Yurong Song

Selective Evolution of Stromal Mesenchyme with p53 Loss in Response to Epithelial Tumorigenesis

Our understanding of cancer has largely come from the analysis of aberrations within the tumor cell population. Yet it is increasingly clear that the tumor microenvironment can significantly influence tumorigenesis. For example, the mesenchyme can support the growth of tumorigenic epithelium. However, whether fibroblasts are subject to genetic/epigenetic changes as a result of selective pressures conferred by oncogenic stress in the epithelium has not been experimentally assessed. Recent analyses of some human carcinomas have shown tumor-suppressor gene mutations within the stroma, suggesting that the interplay among multiple cell types can select for aberrations nonautonomously during tumor progression. We demonstrate that this indeed occurs in a mouse model of prostate cancer where epithelial cell cycle disruption via cell-specific inhibition of pRb function induces a paracrine p53 response that suppresses fibroblast proliferation in associated stroma. This interaction imposes strong selective pressure yielding a highly proliferative mesenchyme that has undergone p53 loss.

Heterogeneous Tumor Evolution Initiated by Loss of pRb Function in a Preclinical Prostate Cancer Model

Because each change in the evolution of a cancer is predicated on the effects of previous events, a full understanding of selective changes and their effect on tumor progression can only be understood in the context of appropriate initiating events. Here, we define the effect of pRb function inactivation in prostate epithelium on both the initiation of prostate cancer and the establishment of selective pressures that lead to diminished Pten function and tumor evolution. Using genetically engineered mice, we show that inactivation of the pRb family proteins (Rb/p107/p130) induces epithelial proliferation and apoptosis and is sufficient to produce prostatic intraepithelial neoplasia (PIN) lesions. Over time, adenocarcinomas develop in all mice with no evidence of neuroendocrine tumors. Apoptosis is dependent on Pten function and not p53, unlike other epithelial cell types tested previously. Consequently, Pten hemizygosity reduces apoptosis by 50%, accelerating progression to adenocarcinomas with heterogeneous composition. Heterogeneity is associated with concurrent Pten haploinsufficiency and focal selective progression to complete Pten loss, which yields distinct tumor properties. Given that this analysis models the apparent timing of highly penetrant events in human prostate cancer, observed effects may recapitulate the natural evolution of prostate cancer development.

Perturbation of Rb, p53 and Brca1 or Brca2 cooperate in inducing metastatic serous epithelial ovarian cancer

The majority of human high-grade serous epithelial ovarian cancer (SEOC) is characterized by frequent mutations in p53 and alterations in the RB and FOXM1 pathways. A subset of human SEOC harbors a combination of germline and somatic mutations as well as epigenetic dysfunction for BRCA1/2. Using Cre-conditional alleles and intrabursal induction by Cre-expressing adenovirus in genetically engineered mice, we analyzed the roles of pathway perturbations in epithelial ovarian cancer initiation and progression. Inactivation of RB-mediated tumor suppression induced surface epithelial proliferation with progression to stage I carcinoma. Additional biallelic inactivation and/or missense p53 mutation in the presence or absence of Brca1/2 caused progression to stage IV disease. As in human SEOC, mice developed peritoneal carcinomatosis, ascites, and distant metastases. Unbiased gene expression and metabolomic profiling confirmed that Rb, p53, and Brca1/2-triple mutant tumors aligned with human SEOC, and not with other intraperitoneal cancers. Together, our findings provide a novel resource for evaluating disease etiology and biomarkers, therapeutic evaluation, and improved imaging strategies in epithelial ovarian cancer.

