Publications by authors named "Anna Chytil"

52 Publications

Cell-programmed nutrient partitioning in the tumour microenvironment.

Nature 2021 05 7;593(7858):282-288. Epub 2021 Apr 7.

Department of Radiology and Radiological Sciences, VUMC, Nashville, TN, USA.

Cancer cells characteristically consume glucose through Warburg metabolism, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.
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http://dx.doi.org/10.1038/s41586-021-03442-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122068PMC
May 2021

Correction: loss sensitizes cells to PI3Kβ and AKT inhibition.

Oncotarget 2019 Oct 29;10(59):6391-6392. Epub 2019 Oct 29.

Vanderbilt-Ingram Cancer Center, Division of Hematology and Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville 37232, TN, USA.

[This corrects the article DOI: 10.18632/oncotarget.26567.].
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http://dx.doi.org/10.18632/oncotarget.27288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824879PMC
October 2019

loss sensitizes cells to PI3Kβ and AKT inhibition.

Oncotarget 2019 Jan 18;10(6):647-659. Epub 2019 Jan 18.

Vanderbilt-Ingram Cancer Center, Division of Hematology and Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville 37232, TN, USA.

Upregulation of the PI3K pathway has been implicated in the initiation and progression of several types of cancer, including renal cell carcinoma (RCC). Although several targeted therapies have been developed for RCC, durable and complete responses are exceptional. Thus, advanced RCC remains a lethal disease, underscoring the need of robust biomarker-based strategies to treat RCC. We report a synthetic lethal interaction between inhibition of phosphatidylinositol 3-kinase beta (PI3Kβ) and loss of methyltransferase. Clear cell RCC (ccRCC)-derived knockout 786-0 and mutant A498 cells treated with TGX221 (PI3Kβ-specific) and AZD8186 (PI3Kβ- and δ-specific) inhibitors displayed decreased cell viability, cell growth, and migration compared to proficient 786-0 cells. Inhibition of the p110 δ and α isoforms alone had modest (δ) and no (α) effect on ccRCC cell viability, growth, and migration. , treatment of mutant A498 cells, but not proficient 786-0 cells, with AZD8186 significantly decreased tumor growth. Interestingly, inhibition of the downstream effector AKT (MK2206) recapitulated the effects observed in AZD8186-treated deficient cells. Our data show that specific inhibition of PI3Kβ causes synthetic lethality with loss and suggest targeting of the AKT downstream effector pathway offers a rationale for further translational and clinical investigation of PI3Kβ-specific inhibitors in ccRCC.
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http://dx.doi.org/10.18632/oncotarget.26567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363018PMC
January 2019

Ultrasound Measurement of Vascular Density to Evaluate Response to Anti-Angiogenic Therapy in Renal Cell Carcinoma.

IEEE Trans Biomed Eng 2019 03 27;66(3):873-880. Epub 2018 Jul 27.

Background: Functional and molecular changes often precede gross anatomical changes, so early assessment of a tumor's functional and molecular response to therapy can help reduce a patient's exposure to the side effects of ineffective chemotherapeutics or other treatment strategies.

Objective: Our intent was to test the hypothesis that an ultrasound microvascular imaging approach might provide indications of response to therapy prior to assessment of tumor size.

Methods: Mice bearing clear-cell renal cell carcinoma xenograft tumors were treated with antiangiogenic and Notch inhibition therapies. An ultrasound measurement of microvascular density was used to serially track the tumor response to therapy.

Results: Data indicated that ultrasound-derived microvascular density can indicate response to therapy a week prior to changes in tumor volume and is strongly correlated with physiological characteristics of the tumors as measured by histology ([Formula: see text]). Furthermore, data demonstrated that ultrasound measurements of vascular density can determine response to therapy and classify between-treatment groups with high sensitivity and specificity.

Conclusion/significance: Results suggests that future applications utilizing ultrasound imaging to monitor tumor response to therapy may be able to provide earlier insight into tumor behavior from metrics of microvascular density rather than anatomical tumor size measurements.
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http://dx.doi.org/10.1109/TBME.2018.2860932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691901PMC
March 2019

Ultrasound Molecular Imaging of VEGFR-2 in Clear-Cell Renal Cell Carcinoma Tracks Disease Response to Antiangiogenic and Notch-Inhibition Therapy.

Theranostics 2018 1;8(1):141-155. Epub 2018 Jan 1.

Joint Department of Biomedical Engineering, The University of North Carolina and North Carolina State University, Chapel Hill, North Carolina.

Metastatic clear-cell renal cell carcinoma (ccRCC) affects thousands of patients worldwide each year. Antiangiogenic therapy has been shown to have beneficial effects initially, but resistance is eventually developed. Therefore, it is important to accurately track the response of cancer to different therapeutics in order to appropriately adjust the therapy to maximize efficacy. Change in tumor volume is the current gold standard for determining efficacy of treatment. However, functional variations can occur much earlier than measurable volume changes. Contrast-enhanced ultrasound (CEUS) is an important tool for assessing tumor progression and response to therapy, since it can monitor functional changes in the physiology. In this study, we demonstrate how ultrasound molecular imaging (USMI) can accurately track the evolution of the disease and molecular response to treatment. A cohort of NSG (NOD/scid/gamma) mice was injected with ccRCC cells and treated with either the VEGF inhibitor SU (Sunitinib malate, Selleckchem, TX, USA) or the Notch pathway inhibitor GSI (Gamma secretase inhibitor, PF-03084014, Pfizer, New York, NY, USA), or started on SU and later switched to GSI (Switch group). The therapies used in the study focus on disrupting angiogenesis and proper vessel development. SU inhibits signaling of vascular endothelial growth factor (VEGF), which is responsible for the sprouting of new vasculature, and GSI inhibits the Notch pathway, which is a key factor in the correct maturation of newly formed vasculature. Microbubble contrast agents targeted to VEGFR-2 (VEGF Receptor) were delivered as a bolus, and the bound agents were imaged in 3D after the free-flowing contrast was cleared from the body. Additionally, the tumors were harvested at the end of the study and stained for CD31. The results show that MI can detect changes in VEGFR-2 expression in the group treated with SU within a week of the start of treatment, while differences in volume only become apparent after the mice have been treated for three weeks. Furthermore, USMI can detect response to therapy in 92% of cases after 1 week of treatment, while the detection rate is only 40% for volume measurements. The amount of targeting for the GSI and Control groups was high throughout the duration of the study, while that of the SU and Switch groups remained low. However, the amount of targeting in the Switch group increased to levels similar to those of the Control group after the treatment was switched to GSI. CD31 staining indicates significantly lower levels of patent vasculature for the SU group compared to the Control and GSI groups. Therefore, the results parallel the expected physiological changes in the tumor, since GSI promotes angiogenesis through the VEGF pathway, while SU inhibits it. This study demonstrates that MI can track disease progression and assess functional changes in tumors before changes in volume are apparent, and thus, CEUS can be a valuable tool for assessing response to therapy in disease. Future work is required to determine whether levels of VEGFR-2 targeting correlate with eventual survival outcomes.
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http://dx.doi.org/10.7150/thno.19658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743465PMC
November 2018

Development of Aggressive Pancreatic Ductal Adenocarcinomas Depends on Granulocyte Colony Stimulating Factor Secretion in Carcinoma Cells.

