Middle T Antigen Research Articles

The Human Polyomavirus Middle and Alternative T-Antigens; Thoughts on Roles and Relevance to Cancer

Approximately 15–20% of human cancer is related to infection, which renders them potentially preventable by antimicrobial or antiviral therapy. Human polyomaviruses (PyVs) are relevant in this regard, as illustrated by the involvement of Merkel cell polyomavirus (MCPyV) in the development of Merkel cell carcinoma. The polyomavirus Small and Large tumor antigen (ST and LT) have been extensively studied with respect to their role in oncogenesis. Recently it was shown that a number of human PyVs, including MCPyV and the trichodysplasia spinulosa polyomavirus (TSPyV), express additional T-antigens called Middle T (MT) and alternative T (ALT). ALT is encoded by ORF5, also known as the alternative T open reading frame (ALTO), which also encodes the second exon of MT, and overlaps out-of-frame with the second exon of LT. Previously, MT was considered unique for oncogenic rodent polyomaviruses, and ALT was still unknown. In this mini-review, we want to point out there are important reasons to explore the involvement of MT and ALT in human cellular transformation. First, just like their rodent equivalents, MT and ALT probably disrupt cellular pathways that control signaling and proliferation. Second, expression of the MT and ALT-encoding ORF5/ALTO characterizes a monophyletic polyomavirus clade that includes human and animal PyVs with known oncogenic potential. And third, ORF5/ALTO is subject to strong positive selection aimed specifically at a short linear motif within MT and ALT that overlaps completely with the RB-binding motif in LT. The latter suggests tight interplay between these T-antigens with possible consequences for cell transformation.

CCL5-CCR5 interactions modulate metabolic events during tumor onset to promote tumorigenesis

BackgroundIn earlier studies we have shown that CCL5 activation of CCR5 induces the proliferation and survival of breast cancer cells in a mechanistic target of rapamycin (mTOR)-dependent manner and that this is in part due to CCR5-mediated increases in glycolytic metabolism.MethodsUsing the MDA-MB-231 triple negative human breast cancer cell line and mouse mammary tumor virus – polyomavirus middle T-antigen (MMTV-PyMT) mouse primary breast cancer cells, we conducted in vivo tumor transplant experiments to examine the effects of CCL5-CCR5 interactions in the context of regulating tumor metabolism. Additionally, we employed Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry imaging (MALDI-FTICR-MSI) to evaluate tumor utilization of cellular metabolites.ResultsWe provide evidence that, in the absence of CCR5, the early events associated with rapid tumor growth in the MMTV-PyMT mouse model of spontaneous breast cancer development, are diminished, as demonstrated by a delay in tumor onset. In tumor transplant studies into immunocompromised mice we identify a direct correlation between reduced tumor proliferation and decreased metabolic activity, specifically associated with tumor expression of CCR5. The reduction in tumorigenesis is accompanied by decreases in glucose uptake, glucose transporter-1 (GLUT-1) cell surface expression, intracellular ATP and lactate levels, as well as reduced CCL5 production. Using MALDI-FTICR-MS, we show that the rapid early tumor growth of CCR5+/+ triple negative breast cancer cells in vivo is attributable to increased levels of glycolytic intermediates required for anabolic processes, in contrast to the slower growth rate of their corresponding CCR5−/− cells, that exhibit reduced glycolytic metabolism.ConclusionsThese findings suggest that CCL5-CCR5 interactions in the tumor microenvironment modulate metabolic events during tumor onset to promote tumorigenesis.

