James T. Forbes

Anti-transforming growth factor (TGF)-beta antibodies inhibit breast cancer cell tumorigenicity and increase mouse spleen natural killer cell activity. Implications for a possible role of tumor cell/host TGF-beta interactions in human breast cancer progression.

TGF-beta effects on angiogenesis, stroma formation, and immune function suggest its possible involvement in tumor progression. This hypothesis was tested using the 2G7 IgG2b, which neutralizes TGF-beta 1, -beta 2, and -beta 3, and the MDA-231 human breast cancer cell line. Inoculation of these cells in athymic mice decreases mouse spleen natural killer (NK) cell activity. Intraperitoneal injections of 2G7 starting 1 d after intraperitoneal inoculation of tumor cells suppressed intraabdominal tumor and lung metastases, whereas the nonneutralizing anti-TGF-beta 12H5 IgG2a had no effect. 2G7 transiently inhibited growth of established MDA-231 subcutaneous tumors. Histologically, both 2G7-treated and control tumors were identical. Intraperitoneal administration of 2G7 resulted in a marked increase in mouse spleen NK cell activity. 2G7 did not inhibit MDA-231 primary tumor or metastases formation, nor did it stimulate NK cell-mediated cytotoxicity in beige NK-deficient nude mice. Finally, serum-free conditioned medium from MDA-231 cells inhibited the NK cell activity of human blood lymphocytes. This inhibition was blocked by the neutralizing anti-TGF-beta 2G7 antibody but not by a nonspecific IgG2. These data support a possible role for tumor cell TGF-beta in the progression of mammary carcinomas by suppressing host immune surveillance.

Conditional Overexpression of Active Transforming Growth Factor β1 In vivo Accelerates Metastases of Transgenic Mammary Tumors

To address the role of transforming growth factor (TGF) β in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-β on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-β1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-β1 for as little as 2 weeks increased lung metastases >10-fold without a detectable effect on primary tumor cell proliferation or tumor size. DOX-induced active TGF-β1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-β and increased metastases. Antisense-mediated inhibition of TGF-β1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Therefore, induction and/or activation of TGF-β in hosts with established TGF-β-responsive cancers can rapidly accelerate metastatic progression.

Early changes in protein expression detected by mass spectrometry predict tumor response to molecular therapeutics.

Biomarkers that predict therapeutic response are essential for the development of anticancer therapies. We have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to directly analyze protein profiles in mouse mammary tumor virus/HER2 transgenic mouse frozen tumor sections after treatment with the erbB receptor inhibitors OSI-774 and Herceptin. Inhibition of tumor cell proliferation and induction of apoptosis and tumor reduction were predicted by a >80% reduction in thymosin β4 and ubiquitin levels that were detectable after 16 hours of a single drug dose before any evidence of in situ cellular activity. These effects were time- and dose-dependent, and their spatial distribution in the tumor correlated with that of the small-molecule inhibitor OSI-774. In addition, they predicted for therapeutic synergy of OSI-774 and Herceptin as well as for drug resistance. These results suggest that drug-induced early proteomic changes as measured by MALDI-MS can be used to predict the therapeutic response to established and novel therapies.

Inhibition of Mammalian Target of Rapamycin Is Required for Optimal Antitumor Effect of HER2 Inhibitors against HER2-Overexpressing Cancer Cells