Cancer Immunol Res 2017 09 3;5(9):718-729. Epub 2017 Aug 3.

Department of Cancer Biology and the Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University, Nashville, Tennessee.

The survival rate for pancreatic ductal adenocarcinoma (PDAC) remains low. More therapeutic options to treat this disease are needed, for the current standard of care is ineffective. Using an animal model of aggressive PDAC (Kras/p48), we discovered an effect of TGFβ signaling in regulation of G-CSF secretion in pancreatic epithelium. Elevated concentrations of G-CSF in PDAC promoted differentiation of Ly6G cells from progenitors, stimulated IL10 secretion from myeloid cells, and decreased T-cell proliferation via upregulation of Arg, iNOS, VEGF, IL6, and IL1b from CD11b cells. Deletion of in PDAC cells or use of a G-CSF-blocking antibody decreased tumor growth. Anti-G-CSF treatment in combination with the DNA synthesis inhibitor gemcitabine reduced tumor size, increased the number of infiltrating T cells, and decreased the number of Ly6G cells more effectively than gemcitabine alone. Human analysis of human datasets from The Cancer Genome Atlas and tissue microarrays correlated with observations from our mouse model experiments, especially in patients with grade 1, stage II disease. We propose that in aggressive PDAC, elevated G-CSF contributes to tumor progression through promoting increases in infiltration of neutrophil-like cells with high immunosuppressive activity. Such a mechanism provides an avenue for a neoadjuvant therapeutic approach for this devastating disease. .
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http://dx.doi.org/10.1158/2326-6066.CIR-16-0311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581681PMC
September 2017

TβRIII Expression in Human Breast Cancer Stroma and the Role of Soluble TβRIII in Breast Cancer Associated Fibroblasts.

Cancers (Basel) 2016 Nov 4;8(11). Epub 2016 Nov 4.

Department of Cancer Biology, Vanderbilt University, Nashville, TN 37232, USA.

The TGF-β pathway plays a major role in tumor progression through regulation of epithelial and stromal cell signaling. Dysfunction of the pathway can lead to carcinoma progression and metastasis. To gain insight into the stromal role of the TGF-β pathway in breast cancer, we performed laser capture microdissection (LCM) from breast cancer patients and reduction mammoplasty patients. Microdissected tumor stroma and normal breast stroma were examined for gene expression. Expression of the TGF-β type III receptor () was greatly decreased in the tumor stroma compared to control healthy breast tissue. These results demonstrated a 44-fold decrease in mRNA in tumor stroma in comparison to control tissue. We investigated publicly available databases, and have identified that mRNA levels are decreased in tumor stroma. We next investigated fibroblast cell lines derived from cancerous and normal breast tissue and found that in addition to mRNA levels, TβRIII protein levels were significantly reduced. Having previously identified that cancer-associated fibroblasts secrete greater levels of tumor promoting cytokines, we investigated the consequences of soluble-TβRIII (sTβRIII) on fibroblasts. Fibroblast conditioned medium was analyzed for 102 human secreted cytokines and distinct changes in response to sTβRIII were observed. Next, we used the fibroblast-conditioned medium to stimulate human monocyte cell line THP-1. These results indicate a distinct transcriptional response depending on sTβRIII treatment and whether it was derived from normal or cancerous breast tissue. We conclude that the effect of TβRIII has distinct roles not only in cancer-associated fibroblasts but that sTβRIII has distinct paracrine functions in the tumor microenvironment.
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http://dx.doi.org/10.3390/cancers8110100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126760PMC
November 2016

Deletion of the BMP receptor BMPR1a impairs mammary tumor formation and metastasis.

Oncotarget 2015 Sep;6(26):22890-904

Department of Cancer Biology, Vanderbilt Ingram Cancer Center, Vanderbilt University, Nashville, TN, USA.

Bone Morphogenetic Proteins (BMPs) are secreted cytokines/growth factors belonging to the Transforming Growth Factor β (TGFβ) family. BMP ligands have been shown to be overexpressed in human breast cancers. Normal and cancerous breast tissue display active BMP signaling as indicated by phosphorylated Smads 1, 5 and 9. We combined mice expressing the MMTV.PyMT oncogene with mice having conditional knockout (cKO) of BMP receptor type 1a (BMPR1a) using whey acidic protein (WAP)-Cre and found this deletion resulted in delayed tumor onset and significantly extended survival. Immunofluorescence staining revealed that cKO tumors co-expressed Keratin 5 and mesenchymal cell markers such as Vimentin. This indicates that epithelial-to-mesenchymal (EMT)-like transitions occurred in cKO tumors. We performed microarray analysis on these tumors and found changes that support EMT-like changes. We established primary tumor cell lines and found that BMPR1a cKO had slower growth in vitro and in vivo upon implantation. cKO tumor cells had reduced migration in vitro. We analyzed human databases from TCGA and survival data from microarrays to confirm BMPR1a tumor promoting functions, and found that high BMPR1a gene expression correlates with decreased survival regardless of molecular breast cancer subtype. In conclusion, the data indicate that BMP signaling through BMPR1a functions as a tumor promoter.
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http://dx.doi.org/10.18632/oncotarget.4413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4673207PMC
September 2015

TGFβ signaling in myeloid cells regulates mammary carcinoma cell invasion through fibroblast interactions.

PLoS One 2015 28;10(1):e0117908. Epub 2015 Jan 28.

Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN, United States of America.

