Mechanical stressors and age as regulators of telomerase

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Mark LaBarge, Ph.D. -
Award Cycle: 2014 (Cycle 20) Grant #: 20IB-0109 Award: $208,900
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2014)

This award is jointly funded with the Anita Tarr Turk Fund for Breast Cancer Research of the Community Foundation for Monterey County.

Non-technical overview of the research topic and relevance to breast cancer
While there is no one cause of breast cancer, we have found that as women age, the cells responsible for maintaining healthy breast tissue stop responding to their immediate surroundings, including mechanical cues that should prompt them to suppress nearby tumors. The proposed research will address primary prevention of breast cancer by examining the microenvironmental regulation of a rate-limiting step in cancer progression, telomerase reactivation. We will examine how mechanical stressors and age regulate telomerase activity in normal human mammary epithelial cells (HMEC) to determine if the errors associated with early stage cancer progression decouple an association between stress and telomerase activity. Normal human breast epithelial cells possess limited replicative capacity, due to the presence of several distinct types of barriers that prevent proliferation and suppress tumor genesis. Breast cancer cells, on the other hand, possess an unlimited replicative potential, or immortality associated with the presence of telomerase, that maintains the chromosomal ends (telomeres). Malignant progression of a finite normal cell requires acquisition of errors that allow these tumor suppressive barriers to be bypassed or overcome. Using the unique, extensive HMEC culture system developed in our lab, our team will study the errors needed to bypass/overcome these barriers. If this hypothesis is true, it can open up new approaches to prevent or reverse how aging alters cellular and molecular functions that contribute to the prevalence of breast cancer in older women. Additionally, since immortality and its kind of telomerase regulation only occurs in abnormal transformed cells, it may be possible to ultimately identify therapeutics with minimal side effects that maintain normal telomerase regulation.

The question(s) or central hypotheses of the research: We are proposing that mechano-tensile forces within a cell, which are influenced by the external microenvironment, normally regulate telomerase; exposure to certain types of mechanical stressors will normally inhibit telomerase expression. Further, we hypothesize that early stage breast cancer progression is associated with errors that alter this normal process. When cells bypass stasis (a situation which would be similar to an initial oncogenic hit in normal breast progenitor cells, postulated to be a cancer cell of origin in vivo), the connection between stress and telomerase expression becomes decoupled, and such post-stasis cells are now vulnerable to errors that induce telomerase expression. Cells that overcame stasis require more errors to reactivate telomerase. Our proposal seeks to demonstrate this connection between stressors and telomerase, and uncover the mechanisms responsible for normal regulation and what changes when this connection is lost.

The general methodology: We will utilize cell culture substrata with tunable stiffness levels, a novel collection of HMEC strains that represent the progression from normal to immortal, and quantitative assays for telomerase expression and activity. With these resources we will determine whether mechanical stressors regulate telomerase in normal epithelial cells, and determine why that mode of control can become decoupled.

Innovative elements of the project: Because expression of telomerase is essential for malignant progression, interventions that could prevent a decoupling of telomerase regulation by stressors, or restore this connection, could have a huge impact on reducing breast cancer incidence. We do not anticipate translational applications within the time frame of this grant; at this point our focus is to understand how mechanical stressors may regulate telomerase. Since the hypothesized decoupling occurs only in abnormal cells, our long-term goal is that once mechanisms are delineated, therapeutic interventions can be identified. Such clinical applications could prevent progression from pre-malignant lesions (DCIS, atypical hyperplasia) to primary or metastatic cancer.




Progress Report 1 (2015)

Using the unique, extensive human mammary epithelial cell (HMEC) culture system developed in our lab, our team has been studying the errors needed to bypass/overcome these barriers. Normal finite lifespan HMEC have been exposed to oncogenic agents thought to play a role in breast cancer etiology in vivo, triggering errors that allow cells to get past senescence barriers and generating cultures representing different stages of breast cancer progression. There are at least three tumor-suppressive barriers. Stasis, results from exposure to certain types of stress that impinge on a key growth control molecule, retinoblastoma (RB); errors in the RB pathway can inactivate this barrier. Post-stasis cultures that acquired such an error prior to exposure to high stress have significantly different properties in response to errors implicated in the next barrier, replicative senescence. Replicative senescence occurs when ongoing proliferation in post-stasis cultures lacking sufficient telomerase leads to critically eroded telomeres, genomic instability, and cessation of growth or death. Errors that reactivate sufficient telomerase are needed to overcome this barrier, and confer immortality. We want to understand the mechanisms and conditions that regulate telomerase in normal cells.

