The Role of the ECM in Breast Cancer DNA Damage Repair

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Albert Davalos, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11IB-0153 Award: $250,560
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease
Biology of the Breast Cell>Biology of the Normal Breast: the starting point



Initial Award Abstract (2005)
Genetic instability is recognized as a leading cause of breast tumor formation and progression. Since the genome encounters endogenous and exogenous factors that damage DNA, cells have evolved complex repair systems to prevent genomic alterations. Similarly, the extracellular matrix (ECM) provides chemical cues that attenuate tumor progression in cells that sustain tumor-initiating alterations. Thus, the extracellular matrix and DNA repair mechanisms act to maintain cellular homeostasis. Recently, much research has focused on how cells respond to damage in S phase (period of DNA synthesis). Due to the inherent vulnerability of cells during this point in the cell cycle, repair needs to be rapid and extremely efficient. Delayed or faulty repair directly promotes genomic instability, which may act as the earliest event in the genesis of breast cancer.

Due to genomic vulnerability during S-phase, multiple repair pathways act to repair DNA lesions. Individuals that lack critical repair proteins, like BRCA1, NBS1, BLM or p53 exhibit an increased cancer risk, which includes breast cancer. Paradoxically, cells derived from these individuals display increased sensitivity to many chemotherapeutic drugs. Our proposal tests the hypothesis that in addition to protecting epithelial cells from genotoxic-induced cell death, the extracellular matrix confers signals that dictate the repair mechanism used to resolve lesions which occur during S-phase damage. Our aims are as follows: 1. Examine the role played by the cell’s microenvironment in activating repair mechanisms that resolve damage encountered during S-phase. 2. Does the loss of a critical “caretaker protein” promote an alternative and less faithful form of repair, which may cooperate with mutant p53 to prime human mammary epithelial cells to undergo transformation following replication stress.

We will grow mammary epithelial cells in 3-D using a technique optimized by Dr. Mina Bissell at LBNL. The role ECM signaling plays in cells lacking critical repair proteins will be examined using RNA interference (RNAi) technology. Using repair protein and phospho-specific antibodies, confocal microscopy, chromosomal analysis and measurement of repair mechanisms, we will assess how the ECM interfaces with critical repair proteins to ensure efficient repair of a damaged genome. For “proof-of-principle” experiments, we will expose mammary epithelial cells to DNA damage and examine their ability to form tumors in mice.

We plan to establish a model that potentially examines the earliest stages of breast cancer. This research merges two separate fields of study. The role that the extracellular matrix plays in maintaining normal breast structure will be linked to the signals that activate the appropriate repair pathway. Breast cancer requires dysregulation of multiple tumor suppressive pathways. Even with early detection, breast tumors have undergone multiple genomic alterations, which manifest in uncontrolled growth. Understanding the most proximal event that may initiate the cascade of aberrations which result in deregulated growth would establish a useful platform for generating diagnostic strategies or interventions that may halt breast tumor progression.


Final Report (2008)
Despite considerable progress in combating breast cancer, early events that initiate tumor formation remain unclear. Breast tumorigenesis requires inactivation or loss of multiple biological checkpoints. These biological safeguards protect the genome from endogenous damage which readily occurs in most tissues. Our proposal sought to investigate whether basement membrane factors which mimic in vivo conditions allowed repair protein deficient mammary epithelial cells to continue to divide following exposure to “replicational stress.”

We successfully generated immortalized human mammary epithelial cells deficient in repair proteins that protect the genome. We found that loss of these proteins sensitized repair deficient mammary epithelial cells to distinct drugs which disrupt replication. Significantly, we discovered the loss of the Breast Cancer Susceptibility Gene (BRCA1) induced mammary epithelial cells to spontaneously mutate the key tumor suppressor protein p53. BRCA1-deficient cells grew more rapidly and failed to die following drug treatment. When we treated mammary epithelial cells depleted of both BRCA1 and p53 with replication inhibiting drugs, we failed to observe cell death. Moreover, surviving cells appear to divide with numerous unresolved DNA breaks. This result suggests loss of BRCA1 predisposes mammary epithelial cells to inactivate the critical tumor suppressor protein, p53 which contributes to genomic changes potentially fueling aggressive breast cancer phenotype. Next, inactivation of the BLM helicase (proteins that “unwind” double-stranded DNA for replications and repair processes), which prevents Bloom syndrome (a rare inherited disorder characterized by a high frequency of breaks and rearrangements in an affected person's chromosomes) also caused mammary epithelial cells to survive drug treatments by inactivating p53 and also divide with unresolved DNA damage. Importantly, repair deficient mammary epithelial cells required –25 population divisions before we observe inactivation of p53.

Our results indicate that loss of critical repair proteins selects cells which disable the critical "gatekeeper" protein. On starting our project, published data indicates that telomerase immortalized mammary epithelial cells fail to synchronize with previously used methods. However, we were unable to examine a cell population at distinct point during the DNA synthesis cycle, because our cells could not be “synchronized.” Thus, we could not anticipate the inability to grow cells in basement membrane factors. Despite numerous obstacles encountered in this project, we successfully engineered mammary epithelial cells which demonstrate loss of key repair proteins (BRCA1 and BLM) predispose loss of p53. Loss of a "caretaker" and "gatekeeper" proteins allows mammary epithelial cells to evade normal cell death mechanism and divide with more DNA damage. Our studies uncovered loss of critical repair proteins alters chromatin organization which promotes the constitutive secretion of HMGB1, a putative nuclear “sentinel” protein. We are currently exploring in clinical samples and cell studies whether HMBG may serve as an early biomarker of genomic instability in breast cancer.

The XIST noncoding RNA functions independently of BRCA1 in X inactivation.
Periodical:Cell
Index Medicus: Cell
Authors: Xiao C, Sharp JA, Kawahara M, Davalos AR, et al
Yr: 2007 Vol: 128 Nbr: 5 Abs: Pg:977-89