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Liver fibrosis is characterized by the persistent deposition of extracellular matrix components by hepatic stellate cell (HSC)-derived myofibroblasts. It is the histological manifestation of progressive, but reversible wound-healing processes. An unabated fibrotic response results in chronic liver disease and cirrhosis, a pathological precursor of hepatocellular carcinoma. We report here that JQ1, a small molecule inhibitor of bromodomain-containing protein 4 (BRD4), a member of bromodomain and extraterminal (BET) proteins, abrogate cytokine-induced activation of HSCs. Cistromic analyses reveal that BRD4 is highly enriched at enhancers associated with genes involved in multiple profibrotic pathways, where BRD4 is colocalized with profibrotic transcription factors. Furthermore, we show that JQ1 is not only protective, but can reverse the fibrotic response in carbon tetrachloride-induced fibrosis in mouse models. Our results implicate that BRD4 can act as a global genomic regulator to direct the fibrotic response through its coordinated regulation of myofibroblast transcription. This suggests BRD4 as a potential therapeutic target for patients with fibrotic complications.

Increased levels of intestinal bile acids (BAs) are a risk factor for colorectal cancer (CRC). Here we show that the convergence of dietary factors (high-fat diet) and dysregulated WNT signaling (APC mutation) alters BA profiles to drive malignant transformations in Lgr5-expressing (Lgr5(+)) cancer stem cells and promote an adenoma-to-adenocarcinoma progression. Mechanistically, we show that BAs that antagonize intestinal Farnesoid X receptor (FXR) function, including tauro-β-muricholic acid (T-βMCA) and deoxycholic acid (DCA), induce proliferation and DNA damage in Lgr5(+) cells. Conversely, selective activation of intestinal FXR can restrict abnormal Lgr5(+) cell growth and curtail CRC progression. This unexpected role for FXR in coordinating intestinal self-renewal with BA levels implicates FXR as a potential therapeutic target for CRC.

The tumor microenvironment plays multiple roles in tumor cell proliferation, differentiation, vascularity, and metastasis through tumor cell-stromal cell interactions. The molecular responses of tumor cells to drug treatment can modify or be modified by the molecular signaling in stromal cells. Therefore, understanding the molecular features of both tumor cells and the tumor microenvironment is crucial for understanding cancer biology and the discovery of targeted therapy. Patient-derived xenograft (PDX) tumor models contain human tumor cells growing in a mouse stromal environment and are widely used models for cancer research and drug discovery. In this study we have applied in situ hybridization to visualize gene expression in human tumor and mouse stroma by developing species-specific probes based on the RNAscope technology. Eight genes were selected for this study based on their roles in a wide range of human cancers: EGFR, ERBB2, FGF19, FGFR1, FGFR4, MET, PECAM1 and TGFB1. We designed human and mouse species-specific probes by targeting the most divergent regions ( In both CRC and liver cancer PDX tumors, human-specific probes detected strong expression of ERBB2, FGFR4, and MET in human tumor cells but no signals in mouse stromal cells. Conversely, the mouse-specific probes only detected expression of Erbb2, Fgfr4, and Met genes in mouse stromal cells. Notably, strong expression of several mouse genes, including Tgfb1, Fgfr1, Egfr, and Pecam1, were detected in the stroma surrounding the human tumor by mouse-specific probes while no human probe signals were detected in the same region. Human TGFB1, FGFR1, EGFR, and PECAM1 probes detected varied levels of gene expression in the human tumor cells from the same sample without cross-detection of the mouse ortholog sequences in the stromal region. We also observed extensive gene expression heterogeneity, both within the same tumor and between tumors. Heterogeneity of FGFR4 and FGF19 expression in the liver cancer model was particularly notable. Our results indicate that RNAscope, a single molecule RNA detection method can robustly detect the expression patterns of FGFR4-FGF19 in PDX tumors and can be utilized for selecting PDX models for mouse trials with therapeutic agents targeting FGFR4 signaling pathway. Overall the findings in this study demonstrate the successful use of RNAscope based in-situ hybridization to examine human tumor specific gene expression in mouse stromal environment in PDX animal models by visualizing gene expression in both the human tumor and mouse stroma with species-specific probes. As human tumor engrafts acquire mouse stromal cells during growth in the murine host, the method presented here will further enable the molecular dissection in PDX tumor models of tumor-host interactions involved in tumor growth, progression, and metastasis as well as responses to cancer drugs and development of drug resistance. Citation Format: Emily Park, Na Li, Katherine Ye, Mingxiao He, Mingxiao He, Zhifu Zhang, Hongzhe Sun, Xin Wang, Courtney Anderson, Yuling Luo, Zhenyu Gu, Xiao-Jun Ma. Dissecting molecular pathways in human tumor vs. mouse stromal environment in patient-derived cancer models. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B03.

