Faculty Bibliography

In cutaneous T cell lymphomas (CTCL), miR-21 is aberrantly expressed in skin and peripheral blood and displays anti-apoptotic properties in malignant T cells. It is, however, unclear exactly which cells express miR-21 and what mechanisms regulate miR-21. Here, we demonstrate miR-21 expression in situ in both malignant and reactive lymphocytes as well as stromal cells. qRT-PCR analysis of 47 patients with mycosis fungoides (MF) and Sezary Syndrome (SS) confirmed an increased miR-21 expression that correlated with progressive disease. In cultured malignant T cells miR-21 expression was inhibited by Tofacitinib (CP-690550), a clinical-grade JAK3 inhibitor. Chromatin immunoprecipitation (ChIP) analysis showed direct binding of STAT5 to the miR-21 promoter. Cytokine starvation ex vivo triggered a decrease in miR-21 expression, whereas IL-2 induced an increased miR-21 expression in primary SS T cells and cultured cytokine-dependent SS cells (SeAx). siRNA-mediated depletion of STAT5 inhibited constitutive- and IL-2-induced miR-21 expression in cytokine-independent and dependent T cell lines, respectively. IL-15 and IL-2 were more potent than IL-21 in inducing miR-21 expression in the cytokine-dependent T cells. In conclusion, we provide first evidence that miR-21 is expressed in situ in CTCL skin lesions, induced by IL-2 and IL-15 cytokines, and is regulated by STAT5 in malignant T cells. Thus, our data provide novel evidence for a pathological role of IL-2Rg cytokines in promoting expression of the oncogenic miR-21 in CTCL.

There is ample evidence that somatic cell differentiation during development is accompanied by extensive DNA demethylation of specific sites that vary between cell types. Although the mechanism of this process has not yet been elucidated, it is likely to involve the conversion of 5mC to 5hmC by Tet enzymes. We show that a Tet2/Tet3 conditional knockout at early stages of B-cell development largely prevents lineage-specific programmed demethylation events. This lack of demethylation affects the expression of nearby B-cell lineage genes by impairing enhancer activity, thus causing defects in B-cell differentiation and function. Thus, tissue-specific DNA demethylation appears to be necessary for proper somatic cell development in vivo.

Microaspiration is a common phenomenon in healthy subjects, but its frequency is increased in chronic inflammatory airway diseases, and its role in inflammatory and immune phenotypes is unclear. We have previously demonstrated that acellular bronchoalveolar lavage samples from half of the healthy people examined are enriched with oral taxa (here called pneumotypeSPT) and this finding is associated with increased numbers of lymphocytes and neutrophils in bronchoalveolar lavage. Here, we have characterized the inflammatory phenotype using a multi-omic approach. By evaluating both upper airway and acellular bronchoalveolar lavage samples from 49 subjects from three cohorts without known pulmonary disease, we observed that pneumotypeSPT was associated with a distinct metabolic profile, enhanced expression of inflammatory cytokines, a pro-inflammatory phenotype characterized by elevated Th-17 lymphocytes and, conversely, a blunted alveolar macrophage TLR4 response. The cellular immune responses observed in the lower airways of humans with pneumotypeSPT indicate a role for the aspiration-derived microbiota in regulating the basal inflammatory status at the pulmonary mucosal surface.

Cutaneous T cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die from infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin-A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of STAT3 and up-regulation of IL-17 in immortalized and primary patient-derived malignant and non-malignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when co-cultured with non-malignant T cells indicating an indirect mode of action. In accordance, malignant T cells expressing a SEA non-responsive T cell receptor V beta chain (TCR-Vb) are non-responsive to SEA in mono-culture, but display strong STAT3 activation and IL-17 expression in co-cultures with SEA-responsive, non-malignant T cells. The response is induced via IL-2Rg cytokines and a Janus kinase 3 (JAK3) - dependent pathway in malignant T cells and blocked by Tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross-talk-dependent activation of STAT3 and expression of IL-17 in malignant T cells suggesting a mechanism where SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis.

A salient feature of pancreatic ductal adenocarcinoma (PDA) is an abundant fibroinflammatory response characterized by the recruitment of immune and mesenchymal cells and the consequent establishment of a pro-tumorigenic microenvironment. Here we report the prominent presence of B cells in human pancreatic intraepithelial neoplasia (PanIN) and PDA lesions as well as in oncogenic K-Ras-driven pancreatic neoplasms in the mouse. The growth of orthotopic pancreatic neoplasms harboring oncogenic K-Ras was significantly compromised in B cell-deficient mice (muMT), and this growth deficiency could be rescued by the reconstitution of a CD1dhighCD5+ B cell subset. The pro-tumorigenic effect of B cells was mediated by their expression of IL-35 through a mechanism involving IL-35-mediated stimulation of tumor cell proliferation. Our results identify a previously unrecognized role for IL-35-producing CD1dhighCD5+ B cells in the pathogenesis of pancreatic cancer and underscore the potential significance of a B cell/IL-35 axis as a therapeutic target.

Breast cancer (BC) is associated with alterations in a number of growth factor and hormone-regulated signaling pathways. Mouse models of metastatic BC typically feature mutated oncoproteins that activate PI3K, Stat3, and Ras signaling, but the individual and combined roles of these pathways in BC progression are poorly understood. In this study, we examined the relationship between oncogenic pathway activation and breast cancer subtype by analyzing mouse mammary tumor formation in which each pathway was activated singly or pairwise. All three oncogenes showed cooperation during primary tumor formation, but efficient dissemination was only dependent on Ras. In addition, transcriptional profiling demonstrated that Ras induced adenocarcinomas with molecular characteristics related to human basal-like and HER2+ tumors. In contrast, Ras combined with PIK3CAH1047R, an oncogenic mutant linked to ERalpha+/luminal BC in humans, induced metastatic luminal B-like tumors. Consistent with these data, elevated Ras signaling was associated with basal-like and HER2+ subtype tumors in humans, and showed a statistically significant negative association with ER-signaling across all BC. Despite this, there are luminal tumors with elevated Ras signaling. Importantly, when considered as a continuous variable, Ras pathway activation was strongly linked to reduced survival of patients with ERalpha+ disease independent of PI3K or Stat3 activation. Therefore, our studies suggest that Ras activation is a key determinant for dissemination and poor prognosis of ERalpha+/luminal BC in humans, and hormone therapy supplemented with Ras-targeting agents may be beneficial for treating this aggressive subtype.