The R-Enantiomer of Ketorolac Delays Mammary Tumor Development in Mouse Mammary Tumor Virus-Polyoma Middle T Antigen (MMTV-PyMT) Mice

Epidemiologic studies report improved breast cancer survival in women who receive ketorolac (Toradol) for postoperative pain relief compared with other analgesic agents. Ketorolac is a racemic drug. The S-enantiomer inhibits cyclooxygenases; R-ketorolac is a selective inhibitor of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), which are signaling molecules up-regulated during breast cancer progression and metastasis. The goal of this study was to determine whether R-ketorolac altered breast cancer development in the mouse mammary tumor virus-polyoma middle T-antigen model. Mice were administered ketorolac orally at 1 mg/kg twice daily to approximate the typical human dose. Mammary glands were analyzed for tumor number and immunohistochemical markers of proliferation and differentiation. R-ketorolac treatment significantly reduced mammary epithelial proliferation, based on Ki67 staining, and suppressed tumor development. Proliferative mammary epithelium from R-ketorolac-treated mice displayed greater differentiation, based on significantly higher total E-cadherin and decreased keratin 5 staining than epithelium of placebo-treated mice. No differences were detected inestrogen receptor, progesterone receptor, β-catenin, or vimentin expression between placebo and R-ketorolac treatment groups. These findings indicate that R-ketorolac treatment slows tumor progression in an aggressive model of breast cancer. R-ketorolac may thus represent a novel therapeutic approach for breast cancer prevention or treatment based on its pharmacologic activity as a Rac1 and Cdc42 inhibitor.

Bromodomain inhibition shows antitumoral activity in mice and human luminal breast cancer.

BET bromodomain inhibitors, which have an antitumoral effect against various solid cancer tumor types, have not been studied in detail in luminal breast cancer, despite the prevalence of this subtype of mammary malignancy. Here we demonstrate that the BET bromodomain inhibitor JQ1 exerts growth-inhibitory activity in human luminal breast cancer cell lines associated with a depletion of the C-MYC oncogene, but does not alter the expression levels of the BRD4 bromodomain protein. Interestingly, expression microarray analyses indicate that, upon JQ1 administration, the antitumoral phenotype also involves downregulation of relevant breast cancer oncogenes such as the Breast Carcinoma-Amplified Sequence 1 (BCAS1) and the PDZ Domain-Containing 1 (PDZK1). We have also applied these in vitro findings in an in vivo model by studying a transgenic mouse model representing the luminal B subtype of breast cancer, the MMTV-PyMT, in which the mouse mammary tumor virus promoter is used to drive the expression of the polyoma virus middle T-antigen to the mammary gland. We have observed that the use of the BET bromodomain inhibitor for the treatment of established breast neoplasms developed in the MMTV-PyMT model shows antitumor potential. Most importantly, if JQ1 is given before the expected time of tumor detection in the MMTV-PyMT mice, it retards the onset of the disease and increases the survival of these animals. Thus, our findings indicate that the use of bromodomain inhibitors is of great potential in the treatment of luminal breast cancer and merits further investigation.

Cytosolic Hsp90α and its mitochondrial isoform Trap1 are differentially required in a breast cancer model.

The Hsp90 family of molecular chaperones includes the cytosolic isoforms Hsp90a and Hsp90β and the mitochondrial isoform Trap1. Hsp90a/βsupport a large number of client proteins in the cytoplasm and the nucleus whereas Trap1 regulates oxidative phosphorylation in mitochondria. Many of the associated proteins and cellular processes are relevant to cancer, and there is ample pharmacological and genetic evidence to support the idea that Hsp90a/βand Trap1 are required for tumorigenesis. However, a direct and comparative genetic test in a mouse cancer model has not been done. Here we report the effects of deleting the Hsp90a or Trap1 genes in a mouse model of breast cancer. Neither Hsp90a nor Trap1 are absolutely required for mammary tumor initiation, growth and metastasis induced by the polyoma middle T-antigen as oncogene. However, they do modulate growth and lung metastasis in vivo and cell proliferation, migration and invasion of isolated primary carcinoma cells in vitro. Without Hsp90a, tumor burden and metastasis are reduced, correlating with impaired proliferation, migration and invasion of cells in culture. Without Trap1, the appearance of tumors is initially delayed, and isolated cells are affected similarly to those without Hsp90a. Analysis of expression data of human breast cancers supports the conclusion that this is a valid mouse model highlighting the importance of these molecular chaperones.