Recent clinical successes with immune checkpoint blockades have provided promising immune-based therapeutic approaches for controlling malignancy. While therapeutic antibodies against CTLA-4 and PD-1/PD-L1 have resulted in potent and durable clinical responses in many patients, there still remains an urgent need to develop biomarker based assays to identify patients who may benefit from these approaches while avoiding toxicity and without compromising efficacy. While gene and protein expression of immune cell and checkpoint markers can be obtained through traditional techniques, insight regarding spatial and cell-specific expression within the tumor microenvironment is lacking, particularly in regards to secreted proteins with key immune functions. RNAscope® is an advanced in situ hybridization assay that allows for the visualization of single-cell gene expression, targeting mRNA sequences directly in tissues. The probes designed for this approach permit the detection of secreted markers, including cytokines and chemokines. In our investigation of 60 archived formalin-fixed paraffin embedded non-small cell lung cancer biopsies, expression profiles of immune checkpoint markers and immune functional molecules were evaluated in the tissue environment by RNAscope®. Each of the tumors displayed distinct co-expression patterns of immune checkpoint markers as well as varying levels of cytokines (TGFβ, IFNγ, and TNFα) and chemokines, which were measured in respect to their relative location in the tumor or stromal regions. Additionally, the detection of immune cell markers were used to pinpoint the location of infiltrating lymphocytes. The methods and findings in this study provide a valuable glimpse into the understanding of key immune functional markers in the tumor microenvironment to potentially advance treatment opportunities in this era of immuno-oncology therapies. Citation Format: Jeffrey Kim, Na Li, Mingxiao He, Bingqing Zhang, Nan Su, Xiao-Jun Ma, Emily Park. Evaluation of the expression of immune functional markers in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A24.

Heat induced target retrieval (HITR) is a widely adopted approach for Immunohistochemistry (IHC) and in situ hybridization (ISH) applications on formalin fixed and paraffin embedded (FFPE) tissues to facilitate target access and increase detection sensitivity. A variety of factors, such as fixative type, fixation time, tissue type, epitope abundance, antibody affinity, and probe length greatly affect the efficiency of HITR. Given such variability, over the decades, multiple HITR buffer formulations and protocols were invented to fit the needs in different experimental scenarios. We have developed a novel HITR solution that allows highly efficient target retrieval for RNA ISH application based on the RNAscope technology. When tested on 15 different tumor types, the new HITR solution resulted in significantly better sensitivity and/or tissue morphology comparing to using traditional HITR buffers, including Citrate, EDTA, and Tris based buffers with various pH. Furthermore, this new HITR solution is highly tolerant to sample variability. When used on multiple human tumor tissue microarrays (TMAs), including breast, colon, lung, and gastric cancers, the new HITR solution improved overall signal-to-noise ratio and reduced core disqualification rate, compared to traditional citrate based HITR buffer for ISH. Moreover, when tested on multiple mouse tissues fixed with different types of fixatives for varying periods of time (6 - 72 hours), a single experimental condition with the new HITR solution showed comparable results in both detection sensitivity and morphological integrity in all the samples. It also allowed detection of a target (Tbp) with extremely low abundance ( Given that all the RNAscope probes are synthetic oligonucleotide probes with length shorter than 60 bases, the new HITR solution, in combination with the RNAscope technology, will allow universal detection of any RNA targets with single molecule detection sensitivity in FFPE tissues with minimal requirement for analytical condition optimization. Citation Format: Li-chong Wang, Liuliu Pan, Kuang-Jung Chang, Daniel Kim, Xingyong Wu, Hongwei Wang, Casey Kernag, Bingqing Zhang, Mingxiao He, Nan Su, Xiao-Jun Ma, Yuling Luo. Developing a universal target retrieval solution and protocol for RNA in situ hybridization based on RNAscope technology. [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 4904. doi:10.1158/1538-7445.AM2015-4904