Purpose: A significant fraction of HER2-overexpressing breast cancers exhibit resistance to the HER2 antibody trastuzumab. Hyperactivity of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway confers trastuzumab resistance, and mammalian target of rapamycin (mTOR) is a major downstream effector of PI3K/AKT. Therefore, we examined whether mTOR inhibitors synergize with trastuzumab. Experimental Design: Immunocompetent mice bearing HER2+ mammary tumors were treated with trastuzumab, the mTOR inhibitor rapamycin, or the combination. Mice were imaged for tumor cell death using an optical Annexin-V probe and with [18F]FDG positron emission tomography. The signaling and growth effects of the mTOR inhibitor RAD001 on HER2+ cells treated with trastuzumab or lapatinib were evaluated. Results: Treatment of mice with trastuzumab plus rapamycin was more effective than single-agent treatments, inducing complete regression of 26 of 26 tumors. The combination induced tumor cell death (Annexin-V binding) and inhibited FDG uptake. Rapamycin inhibited mTOR and tumor cell proliferation as determined by phosphorylated S6 and Ki-67 immunohistochemistry, respectively. In culture, the combination of RAD001 plus trastuzumab inhibited cell growth more effectively than either drug alone. Trastuzumab partially decreased PI3K but not mTOR activity. Knockdown of TSC2 resulted in HER2-independent activation of mTOR and dampened the response to trastuzumab and lapatinib. Treatment with the HER2 inhibitor lapatinib decreased phosphorylated S6 and growth in TSC2-expressing cells but not in TSC2-knockdown cells. Conclusions: Inhibition of PI3K and mTOR are required for the growth-inhibitory effect of HER2 antagonists. These findings collectively support the combined use of trastuzumab and mTOR inhibitors for the treatment of HER2+ breast cancer. (Clin Cancer Res 2009;15(23):7266–76)

Human natural cell-mediated cytotoxicity

SummaryNatural cell-mediated cytotoxicity has been shown to be age-dependent in both rats and mice. The present study was undertaken to study age-related levels of natural cytotoxicity in humans. Mononuclear peripheral blood cells from over 200 normal volunteers were co-incubated with 51Cr-labeled K-562 for 4 h and the supernatants assayed for released isotope. No striking age-related variations in natural cytotoxicity were observed. Cord blood was shown to exhibit both high and low levels of natural cell-mediated cytotoxicity. There was no significant difference in the levels of natural cytotoxicity between males and females. Thoracic duct lymphocytes (TDL) from prospective kidney graft recipients were tested for natural cytotoxicity at intervals during drainage. Natural cytotoxic activity was undetectable in samples from initial drainage but increased as the drainage progressed. This increased activity was correlated with decreased in vitro responsiveness to PHA and decreased levels of E-rosettes in these TDL.

Human recombinant interleukin 2-activated sheep lymphocytes lyse sheep pulmonary microvascular endothelial cells

Administration of lymphokine-activated killer (LAK) cells in combination with interleukin 2 (IL-2) has been effective in reducing tumor mass in humans, but has been accompanied by significant toxicity. We used a chronic awake sheep model to investigate the cause of the vascular leak syndrome associated with IL-2 administration. Sheep repeatedly infused with human recombinant IL-2 (hrIL-2) developed mild pulmonary hypertension, systemic hypotension, acidemia, hypoxemia, and increased flow of protein rich lung lymph. We hypothesized that LAK cells may damage lung endothelium in vivo and cause increased lung vascular permeability. Sheep peripheral blood and lung lymph lymphocytes incubated in vitro with hrIL-2 generated cytotoxic activity for human K-562 cells and sheep pulmonary microvascular endothelial cells. In addition, cytotoxic effector cells were isolated from the peripheral blood of a sheep which had received hrIL-2. These observations suggest that LAK cells possess the ability to damage endothelial cells and may contribute to an increased pulmonary vascular permeability observed following hrIL-2 infusion in sheep.

Effects of orally administered retinol on natural killer cell activity in wild type BALB/c and congenitally athymic BALB/c mice

SummaryRetinoids have been shown to inhibit the growth and development of neoplastic cells in many systems. One mechanism of action may be through activation of the immune system, specifically natural killer (NK) cell activity. The effect of retinol on NK cell cytotoxicity was examined in three groups of mice: BALB/c (wild-type), BALB/c nu/nu (athymic), and BALB/c nu/nu previously injected with human tumor cells. In untreated mice, NK activity was highest in athymic mice without tumors and lowest in wild-type mice, although serum and liver retinol concentrations were identical in all three groups. In mice fed graded, nontoxic doses of retinol daily for 3 weeks, serum retinol levels in all three groups exhibited a sharp peak and decline following daily bolus retinol administration. Retinol stores in the livers showed a dose-dependent increase in all treated animals. However, NK cell activity, differed for each group. Athymic mice without tumors exhibited no change in NK activity as a result of retinol treatment. Athymic mice with tumors had NK levels that tended to increase with increasing retinol doses, but these changes were not statistically significant. Wild-type mice, on the other hand, demonstrated significantly higher NK levels after treatment with retinol doses of 300 and 600 μg/day. In subsequent time course experiments, there was a peak in NK activity 1 h following bolus retinol administration similar to the peak seen in serum retinol concentrations, suggesting either an acute activation or recruitment of cytotoxic cells. Retinol thus appears to increase NK activity in wild-type BALB/c mice, and this activity may be an important component of its antineoplastic activity.

