Effect of Breast Cell Environment on Repair of DNA Damage

Institution: Lawrence Berkeley National Laboratory
Investigator(s): Aylin Rizki, Ph.D. -
Award Cycle: 2002 (Cycle VIII) Grant #: 8FB-0184 Award: $80,656
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point



Initial Award Abstract (2002)
Breast cells become cancerous when they no longer respond to signals that control their growth. Signals regulating cell growth come from both within the cells and from outside. Within the breast tissue, cells contact other cells as well as the scaffold material that surround the cells, called the extracellular matrix (ECM). Many tumors contain cells that have lost cell-ECM interactions. One result of loss of proper cell-ECM communication is manifested in the cell's nucleus in the form of genomic instability, e.g. cells are no longer able to transfer their genetic information with fidelity to the newly formed cells as they divide: they accumulate many mutations. Genomic instability in turn can lead to increased tumor formation. One cellular process that affects genomic instability is DNA double-strand break repair (DSBR). Double-strand breaks are lesions in the DNA that need to be repaired, and if not repaired properly can lead to accumulation of genetic changes, or mutations, in the genome, e.g. to genomic instability. We propose to study the effects of cell-ECM communication on DSBR during the progression of events that push normal breast cells to become tumors in a cell culture system that resembles human breast tissue.

We propose that tissue structure, and more specifically cell-extracellular matrix interactions within the tissue, will affect double-strand break repair in breast cells. We base this hypothesis on observations that cell-ECM interactions affect all aspects of cell structure and function studied, including the functional organization of the nucleus and the stability of the genome.

We will first determine the effect of extracellular matrix signaling on double-strand break repair in normal, or non-cancerous, breast cells by growing cells in the presence or absence of ECM and assaying for DSBR frequency and preferences. This will give us clues as to whether cell-ECM interactions may play a role in keeping DSBR error-free and therefore in keeping the genome stable. Our previous results show that by restoring correct cell-ECM communication to breast cancer cells that had lost such communication we can revert them to non-tumorigenicity. Using this approach, we will ask if reverting tumor cells by restoring cell-ECM interactions also restores normal DSBR properties. This will help us understand if restoring correct tissue structure to tumors can also help normalize cellular processes, such as DSBR, that in turn may help maintain non-tumorigenicity.

Although double-strand break repair has been studied in various mammalian cells in culture, detailed studies in human breast cells are lacking. More importantly, human cell culture studies of DSBR have been performed in culture conditions that do not resemble in vivo tissue environments. We have a unique opportunity to study DSBR in human breast epithelial cells in the presence of extracellular matrix in our well-established cell culture systems. Furthermore, we can manipulate our cell culture environments to dissect the effect of extracellular matrix on DSBR, using our well-established, specialized culture systems. Also, we can make use of our ability to revert tumor cells to normalcy by manipulating their cell-ECM interactions, to ask if restoring correct tissue structure can also restore normal DSBR processes.


Final Report (2004)
Breast cancer occurs mostly in the epithelial cells that are in contact with a basement membrane and other cell types. Cancer is caused when mutations accumulate in the cells and therefore it is important to understand what causes these mutations. It is known that in tissue from breast cancer patients, cell-basement membrane interactions are disrupted and that the cells have many mutations in their genome; i.e. an unstable genome. We asked whether or not these two events are related: Could the basement membrane signal to the cell to maintain a stable genome and prevent mutations? We focused on one mechanism by which mutations can be prevented: the double-strand break repair pathway. Double-strand breaks can be mutagenic in the cell if they are not repaired properly. Breaks can be caused by environmental carcinogens as well as cellular factors such as estrogen intermediates. Double strand breaks are also caused by ionizing radiation, which is used in breast cancer therapy. Therefore, understanding how basement membrane-cell interactions may regulate double-strand break repair would be relevant to both understanding how cells prevent tumor formation as well as how breast cancer patients' response to radiation therapy may be affected by the particular type of basement membrane composition the patient has.

Cells have multiple mechanisms by which they can repair double-strand breaks. Homologous recombination and non-homologous rejoining are the two main pathways. We asked whether the basement membrane can signal to normal breast epithelial cells to determine how well the breaks are repaired, to alter repair pathway choices, and to alter how well cells survive after double-strand break causing ionizing radiation. We find that basement membrane signals to downregulate homologous repair but does not affect the overall repair of ionizing radiation-induced breaks, suggesting that non-homologous end joining may be upregulated. We find that a particular cell surface receptor called betal integrin plays an important role in this regulation. In addition we find that basement membrane can alter the ability of cells to survive long term after ionizing radiation. We find that in single, non-dividing cells, basement membrane signals to enhance survival. In dividing cells, basement membrane signaling results in decreased long-term survival. These results suggest that basement membrane signaling is important in regulating double-strand break repair and the survival response to ionizing radiation.

