Kinome profiling reveals breast cancer heterogeneity and identifies targeted therapeutic opportunities for triple negative breast cancer

Abstract

Our understanding of breast cancer heterogeneity at the protein level is limited despite proteins being the ultimate effectors of cellular functions. We investigated the heterogeneity of breast cancer (41 primary tumors and 15 breast cancer cell lines) at the protein and phosphoprotein levels to identify activated oncogenic pathways and developing targeted therapeutic strategies. Heterogeneity was observed not only across histological subtypes, but also within subtypes. Tumors of the Triple negative breast cancer (TNBC) subtype distributed across four different clusters where one cluster (cluster ii) showed high deregulation of many proteins and phosphoproteins. The majority of TNBC cell lines, particularly mesenchymal lines, resembled the cluster ii TNBC tumors. Indeed, TNBC cell lines were more sensitive than non-TNBC cell lines when treated with targeted inhibitors selected based on upregulated pathways in cluster ii. In line with the enrichment of the upregulated pathways with onco-clients of Hsp90, we found synergy in combining Hsp90 inhibitors with several kinase inhibitors, particularly Erk5 inhibitors. The combination of Erk5 and Hsp90 inhibitors was effective in vitro and in vivo against TNBC leading to upregulation of pro-apoptotic effectors. Our studies contribute to proteomic profiling and improve our understanding of TNBC heterogeneity to provide therapeutic opportunities for this disease.

Figure 1. The Kinex antibody arrays confirm the heterogeneity of breast cancer

Lysates from fresh frozen primary, high grade, breast tumors prior to therapy were subjected to the Kinex™ antibody arrays (Supplementary Fig. S1). (A) Sample-sample correlation plot using all the probes revealed six clusters (i – vi) which were compared by the SAM method and 1.75-fold change cutoff (Supplementary Fig. S1). (B) Hierarchal clustering of patient tumors based on the differentially expressed proteins/phosphoproteins. The numbers on the right of the heat map mark the 14 branches which differentiate the tumor clusters and these are annotated in Supplementary Table 2S. (C) Lysates from established breast cancer cell lines were also subjected to the Kinex™ arrays. Hierarchal clustering of the breast cancer cell lines was supervised by the probes that were differentially expressed across the six clusters of primary patient tumors

Figure 2. Validation of phosphorylations of proteins identified in the Kinex™ antibody arrays using the Proteome Profiler antibody arrays

Lysates from four TNBC cell lines (MDA-MB-231, Hs.578T, BT549 and MDA-MB-435) and four luminal cell lines (MCF7, T47D, MDA-MB-175 and ZR751) were analyzed using the Proteome Profiler™ Array for (A) human phospho-kinases and (B) human phospho-MAPK pathway. Bar graphs show the average ratio of TNBC/luminal normalized intensity for each phosphoprotein (±SEM, n = 4 cell lines each with two technical replicate for each phosphoprotein). Dashed lines mark the 1-fold (no change) and the 2-fold ratios and insets show representative arrays. Red bars: probes which validate exact phosphorylations in the Kinex™ arrays. Orange bars: probes that identify additional phosphorylations of phosphoproteins to those identified in the Kinex™ arrays. Blue bars: probes identifying phosphoproteins that were upregulated at the protein level in the Kinex™ arrays. Black bars: probes that were not present in the Kinex™ arrays or did not meet the cutoff criteria in analysis

Figure 3. Pathway analysis of deregulated proteins and phosphoproteins in TNBC

Pathway analysis in IPA® using only direct protein-protein interaction data was used and the deregulated proteins/phosphoproteins in TNBC cluster ii identified three overlapping networks. These networks were merged and visualized (Supplementary Fig. S3). (A) Canonical signaling pathways using IPA® which were enriched (Fisher's exact test p-value) in the deregulated proteins/phosphoproteins in TNBC cluster ii subgroup. (B) The upregulated phosphoproteins (marked with P) and proteins in cluster ii were analyzed using IPA® and a single network of direct protein-protein interactions was identified. Shades of pink/red reflect the extent of upregulation. At least two nodes, MAPKs and HSPs, were clearly identified in the center of complex signaling cascades.

