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.

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.

Targeted drug approaches have been a major focus for developing new anticancer therapies. Although many such agents approved in the last 20 years have improved outcomes, almost all have underperformed expectations. The full potential of such agents may yet be obtained through novel combinations. Previously, we showed that anti-estrogen drugs combined with a dendritic cell-based anti-HER-2 vaccine known to induce strong Th1-polarized immunity dramatically improved clinical response rates in patients with HER-2 pos /ER pos early breast cancer. Here, we show that the small molecule Akt antagonist MK-2206, when combined with the Th1 cytokines IFN-gamma and TNF-alpha, maximize indicators of apoptotic cell death in a panel of phenotypicallydiverse human breast cancer lines. These findings were mirrored by other, structurallyunrelated Akt-targeting drugs that work through different mechanisms. Interestingly, we found that MK-2206, as well as the other Akt antagonist drugs, also had a tendency to suppress Th1 cytokine expression in stimulated human and murine lymphocytes, potentially complicating their use in conjunction with active immunotherapy. After verifying that MK-2206 plus IFN-gamma could show similar combined effects against breast cancer lines, even in the absence of TNF-alpha, we tested in a rodent HER-2 pos breast cancer model either a HER-2-based DC vaccine, or recombinant IFN-gamma with or without MK-2206 administration. We found that for MK-2206, co-administration of recombinant IFN-gamma outperformed co-administration of DC vaccination for slowing tumor growth kinetics. These findings suggest a combined therapy approach for Akt-targeting drugs that incorporates recombinant Interferon-gamma and is potentially translatable to humans.

BackgroundTransient 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.MethodsHearts 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.ResultsPropofol-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.ConclusionsTRPA1 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.

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