MMTV-PyMT and Derived Met-1 Mouse Mammary Tumor Cells as Models for Studying the Role of the Androgen Receptor in Triple-Negative Breast Cancer Progression.

Triple-negative breast cancer (TNBC) has a faster rate of metastasis compared to other breast cancer subtypes, and no effective targeted therapies are currently FDA-approved. Recent data indicate that the androgen receptor (AR) promotes tumor survival and may serve as a potential therapeutic target in TNBC. Studies of AR in disease progression and the systemic effects of anti-androgens have been hindered by the lack of an AR-positive (AR+) immunocompetent preclinical model. In this study, we identified the transgenic MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mouse mammary gland carcinoma model of breast cancer and Met-1 cells derived from this model as tools to study the role of AR in breast cancer progression. AR protein expression was examined in late-stage primary tumors and lung metastases from MMTV-PyMT mice as well as in Met-1 cells by immunohistochemistry (IHC). Sensitivity of Met-1 cells to the AR agonist dihydrotestosterone (DHT) and anti-androgen therapy was examined using cell viability, migration/invasion, and anchorage-independent growth assays. Late-stage primary tumors and lung metastases from MMTV-PyMT mice and Met-1 cells expressed abundant nuclear AR protein, while negative for estrogen and progesterone receptors. Met-1 sensitivity to DHT and AR antagonists demonstrated a reliance on AR for survival, and AR antagonists inhibited invasion and anchorage-independent growth. These data suggest that the MMTV-PyMT model and Met-1 cells may serve as valuable tools for mechanistic studies of the role of AR in disease progression and how anti-androgens affect the tumor microenvironment.

A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling.

The gene coding for middle T antigen (MT) is the murine polyomavirus oncogene most responsible for tumor formation. Its study has a history of uncovering novel aspects of mammalian cell regulation. The importance of PI3K activity and tyrosine phosphorylation are two examples of insights coming from MT. This study describes new mutants unable to transform like the wild type that point to novel regulation of PI3K signaling. Previous mutants were defective in PI3K because they failed to bind the enzyme and bring the activity to the membrane. These mutants recruit PI3K activity like the wild type, but fail to elevate the cellular level of PIP3, the product used to signal downstream of PI3K. As a result, they fail to activate either Akt or Rac1, explaining the transformation defect.

The glucose transporter GLUT1 is required for ErbB2-induced mammary tumorigenesis.

BackgroundAltered tumor cell metabolism is an emerging hallmark of cancer; however, the precise role for glucose in tumor initiation is not known. GLUT1 (SLC2A1) is expressed in breast cancer cells and is likely responsible for avid glucose uptake observed in established tumors. We have shown that GLUT1 was necessary for xenograft tumor formation from primary mammary cells transformed with the polyomavirus middle-T antigen but that it was not necessary for growth after tumors had formed in vivo, suggesting a differential requirement for glucose depending on the stage of tumorigenesis.MethodsTo determine whether GLUT1 is required early during mammary tumorigenesis, we crossed MMTV-NIC mice, which express activated HER2/NEU/ERBB2 and Cre recombinase, to Slc2a1 Flox/Flox (GLUT1Flox/Flox) mice to generate NIC-GLUT1+/+, NIC-GLUT1Flox/+, and NIC-GLUT1Flox/Flox mice. In addition, we evaluated effects of glucose restriction or GLUT1 inhibition on transformation in MCF10A-ERBB2 breast epithelial cells in three-dimensional culture. Finally, we utilized global gene expression profiling data of primary human breast tumors to determine the relationship between SLC2A1 and stage of tumorigenesis.ResultsAll of the NIC-GLUT1+/+ mice developed tumors in less than 200 days. In contrast, only 1 NIC-GLUT1Flox/Flox mouse and 1 NIC-GLUT1Flox/+ mouse developed mammary tumors, even after 18 months. Mammary gland development was not disrupted in NIC mice lacking GLUT1; however, epithelial content of mature glands was reduced compared to NIC-GLUT1Flox/+ mice. In MCF10A-ERBB2 cells, glucose restriction or GLUT1 inhibition blocked transformation induced by activated ERBB2 through reduced cell proliferation. In human breast cancers, SLC2A1 was higher in ductal carcinoma in situ compared to the normal breast, but lower in invasive versus in situ lesions, suggesting the requirement for GLUT1 decreases as tumors progress.ConclusionsThis study demonstrates a strict requirement for GLUT1 in the early stages of mammary tumorigenesis in vitro and in vivo. While metabolic adaptation has emerged as a hallmark of cancer, our data indicate that early tumor cells rely heavily on glucose and highlight the potential for glucose restriction as a breast cancer preventive strategy.