Evolutionary etiology of high-grade astrocytomas

Significance High-grade astrocytomas (HGAs), including glioblastomas (GBMs), are the most common human brain tumors, and they remain fatal with no effective treatment. The most prevalent form, primary GBM, presents clinically as advanced disease, thus providing no access to or understanding of early stages. We report a comprehensive study in the mouse that establishes causal relationships and an evolutionary etiology in HGA development. Events yielding disease, both engineered and spontaneous, indicate grade-specific roles culminating in the development of GBMs with characteristics of primary GBMs, including molecular alignment with the mesenchymal subclass, asymptomatic early disease, and rapid emergence of high-grade aggressive cancer. These genetically engineered models provide a path to basic understanding of disease etiology and a window into diagnostic and therapeutic discovery. Glioblastoma (GBM), the most common brain malignancy, remains fatal with no effective treatment. Analyses of common aberrations in GBM suggest major regulatory pathways associated with disease etiology. However, 90% of GBMs are diagnosed at an advanced stage (primary GBMs), providing no access to early disease stages for assessing disease progression events. As such, both understanding of disease mechanisms and the development of biomarkers and therapeutics for effective disease management are limited. Here, we describe an adult-inducible astrocyte-specific system in genetically engineered mice that queries causation in disease evolution of regulatory networks perturbed in human GBM. Events yielding disease, both engineered and spontaneous, indicate ordered grade-specific perturbations that yield high-grade astrocytomas (anaplastic astrocytomas and GBMs). Impaired retinoblastoma protein RB tumor suppression yields grade II histopathology. Additional activation of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) network drives progression to grade III disease, and further inactivation of phosphatase and tensin homolog (PTEN) yields GBM. Spontaneous missense mutation of tumor suppressor Trp53 arises subsequent to KRAS activation, but before grade III progression. The stochastic appearance of mutations identical to those observed in humans, particularly the same spectrum of p53 amino acid changes, supports the validity of engineered lesions and the ensuing interpretations of etiology. Absence of isocitrate dehydrogenase 1 (IDH1) mutation, asymptomatic low grade disease, and rapid emergence of GBM combined with a mesenchymal transcriptome signature reflect characteristics of primary GBM and provide insight into causal relationships.

Carcinoma initiation via RB tumor suppressor inactivation: a versatile approach to epithelial subtype-dependent cancer initiation in diverse tissues.

Carcinomas arise in a complex microenvironment consisting of multiple distinct epithelial lineages surrounded by a variety of stromal cell types. Understanding cancer etiologies requires evaluating the relationship among cell types during disease initiation and through progression. Genetically engineered mouse (GEM) models facilitate the prospective examination of early oncogenic events, which is not possible in humans. Since most solid tumors harbor aberrations in the RB network, we developed an inducible GEM approach for the establishment and assessment of carcinoma initiation in a diverse range of epithelial tissues and subtypes upon inactivation of RB-mediated tumor suppression (RB-TS). The system allows independent assessment of epithelial subtypes that express either cytokeratins (K) 18 or 19. By Cre-dependent expression of a protein that dominantly inactivates RB and functionally redundant proteins p107 and p130, neoplasia could be initiated in either K18 or K19 expressing cells of numerous tissues. By design, because only a single pathway aberration was engineered, carcinomas developed stochastically only after long latency. Hence, this system, which allows for directed cell type-specific carcinoma initiation, facilitates further definition of events that can progress neoplasms to aggressive cancers via engineered, carcinogen-induced and/or spontaneous evolution.

RB inactivation in keratin 18 positive thymic epithelial cells promotes non-cell autonomous T cell hyperproliferation in genetically engineered mice

Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia in vivo. To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice (TgK18GT121; K18 mice). We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size.

Abstract 2626: Characterization of the invading cells in glioblastoma using a syngeneic intracranial transplant tumor model.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Local and distant invasion is a hallmark property of glioblastoma (GBM) that prevents curative surgical resection, and may be the key reason for GBM recurrence. Although cancer stem cells (CSCs) have been suggested to cause tumor recurrence based on their resistance to radiotherapy and chemotherapy, it is not clear whether CSCs are also involved in GBM invasiveness. We hypothesize that the invading cells in GBM have stem-like properties. To test our hypothesis, we propose to characterize cells at the invasive front of GBM using a syngeneic intracranial transplant tumor model. Three core regulatory networks are dysregulated in most human GBMs, including the RTK/RAS/PI3K/PTEN, INK/Cyclin D/CDK/RB, and ARF/p53 pathways. We have previously generated a genetically engineered mouse (GEM) model for GBM development via adult-inducible astrocyte-specific perturbation of RB, K-RAS and PTEN. These mice develop astrocytomas that progress to high grade GBM, and faithfully recapitulate the histopathology and molecular characteristics of most human GBMs. We have isolated primary tumor cells from these mice that are maintained in serum-free neural stem cell culture medium and expanded as neurospheres. These primary and low-passage cultures contain a mixed population of cancer stem cells and progenitor cells, and are highly invasive in vitro by matrigel invasion assay. With > 90% penetrance, intracranial injection of 25,000 cells leads to invasive GBM formation and a 32-day median survival in syngeneic host mice. Thus, the model provides an efficient approach for dissecting GBM cell invasion mechanisms in vivo. We will further trace the migration/invasion of GBM cells in the host brain after labeling with lentiviral-expressed mCherry. Host blood vessels are labeled with YFP via Tie2-Cre endothelial expression of a Rosa26-YFP allele. A time course analysis of stem cell marker expression will be used to assess properties of invading cells at the diffusive tumor margin. Laser capture microdissection will be used for invasive rim-cell and non-invasive core-cell isolation, and RNA samples will be compared by unbiased transcriptome studies. Results will provide hypotheses for invasion mechanisms and may identify potential biomarkers for invasive cells. Citation Format: Nailing Zhang, Yurong Song, Sanaz Jansen, Amit Adhikari, Sophie Wang, Lucy Lu, Teresa Sullivan, Terry Van Dyke. Characterization of the invading cells in glioblastoma using a syngeneic intracranial transplant tumor model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2626. doi:10.1158/1538-7445.AM2013-2626