Metastasis is the most devastating aspect of cancer, however we know very little about the mechanisms of local invasion, the earliest step of metastasis. During tumor growth CD11b+ Gr1+ cells, known also as MDSCs, have been shown to promote tumor progression by a wide spectrum of effects that suppress the anti-tumor immune response. In addition to immunosuppression, CD11b+ Gr1+ cells promote metastasis by mechanisms that are currently unknown. CD11b+ Gr1+ cells localize near fibroblasts, which remodel the ECM and leave tracks for collective cell migration of carcinoma cells. In this study we discovered that CD11b+ Gr1+ cells promote invasion of mammary carcinoma cells by increasing fibroblast migration. This effect was directed by secreted factors derived from CD11b+ Gr1+ cells. We have identified several CD11b+ Gr1+ cell secreted proteins that activate fibroblast migration, including CXCL11, CXCL15, FGF2, IGF-I, IL1Ra, Resistin, and Shh. The combination of CXCL11 and FGF2 had the strongest effect on fibroblast migration that is associated with Akt1 and ERK1/2 phosphorylation. Analysis of subsets of CD11b+ Gr1+ cells identified that CD11b+ Ly6Chigh Ly6Glow cells increase fibroblast migration more than other myeloid cell populations. Additionally, tumor-derived CD11b+ Gr1+ cells promote fibroblast migration more than splenic CD11b+ Gr1+ cells of tumor-bearing mice. While TGFβ signaling in fibroblasts does not regulate their migration toward CD11b+ Gr1+ cells, however deletion of TGFβ receptor II on CD11b+ Gr1+ cells downregulates CXCL11, Shh, IGF1 and FGF2 resulting in reduced fibroblast migration. These studies show that TGFβ signaling in CD11b+ Gr1+ cells promotes fibroblast directed carcinoma invasion and suggests that perivascular CD11b+ Ly6Chigh Ly6Glow cells may be the stimulus for localized invasion leading to metastasis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117908PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4309578PMC
February 2016

Age- and pregnancy-associated DNA methylation changes in mammary epithelial cells.

Stem Cell Reports 2015 Feb 22;4(2):297-311. Epub 2015 Jan 22.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Postnatal mammary gland development and differentiation occur during puberty and pregnancy. To explore the role of DNA methylation in these processes, we determined the genome-wide DNA methylation and gene expression profiles of CD24(+)CD61(+)CD29(hi), CD24(+)CD61(+)CD29(lo), and CD24(+)CD61(-)CD29(lo) cell populations that were previously associated with distinct biological properties at different ages and reproductive stages. We found that pregnancy had the most significant effects on CD24(+)CD61(+)CD29(hi) and CD24(+)CD61(+)CD29(lo) cells, inducing distinct epigenetic states that were maintained through life. Integrated analysis of gene expression, DNA methylation, and histone modification profiles revealed cell-type- and reproductive-stage-specific changes. We identified p27 and TGFβ signaling as key regulators of CD24(+)CD61(+)CD29(lo) cell proliferation, based on their expression patterns and results from mammary gland explant cultures. Our results suggest that relatively minor changes in DNA methylation occur during luminal differentiation compared with the effects of pregnancy on CD24(+)CD61(+)CD29(hi) and CD24(+)CD61(+)CD29(lo) cells.
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http://dx.doi.org/10.1016/j.stemcr.2014.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4325231PMC
February 2015

Attenuated transforming growth factor beta signaling promotes metastasis in a model of HER2 mammary carcinogenesis.

Breast Cancer Res 2014 Oct 4;16(5):425. Epub 2014 Oct 4.

Introduction: Transforming growth factor beta (TGFβ) plays a major role in the regulation of tumor initiation, progression, and metastasis. It is depended on the type II TGFβ receptor (TβRII) for signaling. Previously, we have shown that deletion of TβRII in mammary epithelial of MMTV-PyMT mice results in shortened tumor latency and increased lung metastases. However, active TGFβ signaling increased the number of circulating tumor cells and metastases in MMTV-Neu mice. In the current study, we describe a newly discovered connection between attenuated TGFβ signaling and human epidermal growth factor receptor 2 (HER2) signaling in mammary tumor progression.

Methods: All studies were performed on MMTV-Neu mice with and without dominant-negative TβRII (DNIIR) in mammary epithelium. Mammary tumors were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence staining. The levels of secreted proteins were measured by enzyme-linked immunosorbent assay. Whole-lung mount staining was used to quantitate lung metastasis. The Cancer Genome Atlas (TCGA) datasets were used to determine the relevance of our findings to human breast cancer.

Results: Attenuated TGFβ signaling led to a delay tumor onset, but increased the number of metastases in MMTVNeu/DNIIR mice. The DNIIR tumors were characterized by increased vasculogenesis, vessel leakage, and increased expression of vascular endothelial growth factor (VEGF). During DNIIR tumor progression, both the levels of CXCL1/5 and the number of CD11b+Gr1+ cells and T cells decreased. Analysis of TCGA datasets demonstrated a significant negative correlation between TGFBR2 and VEGF genes expression. Higher VEGFA expression correlated with shorter distant metastasis-free survival only in HER2+ patients with no differences in HER2-, estrogen receptor +/- or progesterone receptor +/- breast cancer patients.

Conclusion: Our studies provide insights into a novel mechanism by which epithelial TGFβ signaling modulates the tumor microenvironment, and by which it is involved in lung metastasis in HER2+ breast cancer patients. The effects of pharmacological targeting of the TGFβ pathway in vivo during tumor progression remain controversial. The targeting of TGFβ signaling should be a viable option, but because VEGF has a protumorigenic effect on HER2+ tumors, the targeting of this protein could be considered when it is associated with attenuated TGFβ signaling.
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http://dx.doi.org/10.1186/s13058-014-0425-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303109PMC
October 2014

BMPR2 loss in fibroblasts promotes mammary carcinoma metastasis via increased inflammation.

Mol Oncol 2015 Jan 23;9(1):179-91. Epub 2014 Aug 23.

Department of Cancer Biology, USA. Electronic address:

Bone Morphogenetic Protein (BMP) receptors mediate a diverse range of signals to regulate both development and disease. BMP activity has been linked to both tumor promoting and suppressive functions in both tumor cells and their surrounding microenvironment. We sought to investigate the requirement for BMPR2 in stromal fibroblasts during mammary tumor formation and metastasis. We utilized FSP1 (Fibroblast Specific Protein-1) promoter driven Cre to genetically delete BMPR2 in mice expressing the MMTV.PyVmT mammary carcinoma oncogene. We found that abrogation of stromal BMPR2 expression via FSP1 driven Cre resulted in increased tumor metastasis. Additionally, similar to epithelial BMPR2 abrogation, stromal loss of BMPR2 results in increased inflammatory cell infiltration. We proceeded to isolate and establish fibroblast cell lines without BMPR2 and found a cell autonomous increase in inflammatory cytokine secretion. Fibroblasts were co-implanted with syngeneic tumor cells and resulted in accelerated tumor growth and increased metastasis when fibroblasts lacked BMPR2. We observed that the loss of BMPR2 results in increased chemokine expression, which facilitates inflammation by a sustained increase in myeloid cells. The chemokines increased in BMPR2 deleted cells correlated with poor outcome in human breast cancer patients. We conclude that BMPR2 has tumor suppressive functions in the stroma by regulating inflammation.
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http://dx.doi.org/10.1016/j.molonc.2014.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4277920PMC
January 2015

Role of TGF-β signaling in generation of CD39+CD73+ myeloid cells in tumors.

J Immunol 2014 Sep 15;193(6):3155-64. Epub 2014 Aug 15.

Cancer Biology Department, Vanderbilt-Ingram Cancer Center, Nashville, TN 37232

There is growing evidence that generation of adenosine from ATP, which is mediated by the CD39/CD73 enzyme pair, predetermines immunosuppressive and proangiogenic properties of myeloid cells. We have previously shown that the deletion of the TGF-β type II receptor gene (Tgfbr2) expression in myeloid cells is associated with decreased tumor growth, suggesting protumorigenic effect of TGF-β signaling. In this study, we tested the hypothesis that TGF-β drives differentiation of myeloid-derived suppressor cells into protumorigenic terminally differentiated myeloid mononuclear cells (TDMMCs) characterized by high levels of cell-surface CD39/CD73 expression. We found that TDMMCs represent a major cell subpopulation expressing high levels of both CD39 and CD73 in the tumor microenvironment. In tumors isolated from mice with spontaneous tumor formation of mammary gland and conditional deletion of the type II TGF-β receptor in mammary epithelium, an increased level of TGF-β protein was associated with further increase in number of CD39(+)CD73(+) TDMMCs compared with MMTV-PyMT/TGFβRII(WT) control tumors with intact TGF-β signaling. Using genetic and pharmacological approaches, we demonstrated that the TGF-β signaling mediates maturation of myeloid-derived suppressor cells into TDMMCs with high levels of cell surface CD39/CD73 expression and adenosine-generating capacity. Disruption of TGF-β signaling in myeloid cells resulted in decreased accumulation of TDMMCs, expressing CD39 and CD73, and was accompanied by increased infiltration of T lymphocytes, reduced density of blood vessels, and diminished progression of both Lewis lung carcinoma and spontaneous mammary carcinomas. We propose that TGF-β signaling can directly induce the generation of CD39(+)CD73(+) TDMMCs, thus contributing to the immunosuppressive, proangiogenic, and tumor-promoting effects of this pleiotropic effector in the tumor microenvironment.
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http://dx.doi.org/10.4049/jimmunol.1400578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157098PMC
September 2014

Transforming growth factor beta receptor type III is a tumor promoter in mesenchymal-stem like triple negative breast cancer.

Breast Cancer Res 2014 Jul 1;16(4):R69. Epub 2014 Jul 1.

Introduction: There is a major need to better understand the molecular basis of triple negative breast cancer (TNBC) in order to develop effective therapeutic strategies. Using gene expression data from 587 TNBC patients we previously identified six subtypes of the disease, among which a mesenchymal-stem like (MSL) subtype. The MSL subtype has significantly higher expression of the transforming growth factor beta (TGF-β) pathway-associated genes relative to other subtypes, including the TGF-β receptor type III (TβRIII). We hypothesize that TβRIII is tumor promoter in mesenchymal-stem like TNBC cells.

Methods: Representative MSL cell lines SUM159, MDA-MB-231 and MDA-MB-157 were used to study the roles of TβRIII in the MSL subtype. We stably expressed short hairpin RNAs specific to TβRIII (TβRIII-KD). These cells were then used for xenograft tumor studies in vivo; and migration, invasion, proliferation and three dimensional culture studies in vitro. Furthermore, we utilized human gene expression datasets to examine TβRIII expression patterns across all TNBC subtypes.

Results: TβRIII was the most differentially expressed TGF-β signaling gene in the MSL subtype. Silencing TβRIII expression in MSL cell lines significantly decreased cell motility and invasion. In addition, when TβRIII-KD cells were grown in a three dimensional (3D) culture system or nude mice, there was a loss of invasive protrusions and a significant decrease in xenograft tumor growth, respectively. In pursuit of the mechanistic underpinnings for the observed TβRIII-dependent phenotypes, we discovered that integrin-α2 was expressed at higher level in MSL cells after TβRIII-KD. Stable knockdown of integrin-α2 in TβRIII-KD MSL cells rescued the ability of the MSL cells to migrate and invade at the same level as MSL control cells.

Conclusions: We have found that TβRIII is required for migration and invasion in vitro and xenograft growth in vivo. We also show that TβRIII-KD elevates expression of integrin-α2, which is required for the reduced migration and invasion, as determined by siRNA knockdown studies of both TβRIII and integrin-α2. Overall, our results indicate a potential mechanism in which TβRIII modulates integrin-α2 expression to effect MSL cell migration, invasion, and tumorigenicity.
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http://dx.doi.org/10.1186/bcr3684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4095685PMC
July 2014

Stromally derived lysyl oxidase promotes metastasis of transforming growth factor-β-deficient mouse mammary carcinomas.

Cancer Res 2013 Sep 15;73(17):5336-46. Epub 2013 Jul 15.

Department of Cancer Biology, Vanderbilt University School of Medicine and Vanderbilt-Ingram Cancer Center, Nashville, TN 37212, USA.

