Jian-Xin Gao

ERα signaling through slug regulates E-cadherin and EMT

The ERα signaling pathway is one of the most important and most studied pathways in human breast cancer, yet numerous questions still exist such as how hormonally responsive cancers progress to a more aggressive and hormonally independent phenotype. We have noted that human breast cancers exhibit a strong direct correlation between ERα and E-cadherin expression by immunohistochemistry, suggesting that ERα signaling might regulate E-cadherin and implying that this regulation might influence epithelial–mesenchymal transition (EMT) and tumor progression. To investigate this hypothesis and the mechanisms behind it, we studied the effects of ERα signaling in ERα-transfected ERα-negative breast carcinoma cell lines, the MDA-MB-468 and the MDA-MB-231 and the effects of ERα knockdown in naturally expressing ERα-positive lines, MCF-7 and T47D. When ERα was overexpressed in the ERα-negative lines, 17β-estradiol (E2) decreased slug and increased E-cadherin. Clones maximally exhibiting these changes grew more in clumps and became less invasive in Matrigel. When ERα was knocked down in the ERα-positive lines, slug increased, E-cadherin decreased, cells became spindly and exhibited increased Matrigel invasion. ERα signaling decreased slug expression by two different mechanisms: directly, by repression of slug transcription by the formation of a corepressor complex of ligand-activated ERα, HDAC inhibitor (HDAC1), and nuclear receptor corepressor (N-CoR) that bound the slug promoter in three half-site estrogen response elements (EREs); indirectly by phosphorylation and inactivation of GSK-3β through phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt). The GSK-3β inactivation, in turn, repressed slug expression and increased E-cadherin. In human breast cancer cases, there was a strong inverse correlation between slug and ERα and E-cadherin immunoreactivity. Our findings indicate that ERα signaling through slug regulates E-cadherin and EMT.

Precancerous Stem Cells Have the Potential for both Benign and Malignant Differentiation

Cancer stem cells (CSCs) have been identified in hematopoietic and solid tumors. However, their precursors—namely, precancerous stem cells (pCSCs) —have not been characterized. Here we experimentally define the pCSCs that have the potential for both benign and malignant differentiation, depending on environmental cues. While clonal pCSCs can develop into various types of tissue cells in immunocompetent mice without developing into cancer, they often develop, however, into leukemic or solid cancers composed of various types of cancer cells in immunodeficient mice. The progress of the pCSCs to cancers is associated with the up-regulation of c-kit and Sca-1, as well as with lineage markers. Mechanistically, the pCSCs are regulated by the PIWI/AGO family gene called piwil2. Our results provide clear evidence that a single clone of pCSCs has the potential for both benign and malignant differentiation, depending on the environmental cues. We anticipate pCSCs to be a novel target for the early detection, prevention, and therapy of cancers.

Precancerous Stem Cells Can Serve As Tumor Vasculogenic Progenitors

Tumor neo-vascularization is critical for tumor growth, invasion and metastasis, which has been considered to be mediated by a mechanism of angiogenesis. However, histopathological studies have suggested that tumor cells might be the progenitor for tumor vasculature. Recently, we have reported that the precancerous stem cells (pCSCs) representing the early stage of developing cancer stem cells (CSCs), have the potential for both benign and malignant differentiation. Therefore, we investigated whether pCSCs serve as progenitors for tumor vasculogenesis. Herein, we report that in the pCSC-derived tumors, most blood vessels were derived from pCSCs. Some pCSCs constitutively expressed vasculogenic receptor VEGFR-2, which can be up-regulated by hypoxia and angiogenesis-promoting cytokines, such as GM-CSF, Flt3 ligand, and IL-13. The pCSCs are much more potent in tumor vasculogenesis than the differentiated tumor monocytic cells (TMCs) from the same tumor, which had comparable or even higher capacity to produce some vascular growth factors, suggesting that the potent tumor vasculogenesis of pCSCs is associated with their intrinsic stem-like property. Consistently tumor vasculogenesis was also observed in human cancers such as cervical cancer and breast cancer and xenograft lymphoma. Our studies indicate that pCSCs can serve as tumor vasculogenic stem/progenitor cells (TVPCs), and may explain why anti-angiogenic cancer therapy trials are facing challenge.

Perinatal blockade of b7-1 and b7-2 inhibits clonal deletion of highly pathogenic autoreactive T cells.

