Clinical data support the idea that independence from BM-directed survival is linked to malignant transformation in the breast. Immunologic studies [20
] have shown that invasive breast tumor cells exhibit a reduced level of apoptosis when compared with cells located in benign ductal carcinoma in situ
(DCIS) lesions. Similarly, intense staining for focal adhesion kinase, a tyrosine kinase that can induce anchorage-independent survival in epithelial cells, was detected both in invasive tumor cells and in groups of premalignant cells within adjacent DCIS lesions [21
]. Unfortunately, these data do not establish whether the enhanced survival in the transformed cells is due to genetic selection or is mediated via microenvironmental factors.
Although genetics undoubtedly plays a critical role in driving malignant transformation and apoptosis resistance in the breast, evidence is slowly accumulating that microenvironmental factors must also play a role in these processes. For example, angiogenesis can enhance mammary tumor viability, irrespective of genetic selection [22
], whereas nonmalignant MECs exposed to a reactive stromal ECM can be induced to develop a tumor-like behavior in the absence of genetic events [3
]. Indeed, stromal fibroblasts associated with mammary tumors have been shown to display a 'fetal-like' behavior, and this altered phenotype has been suggested to modify significantly the kinetics of tumor progression [23
]. Interestingly, data show that primary human breast tumors frequently exhibit a decrease in the expression of the 'differentiation-associated' laminin/collagen integrin receptors α2
, and β1
, but they often express the 'invasion and growth-linked' tenascin and fibronectin receptors αv
integrin and α5
]. Some aggressive breast tumors even retain expression of the laminin integrins α6
, and secrete BM proteins [24
Because MECs within DCIS lesions seldom show changes in their integrin expression, this indicates that the dramatic shifts in integrin expression may be necessary to support tumor cell survival and drive malignant transformation. Although one could argue that the changes in integrin expression are solely due to selection of a genetically variant population of cells, it is also possible that the altered integrin expression observed in breast tumors reflects a dynamic adaptive survival response by the tumor cells to the interstitial stromal ECM. This would depend on the ability of the tissue microenvironment to modulate integrin expression and apoptosis resistance epigenetically in MECs.
In support of this concept, significant and rapid changes in integrin expression have been documented in primary tumor cells before (in tumors in situ
that are in contact with a reactive stromal ECM) and after growth within a reconstituted BM ex vivo
]. We and others [19
] have also observed that the ECM microenvironment can dynamically modulate integrin expression in both primary and immortalized MECs. Most recently, we found that malignant transformation and β1
integrin independence in the HMT-3522 tumors occurs in conjunction with, and is dependent on, ligation of α6
integrin and secretion of BM protein (Zahir et al
, unpublished data). These results are consistent with reports that high levels of expression of α6
integrins, and BM proteins in human breast carcinomas correlate with reduced patient survival, and functional deletion of α6
integrin in metastatic breast tumor cells results in a significantly higher rate of apoptosis [24
]. Therefore, a more realistic interpretation regarding the evolution of apoptosis resistance and malignant transformation in the breast is that it arises by a combination of genetic 'mutation/selection' pressures and epigenetic 'adaptation' responses induced by the tissue microenvironment.
Studies conducted with isolated primary human breast tumor cells support the idea that stromal-epithelial interactions are primarily responsible for promoting survival in primary breast tumors in vivo,
as opposed to cell autonomous 'selection' events. 'Tumorigenic' breast cell lines isolated from primary breast tumors that represent earlier, less aggressive breast cancer phenotypes do not always exhibit true anchorage independence for growth and survival. Indeed, the majority of immortalized tumor cells used to study apoptosis regulation and anchorage independence in human breast cancer have been generated from late-stage disease cells isolated from metastatic pleural effusions, in which stromal interactions are minimal and cell–cell interactions are predominant [30
]. Along this vein, Giovanella et al
] reported that a mere 6.1% (16/262) of primary infiltrating duct-cell human breast carcinomas survived and grew following injection into nude mice. Only cells from those tumors that were highly cellular (enhanced cell–cell interactions) and lacked detectable desmoplastic hyperplasia (minimal stromal involvement) could be grown and serially transplanted into nude mice.
Interestingly, primary breast tumor cells that are first embedded within purified extracellular collagen I or a reconstituted BM can be successfully grown and propagated in nude mice [32
]. It has also been observed that primary human breast tumor cells can be maintained and effectively studied ex vivo
if the cells are maintained in the presence of either a reconstituted BM or purified collagen I matrix [33
]. Therefore, a more prudent conclusion regarding malignant transformation and apoptosis resistance in breast cancer is that, rather than acquiring absolute anchorage independence for survival, most primary human mammary tumors probably depend on altered stromal factors and/or adhesive interactions to maintain their viability in vivo