proposed two progression models according to the cell differentiation status. In their study, the progression from ductal carcinoma in situ (DCIS) to invasive carcinoma (IC) was related to an increase of MI with no significant change in AI in well-differentiated lesions and to an increase of MIs and a two-fold decrease in AIs in poorly differentiated tumors. Yamamoto et al. found that advanced breast cancer with distant metastases negative for p73 have low AI and high MI, suggesting an inhibition of apoptosis in those tumors. After analysis of 91 invasive breast carcinomas, Wu and coworkers concluded that low AIs are related to axillary lymph node metastasis and shorter overall survival. Along these lines, transgenic mouse tumor models suggest that in proliferative lesions with high rates of proliferation and apoptosis, progression to frankly malignant aggressive invasive tumors occurs if apoptosis is impaired and proliferation maintained. Therefore, it is likely that the most aggressive metastatic tumors acquire further genetic and epigenetic defects enabling evasion from apoptosis while they invade foreign tissues.

Apoptosis contributes to spontaneous cell death in tumors, but also to cell death induced by various anticancer agents (hormonal therapy, chemotherapy, radiotherapy, immunotherapy). Measurable increases of apoptosis and decreases in proliferation are detected within 24 hours of the start of effective treatments. When those treatments fail, dysregulation of apoptotic pathways may be a cause.

3.2. Molecular dysregulation of apoptosis in breast cancer

3.2.1. Altered expression of death ligands and their receptors in breast cancer

Faulty regulation of the FAS and TNF-related apoptosisinducing ligand (TRAIL) system has been described in a variety of human tumors, including breast carcinomas. Breast carcinomas display an altered expression of death ligands FAS-L and TRAIL and their respective receptors. They generally express much more FAS-L protein and less FAS receptor than normal breast tissue or benign tumors. The differentially expressed FAS-L and FAS are inversely correlated with node status and tumor size. A ratio of Fas-L to Fas mRNA greater than 1 was found to be significantly associated with higher tumor grade, shorter disease-free survival, and increased mortality. Increased expression of FAS-L compared with its receptor would enable breast cancer cells to bypass immune surveillance by inducing apoptosis in FAS-positive infiltrating lymphocytes, thereby facilitating tissue invasion.

In contrast with the FAS/FAS-L system, TRAIL is strongly expressed in 30% to 50% of breast cancers, but the associated death receptors DR4 and DR5 are not downregulated. An immunohistochemistry study of 90 breast cancer patients with invasive ductal carcinoma identified DR4 as the prominent TRAIL receptor expressed and correlated its expression with tumor grade in invasive ductal carcinoma. In the same study, ERBB2-positive tumors were found to express higher levels of both DR5 and TRAIL than ERBB2-negative tissues. The survival of breast tumors that concomitantly express TRAIL and its receptors may be due to altered signal transduction downstream of the death receptors or inactivating mutations in the death receptor itself. Using gene silencing, Day et al. showed that the caspase 8 inhibitor cellular FLICE-like inhibitory protein (c-FLIP) was required for MCF-7 breast cancer cells growth and survival both in vitro and in vivo within tumor xenografts. Inactivating mutations in death receptors have also been found in metastatic breast cancers. TRAIL expression is downregulated by anchorage in breast cancer cells, suggesting that TRAIL-dependent apoptosis may play a role in anoikis of breast epithelial cells. Inactivating mutations in death receptors would therefore contribute to inhibit TRAIL-dependent anoikis of metastatic breast cancer cells.

3.2.2. Deregulation of prosurvival growth factors and their receptors

Growth factors and their receptors function in a complex and interconnected network critical for both the development of the normal breast and the pathogenesis and progression of breast cancer. Their deregulation in cancer leads to the hyperactivation of survival signaling pathways and subsequent evasion from apoptosis.

