Apoptotic Dysregulation in Primary and Metastatic Breast Cancer
By P. Mojica, MD, and E.M. Mora, MD, MS
The process of programmed cell death or apoptosis plays an indispensable role in the development and maintenance of homeostasis within all multicellular organisms. The mechanism is activated when a signal and/or insult, such as viruses, toxins, irradiation, and reactive oxygen species, damage DNA. This process is initiated and completed in an orderly manner through the activation and synthesis of gene products necessary for cellular self-destruction. Developments in the apoptosis field over the past few years have provided a new perspective on how cell populations are normally maintained at equilibrium and have revealed how defects in cell death regulation can also contribute to the development of malignancy.
Apoptic-Cascade Classifications
The apoptotic cascade has been classified in three different groups of proteins. The first is the tumor necrosis factor (TNF) family, which includes membrane-bound proteins that send signals through second messengers to the nucleus for apoptosis to occur. Second, is the cysteine aspartate-specific proteases (caspases) family, which is free in the cytosol and act as intermediates in the apoptotic cascade. Finally, we have the Bcl-2 family of proteins, which are mitochondria-associated proteins that are intermediate between intracellular signaling from the nucleus and the caspase family.
The TNF family of proteins comprehends many different receptors. They interact and coordinate reactions from the environment to the nucleus in an orderly fashion through a signal transduction cascade. One such receptor is CD 95 (also called Apo-1 or Fas receptor), which plays a key role in apoptosis in lymphocytes and other cells.1 This receptor contains a ligand, Fas-L, that interacts with a protein-tyrosine phosphatase (Fap-1)2 and protein tyrosine kinase,3 which activates apoptosis by second messenger mechanisms inside the cell. Other, downstream targets of CD 95 also appear to include FADD/MORT-1, caspase 8, caspase 3, and ICE-like protease, which act on other cytoplasmic components that morphologically demonstrate apoptosis.1 In MCF-7 breast cancer cell line, the estrogen receptor plays a key role in the regulation of the apoptotic cascade. The apoptotic mechanism is inactivated upon estrogen stimulation, which leads to increased levels of Bcl-2 mRNA and increased survival.4 Like CD 95 and estrogen receptor, there are other cell-surface receptors with similar roles that interact with second messengers. What type of receptor participates in this cascade depends on the cell type. In this way, external stimuli contribute to tumorigenesis and apoptotic regulation.
The caspase family of proteins plays a key role in the effector pathway to apoptosis. Ten different proteins in this family have been identified. All caspases are synthesized as an inactive precursor. These proteins are activated when cleavage at specific aspartate residues is followed by assembly into heterotetramers, which results in the active forms of the enzyme.1 These are activated by the liberation of cytochrome C and apoptotic initiation factor (AIF) from the mitochondria upon Bcl-2 family activation or during TNF receptor family activation. Also, the caspase family can be inactivated by the Bcl-xl protein in a tertiary complex with caspase-9 and APAF-1.5 This is the common pathway by which extracellular and intracellular stimulation converge to one end-effector mechanism that ends with the fragmentation of DNA. One of the final substrates of this cascade is poly-ADP-ribose-polymerase (PARP), which is associated with nuclear DNA fragmentation leading to apoptosis. There is a strong correlation between the rate of internucleosomal DNA fragmentation and poly-(ADP-ribosyl)ation of histone H1 during apoptosis, which suggest that PARP activation increases the susceptibility of chromatin to cellular nuclease(s) and DNA degradation.1,6 In fact, inhibition of this end-effector cascade results in increase survival of tumor cells which in turn, promotes tumorigenesis.
The Bcl-2 family of proteins is a critical regulator of cell death pathway. This family of proteins is localized in the mitochondria. To date, 14 cellular homologs in this family have been identified, including the pro-apoptotic proteins (Bax, Bcl-xs, Bad, Bak, Bik, and Bid) and anti-apoptotic proteins (Bcl-2, Bcl-xl, Mcl-1, A1/Bfl-1, Bcl-W, Nr-13, and Ced-9).6 These proteins interact with each other, forming heterodimers that regulate the fate of the cell. Essentially, the pro-apoptotic proteins of this family mediate apoptosis by increasing the plasma membrane permeability, reactive oxygen species, and liberation of cytochrome C and AIF to the cytosol, which are strong caspases activators.7,8 The anti-apoptotic proteins interfere with these events and block the apoptotic cascade.
