Mining the pre-diagnostic antibody repertoire of TgMMTV-neu mice to identify autoantibodies useful for the early detection of human breast cancer.

BACKGROUND: The use of autoantibodies for the early detection of breast cancer has generated much interest as antibodies can be readily assayed in serum when antigen levels are low. Ideally, diagnostic autoantibodies would be identified in individuals who harbored pre-invasive disease/high risk lesions leading to malignancy. Prospectively collected human serum samples from these individuals are rare and not often available for biomarker discovery. We questioned whether transgenic animals could be used to identify cancer-associated autoantibodies present at the earliest stages of the malignant transformation of breast cancer. METHODS: We collected sera from transgenic mice (TgMMTV-neu) from the time of birth to death by spontaneous mammary tumors. Using sera from a time point prior to the development of tumor, i.e. "pre-diagnostic", we probed cDNA libraries derived from syngeneic tumors to identify proteins recognized by IgG antibodies. Once antigens were identified, selected proteins were evaluated via protein arrays, for autoantibody responses using plasma from women obtained prior to the development of breast cancer and matched controls. The ability of the antigens to discriminate cases from controls was assessed using receiver-operating-characteristic curve analyses and estimates of the area under the curve. RESULTS: We identified 6 autoantibodies that were present in mice prior to the development of mammary cancer: Pdhx, Otud6b, Stk39, Zpf238, Lgals8, and Vps35. In rodent validation cohorts, detecting both IgM and IgG antibody responses against a subset of the identified proteins could discriminate pre-diagnostic sera from non-transgenic control sera with an AUC of 0.924. IgG and IgM autoantibodies, specific for a subset of the identified antigens, could discriminate the samples of women who eventually developed breast cancer from case-matched controls who did not develop disease. The discriminatory potential of the pre-diagnostic autoantibodies was enhanced if plasma samples were collected greater than 5 months prior to a breast cancer diagnosis (AUC 0.68; CI 0.565-0.787, p=0.0025). CONCLUSION: Genetically engineered mouse models of cancer may provide a facile discovery tool for identifying autoantibodies useful for human cancer diagnostics.

Drak2 is upstream of p70S6 kinase: its implication in cytokine-induced islet apoptosis, diabetes, and islet transplantation.

Drak2 is a member of the death-associated protein family and a serine threonine kinase. In this study, we investigated its role in beta cell survival and diabetes. Drak2 mRNA and protein were rapidly induced in islet beta cells after stimulation by inflammatory lymphokines known to be present in type 1 diabetes. Drak2 up-regulation was accompanied by increased beta cell apoptosis. beta cell apoptosis caused by the said stimuli was inhibited by Drak2 knockdown using small interfering RNA. Conversely, transgenic Drak2 overexpression led to aggravated beta cell apoptosis triggered by the stimuli. Further in vivo experiments demonstrated that Drak2 transgenic islets were more vulnerable to streptozocin insult. We established that inducible NO synthase was upstream and caspase-9 was downstream of Drak2 in its signaling pathway. Purified Drak2 could phosphorylate ribosomal protein S6 (p70S6) kinase in an in vitro kinase assay. Drak2 overexpression in NIT-1 cells led to enhanced p70S6 kinase phosphorylation, whereas Drak2 knockdown in these cells reduced it. These mechanistic studies proved that p70S6 kinase was a bona fide Drak2 substrate.

Drak2 overexpression results in increased beta-cell apoptosis after free fatty acid stimulation.

Drak2 is a serine threonine kinase in the death-associated protein family. In this study, we investigated its role in free fatty acid (FFA)-induced islet apoptosis. Drak2 mRNA and protein were rapidly induced in islet beta-cells after FFA stimulation. Such Drak2 upregulation was accompanied by increased beta-cell apoptosis, which was inhibited by Drak2 knockdown using siRNA. Conversely, transgenic (Tg) Drak2 overexpression led to aggravated beta-cell apoptosis triggered by FFA. Drak2 overexpression in islets compromised the increase of anti-apoptotic factors, such as Bcl-2, Bcl-xL and Flip, upon FFA assault. Further in vivo experiments demonstrated that Drak2 Tg mice presented compromised glucose tolerance in a diet-induced obesity model. Our data show that Drak2 is detrimental to islet survival in the presence of excessive lipid.