We hypothesized that mechano-tensile forces within a cell, which are influenced by the external microenvironment, normally regulate telomerase; exposure to certain types of mechanical stressors will normally inhibit telomerase expression. Further, we hypothesized that early stage breast cancer progression is associated with errors that alter this normal process. Reasonable progress has been made in our efforts to disprove these hypotheses during the period from March to August 2015. Technical problems in substrate fabrication and isolation of sufficient quantities of RNA and live protein extract have been overcome by optimization.

Due to the short time since funding began, we focused first on transcription of the HTERT gene in response to changes in matrix rigidity. After much optimization, for HTERT is not an abundant transcript, transcription looks to be negatively regulated by rigidity in one pre-menopausal pre-stasis HMEC strain, and immortalization alleviates modulus-imposed repression. HTERT gene expression is remarkably low, so we are considering alternative measurement methods to decrease variation, such as droplet digital PCR, as instruments are available and of costs do not exceed budget. Extracts for telomerase activity have been collected in tandem, though assays have not yet been performed. Multiple strains from pre- and post-menopausal women are being tested at this time. At this time we are not anticipating changes to our originally proffered research design.




Progress Report 2 (2016)

Using the unique, extensive human mammary epithelial cell (HMEC) culture system developed in our lab, our team has been studying the errors needed to bypass/overcome these barriers. Normal finite lifespan HMEC have been exposed to oncogenic agents thought to play a role in breast cancer etiology in vivo, triggering errors that allow cells to get past senescence barriers and generating cultures representing different stages of breast cancer progression. There are at least three tumor-suppressive barriers. Stasis, results from exposure to certain types of stress that impinge on a key growth control molecule, retinoblastoma (RB); errors in the RB pathway can inactivate this barrier. Post-stasis cultures that acquired such an error prior to exposure to high stress have significantly different properties in response to errors implicated in the next barrier, replicative senescence. Replicative senescence occurs when ongoing proliferation in post-stasis cultures lacking sufficient telomerase leads to critically eroded telomeres, genomic instability, and cessation of growth or death. Errors that reactivate sufficient telomerase are needed to overcome this barrier, and confer immortality. We want to understand the mechanisms and conditions that regulate telomerase in normal cells.

We hypothesized that mechano-tensile forces within a cell, which are influenced by the external microenvironment, normally regulate telomerase; exposure to certain types of mechanical stressors will normally inhibit telomerase expression. Further, we hypothesized that early stage breast cancer progression is associated with errors that alter this normal process. Good progress has been made in our efforts to disprove these hypotheses during the period from March 2015 to February 2016. In the 1st, and shortest, reporting period (March –August), we solved a substrate fabrication problem and then focused on measuring transcription of the HTERT gene in response to changes in matrix rigidity because PCR is a rapid and inexpensive form of assay that uses little material. After much optimization, for HTERT is not an abundant transcript, transcription looked to be negatively regulated by rigidity in one pre-menopausal pre-stasis HMEC strain, and immortalization alleviates modulus-imposed repression. In general HTERT gene expression is remarkably low, and measurements were variable in spite of much optimization. Thus we focused on accurately measuring telomerase activity, which is arguably the more important parameter because gene expression is not as informative as protein activity in this case. The gold standard activity assay has been the TRAP, sold as Trapeze TM kit in a semi-quantifiable gel and 96-well formats, which we have used extensively. In order to make rapid and cost-effective progress in this small proposal, we realized that the standard assays used too many cells and did not provide the level of quantification we think is required. Thus we developed a novel quantitative PCR-based TRAP activity assay, qTRAP. We then completed an inhibitor study to determine whether specfic components of the mechanosensing and mechanotransduction apparatuses were involved in regulating telomerase activity. Blockade of YAP/TEAD association, with the small molecule Verteporfin and with YAP siRNA, reduced TRAP activity by 40% in prestasis HMEC on a rigid substrata, implicating the hippo pathway as a mechano-regulator of telomerase. In our opinion the work conducted on this grant proposal was consistent with the work intended by the aims. Even though we did have not yet completed all work, we view the discovery of a new regulator of telomerase activity and the creation of a quantitative telomerase activity as major achievements.

