PIM kinase inhibitor AZD1208 in conjunction with Th1 cytokines potentiate death of breast cancer cellsin vitrowhile also maximizing suppression of tumor growthin vivo when combined with immunotherapy.

PIM kinases are over-expressed by a number of solid malignancies including breast cancer, and are thought to regulate proliferation, survival, and resistance to treatment, making them attractive therapeutic targets. Because PIM kinases sit at the nexus of multiple oncodriver pathways, PIM antagonist drugs are being tested alone and in conjunction with other therapies to optimize outcomes. We therefore sought to test the combination of pharmacological PIM antagonism and Th1-associated immunotherapy. We show that the pan PIM antagonist, AZD1208, when combined in vitro with Th1 cytokines IFN-γ and TNF-α, potentiates metabolic suppression, overall cell death, and expression of apoptotic markers in human breast cancer cell lines of diverse phenotypes (HER-2pos/ERneg, HER-2pos/ERpos and triple-negative). Interestingly, AZD1208 was shown to moderately inhibit IFN-γ secretion by stimulated T lymphocytes of both human and murine origin, suggesting some inherent immunosuppressive activity of the drug. Nonetheless, when multiplexed therapies were tested in a murine model of HER-2pos breast cancer, combinations of HER-2 peptide-pulsed DCs and AZD1208, as well as recombinant IFN-γ plus AZD1208 significantly suppressed tumor outgrowth compared with single-treatment and control groups. These studies suggest that PIM antagonism may combine productively with certain immunotherapies to improve responsiveness.

Murine Dendritic Cells Grown in Serum-Free Culture Show Potent Therapeutic Activity when Loaded with Novel Th Epitopes in an Orthotopic Model of HER2pos Breast Cancer.

Preferred methods for generating mouse dendritic cells (DC) would encompass qualities of consistency, high yield, and potent function. Serum-free culture is also highly desirable, since this is the standard for cell-based therapies used in humans. We report here a serum-free modification of a culture method generating mature, activated DCs from bone marrow precursors. This is achieved through a two-stage culture comprised of 6-day expansion in Flt3 ligand and IL-6 followed by brief differentiation in a medium containing GM-CSF and IL-4, with subsequent activation using TLR ligands ODN1826 and LPS. The serum-free DCs achieve yields and surface phenotype including IL-12p70 secretion similar to standard serum-replete cultures, display a capacity to sensitize in vivo against both MHC class I- and Class II-restricted antigens, and exhibit some aspects of "killer DC" function against tumor cells. We used these DCs to help identify novel CD4pos Th epitopes on the rat ErbB2/HER-2 protein and demonstrated a subset of these as effective immunogens in a DC-based therapeutic model of HER-2pos breast cancer in Balb/c mice, where they induced powerful Th1-polarized immune responses. This method represents a useful way to efficiently produce large numbers of murine dendritic cells with excellent in vivo function well-suited for use in experimental vaccine studies.

Sunitinib Combined with Th1 Cytokines Potentiates Apoptosis in Human Breast Cancer Cells and Suppresses Tumor Growth in a Murine Model of HER-2pos Breast Cancer.

Although immune-based therapies have made remarkable inroads in cancer treatment, they usually must be combined with standard treatment modalities, including cytotoxic drugs, to achieve maximal clinical benefits. As immunotherapies are further advanced and refined, considerable efforts will be required to identify combination therapies that will maximize clinical responses while simultaneously decreasing the unpleasant and sometimes life-threatening side effects of standard therapy. Over the last two decades, evidence has emerged that Th1 cytokines can play a central role in protective antitumor immunity and that combinations of Th1 cytokines can induce senescence and apoptosis in cancer cells. To explore the possibility of combining targeted drugs with Th1-polarizing vaccines, we undertook a study to examine the impact of combining Th1 cytokines with the relatively broad-spectrum receptor tyrosine kinase antagonist, sunitinib. We found that when a panel of five phenotypically diverse human breast cancer cell lines was subjected to treatment with sunitinib plus recombinant Th1 cytokines IFN-γ and TNF-α, synergistic effects were observed across a number of parameters including different aspects of apoptotic cell death. Interestingly, sunitinib was found to have a profoundly suppressive effect of T cell's capacity to secrete IFN-γ, indicating that in vivo use of this drug may hinder robust Th1 responses. Nonetheless, this suppression was circumvented in a mouse model of HER-2pos breast disease by supplying recombinant interferon-gamma to achieve a combination therapy significantly more potent than either agent.

Statin Drugs Plus Th1 Cytokines Potentiate Apoptosis and Ras Delocalization in Human Breast Cancer Lines and Combine with Dendritic Cell-Based Immunotherapy to Suppress Tumor Growth in a Mouse Model of HER-2pos Disease.

