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Proteomic Analysis of Hct116 Colon Cancer Cells Treated with Inhibitors That Bind to the N‐terminal Versus the C‐terminal Domain of Hsp90
Author(s) -
Balch Maurie,
Hartson Steven D,
Blagg Brian S,
Matts Robert L
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.926.12
Subject(s) - hsp90 , heat shock protein , stable isotope labeling by amino acids in cell culture , hsp70 , heat shock , chemistry , proteome , cancer research , cancer cell , cancer , biochemistry , biology , microbiology and biotechnology , proteomics , genetics , gene
Cancer cells rely on the chaperone protein Hsp90 to maintain oncoproteins in a functional state to promote proliferation and evade cell death. Because Hsp90 supports many proteins involved in tumorigenseis and progression, Hsp90 inhibitors are thought to have the potential to be very effective anti‐cancer drugs. Hsp90 inhibitors currently in clinical trials target the N‐terminal ATPase domain of Hsp90. However, these N‐terminal inhibitors have not been as effective as hypothesized because they induced the heat shock response, which is anti‐apoptotic, and other untoward toxicites. The upregulation of the heat shock response, which most significantly raises Hsp70 levels, requires dose escalation, and Hsp90 inhibitors with superior efficacy are still being sought. Hsp90 inhibitors that bind to the C‐terminal domain of Hsp90 do not induce the heat shock response. Novobiocin analogues are being synthesized to elucidate structure activity relationships that will lead to superior derivatives that target the C‐terminal domain of Hsp90. In this study, we compare and contrast the effects of 17‐DMAG, a traditional N‐terminal inhibitor to those of KU 174, a new cytotoxic C‐terminal inhibitor on the proteomes of HCT166 colon cancer cells. Using SILAC and high pH MudPIT coupled to an Orbitrap Fusion Tribrid mass spectrometer, we identified 4,235 protein from the KU 174 dataset and 5,520 from the 17‐DMAG dataset. Significant protein expression changes were determined by a one‐sample two‐sided t‐test, using Benjamini‐Hochberg FDR set to 0.01. Results showed that KU 174 induced 635 significant protein expression changes with 138 being upregulated and 497 proteins downregulated. 17‐DMAG induced 1,700 protein expression changes with 596 being upregulated and 1104 being downregulated. In both sets, the majority of the proteins were downregulated, which is consistent with other Hsp90 inhibitor studies. There were 266 proteins downregulated by both drugs, while 43 proteins were upregulated by both drugs. The Pearson correlation coefficient was 0.599 when comparing significant expression changes by KU 174 to those changed by 17‐DMAG. A coefficient of 0.728 was calculated when only the significant downregulated proteins in both sets were analyzed. Therefore, KU 174 does not share an identical mechanism with 17‐DMAG, but shares some similarity. Individual effected pathways will be discussed in more detail, but overall, results show that KU 174 induces ER and mitochondrial stress, but does not induce a traditional HSF‐1‐dependent heat shock response like N‐terminal inhibitors. In addition, KU 174 downregulates a subset of Hsp90 client proteins that are involved in multiple tumor cell survival pathways. These results show that KU 174 gives a distinct proteomic fingerprint from that of 17‐DMAG, and may offer some improved effectiveness over traditional N‐terminal Hsp90 inhibitors in the clinic. Support or Funding Information This research was supported by an OU Cancer Institute Breast Cancer Research Seed Grant (RLM) the OAES projects 1970 and 2959 (RLM) and NIH CA120458 (BSB).