The tumor stromal environment can dictate many aspects of tumor progression. A complete understanding of factors driving stromal activation and their role in tumor metastasis is critical to furthering research with the goal of therapeutic intervention. Polyoma middle T-induced mammary carcinomas lacking the type II TGF-β receptor (PyMT(mgko)) are highly metastatic compared with control PyMT-induced carcinomas (PyMT(fl/fl)). We hypothesized that the PyMT(mgko)-activated stroma interacts with carcinoma cells to promote invasion and metastasis. We show that the extracellular matrix associated with PyMT(mgko) tumors is stiffer and has more fibrillar collagen and increased expression of the collagen crosslinking enzyme lysyl oxidase (LOX) compared with PyMT(fl/fl) mammary carcinomas. Inhibition of LOX activity in PyMT(mgko) mice had no effect on tumor latency and size, but significantly decreased tumor metastasis through inhibition of tumor cell intravasation. This phenotype was associated with a decrease in keratin 14-positive myoepithelial cells in PyMT(mgko) tumors following LOX inhibition as well as a decrease in focal adhesion formation. Interestingly, the primary source of LOX was found to be activated fibroblasts. LOX expression in these fibroblasts can be driven by myeloid cell-derived TGF-β, which is significantly linked to human breast cancer. Overall, stromal expansion in PyMT(mgko) tumors is likely caused through the modulation of immune cell infiltrates to promote fibroblast activation. This feeds back to the epithelium to promote metastasis by modulating phenotypic characteristics of basal cells. Our data indicate that epithelial induction of microenvironmental changes can play a significant role in tumorigenesis and attenuating these changes can inhibit metastasis. Cancer Res; 73(17); 5336-46. ©2013 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-0012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3766496PMC
September 2013

Lack of transforming growth factor-β signaling promotes collective cancer cell invasion through tumor-stromal crosstalk.

Breast Cancer Res 2012 Jul 2;14(4):R98. Epub 2012 Jul 2.

Introduction: Transforming growth factor beta (TGF-β) has a dual role during tumor progression, initially as a suppressor and then as a promoter. Epithelial TGF-β signaling regulates fibroblast recruitment and activation. Concurrently, TGF-β signaling in stromal fibroblasts suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Much is known about the contribution of TGF-β signaling to tumorigenesis, yet the role of TGF-β in epithelial-stromal migration during tumor progression is poorly understood. We hypothesize that TGF-β is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion.

Methods: Fluorescently labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT transforming growth factor-beta receptor II knockout (TβRII KO) or TβRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. These combinatorial xenografts were used as a model to study epithelial-stromal crosstalk. Intravital imaging of migration was monitored ex ovo, and metastasis was investigated in ovo. Epithelial RNA from in ovo tumors was isolated by laser capture microdissection and analyzed to identify gene expression changes in response to TGF-β signaling loss.

Results: Intravital microscopy of xenografts revealed that mammary fibroblasts promoted two migratory phenotypes dependent on epithelial TGF-β signaling: single cell/strand migration or collective migration. At epithelial-stromal boundaries, single cell/strand migration of TβRIIfl/fl carcinoma cells was characterized by expression of α-smooth muscle actin and vimentin, while collective migration of TβRII KO carcinoma cells was identified by E-cadherin+/p120+/β-catenin+ clusters. TβRII KO tumors also exhibited a twofold greater metastasis than TβRIIfl/fl tumors, attributed to enhanced extravasation ability. In TβRII KO tumor epithelium compared with TβRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Immunoblotting and quantitative PCR analyses on cultured cells validated these targets and correlated Tmeff1 expression with disease progression of TGF-β-insensitive mammary cancer.

Conclusion: Fibroblast-stimulated carcinoma cells utilize TGF-β signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-β signaling. These migration patterns involve the signaling regulation of several epithelial-to-mesenchymal transition pathways. Our findings concerning TGF-β signaling in epithelial-stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.
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http://dx.doi.org/10.1186/bcr3217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680921PMC
July 2012

Deletion of TGF-β signaling in myeloid cells enhances their anti-tumorigenic properties.

J Leukoc Biol 2012 Sep 8;92(3):641-51. Epub 2012 Jun 8.

Department of Cancer Biology, Vanderbilt University School of Medicine and Vanderbilt-Ingram Comprehensive Cancer Center, Nashville, TN 37232, USA.

By crossing LysM-Cre and TGF-β type II receptor (Tgfbr2) floxed mice we achieved specific deletion of Tgfbr2 in myeloid cells (Tgfbr2(MyeKO) mice). S.c.-injected (LLC, EL4-OVA) and implanted (MMTV-PyMT) carcinoma cells grow slower in Tgfbr2(MyeKO) mice. The number of CD45(+) cells in the tumor tissue was the same in both genotypes of mice, but upon analysis, the percentage of T cells (CD45(+)CD3(+)) in the KO mice was increased. By flow cytometry analysis, we did not detect any differences in the number and phenotype of TAMs, CD11b(+)Gr1(+), and DCs in Tgfbr2(MyeKO) compared with Tgfbr2(MyeWT) mice. ELISA and qRT-PCR data showed differences in myeloid cell functions. In Tgfbr2(MyeKO) TAMs, TNF-α secretion was increased, basal IL-6 secretion was down-regulated, TGF-β did not induce any VEGF response, and there was decreased MMP9 and increased MMP2 and iNOS expression. TGF-β did not have any effect on CD11b(+)Gr1(+) cells isolated from Tgfbr2(MyeKO) mice in the regulation of Arg, iNOS, VEGF, and CXCR4, and moreover, these cells have decreased suppressive activity relative to T cell proliferation. Also, we found that DCs from tumor tissue of Tgfbr2(MyeKO) mice have increased antigen-presented properties and an enhanced ability to stimulate antigen-specific T cell proliferation. We conclude that Tgfbr2 in myeloid cells has a negative role in the regulation of anti-tumorigenic functions of these cells, and deletion of this receptor decreases the suppressive function of CD11b(+)Gr1(+) cells and increases antigen-presenting properties of DCs and anti-tumorigenic properties of TAMs.
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http://dx.doi.org/10.1189/jlb.1211639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427612PMC
September 2012

TGF-β receptor II loss promotes mammary carcinoma progression by Th17 dependent mechanisms.

Cancer Discov 2011 Oct;1(5):430-41

Department of Cancer Biology, Vanderbilt University, Nashville, TN 37212, USA.

We report that IL-17 significantly increases the secretion of CXCL1 and CXCL5 from mammary carcinoma cells, which is downregulated by TGF-β through the type II TGF-β receptor (TβRII). Carcinoma cells with conditional knockout of TβRII (Tgfbr2(KO)) have enhanced sensitivity to IL-17a in the stimulation of chemokine secretion. During polyoma middle T (PyMT) induced tumor progression, levels of Th17 inducing cytokines TGF-β, IL-6, IL-23 were increased in PyMT/Tgfbr2(KO) tumors, which was associated with an increased number of Th17 cells. IL-17 increased the suppressive function of MDSCs on T cells through the upregulation of Arg, IDO, and COX2. Treatment of PyMT/Tgfbr2(KO) mice with anti-IL-17 Ab decreased carcinoma growth and metastatic burden. Analysis of human breast cancer transcriptome databases showed a strong association between IL-17 gene expression and poor outcome in lymph node positive, estrogen receptor negative or luminal B subtypes suggesting potential therapeutic approaches.
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http://dx.doi.org/10.1158/2159-8290.CD-11-0100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3297196PMC
October 2011

Inhibiting Cxcr2 disrupts tumor-stromal interactions and improves survival in a mouse model of pancreatic ductal adenocarcinoma.