Armc5 deletion causes developmental defects and compromises T-cell immune responses.

Armadillo repeat containing 5 (ARMC5) is a cytosolic protein with no enzymatic activities. Little is known about its function and mechanisms of action, except that gene mutations are associated with risks of primary macronodular adrenal gland hyperplasia. Here we map Armc5 expression by in situ hybridization, and generate Armc5 knockout mice, which are small in body size. Armc5 knockout mice have compromised T-cell proliferation and differentiation into Th1 and Th17 cells, increased T-cell apoptosis, reduced severity of experimental autoimmune encephalitis, and defective immune responses to lymphocytic choriomeningitis virus infection. These mice also develop adrenal gland hyperplasia in old age. Yeast 2-hybrid assays identify 16 ARMC5-binding partners. Together these data indicate that ARMC5 is crucial in fetal development, T-cell function and adrenal gland growth homeostasis, and that the functions of ARMC5 probably depend on interaction with multiple signalling pathways.

An Autoimmune Response Signature Associated with the Development of Triple-Negative Breast Cancer Reflects Disease Pathogenesis.

The repertoire of antigens associated with the development of an autoimmune response in breast cancer has relevance to detection and treatment strategies. We have investigated the occurrence of autoantibodies associated with the development of triple-negative breast cancer (TNBC) in the before diagnosis setting and in samples collected at the time of diagnosis of TNBC. Lysate arrays containing protein fractions from the TNBC MDA-MB-231 cell line were hybridized with TNBC plasmas from the Women's Health Initiative cohort, collected before clinical diagnosis and with plasmas from matched controls. An immune response directed against spliceosome and glycolysis proteins was observed with case plasmas as previously reported in estrogen receptor(+) breast cancer. Importantly, autoantibodies directed against networks involving BRCA1, TP53, and cytokeratin proteins associated with a mesenchymal/basal phenotype were distinct to TNBC before diagnosis samples. Concordant autoantibody findings were observed with mouse plasma samples collected before occurrence of palpable tumors from a C3(1)-T triple negative mouse model. Plasma samples collected at the time of diagnosis of stage II TNBC and from matched healthy controls were subjected to proteomic analysis by mass spectrometry to identify Ig-bound proteins yielding a predominance of cytokeratins, including several associated with a mesenchymal/basal phenotype among cases compared with controls. Our data provide evidence indicative of a dynamic repertoire of antigens associated with a humoral immune response reflecting disease pathogenesis in TNBC.

Investigation of tissue-specific expression and functions of MLF1-IP during development and in the immune system.

Myeloid leukemia factor 1-interacting protein (MLF1-IP) has been found to exert functions in mitosis, although studies have been conducted only in cell lines up to now. To understand its roles during ontogeny and immunity, we analyzed its mRNA expression pattern by in situ hybridization and generated MLF1-IP gene knockout (KO) mice. MLF1-IP was expressed at elevated levels in most rudimentary tissues during the mid-gestation stage, between embryonic day 9.5 (e9.5) and e15.5. It declined afterwards in these tissues, but was very high in the testes and ovaries in adulthood. At post-natal day 10 (p10), the retina and cerebellum still expressed moderate MLF1-IP levels, although these tissues do not contain fast-proliferating cells at this stage. MLF1-IP expression in lymphoid organs, such as the thymus, lymph nodes, spleen and bone marrow, was high between e15.5 and p10, and decreased in adulthood. MLF1-IP KO embryos failed to develop beyond e6.5. On the other hand, MLF1-IP(+/-) mice were alive and fertile, with no obvious anomalies. Lymphoid organ size, weight, cellularity and cell sub-populations in MLF1-IP(+/-) mice were in the normal range. The functions of MLF1-IP(+/-) T cells and naïve CD4 cells, in terms of TCR-stimulated proliferation and Th1, Th17 and Treg cell differentiation in vitro, were comparable to those of wild type T cells. Our study demonstrates that MLF1-IP performs unique functions during mouse embryonic development, particularly around e6.5, when there was degeneration of epiblasts. However, the cells could proliferate dozens of rounds without MLF1-IP. MLF1-IP expression at about 50% of its normal level is sufficient to sustain mice life and the development of their immune system without apparent abnormalities. Our results also raise an intriguing question that MLF1-IP might have additional functions unrelated to cell proliferation.