Figure 4. Targeted inhibition of activated kinases in cluster ii kills TNBC cell lines

(A) Activated cross-talking canonical signaling pathways in TNBC tumors in cluster ii. (B) Six TNBC cell lines, four non-TNBC cell lines and the “near-normal” MCF10A cell line were treated (in duplicates) with escalating doses of the specified drugs and survival was measured six days after treatment using the CellTitre™ assay. Data shown is -log10[IC50], a measure of sensitivity, where red color denotes relative increase in sensitivity. The red box marks drugs which specifically killed TNBC cell lines in comparison to non-TNBC cell lines. The raw data of dose response curves are shown in Supplementary Fig. S4. (C & D) Two Hsp90 inhibitors (PF04929113 and AUY922) were separately used as single agents or in combination with the specified kinase inhibitors. The combinations were performed using the IC25 of each drug against the specified TNBC cell lines. Data shown is the survival of cells where red color represents more killing than green color. Raw data for panels C and D are shown in Supplementary Fig. S5.

Figure 5. The combination of Hsp90 inhibition and Erk5 inhibition is synergistic in TNBC

(A&B) Cultures of six TNBC cell lines were treated with escalating doses of ERK5i XMD 8-92 combined with Hsp90i PF-04929113 (A) or the Hsp90i AUY922 (B) to calculate the combination index (CI, < 1 is synergistic). (C) Graph of CI for the Hsp90 inhibitors at the lowest doses with Erk5 inhibition. (D) Immunoblot analysis of MDA-MB-231 cells treated with 1 µM Erk5i (XMD 8-92) alone, 2 nM of Hsp90i (AUY922) alone, chemotherapy alone (2.5 nM docetaxel and 10 nM doxorubicin) or the combinations at 24 hours after treatment with indicated antibodies. (E) Clonogenic survival of MDA-MB-231 cells when subjected to treatments as indicated in D at 72 hours or 96 hours.

Figure 6. The combination of Erk5 and Hsp90 inhibitors in vivo

Female nude mice bearing the TNBC MDA-MB-231 xenografts (50mm³ volume) were untreated (vehicle control) or treated with chemotherapy (chemo; 2 mg/kg docetaxel and 10 mg/kg doxorubicin administered i.p. on days 1 and 8). Additional groups of mice were treated with Erk5 inhibitor alone (Erk5i XMD 8-92 at 25 mg/kg) or Hsp90 inhibitor alone (Hsp90i AUY922 at 25 mg/kg) daily on days 1-5 and days 8-12. Additional groups of mice were treated with the combinations; chemo+Erk5i or Hsp90i+Erk5i. Six tumors were excised on day 13 (after treatment completion) for ex vivo studies. (A) Tumor growth curves based on change in tumor volume compared to day 0 prior to treatment (n=10 tumors/group). (B) Representative immunoblots from two tumors per group with indicated antibodies (cPARP1 – cleaved PARP1). Blots from additional four more tumors/group showed similar results (Supplementary Fig. S7). (D&E) Representative images (20× magnification, scale bar 100 µm) of tumor histology (H&E and trichrome staining) and IHC staining for DNA double strand breaks (DSBs, γH2AX staining) and apoptosis (ApopTag) from control and Hsp90i+Erk5i treated mice on day 13. Quantification of staining from six tumors/group was performed as in Methods; (E) ApopTag staining, (F) γH2AX staining, (G) cellularity: ratio of pink (cells) to blue (collagen) staining from trichrome staining. Error bars represent the standard error of the mean, * p<0.05, ** p<0.01 and *** p<0.001 from One-Way ANOVA (GraphPad® Prism).