J Clin Invest 2011 Oct 19;121(10):4106-17. Epub 2011 Sep 19.

Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal neoplasms, is characterized by an expanded stroma with marked fibrosis (desmoplasia). We previously generated pancreas epithelium-specific TGF-β receptor type II (Tgfbr2) knockout mice in the context of Kras activation (mice referred to herein as Kras+Tgfbr2KO mice) and found that they developed aggressive PDAC that recapitulated the histological manifestations of the human disease. The mouse PDAC tissue showed strong expression of connective tissue growth factor (Ctgf), a profibrotic and tumor-promoting factor, especially in the tumor-stromal border area, suggesting an active tumor-stromal interaction. Here we show that the PDAC cells in Kras+Tgfbr2KO mice secreted much higher levels of several Cxc chemokines compared with mouse pancreatic intraepithelial neoplasia cells, which are preinvasive. The Cxc chemokines induced Ctgf expression in the pancreatic stromal fibroblasts, not in the PDAC cells themselves. Subcutaneous grafting studies revealed that the fibroblasts enhanced growth of PDAC cell allografts, which was attenuated by Cxcr2 inhibition. Moreover, treating the Kras+Tgfbr2KO mice with the CXCR2 inhibitor reduced tumor progression. The decreased tumor progression correlated with reduced Ctgf expression and angiogenesis and increased overall survival. Taken together, our data indicate that tumor-stromal interactions via a Cxcr2-dependent chemokine and Ctgf axis can regulate PDAC progression. Further, our results suggest that inhibiting tumor-stromal interactions might be a promising therapeutic strategy for PDAC.
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http://dx.doi.org/10.1172/JCI42754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3195452PMC
October 2011

Disruption of bone morphogenetic protein receptor 2 (BMPR2) in mammary tumors promotes metastases through cell autonomous and paracrine mediators.

Proc Natl Acad Sci U S A 2012 Feb 16;109(8):2814-9. Epub 2011 May 16.

Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Nashville, TN 37232, USA.

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily of signaling molecules. BMPs can elicit a wide range of effects in many cell types and have previously been shown to induce growth inhibition in carcinoma cells as well as normal epithelia. Recently, it has been demonstrated that BMP4 and BMP7 are overexpressed in human breast cancers and may have tumor suppressive and promoting effects. We sought to determine whether disruption of the BMP receptor 2 (BMPR2) would alter mammary tumor progression in mice that express the Polyoma middle T antigen. Mice expressing Polyoma middle T antigen under the mouse mammary tumor virus promoter were combined with mice that have doxycycline-inducible expression of a dominant-negative (DN) BMPR2. We did not observe any differences in tumor latency. However, mice expressing the BMPR2-DN had a fivefold increase in lung metastases. We characterized several cell autonomous changes and found that BMPR2-DN-expressing tumor cells had higher rates of proliferation. We also identified unique changes in inflammatory cells and secreted chemokines/cytokines that accompanied BMPR2-DN-expressing tumors. By immunohistochemistry, it was found that BMPR2-DN primary tumors and metastases had an altered reactive stroma, indicating specific changes in the tumor microenvironment. Among the changes we discovered were increased myeloid derived suppressor cells and the chemokine CCL9. BMP was shown to directly regulate CCL9 expression. We conclude that BMPR2 has tumor-suppressive function in mammary epithelia and microenvironment and that disruption can accelerate mammary carcinoma metastases.
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http://dx.doi.org/10.1073/pnas.1101139108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286911PMC
February 2012

Loss of one Tgfbr2 allele in fibroblasts promotes metastasis in MMTV: polyoma middle T transgenic and transplant mouse models of mammary tumor progression.

Clin Exp Metastasis 2011 Apr 4;28(4):351-66. Epub 2011 Mar 4.

Department of Pathology and Laboratory Medicine, University of Kansas Cancer Center, Kansas City, 66160, USA.

Accumulation of fibroblasts is a phenomenon that significantly correlates with formation of aggressive cancers. While studies have shown that the TGF-β signaling pathway is an important regulator of fibroblast activation, the functional contribution of TGF-β signaling in fibroblasts during multi-step tumor progression remains largely unclear. In previous studies, we used a sub-renal capsule transplantation model to demonstrate that homozygous knockout of the Tgfbr2 gene (Tgbr2(FspKO)) enhanced mammary tumor growth and metastasis. Here, we show for the first time a significant role for loss of one Tgfbr2 allele during multi-step mammary tumor progression. Heterozygous deletion of Tgfbr2 in stromal cells in MMTV-PyVmT transgenic mice (PyVmT/Tgfbr2(hetFspKO) mice) resulted in earlier tumor formation and increased stromal cell accumulation. In contrast to previous studies of Tgbr2(FspKO) fibroblasts, Tgfbr2(hetFspKO) fibroblasts did not significantly increase tumor growth, but enhanced lung metastasis in PyVmT transgenic mice and in co-transplantation studies with PyVmT mammary carcinoma cells. Furthermore, Tgfbr2(hetFspKO) fibroblasts enhanced mammary carcinoma cell invasiveness associated with expression of inflammatory cytokines including CXCL12 and CCL2. Analyses of Tgbr2(FspKO) and Tgfbr2(hetFspKO) fibroblasts revealed differences in the expression of factors associated with metastatic spread, indicating potential differences in the mechanism of action between homozygous and heterozygous deletion of Tgfbr2 in stromal cells. In summary, these studies demonstrate for the first time that loss of one Tgfbr2 allele in fibroblasts enhances mammary metastases in a multi-step model of tumor progression, and demonstrate the importance of clarifying the functional contribution of genetic alterations in stromal cells in breast cancer progression.
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http://dx.doi.org/10.1007/s10585-011-9373-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3373018PMC
April 2011

Gr-1+CD11b+ myeloid cells tip the balance of immune protection to tumor promotion in the premetastatic lung.

Cancer Res 2010 Aug 14;70(15):6139-49. Epub 2010 Jul 14.

Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20876-4258, USA.

