Chang-Il Hwang

Organoid Models of Human and Mouse Ductal Pancreatic Cancer

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

Frequent Downregulation of miR-34 Family in Human Ovarian Cancers

Purpose: The miR-34 family is directly transactivated by tumor suppressor p53, which is frequently mutated in human epithelial ovarian cancer (EOC). We hypothesized that miR-34 expression would be decreased in EOC and that reconstituted miR-34 expression might reduce cell proliferation and invasion of EOC cells. Experimental Designs: miR-34 expression was determined by quantitative reverse transcription-PCR and in situ hybridization in a panel of 83 human EOC samples. Functional characterization of miR-34 was accomplished by reconstitution of miR-34 expression in EOC cells with synthetic pre-miR molecules followed by determining changes in proliferation, apoptosis, and invasion. Results: miR-34a expression is decreased in 100%, and miR-34b*/c in 72%, of EOC with p53 mutation, whereas miR-34a is also downregulated in 93% of tumors with wild-type p53. Furthermore, expression of miR-34b*/c is significantly reduced in stage IV tumors compared with stage III (P = 0.0171 and P = 0.0029, respectively). Additionally, we observed promoter methylation and copy number variations at mir-34. In situ hybridization showed that miR-34a expression is inversely correlated with MET immunohistochemical staining, consistent with translational inhibition by miR-34a. Finally, miR-34 reconstitution experiments in p53 mutant EOC cells resulted in reduced proliferation, motility, and invasion, the latter of which was dependent on MET expression. Conclusions: Our work suggests that miR-34 family plays an important role in EOC pathogenesis and reduced expression of miR-34b*/c may be particularly important for progression to the most advanced stages. Part of miR-34 effects on motility and invasion may be explained by regulation of MET, which is frequently overexpressed in EOC. Clin Cancer Res; 16(4); 1119–28

Ovarian surface epithelium at the junction area contains a cancer-prone stem cell niche

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer deaths among women in the United States, but its pathogenesis is poorly understood. Some epithelial cancers are known to occur in transitional zones between two types of epithelium, whereas others have been shown to originate in epithelial tissue stem cells. The stem cell niche of the ovarian surface epithelium (OSE), which is ruptured and regenerates during ovulation, has not yet been defined unequivocally. Here we identify the hilum region of the mouse ovary, the transitional (or junction) area between the OSE, mesothelium and tubal (oviductal) epithelium, as a previously unrecognized stem cell niche of the OSE. We find that cells of the hilum OSE are cycling slowly and express stem and/or progenitor cell markers ALDH1, LGR5, LEF1, CD133 and CK6B. These cells display long-term stem cell properties ex vivo and in vivo, as shown by our serial sphere generation and long-term lineage-tracing assays. Importantly, the hilum cells show increased transformation potential after inactivation of tumour suppressor genes Trp53 and Rb1, whose pathways are altered frequently in the most aggressive and common type of human EOC, high-grade serous adenocarcinoma. Our study supports experimentally the idea that susceptibility of transitional zones to malignant transformation may be explained by the presence of stem cell niches in those areas. Identification of a stem cell niche for the OSE may have important implications for understanding EOC pathogenesis.

Identification of radiation-specific responses from gene expression profile.

The responses to ionizing radiation (IR) in tumors are dependent on cellular context. We investigated radiation-related expression patterns in Jurkat T cells with nonsense mutation in p53 using cDNA microarray. Expression of 2400 genes in γ-irradiated cells was distinct from other stimulations like anti-CD3, phetohemagglutinin (PHA) and concanavalin A (ConA) in unsupervised clustering analysis. Among them, 384 genes were selected for their IR-specific changes to make ‘RadChip’. In spite of p53 status, every type of cells showed similar patterns in expression of these genes upon γ-radiation. Moreover, radiation-induced responses were clearly separated from the responses to other genotoxic stress like UV radiation, cisplatin and doxorubicin. We focused on two IR-related genes, phospholipase Cγ2 (PLCG2) and cytosolic epoxide hydrolase (EPHX2), which were increased at 12 h after γ-radiation in RT–PCR. TPCK could suppress the induction of these two genes in either of Jurkat T cells and PBMCs, which might suggest the transcriptional regulation of PLCG2 and EPHX2 by NF-κB upon γ-radiation. From these results, we could identify the IR-specific genes from expression profiling, which can be used as radiation biomarkers to screen radiation exposure as well as probing the mechanism of cellular responses to ionizing radiation.

Wild-type p53 controls cell motility and invasion by dual regulation of MET expression

Recent observations suggest that p53 mutations are responsible not only for growth of primary tumors but also for their dissemination. However, mechanisms involved in p53-mediated control of cell motility and invasion remain poorly understood. By using the primary ovarian surface epithelium cell culture, we show that conditional inactivation of p53 or expression of its mutant forms results in overexpression of MET receptor tyrosine kinase, a crucial regulator of invasive growth. At the same time, cells acquire increased MET-dependent motility and invasion. Wild-type p53 negatively regulates MET expression by two mechanisms: (i) transactivation of MET-targeting miR-34, and (ii) inhibition of SP1 binding to MET promoter. Both mechanisms are not functional in p53 absence, but mutant p53 proteins retain partial MET promoter suppression. Accordingly, MET overexpression, cell motility, and invasion are particularly high in p53-null cells. These results identify MET as a critical effector of p53 and suggest that inhibition of MET may be an effective antimetastatic approach to treat cancers with p53 mutations. These results also show that the extent of advanced cancer traits, such as invasion, may be determined by alterations in individual components of p53/MET regulatory network.

Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer

Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of &agr;-smooth muscle actin (&agr;SMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated &agr;SMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development.

FOXO3a Turns the Tumor Necrosis Factor Receptor Signaling Towards Apoptosis Through Reciprocal Regulation of c-Jun N-Terminal Kinase and NF-κB

Objective—We evaluated the full range effects of FOXO3a in endothelial cells (ECs) by microarray analysis and investigated the role of FOXO3a regulating TNF receptor signaling pathway. Methods and Results—Human umbilical vein endothelial cells (HUVECs) were transfected with adenoviral vectors expressing constitutively active FOXO3a (Ad-TM-FOXO3a). Ad-TM-FOXO3a transfection caused remarkable apoptosis, which were accompanied with upregulation of genes related with TNF receptor signaling, such as TNF-α, TANK (TRAF-associated NF-&kgr;B activator), and TTRAP (TRAF and TNF receptor-associated protein). Furthermore, &kgr;B-Ras1 (I&kgr;B-interacting Ras-like protein-1) which is known to block I&kgr;B degradation was found increased, and intranuclear translocation of NF-&kgr;B was inhibited. GADD45β and XIAP, negative regulators of c-Jun N-terminal kinase (JNK), were suppressed and JNK activity was increased. Attenuation of TNF signaling pathway either by blocking antibody for TNF receptor or by blocking JNK with DMAP (6-dimethylaminopurine) or Ad-TAM67 (dominant negative c-Jun) cotransfection, significantly reduced FOXO3a-induced apoptosis. Finally, treatment of vasculature with heat shock, an activator of endogenous FOXO3a, resulted in EC apoptosis, which was completely rescued by Ad-TAM67. Conclusion—FOXO3a promotes apoptosis of ECs, through activation of JNK and suppression of NF-&kgr;B. These data identify a novel role of FOXO3a to turn TNF receptor signaling to a proapoptotic JNK-dependent pathway.

Adenovirus-TRAIL can overcome TRAIL resistance and induce a bystander effect

TRAIL is a cytokine with a unique ability to induce apoptosis selectively in many transformed cell lines. The instability of TRAIL and the resistance of some cancer cells to TRAIL present the main obstacles for clinical experimentation. We generated an adenovirus expressing full-length TRAIL and tested its efficacy in several cancer cell lines. Ad-TRAIL-infected cancer cells localized full-length TRAIL protein to the cytoplasm and released same-sized TRAIL in the media. Ad-TRAIL was found to induce apoptotic cell death in several cancer cell lines resistant to soluble TRAIL (A549, SKOV3, HT-29 and LNCap) and in TRAIL-sensitive cell lines. Ad-TRAIL, but not soluble TRAIL, induced apoptotic cell death in TRAIL-resistant cell lines, manifested by an increased sub-G1 proportion, caspase-3 activation and PARP cleavage. Ad-TRAIL also induced a media-transferable bystander effect, but only in soluble TRAIL-sensitive cell lines. In conclusion, two novel characteristics of ad-TRAIL were found during this study. First, that ad-TRAIL can induce apoptotic cell death in several cancer cell lines resistant to sTRAIL. Second, that ad-TRAIL induces a media-transferable bystander effect, which is expected to increase its therapeutic value by allowing TRAIL to overcome the locally acting nature and low transduction rate commonly encountered in clinical situation.

Caveolin-1 upregulation in senescent neurons alters amyloid precursor protein processing.

Lipid rafts provide a platform for regulating cellular functions and participate in the pathogenesis of several diseases. However, the role of caveolin-1 in this process has not been elucidated definitely in neuron. Thus, this study was performed to examine whether caveolin-1 can regulate amyloid precursor protein (APP) processing in neuronal cells and to identify the molecular mechanisms involved in this regulation. Caveolin-1 is up-regulated in all parts of old rat brain, namely hippocampus, cerebral cortex and in elderly human cerebral cortex. Moreover, detergent-insoluble glycolipid (DIG) fractions indicated that caveolin-1 was co-localized with APP in caveolae-like structures. In DIG fractions, bAPP secretion was up-regulated by caveolin-1 over-expression, which was modulated via protein kinase C (PKC) in neuroblastoma cells. From these results we conclude that caveolin-1 is selectively expressed in senescent neurons and that it induces the processing of APP by β-secretase via PKC downregulation.

Preclinical models of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is one of the most difficult human malignancies to treat. The 5‐year survival rate of PDA patients is 7% and PDA is predicted to become the second leading cancer‐related cause of death in the USA. Despite intensive efforts, the translation of findings in preclinical studies has been ineffective, due partially to the lack of preclinical models that faithfully recapitulate features of human PDA. Here, we review current preclinical models for human PDA (eg human PDA cell lines, cell line‐based xenografts and patient‐derived tumour xenografts). In addition, we discuss potential applications of the recently developed pancreatic ductal organoids, three‐dimensional culture systems and organoid‐based xenografts as new preclinical models for PDA. Copyright