Immunological studies in a double blind randomized trial comparing intrapleural BCG against placebo in patients with resected stage I non-small cell lung cancer

SummaryImmunological data were obtained during the course of a randomized trial comparing intrapleural BCG plus oral isoniazid (INH) with intrapleural saline plus oral placebo after resection of stage I non-small cell lung cancer. Immunological testing with a variety of assays was carried out with good standardization among six collaborating laboratories and with good reproducibility within each laboratory. Those patients with larger tumors tended to have higher initial white cell counts. The percentage of lymphocytes in the differential was greatest in those with non-squamous cancer histology. Otherwise, no associations were found between initial immunologic parameters and baseline variables. The main effect of BCG/INH therapy was to cause statistically significant increases in purified protein derivative (PPD) skin test induration and PPD in vitro blastogenesis compared with controls. Other skin tests and in vitro assays increased more in the saline/placebo control group, but these treatment differences were usually not statistically significant. Initial white count and neutrophil count elevations were found to be associated with increased risk of recurrence. Even after adjustment for treatment and tumor stage, initial neutrophil count elevation was associated with increased risk of recurrence. Surprisingly, a low 29° C T cell rosette index was associated with a decreased risk of recurrence, though the differences were minimal. Serial immunological tests were carried out to evaluate their potential for monitoring disease recurrence. White count elevations continued to be significantly associated with increased risk of recurrence, but more follow-up data are needed before other associations can be assessed.

A functional cyclooxygenase enzyme is not required for mediation of the pleiotropic effects of human alpha or beta interferon

Aspirin, indomethacin, and phenbutazone at 50 microM concentration inhibit cyclooxygenase in cultured human foreskin fibroblasts as evidenced by the suppression of the major prostaglandin species which accumulate in the culture medium. In contrast to data reported for mouse interferon on target mouse cells, these agents have no effect on the induction of antiviral activity by human alpha or beta interferons. Similarly, these agents have no effect on interferon induced inhibition of cell growth in vitro or on interferon induced natural killer cell activity.

MODIFICATION OF HUMAN NATURAL CELL-MEDIATED CYTOTOXICITY BY MVE-2

Publisher Summary This chapter examines the modification of human natural cell-mediated cytotoxicity by maleic anhydride and divinyl ether (MVE-2). In a study described in the chapter, a total of 21 evaluable patients with advanced refractory neoplastic disease were entered into the Phase I study of MVE-2. MVE-2 at the appropriate dose level was infused intravenously over two hours at weekly intervals for a total of six weeks. Three patients were entered at 150 mg/m 2 , four at 300 mg/m 2 , four at 350 mg/m 2 , one at 400 mg/m 2 , four at 450 mg/m 2 , and five at 600 mg/m 2 . Patients were monitored for biological response modification by measuring 29°C E-rosettes, 4°C E-rosettes, phytohemagglutinin (PHA)-induced blast transformation, pokeweed mitogen (PWM)–induced blast transformation, natural cytotoxicty to K-562, plasma interferon levels, and plasma lysozyme levels in the peripheral blood. All tests on cells were performed on Ficoll–Hypaque separated cells. All assays were performed twice prior to administration of MVE-2 and weekly thereafter for as long as the patient remained in the study. No significant changes in the levels of response to the T-cell mitogen PHA were noted during MVE-2 administration. However, nine of eighteen patients tested showed increased responses to pokeweed mitogen.