The mechanisms by which a primary tumor affects a selected distant organ before tumor cell arrival remain to be elucidated. This report shows that Gr-1+CD11b+ cells are significantly increased in lungs of mice bearing mammary adenocarcinomas before tumor cell arrival. In the premetastatic lungs, these immature myeloid cells significantly decrease IFN-gamma production and increase proinflammatory cytokines. In addition, they produce large quantities of matrix metalloproteinase 9 (MMP9) and promote vascular remodeling. Deletion of MMP9 normalizes aberrant vasculature in the premetastatic lung and diminishes lung metastasis. The production and activity of MMP9 is selectively restricted to lungs and organs with a large number of Gr-1+CD11b+ cells. Our work reveals a novel protumor mechanism for Gr-1+CD11b+ cells that changes the premetastatic lung into an inflammatory and proliferative environment, diminishes immune protection, and promotes metastasis through aberrant vasculature formation. Thus, inhibition of Gr-1+CD11b+ cells could normalize the premetastatic lung environment, improve host immunosurveillance, and inhibit tumor metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-0706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675145PMC
August 2010

Microdialysis combined with proteomics for protein identification in breast tumor microenvironment in vivo.

Cancer Microenviron 2010 Apr 13;4(1):61-71. Epub 2010 Apr 13.

Unlabelled: Tumor microenvironment constitutes a reservoir for proteins released from tumor cells and the host, which can contribute significantly to tumor growth and invasion. This study aims to apply a method of combining in vivo microdialysis and proteomics to identify proteins in mammary tumor interstitial fluids, a major component of tumor microenvironment. In vivo microdialysis was performed in polyomavirus middle T antigen (PyVmT) transgenic mouse mammary tumors and age-matched control wild-type mammary glands. Over four hundred proteins were identified from the microdialysis perfusates, using the Multidimensional Protein Identification Technology. Osteopontin (OPN) is one of the proteins overexpressed in breast tumor perfusates, as confirmed with immunoassays. OPN was also found to be present in tumor-associated stroma in both PyVmT and human breast tumors, using immunohistochemistry. Specifically, fibroblasts were further shown to express OPN at both mRNA and protein levels. In vitro assays showed that OPN can stimulate PyVmT breast carcinoma cell proliferation and migration. Finally, the expression of OPN was significantly higher in the peripheral blood of mice bearing breast tumors, compared to wild-type mice. Overall, microdialysis combined with proteomics is a unique technique for identifying proteins in a tumor microenvironment in vivo. Mammary fibroblasts can secrete OPN, and its overexpression in mammary tumor microenvironment may contribute significantly to mammary tumor progression.

Electronic Supplementary Material: The online version of this article (doi:10.1007/s12307-010-0046-3) contains supplementary material, which is available to authorized users.
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http://dx.doi.org/10.1007/s12307-010-0046-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3047629PMC
April 2010

Abrogation of TGF-beta signaling enhances chemokine production and correlates with prognosis in human breast cancer.

J Clin Invest 2009 Jun 18;119(6):1571-82. Epub 2009 May 18.

Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee, USA.

In human breast cancer, loss of carcinoma cell-specific response to TGF-beta signaling has been linked to poor patient prognosis. However, the mechanisms through which TGF-beta regulates these processes remain largely unknown. In an effort to address this issue, we have now identified gene expression signatures associated with the TGF-beta signaling pathway in human mammary carcinoma cells. The results strongly suggest that TGF-beta signaling mediates intrinsic, stromal-epithelial, and host-tumor interactions during breast cancer progression, at least in part, by regulating basal and oncostatin M-induced CXCL1, CXCL5, and CCL20 chemokine expression. To determine the clinical relevance of our results, we queried our TGF-beta-associated gene expression signatures in 4 human breast cancer data sets containing a total of 1,319 gene expression profiles and associated clinical outcome data. The signature representing complete abrogation of TGF-beta signaling correlated with reduced relapse-free survival in all patients; however, the strongest association was observed in patients with estrogen receptor-positive (ER-positive) tumors, specifically within the luminal A subtype. Together, the results suggest that assessment of TGF-beta signaling pathway status may further stratify the prognosis of ER-positive patients and provide novel therapeutic approaches in the management of breast cancer.
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http://dx.doi.org/10.1172/JCI37480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2689133PMC
June 2009

GFAP-Cre-mediated activation of oncogenic K-ras results in expansion of the subventricular zone and infiltrating glioma.

Mol Cancer Res 2009 May 12;7(5):645-53. Epub 2009 May 12.

Department of Pathology, Vanderbilt University Medical Center, MCN C-2318, Nashville, TN 37232-2561, USA.

A subset of neoplastic cells within human high-grade gliomas has features associated with stem cells. These cells may sustain glioma growth, and their stem-like properties may confer resistance to standard glioma treatments. Whether glioma stem cells derive from indigenous neural stem cells (NSC), or from tumor cells that have reacquired stem cell-like properties, is unknown. However, signaling pathways that are tightly regulated and central to NSC biology, including the Ras/Raf/Erk pathway, are hyperactive and pathogenic in gliomagenesis. Furthermore, data in animal models suggests that, in some cases, tumors are initiated in the subventricular zone (SVZ), a stem/progenitor cell niche in the mature brain. We activated oncogenic K-ras in mouse glioneuronal precursor cells and adult SVZ cells using GFAP-Cre. GFAP-Cre+/K-ras(G12D) mice showed a marked expansion of glial fibriallary acidic protein (GFAP)- and TUJ1-expressing cell populations in the SVZ. In addition, mice developed intermediate grade, infiltrating glioma with 100% penetrance. Tumors were consistently located in the amygdalohippocampal region and nearby cortex, often in association with the lateral ventricle and expanded SVZ. Tumor cells expressed markers associated with neural progenitor cells, including Olig2, Bmi-1, and PDGFR-alpha. These data suggest that infiltrating tumor cells may arise from NSC transformed by activation of oncogenic K-ras in vivo.
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http://dx.doi.org/10.1158/1541-7786.MCR-08-0477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3406747PMC
May 2009

Transforming growth factor-beta signaling-deficient fibroblasts enhance hepatocyte growth factor signaling in mammary carcinoma cells to promote scattering and invasion.

Mol Cancer Res 2008 Oct;6(10):1521-33

Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-6838, USA.