ApoA-I mimetic administration, but not increased apoA-I-containing HDL, inhibits tumour growth in a mouse model of inherited breast cancer.

Low levels of high-density lipoprotein cholesterol (HDLc) have been associated with breast cancer risk, but several epidemiologic studies have reported contradictory results with regard to the relationship between apolipoprotein (apo) A-I and breast cancer. We aimed to determine the effects of human apoA-I overexpression and administration of specific apoA-I mimetic peptide (D-4F) on tumour progression by using mammary tumour virus-polyoma middle T-antigen transgenic (PyMT) mice as a model of inherited breast cancer. Expression of human apoA-I in the mice did not affect tumour onset and growth in PyMT transgenic mice, despite an increase in the HDLc level. In contrast, D-4F treatment significantly increased tumour latency and inhibited the development of tumours. The effects of D-4F on tumour development were independent of 27-hydroxycholesterol. However, D-4F treatment reduced the plasma oxidized low-density lipoprotein (oxLDL) levels in mice and prevented oxLDL-mediated proliferative response in human breast adenocarcinoma MCF-7 cells. In conclusion, our study shows that D-4F, but not apoA-I-containing HDL, hinders tumour growth in mice with inherited breast cancer in association with a higher protection against LDL oxidative modification.

Activation of DNA damage repair pathways by murine polyomavirus

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble.

Transformation by Polyomavirus Middle T Antigen Involves a Unique Bimodal Interaction with the Hippo Effector YAP.

Murine polyomavirus has repeatedly provided insights into tumorigenesis, revealing key control mechanisms such as tyrosine phosphorylation and phosphoinositide 3-kinase (PI3K) signaling. We recently demonstrated that polyomavirus small T antigen (ST) binds YAP, a major effector of Hippo signaling, to regulate differentiation. Here we characterize YAP as a target of middle T antigen (MT) important for transformation. Through a surface including residues R103 and D182, wild-type MT binds to the YAP WW domains. Mutation of either R103 or D182 of MT abrogates YAP binding without affecting binding to other signaling molecules or the strength of PI3K or Ras signaling. Either genetic abrogation of YAP binding to MT or silencing of YAP via short hairpin RNA (shRNA) reduced MT transformation, suggesting that YAP makes a positive contribution to the transformed phenotype. MT targets YAP both by activating signaling pathways that affect it and by binding to it. MT signaling, whether from wild-type MT or the YAP-binding MT mutant, promoted YAP phosphorylation at S127 and S381/397 (YAP2/YAP1). Consistent with the known functions of these phosphorylated serines, MT signaling leads to the loss of YAP from the nucleus and degradation. Binding of YAP to MT brings it together with protein phosphatase 2A (PP2A), leading to the dephosphorylation of YAP in the MT complex. It also leads to the enrichment of YAP in membranes. Taken together, these results indicate that YAP promotes MT transformation via mechanisms that may depart from YAP's canonical oncogenic transcriptional activation functions. The highly conserved Hippo/YAP pathway is important for tissue development and homeostasis. Increasingly, changes in this pathway are being associated with cancer. Middle T antigen (MT) is the primary polyomavirus oncogene responsible for tumor formation. In this study, we show that MT signaling promotes YAP phosphorylation, loss from the nucleus, and increased turnover. Notably, MT genetics demonstrate that YAP binding to MT is important for transformation. Because MT also binds PP2A, YAP bound to MT is dephosphorylated, stabilized, and localized to membranes. Taken together, these results indicate that YAP promotes MT transformation via mechanisms that depart from YAP's canonical oncogenic transcriptional activation functions.

