Chen-Hua Chuang

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.

Upregulation of the microRNA cluster at the Dlk1-Dio3 locus in lung adenocarcinoma

Mice in which lung epithelial cells can be induced to express an oncogenic KrasG12D develop lung adenocarcinomas in a manner analogous to humans. A myriad of genetic changes accompany lung adenocarcinomas, many of which are poorly understood. To get a comprehensive understanding of both the transcriptional and post-transcriptional changes that accompany lung adenocarcinomas, we took an omics approach in profiling both the coding genes and the non-coding small RNAs in an induced mouse model of lung adenocarcinoma. RNAseq transcriptome analysis of KrasG12D tumors from F1 hybrid mice revealed features specific to tumor samples. This includes the repression of a network of GTPase-related genes (Prkg1, Gnao1 and Rgs9) in tumor samples and an enrichment of Apobec1-mediated cytosine to uridine RNA editing. Furthermore, analysis of known single-nucleotide polymorphisms revealed not only a change in expression of Cd22 but also that its expression became allele specific in tumors. The most salient finding, however, came from small RNA sequencing of the tumor samples, which revealed that a cluster of ∼53 microRNAs and mRNAs at the Dlk1-Dio3 locus on mouse chromosome 12qF1 was markedly and consistently increased in tumors. Activation of this locus occurred specifically in sorted tumor-originating cancer cells. Interestingly, the 12qF1 RNAs were repressed in cultured KrasG12D tumor cells but reactivated when transplanted in vivo. These microRNAs have been implicated in stem cell pleuripotency and proteins targeted by these microRNAs are involved in key pathways in cancer as well as embryogenesis. Taken together, our results strongly imply that these microRNAs represent key targets in unraveling the mechanism of lung oncogenesis.

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.

A quantitative and multiplexed approach to uncover the fitness landscape of tumor suppression in vivo

Cancer growth is a multistage, stochastic evolutionary process. While cancer genome sequencing has been instrumental in identifying the genomic alterations that occur in human tumors, the consequences of these alterations on tumor growth remain largely unexplored. Conventional genetically engineered mouse models enable the study of tumor growth in vivo, but they are neither readily scalable nor sufficiently quantitative to unravel the magnitude and mode of action of many tumor-suppressor genes. Here, we present a method that integrates tumor barcoding with ultradeep barcode sequencing (Tuba-seq) to interrogate tumor-suppressor function in mouse models of human cancer. Tuba-seq uncovers genotype-dependent distributions of tumor sizes. By combining Tuba-seq with multiplexed CRISPR–Cas9-mediated genome editing, we quantified the effects of 11 tumor-suppressor pathways that are frequently altered in human lung adenocarcinoma. Tuba-seq enables the broad quantification of the function of tumor-suppressor genes with unprecedented resolution, parallelization, and precision.

An integrative approach unveils FOSL1 as an oncogene vulnerability in KRAS-driven lung and pancreatic cancer

KRAS mutated tumours represent a large fraction of human cancers, but the vast majority remains refractory to current clinical therapies. Thus, a deeper understanding of the molecular mechanisms triggered by KRAS oncogene may yield alternative therapeutic strategies. Here we report the identification of a common transcriptional signature across mutant KRAS cancers of distinct tissue origin that includes the transcription factor FOSL1. High FOSL1 expression identifies mutant KRAS lung and pancreatic cancer patients with the worst survival outcome. Furthermore, FOSL1 genetic inhibition is detrimental to both KRAS-driven tumour types. Mechanistically, FOSL1 links the KRAS oncogene to components of the mitotic machinery, a pathway previously postulated to function orthogonally to oncogenic KRAS. FOSL1 targets include AURKA, whose inhibition impairs viability of mutant KRAS cells. Lastly, combination of AURKA and MEK inhibitors induces a deleterious effect on mutant KRAS cells. Our findings unveil KRAS downstream effectors that provide opportunities to treat KRAS-driven cancers.

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.

Tumor Suppressor Activity of Selenbp1, a Direct Nkx2-1 Target, in Lung Adenocarcinoma

The Nkx2-1 transcription factor promotes differentiation of lung epithelial lineages and suppresses malignant progression of lung adenocarcinoma. However, targets of Nkx2-1 that limit tumor growth and progression remain incompletely understood. Here, direct Nkx2-1 targets are identified whose expression correlates with Nkx2-1 activity in human lung adenocarcinoma. Selenium-binding protein 1 (Selenbp1), an Nkx2-1 effector that limits phenotypes associated with lung cancer growth and metastasis, was investigated further. Loss- and gain-of-function approaches demonstrate that Nkx2-1 is required and sufficient for Selenbp1 expression in lung adenocarcinoma cells. Interestingly, Selenbp1 knockdown also reduced Nkx2-1 expression and Selenbp1 stabilized Nkx2-1 protein levels in a heterologous system, suggesting that these genes function in a positive feedback loop. Selenbp1 inhibits clonal growth and migration and suppresses growth of metastases in an in vivo transplant model. Genetic inactivation of Selenbp1, using CRISPR/Cas9, also enhanced primary tumor growth in autochthonous lung adenocarcinoma mouse models. Collectively, these data demonstrate that Selenbp1 is a direct target of Nkx2-1, which inhibits lung adenocarcinoma growth in vivo. Implications: Selenbp1 is an important suppressor of lung tumor growth that functions in a positive feedback loop with Nkx2-1, and whose loss is associated with worse patient outcome. Mol Cancer Res; 16(11); 1737–49. ©2018 AACR.

