Mark A. LaBarge, Ph.D. - US grants

The mechanisms underlying the exponentially increased incidence of breast cancer in women >55 years of age are poorly understood. A useful conceptual framework from which to build hypotheses is that agingrelated phenotypes are etiologically rooted in changes In tissue-specific stem cells or in their regulation. Indeed, common aging-related phenotypes i.e. cancers and deficits in tissue regeneration both have been linked to stem cells. A number of reports that studied stem cells as a function of age have suggested that age-related phenotypes can be due to stem cell-intrinsic or-extrinsic factors, but that delineation appears to be tissue specific. The Bissell laboratory and others have shown that the mammary microenvironment is as important as are the mutations in epithelial tumor cells for development of breast cancers. In a number of cases it has even been shown that the microenvironment can be dominant over strong oncogenes. In the aging breast, does the microenvironment change so as to catalyze tumorigenesis? Do damaged or aged mammary stem cells cease listening to, or misinterpret regulatory cues from their microenvironment? Or is it a combination? We are now uniquely poised to address these questions for the breast. Over several decades, 3-dimensional culture models that mimic many aspects ofthe human mammary gland and breast cancer microenvironments were developed in the Bissell laboratory. Recently, we also have developed a cell-based microenvironment microarray technology that facilitates elucidation of the functional roles that are played by individual microenvironmental constituents and combinations thereof. We have used these models together with primary human mammary progenitor cells to demonstrate that the microenvironment can dictate mammary progenitor cell fate decisions. Here we propose to combine these assets to address the following specific aims: (1) To identify age-dependent functional responses in microenvironment-directed mammary progenitor cell regulation, and the genetic circuitry that underlies them. (2) To determine whether mutations characteristic of breast cancers endow normal mammary progenitor cells with tumor-forming potential, or shifts their spectrum of response to mammary microenvironments in an age-dependent manner. (3) To design and test a therapeutic strategy based on age-related differences in mammary microenvironments and stem cell behavior using physiologically relevant 3D organotypic assays.

DESCRIPTION (provided by applicant): The association of aging with increased incidence of breast cancer is well known, but little understood. Emphasis has been placed on differences among tumors as a function of age, while considerably less attention is given to changes that occur normally in mammary epithelium during the process of aging, which may facilitate tumorigenesis. A commonly invoked mechanism to help explain age-related cancer is the gradual accumulation of mutations and epigenetic changes. However, it is known that cells harboring even the strongest oncogenes can appear phenotypically normal when held in check by normal tissue architecture. Most tissues exhibit functional and regenerative decline with advancing age. Our over-arching hypothesis is that age-associated breast cancer may partly result from loss-of-function alterations, e.g. changes to structural and architectural gatekeepers that maintain normal tissue organization and polarity, which leads to deleterious imbalances of or changes in the activity of progenitors and more differentiated epithelial lineages. That the majority of women live healthy cancer-free lives suggests age-related changes to the breast tissue are usually benign, but in cases when deleterious genomic changes also are present the combination with deteriorating microenvironments may be catastrophic. Some age-associated changes in breast includes increased fat and estrogen receptor expression;decreased connective tissue, numbers of alveoli and overall breast density;changes in collagen-type expression, and discontinuities in the basement membrane of the mammary gland. We simply do not know what impact these changes have on epithelial cell biology or on the architecture and organization with the gland. This proposal will use the Human Mammary Epithelial Cell (HMEC) Aging Resource, which is a large collection of normal finite-life span HMEC strains that were established from patients ranging in age from 16-91, to perform heretofore impossible quantitative and functional analysis of normal HMEC as a function of age. Changes in populations and functional properties of stem, progenitor, and more differentiated lineages will be measured, as will the ability of HMEC to form and maintain a normal organized bilayered architecture, an ability that is lost early in tumorigenesis, is affected by the aging process. In parallel with our functional analysis, we will perform an automated quantitative analysis of over 500 histological sections of normal breast to generate an atlas of aging-associated changes in the breast tissue, and to identify changes in organization in vivo among the different HMEC lineages. Our goal is to identify potentially deleterious changes that occur in most women during the aging process that could be targets of future prophylactic and preventative strategies. PUBLIC HEALTH RELEVANCE: It has been known for decades that aging is one of the principle risk factors for breast cancer, yet the impact of the normal aging process on function and architecture of normal breast epithelium is not well characterized. This proposal uses unique biological and technological assets that were developed at the Lawrence Berkeley National Laboratory to generate a previously unrealizable atlas of functional and architectural changes that occur during the process of aging. We will identify normal age-associated changes that may create a predisposition towards malignant states when combined with additional genetic mutations, with a goal of informing future prophylactic or preventative strategies.

