Resistance to paclitxel in breast carcinoma cells requires a quality control of mitochondrial antiapoptotic proteins by TRAP1

Abstract

TRAP1 is a mitochondrial antiapoptotic protein up-regulated in several human malignancies. However, recent evidences suggest that TRAP1 is also localized in the endoplasmic reticulum (ER) where it is involved in ER stress protection and protein quality control of tumor cells. Based on the mechanistic link between ER stress, protection from apoptosis and drug resistance, we questioned whether these novel roles of TRAP1 are relevant for its antiapoptotic function. Here, we show for the first time that: i) TRAP1 expression is increased in about 50% of human breast carcinomas (BC), and ii) the ER stress protecting activity of TRAP1 is conserved in human tumors since TRAP1 is co-upregulated with the ER stress marker, BiP/Grp78. Notably, ER-associated TRAP1 modulates mitochondrial apoptosis by exerting a quality control on 18 kDa Sorcin, a TRAP1 mitochondrial client protein involved in TRAP1 cytoprotective pathway. Furthermore, this TRAP1 function is relevant in favoring resistance to paclitaxel, a microtubule stabilizing/ER stress inducer agent widely used in BC therapy. Indeed, the transfection of a TRAP1 deletion mutant, whose localization is restricted to the ER, in shTRAP1 cells enhances the expression of mitochondrial Sorcin and protects from apoptosis induced by ER stress agents and paclitaxel. Furthermore, BC cells adapted to paclitaxel or ER stress inducers share common resistance mechanisms: both cell models exhibit cross-resistance to single agents and the inhibition of TRAP1 by siRNAs or gamitrinib, a mitochondria-directed HSP90 family inhibitor, in paclitaxel-resistant cells rescues the sensitivity to paclitaxel. These results support the hypothesis that ER-associated TRAP1 is responsible for an extramitochondrial control of apoptosis and, therefore, an interference of ER stress adaptation through TRAP1 inhibition outside of mitochondria may be considered a further compartment-specific molecular approach to rescue drug-resistance.

Keywords: Apoptosis; Breast carcinoma; Drug resistance; ER stress; Paclitaxel; TRAP1.

Figure 1

TRAP1 and BiP/Grp78 expression in human breast carcinomas and the role of ER stress induction in paclitaxel cytotoxicity. A. Total cell lysates from 8 human breast carcinomas (T) and the respective non‐infiltrated peritumoral gland (C) were separated by SDS–PAGE and immunoblotted with anti‐TRAP1 and anti‐GAPDH antibodies. BiP/Grp78 mRNA levels are reported as time increase in tumors compared to the respective control. B. Apoptotic levels in MCF7 cells treated with increasing concentrations of paclitaxel or 1 μM thapsigargin (Tg) for 24 h. p‐values versus vehicle‐treated cells: *p < 0.02; **p = 0.001; ***p < 0.0001. C. Bip/Grp78 mRNA levels in MCF7 cells treated with increasing concentrations of paclitaxel or 1 μM thapsigargin (Tg) for 24 and 48 h. p‐values versus vehicle‐treated cells: *p < 0.0001. D. Total cell lysates from MCF7 cells exposed to 1 and 10 μM paclitaxel for 24 h were separated by SDS–PAGE and immunoblotted with anti‐TRAP1, anti‐Grp94, anti‐Caspase12 and anti‐GAPDH antibodies. Error bars, ±SD.

Figure 2

TRAP1 protects from paclitaxel‐induced apoptosis and ER stress. A. Apoptotic levels in scramble and shTRAP1 MCF7 cells and in shTRAP1 cells transfected with pMock or TRAP1 cDNA (pTRAP1) treated with increasing concentrations of paclitaxel or 1 μM thapsigargin (Tg) for 24 h. p‐values versus scramble cells (*p = 0.014; **p < 0.0001; °°p = 0.004) or versus shTRAP1 cells transfected with pMock (#p < 0.0001; ***p < 0.006; °p = 0.003). B. BiP/Grp78 mRNA levels in scramble, shTRAP1 MCF7 cells transfected with pMock or TRAP1 cDNA (pTRAP1) treated with 10 μM paclitaxel or 1 μM thapsigargin (Tg) for 24 h p‐values versus scramble cells (*p < 0.0001) or versus shTRAP1 cells transfected with pMock (**p < 0.0001; ***p = 0.003). C. Total cell lysates from scramble and shTRAP1 cells transfected with pMock or TRAP1 cDNA (pTRAP1) exposed to 10 μM paclitaxel (paclit) for 24 h were separated by SDS–PAGE and immunoblotted with anti‐TRAP1, anti‐Grp94, anti‐Caspase12 and anti‐GAPDH antibodies.

Figure 3

TRAP1 is responsible for resistance to paclitaxel and ER stress agents in paclitaxel‐resistant MCF7 cells. A. Total cell lysates from scramble and paclitaxel‐resistant (Paclit‐R) MCF7 cells were separated by SDS–PAGE and immunoblotted with anti‐Grp94, anti‐TRAP1, and anti‐GAPDH antibodies. B. BiP/Grp78 mRNA levels in scramble and paclitaxel‐resistant (Paclit‐R) MCF7 cells. p‐values versus scramble cells: *p = 0.004. C. Apoptotic levels in scramble and paclitaxel‐resistant MCF7 cells upon transfection with control siRNA (Neg siRNA) and TRAP1 siRNA treated for 24 h with 1 μM paclitaxel, 1 uM thapsigargin (Tg) or 100 nM Bortezomib. p‐values versus scramble cells (*p < 0.0001; **p = 0.001) or versus paclitaxel‐resistant cells transfected with negative siRNA (***p < 0.0001). Insert: TRAP1 levels in paclitaxel‐resistant MCF7 cells upon transfection with negative (1) and TRAP1 siRNA (2).