The EGF receptor (EGFR) family consists of four related receptor tyrosine kinases (EGFR, ERBB2/HER2, ERBB3/HER3, and ERBB4/HER4). HER2 is the preferred heterodimerization partner of all ERBB receptors and can mediate signal transduction of all ERBB members when they bind to their cognate ligands such as EGF, TGF-α, amphiregulin, or neuroregulins. Over-expression of EGFR and ERBB2, which causes growth factor independence, is frequent in breast cancer and is linked with a more aggressive course of the disease. Over-expression of ERBB receptors leads to the enhanced activation of two main survival pathways: the MAPK and PI3K/AKT pathways. Hyperactivation of survival signaling has been shown to confer resistance to apoptosis induced by hormonal or chemotherapy, whereas inhibition of this downstream signaling by trastuzumab, a monoclonal

antibody against HER2, effectively induces apoptosis in HER2–over-expressing metastatic breast cancer.

The IGF/IGF-1R system exerts an antiapoptotic function in both the normal and neoplastic breast. Among all growth factor receptors, IGF-1R displays one of the most potent antiapoptotic activities because it protects cells from apoptosis via multiple signaling pathways, all leading to the phosphorylation of BAD.

A disrupted balance in the IGF system can cause excessive survival signals as observed in breast tumors. A change in IGF expression has been found in breast carcinomas. High endocrine circulating levels of IGF-1 have been associated with an increased risk of breast cancer in premenopausal women. Moreover, stromal cells surrounding the normal breast epithelium secrete IGF- 1, whereas those surrounding the malignant epithelium secrete IGF-2, suggesting that transformation of breast cells may be associated with a switch from stromal IGF- 1 to IGF-2 expression. IGF-1R is over-expressed and/or constitutively activated in breast cancer tissue compared with normal or benign tumoral tissue. This overexpression protects breast cancer cells from apoptosis and enhances their survival in vitro and in vivo, whereas downregulation or functional inactivation of IGF-1R causes massive apoptosis in tumor xenografts. Inactivating mutations of tumor suppressors and cross-talk with hormone and growth factor receptors signaling contribute to the regulation of IGF-1R expression and activity in breast neoplasia. Indeed, expression of IGF-1R is inhibited by tumor suppressors such as wild-type p53 and breast cancer 1 (BRCA-1) but upregulated by mutant p53. The expression and activity of IGFR is also regulated by steroid hormone receptors (SR) and EGF receptors via a cross-talk in signaling networks. Growth factors such as EGF and IGF are known to influence the expression and activity of SRs. In turn, the expression of growth factor receptors, their ligands, and signaling molecules is often controlled by SRs. For instance, estrogens induce expression of IGFs, IGF-1R, and IRS-1 and IRS-2, whereas IGF- 1 enhances the expression and activation of ER in breast cancer cells. EGF also stimulates IGF-1R expression, and its receptor EGFR can interact with IGF-1R and regulate its stability. IGF-1R/HER2 heterodimerization was shown to contribute to trastuzumab resistance of breast cancer cells. Thus both the IGF ligand and receptor play a major role in tumor cell survival and resistance to apoptosis induced by anticancer therapies.

TGFβs are well-known growth inhibitory and proapoptotic factors contributing to normal mammary development. In breast cancer, TGFβs play a dual role in mammary tumorigenesis, acting as a tumor suppressor in early stages of cancer and promoting invasion and metastasis at later stages. One proposed mechanism

to explain TGFβ tumor suppressor function involves the TGFβ1-dependent repression of human telomerase reverse transcriptase causing cellular senescence in the mammary stem cell population. Enhanced expression of TGFβ and downregulation of TGFβ receptor 2 have been associated with breast cancer progression and aggressiveness of the disease. The shift from tumor suppressor to tumor promoter function could be due to mutations in Tgfβ2 acquired during the course of the disease. Indeed, an insertion polymorphism in Tgfβ2 leading to an increased Tgfβ2 promoter activity was associated with lymph node metastases and advanced breast tumor stage independently of estrogen and progesterone receptor status. This study suggests that increased TGFβ2 expression in breast tumors bearing this allele may promote metastasis. According to Yu et al., activation of latent TGFβ2 in CD44- MMP-9 complexes on the surface of mouse mammary carcinoma would be required for tumor cell survival during metastatic colony formation.

