We have demonstrated that the small heat shock protein αB-crystallin is a novel oncoprotein: overexpression of WT αB-crystallin was sufficient to transform immortalized human MCF-10A MECs and to induce mammary carcinomas in vivo. Specifically, overexpression of WT αB-crystallin in MCF-10A cells induced profound abnormalities in mammary acini that resembled features of preinvasive tumors: enlarged masses with filled lumens, loss of polarity, increased proliferation, and diminished luminal apoptosis (19
). Notably, the abnormal morphology of αB-WT acini was suppressed by silencing αB-crystallin by RNAi. αB-Crystallin overexpression also induced neoplastic changes in immortalized MCF-12A MECs grown in 3D culture, indicating that the observed oncogenic effects are not cell type–specific. Similar neoplastic abnormalities were induced in mammary acini by activating the ErbB2/HER-2 oncogene, which, like αB-crystallin, inhibits apoptosis and promotes proliferation (19
). Moreover, overexpression of αB-crystallin in MCF-10A cells induced EGF- and anchorage-independent growth and promoted cell migration and invasion in vitro, which are defining characteristics of malignant neoplasms. Furthermore, MCF-10A pools stably expressing WT αB-crystallin formed invasive mammary carcinomas in nude mice, thereby confirming the malignant nature in vivo of MCF-10A cells transformed by αB-crystallin. Importantly, the levels of ectopically expressed αB-crystallin observed in mammary acini are comparable to those previously observed in human DCIS (13
). In contrast, overexpression of similar levels of a αB-crystallin phosphorylation mutant that mimics stress-induced phosphorylation and is impaired in its cytoprotective function was largely incapable of transforming MECs or initiating mammary tumors, which suggests that the transforming activity of αB-crystallin may be negatively regulated by phosphorylation. These data indicate that αB-crystallin is a bona fide oncoprotein, which to our knowledge was previously unrecognized.
How then does αB-crystallin transform MECs? Although overexpression of αB-WT, but not the 3XSE mutant, induced EGF-independent activation of multiple signaling pathways, the transformed phenotype of MCF-10A–αB-WT cells was selectively suppressed by MEK inhibitors. Indeed, treatment of αB-WT mammary acini with MEK inhibitors restored apicobasal polarity, suppressed proliferation, and promoted apoptosis of centrally located MECs, resulting in morphologically normal acini despite their transformed genotype. These findings are consistent with previously published reports indicating that MEK inhibition suppresses, at least in part, the neoplastic phenotype of breast cancer cells grown in 3D basement membrane culture (28
). Constitutively active MEK transforms NIH 3T3 cells, while a dominant-negative MEK inhibitor suppresses transformation by v-Src or H-Ras, indicating a critical role for the Raf/MEK/ERK pathway in transformation (30
). Overexpression of αB-crystallin increased levels of total and phosphorylated ERK1/2 protein, a pattern commonly observed in human breast cancer and in MCF-10A cells transformed by H-Ras (32
). Although ERK is activated by EGFR/HER family members and integrins in breast cancer (19
), our results suggest that αB-crystallin contributes to ERK activation in basal-like tumors. Consistent with this notion, activation of the MEK/ERK pathway produces many of the same consequences we observed by overexpressing αB-crystallin, namely, increased proliferation, reduced apoptosis, and increased cell migration and invasion (34
). Clearly, the mechanisms by which αB-crystallin activates ERK have yet to be elucidated. As a molecular chaperone, αB-crystallin may regulate ERK1/2 protein stability and/or phosphorylation/dephosphorylation as Hsp90 regulates Akt and Raf kinase activity (35
). Nevertheless, our data indicate unequivocally that the MEK/ERK pathway plays a key role in MEC transformation by αB-crystallin.
