Shin-Heng Chiou

Up-regulation of Inhibitory Natural Killer Receptors CD94/NKG2A with Suppressed Intracellular Perforin Expression of Tumor-Infiltrating CD8+ T Lymphocytes in Human Cervical Carcinoma

Inhibitory signals that govern the cytolytic functions of CD8(+) T lymphocytes have been linked to the expression of natural killer cell receptors (NKRs) on CTLs. There is limited knowledge about the induction of inhibitory NKR (iNKR) expression in vivo. Up-regulation of iNKRs has been linked to the modulation of the virus- and/or tumor-specific immune responses in animal models. In the present study, we directly examined the expression of various NKRs on tumor-infiltrating lymphocytes (TILs) derived from human cervical cancer. We found that in human cervical cancer, the percentage expression of immunoglobulin-like NKR(+)CD8(+) T lymphocytes were similar in gated CD8(+)-autologous TILs and peripheral blood mononuclear cells. On the contrary, cervical cancer-infiltrating CD8(+) T lymphocytes expressed up-regulated C-type lectin NKRs CD94/NKG2A compared with either peripheral blood CD8(+) T cells or normal cervix-infiltrating CD8(+) T lymphocytes. Dual NKR coexpression analyses showed that CD94 and NKG2A were mainly expressed on CD56(-)CD161(-)CD8(+) TILs within the cancer milieu. Immunohistochemical study showed that cervical cancer cells expressed abundant interleukin 15 (IL-15) and transforming growth factor-beta (TGF-beta). In kinetic coculture assay, cervical cancer cells can promote the expression of CD94/NKG2A on CD8(+) T lymphocytes. The cancer-derived effects can be reversed by addition of rIL-15Ralpha/Fc and anti-TGF-beta antibody. Functional analyses illustrated that intracellular perforin expression of CD8(+) T cells was minimal upon up-regulation of CD94/NKG2A. Kinetic cytotoxicity assays showed that up-regulated expressions of CD94/NKG2A restrain CD8(+) T lymphocyte cytotoxicity. Our study strongly indicated that cervical cancer cells could promote the expression of iNKRs via an IL-15- and possibly TGF-beta-mediated mechanism and abrogate the antitumor cytotoxicity of TILs.

Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing

Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.

Obligate progression precedes lung adenocarcinoma dissemination.

UNLABELLED Despite its clinical importance, very little is known about the natural history and molecular underpinnings of lung cancer dissemination and metastasis. Here, we used a genetically engineered mouse model of metastatic lung adenocarcinoma in which cancer cells are fluorescently marked to determine whether dissemination is an inherent ability or a major acquired phenotype during lung adenocarcinoma metastasis. We find very little evidence for dissemination from oncogenic KRAS-driven hyperplasias or most adenocarcinomas. p53 loss is insufficient to drive dissemination but rather enables rare cancer cells in a small fraction of primary adenocarcinomas to gain alterations that drive dissemination. Molecular characterization of disseminated tumor cells indicates that downregulation of the transcription factor Nkx2-1 precedes dissemination. Finally, we show that metastatic primary tumors possess a highly proliferative subpopulation of cells with characteristics matching those of disseminating cells. We propose that dissemination is a major hurdle during the natural course of lung adenocarcinoma metastasis. SIGNIFICANCE Because of its aggressively metastatic nature, lung cancer is the top cancer killer of both men and women in the United States. We show that, unlike in other cancer types, lung cancer dissemination is a major initial barrier to metastasis. Our findings provide insight into the effect of p53 deficiency and downregulation of Nkx2-1 during lung adenocarcinoma progression.

