In an attempt to identify detailed molecular mechanisms of drug response and resistance to treatment with 17AAG we performed gene expression profiling of eight breast cancer cell lines treated with the drug. Although there are many studies analyzing the molecular effect of HSP90 inhibition, global expression changes after 17AAG in breast cancer have not been analyzed in-depth. In this study we examined the expression changes in the sensitive (MCF-7, MDA-MB-157, Hs578T, UACC3199, HCC1937, MDA-MB-436) and two resistant (MDA-MB-231, T47D) breast cancer cell lines to the HSP90 inhibitor, 17AAG.
Gene expression profiling after 17AAG, showed different number of 17AAG responsive genes associated to the different cell lines. As HSP90 inhibition results in degradation of client proteins, it is possible that intrinsic differences in the abundance of HSP90 clients in the cells cause subsequent transcriptional changes in a cell line dependent manner. Although it was clear that HSP90 inhibition produced cell line dependent changes we could not associate them to the fact that these cell lines belong to different molecular breast cancer subtypes. Since one of the resistant cell lines, MDA-MB-231, is reported to be basal B and the other, T47 D, as luminal, and the rest of the sensitive cell lines being all basal B except for MCF7 (luminal) and HCC 1937 (basal A) [36
] it is likely that sensitivity to 17AAG is not connected to known breast cancer subtypes.
However, there were a group of genes commonly regulated in all sensitive cell lines upon treatment. We have identified a breast cancer associated molecular signature of response to 17AAG, consisting of 35 17AAG-responsive genes. This gene signature of 17AAG response included, similar as previously reported in other studies [24
], members of the chaperon complex, HSP90
itself and HSP70 (HSPA8)
. These changes were identified in a study done in an ovarian cancer cell line as likely on-target effects of the drug, which are induced as a direct consequence of HSP90 inhibition [25
]. Importantly, HSP70 isoforms have been used as pharmacodynamic end point in clinical trials [27
]. In addition to these chaperons, we also identified up-regulation of other members of heat shock response family such as HSPA4L, HSPA1L, HSP40 (DNAJA1
and in CHORDC1
, another HSP90 binding protein. Interestingly, we found transcriptional induction of HSP90, HSP70 (HSPA8)
in one of the resistant cell lines analyzed, MDA-MB-231. Moreover, HSC70/HSPA8 and HSP72 induction was confirmed by western blot in these cells. The up-regulation after treatment of HSP90 and HSP70 (both HSC70 and HSP72) might suggest that HSP90 is inhibited at 500 nM 17AAG in both sensitive and MDA-MB-231 resistant cell line. Yet, the resistance in the MDA-MB-231cell lines might be caused by very low levels of NQO1, as reported previously [37
]. HSC70 and HSP72 also have an antiapoptotic role [38
], so their induction in MDA-MB-231 might also contribute to the 17AAG resistance in these cells. The other resistant T47 D cells seem not to show the induction of Hsp70 isoforms following treatment what could suggest lack of HSP90 target inhibition by 17AAG. Interestingly, T47 D cells have some expression of NQO1 and probably an alternative mechanism of resistance.
The analysis of a number of HSP70 isoforms by immunoblotting revealed, in addition to induction of HSC70 and HSP72, that some other HSP70 isoforms were also induced by 17AAG. Up-regulation of HSPA1L and HSPA2 were found in sensitive cells following exposure to 17AAG. However, they showed lack of induction in resistant MDA-MB-231 cells. We suggest that HSPA1L and HSPA2 could represent potential biomarkers to follow up the effectiveness of 17AAG in breast cancer, although the mechanism underlying this effect is still unclear.
Other genes from the signature also exhibiting increased expression in response to 17AAG were Rac GTP-ase activating protein (RACGAP1
), ubiquitin conjugating enzyme E2C (UBE2C
), zinc fingers proteins (ZNF473, ZNF587
) and MHC class I antigen (MICB
). These data are in line with previous work done on 17AAG treated ovarian cancer cell lines [25
]. Additionally, in a recent microarray study of novel HSP90 inhibitor (IPI-504) in pancreatic cancer, Song and colleagues [26
] identified similar class of up-regulated genes following treatment along with GTPase activating proteins, zinc finger proteins, heat shock proteins and ribosomal proteins.
