Katie Thies

CSF1-ETS2-induced microRNA in myeloid cells promote metastatic tumor growth

Metastasis of solid tumors is associated with poor prognosis and bleak survival rates. Tumor-infiltrating myeloid cells (TIMs) are known to promote metastasis, but the mechanisms underlying their collaboration with tumor cells remain unknown. Here, we report an oncogenic role for microRNA (miR) in driving M2 reprogramming in TIMs, characterized by the acquisition of pro-tumor and pro-angiogenic properties. The expression of miR-21, miR-29a, miR-142-3p and miR-223 increased in myeloid cells during tumor progression in mouse models of breast cancer and melanoma metastasis. Further, we show that these miRs are regulated by the CSF1-ETS2 pathway in macrophages. A loss-of-function approach utilizing selective depletion of the miR-processing enzyme Dicer in mature myeloid cells blocks angiogenesis and metastatic tumor growth. Ectopic expression of miR-21 and miR-29a promotes angiogenesis and tumor cell proliferation through the downregulation of anti-angiogenic genes such as Col4a2, Spry1 and Timp3, whereas knockdown of the miRs impedes these processes. miR-21 and miR-29a are expressed in Csf1r+ myeloid cells associated with human metastatic breast cancer, and levels of these miRs in CD115+ non-classical monocytes correlates with metastatic tumor burden in patients. Taken together, our results suggest that miR-21 and miR-29a are essential for the pro-tumor functions of myeloid cells and the CSF1-ETS2 pathway upstream of the miRs serves as an attractive therapeutic target for the inhibition of M2 remodeling of macrophages during malignancy. In addition, miR-21 and miR-29a in circulating myeloid cells may potentially serve as biomarkers to measure therapeutic efficacy of targeted therapies for CSF1 signaling.

Stromal PDGFR-α Activation Enhances Matrix Stiffness, Impedes Mammary Ductal Development, and Accelerates Tumor Growth

The extracellular matrix (ECM) is critical for mammary ductal development and differentiation, but how mammary fibroblasts regulate ECM remodeling remains to be elucidated. Herein, we used a mouse genetic model to activate platelet derived growth factor receptor-alpha (PDGFRα) specifically in the stroma. Hyperactivation of PDGFRα in the mammary stroma severely hindered pubertal mammary ductal morphogenesis, but did not interrupt the lobuloalveolar differentiation program. Increased stromal PDGFRα signaling induced mammary fat pad fibrosis with a corresponding increase in interstitial hyaluronic acid (HA) and collagen deposition. Mammary fibroblasts with PDGFRα hyperactivation also decreased hydraulic permeability of a collagen substrate in an in vitro microfluidic device assay, which was mitigated by inhibition of either PDGFRα or HA. Fibrosis seen in this model significantly increased the overall stiffness of the mammary gland as measured by atomic force microscopy. Further, mammary tumor cells injected orthotopically in the fat pads of mice with stromal activation of PDGFRα grew larger tumors compared to controls. Taken together, our data establish that aberrant stromal PDGFRα signaling disrupts ECM homeostasis during mammary gland development, resulting in increased mammary stiffness and increased potential for tumor growth.

Stromal PTEN inhibits the expansion of mammary epithelial stem cells through Jagged-1

Fibroblasts within the mammary tumor microenvironment are active participants in carcinogenesis mediating both tumor initiation and progression. Our group has previously demonstrated that genetic loss of phosphatase and tensin homolog (PTEN) in mammary fibroblasts induces an oncogenic secretome that remodels the extracellular milieu accelerating ErbB2-driven mammary tumor progression. While these prior studies highlighted a tumor suppressive role for stromal PTEN, how the adjacent normal epithelium transforms in response to PTEN loss was not previously addressed. To identify these early events, we have evaluated both phenotypic and genetic changes within the pre-neoplastic mammary epithelium of mice with and without stromal PTEN expression. We report that fibroblast-specific PTEN deletion greatly restricts mammary ductal elongation and induces aberrant alveolar side-branching. These mice concomitantly exhibit an expansion of the mammary epithelial stem cell (MaSC) enriched basal/myoepithelial population and an increase in in vitro stem cell activity. Further analysis revealed that NOTCH signaling, specifically through NOTCH3, is diminished in these cells. Mechanistically, JAGGED-1, a transmembrane ligand for the NOTCH receptor, is downregulated in the PTEN-null fibroblasts leading to a loss in the paracrine activation of NOTCH signaling from the surrounding stroma. Reintroduction of JAGGED-1 expression within the PTEN-null fibroblasts was sufficient to abrogate the observed increase in colony forming activity implying a direct role for stromal JAGGED-1 in regulation of MaSC properties. Importantly, breast cancer patients whose tumors express both low stromal JAG1 and low stromal PTEN exhibit a shorter time to recurrence than those whose tumors express low levels of either alone suggesting similar stromal signaling in advanced disease. Combined, these results unveil a novel stromal PTEN-to-JAGGED-1 axis in maintaining the MaSC niche, and subsequently inhibiting breast cancer initiation and disease progression.