To investigate the necessity of STRA6 upregulation in T cells during T cell immune responses.

Our earlier study revealed that STRA6 (stimulated by retinoic acid gene 6) was up-regulated within 3 h of TCR stimulation. STRA6 is the high-affinity receptor for plasma retinol-binding protein (RBP) and mediates cellular vitamin A uptake. We generated STRA6 knockout (KO) mice to assess whether such up-regulation was critical for T-cell activation, differentiation and function. STRA6 KO mice under vitamin A sufficient conditions were fertile without apparent anomalies upon visual inspection. The size, cellularity and lymphocyte subpopulations of STRA6 KO thymus and spleen were comparable to those of their wild type (WT) controls. KO and WT T cells were similar in terms of TCR-stimulated proliferation in vitro and homeostatic expansion in vivo. Naive KO CD4 cells differentiated in vitro into Th1, Th2, Th17 as well as regulatory T cells in an analogous manner as their WT counterparts. In vivo experiments revealed that anti-viral immune responses to lymphocytic choriomeningitis virus in KO mice were comparable to those of WT controls. We also demonstrated that STRA6 KO and WT mice had similar glucose tolerance. Total vitamin A levels are dramatically lower in the eyes of KO mice as compared to those of WT mice, but the levels in other organs were not significantly affected after STRA6 deletion under vitamin A sufficient conditions, indicating that the eye is the mouse organ most sensitive to the loss of STRA6. Our results demonstrate that 1) in vitamin A sufficiency, the deletion of STRA6 in T cells does no affect the T-cell immune responses so-far tested, including those depend on STAT5 signaling; 2) STRA6-independent vitamin A uptake compensated the lack of STRA6 in lymphoid organs under vitamin A sufficient conditions in mice; 3) STRA6 is critical for vitamin A uptake in the eyes even in vitamin A sufficiency.

Autoantibody signatures involving glycolysis and splicesome proteins precede a diagnosis of breast cancer among postmenopausal women.

We assessed the autoantibody repertoire of a mouse model engineered to develop breast cancer and the repertoire of autoantibodies in human plasmas collected at a preclinical time point and at the time of clinical diagnosis of breast cancer. In seeking to identify common pathways, networks, and protein families associated with the humoral response, we elucidated the dynamic nature of tumor antigens and autoantibody interactions. Lysate proteins from an immortalized cell line from a MMTV-neu mouse model and from MCF7 human breast cancers were spotted onto nitrocellulose microarrays and hybridized with mouse and human plasma samples, respectively. Immunoglobulin-based plasma immunoreactivity against glycolysis and spliceosome proteins was a predominant feature observed both in tumor-bearing mice and in prediagnostic human samples. Interestingly, autoantibody reactivity was more pronounced further away than closer to diagnosis. We provide evidence for dynamic changes in autoantibody reactivity with tumor development and progression that may depend, in part, on the extent of antigen-antibody interactions.

Transgenic drak2 overexpression in mice leads to increased T cell apoptosis and compromised memory T cell development.

