Yi-Chieh Nancy Du

Seeding and propagation of untransformed mouse mammary cells in the lung.

The acquisition of metastatic ability by tumor cells is considered a late event in the evolution of malignant tumors. We report that untransformed mouse mammary cells that have been engineered to express the inducible oncogenic transgenes MYC and KrasD12, or polyoma middle T, and introduced into the systemic circulation of a mouse can bypass transformation at the primary site and develop into metastatic pulmonary lesions upon immediate or delayed oncogene induction. Therefore, previously untransformed mammary cells may establish residence in the lung once they have entered the bloodstream and may assume malignant growth upon oncogene activation. Mammary cells lacking oncogenic transgenes displayed a similar capacity for long-term residence in the lungs but did not form ectopic tumors.

Yph1p, an ORC-interacting protein: potential links between cell proliferation control, DNA replication, and ribosome biogenesis.

Immunoprecipitation of the origin recognition complex (ORC) from yeast extracts identified Yph1p, an essential protein containing a BRCT domain. Two Yph1p complexes were characterized. Besides ORC, MCM proteins, cell-cycle regulatory proteins, checkpoint proteins, 60S ribosomal proteins, and preribosome particle proteins were found to be associated with Yph1p. Yph1p is predominantly nucleolar and is required for 60S ribosomal subunit biogenesis and possibly for translation on polysomes. Proliferating cells depleted of Yph1p arrest in G(1) or G(2), with no cells in S phase, or significantly delay S phase progression after release from a hydroxyurea arrest. Yph1p levels decline as cells commit to exit the cell cycle, and levels vary depending on energy source. Yph1p may link cell proliferation control to DNA replication, ribosome biogenesis, and translation on polysomes.

Assessing Tumor Progression Factors by Somatic Gene Transfer into a Mouse Model: Bcl-xL Promotes Islet Tumor Cell Invasion

Tumors develop through multiple stages, implicating multiple effectors, but the tools to assess how candidate genes contribute to stepwise tumor progression have been limited. We have developed a novel system in which progression of phenotypes in a mouse model of pancreatic islet cell tumorigenesis can be used to measure the effects of genes introduced by cell-type-specific infection with retroviral vectors. In this system, bitransgenic mice, in which the rat insulin promoter (RIP) drives expression of both the SV40 T antigen (RIP-Tag) and the receptor for subgroup A avian leukosis virus (RIP-tva), are infected with avian viral vectors carrying cDNAs encoding candidate progression factors. Like RIP-Tag mice, RIP-Tag; RIP-tva bitransgenic mice develop isolated carcinomas by ∼14 wk of age, after progression through well-defined stages that are similar to aspects of human tumor progression, including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. When avian retroviral vectors carrying a green fluorescent protein marker were introduced into RIP-Tag; RIP-tva mice by intra-cardiac injection at the hyperplastic or early dysplastic stage of tumorigenesis, approximately 20% of the TVA-positive cells were infected and expressed green fluorescent proteins as measured by flow cytometry. Similar infection with vectors carrying cDNA encoding either of two progression factors, a dominant-negative version of cadherin 1 (dnE-cad) or Bcl-xL, accelerated the formation of islet tumors with invasive properties and pancreatic lymph node metastasis. To begin studying the mechanism by which Bcl-xL, an anti-apoptotic protein, promotes invasion and metastasis, RIP-Tag; RIP-tva pancreatic islet tumor cells were infected in vitro with RCASBP-Bcl-xL. Although no changes were observed in rates of proliferation or apoptosis, Bcl-xL altered cell morphology, remodeled the actin cytoskeleton, and down-regulated cadherin 1; it also induced cell migration and invasion, as evaluated using two-chamber transwell assays. In addition, myosin Va was identified as a novel Bcl-xL-interacting protein that might mediate the effects of Bcl-xL on tumor cell migration and invasion.

Attenuation of the Retinoblastoma Pathway in Pancreatic Neuroendocrine Tumors Due to Increased Cdk4/Cdk6