Abstract 1809: Analysis of molecular networks that drive astrocytoma initiation and progression.

High grade astrocytomas, anaplastic astrocytoma and glioblastoma multiforme (GBM) remain incurable in spite of advanced aggressive treatments including surgery, radiation and chemotherapy. To study pathways and mechanisms involved in the development of high grade astrocytomas, we used mouse models wherein key molecular pathways perturbed in human GBMs were inactivated or induced via Cre-driven adult astrocyte-specific system. Inhibition of Rb pathway via expression of T 121 , a N-terminal fragment of SV40 large T antigen (T: TgGZT 121, GFAP-CreER TM ) initiated diffuse grade II astrocytoma formation by 2 months after tamoxifen treatment which in some cases developed to grade III pathology 1.5 year after induction. Activation of KRas pathway (TR: TgGZT 121 , Kras +/lsl-G12D , GFAP-CreER TM ) facilitated progression to grade III anaplastic astrocytoma tumor masses 4-5 months post induction which in a few cases developed to grade IV glioblastoma with some features of human disease. Additional PTEN loss or haploinsufficiency (TRPhet: TgGZT 121 , Kras +/lsl-G12D , PTEN +/fl , GFAP-CreER TM ; TRPnull: TgGZT 121 , Kras +/lsl-G12D , PTEN fl/fl , GFAP-CreER TM ) led to rapid development of glioblastoma with characteristic features of angiogenesis and necrosis observed in human disease. Transcriptome analyses of GBM in our mouse models showed concordance with highly aggressive mesenchymal and proneural subclasses. To identify disease-specific gene networks involved in astrocytoma initiation and progression, we analyzed more than 300 brain samples from tamoxifen-induced T, TR, TRPhet and TRPnull mice and corresponding controls at different time points after induction for gene and miRNA expression by microarray and Nanostring technology. The genes that were induced early and gradually increased in expression with tumor grade belonged to several key molecular networks: DNA replication and repair, cell division and chromosome transmission fidelity, cell cycle progression, metabolism, and pathways important for embryonic stem cell biology. Pathways significantly induced at later stages of disease (grades III-IV) included Notch and Wnt signaling, inflammatory response genes, p53 signaling, and RNA processing. Significantly downregulated pathways were related to neuronal development and function. We have confirmed expression of several candidate genes in mouse tumor samples and cell lines derived from low and high grade tumors. Currently we are investigating potential role of the selected candidates in astrocytoma development by in vitro and in vivo functional analysis. In summary, we utilized mouse models to determine global molecular changes during astrocytoma initiation and progression to high grade. These studies are important for understanding the mechanisms of the disease and may facilitate the development of new therapies. Citation Format: Yaroslava Ruzankina, Burak Kutlu, Sophie Wang, Yurong Song, Deborah Householder, Philip Martin, Maureen Baran, Simone Difilippantonio, Leroy Hood, Terry Van Dyke. Analysis of molecular networks that drive astrocytoma initiation and progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1809. doi:10.1158/1538-7445.AM2013-1809

Abstract 1577: Subtype dependent carcinoma initiation by Rb tumor suppressor inactivation in multiple epithelial tissues.