We are currently exploring effects of such signaling on mutagenesis, as well as pursuing candidate molecules that relay signals from the basement membrane to the double-strand break repair machinery.


Symposium Abstract (2003)
Breast cells become cancerous when they no longer respond to signals that control their growth. Signals regulating cell growth come from both within the cells and from outside. Within the breast tissue, cells contact other cells as well as the scaffold material that surround the cells, called the extracellular matrix (ECM). Many tumors contain cells that have lost cell-ECM interactions. Such tumors also exhibit genomic instability e.g. cells are no longer able to transfer their genetic information with fidelity to the newly formed cells as they divide: they accumulate many mutations. One cellular process that affects genomic instability is DNA double-strand break repair (DSBR). Double-strand breaks are lesions in the DNA that need to be repaired, and if not repaired properly can lead to accumulation of genetic changes, or mutations, in the genome, e.g. to genomic instability. We proposed to study the effects of cell-ECM communication on DSBR during the progression of events that push normal breast cells to become tumors in a cell culture system that resembles human breast tissue.

We determined the effect of extracellular matrix signaling on double-strand break repair in normal, or non-cancerous, human breast cells by growing cells in the presence or absence of ECM and assaying for DSBR frequency and preferences. We found that addition of extracellular matrix to breast epithelial cells in culture decreases the frequency with which they repair a DSBR by homologous recombination, a form of DSBR repair that is mostly error-free. We observed this effect in both dividing and non-dividing cells. We also found that introducing DSBs to cells in the presence of ECM results in a lower frequency of survival than in the absence of ECM. These results provide the first observation that ECM signaling can influence DNA repair. In addition, we speculate that within the tissue, repair of double-strand break damage may be downregulated by cell-extracellular matrix interactions and that proper ECM signaling may be necessary to discard cells with badly damaged DNA.


Symposium Abstract (2005)
The ability to invade a basement membrane is a defining step of premalignant to malignant conversion in breast cancer. To delineate molecular alterations necessary for this transition, we utilized loss of differentiation in three-dimensional laminin-rich basement membrane (3DlrBM) cultures as a screening tool and isolated cell lines that have lost the ability to form organized acini but remain non-invasive. These cells displayed high potential for acquiring invasiveness, low frequency of non-invasive tumor formation in vivo, and similarities to premalignant lesions in vivo as shown by their patterns of genomic aberration and gene expression. Inhibiting matrix metalloproteinases and integrins that were upregulated in the malignant derivatives abrogated their invasiveness, implicating aberrant extracellular matrix signaling as a crucial step in the transition to malignancy.

In addition to alterations in extracellular matrix signaling, genomic instability is a hallmark of most breast cancers. One cause of genomic instability is altered double-strand break (DSB) repair. DSBs can be repaired either by homologous recombination or non-homologous end-joining (NHEJ). Here we showed that lrBM signals to downregulate HR of an I-SceI induced DSB in non-tumorigenic breast epithelial cells. Blocking β1 integrin stimulated HR and stimulating β1 integrin downregulated HR. Kinetics of overall induction or repair of breaks induced by 40Gy IR was not significantly affected by lrBM as measured by PFGE, however, kinetics of γ-H2AX foci formation after 3 or 6Gy IR is significantly altered by lrBM. Clonogenic survival after 3, 6, or 10 Gy IR was either enhanced or inhibited by lrBM, depending on the growth and polarity of cells. These results implicate extracellular matrix signaling as a modifier of cellular response to radiation therapy in breast cancer via effects on DNA repair.

Defects in mismatch repair promote telomerase-independent proliferation.
Periodical:Nature
Index Medicus: Nature
Authors: Rizki A, Lundblad V
Yr: 2001 Vol: 411 Nbr: Abs: Pg:713-6

The organizing principle: microenvironmental influences in the normal and malignant breast.
Periodical:Differentiation
Index Medicus: Differentiation
Authors: Bissell MJ, Radisky DC, Rizki A, Weaver VM, Petersen OW
Yr: 2002 Vol: 70 Nbr: Abs: Pg:537-46

Simultaneous classification and relevant feature identification in high-dimensional spaces: application to molecular profiling data.
Periodical:Signal Processing
Index Medicus: Signal Processing
Authors: Kenny PA, Rizki A
Yr: 2003 Vol: 83 Nbr: Abs: Pg:729-743

Meeting report. 42nd Annual Meeting of the Am. Soc. for Cell Biology, San Francisco, CA, December 14-18, 2002.
Periodical:Signal Processing
Index Medicus: Signal Processing
Authors: Kenny PA, Rizki A
Yr: 2003 Vol: 5 Nbr: Abs: Pg:147-153