Purpose: In mice, genetic changes that inactivate the retinoblastoma tumor suppressor pathway often result in pancreatic neuroendocrine tumors (Pan-NETs). Conversely, in humans with this disease, mutations in genes of the retinoblastoma pathway have rarely been detected, even in genome-wide sequencing studies. In this study, we took a closer look at the role of the retinoblastoma pathway in human Pan-NETs. Experimental Design: Pan-NET tumors from 92 patients were subjected to immunohistochemical staining for markers of the retinoblastoma pathway. To search for amplifications of retinoblastoma pathway genes, genomic DNAs from 26 tumors were subjected to copy number analysis. Finally, a small-molecule activator of the retinoblastoma pathway was tested for effects on the growth of two Pan-NET cell lines. Results: A majority of tumors expressed high amounts of Cdk4 or its partner protein cyclin D1. High amounts of phosphorylated Rb1 were present in tumors that expressed high levels of Cdk4 or cyclin D1. The copy numbers of Cdk4 or the analogous kinase gene Cdk6 were increased in 19% of the tumors. Growth of the human Pan-NET cell line QGP1 was inhibited in a xenograft mouse model by the Cdk4/6 inhibitor, PD 0332991, which reactivates the retinoblastoma pathway. Conclusions: Inactivation of the retinoblastoma pathway was indicated for most Pan-NETs. Gene amplification and overexpression of Cdk4 and Cdk6 suggests that patients with Pan-NETs may respond strongly to Cdk4/6 inhibitors that are entering clinical trials. Clin Cancer Res; 18(17); 4612–20. ©2012 AACR.

Defining the ATM-mediated barrier to tumorigenesis in somatic mammary cells following ErbB2 activation

p53, apoptosis, and senescence are frequently activated in preneoplastic lesions and are barriers to progression to malignancy. These barriers have been suggested to result from an ATM-mediated DNA damage response (DDR), which may follow oncogene-induced hyperproliferation and ensuing DNA replication stress. To elucidate the currently untested role of DDR in breast cancer initiation, we examined the effect of oncogene expression in several murine models of breast cancer. We did not observe a detectable DDR in early hyperplastic lesions arising in transgenic mice expressing several different oncogenes. However, DDR signaling was strongly induced in preneoplastic lesions arising from individual mammary cells transduced in vivo by retroviruses expressing either PyMT or ErbB2. Thus, activation of an oncogene after normal tissue development causes a DDR. Furthermore, in this somatic ErbB2 tumor model, ATM, and thus DDR, is required for p53 stabilization, apoptosis, and senescence. In palpable tumors in this model, p53 stabilization and apoptosis are lost, but unexpectedly senescence remains in many tumor cells. Thus, this murine model fully recapitulates early DDR signaling; the eventual suppression of its endpoints in tumorigenesis provides compelling evidence that ErbB2-induced aberrant mammary cell proliferation leads to an ATM-mediated DDR that activates apoptosis and senescence, and at least the former must be overcome to progress to malignancy. This in vivo study also uncovers an unexpected effect of ErbB2 activation previously known for its prosurvival roles, and suggests that protection of the ATM-mediated DDR-p53 signaling pathway may be important in breast cancer prevention.

Receptor for hyaluronan-mediated motility isoform B promotes liver metastasis in a mouse model of multistep tumorigenesis and a tail vein assay for metastasis

The gene encoding the receptor for hyaluronan-mediated motility (RHAMM) is overexpressed in many human cancers. However, it is unclear whether RHAMM plays a causal role in tumor initiation or progression. Using somatic gene transfer in a mouse model of islet cell tumorigenesis, we demonstrate that RHAMM isoform B (RHAMMB) promotes tumor growth and metastases to lymph nodes and the liver. The propensity of RHAMMB-expressing cells to metastasize to the liver was confirmed using an experimental metastasis assay in which cells were injected into the tail vein of immunodeficient mice. However, RHAMMB did not increase cell migration or proliferation in culture. In initial efforts to identify signaling pathways activated by RHAMMB, we found that RHAMMB induced phosphorylation of epidermal growth factor receptor (EGFR), Erk1/2, and STAT3 and conferred susceptibility to apoptosis after treatment with an EGFR inhibitor, gefitinib. Taken together, the results indicate that RHAMMB promotes hepatic metastasis by islet tumor cells, perhaps through growth factor receptor-mediated signaling.

Bcl-xL promotes metastasis independent of its anti-apoptotic activity

Bcl-xL suppresses mitochondria-mediated apoptosis and is frequently overexpressed in cancer to promote cancer cell survival. Bcl-xL also promotes metastasis. However, it is unclear whether this metastatic function is dependent on its anti-apoptotic activity in the mitochondria. Here we demonstrate that Bcl-xL promotes metastasis independent of its anti-apoptotic activity. We show that apoptosis-defective Bcl-xL mutants and an engineered Bcl-xL targeted to the nucleus promote epithelial–mesenchymal transition, migration, invasion and stemness in pancreatic neuroendocrine tumour (panNET) and breast cancer cell lines. However, Bcl-xL proteins targeted to the mitochondria or outside of the nucleus do not have these functions. We confirm our findings in spontaneous and xenograft mouse models. Furthermore, Bcl-xL exerts metastatic function through epigenetic modification of the TGFβ promoter to increase TGFβ signalling. Consistent with these findings, we detect nuclear Bcl-xL in human metastatic panNETs. Taken together, the metastatic function of Bcl-xL is independent of its anti-apoptotic activity and its residence in the mitochondria.

