Development and Preclinical Application of an Immunocompetent Transplant Model of Basal Breast Cancer with Lung, Liver and Brain Metastases.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is associated with a poor prognosis and for which no targeted therapies currently exist. In order to improve preclinical testing for TNBC that relies primarily on using human xenografts in immunodeficient mice, we have developed a novel immunocompetent syngeneic murine tumor transplant model for basal-like triple-negative breast cancer. The C3(1)/SV40-T/t-antigen (C3(1)/Tag) mouse mammary tumor model in the FVB/N background shares important similarities with human basal-like TNBC. However, these tumors or derived cell lines are rejected when transplanted into wt FVB/N mice, likely due to the expression of SV40 T-antigen. We have developed a sub-line of mice (designated REAR mice) that carry only one copy of the C3(1)/Tag-antigen transgene resulting from a spontaneous transgene rearrangement in the original founder line. Unlike the original C3(1)/Tag mice, REAR mice do not develop mammary tumors or other phenotypes observed in the original C3(1)/Tag transgenic mice. REAR mice are more immunologically tolerant to SV40 T-antigen driven tumors and cell lines in an FVB/N background (including prostate tumors from TRAMP mice), but are otherwise immunologically intact. This transplant model system offers the ability to synchronously implant the C3(1)/Tag tumor-derived M6 cell line or individual C3(1)/Tag tumors from various stages of tumor development into the mammary fat pads or tail veins of REAR mice. C3(1)/Tag tumors or M6 cells implanted into the mammary fat pads spontaneously metastasize at a high frequency to the lung and liver. M6 cells injected by tail vein can form brain metastases. We demonstrate that irradiated M6 tumor cells or the same cells expressing GM-CSF can act as a vaccine to retard tumor growth of implanted tumor cells in the REAR model. Preclinical studies performed in animals with an intact immune system should more authentically replicate treatment responses in human patients.

Cross-species genomic and functional analyses identify a combination therapy using a CHK1 inhibitor and a ribonucleotide reductase inhibitor to treat triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is diagnosed in approximately 15% of all human breast cancer (BrCa) patients. Currently, no targeted therapies exist for this subtype of BrCa and prognosis remains poor. Our laboratory has previously identified a proliferation/DNA repair/cell cycle gene signature (Tag signature) that is characteristic of human TNBC. We hypothesize that targeting the dysregulated biological networks in the Tag gene signature will lead to the identification of improved combination therapies for TNBC. METHODS: Cross-species genomic analysis was used to identify human breast cancer cell lines that express the Tag signature. Knock-down of the up-regulated genes in the Tag signature by siRNA identified several genes that are critical for TNBC cell growth. Small molecule inhibitors to two of these genes were analyzed, alone and in combination, for their effects on cell proliferation, cell cycle, and apoptosis in vitro and tumor growth in vivo. Synergy between the two drugs was analyzed by the Chou-Talalay method. RESULTS: A custom siRNA screen was used to identify targets within the Tag signature that are critical for growth of TNBC cells. Ribonucleotide reductase 1 and 2 (RRM1 and 2) and checkpoint kinase 1 (CHK1) were found to be critical targets for TNBC cell survival. Combination therapy, to simultaneously attenuate cell cycle checkpoint control through inhibition of CHK1 while inducing DNA damage with gemcitabine, improved therapeutic efficacy in vitro and in xenograft models of TNBC. CONCLUSIONS: This combination therapy may have translational value for patients with TNBC and improve therapeutic response for this aggressive form of breast cancer.

