The results presented here using breast cancer cell lines identify differential sensitivity to TRAIL-mediated apoptosis based on the molecular phenotype of the breast cancer cells. The majority of triple-negative breast cancer cell lines, and particularly all of those with a mesenchymal phenotype, are TRAIL-sensitive (, ). The three TRAIL-resistant triple-negative breast cancer cell lines do not have a mesenchymal phenotype, both based on our expression analysis of vimentin and E-cadherin and the published cDNA microarray expression data ( and [6
]). Of note, two of the three TRAIL-resistant triple-negative breast cancer cell lines (MB468 and BT20) have amplification of the EGFR and thus may represent a unique subset of triple-negative breast cancers [22
The mesenchymal cytoskeletal protein, vimentin, was expressed predominantly in the majority of the TRAIL-sensitive triple-negative cell lines. The expression of vimentin protein is highly predictive of TRAIL-sensitivity among the triple-negative breast cancer cell lines (seven of seven vimentin protein positive cell lines were TRAIL-sensitive). In contrast, only one of the four triple-negative breast cancer cell lines that do not express vimentin protein is sensitive to TRAIL-induced apoptosis (i.e.,
SUM149). This cell line expresses vimentin mRNA and clusters with the mesenchymal cell lines by transcriptional profiling ( and [6
While previous transcriptional profiling data has identified triple-negative breast cancer cell lines with a mesenchymal phenotype, this classification has not been identified in transcriptional profiling of primary breast cancer samples [6
]. The reason for this discrepancy is not clear. These mesenchymal cell lines may represent a transformation that occurs in culture due, for example, to the lack of interactions with the tumor stroma. Alternatively, these cells may be a minor population that is better able to grow under cell culture conditions. However, immunohistochemical analysis of primary tumors has identified tumors in which the cancer cells express vimentin [17
]. In the largest study (>2000 samples), approximately 14% of breast tumors expressed vimentin in the tumor cells [27
]. Also, in this study, 35% of the ER negative tumors were vimentin positive [27
]. The enrichment of vimentin-positive tumors among the ER negative tumors is consistent with vimentin expression in triple-negative tumors. However, this work did not simultaneously characterize HER-2 amplification and so the tumors can not be accurately classified as triple-negative [27
]. Two small studies demonstrated vimentin expression in 17 of 18 and 4 of 11 triple-negative breast cancer samples [17
]. In one of these studies, all of the samples had been classified as basal-type tumors by microarray analysis and the vimentin staining observed was strong and diffuse in the tumors [17
]. Thus, vimentin-positive triple-negative tumors are a clinical entity and not a cell culture artifact.
Recently moesin has been identified as a marker of triple-negative breast cancer cells in primary breast cancer samples and breast cancer cell lines [14
]. This protein is a member of the ezrin, radixin, moesin family of proteins which play important roles in cytoskeletal function, cell adhesion, and motility [28
]. In our work, moesin protein was expressed in all of the mesenchymal TRAIL-sensitive triple-negative cell lines and in one of the two TRAIL- sensitive HER-2 amplified cell lines. Thus 9 of 10 TRAIL-sensitive cell lines express moesin - making moesin a potential biomarker for TRAIL sensitivity.
Together, these data suggest that characterization of vimentin and moesin expression by immunohistochemistry may be useful biomarkers for TRAIL sensitivity. Measurements of vimentin and moesin mRNA and/or protein expression could be incorporated readily into future clinical trials of TRAIL agonists to investigate whether these proteins have predictive value for TRAIL response in patients with breast cancer.
The mechanism underlying the differential TRAIL sensitivity of the mesenchymal triple-negative breast cancer cell lines is unknown. siRNA knockdown experiments indicate that while vimentin, moesin, and E-cadherin protein expression are predictive of TRAIL sensitivity or resistance, they do not regulate TRAIL function (). Recent analysis in pancreatic cancer, colorectal cancer, non-small-cell lung cancer, and melanoma cell lines has identified low expression of O-glycosylation genes as a potential mechanism of TRAIL-resistance [29
]. However our gene expression analysis did not find a correlation between the genes described in that work for O-glycosylation and TRAIL sensitivity in the breast cancer cell lines (unpublished observation). Our analysis of gene expression identified 77 genes that classified the cell lines into two clusters based on TRAIL sensitivity. None of these genes have been implicated as regulators of TRAIL activity (). Several of the genes that are expressed at a higher level in the TRAIL-sensitive triple-negative cell lines have been described previously in breast cancer. For example, the receptor tyrosine kinase Axl, also preferentially expressed in the mesenchymal breast cancer cells ( and [30
]), has been implicated in angiogenesis and in breast cancer tumorigenesis [31
]. The membrane protein EMP3 was found highly expressed in the mesenchymal cell lines but its function is unknown [32
]. PROCR has been described as expressed in breast cancer cells with a “stem cell” phenotype [33
]. Interestingly, the mesenchymal breast cancer cell lines have characteristics consistent with putative breast cancer “stem cells” (e.g.