Recent studies have identified possible molecular mechanisms by which TGFβ promotes survival. Ehata and colleagues report that TGFβ promotes survival of mammary carcinoma cells through induction of antiapoptotic transcription factor DEC1. Several groups also demonstrate that activation of the PI3K/AKT is involved. Yi and coworkers showed that type 1 TGFβ receptors could bind and activate PI3K in COS-7 epithelial cells, suggesting that PI3K/AKT signaling can be an effector of the oncogenic function of TGFβ. Dumont et al. used MDA-MB-231 breast cancer cells stably expressing a kinase-inactive type II TGFβ receptor to show that TGFβ promotes motility through mechanisms independent of SMAD signaling, possibly involving the activation of the PI3K/AKT and/or MAPK pathways. A recent study from Wang et al. suggests that TGFβ-dependent activation of the PI3K/AKT pathway could be involved in trastuzumab/herceptin resistance. In HER2–over- expressing cancer cells, TGFβ indeed synergized with the HER2 signaling network to activate the PI3K/AKT pathway and promote cancer cell survival, migration, and resistance to trastuzumab. Finally, TGFβ signaling is regulated by estrogens in ER-positive tumors. Antiestrogens such as tamoxifen can induce TGFβ1 expression and proapoptotic activity in human MCF-7 breast cancer cells in vitro.

3.2.3. Alterations in cell adhesion and resistance to anoikis

A hallmark of cancer is anchorage-dependent growth, which allows cancer cells to survive when they pile up and detach from the basement membrane. This

resistance to anoikis is mediated in part by alterations in the adhesion system and its related signaling. Breast cancer progression is associated with a change in integrin and cadherin expression profiles, referred as the integrin and cadherin switches. Invasive breast cancer cells tend to downregulate the integrins such as α2β1 that mediate their adhesion to the basement membrane and to upregulate integrins such as αvβ6 or α6β4, which promote survival, migration, and invasion during metastasis. In breast cancer cells over-expressing ERBB2, integrins α6β4 colocalize and associate with ERBB2, which potentiates the survival, proliferative, and invasive capacities of those neoplastic cells. A cadherin switch also occurs during breast cancer progression, with downregulation of the invasion suppressor E-cadherin and upregulation of N- and P-cadherins. E-cadherin is considered as an invasion suppressor because loss of E-cadherin cell adhesion leads to anoikis and thus prevents tumor cells from invading surrounding tissues. Reduced E-cadherin expression therefore favors dissemination and in general correlates with noninvasive phenotypes. On the other hand, N- and P-cadherins would facilitate invasion and metastasis by promoting tumor cell affinity for the stromal and endothelial cells of distant sites. Regain of E-cadherin expression is sometimes observed in some metastases to favor survival and reattachment of metastases.

β-catenin is often upregulated and stabilized in breast cancer, thereby providing additional survival cues. MMPs are also commonly expressed and secreted at high levels by stromal cells (fibroblasts and tumor-infiltrating macrophages) in invasive breast cancer. They are known to stimulate proliferation, activation of growth factors and their receptors, and resistance to apoptosis. By degrading the ECM of primary neoplastic cells, MMPs would favor the anoikis of a majority of epithelial cells but also help to select anoikis-resistant clones. Moreover, several MMPs promote tumor cell proliferation and survival by releasing growth factors and death ligand FAS-L bound to ECM components.

Overall, dysregulated cell adhesion is one of the features acquired by breast tumor cells to bypass anoikis and which allows them to survive in an unpolarized state in foreign microenvironments.