Mediators of Tumorigenesis
Studies have shown that cells of normal breast tissue pass through apoptotic events during the menstrual cycle.4 This process takes place during the peak levels of estrogen and progesterone stimulation. It is during this time that dysregulation of apoptosis can lead to tumor transformation and proliferation. Studies using breast cancer cell lines have been used as a primary model system in vitro and in vivo to examine apoptotic dysregulation and its relation to the pathogenesis of cancer.4 Some studies demonstrate that the ratio between levels of pro-apoptotic proteins and anti-apoptotic proteins correlates with tumor formation. Bargou and associates found that strong expression of Bax was observed in normal breast tissue;9 in contrast, only weak expression was found in its malignant counterpart. This correlates with prolonged survival and decreased apoptosis. Also, the anti-apoptotic proteins of the Bcl-2 family have been implicated in the pathogenesis of tumorigenesis in breast tissue. Bcl-xl protein was overexpressed in invasive breast cancers when compared with adjacent breast tissue.10 Other mediators of tumorigenesis are the tyrosine kinases, epidermal growth factor, and Her-2, which are overexpressed in up to one-third of breast cancers. Their overexpression had been associated with poor clinical outcome.4
Dysregulation of apoptosis is not only implicated in the tumorigenesis of breast tissue, but also has important implications in treatment response. One of the proposed mechanisms by which apoptotic dysregulation contributes to decreased sensitivity to chemotherapy is that the resulting increase in cell survival provides the cell with time to repair any damage done by chemotherapeutic agents. Overexpression of anti-apoptotic proteins, like Bcl-2 or Bcl-xl, has been shown to result in reduced sensitivity to the inhibitory effects of chemotherapy.11 These findings also support the fact that not only the absolute number of anti-apoptotic proteins, but also the balance between pro-apoptotic and anti-apoptotic proteins determines the final fate of the cell. In fact, a decreased ratio of pro-apoptotic to anti-apoptotic proteins had been correlated with a decreased sensitivity to various chemotherapeutic agents. Manipulation of the expression of the apoptotic proteins in vitro has changed the sensitivity of some cancer cell lines to chemotherapy. One example is the overexpression of the pro-apoptotic protein Bcl-xs in the MCF-7 cell line, which is associated with increased sensitivity to chemotherapy-induced apoptosis.12 Also, upregulation of the death-promoting gene Bax in breast cancer cells sensitizes the cells to drug-induced apoptosis.13 In the future, apoptotic manipulation by increasing the pro-apoptotic proteins or decreasing the anti-apoptotic proteins may give new targets for adjuvant therapy in cancer patients by increasing the sensitivity to chemotherapy drugs.
Possible Role of Bcl-xl
In our laboratory, we have studied the role of apoptotic dysregulation and metastatic potential of breast cancer cells. Comparing metastatic breast cancer cell lines from pleura and bone, we found that the protein Bcl-xl was overexpressed in the bone-metastatic cell line compared to the pleural-metastatic cell lines MDA-231 and MCF-7. Other Bcl-2 family proteins (Bcl-2, Bcl-xs, Bad, and Bak) did not show any significant difference in their expression in cancer cell lines. This finding suggests that the anti-apoptotic protein Bcl-xl may play an important role in the mechanism of metastases. Recent studies by Olopade and colleagues has shown that overexpression of Bcl-xl is associated with high tumor grade and nodal metastases in breast cancer cell lines and primary untreated breast carcinoma.10 This suggests that Bcl-xl may have a key role in the progression from tumorigenesis to metastases. We still need to delineate the mechanism by which Bcl-xl contributes to breast cancer metastases to bone. Future experiments will focus on the manipulation of the apoptotic cascade.
In summary, studies have shown that apoptosis not only plays an important role in the normal development of breast tissue during the menstrual cycle, but also is intimately related in the process of tumorigenesis and metastatic potential in breast cancer. Several studies suggest that important components of the apoptotic cascade are differentially expressd between normal, primary, and metastatic malignant cells. This difference contributes to the aggresiveness of the tumor, making it more resistant to current chemotherapy, which sometimes can be indicative of poor clinical outcome. The current challenge is to identify the apoptotic mechanism, activators, and inhibitors that are important in the pathogenesis of breast cancer, since their identification could provide opportunities for the development of new preventive and therapeutic approaches. Also, the identification of important mechanisms in the progression of tumorigenesis has the potential to define metastases and apoptotic markers that can serve as prognostic or predictive factors in breast cancer diagnosis and treatment. (Dr. Majica and Dr. Mora, University of Puerto Rico, Medical Sciences Campus, School of Medicine, Department of Surgery. San Juan, Puerto Rico.)
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Apoptotic cascades have been shown to involve all of the following patterns except:
-
a. caspases.
b. tumor necrosis factor.
c. MCF-7.
d. Bcl-2.
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