, high CD44 and low CD24 expression) ( and [6
]). A role for these proteins in TRAIL activity has not been studied.
We also investigated inhibition of the EGFR as a means to overcome TRAIL resistance in the epithelial triple-negative breast cancer cell lines. EGFR is frequently expressed in triple-negative breast cancer and we found that it was expressed in all of the triple-negative breast cancer cell lines tested whether they are TRAIL-sensitive or resistant ([16
] and ). While we found that EGFR inhibition can enhance TRAIL-mediated apoptosis in EGFR-expressing breast cancer cells that are already sensitive to TRAIL (i.e.,
the mesenchymal triple-negative breast cancer cell lines), EGFR inhibition does not overcome the resistance in the epithelial triple-negative breast cancer cell lines ().
The patients with triple-negative breast cancer have no clinically validated molecularly targeted therapies and have a poor prognosis relative to those patients having other breast cancer subtypes [4
]. The data here provide a strong rationale for testing TRAIL receptor agonists in this population - especially those with tumors that have mesenchymal features (i.e.,
vimentin expression). In addition, combination of EGFR inhibitors with TRAIL may be particularly effective for the treatment of triple-negative breast cancers with mesenchymal features.
HER-2 amplified tumors represent a second population that may be sensitive to TRAIL. However, only two of five cell lines tested here with HER-2 amplification are sensitive to TRAIL-induced apoptosis and three additional lines reported in the literature are resistant to TRAIL [2
]. In addition, the IC50 in these cell lines is higher than the IC50 in the sensitive triple-negative cell lines (). These cell lines cluster with the resistant cell lines by transcriptional profiling and they do not express the vimentin protein (). Thus, they do not have mesenchymal markers. One of the TRAIL-resistant HER-2 amplified cell lines (BT474) also expresses ER, and all of the ER-positive cell lines tested are resistant to TRAIL-mediated apoptosis (). Thus, ER expression appears to identify TRAIL-resistant cell lines. It is not clear what distinguishes the other TRAIL-resistant HER-2 amplified cell lines from the two TRAIL-sensitive HER-2 amplified cell lines. Interestingly, our previous work suggests that TRAIL-induced apoptosis could be enhanced in the resistant cell lines with HER-2 amplification (e.g.
, SKBR3 and MB453) when TRAIL was combined with trastuzumab pre-treatment [5
]. Thus, tumors with HER-2 amplification may benefit from combined molecularly targeted therapies of TRAIL and trastuzumab.
Expression levels of TRAIL receptors do not predict whether a cell will be sensitive or resistant to TRAIL (). TRAIL agonists which target both TRAIL receptors and those which selectively target either TRAIL-R1 or TRAIL-R2 are currently in phase I and phase II clinical trials. Previous work has demonstrated that TRAIL induces apoptosis predominantly via TRAIL-R1 in some tumors and via TRAIL-R2 in others [34
]. Using RNAi to selectively reduce expression of either TRAIL-R1 or TRAIL-R2, our data indicate that TRAIL-R2 is the predominant death-inducing receptor in the sensitive breast cancer cell lines (in both the TRAIL-sensitive triple-negative and HER-2 amplified cells (). Thus, agonists that activate both TRAIL-R1 and TRAIL-R2 or those that selectively activate TRAIL-R2 (but not those that selectively activate TRAIL-R1) should be tested in breast cancer patients.
Overall, our work suggests that TRAIL receptor agonists will have differential effects on breast cancer tumors with different molecular phenotypes. This work suggests that patients with triple-negative tumors with mesenchymal features, a group of patients with a poor prognosis, are most likely to benefit from TRAIL receptor agonists. Importantly, these data provide a basis for designing clinical studies using these agents in triple-negative breast cancer patients.