Fibroblasts are major cellular components of the tumor microenvironment, regulating tumor cell behavior in part through secretion of extracellular matrix proteins, growth factors, and angiogenic factors. In previous studies, conditional deletion of the type II transforming growth factor-beta (TGF-beta) receptor in fibroblasts (Tgfbr2FspKO) was shown to promote mammary tumor metastasis in fibroblast-epithelial cell cotransplantation studies in mice, correlating with increased expression of hepatocyte growth factor (HGF). Here, we advance our findings to show that Tgfbr2(FspKO) fibroblasts enhance HGF/c-Met and HGF/Ron signaling to promote scattering and invasion of mammary carcinoma cells. Blockade of c-Met and Ron by small interfering RNA silencing and pharmacologic inhibitors significantly reduced mammary carcinoma cell scattering and invasion caused by Tgfbr2FspKO fibroblasts. Moreover, neutralizing antibodies to c-Met and Ron significantly inhibited HGF-induced cell scattering and invasion, correlating with reduced Stat3 and p42/44MAPK phosphorylation. Investigation of the signal transducer and activator of transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) signaling pathways by pharmacologic inhibition and small interfering RNA silencing revealed a cooperative interaction between the two pathways to regulate HGF-induced invasion, scattering, and motility of mammary tumor cells. Furthermore, whereas c-Met was found to regulate both the Stat3 and MAPK signaling pathways, Ron was found to regulate Stat3 but not MAPK signaling in mammary carcinoma cells. These studies show a tumor-suppressive role for TGF-beta signaling in fibroblasts, in part by suppressing HGF signaling between mammary fibroblasts and epithelial cells. These studies characterize complex functional roles for HGF and TGF-beta signaling in mediating tumor-stromal interactions during mammary tumor cell scattering and invasion, with important implications in the metastatic process.
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http://dx.doi.org/10.1158/1541-7786.MCR-07-2203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2740918PMC
October 2008

Identification of novel Smad2 and Smad3 associated proteins in response to TGF-beta1.

J Cell Biochem 2008 Oct;105(2):596-611

Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

Transforming growth factor-beta 1 (TGF-beta1) is an important growth inhibitor of epithelial cells and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. TGF-beta1 signals through the TGF-beta type I and type II receptors, and activates the Smad pathway via phosphorylation of Smad2 and Smad3. Since little is known about the selective activation of Smad2 versus Smad3, we set out to identify novel Smad2 and Smad3 interacting proteins in epithelial cells. A non-transformed human cell line was transduced with Myc-His(6)-Smad2 or Myc-His(6)-Smad3-expressing retrovirus and was treated with TGF-beta1. Myc-His(6)-Smad2 or Myc-His(6)-Smad3 was purified by tandem affinity purification, eluates were subject to SDS-PAGE and Colloidal Blue staining, and select protein bands were digested with trypsin. The resulting tryptic peptides were analyzed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS) and the SEQUEST algorithm was employed to identify proteins in the bands. A number of proteins that are known to interact with Smad2 or Smad3 were detected in the eluates. In addition, a number of putative novel Smad2 and Smad3 associated proteins were identified that have functions in cell proliferation, apoptosis, actin cytoskeleton regulation, cell motility, transcription, and Ras or insulin signaling. Specifically, the interaction between Smad2/3 and the Cdc42 guanine nucleotide exchange factor, Zizimin1, was validated by co-immunoprecipitation. The discovery of these novel Smad2 and/or Smad3 associated proteins may reveal how Smad2 and Smad3 are regulated and/or uncover new functions of Smad2 and Smad3 in TGF-beta1 signaling.
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http://dx.doi.org/10.1002/jcb.21860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2700048PMC
October 2008

Transforming growth factor-beta regulates mammary carcinoma cell survival and interaction with the adjacent microenvironment.

Cancer Res 2008 Mar;68(6):1809-19

Department of Cancer Biology, Vanderbilt University, Nashville, TN 37232-6838, USA.

Transforming growth factor (TGF)-beta signaling has been associated with early tumor suppression and late tumor progression; however, many of the mechanisms that mediate these processes are not known. Using Cre/LoxP technology, with the whey acidic protein promoter driving transgenic expression of Cre recombinase (WAP-Cre), we have now ablated the type II TGF-beta receptor (T beta RII) expression specifically within mouse mammary alveolar progenitors. Transgenic expression of the polyoma virus middle T antigen, under control of the mouse mammary tumor virus enhancer/promoter, was used to produce mammary tumors in the absence or presence of Cre (T beta RII((fl/fl);PY) and T beta RII((fl/fl);PY;WC), respectively). The loss of TGF-beta signaling significantly decreased tumor latency and increased the rate of pulmonary metastasis. The loss of TGF-beta signaling was significantly correlated with increased tumor size and enhanced carcinoma cell survival. In addition, we observed significant differences in stromal fibrovascular abundance and composition accompanied by increased recruitment of F4/80(+) cell populations in T beta RII((fl/fl);PY;WC) mice when compared with T beta RII((fl/fl);PY) controls. The recruitment of F4/80(+) cells correlated with increased expression of known inflammatory genes including Cxcl1, Cxcl5, and Ptgs2 (cyclooxygenase-2). Notably, we also identified an enriched K5(+) dNp63(+) cell population in primary T beta RII((fl/fl);PY;WC) tumors and corresponding pulmonary metastases, suggesting that loss of TGF-beta signaling in this subset of carcinoma cells can contribute to metastasis. Together, our current results indicate that loss of TGF-beta signaling in mammary alveolar progenitors may affect tumor initiation, progression, and metastasis through regulation of both intrinsic cell signaling and adjacent stromal-epithelial interactions in vivo.
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http://dx.doi.org/10.1158/0008-5472.CAN-07-5597DOI Listing
March 2008

Abrogation of TGF beta signaling in mammary carcinomas recruits Gr-1+CD11b+ myeloid cells that promote metastasis.

Cancer Cell 2008 Jan;13(1):23-35

Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

Aberrant TGFbeta signaling is common in human cancers and contributes to tumor metastasis. Here, we demonstrate that Gr-1+CD11b+ myeloid cells are recruited into mammary carcinomas with type II TGF beta receptor gene (Tgfbr2) deletion and directly promote tumor metastasis. Gr-1+CD11b+ cells infiltrate into the invasive front of tumor tissues and facilitate tumor cell invasion and metastasis through a process involving metalloproteinase activity. This infiltration of Gr-1+CD11b+ cells also results in increased abundance of TGF beta 1 in tumors with Tgfbr2 deletion. The recruitment of Gr-1+CD11b+ cells into tumors with Tgfbr2 deletion involves two chemokine receptor axes, the SDF-1/CXCR4 and CXCL5/CXCR2 axes. Together, these data indicate that Gr-1+CD11b+ cells contribute to TGFbeta-mediated metastasis through enhancing tumor cell invasion and metastasis.
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http://dx.doi.org/10.1016/j.ccr.2007.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2245859PMC
January 2008
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