Conference Abstract (2016)

Mechanical Stressors and Age as Mediators of Telomerase Regulation

Masaru Miyano, Jessica Bloom, Susan Samson, Martha R Stampfer, and Mark A LaBarge
Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley CA
University of California, San Francisco, Breast Science Advocacy Core

Background: The ultimate objective of this proposal is to facilitate identification of safe effective interventions that can prevent breast cancer progression from pre-malignant lesions to primary or metastatic cancer. The immediate aim is to explore a novel hypothesis about early stage breast cancer that can radically alter our understanding of the mechanisms underlying carcinoma progression, and may offer specific targets for prevention. Multi-step progression of a normal mammary epithelial cell (HMEC) to breast cancer requires acquisition of errors that permit it to bypass or overcome several distinct tumor-suppressive senescence barriers. Virtually all breast cancers have an error in the RB pathway, have lost vulnerability to oncogene-induced senescence (OIS), and have reactivated sufficient telomerase activity to maintain stable telomere lengths and become immortal. Our research team’s studies with the unique, extensive HMEC culture system developed in our labs have shown that errors in the RB pathway are needed to get past a stress-associated senescence barrier, stasis. Errors that reactivate telomerase not only overcome replicative senescence but also eliminate vulnerability to OIS and provide additional malignancy-promoting changes. Consequently, telomerase reactivation is critical for cancer progression.

Hypothesis: We propose that mechano-tensile forces within a cell, which are influenced by the external microenvironment, normally regulate telomerase; exposure to certain types of mechanical stressors will normally inhibit telomerase expression. Further, we hypothesize that early stage breast cancer progression is associated with errors that alter this normal process. When cells bypass stasis (a situation which would be similar to an initial oncogenic hit in normal breast progenitor cells, postulated to be a cancer cell of origin in vivo), the connection between stress and telomerase expression becomes decoupled, and such post-stasis cells are now vulnerable to errors that induce telomerase expression. Cells that overcame stasis require more errors to reactivate telomerase. Our proposal seeks to demonstrate this connection between stressors and telomerase, and uncover the mechanisms responsible for normal regulation and what changes when this connection is lost.

Approach & Results: We are taking advantage of our large bank of normal pre-stasis HMEC from women who ranged in age from 16 to 91 years, and a unique cancer progression series in which the stages of progression from normal finite to immortal malignant have been delineated. We are dissecting the mechanistic basis of mechanical hTERT regulation by culturing cells at different stages in progression on polymer surfaces with tuned elastic modulus, and by identifying key nodes involved in mechano-regulation of hTERT using siRNA, ectopic expression, and pharmacological inhibition of potential signaling nodes. Here we present a description of the HMEC cell system, and the earliest incarnations of these mechano-regulation experiments, which includes the development of a novel quantitative PCR based telomerase activity assay called qTRAP that has enabled preliminary identification of mechano-sensitive pathways involved in telomerase regulation.

Potential for Translation: Because expression of telomerase is essential for malignant progression, interventions that could prevent a decoupling of telomerase regulation by stressors, or restore this connection, could reduce breast cancer incidence, such as by preventing progression from pre-malignant lesions to primary or metastatic cancer.



Breast Cancer beyond the Age of Mutation (DOI:10.1159/000441030)
Periodical:Gerontology
Index Medicus: Gerontology
Authors: LaBarge MA, Mora-Blanco EL, Samson S, Miyano M.
Yr: 2015 Vol: Nbr: Abs: Pg:

Microenvironment rigidity modulates responses to the HER2 receptor tyrosine kinase inhibitor lapatinib via YAP and TAZ transcription factors
Periodical:Molecular Biology of the Cell
Index Medicus: Mol Biol Cell
Authors: Lin CH, Pelissier FA, Zhang H, Lakins J, Weaver VM, Park C, LaBarge MA
Yr: 2015 Vol: 26 Nbr: 22 Abs: Pg:3946-53

Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells
Periodical:Frontiers in Cell and Developmental Biology
Index Medicus:
Authors: Lee JK, Garbe JC, Vrba L, Miyano M, Futscher BW, Stampfer MR, LaBarge MA
Yr: 2015 Vol: 3 Nbr: 13 Abs: Pg: