Stephen P. Ethier - US grants

The overall objective of these studies is to examine the alterations in cellular growth control that occur during neoplastic transformation of rat mammary epithelial (RME) cells. The working hypothesis is that normal cells have a finite proliferative lifespan and that an important alteration in cellular growth potential occurs during cancer development which results in the acquisition of an enhanced, perhaps indefinite, proliferative lifespan. Acquisition of this enhanced proliferative potential is necessary but insufficient for complete neoplastic transformation and therefore represents a truly preneoplastic phenotype. The experiments will employ a newly developed culture system in which normal RME cells undergo 15 to 20 population doublings over 4 to 5 in vitro passages before exhibiting signs of senescence. This culture system will be used; a) to examine the hormone and growth factor requirements that induce long term proliferation of normal RME cells in vitro, b) to isolate populations of preneoplastic cells from mammary tissues of carcinogen treated rats and populations of neoplastic cells from mammary carcinomas based on their enhanced proliferative lifespan, 3) to examine the hormone and growth factor requirements of preneoplastic and neoplastic RME cells relative to those of normal RME cells in vitro. These studies address the mechanisms of the alterations in growth control that occur in RME cells during the stepwise acquisition of enhanced proliferative potential and neoplastic potential.

The broad objective of this project is to identify specific alterations in growth control mechanisms that are causally related to the neoplastic growth potential of rat mammary carcinoma (RMT) cells. To accomplish this, experiments have focused on understanding growth control mechanisms operative for normal rat mammary epithelial (RME) cells and from that perspective examining for normal rat mammary epithelial (RME) cells and from that perspective examining growth control in corresponding tumor derived cells. We have found that, whereas RME cells have a finite and reproducible proliferative lifespan in culture, every primary tumor examined has cells that are immortal with respect to their proliferative potential in culture. Despite this difference, no all immortal RMT cells express neoplastic potential when transplanted into syngeneic hosts. However, immortal RMT cells that also have become independent of growth factors strictly required by RME cells for growth in serum-free culture express high neoplastic potential upon transplantation to syngeneic recipients. Therefore, this project is aimed at determining the cellular and molecular basis for the acquisition of growth factor independence by neoplastic cells and to test directly the hypothesis that growth factor independence is causally related to neoplastic potential. The specific aims are: 1) To determine if the loss of specific growth factor requirements by growth factor independent RMT cells results from growth factor synthesis by these cells and if so, to identify and characterize the autocrine factors responsible for this altered phenotype. 2) To determine if induction of growth factor independence in normal RME cells or in immortalized growth factor dependent RMT cells by chemical carcinogen treatment in vitro results in concomitant acquisition of neoplastic potential in vivo; and 3) To test the hypothesis that some (or all) growth factor independent RMT cells became so by mechanism not directly involving growth factor synthesis, but via activation of proto- oncogenes that are homologous to growth factor receptors, (e.g. c- erbB or e.g. c-erbB-2) or that act in growth factor mediated signal transduction (e.g. c-ras).

DESCRIPTION: The erbB-2 gene is amplified and overexpressed in approximately one-fourth of breast cancer specimens, and it is overexpressed without gene amplification in an additional 5% to 10% of patients. The repeated observation that erbB-2 overexpression is an indicator of poor prognosis, coupled with the evidence which indicates that erbB-2 has transforming potential both in vivo and in vitro, provide strong evidence that this gene plays a direct and causal role in human breast cancer progression. In studies with both rodent and human breast cancer cells, the principal investigator has repeatedly observed that constitutive activation of erbB-2 is associated with growth factor independent proliferation of the cells. This suggests that overexpression of erbB-2 in breast cancer is a likely mechanism by which cells become autonomous of growth factors for proliferation. Nevertheless, a direct and causal link between overexpression of erbB-2 and growth factor independent proliferation has yet to be established. To examine this, the following studies will be carried out. Aim 1. To determine if progressive overexpression of erbB-2 in normal human mammary epithelial cells that are strictly growth factor dependent for proliferation results in the gradual acquisition of growth factor independent proliferation. Further, the principal investigator will determine if the level of erbB-2 overexpression determines the extent of growth factor independent proliferation. Aim 2. To determine the role of a stimulatory ligand in constitutive activation of erbB-2 in cells that overexpress the receptor. It is clear that overexpression of erbB-2 in breast cancer cells yields receptors that are constitutively tyrosine phosphorylated to high levels in the absence of exogenous stimulatory ligand. What is unclear is whether autocrine or juxtacrine activation of these receptors is necessary for this high level activation, or whether receptor overexpression alone is sufficient to yield constitutive activation of these receptors. Aim 3. To determine the role of erbB-3 in constitutive activation of erbB-2 and growth factor independent proliferation of human breast cancer cells that overexpress erbB-2. It is known that in normal cells, heregulin-induced activation of erbB-2 requires the presence of erbB-3. However, it is not known whether erbB-3 plays a role in erbB-2 activation and signal transduction when erbB-2 is overexpressed or whether erbB-2 acts independently of erbB-3 when the former is expressed to high levels.