A number of in vitro studies have suggested that costimulatory molecules B7-1 and B7-2 and their receptor CD28 can promote clonal deletion, and limited in vivo studies have indicated that CD28 is involved in the clonal deletion of some T cells. However, the significance of B7-mediated clonal deletion in preventing autoimmune diseases has not been studied systematically. Here we report that the perinatal blockade of B7-1 and B7-2 substantially inhibits the clonal deletion of T cells in the thymus and leads to an accumulation of T cells capable of inducing fatal multiorgan inflammation. These results reveal a critical role for costimulatory molecules B7-1 and B7-2 in deleting pathogenic autoreactive T cells in the thymus. The critical role of B7-1 and B7-2 in T cell clonal deletion may explain, at least in part, the paradoxical increase of autoimmune disease in mice deficient for this family of costimulatory molecules, such as cytotoxic T lymphocyte associated molecule 4, CD28, and B7-2. The strong pathogenicity of the self-reactive T cells supports a central hypothesis in immunology, which is that clonal deletion plays an important role in preventing autoimmune diseases.

Clonal Deletion of Simian Virus 40 Large T Antigen-Specific T Cells in the Transgenic Adenocarcinoma of Mouse Prostate Mice: An Important Role for Clonal Deletion in Shaping the Repertoire of T Cells Specific for Antigens Overexpressed in Solid Tumors

In addition to their overexpression in cancer cells, most of the tumor-associated Ags are expressed at low but detectable levels in normal tissues. It is not clear whether the repertoire of T cells specific for unmutated tumor Ags is shaped by negative selection during T cell development. The transgenic adenocarcinoma of mouse prostate (TRAMP) model is transgenic for the SV40 large T Ag (Tag) under the control of the rat probasin regulatory elements. Although it has been established that T lymphocytes from TRAMP mice are tolerant to SV40 Tag, the mechanism of the tolerance is largely unknown. To examine whether the T cell clonal deletion is responsible for the tolerance, we crossed the TRAMP mice with mice transgenic for a rearranged TCR specific for SV40 Tag presented by the H-2Kk. Double transgenic TRAMP/TCR mice showed profound thymic deletion of SV40 Tag-reactive T cells, including a 6- to 10-fold reduction in the total thymocyte numbers and a >50-fold reduction in phenotypically mature T cells. Consistent with this finding, we observed that the SV40 Tag and endogenous mouse probasin genes are expressed at low levels in the thymus. These results demonstrate that clonal deletion is a major mechanism for tolerance to Ags previously regarded as prostate-specific, and provide direct evidence that the T cell repertoire specific for an unmutated tumor Ag can be shaped by clonal deletion in the thymus.

B7-CD28 Interaction Promotes Proliferation and Survival but Suppresses Differentiation of CD4−CD8− T Cells in the Thymus

Costimulatory molecules play critical roles in the induction and effector function of T cells. More recent studies reveal that costimulatory molecules enhance clonal deletion of autoreactive T cells as well as generation and homeostasis of the CD25+CD4+ regulatory T cells. However, it is unclear whether the costimulatory molecules play any role in the proliferation and differentiation of T cells before they acquire MHC-restricted TCR. In this study, we report that targeted mutations of B7-1 and B7-2 substantially reduce the proliferation and survival of CD4−CD8− (double-negative (DN)) T cells in the thymus. Perhaps as a result of reduced proliferation, the accumulation of RAG-2 protein in the DN thymocytes is increased in B7-deficient mice, which may explain the increased expression of TCR gene and accelerated transition of CD25+CD44− (DN3) to CD25−CD44− (DN4) stage. Qualitatively similar, but quantitatively less striking effects were observed in mice with a targeted mutation of CD28, but not CTLA4. Taken together, our results demonstrate that the development of DN in the thymus is subject to modulation by the B7-CD28 costimulatory pathway.

Differentiation of Monocytic Cell Clones into CD8α+ Dendritic Cells (DC) Suggests that Monocytes Can Be Direct Precursors for Both CD8α+ and CD8α− DC in the Mouse

Dendritic cells (DC) are the professional APCs that initiate T cell immune responses. DC can develop from both myeloid and lymphoid progenitors. In the mouse, the CD8α+ DC had been designated as “lymphoid” DC, and CD8α− DC as “myeloid” DC until recently when it was demonstrated that common myeloid progenitors can also give rise to CD8α+ DC in bone marrow chimera mice. However, it is still not clear which committed myeloid lineages differentiate into CD8α+ DC. Because monocytes can differentiate into DC in vivo, the simplest hypothesis is that the CD8α+ DC can be derived from the monocyte/macrophage. In this study we show that cell clones, isolated from CD8α+ DC lymphoma but with a monocytic phenotype (CD11clow/−D11bhighCD8α−I-Alow), can redifferentiate into CD8α+ DC either when stimulated by LPS and CD40L or when they migrate into the lymphoid organs. Maturation of DC in vivo correlated with strong priming of allogeneic T cells. Moreover, the monocytes from cultured splenocytes or peritoneal exudates macrophages of wild-type mice are also capable of differentiating into CD11c+CD8α+ DC after their migration into the draining lymph nodes. Our results suggest that monocytes can be direct precursors for CD11c+CD8α+ DC in vivo. In addition, the monocyte clones described in this study may be valuable for studying the differentiation and function of CD8α+ DC that mediate cross-presentation of Ag to CD8 T cells specific for cell-associate Ags.