Drak2 is a death-associated protein family serine-threonine kinase. Its expression and roles in the immune system were investigated in this study. According to in situ hybridization, Drak2 expression was ubiquitous at the mid-gestation stage in embryos, followed by more focal expression in various organs in the perinatal period and adulthood, notably in the thymus, spleen, lymph nodes, cerebellum, suprachiasmatic nuclei, pituitary, olfactory lobes, adrenal medulla, stomach, skin, and testes. Drak2 transgenic (Tg) mice were generated using the human beta-actin promoter. These Tg mice showed normal T cell versus B cell and CD4 versus CD8 populations in the spleen, but their spleen weight cellularity was lower in comparison with wild type mice. After TCR activation, the proliferation response in Drak2 Tg T cells was normal, although their interleukin (IL)-2 and IL-4 but not interferon-gamma production was augmented. Activated Drak2 Tg T cells demonstrated significantly enhanced apoptosis in the presence of exogenous IL-2. At the molecular level, Drak2 Tg T cells manifested a lower increase of anti-apoptotic factors during activation; such a change probably rendered the cells vulnerable to subsequent IL-2 insults. The compromised apoptosis in Drak2 Tg T cells was associated with reduced numbers of T cells with the memory cell phenotype (CD62L(lo)) and repressed secondary T cell responses in delayed type hypersensitivity. Our study demonstrates that Drak2 expresses in the T cell compartment but is not T cell-specific; it plays critical roles in T cell apoptosis and memory T cell development.

Ephrin-B1 is critical in T-cell development.

Eph kinases are the largest family of receptor tyrosine kinases, and their ligands, ephrins (EFNs), are also cell surface molecules. In this study, we investigated the role of EFNB1 and the Ephs it interacts with (collectively called EFNB1 receptors) in mouse T-cell development. In the thymus, CD8 single positive (SP) and CD4CD8 double positive (DP) cells expressed high levels of EFNB1 and EFNB1 receptors, whereas CD4 SP cells had moderate expression of both. Soluble EFNB1-Fc in fetal thymus organ culture caused significant subpopulation ratio skew, with increased CD4 SP and CD8 SP and decreased DP percentage, while the cellularity of the thymus remained constant. Moreover, in EFNB1-treated fetal thymus organ culture, CD117(+), CD25(+), DP, CD4 SP, and CD8 SP cells all had significantly enhanced proliferation history, according to bromodeoxyuridine uptake. In vitro culture of isolated thymocytes revealed that EFNB1-Fc on solid-phase protected thymocytes from anti-CD3-induced apoptosis, with concomitant augmentation of several antiapoptotic factors, particularly in CD4 SP and CD8 SP cells; on the other hand, soluble EFNB1-Fc promoted anti-CD3-induced apoptosis, as was the case in vivo. This study reveals that EFNB1 and EFNB1 receptors are critical in thymocyte development.

Tumor vaccine based on cell surface expression of DcR3/TR6.

DcR3/TR6, a secreted protein belonging to the TNF receptor superfamily, interacts with lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entrance mediator (LIGHT), Fas ligand (FasL), and TL1A, all members of the TNF superfamily. Solid-phase TR6 can trigger reverse signaling of LIGHT and FasL expressed on T cells, and lead to T cell costimulation. In this study, we engineered tumor cells to express cell surface TR6 and used these cells as a tumor vaccine. We demonstrated that mastocytoma P815 cells expressing surface TR6 (TR6-P815) effectively augmented the T cells response in vitro and ex vivo in terms of proliferation, as well as IL-2 and IFN-gamma secretion. TR6-P815 cells had reduced tumorigenicity compared with parental P815 cells. When inactivated TR6-P815 cells were employed as a vaccine, they protected the mice from challenge with live parental P815 cells, and eliminated established P815 tumors. The cell surface TR6-based tumor vaccine was also effective against low antigenicity tumors, such as B16 melanoma; co-administration of bacillus Calmette-Guérin further enhanced the vaccine's efficacy. Thus, cell surface TR6 expression is a useful addition to our tumor vaccine arsenal.