Adenoviridae; viral vaccines; neoplastic cell; neoplasm /cancer immunotherapy; breast neoplasms; neoplasm /cancer vaccine; vector vaccine; cooperative study; antigen presenting cell; clinical trials; lymphocyte proliferation; tumor antigens; cryopreservation; laboratory mouse; female; monoclonal antibody; human subject;

An important subset of human breast cancer is characterized by over- expression of the epidermal growth factor receptor (EGFR). This sub-group constitutes about 30% of all breast cancers, and over-expression of EGFR has been repeatedly shown to be an indicator of poor prognosis. The overall goal of this grant is to use a novel expression cloning strategy, in conjunction with a unique panel of breast cancer and normal breast epithelial cell lines, to determine the causal molecular alterations that induce specific altered growth phenotypes pf EGFR-positive human breast cancer cells. The specific aims of this proposal are: 1.) To functionally clone and identify genes from retroviral expression libraries derived from EGFR over-expressing breast cancer cells that induce EGFR- independent proliferation of normal and immortalized human mammary epithelial cells. 2.) To functionally clone and identify genes from retroviral expression libraries derived from EGFR over-expressing breast cancer cells that induce anchorage independent growth capacity in a panel of human mammary epithelial cells with varying abilities to survive in soft agar. 3.) To use differential display PCR, and high density oligonucleotide arrays, to analyze changes in gene expression in growth factor independent or anchorage independent clones isolated in these experiments. High titer retroviral expression libraries will be developed, using HBC cell lines developed in our lab, that over-express EGFR without gene amplification. These cells do not have amplifications of the common breast oncogenes, but do have amplified regions of their genome as determined by comparative genomic hybridization. Retroviral expression libraries will be transduced into a panel of well characterized human mammary epithelial cells, and transformants will be selected on the basis growth factor or anchorage independent growth. Inserts will be rescued by PCR using primers that bind to vector sequences, and then sequenced. This approach will allow us to identify and functionally clone breast cancer oncogenes directly from well characterized human breast cancer cells.

DESCRIPTION (As Adapted From the Investigator's Abstract): The Specific Aims are: Specific Aim 1 is designed to test the hypothesis that constitutive activation of PI3 kinase and its downstream signaling pathways mediate the alter phenotypes expressed by transformed HME cells expressing varying levels of erbB-2. Specific Aim 2 will be used to determine the mechanisms by which other oncogenes, that are amplified and/or overexpressed in human breast cancer cells, transform HME cells. This will be done by examining, in the context of the model recently developed, the ability of specific known and candidate oncogenes to induce phenotypes that are induced by erbB-2, when overexpressed to levels similar to that of breast cancer cell lines. The phenotypes to be studied include; factor independent survival/proliferation, growth in soft agar, and invasion of basement membranes. Specific Aim 3 is designed to examine the epigenetic consequences of overexpression of erbB-2 and other putative breast cancer oncogenes in order to identify common transcriptional events mediated by these genes, and to determine their role in the acquisition of specific altered phenotypes.

