Our Science – Vonderhaar Website
Barbara K. Vonderhaar, Ph.D.
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Biography
Dr. Vonderhaar received her Ph.D. from the University of Wisconsin-Madison. After postdoctoral training in mammary gland biology with Yale Topper at the NIH, she joined the NCI where she studies prolactin action in breast cancer. She was the first to purify a prolactin receptor from any source and the first to characterize a monoclonal antibody directed against the human prolactin receptor. She was also the first to demonstrate that human breast cancer cells synthesize and secrete significant amounts of biologically active prolactin. She serves as Chief of the Mammary Biology and Tumorigenesis Laboratory, Co-Chair of the Breast and Gynecologic Malignancies Faculty, Co-Chair of the Cancer Stem Cell Consortium and Co-Chair of the Intramural Program for Research on Women's Health. Dr. Vonderhaar is a member of the Senior Biomedical Research Service.Research
Prolactin Action in Mammary Gland Development and Tumorigenesis
The emphasis of our research is on understanding the mechanisms of prolactin (PRL) action in concert with estrogen (E), progesterone (P), epidermal growth factor (EGF), and insulin-like growth factors (IGF-I and IGF-II) in mammary gland development, differentiation, and tumorigenesis. Both in vivo and in vitro approaches are used to confirm physiological relevance.
Development of the Normal Gland
The mammary gland is a complex organ whose growth and development are controlled by the interaction of a wide variety of hormones and growth factors also involved in the etiology and progression of the cancerous state. Our emphasis has been on the interactions of prolactin (PRL), estrogen (E), and progesterone (P) during the peripubertal period and the lobulo-alveolar development of pregnancy as well as during tumorigenesis. We have shown that E and P are required to promote development of the primary/secondary ductal network in addition to other endocrine growth factor(s), and that P facilitates the formation of tertiary side-branches. The Hox-related homeobox containing gene, Msx2, is highly expressed during branching morphogenesis where our studies in vivo and in vitro showed that its expression is regulated by P in the presence of E. Concurrent with these morphological changes, progesterone receptor (PR) localizes at early branch points. During peripubertal morphogenesis PR distribution shifted from a homogeneous to a heterogeneous pattern. Concomitantly the PRL receptor (PRLR) undergoes a similar shift in pattern. The transcription factor, C/EBP-beta appears to regulate mammary epithelial cell fate resulting in the correct spatial pattern of gene expression required to permit steroid hormone regulated cell proliferation.We demonstrated differential transcription of the four PRLR isoforms by stromal as well as epithelial cells throughout development. The distribution of the PRLR in the epithelium, like that of the PR, progressed from a homogeneous to a heterogeneous pattern. Hence, while exogenous P or PRL alone was without effect on epithelial proliferation in ovariectomized mice, these hormones synergize to stimulate epithelial and stromal proliferation. We are studying changes in the vascular network that facilitates lactogenesis and tumorigenesis in the mammary gland. Our data support the conclusion that specific cell types within the mammary gland differentially transcribe VEGF and that it functions as an autocrine/paracrine endothelial growth factor under hormonal regulation. We have identified that PRL induces expression of VEGF in breast cells through an increase in the transcription factor Egr-1.
PRL and its Receptors in Breast Cancer
Additional studies aim to understand the role of PRL in the etiology and progression of human breast cancer. Specifically, we are examining the role of PRLR isoforms and autocrine/paracrine PRL in tumorigenesis and carcinogenic susceptibility. Comparisons between cancerous and adjacent, noninvolved tissue from the same breast of 23 patients showed that, on average, both PRL and PRLR mRNA expression was significantly higher in the cancerous tissue compared to the noninvolved tissue. The various forms of the PRLR differ in their cytoplasmic domains due to alternate splicing. Using 3' RACE we isolated five splice variants of the hPRLR, three of which encode the complete extracellular binding domain. Two of these isoforms, short form 1a (SF1a) and short form 1b (SF1b), possess unique intracellular domains encoded by splicing to exon 11 from exons 10 and 9, respectively. A third novel isoform (delta7/11) reflects alternative splicing from exon 7 to exon 11 and encodes a secreted soluble PRL-binding protein. Additional splice variants of SF1b and delta7/11 that lacked exon 4 (delta4-SF1b and delta4-delta7/11) were also identified. Functional analyses indicated that hPRLR-SF1b is a strong dominant negative to the differentiative function of the PRLR long form while hPRLR-SF1a is a weaker dominant negative. Differential abundance of SF1a, SF1b and delta7/11 expression was detected in normal breast, colon, placenta, kidney, liver, ovary and pancreas, and breast and colon tumors. Taken together, these data indicate the presence of multiple isoforms of the hPRLR that may function to modulate the endocrine and autocrine effects of PRL in normal human tissue and cancer.
Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal and monoclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. Antibodies were validated by Western blot, immunoprecipitation and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. Our antibodies have high titer and can specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. While nearly all tumors contained LF and SF1b, the majority (76%) of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72%) and 27% had only low levels of expression. Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in treatment of this disease by providing new reagents to study the protein expression of the human PRLR.
Several isoforms of PRL exist in human circulation, including a 16 kDa isoform that is an N-terminal fragment of the full-length 23 kDa PRL. 16 kDa PRL has been shown to be anti-angiogenic in vitro and in vivo, and to reduce formation of tumors from prostate, colon and melanoma cancer cell lines. We explored the effect of 16 kDa PRL expression in vitro and in vivo using two breast cancer cell line models (MCF-7 and MDA-MB-231) and also the HCT-116 colon cancer cell line. In all three cell lines, 16 kDa PRL expression inhibited cell proliferation in vitro compared to empty vector controls. In vivo results were markedly different between the two types of cell lines. HCT-116 cells expressing 16 kDa PRL exhibited reduced vascularization and tumor formation, consistent with published results. The breast cancer cell lines expressing 16 kDa PRL also exhibited inhibition of angiogenesis in vivo but no reduction in tumor size or formation. These results suggest that the effects of 16 kDa PRL on tumor formation may vary across tissue types. The unique sensitivity of breast cancer to PRL as a mitogen and/or additional factors in the mammary gland environment (e.g. local hormone/mitogen concentration) may play a dominant role in tumor formation in vivo, thus outweighing the anti-angiogenesis effects and in vitro reduction in cell proliferation induced by 16 kDa PRL.
Breast tumor microenvironment
While breast cancer studies frequently focus on the role of the tumor microenvironment in the promotion of cancer; however, the influence of the normal breast microenvironment on cancer cells remains relatively unknown. Breast cancer studies implant human cancer cells under the renal capsule, subcutaneously, or orthotopically and often use estrogen supplementation and immune suppressants (etoposide) in xenograft mouse models. However, cell behavior is significantly impacted by signals from the local microenvironment. Therefore, we investigated how the combinatorial effect of the microenvironment and procedural differences affected xenograft characteristics. Patient-derived breast cancer cells were in the presence or absence of estrogen and/or etoposide pretreatment. Abdominal xenografts had increased tumor incidence and volume, and decreased latency compared to thoracic tumors. No statistically significant difference in tumor volume was found in abdominal xenografts treated in the presence or absence estrogen or etoposide; however, etoposide suppressed tumor volume in thoracic xenografts. The combination of estrogen and etoposide significantly decreased tumor incidence in both sites. In addition, mice treated in the presence or absence of estradiol were injected orthotopically or subcutaneously with well-characterized breast cancer cell lines (MCF7, ZR75-1, MDA MB-231, or MCF10Ca1h). Orthotopic injection increased tumor volume; growth varied with estrogen supplementation. Location also altered methylation status of several breast cancer-related gene promoters. Lastly, vascularization of orthotopic tumors was significantly enhanced compared to subcutaneous tumors. These data suggest that optimal xenograft success occurs with orthotopic abdominal injections and illustrate molecular details of the compelling influence of the local microenvironment on in vivo models.