Project Summary Early detection of breast cancer, especially those with high risk, would greatly improve outcomes. One method under current evaluation for early breast-cancer detection is random periareolar fine needle aspiration (RPFNA). RPFNA is based on the assumption that widespread cellular changes in the breast can be detected by random-tissue sampling. Cytopathology is performed on the collected epithelial cells to determine pre- cancerous changes; however, this analysis is only semi-quantitative. We propose to develop a point-of-care (POC), label-free platform to mechanically phenotype epithelial cells collected from RPFNAs to thus determine the presence of cancer cells and/or changes in cells that would indicate the likelihood of cancer. Our platform will be based on a novel microfluidic method we call ?mechano-Node-Pore Sensing? (mechano-NPS). Mechano-NPS utilizes a node-pore sensor with a microfluidic contraction channel to measure simultaneously a single cell?s diameter, resistance to compressive deformation, transverse deformation, and recovery from deformation. We have used this multi- dimensional method of mechanical phenotyping to differentiate malignant vs. non-malignant epithelial cells, distinguish cells treated or untreated with cytoskeletal-perturbing small molecules, and discriminate between sub-lineages of normal primary human epithelial cells (HMECs). Importantly, we have used mechano-NPS to identify mechanical phenotypes that correlate with chronological age and malignant progression. Thus, we hypothesize that mechano-NPS and its ability to mechanically phenotype cells could potentially be used for early disease detection. We intend to demonstrate the full potential of our platform ability to distinguish normal cells from transformed ones by screening de-identified RPFNA patient samples. PI Lydia L. Sohn, Professor of Mechanical Engineering at UC Berkeley and Core Member of the UCSF-UC Berkeley Joint Graduate Group in Bioengineering will lead this NIH R01 project with PI, Mark LaBarge, who is a Professor of Population Science and an expert in breast-cancer biology at City of Hope. Sohn will lead the development of the platform with Key Personnel, Michael Lustig, Associate Professor of Electrical Engineering & Computer Sciences at UC Berkeley. LaBarge will provide guidance on sample choice, experimental design, data analysis, and clinical relevance.

ABSTRACT A progressive breakdown in the bilayered structure of the mammary gland is the hallmark of all breast cancers, but the structural change that occurs between ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) is of particular importance because it represents a major inflection point in risk for patients. Breast cancers originate in the inner luminal layer of the mammary epithelium, where transformed luminal epithelial cells (LEP) proliferate to fill the ducts and lobules in DCIS. Surprisingly, LEP in DCIS have acquired all the necessary genetic aberrations to invade, but remain constrained within the tissue by an intact outer myoepithelial (MEP) layer?a group of cells that forms a dynamic barrier blocking access of the in situ tumor to the basement membrane (BM, the specialized extracellular matrix (ECM) that surrounds the mammary epithelium). Thus, we propose that translocation of transformed LEP past the MEP layer, and not genetic mutations, is a key rate- limiting step in progression to IDC. Here, we aim to identify the physical and molecular changes that must occur in LEP to facilitate this structural transition. We approach this challenge through the lens of mammary epithelial self-organization. We previously demonstrated that normal human LEP and MEP can self-organize in vitro, and that the capacity of MEP to exclude LEP from the BM is determined by hard-wired and lineage-specific interfacial tensions at each cell-cell and cell-ECM interface. We showed using experiments and mathematical modeling that the LEP-ECM interface is highly unfavorable energetically compared to the MEP-ECM interface, which prevents LEP from positioning themselves next to the BM. We hypothesize the existence of a rate-limiting and high-energy structural intermediate during the progression of DCIS to IDC, where LEP translocate into the MEP layer, next to the BM. We propose a statistical mechanical framework for understanding how perturbations to the interfacial properties and dynamics of tumor cells facilitate the formation of this intermediate. Specifically, we predict that changes to the LEP-ECM interfacial energy are a critical physical change necessary to promote basal translocation of transformed LEP. Preliminary studies support this hypothesis: we found that a frequently dysregulated gene?PIK3CA?disrupts self-organization when activated in LEP by rendering the LEP-ECM interface more energetically favorable. In this proposal, we will determine whether this and other physical changes to LEP are necessary for their basal translocation, and identify the molecular changes downstream of PIK3CA that give rise to these physical changes. We will test our hypothesis using complementary in vitro and in vivo experimental systems: using organoids reconstituted from human reduction mammoplasty tissues and genetically engineered mouse models. Our long-term goal is to reveal the changes that promote and inhibit progression from DCIS to IDC. Better physical and molecular predictors of progression would benefit DCIS patients who would otherwise be over-treated, as only a third of DCIS cases progress to IDC. Further, blocking LEP translocation would represent a therapeutic strategy to prevent breast cancer progression.