Abstract 2255: Mechanisms governing lung adenocarcinoma metastasis

Lung cancer is the leading cause of cancer deaths in men and women in the United States. Most lung cancer patients are diagnosed at a stage when they already have inoperable metastases. Identifying the drivers of lung cancer metastatic potential and the underlying mechanisms by which this state is induced, reversed or maintained is a question of immense clinical importance. Understanding the metastatic process in humans is limited, as early tumors exist in undiagnosed patients. Genetically engineered mouse models of lung adenocarcinoma provide an opportunity to study lung tumor progression and metastasis in vivo. We have recently improved upon existing lung adenocarcinoma mouse models enabling us to alter genes in developing tumors in vivo using lentiviral vectors that co-deliver Cre-recombinase and a cDNA. Additionally, the introduction of a tdTomato Cre reporter allows us to identify and isolate cells from all stages of cancer progression including primary tumors, disseminating tumor cells, circulating tumor cells, and micro-and macro-metastases. We hypothesize that differential gene expression between primary tumors and their metastases will identify key determinants of lung cancer metastatic progression. To this end, we have used RNA-Seq to identify differentially expressed genes between tumors and their metastases and validated a subset of these genes by Fluidigm® microfluidics qPCR. Our validated genes include the lysyl hydroxylase Plod2 (a collagen modifier) and the transcription factor Arntl2, both recapitulating gene expression correlations observed in human metastatic lung adenocarcinoma. Our data demonstrate that knockdown of Plod2 decreases the metastatic properties of mouse metastatic cell lines, specifically migration and the ability to give rise to metastases in transplantation assays. Migration and colony morphology defects upon knockdown of Plod2 could be rescued by the addition of normal collagen. Further in vitro experiments using soft agar assays demonstrated that an additional candidate gene, the transcription factor Arntl2, is required for anchorage independent growth of mouse metastatic cell lines. These findings were extended to in vivo transplant experiments, demonstrating that Arntl2 is critical for metastatic seeding. This in vivo phenotype was rescued by expression of a hairpin insensitive Arntl2 cDNA, demonstrating the specific role of Arntl2 in metastatic progression. Current efforts are focused on confirming the role of Arntl2 in human lung adenocarcinoma and determining the molecular mechanism of how Arntl2 modulates cancer progression and metastasis using RNA-Seq. Together, these studies will increase our knowledge of factors that mediate human lung adenocarcinoma progression with the potential to identify new targets for intervention at distinct steps of the metastatic cascade. Citation Format: Jennifer J. Brady, Chen-Hua Chuang, Deborah R. Caswell, Monte M. Winslow. Mechanisms governing lung adenocarcinoma metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2255. doi:10.1158/1538-7445.AM2015-2255

Abstract B230: Targeting mitochondria for metastatic lung adenocarcinoma specific lethality.

Introduction: Lung cancer is the leading cause of cancer deaths in the United States. Its ability to leave the primary tumor and establish inoperable metastasis impairs successful therapy and is a major contributor to its lethality. A genetically engineered mouse model recapitulates the human disease very well, incorporating conditional alleles of the Kras oncogene and p53 tumor suppressor. Recently, cell lines generated from non-metastatic primary lung tumors (TnonMet) and metastases (Met) in this model were shown to maintain their metastatic state and exhibit dramatic differences in gene expression programs. This unique set of cell lines from early stage non-metastatic tumors and late-stage metastases provides an opportunity to understand whether cancer progression also creates unique vulnerabilities. Experimental design: A pooled lentiviral-shRNA library screen which targeted ∼20,000 genes with >92,000 shRNA in two TnonMet and two Met cell lines was used to systematically uncover genes that are specifically required to sustain metastasis survival or growth. High-throughput sequencing of the shRNA in the cancer cell populations before and after 20 population doublings in culture identified shRNAs that disadvantaged each cell line. Gene-set enrichment algorithm was utilized to analyse the data. qRT-PCR, cell competition assays and mitochondria-specific dyes were used to verify the results and unravel the underlying mechanism. Results and Conclusion: The analysis of our pooled shRNA screen revealed mitochondrial ribosomal protein (Mrp) complexes as the top two gene-sets; in fact individual shRNA targeting more than 15 Mrp genes selectively disadvantaging Met cells with minimal impact on TnonMet cells. These results were individually verified with qRT-PCR. Interestingly, Met cells showed increased expression of mitochondria encoded genes, pointing to a strong imbalance in mitochondrial homeostasis. Furthermore, mitochondria-targeted therapy blocking mitochondrial translation or replication significantly inhibited Met cell growth more than TnonMet cell growth. Taken together, these results point to mitochondria as a metastasis-specific therapeutic target in lung adenocarcinoma and need to be further investigated. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B230. Citation Format: Barbara M. Gruener, Chen-Hua Chuang, Ian Linde, Shin-Heng Chiou, Ben Readhead, Joel Dudley, John Doench, David Root, Monte M. Winslow. Targeting mitochondria for metastatic lung adenocarcinoma specific lethality. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B230.