To investigate the role of the normal breast microenvironment on breast cancer cell tumorigenicity, we examined whether extracellular matrix molecules (ECM) derived from pre-menopausal African-American (AA) or Caucasian-American (CAU) breast tissue would affect the tumorigenicity of cancer cells in vitro and in vivo. We chose these two populations because of the well-documented predisposition of AA to develop aggressive, highly metastatic breast cancer compared to CAU women. The effects of primary breast fibroblasts on tumorigenicity were analyzed via real-time PCR arrays and mouse xenograft models. Whole breast-ECM was isolated, analyzed via zymography, and its effects on breast cancer cell aggressiveness were tested in vitro via soft agar and invasion assays, and in vivo via xenograft models. Breast ECM and hormone metabolites were analyzed via mass spectrometry. Mouse mammary glands humanized with pre-menopausal CAU-fibroblasts and injected with primary breast cancer cells developed significantly larger tumors compared to AA-humanized glands. Examination of 164 ECM molecules and cytokines from CAU -derived fibroblasts demonstrated a differentially regulated set of ECM proteins and increased cytokine expression. Whole breast-ECM was isolated; invasion and soft agar assays demonstrated that ER-/PR- cells were significantly more aggressive when in contact with AA ECM, as were ER+/PR+ cells with CAU ECM. Using zymography, protease activity was comparatively upregulated in CAU ECM. In xenograft models, CAU ECM significantly increased the tumorigenicity of ER+/PR+ cells and enhanced metastases. Mass spectrometry analysis of ECM proteins showed that only 1,759 of ~8,000 identified were in common. In the AA dataset, proteins associated with breast cancer were primarily related to tumorigenesis/neoplasia, while CAU -unique proteins were involved with growth/metastasis. Using a novel mass spectrometry method, 17 biologically-active hormones were measured; estradiol, estriol and 2-methoxyestrone were significantly higher in CAU breast tissue.
Breast cancer stem cells
The invasive, mesenchymal phenotype of CD44posCD24neg breast cancer cells has made them a promising target for eliminating the metastatic capacity of primary tumors. It has been demonstrated previously that CD44neg/dimCD24pos breast cancer cells lack the ability to give rise to their invasive CD44posCD24neg counterpart. We have demonstrated that noninvasive, epithelial-like CD44posCD24pos cells readily give rise to invasive, mesenchymal CD44posCD24neg progeny in vivo and in vitro. This interconversion was found to be dependent upon Activin/Nodal signaling. We sorted breast cancer cell lines into CD44posCD24pos and CD44posCD24neg populations to evaluate their progeny for the expression of CD44, CD24, and markers of a mesenchymal phenotype. The FACS sorted populations were injected into immunocompromised mice to evaluate their tumorigenicity and invasiveness of the resulting xenografts. CD24 expression was dynamically regulated in vitro in all evaluated breast cancer cell lines. Furthermore, a single noninvasive, epithelial-like CD44posCD24pos cell had the ability to give rise to invasive, mesenchymal CD44posCD24neg progeny. Importantly, this interconversion occurred in vivo as CD44posCD24pos cells gave rise to xenografts with a capacity for local invasion similar to that seen with xenografts initiated with CD44posCD24neg cells. Lastly, the ability of CD44posCD24pos cells to give rise to mesenchymal progeny, and vice versa, was blocked upon ablation of Activin/Nodal signaling. Our data demonstrate that the invasive, mesenchymal CD44posCD24neg phenotype is under dynamic control in breast cancer cell lines both in vivo and in vitro and suggest that targeted therapy against CD44posCD24neg tumor cells may have limited success in preventing primary tumor metastasis unless Activin/Nodal signaling is arrested.
It has been speculated that between estrogen receptor (ER) negative and ER positive breast cancer originate from different cellular subpopulations of the normal gland. We have demonstrated that in ER negative breast tumors and cancer xenografts, CD44posCD24neg and CD44posCD24pos populations are equally tumorigenic. Instead, xenograft-initiating cells (XIC) for ER negative breast cancer are enriched in the CD44posCD133hiCD49fhi population. In addition to their heightened tumorigenicity, CD44posCD133hiCD49fhi cells are capable of self-renewal in vivo and give rise to functional and molecular heterogeneity. Consistent with their capacity for self-renewal, CD44posCD133hiCD49fhi cells express elevated levels of Sox2, Bmi-1, and/or Nanog. We observed that relative to non-tumorigenic cells, CD44posCD133hiCD49fhi cells are globally hypomethylated while CpG islands are hypermethylated. These differences in methylome regulation may be responsible for the dramatic functional differences between the two populations. The identification of CD44posCD133hiCD49fhi TIC for ER negative tumors may lead to expanded understanding of these tumors and ultimately the development of therapeutics designed to specifically target these cells.
Our collaborators are Paul Goldsmith, Antibody and Protein Purification Unit, CCR, NCI; Charles Brooks, Ohio State University; Patricia Berg, George Washington University; Russell Hovey, UC-Davis; Jessica Faupel-Badger, Cancer Prevention Fellowship Program, NCI; Mark Sherman, DCEG, NCI.
This page was last updated on 3/5/2013.