Activation of PyMT in β Cells Induces Irreversible Hyperplasia, but Oncogene-Dependent Acinar Cell Carcinomas When Activated in Pancreatic Progenitors

It is unclear whether the cellular origin of various forms of pancreatic cancer involves transformation or transdifferentiation of different target cells or whether tumors arise from common precursors, with tumor types determined by the specific genetic alterations. Previous studies suggested that pancreatic ductal carcinomas might be induced by polyoma middle T antigen (PyMT) expressed in non-ductal cells. To ask whether PyMT transforms and transdifferentiates endocrine cells toward exocrine tumor phenotypes, we generated transgenic mice that carry tetracycline-inducible PyMT and a linked luciferase reporter. Induction of PyMT in β cells causes β-cell hyperplastic lesions that do not progress to malignant neoplasms. When PyMT is de-induced, β cell proliferation and growth cease; however, regression does not occur, suggesting that continued production of PyMT is not required to maintain the viable expanded β cell population. In contrast, induction of PyMT in early pancreatic progenitor cells under the control of Pdx1 produces acinar cell carcinomas and β-cell hyperplasia. The survival of acinar tumor cells is dependent on continued expression of PyMT. Our findings indicate that PyMT can induce exocrine tumors from pancreatic progenitor cells, but cells in the β cell lineage are not transdifferentiated toward exocrine cell types by PyMT; instead, they undergo oncogene-dependent hyperplastic growth, but do not require PyMT for survival.

Expression of the receptor for hyaluronic acid mediated motility (RHAMM) is associated with poor prognosis and metastasis in non-small cell lung carcinoma

The receptor for hyaluronic acid-mediated motility (RHAMM) is upregulated in various cancers, but its role in primary and metastatic non-small cell lung carcinoma (NSCLC) remains to be determined. Here, we investigate the clinical relevance of RHAMM expression in NSCLC. RHAMM protein expression correlates with histological differentiation stages and extent of the primary tumor (T stages) in 156 patients with primary NSCLC. Importantly, while focal RHAMM staining pattern is present in 57% of primary NSCLC, intense RHAMM protein expression is present in 96% of metastatic NSCLC cases. In a publicly available database, The Cancer Genome Atlas (TCGA), RHAMM mRNA expression is 12- and 10-fold higher in lung adenocarcinoma and squamous lung carcinoma than in matched normal lung tissues, respectively. RHAMM mRNA expression correlates with stages of differentiation and inferior survival in more than 400 cases of lung adenocarcinoma in the Directors Challenge cohort. Of 4 RHAMM splice variants, RHAMMv3 (also known as RHAMMB) is the dominant variant in NSCLC. Moreover, shRNA-mediated knockdown of RHAMM reduced the migratory ability of two lung adenocarcinoma cell lines, H1975 and H3255. Taken together, RHAMM, most likely RHAMMv3 (RHAMMB), can serve as a prognostic factor for lung adenocarcinomas and a potential therapeutic target in NSCLC to inhibit tumor migration.

p53 and p16Ink4a/p19Arf Loss Promotes Different Pancreatic Tumor Types from PyMT-Expressing Progenitor Cells

In human studies and mouse models, the contributions of p53 and p16Ink4a/p19Arf loss are well established in pancreatic ductal adenocarcinoma (PDAC). Although loss of functional p53 pathway and loss of Ink4a/Arf in human pancreatic acinar cell carcinoma (PACC) and pancreatic neuroendocrine tumor (PanNET) are identified, their direct roles in tumorigenesis of PACC and PanNET remain to be determined. Using transgenic mouse models expressing the viral oncogene polyoma middle T antigen (PyMT), we demonstrate that p53 loss in pancreatic Pdx1+ progenitor cells results in aggressive PACC, whereas Ink4a/Arf loss results in PanNETs. Concurrent loss of p53 and Ink4a/Arf resembles loss of p53 alone, suggesting that Ink4a/Arf loss has no additive effect to PACC progression. Our results show that specific tumor suppressor genotypes provocatively influence the tumor biological phenotypes in pancreatic progenitor cells. Additionally, in a mouse model of β-cell hyperplasia, we demonstrate that p53 and Ink4a/Arf play cooperative roles in constraining the progression of PanNETs.