DESCRIPTION (provided by applicant): Focal amplifications involving chromosome 8p11-p12 occur in approximately 15% of human breast cancers. The fibroblast growth factor receptor 1 (FBFR1) gene has long been considered to be the best candidate oncogene at that locus. Three breast cancer cell lines developed in our lab; SUM-44, SUM-52 and SUM- 225, have overlapping amplicons centered around chromosome 8p11.2. FGFR1 is amplified in two of these cell lines, but is over expressed in only one of them. A second gene that maps to this region, transformation associated coiled-coiled protein 1 (TACC1), has recently been shown to be over expressed in some breast cancer cells and to have transforming activity in NIH3T3 cells. TACC1 is amplified and over expressed in all three of our cell lines. In addition to the FGFR1/TACC1 locus, SUM-52 and SUM-225 cells may each have distinct amplicons that center around 8p12 and 8q11.1 respectively. These regions contain several genes that are highly over expressed. Therefore, the broad aim of this proposal is to examine the prognostic and predictive significance of the 8p11-p12 amplicon in human breast cancer, and to identify genes therein that may be good targets for the development of novel therapeutics. The specific aims of this project are: 1) To complete the fine mapping of the 8p11-p12 amplicon in the SUM-44, SUM-52, and SUM-225 cell lines by Southern blotting using probes for genes known to map to the region of gene amplification in each line, 2) To examine the expression levels of the amplified genes in each cell line by northern blot and RT-PCR, 3) To determine the predictive and/or prognostic significance of amplification of distinct regions of the 8p12-8q11 amplicon, and of specific candidate breast cancer genes, in breast cancer specimens using tissue micro arrays, 4) To test the mechanistic significance of genes found to be amplified and over expressed in the three breast cancer cell lines by transduction of candidate genes into immortalized human mammary epithelial cells, and by antisense techniques to down-regulate over expressed genes in breast cancer cells. Thus, these studies are aimed at defining new and better prognostic and predictive markers for an important subset of breast cancer, and at identifying causally relevant genes the products of which could be good targets for novel therapeutics.

We have found that approximately half of basal type breast cancers show evidence for autocrine activation of the EGFR by amphiregulin (AREG). Furthermore, our data indicate that when AREG is the activating ligand for EGFR, receptor trafficking and down stream signaling is dramatically altered resulting in the establishment of a positive feedback loop involving NF-B and IL-1, the expression of a distinctive transcription profile, and the acquisition of motile and invasive capacity. The over arching goal of the work proposed in this application is to elucidate how AREG fundamentally alters the biology of human breast cancer cells and contributes to the expression of phenotypes characteristic of aggressive human breast cancer, and to test the hypothesis that AREG's effects are mediated via activation of NF-B and IL-1. The specific aims of this project are: 1) To test the hypothesis that accumulation of EGFR at the cell surface, which occurs in AREG stimulated cells and not EGF-stimulated cells, is the proximate mechanism for the altered EGFR signaling that results in activation of NF-B and expression of IL-1, 2) To determine the components of the EGFR signalosome in AREG stimulated cells, to elucidate the signaling pathway from AREG-activated EGFR to the nuclear accumulation of NF-B, and to analyze the role of this pathway in the expression of aggressive growth phenotypes of basal breast cancer cells, 3) To determine how IL-1 signaling modulates EGFR tyrosine phosphorylation in breast cancer cells and mammary epithelial cells with an AREG/EGFR autocrine loop by influencing the activity of tyrosine phosphatases and/or tyrosine kinases that target EGFR, and 4) to determine if EGFR-inhibition in breast cancers with AREG/EGFR autocrine loops results in down- regulation of IL-1, and loss of nuclear of NF-kB. AREG activation of EGFR fundamentally alters the biology of HME cells and induces phenotypes expressed by highly aggressive and drug-resistant breast cancer cells. Understanding the mechanistic basis for this difference, and understanding the downstream consequences of AREG mediated activation of the EGFR will improve our understanding of the biology of a particularly aggressive subclass of breast cancer for which there are no therapeutic options beyond standard chemotherapy.

