Combination therapy using three novel prolactin receptor antagonist-based fusion proteins effectively inhibits tumor recurrence and metastasis in HER2/neu transgenic mice.

Previously, prolactin receptor antagonist (G129R)- based fusion proteins were developed including G129R fusions with an angiogenesis inhibitor (endostatin), an immune system modulator (interleukin 2), and a modified truncated cytotoxin (PE38KDEL). Each fusion protein was designed to target the PRLR-positive cells via the G129R moiety and at the same time attack a hallmark common to cancer cells via the second moiety. In this study, we tested the efficacy of the three fusion proteins as a combination therapy in an aggressive but clinically relevant mouse tumor model. To test the feasibility and to optimize a treatment regimen, allografts of a mammary carcinoma cell line (McNeuA) derived from an MMTV-neu transgenic mouse were first used. Growth of the allografts was significantly retarded by regimens which combined all three fusion proteins. In addition, a significant increase in cytotoxic CD8+ T cells was observed within the tumors of the combination treated groups. After establishing the dosing regimen, two doses of cocktail treatment (low and high doses administered twice weekly) along with individual component controls were administered to female MMTV-neu transgenic mice after surgical removal of a naturally occurring tumor. The average tumor recurrence time was significantly delayed in both low and high combination treatment groups in comparison to the no treatment control group (34, 50 and 18 days, respectively). The total number of lung metastases was also significantly decreased in both combination treatment groups. In conclusion, using G129R-based fusion proteins to target mammary carcinomas and to tackle multiple hallmarks of cancer at the same time was an effective strategy for treating HER2-postive mammary cancer in this mouse tumor model.

Rassf3 is responsible in part for resistance to mammary tumor development in neu transgenic mice.

MMTV/neu transgenic mouse line is a well-documented model for studying HER2/neu-related breast cancer. Approximately 80% of these mice develop mammary tumors by 11 months of age, whereas a small percentage appears to have naturally acquired resistance to HER2/neu tumorigenesis. To identify factors responsible for tumor resistance in these transgenic mice, comparative genetic profiling was used to screen alterations in gene expression in the mammary gland. A novel gene, the RAS association domain (RalGDS/AF-6) family 3 (Rassf3), which belongs to a family of RAS effectors and tumor suppressor genes, was identified. Data indicated 1) that Rassf3 is overexpressed in mammary gland of tumor-resistant MMTV/neu mice compared to tumor-susceptible MMTV/neu littermates or non-transgenic mice, and 2) Rassf3 is significantly up-regulated in neu-specific mouse mammary tumors compared to adjacent normal tissues. In vitro overexpression of RASSF3 inhibited cell proliferation in HER2/neu positive human and mouse breast cancer cell lines, possibly through induction of apoptosis. A novel MMTV/Rassf3-neu bi-transgenic mouse line, overexpressing Rassf3 and neu genes in mammary glands, was established. Mammary tumor incidence in bi-transgenic mice was delayed compared to their MMTV/neu+/- littermates. These data suggest that Rassf3 may influence mammary tumor incidence in MMTV/neu transgenic mice.

Effects of targeted deletion of cannabinoid receptors CB1 and CB2 on immune competence and sensitivity to immune modulation by Delta9-tetrahydrocannabinol.

The role of cannabinoid receptors, CB1 and CB2, in immune competence and modulation by Delta9-tetrahydrocannabinol (Delta9-THC) was investigated in CB1(-/-)/CB2(-/-) mice. Immunofluorescence analysis of splenic leukocytes showed no significant differences in the percentage of T cell subsets, B cells, or macrophages between wild-type and CB1(-/-)/CB2(-/-) mice. Lymphoproliferative control responses to PHA, phorbol ester plus ionomycin, or LPS and sensitivity to suppression by Delta9-THC showed no profound differences between the two genotypes, although some differences were observed in control baseline responses. Likewise, similar control responses and sensitivity to Delta9-THC were observed in mixed lymphocyte responses (MLR) and in IL-2 and IFN-gamma production in both genotypes. Conversely, humoral immune responses showed a markedly different profile of activity. Delta9-THC suppressed the in vivo T cell-dependent, anti-sheep RBC (anti-sRBC) IgM antibody-forming cell (AFC) response in wild-type but not in CB1(-/-)/CB2(-/-) mice, and the in vitro anti-sRBC IgM response in CB1(-/-)/CB2(-/-) splenocytes was too low to rigorously assess CB1/CB2 involvement in modulation by Delta9-THC. Conversely, comparable in vitro IgM AFC control responses to LPS and CD40 ligand (CD40L) activation were observed in the two genotypes. Interestingly, LPS-induced IgM responses were refractory to suppression by Delta9-THC, regardless of genotype, and CD40L-induced IgM responses were only suppressed by Delta9-THC in wild-type but not in CB1(-/-)/CB2(-/-) B cells. Collectively, we demonstrate differential involvement of CB1 and/or CB2 in immune modulation by Delta9-THC and in some control responses. Moreover, CB1/CB2 involvement was observed in humoral responses requiring CD40-initiated signaling for suppression by Delta9-THC.

The profile of immune modulation by cannabidiol (CBD) involves deregulation of nuclear factor of activated T cells (NFAT).

Cannabidiol (CBD) is a cannabinoid compound derived from Cannabis Sativa that does not possess high affinity for either the CB1 or CB2 cannabinoid receptors. Similar to other cannabinoids, we demonstrated previously that CBD suppressed interleukin-2 (IL-2) production from phorbol ester plus calcium ionophore (PMA/Io)-activated murine splenocytes. Thus, the focus of the present studies was to further characterize the effect of CBD on immune function. CBD also suppressed IL-2 and interferon-gamma (IFN-gamma) mRNA expression, proliferation, and cell surface expression of the IL-2 receptor alpha chain, CD25. While all of these observations support the fact that CBD suppresses T cell function, we now demonstrate that CBD suppressed IL-2 and IFN-gamma production in purified splenic T cells. CBD also suppressed activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) transcriptional activity, which are critical regulators of IL-2 and IFN-gamma. Furthermore, CBD suppressed the T cell-dependent anti-sheep red blood cell immunoglobulin M antibody forming cell (anti-sRBC IgM AFC) response. Finally, using splenocytes derived from CB1(-/-)/CB2(-/-) mice, it was determined that suppression of IL-2 and IFN-gamma and suppression of the in vitro anti-sRBC IgM AFC response occurred independently of both CB1 and CB2. However, the magnitude of the immune response to sRBC was significantly depressed in CB1(-/-)/CB2(-/-) mice. Taken together, these data suggest that CBD suppresses T cell function and that CB1 and/or CB2 play a critical role in the magnitude of the in vitro anti-sRBC IgM AFC response.

Additive effects of a prolactin receptor antagonist, G129R, and herceptin on inhibition of HER2-overexpressing breast cancer cells.

Breast cancers overexpressing human epidermal growth factor receptor 2 (HER2) have been reported to have higher proliferative and metastatic activity in the presence of autocrine prolactin (PRL), indicating potential cooperation between HER2 and the PRL receptor (PRLR) during breast cancer progression. PRL can induce the tyrosine phosphorylation of HER2 which stimulates mitogen-activated protein kinase (MAPK) activity. To determine if this transactivation of HER2 by PRL contributes to anti-HER2 therapy resistance we examined the potential of combining Herceptin with a PRLR antagonist, G129R, which inhibits PRL-induced signaling, as a novel therapeutic strategy. Two PRL-expressing human breast cancer cell lines (T-47D and BT-474) that overexpress PRLR and HER2 to different degrees were chosen for this study. The phosphorylation status of HER2 and activation of MAPK, signal transducers and activators of transcription (STAT), as well as phosphatidylinositol-3 kinase (PI3K) signaling cascades were examined in response to Herceptin, G129R or a combination of the two in either the absence or presence of exogenous PRL. As a single agent, Herceptin was more effective than G129R at inhibiting AKT phosphorylation; whereas, G129R was superior at blocking STAT3 and STAT5 activation. G129R was also able to directly inhibit the HER2 phosphorylation. The combination of Herceptin and G129R had an additive inhibitory effect on HER2 and MAPK phosphorylation, confirming that the MAPK signaling is a converging pathway shared by both HER2 and the PRLR. Combination of Herceptin and G129R also additively inhibited cell proliferation in vitro and in vivo as measured by inhibition of the growth of T-47D and BT-474 xenografts in athymic nude mice. We conclude that an anti-HER2 and anti-PRLR regimen may offer a new approach to treat HER2-overexpressing breast cancers.

The Effects of Indirubin-3'-Monoxime, A Novel AHR Ligand, on Stress and Toxicity-Related Gene/Protein Expression in Human U937 Cells Undergoing Differentiation and Activation.

Indirubin-3-monoxime is an aryl hydrocarbon receptor (AhR)-binding, idole-derived compound known to be a potent inducer of CYP1A1, at least in hepatic cell lines. To date, very little is known about the effects of indirubin-3-monoxime on cells relevant to the immune system. We treated human U937 histiocytic lymphoma cells with indirubin-3'-monoxime in four different regimens, representing various stages of PMA-induced differentiation and further activation with LPS. Gene expression profiles were monitored using commercial macroarrays for 96 select stress and toxicity genes. The four treatment regimens were (A) the monocyte model: U937 cells were treated with 1 muM indirubin-3'-monoxime or carrier control for 24 hrs; (B) the differentiated model: PMA-differentiated U937 cells were treated with 1 muM indirubin-3'-monoxime or carrier control for 24 hrs; (C) the differentiation process model: U937 cells were treated for 24 hours with 1 muM indirubin-3'-monoxime or carrier control, followed by PMA-induced differentiation over a 24-hr period; and (D) the LPS-activated model: PMA-differentiated and LPS-activated U937 cells were treated with 1 muM indirubin-3'-monoxime or carrier control for 24 hrs. Indirubin-3'-monoxime treatment caused a 12.7-, 2.1-, 3.2-, and 4.5-fold increases in CYP1A1 in regimes A, B, C, and D, respectively. Indirubin-3'-monoxime treatment also enhanced cyclo-oxygenase 2 expression in PMA-differentiated cells, but reduced the expression of macrophage inflammatory protein, catalase, heme oxygenase-1, glutathione peroxidase, and manganese superoxide dismutase; a scenario consistent with oxidative stress. Macroarray validity was confirmed by RT-PCR using CYP1A1, COX-2, MIP-beta, and GADPH as target genes, and at the protein level for CYP1A1, COX-2, and beta-actin. To our knowledge, this is the first study to examine the effects of indirubin-3-monoxime in a human cell line relevant to the immune system. Overall, our study suggests that indirubin-3'-monoxime is either a classical AhR ligand, or that it modulates endogenous AhR-regulated activity.