3.2.4. Enhanced activation of the PI3K/AKT pathway in breast cancer

The PI3K/AKT pathway is frequently activated in breast cancer through diverse mechanisms, including membrane receptor signaling and/or mutations in PI3K or its negative regulator PTEN. PI3K/AKT is activated by upstream membrane receptor pathways (i.e., ER, ERBB2,

EGFR, integrin receptor signaling) that are often dysregulated in cancer. Activating mutations in PI3K are common in invasive ductal carcinomas of the breast and are associated with poor prognosis. Moreover, inactivating mutations in the tumor suppressor PTEN occur in up to 50% of breast cancer.

Zhou et al. showed that phosphorylation of AKT increases progressively during breast cancer progression from normal epithelium to hyperplasia and invasive carcinoma. AKT activity increases as breast cancer malignancy intensifies, resulting in a poor prognosis. Several reports indicate a positive correlation between active AKT, over-expression of ERBB2, and histological grade of the tumor. AKT activation has also been involved in breast cancer cells’ resistance to many anticancer therapies; thus inhibition of the PI3K/AKT pathway is being investigated as a new therapeutic strategy for breast cancer patients.

Therefore, activation of the survival kinase AKT significantly contributes to the progression of breast cancer and resistance to radioand chemotherapy.

3.2.5. p53 inactivation in breast cancer

Wild-type p53 is a tumor suppressor that plays a central role in maintaining cellular genetic integrity by preventing DNA-damaged cells from further proliferation. Inactivation of p53 is a major event in tumorigenesis.

Mutation and deletion of p53 are the most common genetic defects seen in clinical cancer. Somatic mutations of p53 are found with high frequency in both the epithelium and stroma of invasive breast carcinomas. p53 mutations generally result in impaired transcriptional activity and increased protein stability. Large case studies demonstrated that p53 mutations are independent markers of poor prognosis in breast cancer and that the exact type and position of the mutation influences disease outcome. Accumulation of stabilized p53 can be detected in early breast lesions, and its occurrence increases with tumor progression.

Many tumors with wild-type p53 do not have normal p53 function, suggesting that some oncogenic signals suppress the function of p53. Zhou et al. showed that HER2/neu-mediated resistance to DNAdamaging agents requires the activation of AKT, which enhances MDM2-mediated ubiquitination and degradation of p53. In a recent study, Danes et al. demonstrated that 14–3–3 zeta over-expression is a critical event in early breast disease conferring resistance to anoikis via the downregulation of p53 expression. Mechanistically, 14–3–3 zeta induced hyperactivation of the PI3K/AKT pathway, which led to phosphorylation

and translocation of the MDM2 E3 ligase, resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14–3–3 zeta–over- expressing MCF10A acini in 3D cultures. The authors concluded that downregulation of p53 is one of the mechanisms by which 14–3–3 zeta alters mammary epithelial cells acini structure and increases the risk of breast cancer. Recent publications also suggest that loss of p53 permits expansion of cancer stem cells in mouse mammary tumors and in human breast cell lines.

Cell and animal experimental studies have linked p53 status with response to therapy. They support a role for wild-type p53 in drug sensitivity and a role for mutant p53 in chemoresistance, but the predictive value of p53 in response to chemotherapy remains unclear in patient studies in which treatment, tumor, and mutation types are often heterogeneous.

3.2.6. Altered expression of BCL-2 family of proteins in breast cancer

Dysregulation of apoptosis contributes to the pathogenesis of breast cancer, at least in part, through an imbalance in BCL-2 family members between antiapoptosis (such as BCL-2/BCL-X) and apoptosis-promoting proteins (BAX).