Critical role for endocytosis in the regulation of signaling by the Kaposi's sarcoma-associated herpesvirus K1 protein.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family. KSHV is the etiologic agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The first open reading frame of the KSHV genome encodes a type 1 transmembrane glycoprotein named K1. K1 is structurally similar to the B-cell receptor (BCR), and its cytoplasmic tail contains an immunoreceptor tyrosine-based activation motif that can activate Syk kinase and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Recent evidence suggests that receptor signaling occurs not only at the cell membrane, but from intracellular compartments as well. We have found that K1 is internalized in a clathrin-dependent manner, and efficient internalization is coupled to its signaling function. Once internalized, K1 traffics from the early endosome to the recycling endosome. Interestingly, blocking K1's activation of Syk and PI3K prevents K1 from internalizing. We have also found that blocking clathrin-mediated endocytosis prevents downstream signaling by K1. These results strongly suggest that internalization of K1 is intimately associated with normal signaling. When K1 internalization was examined in B lymphocytes, we found that K1 cointernalized with the BCR. Altogether, these results suggest that K1's signaling function is tightly coupled to its internalization.

Immortalization of primary endothelial cells by the K1 protein of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to three different human cancers: Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The Kaposi's sarcoma lesion expresses high levels of angiogenic factors and is comprised of a mixed cell population, including endothelial cells that are infected with KSHV. We find that the KSHV K1 protein is expressed in Kaposi's sarcoma lesions and can immortalize and extend the life span of primary human umbilical vein endothelial cells in culture. Vascular endothelial growth factor (VEGF) is critical for the survival of endothelial cells, and we show that expression of K1 in endothelial cells resulted in increased levels of secreted VEGF and the activation of key signaling pathways, including the VEGF/VEGF receptor and the phosphatidylinositol-3'-OH-kinase (PI3K) pathway. The SH2 binding motifs present in the cytoplasmic tail of K1 were critical for K1's ability to activate these pathways. Activation of PI3K by K1 results in activation of Akt kinase and mammalian target of rapamycin and inactivation of the proapoptotic proteins FKHR, glycogen synthase kinase-3, and Bad, which are events indicative of cell survival. Because activation of the PI3K pathway is critical for transformation of many human cells, we suggest that PI3K activation by K1 is involved in endothelial cell immortalization and contributes to KSHV-associated tumorigenesis. We also report that K1 enhances angiogenesis in vivo and increases tumor vasculature and tumor size.

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) K1 protein induces expression of angiogenic and invasion factors.

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) has been linked to Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. In addition to endothelial cells and B lymphocytes, KSHV also has been shown to infect epithelial cells and keratinocytes. The transmembrane glycoprotein K1, encoded by the first open reading frame of KSHV, is a signaling protein capable of eliciting B-cell activation. We show that KSHV K1 can induce expression and secretion of vascular endothelial growth factor (VEGF) in epithelial and endothelial cells. Up-regulation of VEGF was mediated at the transcriptional level because expression of K1 resulted in VEGF promoter activation. We also show that K1 induces expression of matrix metalloproteinase-9 (MMP-9) in endothelial cells. Additional analyses with K1 mutant proteins revealed that the SH2 binding motifs present in the K1 cytoplasmic tail are necessary for VEGF secretion and MMP-9 induction. These results indicate that K1 signaling may contribute to KSHV-associated pathogenesis through a paracrine mechanism by promoting the secretion of VEGF and MMP-9 into the surrounding matrix.

Fatty acid synthase expression is induced by the Epstein-Barr virus immediate-early protein BRLF1 and is required for lytic viral gene expression.