We have also demonstrated that MCF-10A–αB-WT cells induced mammary carcinomas in nude mice. In contrast, MCF-10A cells constitutively overexpressing oncogenic H-RasV12, cyclin D1, or ErbB2 do not induce tumors in nude mice, despite promoting anchorage-independent growth in vitro (26
), thereby underscoring the tumorigenic potency of αB-crystallin. Early-stage MCF-10A–αB-WT carcinomas had prominent mesenchymal-like spindle cells that expressed αB-crystallin and both mesenchymal (vimentin) and epithelial markers (cytokeratins). These characteristics are strongly suggestive of epithelial-mesenchymal transition (EMT), a process implicated in carcinoma progression, whereby epithelial cells lose polarity and cell-cell adhesion and acquire mesenchymal characteristics such as motility (39
). Intriguingly, late-stage carcinomas continued to express αB-crystallin, vimentin, and cytokeratins and retained a less prominent spindle cell component. Although it remains to be determined whether the observed differences in early and late mammary carcinomas represent a temporal progression, it is striking that overexpression of αB-crystallin promoted some aspects of EMT in vitro (disruption of epithelial polarity and enhanced cell migration and invasion) and corresponding histological features in vivo (spindle cells and vimentin expression). Consistent with its potential role in neoplastic EMT, αB-crystallin expression is induced at an early stage during cardiac and skeletal muscle differentiation (mesodermal tissue) and protects myoblasts from apoptosis (10
). Both early and late tumors were ER-negative, progesterone receptor–negative (PR-negative), and ErbB2/HER-2–negative (data not shown) and resembled human metaplastic breast carcinomas, a histopathologic subtype notable for mesenchymal spindle cells mixed with epithelial glandular elements (41
). Metaplastic carcinomas are also predominantly ER-, PR-, and ErbB2/HER-2–negative and frequently express vimentin and CK5 (43
), an expression pattern shared by basal-like breast carcinomas. Hence, the mammary carcinomas induced by αB-crystallin overexpression recapitulated aspects of human basal-like breast tumors, and this xenograft model may be useful for testing new breast cancer therapies. In addition to its role in transformation and tumorigenesis, we recently demonstrated that overexpression of αB-crystallin in breast carcinoma cells promotes their growth as xenograft mammary tumors (11
), suggesting that αB-crystallin may also play a role in breast cancer progression.
is commonly mutated in basal-like breast tumors, and BRCA1
carriers tend to develop these tumors (4
), additional genes likely contribute to their aggressive nature. The potent transforming and tumorigenic activity of αB-crystallin, coupled with our observation that αB-crystallin was expressed in approximately half of basal-like breast tumors, suggests that αB-crystallin may contribute to the aggressive behavior of these basal-like tumors. Indeed, we observed that αB-crystallin predicted shorter disease-specific survival independent of tumor grade, lymph node status, and ER and ErbB2/HER-2 status, suggesting that αB-crystallin may provide additional prognostic information for breast cancer patients independent of these established factors. Our findings agree in part with those of a recent study indicating that αB-crystallin expression in breast cancer was associated with lymph node involvement and shorter survival in univariate analyses (14
). In contrast to our findings, αB-crystallin expression was not predictive of survival independent of lymph node status in the study by Chelouche-Lev et al. (14
). These investigators observed that 88% of breast carcinomas expressed αB-crystallin by IHC, in contrast to the 11% we noted. It is unclear whether these disparities reflect methodological or patient cohort differences. However, our IHC results are consistent with gene expression data from an independent breast cancer cohort indicating that only a minority of breast carcinomas highly express the αB-crystallin
gene. We are currently examining the expression of αB-crystallin in additional breast cancer cohorts to determine whether αB-crystallin may be a clinically useful predictor of prognosis or drug response. Indeed, the suppression of the transformed phenotype of MCF-10A–αB-crystallin cells by MEK inhibitors in vitro suggest that MEK inhibitors may be an effective therapy for basal-like breast tumors expressing αB-crystallin. Orally active MEK inhibitors have been shown to potently suppress colon cancer growth and melanoma metastasis in xenograft models (45
), underscoring the potential feasibility of this therapeutic strategy.