Abstract B28: Identification of breast cancer metastasis promoting genes by next generation sequencing

Abstract Breast cancer is a leading cause of cancer-related mortality of women in the United States. The majority of cancer deaths are due to metastases rather than the primary tumor, therefore a better understanding of the mechanisms that lead to metastatic disease is critical to reducing breast cancer-related mortality. A majority of cancer cells that leave the primary tumor are either removed by the immune system or fail to survive in distant organs. However, some cancer cells acquire molecular features that allow them to survive and proliferate in secondary sites, such as lung, liver, brain, and bone. In order to design highly efficient anti-metastatic therapies, it is crucial to address metastasis-specific mechanisms of growth and survival. One of the major hurdles to developing these therapies is the genotypic and phenotypic heterogeneity between primary tumors and metastases, as well as between individual metastases. The goal of this project is to characterize the transcriptomic landscape of breast cancer metastases using Next Generation Sequencing (NGS) tools in order to identify potential genes that drive (metastasis promoters) or inhibit (metastasis suppressors) the metastatic outgrowth in distant organs. Therefore, RNA sequencing was performed on breast cancer primary tumors and matching lung metastases from two different mouse models: Polyomavirus Middle-T antigen (PyMT) transgenic mice, a model of spontaneous and highly aggressive breast cancer, as well as from mouse mammary cancer cells orthotopically injected into FVB mice. RNA sequencing using the NGS platform revealed genes that were highly expressed in metastases compared to primary tumors. Interestingly, overlapping the RNA-seq datasets from these two distinct models revealed a shared subset of upregulated genes, strongly suggesting that these genes are likely to be common metastatic outgrowth promoters. To validate these candidate genes, we are currently developing a CRISPR sgRNA library for orthotopic transplant experiments. The genes displaying in vivo effects on metastatic outgrowth in the lungs will be further explored for the development of targeted anti-metastatic therapies. Citation Format: Karol Szczepanek, Kent Hunter. Identification of breast cancer metastasis promoting genes by next generation sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B28.

A truncated phosphorylated p130Cas substrate domain is sufficient to drive breast cancer growth and metastasis formation in vivo.

Elevated p130Cas (Crk-associated substrate) levels are found in aggressive breast tumors and are associated with poor prognosis and resistance to standard therapeutics in patients. p130Cas signals majorly through its phosphorylated substrate domain (SD) that contains 15 tyrosine motifs (YxxP) which recruit effector molecules. Tyrosine phosphorylation of p130Cas is important for mediating migration, invasion, tumor promotion, and metastasis. We previously developed a Src*/SD fusion molecule approach, where the SD is constitutively phosphorylated. In a polyoma middle T-antigen (PyMT)/Src*/SD double-transgenic mouse model, Src*/SD accelerates PyMT-induced tumor growth and promotes a more aggressive phenotype. To test whether Src*/SD also drives metastasis and which of the YxxP motifs are involved in this process, full-length and truncated SD molecules fused to Src* were expressed in breast cancer cells. The functionality of the Src*/SD fragments was analyzed in vitro, and the active proteins were tested in vivo in an orthotopic mouse model. Breast cancer cells expressing the full-length SD and the functional smaller SD fragment (spanning SD motifs 6-10) were injected into the mammary fat pads of mice. The tumor progression was monitored by bioluminescence imaging and caliper measurements. Compared with control animals, the complete SD promoted primary tumor growth and an earlier onset of metastases. Importantly, both the complete and truncated SD significantly increased the occurrence of metastases to multiple organs. These studies provide strong evidence that the phosphorylated p130Cas SD motifs 6-10 (Y236, Y249, Y267, Y287, and Y306) are important for driving mammary carcinoma progression.

Abstract PR03: GLUT1 is required for induction of mammary tumorigenesis by activated ErbB2/HER2/Neu

Abstract Alterations in tumor cell metabolism have been investigated for decades, and include changes in glucose utilization, as well as changes in mitochondrial and fatty acid metabolism; however, it is unclear at what point during the process of tumorigenesis these metabolic changes occur. We hypothesized that elevated glucose uptake represents the first metabolic adaptation to transformation, and thus may be a target for the prevention of breast cancer. The GLUT1 transporter is expressed in breast cancer cells and is responsible for the majority of their glucose uptake. Not surprisingly, breast cancer patients with high levels of GLUT1 have a poorer clinical outcome than those with lower levels, likely due to the association between glucose uptake and high tumor grade. We previously showed that the reduction of GLUT1 in established tumor cells resulted in a 50% decrease in glucose consumption and lactate generation. There was a corresponding decrease in proliferation in both two and three-dimensional culture, and in tumor growth in immunodeficient mice. Mammary epithelial cells from GLUT1fl/fl mice were transformed using polyoma middle tumor antigen (PyMT), and GLUT1 excised using Cre recombinase. Cells expressing GLUT1 were tumorigenic in immunodeficient mice, while cells lacking GLUT1 were not tumorigenic. This suggests that in mammary epithelial cells transformed in vitro, loss of GLUT1 was sufficient to prevent tumor outgrowths when injected into the mammary fat pad of nude mice. This indicates that GLUT1 and by extension glucose, are critical at an early stage of mammary tumorigenesis. To test the hypothesis that elevated glucose uptake is required very early in the process of tumor formation, we crossed the MMTV-NIC (NIC) mice, which contain activated NeuNT, an internal ribosome entry site (IRES), and Cre recombinase, to mice bearing floxed alleles of GLUT1, and monitored tumor development. All of the control MMTV-NIC mice developed tumors in less than 200 days. In contrast, none of the NIC-GLUT1fl/fl mice developed mammary tumors even after eighteen months. We anticipated that loss of one allele of GLUT1 would significantly delay mammary tumorigenesis; however, we did not observe any tumors in these mice, even at eighteen months of age. Tumors that formed in the NIC mice were typical Neu-dependent tumors observed in similar transgenic mice with a high proliferative index (38% by Ki67 staining), and there was evidence of preneoplastic lesions in the non tumor-bearing mammary glands. Small Cre-positive lesions were also positive for GLUT1 indicating GLUT1 expression increases very early in Neu-mediated tumorigenesis. The mammary ductal tree developed normally in NIC-Glut1fl/fl and NIC-Glut1fl/+ mice. Mammary glands from mice lacking one allele of GLUT1 had increased epithelial content and higher proliferative index, compared to mice lacking both Glut1 alleles; however, no palpable tumors ever formed in either group. These results suggest that loss of a single allele of GLUT1 is sufficient to suppress Neu-induced mammary tumorigenesis and that there is an absolute requirement for GLUT1 at the earliest stages of tumorigenesis. Citation Format: Elizabeth A. Wellberg, Angelo D'Allessandro, Andrew S. Lewis, Kristina Terrell, E. Dale Abel, William A. Muller, Kirk A. Hansen, Steven M. Anderson. GLUT1 is required for induction of mammary tumorigenesis by activated ErbB2/HER2/Neu. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr PR03.