Gpr124 is essential for blood–brain barrier integrity in central nervous system disease

Although blood–brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt–β-catenin signaling. Constitutive activation of Wnt–β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

Molecular definition of a metastatic lung cancer state reveals a targetable CD109-Janus kinase-Stat axis

Lung cancer is the leading cause of cancer deaths worldwide, with the majority of mortality resulting from metastatic spread. However, the molecular mechanism by which cancer cells acquire the ability to disseminate from primary tumors, seed distant organs, and grow into tissue-destructive metastases remains incompletely understood. We combined tumor barcoding in a mouse model of human lung adenocarcinoma with unbiased genomic approaches to identify a transcriptional program that confers metastatic ability and predicts patient survival. Small-scale in vivo screening identified several genes, including Cd109, that encode novel pro-metastatic factors. We uncovered signaling mediated by Janus kinases (Jaks) and the transcription factor Stat3 as a critical, pharmacologically targetable effector of CD109-driven lung cancer metastasis. In summary, by coupling the systematic genomic analysis of purified cancer cells in distinct malignant states from mouse models with extensive human validation, we uncovered several key regulators of metastatic ability, including an actionable pro-metastatic CD109–Jak–Stat3 axis.

BLIMP1 Induces Transient Metastatic Heterogeneity in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. By generating a genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor BLIMP1 as a driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes, and hypoxia-mediated induction of BLIMP1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which upregulation of BLIMP1 links microenvironmental cues to a metastatic stem cell character.Significance: PDAC is an almost uniformly lethal cancer, largely due to its tendency for metastasis. We define a highly metastatic subpopulation of cancer cells, uncover a key transcriptional regulator of metastatic ability, and define hypoxia as an important factor within the tumor microenvironment that increases metastatic proclivity. Cancer Discov; 7(10); 1184-99. ©2017 AACR.See related commentary by Vakoc and Tuveson, p. 1067This article is highlighted in the In This Issue feature, p. 1047.

An in vivo multiplexed small-molecule screening platform

Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo.

The E3 ligase c‐Cbl regulates dendritic cell activation

The activation of innate and adaptive immunity is always balanced by inhibitory signalling mechanisms to maintain tissue integrity. We have identified the E3 ligase c‐Cbl––known for its roles in regulating lymphocyte signalling––as a modulator of dendritic cell activation. In c‐Cbl‐deficient dendritic cells, Toll‐like receptor‐induced expression of proinflammatory factors, such as interleukin‐12, is increased, correlating with a greater potency of dendritic‐cell‐based vaccines against established tumours. This proinflammatory phenotype is accompanied by an increase in nuclear factor (NF)‐κB activity. In addition, c‐Cbl deficiency reduces both p50 and p105 levels, which have been shown to modulate the stimulatory function of NF‐κB. Our data indicate that c‐Cbl has a crucial, RING‐domain‐dependent role in regulating dendritic cell maturation, probably by facilitating the regulatory function of p105 and/or p50.

A Conditional System to Specifically Link Disruption of Protein-Coding Function with Reporter Expression in Mice

Conditional gene deletion in mice has contributed immensely to our understanding of many biological and biomedical processes. Despite an increasing awareness of nonprotein-coding functional elements within protein-coding transcripts, current gene-targeting approaches typically involve simultaneous ablation of noncoding elements within targeted protein-coding genes. The potential for protein-coding genes to have additional noncoding functions necessitates the development of novel genetic tools capable of precisely interrogating individual functional elements. We present a strategy that couples Cre/loxP-mediated conditional gene disruption with faithful GFP reporter expression in mice in which Cre-mediated stable inversion of a splice acceptor-GFP-splice donor cassette concurrently disrupts protein production and creates a GFP fusion product. Importantly, cassette inversion maintains physiologic transcript structure, thereby ensuring proper microRNA-mediated regulation of the GFP reporter, as well as maintaining expression of nonprotein-coding elements. To test this potentially generalizable strategy, we generated and analyzed mice with this conditional knockin reporter targeted to the Hmga2 locus.

Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity

Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded KrasHDR alleles from bulk lung and pancreas reveals surprising diversity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis.Genome editing technologies enable the rapid interrogation of genetic alterations. Here, the authors present a CRISPR/Cas9-based platform to simultaneously investigate multiple activating point mutations in de novo cancers in mice; and generate panels of Kras-variants in different tissues to induce cancer.