B and of CTL producing IFN-γ was augmented by doxorubicin administration. Of therapeutic relevance, this drug efficiently combined with Th1 or Th17 lymphocytes to suppress tumor development and metastatic disease. MDSC isolated from patients with different types of cancer were also sensitive to doxorubicin-mediated cytotoxicity in vitro. These results thus indicate that doxorubicin may be used not only as a direct cytotoxic drug against tumor cells, but also as a potent immunomodulatory agent that selectively impairs MDSC-induced immunosuppression, thereby fostering the efficacy of T-cell-based immunotherapy.

Myeloid-derived suppressor cells from tumor-bearing mice impair TGF-β-induced differentiation of CD4+CD25+FoxP3+ Tregs from CD4+CD25−FoxP3− T cells

MDSCs and Tregs play an essential role in the immunosuppressive networks that contribute to tumor‐immune evasion. The mechanisms by which tumors promote the expansion and/or function of these suppressive cells and the cross‐talk between MDSC and Treg remain incompletely defined. Previous reports have suggested that MDSC may contribute to Treg induction in cancer. Herein, we provide evidence that tumor‐induced gr‐MDSCs, endowed with the potential of suppressing conventional T Lc, surprisingly impair TGF‐β1‐mediated generation of CD4+CD25+FoxP3+ iTregs. Furthermore, gr‐MDSCs impede the proliferation of nTregs without, however, affecting FoxP3 expression. Suppression of iTreg differentiation from naïve CD4+ cells by gr‐MDSC occurs early in the polarization process, requires inhibition of early T cell activation, and depends on ROS and IDO but does not require arginase 1, iNOS, NO, cystine/cysteine depletion, PD‐1 and PD‐L1 signaling, or COX‐2. These findings thus indicate that gr‐MDSCs from TB hosts have the unanticipated ability to restrict immunosuppressive Tregs.

IL-6 stimulates STAT3 and Pim-1 kinase in pancreatic cancer cell lines

Objectives We investigated the signaling pathways activated in response to interleukin 6 (IL-6) in pancreatic cell lines, with a focus on signal transducer and activator of transcription 3 (STAT3) and proto-oncogene serine/threonine-protein (Pim-1) kinase. Methods Interleukin 6 receptor (IL-6R) expression and IL-6–induced cell signaling was measured by Western blotting in human pancreatic cell lines. Cucurbitacin I was used as a pharmacological tool to investigate the role of STAT3 in Pim-1 activation. Stably overexpressing Pim-1 kinase cell lines were characterized for their response to IL-6 in vitro and for their growth rate as flank tumors in scid mice. Results Interleukin 6 receptor was expressed across multiple cancer cell lines. In Panc-1 cells, IL-6 treatment increased expression of phosphorylation of signal transducer and activator of transcription 3 and Pim-1 kinase. Cucurbitacin I treatment alone increased pErk1/2 expression in wild-type and Pim-1–overexpressing cell lines and resulted in exaggerated Pim-1 kinase protein levels in control and IL-6–stimulated cells, suggesting that up-regulation of Pim-1 may be partially STAT3 independent. Pim-1 overexpression did not significantly affect growth rate in vitro or in vivo in Panc-1 or MiaPaCa2 cell lines. Conclusions Interleukin 6 activates STAT3 and stimulates Pim-1 kinase in pancreatic cell line models. The regulation and consequence of Pim-1 expression seems to be highly context dependent.

Th-1 Lymphocytes Induce Dendritic Cell Tumor Killing Activity by an IFN-γ–Dependent Mechanism

Dendritic cells (DCs) encompass a heterogeneous population of cells capable of orchestrating innate and adaptive immune responses. The ability of DCs to act as professional APCs has been the foundation for the development and use of these cells as vaccines in cancer immunotherapy. DCs are also endowed with the nonconventional property of directly killing tumor cells. The current study investigates the regulation of murine DC cytotoxic function by T lymphocytes. We provide evidence that CD4+ Th-1, but not Th-2, Th-17 cells, or regulatory T cells, are capable of inducing DC cytotoxic function. IFN-γ was identified as the major factor responsible for Th-1–induced DC tumoricidal activity. Tumor cell killing mediated by Th-1–activated killer DCs was dependent on inducible NO synthase expression and NO production. Importantly, Th-1–activated killer DCs were capable of presenting the acquired Ags from the killed tumor cells to T lymphocytes in vitro or in vivo. These observations offer new possibilities for the application of killer DCs in cancer immunotherapy.