Limited variation during circulation of a polyomavirus in the human population involves the COCO-VA toggling site of Middle and Alternative T-antigen(s)

We have recently shown that the trichodysplasia spinulosa-associated polyomavirus (TSPyV) belongs to a large monophyletic group of mammalian polyomaviruses that experienced accelerated codon-constrained Val-Ala (COCO-VA) toggling at a protein site common to both Middle and Alternative T-antigens (MT/ALTO). Here we analyzed thirteen, mostly newly sequenced TSPyV genomes, representing ~40% of reported TS disease cases world-wide. We found two deletions and 30 variable sites (≤0.6%) that included only four sites with non-synonymous substitutions (NSS). One NSS site was under positive selection in the exon shared by Small and Middle T antigens, while three others were segregated in MT/ALTO. Two MT/ALTO sites covaried with five sites elsewhere in the genome and determined separation of twelve TSPyVs into two most populous phylogenetic lineages. The other, most distant TSPyV was distinguished by NSS at the COCO-VA site, observed for the first time during intra-species evolution. Our findings reveal a connection between micro- and macro-evolution of polyomaviruses.

Host JDP2 expression in the bone marrow contributes to metastatic spread.

The c-Jun Dimerization Protein 2, JDP2, is a basic leucine zipper protein member of the activator protein-1 (AP-1) family of transcription factors. JDP2 typically suppresses gene transcription through multiple mechanisms and plays a dual role in multiple cellular processes, including cell differentiation and proliferation which is dependent on AP-1 function. Whereas the role of JDP2 expression within cancer cells has been studied, its role in stromal cells at the tumor microenvironment is largely unknown. Here we show that mice lacking JDP2 (JDP2-/-) display a reduced rate of metastasis in Lewis lung carcinoma (LLC) and polyoma middle T-antigen (PyMT) breast carcinoma mouse models. The replacement of wild-type bone marrow derived cells (BMDCs) with JDP2-deficient BMDCs recapitulates the metastatic phenotype of JDP2-/- tumor-bearing mice. In vitro, conditioned medium of wild-type BMDCs significantly potentiates the migration and invasion capacity of LLC cells as compared to that of JDP2-/- BMDCs. Furthermore, wild-type BMDCs secrete CCL5, a chemokine known to contribute to metastasis, to a greater extent than JDP2-/- BMDCs. The supplementation of CCL5 in JDP2-/- BMDC conditioned medium was sufficient to potentiate the invasion capacity of LLC. Overall, this study suggests that JDP2-expressing BMDCs within the tumor microenvironment contribute to metastatic spread.

Differential effects of polyoma virus middle tumor antigen mutants upon gap junctional, intercellular communication

Gap junctions are channels that connect the cytoplasm of adjacent cells. Oncogenes such as the middle Tumor antigen of polyoma virus (mT) are known to suppress gap junctional, intercellular communication (GJIC). mT associates with and is tyrosine-phosphorylated by cSrc family members. Specific mT phosphotyrosines provide docking sites for the phosphotyrosine binding domain of Shc (mT-tyr250) or the SH2 domain of the regulatory subunit of the phosphatidylinositol-3 kinase (PI3k, mT-tyr315). Binding results in the activation of their downstream signaling cascades, Ras/Raf/Erk and PI3 kinase/Akt, respectively, both of which are needed for full neoplastic transformation.To examine the effect of mT-initiated pathways upon gap junctional communication, GJIC was quantitated in rat liver epithelial T51B cells expressing mT-mutants, using a novel technique of in situ electroporation. The results demonstrate for the first time that, although even low levels of wild-type mT are sufficient to interrupt gap junctional communication, GJIC suppression still requires an intact tyr-250 site, that is activation of the Ras pathway. In sharp contrast, activation of the PI3k pathway is not required for GJIC suppression, indicating that GJIC suppression is independent of full neoplastic conversion and the concomitant morphological changes. Interestingly, expression of a constitutively active, myristylated form of the catalytic subunit of PI3k, p110, or the constitutively active mutants E545K and H1047R increased GJIC, while pharmacological inhibition of PI3k eliminated communication. Therefore, although PI3k is growth promoting and in an activated form it can act as an oncogene, it actually plays a positive role upon gap junctional, intercellular communication.