BCL-2, BCL-X, MCL-1, BAX, BAK, and BAG-1 are expressed in human breast cancers. BAXα, a splice variant of BAX, is expressed in normal breast tissue but only weakly in breast tumors. BCL-2 expression is higher in normal breast tissue from a cancer-containing breast in comparison with controls, suggesting that an increase in BCL-2 antiapoptotic protein favors tumorigenesis. BCL- 2 expression is more common in tumors that express ER, whereas BCL-XL is more commonly found among HER2-positive tumors. Bcl-2 associated athanogene-1 (BAG-1) is an antiapoptotic protein that binds to and enhances the antiapoptotic activity of BCL-2. It was shown to modulate the interactions of HSP70 chaperones with other proteins, thereby enhancing their biological activity. BAG-1 interacts with several prosurvival proteins important to tumorigenesis (i.e., BCL-2, RAF-1, steroid hormone receptors, some tyrosine kinase receptors, hepatocyte growth factor receptor, and plateletderived growth factor receptor). BAG-1 expression is high in the majority of invasive breast carcinomas and is correlated with BCL-2 expression and SR positivity.

Alterations in the relative expression levels of individual BCL-2 family members appear to influence breast cancer progression. Over-expression of BCL-X protein in primary breast cancer is associated with high tumor grade and nodal metastases, suggesting that upregula-

tion of BCL-X protein may be a marker of tumor progression. BCL-XL protein would promote survival of cells in metastatic foci by counteracting the proapoptotic signals in the microenvironment. According to Sierra and colleagues, BCL-XL indeed mediates a change in metabolic pathways to protect breast cancer metastatic cells during transit from the primary tumor to the metastatic site.

BCL-2 immunostaining has been correlated with low AI, low histological grade, SR positivity, p53 expression, absence of c-ERBB-2, and better overall survival. It is surprising to find an inhibitor of apoptosis associated with better prognosis. Several explanations for these seemingly paradoxical results can be proposed: (1) regulation of BCL-2 expression by estrogen; (2) inhibitory effect of BCL-2 on cell cycle progression; (3) downregulation of BCL-2 by mutant p53; and (4) the presence of BCL-2 antagonists such as BAX or BAK, which negatively regulate its cytoprotective function. First, expression of BCL-2 is regulated by estrogens in mammary epithelial cells and ER-positive breast cancer cell lines. It gradually decreases during the development of breast cancers in relation with the loss of ER. Second, BCL-2 not only blocks cell death, but also has an independent inhibitory effect on cell division. The loss of BCL-2 can therefore enable high proliferation rates and high histological grade. Third, the inverse relationship between BCL-2 and p53 expression suggests that mutations in p53 could be related to the regulation of BCL-2 gene expression in breast cancers. Consistent with this hypothesis, transfection of mutant p53 into a wild-type p53 breast cancer cell line suppressed the expression of BCL-2. Finally, reduction in antiapoptotic BCL-2 can be compensated by a reduction in expression levels of the proapoptotic members BAX and BAK in some breast tumors. BAX immunostaining is associated with c-ERBB2 immunopositivity in invasive ductal carcinoma while reduced expression of both BCL-2 and BAX strongly correlates with the development of distant metastases. Reduction in BAK protein is associated with the conversion to hormone-independent ER-negative breast cancer and may play an important role in malignant progression by counteracting the reduced levels of BCL-2.

Anticancer therapies trigger apoptosis in part by modulating the expression of several members of the BCL-2 family and tipping the balance toward cell death. Paclitaxel acts by upregulating several proapoptotic BCL-2 proteins and downregulating antiapoptotic members. Similarly, doxorubicin causes a decrease in BCL-2 and an increase in BAX expression. Increased levels of BAX were correlated with a good response to chemotherapy in some studies. In the MCF-7 cell line, susceptibility

to drug-induced apoptosis was correlated with differential modulation of BAD, BCL-2, and BCL-XL protein levels, with Bad upregulation being an early indicator of a cell death outcome.

In summary, several members of the BCL-2 family regulate apoptosis in breast carcinomas. Alterations in their expression levels occur during tumorigenesis, tumor progression, and anticancer therapies. A reduction in BCL-2, BAX, and BAK, together with an upregulation of BCL-XL, are often seen in high-grade breast tumors. BCL-2 expression is associated with a number of favorable prognostic factors, whereas BAX expression seems to have a predictive value for positive response to chemotherapy in lymph node-negative breast cancer patients.