Changes in BAI1 and Nestin Expression Are Prognostic Indicators for Survival and Metastases in Breast Cancer and Provide Opportunities for Dual Targeted Therapies

The 2-year survival rate of patients with breast cancer brain metastases is less than 2%. Treatment options for breast cancer brain metastases are limited, and there is an unmet need to identify novel therapies for this disease. Brain angiogenesis inhibitor 1 (BAI1) is a GPCR involved in tumor angiogenesis, invasion, phagocytosis, and synaptogenesis. For the first time, we identify that BAI1 expression is significantly reduced in breast cancer and higher expression is associated with better patient survival. Nestin is an intermediate filament whose expression is upregulated in several cancers. We found that higher Nestin expression significantly correlated with breast cancer lung and brain metastases, suggesting both BAI1 and Nestin can be therapeutic targets for this disease. Here, we demonstrate the ability of an oncolytic virus, 34.5ENVE, to target and kill high Nestin-expressing cells and deliver Vstat120 (extracellular fragment of BAI1). Finally, we created two orthotopic immune-competent murine models of breast cancer brain metastases and demonstrated 34.5ENVE extended the survival of immune-competent mice bearing intracranial breast cancer tumors. Mol Cancer Ther; 14(1); 307–14. ©2014 AACR.

Generation of a pancreatic cancer model using a Pdx1-Flp recombinase knock-in allele

The contribution of the tumor microenvironment to the development of pancreatic adenocarcinoma (PDAC) is unclear. The LSL-KrasG12D/+;LSL-p53R172H/+;Pdx-1-Cre (KPC) tumor model, which is widely utilized to faithfully recapitulate human pancreatic cancer, depends on Cre-mediated recombination in the epithelial lineage to drive tumorigenesis. Therefore, specific Cre-loxP recombination in stromal cells cannot be applied in this model, limiting the in vivo investigation of stromal genetics in tumor initiation and progression. To address this issue, we generated a new Pdx1FlpO knock-in mouse line, which represents the first mouse model to physiologically express FlpO recombinase in pancreatic epithelial cells. This mouse specifically recombines Frt loci in pancreatic epithelial cells, including acinar, ductal, and islet cells. When combined with the Frt-STOP-Frt KrasG12D and p53Frt mouse lines, simultaneous Pdx1FlpO activation of mutant Kras and deletion of p53 results in the spectrum of pathologic changes seen in PDAC, including PanIN lesions and ductal carcinoma. Combination of this KPF mouse model with any stroma-specific Cre can be used to conditionally modify target genes of interest. This will provide an excellent in vivo tool to study the roles of genes in different cell types and multiple cell compartments within the pancreatic tumor microenvironment.

Stromal PTEN determines mammary epithelial response to radiotherapy

The importance of the tumor–associated stroma in cancer progression is clear. However, it remains uncertain whether early events in the stroma are capable of initiating breast tumorigenesis. Here, we show that in the mammary glands of non-tumor bearing mice, stromal-specific phosphatase and tensin homolog (Pten) deletion invokes radiation-induced genomic instability in neighboring epithelium. In these animals, a single dose of whole-body radiation causes focal mammary lobuloalveolar hyperplasia through paracrine epidermal growth factor receptor (EGFR) activation, and EGFR inhibition abrogates these cellular changes. By analyzing human tissue, we discover that stromal PTEN is lost in a subset of normal breast samples obtained from reduction mammoplasty, and is predictive of recurrence in breast cancer patients. Combined, these data indicate that diagnostic or therapeutic chest radiation may predispose patients with decreased stromal PTEN expression to secondary breast cancer, and that prophylactic EGFR inhibition may reduce this risk.The tumor microenvironment influences tumor progression. Here the authors show that lack of stromal PTEN phosphatase induces DNA repair defects in the neighboring mammary gland epithelial cells via hyperactivation of EGF-receptor signaling, resulting in higher radiation-induced DNA damage and hyperplasia.