A dendritic cell-based, Type 1 Helper T cell (Th1)-polarizing anti-Human Epidermal Growth Factor Receptor-2 (HER-2) vaccine supplied in the neoadjuvant setting eliminates disease in up to 30% of recipients with HER-2-positive (HER-2pos) ductal carcinoma in situ (DCIS). We hypothesized that drugs with low toxicity profiles that target signaling pathways critical for oncogenesis may work in conjunction with vaccine-induced immune effector mechanisms to improve efficacy while minimizing side effects. In this study, a panel of four phenotypically diverse human breast cancer lines were exposed in vitro to the combination of Th1 cytokines Interferon-gamma (IFN-γ) and Tumor Necrosis Factor-alpha (TNF-α) and lipophilic statins. This combination was shown to potentiate multiple markers of apoptotic cell death. The combination of statin drugs and Th1 cytokines minimized membrane K-Ras localization while maximizing levels in the cytoplasm, suggesting a possible means by which cytokines and statin drugs might cooperate to maximize cell death. A combined therapy was also tested in vivo through an orthotopic murine model using the neu-transgenic TUBO mammary carcinoma line. We showed that the combination of HER-2 peptide-pulsed dendritic cell (DC)-based immunotherapy and simvastatin, but not single agents, significantly suppressed tumor growth. Consistent with a Th1 cytokine-dependent mechanism, parenterally administered recombinant IFN-γ could substitute for DC-based immunotherapy, likewise inhibiting tumor growth when combined with simvastatin. These studies show that statin drugs can amplify a DC-induced effector mechanism to improve anti-tumor activity.

Th1 cytokines sensitize HER-expressing breast cancer cells to lapatinib.

The HER family of receptor tyrosine kinases has been linked to deregulation of growth and proliferation for multiple types of cancer. Members have therefore become thefocus of many drug and immune-based therapy innovations. The targeted anti-cancer agent, lapatinib, is a small molecule inhibitor that directly interferes with EGFR (HER-1)and HER-2 signaling, and indirectly reduces HER-3 signaling, thus suppressing important downstream events. A recently-developed dendritic cell-based vaccine against early breast cancer (ductal carcinoma in situ; DCIS) that generates strong Th1-dominated immunity against HER-2 has induced pathologic complete response in about one-third of immunized individuals. In vitro studies suggested cytokines secreted by Th1 cells could be major contributors to the vaccine effects including induction of apoptosis and suppression of HER expression. With a view toward improving complete response rates, we investigated whether the principle Th1 cytokines (IFN-γ and TNF-α) could act in concert with lapatinib to suppress activity of breast cancer lines in vitro. Lapatinib-sensitive SKBR3, MDA-MB-468 and BT474 cells were incubated with Th1 cytokines, lapatinib, or both. It was found that combined treatment maximized metabolic suppression(Alamar Blue assay), as well as cell death (Trypan Blue) and apoptosis(Annexin V/Propidium Iodide and TMRE staining). Combined drug plus cytokine treatment also maximized suppression of both total and phosphorylated forms of HER-2 and HER-3. Interestingly, when lapatinib resistant lines MDA-MB-453 and JIMT-1 were tested, it was found that the presence of Th1 cytokines appeared to enhance sensitivity for lapatinib-induced metabolic suppression and induction of apoptotic cell death, nearly abrogating drug resistance. These studies provide pre-clinical data suggesting the possibility that targeted drug therapy may be combined with vaccination to enhance anti-cancer effects, and furthermore that robust immunity in the form of secreted Th1 cytokines may have the capacity to mitigate resistance to targeted drugs.

TRPA1 and TRPV1 contribute to propofol-mediated antagonism of U46619-induced constriction in murine coronary arteries.

BACKGROUND: Transient receptor potential (TRP) ion channels have emerged as key components contributing to vasoreactivity. Propofol, an anesthetic is associated with adverse side effects including hypotension and acute pain upon infusion. Our objective was to determine the extent to which TRPA1 and/or TRPV1 ion channels are involved in mediating propofol-induced vasorelaxation of mouse coronary arterioles in vitro and elucidate the potential cellular signal transduction pathway by which this occurs. METHODS: Hearts were excised from anesthetized mice and coronary arterioles were dissected from control C57Bl/6J, TRPA1-/-, TRPV1-/- and double-knockout mice (TRPAV-/-). Isolated microvessels were cannulated and secured in a temperature-controlled chamber and allowed to equilibrate for 1 hr. Vasoreactivity studies were performed in microvessels pre-constricted with U46619 to assess the dose-dependent relaxation effects of propofol on coronary microvascular tone. RESULTS: Propofol-induced relaxation was unaffected in vessels obtained from TRPV1-/- mice, markedly attenuated in pre-constricted vessels obtained from TRPA1-/- mice and abolished in vessels obtained from TRPAV-/- mice. Furthermore, NOS inhibition with L-NAME or endothelium denuding abolished the proporfol-induced depressor response in pre-constricted vessels obtained from all mice. In the absence of L-NAME, BKCa inhibition with penitrem A markedly attenuated propofol-mediated relaxation in vessels obtained from wild-type mice and to a lesser extent in vessels obtained from TRPV1-/-, mice with no effect in vessels obtained from TRPA1-/- or TRPAV-/- mice. CONCLUSIONS: TRPA1 and TRPV1 appear to contribute to the propofol-mediated antagonism of U46619-induced constriction in murine coronary microvessels that involves activation of NOS and BKCa.