4. NONAPOPTOTIC TYPES OF CELL DEATH IN NORMAL

AND NEOPLASTIC BREAST

Increasing evidence suggests that nonapoptotic modes of cell death also play a role in breast physiology and pathology. These distinct modes of cell death can occur concomitantly with apoptosis or provide alternative death pathways when apoptosis is impaired. Three types of nonapoptotic cell death have been described thus far in the normal and neoplastic breast: autophagy, necrosis, and entosis.

Autophagy (type 2 cell death) is a catabolic process of cellular “self-eating” involving the engulfment of the cell’s components (cytoplasm, organelles, and long-lived proteins) in double-membrane vacuoles (autophagosomes) and their degradation in lysosomes. Autophagy plays a dual role, prosurvival or pro-death, in both normal and neoplastic cells. It functions as a survival mechanism conferring temporary cytoprotection during nutrient starvation or metabolic stress. It helps maintain cellular homeostasis by preventing the accumulation of deleterious products and organelles and by supplying energy and amino acids through catabolism. Autophagy has also been described as a nonapoptotic type of cellular demise because extended autophagy ultimately results in type 2 programmed cell death. The role of autophagy, cell survival versus cell death, is both stimulusand context-dependent. When needed, autophagic cell death can be used as an alternative to apoptosis to eliminate unwanted, damaged, or transformed cells. Regulators of apoptosis (e.g., BCL-2 family members, p53, AKT) also modulate autophagy, suggesting an intimate cross-talk between these two death pathways.

The role of autophagy in mammary development was shown by autophagosomes detection during lumen

formation and early stages of involution. According to Fung and coworkers, autophagy during lumen formation would promote epithelial cell survival during anoikis. Autophagy observed in the involuting mammary tissue could be the natural cell defense against the transient nutrient and hormone deprivation after lactation and the energy supply for the apoptotic process.

Autophagy exerts a tumor suppressor function by preventing the accumulation of DNA-damaged cells or deleterious organelles such as reactive oxygen species– generating mitochondria. Defects in autophagy thus favor breast tumorigenesis by promoting genome damage and instability. Indeed, heterozygous disruption of the autophagy regulator beclin-1 in mice results in reduced autophagy in vivo and development of various tumors. Monoallelic deletion in BECLIN-1 is frequent in human breast carcinomas, which then express decreased beclin-1 protein levels when compared with adjacent normal tissue. Autophagy, which initially prevents tumorigenesis, plays an opposite survival role in later stages of breast cancer. It helps prevent the anoikis of metastatic breast tumor cells that lack ECM contact. Moreover, autophagy allows hypoxic inner regions within the tumor to survive metabolic stress. Defects in both autophagy and apoptosis in those hypoxic areas lead to cell death by necrosis.

Necrosis (type 3 cell death) is an irreversible inflammatory form of cell death induced by accidental cellular damage and characterized by the rupture of the plasma membrane and the release of the intracellular components to the surrounding tissues. Necrosis can also act as the ultimate backup mechanism for cell death when both apoptosis and autophagy are impaired. By provoking an inflammatory response similar to wound healing, necrosis stimulates angiogenesis and tumor growth. Necrosis is therefore a common feature of aggressive breast tumors associated with poor prognosis.

Apoptosis is not the only mode of cell death induced by anticancer therapies. Heterogeneous modes of cellular demise are observed when breast cancer cells are exposed to anticancer agents. Apoptosis prevails in most cases, but autophagy dominates in MCF-7 breast cancer cells treated with the antiestrogen tamoxifen, aromatase inhibitors, or new sesquiterpene analogs of paclitaxel. The mitotic inhibitor paclitaxel induces a biphasic death response in breast cancer cell lines: apoptosis at low concentrations and necrosis at high concentrations. The topoisomerase inhibitor camptothecin triggers both apoptosis and autophagy in MCF-7 cells. Silencing of BID in those cells led to a shift of cell death from apoptosis to autophagy, suggesting that BID could serve as