Cas is overexpressed in a large number of breast cancers, coincident with increased resistance to tamoxifen and poor relapse-free and overall survival rates (11
). Here, we have examined the putative role of Cas in resistance to the cytotoxic agent adriamycin. We show for the first time that MCF-7 cells overexpressing Cas are less sensitive to the growth-inhibitory and pro-apoptotic effects of adriamycin. The kinase activity of c-Src, but not that of the EGFR, is required for Cas-mediated enhancement of cell proliferation and survival in the presence of adriamycin. Akt and ERK1/2 activation are upregulated when cells overexpressing Cas are treated with adriamycin, coincident with a down-regulation in Bak expression. This results in a shift toward pro-survival/proliferation signals stemming from Akt and ERK1/2 and away from pro-apoptotic Bcl-2 proteins. Based on these findings, we propose that Cas overexpression in sensitive cells activates growth and survival pathways that are regulated by c-Src, Akt, and ERK1/2 and lead to the inhibition of mitochondrial-mediated cell death and promotion of continued proliferation in the presence of adriamycin (). Conversely, Cas depletion in more resistant cell lines results in a shift toward pro-apoptotic Bcl-2 proteins, coincident with decreased R123 incorporation in the presence of adriamycin (). However, residual expression of hyperphosphorylated Cas, which is seen when siControl-treated cells are treated with adriamycin, may protect cells from the cytotoxic effects of adriamycin in the presence of a similar shift toward pro-apoptotic Bcl-2 signals (). These results bring to light a novel association between Cas overexpression and adriamycin resistance, a finding that is relevant to human breast cancer since Cas is often found to be expressed at high levels in breast tumors (11
Figure 6 Models for Cas-mediated resistance to adriamycin. A, Effect of Cas overexpression in “sensitive” cell lines treated with adriamycin (MCF-7 cells expressing 6X Cas). B, Effect of Cas depletion in “resistant” cell lines treated (more ...)
The growth and survival pathways that promote resistance to adriamycin in the presence of high Cas expression share many features with those pathways that regulate Cas-dependent estrogen signaling and antiestrogen resistance. Cabodi et al.
showed that Cas overexpression in T47D cells caused increased c-Src and ERK1/2 activities in response to estrogen (22
). More recently, our group showed that Cas-dependent resistance to tamoxifen involved a signaling axis that included c-Src, EGFR, and STAT5b (13
). Interestingly, while the data presented above indicate that c-Src kinase activity is important for Cas-dependent adriamycin resistance, the catalytic activity of the EGFR appears to be less important. These data are not necessarily inconsistent with our previous data on tamoxifen resistance since the earlier study implicated EGFR functions that could have been independent of its catalytic activity. It would appear from the sum of the data that high Cas expression in breast cancer cells activates potent proliferation/survival programs that override a variety of inputs that would otherwise induce cell cycle arrest and/or death.
One component of the Cas signaling axis that appears to control resistance to adriamycin is Akt. Others have found a correlation between transient Akt activation and chemoresistance of human breast tumors (8
). Cas binds to the p85 subunit of PI3K under a number of conditions, providing a potential link between Cas overexpression and Akt activation Akt (21
). In addition to Akt, ERK1/2 activity was found to be up-regulated in Cas-overexpressing cells. Evidence for an association between ERK1/2 activation and chemotherapeutic resistance is somewhat conflicting. Two groups have shown that ERK1/2 activation correlates with protection from cytotoxic drugs (30
). However, others report that ERK activation facilitates DNA damage-induced apoptosis (32
). It would seem from these conflicting data that the role played by ERK1/2 in therapeutic resistance can vary, depending on both the nature of the cellular insult and the cell type.
The data presented above are the first to show a link between Cas overexpression and protection from mitochondrial-mediated cell death. Results from the R123 incorporation studies suggest that Cas overexpression contributes to the maintenance of mitochondrial membrane integrity in the presence of adriamycin. The pro-apoptotic Bcl-2 family members, Bak and Bax, are considered to be the gatekeepers of this cell death pathway. In cells expressing endogenous Cas levels, Bak expression remained unchanged 48 hours post-adriamycin treatment (). In contrast, Bak expression was reduced when Cas was overexpressed, and significantly more decreased when the cells were treated with adriamycin. Because other Bcl-2 members were unaffected by Cas levels in the MCF-7 model, this reduction in Bak expression would result in a decrease in the ratio of pro- to anti-apoptotic Bcl-2 family members, ultimately leading to reduced activation of the mitochondrial death pathway following adriamycin treatment. Since Akt and/or ERK1/2 activity have been shown to influence the intrinsic cell death pathway through the regulation of Bcl-2 family proteins (35
), we propose that Cas overexpression may inhibit mitochondrial-mediated cell death by maintaining a low level of the pro-apoptotic protein Bak through the combined activation of Akt and ERK1/2. Results from the MDA-MB-231 and T47D models suggest that additional factors may also play a role. Specifically, it appears that expression of hyperphosphorylated Cas, which has been shown to regulate cell growth and survival pathways (14
), may override pro-apoptotic signals stemming from Bcl-2 family members.
Overexpression of human enhancer of filamentation 1 (HEF1), which belongs to the Cas family of adaptor proteins, results in the activation of caspases, cleavage of HEF1, and apoptosis of MCF-7 cells in the absence of any exogenous stress (39
). It is noteworthy to mention that the caspase activity in this instance is likely due to caspase 7 or one of the other redundant caspases active in MCF-7 cells, as these cells have been reported to be deficient of caspase 3 (40
). Based on the data reported herein, it would appear that Cas and HEF1 function quite differently in controlling cell survival. Rather than inducing cell death, Cas overexpression had positive effects on cell proliferation and survival both in the presence and absence of adriamycin (). Coincidentally, Cas overexpression did not result in PARP cleavage, suggesting that caspase activity was not induced under these conditions (). Nonetheless, Cas has been previously identified as a substrate for caspases during treatment with etoposide, staurosporine and cisplatin (26
). Indeed, we have observed a decrease in full-length Cas following treatment of cells with adriamycin, and this coincided with the appearance of a 31 kDa carboxy-terminal fragment that has been reported to accumulate in response to caspase cleavage of Cas and induce apoptosis in MCF-7 cells (26
). Interestingly, the 31 kDa species was detected in Cas-overexpressing cells and may contribute to the low level of apoptosis exhibited by these cells in the presence of adriamycin (see ). However, full-length Cas remains high under these conditions coincident with the cells exhibiting greater resistance to the cytotoxic effects of adriamycin. This suggests that the pro-growth and survival activities of full-length Cas may play a dominant role over the apoptotic activities of the 31 kDa fragment in the presence of adriamycin.
Together, these data support a model whereby the cellular response to cytotoxic therapies such as adriamycin is governed by the balance and integration of proliferation, survival, and death pathways. Since Cas overexpression in human breast tumors is associated with poor prognosis, this study identifies the Cas signaling axis as a potential target that can be exploited to enhance the efficacy of adriamycin treatment and/or prevent resistance.