Figure 4

Breast carcinoma cells adapted to thapsigargin and bortezomib are cross‐resistant to paclitaxel. A. Total cell lysates from scramble, thapsigargin (Tg)‐ and bortezomib (Bort)‐resistant MCF7 cells were separated by SDS–PAGE and immunoblotted with anti‐Grp94, anti‐TRAP1, and anti‐GAPDH antibodies. B. Apoptotic levels in scramble, bortezomib‐resistant (Bort‐R) and bortezomib‐resistant MCF7 cells transfected with TRAP1 siRNA treated with 100 nM bortezomib or 1 μM paclitaxel for 24 h. C. Apoptotic levels in scramble, thapsigargin‐resistant (Tg‐R) and thapsigargin‐resistant MCF7 cells transfected with TRAP1 siRNA treated with 1 μM thapsigargin or 1 μM paclitaxel for 24 h. B and C. p‐values versus scramble cells (*p < 0.0001; **p = 0.002) or versus drug‐resistant cells transfected with negative siRNA (***p < 0.0001) each treated with the same agent. Inserts (B–C): TRAP1 levels in bortezomib‐resistant (B) and thapsigargin‐resistant (C) MCF7 cells upon transfection with negative (1) and TRAP1 siRNA (2).

Figure 5

The ER stress‐protecting role of TRAP1 is relevant for its antiapoptotic activity. A. Total cell lysates and mitochondrial fractions from shTRAP1 MCF7 cells transfected with pMock, wild type TRAP1 cDNA or the Δ1‐59TRAP1 mutant were separated by SDS–PAGE and immunoblotted with anti‐TRAP1 and anti‐Myc antibodies. B. BiP/Grp78 mRNA levels in shTRAP1 MCF7 cells transfected with pMock, wild type TRAP1 cDNA or the Δ1‐59TRAP1 mutant treated for 24 h with 10 μM paclitaxel (Paclit) and 1 μM thapsigargin (Tg). p‐values versus shTRAP1 cells transfected with pMock: *p < 0.0001. C and D. Apoptotic levels in shTRAP1 MCF7 cells transfected with pMock, TRAP1 cDNA or the Δ1‐59TRAP1 mutant upon treatment with 1 mM tunicamicin (Tun), 100 nM Bortezomib (Bort) or 100 nM Mg132 (C) and with 10 μM paclitaxel (Paclit) or 1 μM thapsigargin (Tg) (D). p‐values versus shTRAP1 cells transfected with pMock each treated with the same agent: *p < 0.0001; **p = 0.001; ***p = 0.002; °p = 0.012.

Figure 6

The TRAP1‐dependent quality control on 18 kDa Sorcin is relevant for its regulation of mitochondrial apoptotic pathway. A. Apoptotic levels in shTRAP1 MCF7 cells transfected with pMock and treated with 10 μM paclitaxel (Paclit) in the presence and the absence of 1 μM cyclosporine A (CsA), and in shTRAP1 MCF7 cells transfected the Δ1‐59TRAP1 mutant or 18 kDa Sorcin cDNA and treated with 10 μM paclitaxel. p‐values versus shTRAP1 cells exposed to paclitaxel (*p < 0.0001) or versus shTRAP1 cells transfected with pMock each treated with paclitaxel (**p < 0.0001; ***p = 0.001). B. Mitochondrial fractions from scramble and shTRAP1 MCF7 cells transfected with pMock, TRAP1 cDNA, the Δ1‐59TRAP1 mutant or 18 kDa Sorcin cDNA were separated by SDS–PAGE and immunoblotted with anti‐TRAP1, anti‐Sorcin, and anti‐Cox 4 antibodies. C. Mitochondrial fractions from scramble and shTRAP1 MCF7 cells treated with 250 nM Mg132 upon transfection with pMock, TRAP1 cDNA, and the Δ1‐59TRAP1 mutant were separated by SDS–PAGE and immunoblotted with anti‐TRAP1, anti‐Sorcin, and anti‐Cox 4 antibodies. D. Total cell lysates from scramble and shTRAP1 MCF7 cells, treated with 1 μM or 10 μM paclitaxel, were separated by SDS–PAGE and immunoblotted with anti‐TRAP1, anti‐phosphoPERK, anti‐PERK, anti‐phospho eIF2α, anti‐eIF2α and anti‐βactin antibodies.

Figure 7

The cytotoxic activity of paclitaxel is enhanced by TRAP1 inhibition. A and B. Apoptotic levels in MCF7 cells (A) and paclitaxel‐resistant MCF7 cells (B) exposed to 10 μM paclitaxel, 10 μM gamitrinibs or the combination of the 2 agents for 24 h. p‐values versus vehicle‐treated cells (*p = 0.006; **p < 0.0001) or versus paclitaxel‐treated cells (***p < 0.0001). C. Total cell lysates from MCF7 cells exposed to 10 μM paclitaxel, 10 μM gamitrinibs or the combination of the 2 agents for 12 h were separated by SDS–PAGE and immunoblotted with anti‐TRAP1, anti‐Grp94, anti‐caspase 12 and anti‐GAPDH antibodies.