There were also genes with decreased expression following HSP90 inhibition by 17AAG. Some of them clearly represent cell cycle regulators (CCND1, PLK3) and important proliferation signaling pathways mediators (JUNB, NFKBIA). The decreased expression of them might be a consequence of cell cycle arrest produced after 17AAG.
The fact that the expression changes seen in the primary tumor sample after treatment with 17AAG resembled the changes in cell lines, suggests that this set of genes would constitute a robust signature of response in breast cancer. Further studies in additional tumor biopsies are required to better establish the value of the biomarkers identified in this study.
Since effects of 17AAG are driven by HSP90 client proteins degradation, we were interested in studying whether protein depletion also results in transcriptional changes of known client proteins following treatment. Changes in the mRNA levels of a number of client proteins were evident in cell lines responsive to 17AAG, while resistant cell lines demonstrated insignificant variations in transcriptional levels of HSP90 interactors. This observation suggests that the use of transcriptional changes of HSP90 client proteins may facilitate the selection of potentially responsive patients to 17AAG therapy. It is known that client proteins are variable in different types of tumor [10
]. It is reasonable then, to find cell line specific transcriptional changes profiles in the 17AAG sensitive cell lines. This finding could be of interest in order to define, in further studies, key client proteins for specific tumor subtypes, with potential clinical significance. In addition, consistently up or down-regulated HSP90 client transcripts following treatment were identified shared by some of the cell lines analyzed (AHSA1
, CCNB1, IRAK1)
, that could represent important HSP90 clients in breast cancer. It is clear that biological processes are regulated not only at transcriptional level, but also protein levels or posttranscriptional modifications of proteins are important when analyzing the effects of HSP90 inhibitors. However, mRNA changes could be helpful in order to evaluate the effect of the drug in clinical samples.
Mechanisms of resistance to 17AAG remain largely unknown. We analyze global expression changes after 17AAG occurring in resistant cells, to define genes or pathways commonly involved in insensitivity to this drug. The identification of pathways in relation to 17AAG resistance would be important to develop in future candidate treatments to be used in combination with 17AAG to induce growth inhibitory effects in the insensitive cell lines. Functional studies with the pathway-focused reporter assays shown significant up-regulation of NF-κB pathway in resistant cells after exposure to 17AAG. It is interesting that GSEA also showed NF-κB pathway as potentially involved in resistance to 17AAG. In addition, there are two other resistance associated pathways in the top-ten list that were related to inflammation/immune response (NTHI_PATHWAY and INFLAM_PATHWAY) (Additional file 1
: Supplemental Table S3). Thus it is possible that resistant breast cancer cell lines make use of the inflammation/immune response machinery to evade cell cycle arrest or apoptosis after 17AAG treatment. NF-κB activation was already observed after treatment with cancer chemotherapeutic agents such as gemcitabine [39
], thereby inducing resistance to apoptosis which results in poor clinical outcome. NF-κB is a ubiquitously expressed transcription factor that is involved in a wide spectrum of cellular functions including cell cycle control, stress adaptation, inflammation and control of apoptosis [42
]. Activation of NF-κB has been implicated in the development of a number of human malignancies, and it appears to be important for the survival of cancer cells, as well as the conferring of more aggressive tumor phenotype and resistance to drug therapies [43
]. Moreover, high basal levels of NF-kB have been related with resistance to gemcitabine in pancreatic carcinoma cell lines [41
]. The finding of NF-kB pathway activation after 17AAG treatment significantly associated to resistant cell lines suggests that simultaneous use of anti-tumor agents that block NF-κB activity together with HSP90 inhibitors may have a greater therapeutic value. MAPK/JNK, and also MAPK/ERK pathway were activated in the resistance cell lines, suggesting these pathways may be important for survival of cells after 17AAG treatment.
In summary, in this study we have established a 35 gene-based molecular signature of response to 17AAG in breast cancer, which revealed novel pharmacodynamic markers of drug response. Secondly, we have defined transcriptional changes in known HSP90 client proteins, which may be useful to monitor drug efficacy and finally, we have identified signaling pathways differentially activated following 17AAG in resistant cell lines.