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. 2024 Sep;14(9):1559-1569.
doi: 10.1002/2211-5463.13843. Epub 2024 Jul 24.

Bag-1-mediated HSF1 phosphorylation regulates expression of heat shock proteins in breast cancer cells

Tugba Kizilboga  1   2 , Can Özden  1 , Nisan Denizce Can  1 , Evren Onay Ucar  3 , Gizem Dinler Doganay  1
Affiliations

Bag-1-mediated HSF1 phosphorylation regulates expression of heat shock proteins in breast cancer cells

Tugba Kizilboga et al. FEBS Open Bio. 2024 Sep.

Abstract

According to the World Health Organization in 2022, 2.3 million women were diagnosed with breast cancer. Investigating the interaction networks between Bcl-2-associated athanogene (Bag)-1 and other chaperone proteins may further the current understanding of the regulation of protein homeostasis in breast cancer cells and contribute to the development of treatment options. The present study aimed to determine the interactions between Bag-1 and heat shock proteins (HSPs); namely, HSP90, HSP70 and HSP27, to elucidate their role in promoting heat shock factor-1 (HSF1)-dependent survival of breast cancer cells. HER2-negative (MCF-7) and HER2-positive (BT-474) cell lines were used to examine the impact of Bag-1 expression on HSF1 and HSPs. We demonstrated that Bag-1 overexpression promoted HER2 expression in breast cancer cells, thereby resulting in the concurrent constitutive activation of the HSF1-HSP axis. The activation of HSP results in the stabilization of several tumor-promoting HSP clients such as AKT, mTOR and HSF1 itself, which substantially accelerates tumor development. Our results suggest that Bag-1 can modulate the chaperone activity of HSPs, such as HSP27, by directly or indirectly regulating the phosphorylation of HSF1. This modulation of chaperone activity can influence the activation of genes involved in cellular homeostasis, thereby protecting cells against stress.

Keywords: Bag‐1; HER2; HSF1; breast cancer; chaperones.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
HER2 and PI3K pathways are upregulated by Bag‐1 expression. (A) MCF‐7 and (B) BT‐474 cells were transfected with the Bag‐1L vector and Bag‐1 siRNA for 48 h. Total protein was analyzed by immunoblotting with specific antibodies against HER2, PI3K, AKT, phospho‐AKTSer473, mTOR and phospho‐mTORS2448. The all data are presented as the mean ± SD from three biological independent experiments. Expression levels were analyzed with a t‐test after normalization to vinculin. The t‐test was used to calculate P values (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).
Fig. 2
Fig. 2
Expression of Bag‐1 increases the expression of heat shock proteins by affecting HSF1. Immunoblot analysis of total heat shock protein levels in cell lysates from Bag‐1 overexpressed, Bag‐1‐silenced and untransfected (A) MCF‐7 and (B) BT‐474 cells. Total protein was analyzed by immunoblotting with specific antibodies against HSF‐1, phospho‐HSF1Ser32 6, HSP90, HSP70, HSP27 and phospho‐HSPSer78. The all data are presented as the mean ± SD from three biological independent experiments. Expression levels were analyzed with a t‐test after normalization to vinculin. The t‐test was used to calculate P values (*P < 0.05; ***P < 0.001 and ****P < 0.0001).
Fig. 3
Fig. 3
Subcellular localization of Bag‐1 and other proteins in breast cancer cells. Immunocytochemical detection of Bag‐1 and its chaperone partners in (A) MCF‐7 and (B) BT‐474 cells. Bag‐1 was stained with AlexaFlour647 goat anti‐mouse (green). HER2, HSF1, phospho‐HSF1Ser336, HSP70 and phospho‐HSP27Ser78 were stained with AlexaFlour488 goat anti‐rabbit (red). Nuclei were stained with DAPI (blue). Magnification: 63×.
Fig. 4
Fig. 4
Bag‐1 interacts with HSP70 and HSP27 in BT‐474 cells. (A) Immunoprecipitated (IP) BT‐474 cell lysates with anti‐Bag‐1, anti‐HSP70 and anti‐HSP27 antibodies were analyzed by immunoblots with Bag‐1, HSP70, HSP27 and HSF1 antibodies. (B) string predictions for the complexes between Bag‐1 and its interaction partners, and models for their cell stress response mechanisms (HSP90AA1: HSP90, HSPA4: HSP70, HSPB1: HSP27). (C) Structural predictions for the surfaces of Bag‐1 (BAG domain, PDB ID: 1HXB), HSF1 (PDB ID: 5HDG), HSP90 (PDB ID: 2YI5:A, 1UYM:B), HSP70 (PDB ID: NBD: 3A8Y, SBD:4PO2) and HSP27 (PDB ID: 4MJH) proteins using crystal structures in the PDB database were analyzed by the PRISM algorithm. Interaction of the BAG domain of Bag‐1 (turquoise) with the nucleotide binding and substrate binding domain of HSP70 (pink), the HSP90 alfa protein (magenta), the HSP90 beta protein (purple) and the HSP27 protein (blue). BES, binding energy score.
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