Disruption of stromal hedgehog signaling initiates RNF5-mediated proteasomal degradation of PTEN and accelerates pancreatic tumor growth

Disrupting paracrine Hedgehog signaling in pancreatic cancer stroma through genetic deletion of fibroblast Smoothened leads to proteasomal degradation of fibroblast PTEN and accelerates tumor growth. The contribution of the tumor microenvironment to pancreatic ductal adenocarcinoma (PDAC) development is currently unclear. We therefore examined the consequences of disrupting paracrine Hedgehog (HH) signaling in PDAC stroma. Herein, we show that ablation of the key HH signaling gene Smoothened (Smo) in stromal fibroblasts led to increased proliferation of pancreatic tumor cells. Furthermore, Smo deletion resulted in proteasomal degradation of the tumor suppressor PTEN and activation of oncogenic protein kinase B (AKT) in fibroblasts. An unbiased proteomic screen identified RNF5 as a novel E3 ubiquitin ligase responsible for degradation of phosphatase and tensin homolog (PTEN) in Smo-null fibroblasts. Ring Finger Protein 5 (Rnf5) knockdown or pharmacological inhibition of glycogen synthase kinase 3β (GSKβ), the kinase that marks PTEN for ubiquitination, rescued PTEN levels and reversed the oncogenic phenotype, identifying a new node of PTEN regulation. In PDAC patients, low stromal PTEN correlated with reduced overall survival. Mechanistically, PTEN loss decreased hydraulic permeability of the extracellular matrix, which was reversed by hyaluronidase treatment. These results define non-cell autonomous tumor-promoting mechanisms activated by disruption of the HH/PTEN axis and identifies new targets for restoring stromal tumor-suppressive functions.

Abstract C30: The CSF1-PU.1 pathway in tumor associated macrophages promotes breast cancer growth and progression

Purpose of Study: Research over the past decade has established a critical role for the tumor microenvironment in facilitating tumor growth and promoting invasiveness. Cells of the myeloid lineage, including macrophages, have been known to be key mediators of tumor progression and facilitate metastasis. Recent work from our lab highlights the Ets transcription factors, PU.1 and ETS2, as important players in tumor associated macrophages (TAMs) during breast cancer progression. Both transcription factors are downstream effectors of the Colony Stimulating Factor 1 (CSF1) signaling pathway, which is not only important for myeloid cell survival, but also has a well-established role within the tumor microenvironment. Here, we investigated the requirement of PU.1 in TAMs in a mouse mammary tumor model and have begun to address the role of PU.1 during metastasis. Research Method: We employed cre-loxP technology to conditionally delete PU.1 in the myeloid cell compartment. Syngeneic mice were orthotopically injected with mammary tumor cells directly into the fat pad. To model breast cancer brain metastasis in the mouse, we employed an intracranial injection model which recapitulates the biology of the human disease. We were able to isolate tumor associated macrophages from either site (mammary or brain tumor) for downstream applications. Novel Findings: Macrophage-specific deletion of PU.1 resulted in a significant reduction in mammary tumor growth as well as tumor angiogenesis. Our results suggest that ETS2 and PU.1, acting downstream of the CSF1 signaling pathway, cooperatively regulate the expression of pro-tumor genes along with ‘oncogenic’ microRNA. Conventional ChIP assays show that both transcription factors occupy enhancer regions adjacent to a set of selected genes and cooperatively regulate expression in tumor infiltrating macrophages. We focused on miR-21 and miR-29a for a more detailed investigation as both are expressed in CSF1R+ myeloid cells within the brain metastatic microenvironment. In our intracranial injection model, macrophages (F4/80 positive cells) are recruited to the growing tumor, and both miR-21 and miR-29a are upregulated in TAMs isolated by FACS from tumor-bearing mice as compared to controls. Conclusions and Implications: Given our present data, we believe that the Ets factors, PU.1 and Ets2, regulate a transcriptional program, including microRNAs, that is critical for CSF1 action in tumor infiltrating macrophages. Currently, clinical trials are underway using CSF1R inhibitors for several solid tumor types [clinicaltrials.gov]. The macrophage-specific microRNA, including miR-21 and miR-29a, may serve as indicators for to the efficacy of CSF1R inhibition. Citation Format: Katie Thies, David A. Taffany, Haritha Mathsyaraja, Sudarshana M. Sharma, Walter Hans Meisen, Tom Liu, Cynthia Timmers, Jose Otero, Balveen Kaur, Michael C. Ostrowski. The CSF1-PU.1 pathway in tumor associated macrophages promotes breast cancer growth and progression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C30.