The anti-tumor efficacy of 2-deoxyglucose and D-allose are enhanced with p38 inhibition in pancreatic and ovarian cell lines

PurposeThe anti-tumor activity of glucose analogs 2-deoxy-glucose (2-DG) and D-allose was investigated alone or in combination with p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 or platinum analogs as a strategy to pharmacologically target glycolytic tumor phenotypes.MethodsHypoxia inducible factor-1 alpha (HIF-1α) protein accumulation in pancreatic cell lines treated with SB202190 alone and in combination with glucose analogs was analyzed by Western blot. HIF-1α transcriptional activity was measured in MIA PaCa-2 cells stably transfected with a hypoxia response element luciferase reporter following treatment with glucose analogs alone, and in combination with SB202190. Induction of cleaved poly(ADP-ribose) polymerase (PARP) was measured by Western blot in the MIA PaCa-2 cells. In vitro anti-proliferative activity of 2-DG and D-allose alone, or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines), or with SB202190 were investigated using the MTT assay.ResultsSB202190 decreased HIF-1α protein accumulation and transcriptional activity. 2-DG demonstrated greater anti-proliferative activity than D-allose. Pre-treatment with SB202190 enhanced activity of both 2-DG and D-allose in MIA PaCa-2, BxPC-3, ASPC-1, and SK-OV-3 cells. The combination of D-allose and platinum agents was additive to moderately synergistic in all but the OVCAR-3 and HEY cells. SB202190 pre-treatment further enhanced activity of D-allose and 2-DG with platinum agents in most cell lines investigated.ConclusionsSB202190 induced sensitization of tumor cells to 2-DG and D-allose may be partially mediated by inhibition of HIF-1α activity. Combining glucose analogs and p38 MAPK inhibitors with chemotherapy may be an effective approach to target glycolytic tumor phenotypes.

Loss of catalase increases malignant mouse keratinocyte cell growth through activation of the stress activated JNK pathway

A cell line that produces mouse squamous cell carcinoma (6M90) was modified to develop a cell line with an introduced Tet‐responsive catalase transgene (MTOC2). We have previously reported that the overexpressed catalase in the MTOC2 cells reverses the malignant phenotype in part by decreasing epidermal growth factor receptor (EGFR) signaling. With this work we expanded the investigation into the differences between these two cell lines. We found that the decreased EGFR pathway activity of the MTOC2 cells is not because of reduced autocrine secretion of an epidermal growth factor (EGF) ligand but rather because of lower basal receptor activity. Phosphorylated levels of the mitogen‐activated protein kinase (MAPK) members JNK and p38 were both higher in the 6M90 cells with low catalase when compared with the MTOC2 cell line. Although treatment with an EGFR inhibitor, AG1478, blocked the increased activity of JNK in the 6M90 cells, a similar effect was not observed for p38. Basal levels of downstream c‐jun transcription were also found to be higher in the 6M90 cells versus MTOC2 cells. Activated p38 was found to down‐regulate the JNK MAPK pathway in the 6M90 cells. However, the 6M90 cells contain constitutively high levels of phosphorylated JNK, generating higher levels of phosphorylated c‐jun and total c‐jun than those in the MTOC2 cells. Inhibition of JNK activity in the 6M90 cells reduced AP‐1 transcription and cell proliferation. The data confirm the inhibitory effects of catalase on tumor cell growth, specifically through a ligand‐independent decrease in the stress activated JNK pathway.

PIAS1 and STAT-3 impair the tumoricidal potential of IFN-γ-stimulated mouse dendritic cells generated with IL-15

Primarily defined by their antigen‐presenting property, dendritic cells (DCs) are being implemented as cancer vaccines in immunotherapeutic interventions. DCs can also function as direct tumor cell killers. How DC cytotoxic activity can be efficiently harnessed and the mechanisms controlling this nonconventional property are not fully understood. We report here that the tumoricidal potential of mouse DCs generated from myeloid precursors with GM‐CSF and IL‐15 (IL‐15 DCs) can be triggered with the Toll‐like receptor (TLR) 4 ligand lipopolysaccharide to a similar extent compared with that of their counterparts, conventionally generated with IL‐4 (IL‐4 DCs). The mechanism of tumor cell killing depends on the induction of iNOS expression by DCs. In contrast, interferon (IFN)‐γ induces the cytotoxic activity of IL‐4 but not IL‐15 DCs. Although the IFN‐γ‐STAT‐1 signaling pathway is overall functional in IL‐15 DCs, IFN‐γ fails to induce iNOS expression in these cells. iNOS expression is negatively controlled in IFN‐γ‐stimulated IL‐15 DCs by the cooperation between the E3 SUMO ligase PIAS1 and STAT‐3, and can be partially restored with PIAS1 siRNA and STAT‐3 inhibitors.