A virus-encoded miRNA in mouse polyomavirus regulates viral replication in the kidneys in a CD8 T cell dependent manner. (VIR6P.1166)

Abstract The polyomaviruses JCV and BKV are ubiquitous DNA viruses in the human population which persist as low-level infections in host kidneys and give rise to disease upon immunosuppression, resulting in significant morbidity and mortality. The polyomaviruses encode one endogenous miRNA, with both the 5p and 3p mature miRNAs functioning to reduce levels of target transcripts. The mouse polyomavirus (MPyV) also encodes a miRNA within the sequence for the early nonstructural proteins large (LT) and middle (MT) T-antigen, both of which have been established as miRNA targets. Other targets of the MPyV miRNA and their physiologic consequences have been poorly defined. The dl1013 virus is a deletion mutant of MPyV which eliminates expression of the endogenous miRNA while preserving LT and MT functions vital to the viral life cycle. We have shown that infecting immunocompetent C57BL/6 mice with dl1013 results in lower viral levels in the kidneys than infection with parental virus, but not in other organs. No difference in kidney viral levels between parental and dl1013 virus is seen in CD8α-/- mice, suggesting that the effect of the miRNA on viral replication is dependent on CD8 T cells. We are currently investigating whether this MPyV miRNA affects recruitment of myeloid and lymphoid cells into the kidney and their role in controlling MPyV in the dominant reservoir of viral persistence of both human and mouse polyomaviruses.

C-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression.

Breast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis.

Abstract PR01: Rebastinib, a selective TIE2 kinase inhibitor, decreases TIE2-expressing macrophages, reduces metastasis, and increases survival in murine cancer models

Abstract In the tumor microenvironment, TIE2 expression on tissue macrophages, bone marrow derived TIE2-expressing monocytes (TEMs), osteoclasts, and vascular endothelial cells promotes tumor invasiveness, dissemination, and metastasis. Additionally, a subset of TIE2-expressing macrophages, located within specialized vascular structures known as tumor microenvironment for metastases (TMEMs), are linked to intravasation of cancer cells into circulation and dissemination to metastatic sites. Rebastinib is a picomolar inhibitor of TIE2 kinase, and exhibits an extraordinarily long off-rate from TIE2, measured to be over 24 hours in a cell-based assay. Herein, we examine the efficacy of rebastinib in the polyoma middle-T antigen (PyMT) syngeneic mouse breast cancer model. In this model, PyMT breast cancer cells are implanted in the mammary fat pad, and primary tumor growth leads to lung metastasis, which is known to be modulated by TEMs and TMEM vascular structures. We examined multiple dosing schedules of rebastinib in combination with anti-tubulin agents (ATAs). Rebastinib treatment in this model significantly ablated TEMs in the primary tumor stroma and caused a significant decrease in lung metastases. Furthermore, the combination of rebastinib with ATAs, even with once or twice weekly oral dosing of rebastinib, led to a significant further decrease in lung metastases compared to single-agent treatment with ATAs. Rebastinib also enhanced the activity of ATAs in reducing primary tumor growth and regrowth of tumor post-resection. TIE2 inhibition with targeted therapy represents a novel treatment approach for metastatic breast cancer and other cancers that rely on TEMs and TMEMs for growth and metastasis. As such, rebastinib has been selected for further clinical development in solid tumors with a Phase 1b trial being planned for 2014. This abstract is also presented as Poster A5. Citation Format: Daniel L. Flynn, Michael D. Kaufman, Cynthia B. Leary, Molly M. Hood, Wei-Ping Lu, Benjamin A. Turner, Scott C. Wise, Marc S. Rudoltz, Bryan D. Smith. Rebastinib, a selective TIE2 kinase inhibitor, decreases TIE2-expressing macrophages, reduces metastasis, and increases survival in murine cancer models. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR01. doi:10.1158/1538-7445.CHTME14-PR01