Abstract C28: Platelet-derived growth factor receptor-β (PDGFRβ) in the breast metastatic tumor microenvironment

A role for the tumor microenvironment (TME) in cancer progression is irrefutable and our laboratory has been at the forefront of this field providing evidence for both tumor suppressive and oncogenic roles of the TME. The PDGF pathway is an exemplar for the study of tumor-stroma interaction as PDGF receptors (PDGFR) are frequently expressed in the fibroblasts and pericytes within the tumor-associated stroma of epithelial tumors including breast cancer. In contrast, PDGF ligands are expressed by the epithelial tumor cells themselves. However, beyond a few descriptive studies, the role of interactive PDGFRβ signaling in the TME during breast cancer initiation, progression and metastases is not understood. This can be attributed in part to limited in vivo models to study the complex TME, especially for breast cancer associated metastases. To overcome this limitation, we have established a transgenic knock-in mouse model that expresses constitutively active PDGFRβ in the stroma of the mammary gland as well in the lung and the brain, two common sites of metastatic breast cancer dissemination. We have found that these mice develop mammary gland hyperplasia highlighting the importance of PDGFRβ in the TME in driving mammary epithelial cell growth. To test whether activation of mutant PDGFRβ in either the lung or the brain increases metastatic growth at either site, two experimental metastases assays were performed: (1) tail vein and (2) intracranial injections to test for lung and brain metastatic outgrowth, respectively. Tail vein injection of the non-metastatic murine mammary cancer cell line DB7 cells led to pronounced lung metastases in PDGFRβ knock-in mice in less than 4 weeks. No macrometastases were seen in the control at this same time point. Similar to the surge in lung metastasis, intracranial injection of DB7 cells led to an increase in tumor growth in brains of the mutant versus wild type controls, revealing an important role for PDGFRβ signaling in the breast cancer metastatic microenvironment. In addition, knockdown of PDGF-B in mammary cancer cells represses intracranial growth in wild type animals. Combined these data support a role for PDGFRβ signaling in the breast cancer metastatic microenvironment. Ongoing investigation is aimed to delineate how activated PDGF-B to PDGFRβ signaling primes the metastatic niche. Citation Format: Gina Sizemore, Anisha Mathur, Katie Thies, Chelsea Bolyard, Steven Sizemore, Raleigh Kladney, Anthony Trimboli, Balveen Kaur, Gustavo Leone, Michael Ostrowski. Platelet-derived growth factor receptor-β (PDGFRβ) in the breast metastatic tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C28.

Abstract P6-16-02: Oncolytic viral therapy enhances the survival of mice in a novel model of breast cancer brain metastases

Breast cancer [BC] is one of the leading causes of brain metastases. The 2 year survival rate of patients with breast cancer brain metastases [BM] is less than 2%. Oncolytic viruses exploit the aberrant molecular and genetic pathways found in cancer cells to selectively replicate in and destroy tumors while sparing normal tissues. Here, we demonstrate the oncolytic Herpes Simplex Virus [HSV-1], 34.5ENVE, can specifically target and destroy BC brain metastases. The 34.5ENVE virus expresses anti-angiogenic Vstat120 and its replication is transcriptionally driven by the cancer specific promoter Nestin. Vstat120 expression is reduced in brain, renal, and gastric cancers, however its expression status in BC is not known. Analysis of The Cancer Genome Atlas revealed a 52% reduction in Vstat120 expression in invasive ductal breast carcinomas [n=69] compared to normal breast tissue [n=389; P Citation Format: W Hans Meisen, Samuel Dubin, Steven Sizemore, Haritha Mathsyaraja, Katie Thies, Norm Lehman, Peter Boyer, Alena C Jaime-Ramirez, J Bradley Elder, Kimerly Powell, Michael Ostrowski, Balveen Kaur. Oncolytic viral therapy enhances the survival of mice in a novel model of breast cancer brain metastases [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-16-02.