Characterization of carfilzomib-resistant non-small cell lung cancer cell lines

PurposeWe previously showed that carfilzomib (CFZ) has potent anti-proliferative and cytotoxic activity in a broad range of lung cancer cell lines. Here we investigate possible mechanisms of CFZ acquired resistance in lung cancer cell lines.MethodsCFZ-resistant non-small cell lung cancer (NSCLC) cell lines were developed by exposing A549 and H520 cells to stepwise increasing concentrations of CFZ. Resistance to CFZ and cross-resistance to bortezomib and other chemotherapy drugs was measured using the MTT assay. Cytotoxicity to CFZ was determined using a CytoTox assay. Western blot was used to measure apoptosis, autophagy, and drug efflux transporter-related proteins. Quantitative targeted whole transcriptome sequencing and quantitative RT-PCR was used to measure gene expression. Flow cytometry was used to analyze intracellular accumulation of doxorubicin.ResultsThe CFZ IC50 value of the resistant cells increased versus parental lines (2.5-fold for A549, 122-fold for H520). Resistant lines showed reduced expression of apoptosis and autophagy markers and reduced death versus parental lines following CFZ treatment. Both resistant lines exhibited higher P-glycoprotein (Pgp) gene (TempO-Seq® analysis, increased 1.2-fold in A549, > 9000-fold in H520) and protein expression levels versus parental lines. TempO-Seq® analysis indicated other drug resistance pathways were upregulated. The resistant cell lines demonstrated less accumulation of intracellular doxorubicin, and were cross-resistant to other Pgp client drugs: bortezomib, doxorubicin, and paclitaxel, but not cisplatin.ConclusionsUpregulation of Pgp appears to be an important, but not the only, mechanism of CFZ resistance in NSCLC cell lines.

Abstract 2930: IL-6 stimulates STAT-3 and Pim-1 kinase in pancreatic cancer cell lines

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: IL-6 has both diagnostic and prognostic significance in pancreatic cancer. We investigated the signaling pathways activated in response to IL-6 in pancreatic cell lines, with a focus on STAT-3 and Pim-1 kinase. Methods: IL-6 receptor (IL-6R) expression was evaluated by Western blot analysis across a panel of human pancreatic cell lines. Panc-1 and MiaPaCa2 cell lines were serum starved for 24 hrs and then treated with 100 ng/mL of IL-6 for various time courses from 0 to 6 hr. STAT-3 activation and Pim-1 kinase protein levels were assessed in the presence and absence of IL-6 by Western blot analysis. The ability of cucurbitacin B to block both STAT-3 and Pim-1 activation, and inhibit cell proliferation was investigated. Stably transfected Pim-1 kinase over-expressing cell lines were generated and characterized for their response to IL-6 in vitro, and their growth rate in vivo as flank tumors in scid mice. Results: IL-6R is broadly expressed across multiple cancer cell lines, with the highest expression observed in the Panc-1 cell line. IL-6 stimulation resulted in increased STAT-3 phosphorylation in Panc-1, and MiaPaCa2 tumor cell lines as well as the HP-DEV transformed pancreatic ductal cell line. Treatment with IL-6 resulted in increased Pim-1 protein expression in the Panc-1 but not the MiaPaCa2 parent cell lines. Induction of Pim-1 protein was also observed in both Panc-1 and MiaPaCa2 stably transfected over-expressing cell lines. Pre-treatment with the STAT-3 inhibitor cucurbitacin B resulted in inhibition of STAT-3 phosphorylation and decreased cell proliferation, but was not associated with suppression of Pim-1 protein expression in the Panc-1 cell lines. Over-expression of Pim-1 in the Panc-1 cell line was associated with no significant changes in basal apoptosis rate, cell growth rate in vitro or in flank xenografts, or IL-6 induced expression of VEGF. Conclusions: IL-6 stimulation activates both STAT-3 and Pim-1 kinase expression in pancreatic cell line models. Induction of Pim-1 kinase was cell line specific, suggesting that the molecular context is essential for this pathway to be activated. Suppression of STAT-3 with a pharmacological inhibitor resulted in exaggerated Pim-1 expression in over-expressing cell lines implying Pim-1 up-regulation may be one of the compensatory and/or stress pathways activated when STAT-3 is inhibited. Stable over-expressing Pim-1 kinase Panc-1 and MiaPaCa2 cells did not exhibit significant changes in growth or apoptosis, unlike previously reported studies in the literature with other pancreatic cancer cell line models. This may be due to the multiple pathways that are dysregulated in these cell lines. Both STAT-3 and Pim-1 kinase have previously been identified as drug targets in multiple malignancies, including leukemia, lymphoma, prostate, and pancreatic cancer. Our study suggests that both pathways may be activated in patients with elevated IL-6 levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2930. doi:10.1158/1538-7445.AM2011-2930