The Epstein-Barr virus (EBV) immediate-early (IE) protein BRLF1 (R) is a transcription factor that induces the lytic form of EBV infection. R activates certain early viral promoters through a direct binding mechanism but induces transcription of the other EBV IE gene, BZLF1 (Z), indirectly through cellular factors binding to a CRE motif in the Z promoter (Zp). Here we demonstrate that R activates expression of the fatty acid synthase (FAS) cellular gene through a p38 stress mitogen-activated protein kinase-dependent mechanism. B-cell receptor engagement of Akata cells also increases FAS expression. The FAS gene product is required for de novo synthesis of the palmitate fatty acid, and high-level FAS expression is normally limited to liver, brain, lung, and adipose tissue. We show that human epithelial tongue cells lytically infected with EBV (from oral hairy leukoplakia lesions) express much more FAS than uninfected cells. Two specific FAS inhibitors, cerulenin and C75, prevent R activation of IE (Z) and early (BMRF1) lytic EBV proteins in Jijoye cells. In addition, cerulenin and C75 dramatically attenuate IE and early lytic gene expression after B-cell receptor engagement in Akata cells and constitutive lytic viral gene expression in EBV-positive AGS cells. However, FAS inhibitors do not reduce lytic viral gene expression induced by a vector in which the Z gene product is driven by a strong heterologous promoter. In addition, FAS inhibitors do not reduce R activation of a naked DNA reporter gene construct driven by the Z promoter (Zp). These results suggest that cellular FAS activity is important for induction of Z transcription from the intact latent EBV genome, perhaps reflecting the involvement of lipid-derived signaling pathways or palmitoylated proteins. Furthermore, using FAS inhibitors may be a completely novel approach for blocking the lytic form of EBV replication.

The K1 protein of Kaposi's sarcoma-associated herpesvirus activates the Akt signaling pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) has been implicated in Kaposi's sarcoma, as well as in primary effusion lymphoma and multicentric Castleman's disease. The K1 protein of KSHV has been shown to induce cellular transformation and focus formation and to deregulate B-lymphocyte signaling pathways by functionally mimicking the activated B-cell receptor complex. Here we show that expression of K1 in B lymphocytes targets the phosphatidylinositol-3 kinase pathway, leading to the activation of the Akt kinase and the inhibition of the phosphatase PTEN. We also demonstrate that activation of Akt by the K1 protein leads to the phosphorylation and inhibition of members of the forkhead (FKHR) transcription factor family, which are key regulators of cell cycle progression and apoptosis. We demonstrate that K1 can inhibit apoptosis induced by the FKHR proteins and by stimulation of the Fas receptor. Our observations suggest that the K1 viral protein promotes cell survival pathways and may contribute to KSHV pathogenesis by preventing virally infected cells from undergoing apoptosis prematurely.

Intravitreal injection of the attenuated pseudorabies virus PRV Bartha results in infection of the hamster suprachiasmatic nucleus only by retrograde transsynaptic transport via autonomic circuits.

Intravitreal injection of the attenuated strain of pseudorabies virus (PRV Bartha) results in transneuronal spread of virus to a restricted set of central nuclei in the rat and mouse. We examined the pattern of central infection in the golden hamster after intravitreal inoculation with a recombinant strain of PRV Bartha constructed to express enhanced green fluorescent protein (PRV 152). Neurons in a subset of retinorecipient nuclei [i.e., suprachiasmatic nucleus (SCN), intergeniculate leaflet, olivary pretectal nucleus (OPN), and lateral terminal nucleus] and autonomic nuclei [i.e., paraventricular hypothalamic nucleus and Edinger-Westphal nucleus (EW)] are labeled by late stages of infection. Infection of the EW precedes infection in retinorecipient structures, raising the possibility that the SCN becomes infected by retrograde transsynaptic infection via autonomic (i.e., EW) circuits. We tested this hypothesis in two ways: (1) by removing the infected eye 24 hr after PRV 152 inoculation, well before viral infection first appears in the SCN; and (2) by examining central infection after intravitreal PRV 152 injection in animals with ablation of the EW. The pattern and time course of central infection were unchanged after enucleation, whereas EW ablation before intravitreal inoculation eliminated viral infection in the SCN. The results of EW lesions along with known connections between EW, OPN, and SCN indicate that intravitreal injection of PRV Bartha produces a retrograde infection of the autonomic innervation of the eye, which subsequently labels a restricted set of retinorecipient nuclei via retrograde trans-synaptic infection. These results, taken together with other genetic data, indicate that the mutations in PRV Bartha render the virus incapable of anterograde transport. PRV Bartha is thus a retrograde transsynaptic marker in the CNS.