Carcinomas arise in a complex microenvironment consisting of multiple distinct epithelial lineages surrounded by mesenchymal cells. Of major importance is to understand the relationship among cell types, initiation event, and ultimate cancer outcome. Mouse models facilitate the prospective examination of early oncogenic events, which is not feasible in humans, permitting an analysis of epithelial subtype susceptibility. Since most solid tumors harbor aberrations in the pRb network, we assessed the susceptibility of epithelial subtypes expressing cytokeratin (K) 18 or K19 to the disruption of Rb tumor suppression (Rb-TS) in vivo. Tumorigenesis could be initiated in either K18- or K19-expressing cells. However, the susceptibility of epithelial tissues was subtype-dependent. K19 cells were more prone to hyperplasia and neoplasia than K18 targeted cells. Given that a single genetic event was engineered, a small percentage of animals developed carcinoma/adenocarcinoma, providing the opportunity to define progression events via engineering or spontaneous evolution. To extend our analysis to a specific tissue, we employed a prostate-specific Cre line to target either K18- or K19-expressing prostate epithelial cells. Differential responses observed in the frequency and extent of premalignant hyperplastic lesions strongly supported the notion of cell type-dependent susceptibility to Rb-TS inactivation. Our data suggest that the tumorigenic consequences of a single initiation event are dictated by cellular subtype distinctiveness, underscoring the importance of the tumor cell of origin. Citation Format: Yurong Song, Chunyu Yang, Wenqi Pan, Alisan Fathalizadeh, Lucy Lu, Debra Gilbert, T. Norene O9Sullivan, Diana C. Haines, Philip L. Martin, Terry Van Dyke. Subtype dependent carcinoma initiation by Rb tumor suppressor inactivation in multiple epithelial tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1577. doi:10.1158/1538-7445.AM2013-1577

Abstract 5014: Reprogramming of mature astrocytes by abrogation of Rb tumor suppression leads to astrocytoma initiation in GEMMs.

Astrocytomas are the most common malignant brain tumors which maintain a poor survival rate despite decades of research effort. Understanding the initiation process and identifying astrocytoma cell(s) of origin have been of immense interest. Genetically engineered mouse models (GEMMs) are ideal for studying the initiation process compared to evolved human tumor cells. Our lab has developed an adult inducible GEMM that alters key human glioblastoma (Grade IV) pathways. Inactivation of Rb tumor suppression (TS) by expressing N-terminal 121aa of large T antigen (T121) under GFAP promoter is sufficient to initiate grade II astrocytoma. Addition of mutant KrasG12D leads to grade III and further PTEN deletion to glioblastomas. KrasG12D activation and PTEN deletion alone or in combination failed to initiate astrocytomas, suggesting that Rb TS inactivation is essential for its initiation. Studies from several cancers have identified tumor-initiating cells, a subset of tumor cells with stem cell-like properties responsible for generating the entire tumor mass. Their origin remains a mystery. We hypothesize that Rb TS inactivation reprograms astrocytes into stem/progenitor-like state generating potential cell(s) of origin for astrocytoma. Immunostaining of progenitor and proliferation markers (e.g. nestin and Ki-67) showed their co-expression in the cortex with T121, suggesting a possible reprogramming of mature astrocytes. Moreover, astrocyte differentiation marker S100beta was downregulated in T121 cells. To rule out the possibility of migration resulting in the observed cortical progenitor cells, focal induction of T121 in cortex using lenti-cre injection was performed. We found expression of Ki-67 and nestin and reduced expression of S100beta, suggesting that the reprogramming was independent of the germinal zones in the brain. Furthermore, cortical T121 expressing astrocytes were able to generate neurospheres but astrocytes from wildtype cortex failed to do so. These cells also showed self-renewability and multilineage ability, emphasizing their dedifferentiated status upon Rb-TS inactivation. We were able to achieve in-vitro reprogramming of mature astrocytes upon T121 expression suggesting that the reprogramming does not require brain microenvironment. Sphere assays using cortical cells from mice where individual Rb family member was inactivated suggests that loss of individual Rb family members alone is insufficient in reprogramming the mature astrocytes. With the Rb pathway known to be altered in more than 75% of glioblastomas, our results are a step in the direction of unraveling the process of tumor initiation for this deadly disease. Citation Format: Amit S. Adhikari, Teresa Sullivan, Yurong Song, Xiuyun Lu, Norene O9Sullivan, Terry Van Dyke. Reprogramming of mature astrocytes by abrogation of Rb tumor suppression leads to astrocytoma initiation in GEMMs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5014. doi:10.1158/1538-7445.AM2013-5014