DESCRIPTION (provided by applicant): In the previous project period, we performed an extensive analysis of the 8p11-p12 amplicon in human breast cancer cell lines and tissues to identify and validate novel breast cancer oncogenes. Using statistical analysis of copy number increase and over expression, we identified a subset of 21 genes as candidate oncogenes. Next, we directly tested the transforming function of these genes in human mammary epithelial cells. From these experiments, we identified four genes that are potently transforming in MCF-10A cells and three other genes with more modest transforming function. The most potently transforming genes identified, which include DDHD2, SPFH2, LSM1 and WHSC1L1, induce growth factor independent proliferation, anchorage-independent growth, invasive capacity, and altered morphogenesis in Matrigel. In addition, we identified gene combinations that effect the expression of transformed phenotypes. In the next phase of this work, we will perform experiments to understand the mechanistic basis for the transforming potential of these oncogenes, and we will examine their transforming function in human breast cancer cells in vitro, and in human and mouse mammary epithelial cells in vivo. The specific aims of the work in the next project period are: 1) To determine if the seven genes that induce transformed phenotypes in MCF-10A cells are directly transforming, and to determine if transformation is a common or a rare event in cells over expressing the oncogene; 2) To determine if the genes from the 8p11 region that are amplified and over expressed in human breast cancer cell lines are required for growth and survival of these breast cancer cells compared with normal mammary epithelial cells or breast cancer cells without the amplicon. We will also test the hypothesis that some oncogenes on the 8p11 amplicon cooperate to influence the transformed growth potential of human breast cancer cells; 3) To determine the influence of 8p11 oncogene over expression in the in vivo growth potential of human mammary epithelial cells, and to determine if these oncogenes can transform mouse mammary epithelial cells in vivo; 4) To test the hypothesis that over expression of the short isoform of WHSC1L1 alters the histone methylation code and gene expression profile, resulting in cells that exhibit properties of tumor initiating cells, including enhanced self-renewal capacity, expression of markers of cancer stem cells, and ability to form mammospheres in culture. We will also test the hypothesis that induction of these altered phenotypes requires an intact PWWP domain and does not require the SET domain of the protein. It is essential to demonstrate unequivocally that newly discovered 8p11 transforming genes are bone fide breast cancer oncogenes, and to elucidate the mechanism by which they induce cell transformation in order to develop therapeutic strategies that target these oncogenes.

? DESCRIPTION (provided by applicant): The overarching goal of the Medical University of South Carolina (MUSC) Transdisciplinary Collaborative Center (TCC) in Precision Medicine for Minority Men's Health is to determine the most effective ways to integrate, interpret, and apply biological, social, psychological, and clinical determinants of disease risks and outcomes into more precise medical strategies to prevent, diagnose, and treat chronic health conditions and diseases. To accomplish this goal, the MUSC TCC will: (1) utilize a multi-regional consortium network representing academic medical centers, community-based health organizations, public health agencies, and community stakeholders who will continually shape research priorities and strategies by embracing the principles of community-based participatory research; (2) conduct novel transdisciplinary research examining the synergistic effects of the multi-level, individual-specific characteristics that impact disease risks and outcomes; and (3) determine and disseminate best practices for delivering precision medicine-based care in an effort to reverse health care disparities among minority men in different clinical and community settings. Center partners will include NIMHD-funded centers at the University of Pennsylvania, Hampton University, and the University of Texas Health Sciences Center, San Antonio as well as forefront thought-leaders in minority health from the Hope Institute, the National Black Leadership Initiative on Cancer, Southeast Health Equity Council, and regional HRSA-funded Area Health Education Centers. Key elements of the MUSC TCC organizational structure include a well-defined academic home at MUSC with synergistic health disparity- focused missions and support from an NCI-designated Cancer Center and an NCATS-funded Clinical and Translational Science Award; three initial precision medicine research projects linked by a novel theoretical framework; a Data Integration Core led by nationally-recognized bioinformatic and medical informatic experts charged with creating integrated data sets that include clinical health outcomes data, biospecimen data, and data on social, psychological, and behavioral determinants; an Implementation Core that will deploy the latest approaches in dissemination research through the use of regional Evidence Academies; and a governance structure comprised of three highly qualified multi-PIs with expertise in health disparities, precision medicine, and translational medicine who set the tone for the transdisciplinary underpinnings of this TCC. The MUSC TCC and its partners across Health and Human Services Regions III, IV, and VI are poised to significantly impact the health outcomes of minority men throughout their regional communities utilizing innovative precision medicine-based insights and interventions.