CD28/B7-Mediated Co-stimulation Is Critical for Early Control of Murine Cytomegalovirus Infection

Control of acute murine cytomegalovirus (MCMV) infection is dependent upon both innate and adaptive immune responses, relying primarily upon natural killer (NK) and T-cell responses for control. Although CD28/B7 plays a clear role in T-cell responses in many antigen systems including some viral infections, the importance of co-stimulation during MCMV infection is unconfirmed. In addition, recent data suggest that CD28/B7 co-stimulation might also be important to Ly49H+ NK-cell expansion. We therefore hypothesized that CD28/B7 co-stimulation is critical to viral control after MCMV infection, and further that CD28/B7 co-stimulation plays a role in MCMV-specific T- and NK-cell responses. To test these hypotheses, we utilized C57BL/6 mice lacking the co-stimulatory molecules B7-1 and B7-2 or CD28. After primary infection with MCMV, viral titers are significantly elevated in mice lacking CD28 or B7 compared with wild-type mice. Impaired viral control is associated with significant defects in peripheral T-cell responses to MCMV, which appear to be dependent upon CD28/B7 co-stimulation. Abnormal hepatic T-cell responses in CD28(-/-) mice are preceded by impaired MCMV-specific Ly49H+ NK-cell responses. Cytokine evaluations confirm that CD28/B7 co-stimulation is not required for non-specific antiviral responses. We conclude that CD28-mediated co-stimulation is critical for early viral control during acute MCMV infection.

Altered thymic selection by overexpressing cellular FLICE inhibitory protein in T cells causes lupus-like syndrome in a BALB/c but not C57BL/6 strain

The cellular FLICE inhibitory protein (c-FLIP) is an endogenous inhibitor of the caspase-8 proapoptotic signaling pathway downstream of death receptors. Recent evidence indicates that the long form of c-FLIP (c-FLIPL) is required for proliferation and effector T-cell development. However, the role of c-FLIPL in triggering autoimmunity has not been carefully analyzed. We now report that c-FLIPL transgenic (Tg) mice develop splenomegaly, lymphadenopathy, multiorgan infiltration, high titers of auto-antibodies, and proliferative glomerulonephritis with immune complex deposition in a strain-dependent manner. The development of autoimmunity requires CD4+ T cells and may result from impaired thymic selection. At the molecular level, c-FLIPL overexpression inhibits the ζ chain-associated protein tyrosine kinase of 70 kDa (ZAP-70) activation, thus impairing the signaling pathway derived from ZAP-70 required for thymic selection. Therefore, we have identified c-FLIPL as a susceptibility factor under the influence of epistatic modifiers for the development of autoimmunity.

Expression of a mutant p53 results in an age-related demographic shift in spontaneous lung tumor formation in transgenic mice.

Background Mutations in the P53 gene are among the most common genetic abnormalities in human lung cancer. Codon 273 in the sequence-specific DNA binding domain is one of the most frequently mutated sites. Methodology To investigate the role of mutant p53 in lung tumorigenesis, a lung specific p53(273H) transgenic mouse model was developed. Rates of lung cancer formation in the transgenic animals and their littermates were evaluated by necropsy studies performed in progressive age cohorts ranging from 4 to 24 months. In order to establish the influence of other common genetic abnormalities in lung tumor formation in the animals, K-Ras gene mutation and p16INK4a (p16) promoter methylation were evaluated in a total of 281 transgenic mice and 189 non-transgenic littermates. Principal Findings At the age extremes of 4–12 and 22–24 months no differences were observed, with very low prevalence of tumors in animals younger than 12 months, and a relatively high prevalence at age 22 months or older. However, the transgenic mice had a significant higher lung tumor rate than their non-transgenic counterparts during the age of 13–21 months, suggesting an age-related shift in lung tumor formation induced by the lung-specific expression of the human mutant p53. Histopathology suggested a more aggressive nature for the transgenic tumors. Older mice (>13 months) had a significantly higher rate of p16 promoter methylation (17% v 82%). In addition, an age related effect was observed for K-Ras codons 12 or 13 mutations, but not for codon 61 mutations. Conclusions/Significance These results would suggest that the mutant p53(273H) contributes to an acceleration in the development of spontaneous lung tumors in these mice. Combination with other genetic and epigenetic alterations occurring after the age of 13 months is intimately linked to its oncogenic potential.