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. 2014 Dec 15;74(24):7498-509.
doi: 10.1158/0008-5472.CAN-14-0844. Epub 2014 Nov 6.

Mitochondrial MKP1 is a target for therapy-resistant HER2-positive breast cancer cells

Demet Candas  1 , Chung-Ling Lu  1 , Ming Fan  1 , Frank Y S Chuang  2 , Colleen Sweeney  3 , Alexander D Borowsky  4 , Jian Jian Li  5
Affiliations

Mitochondrial MKP1 is a target for therapy-resistant HER2-positive breast cancer cells

Demet Candas et al. Cancer Res. .

Abstract

The MAPK phosphatase MKP1 (DUSP1) is overexpressed in many human cancers, including chemoresistant and radioresistant breast cancer cells, but its functional contributions in these settings are unclear. Here, we report that after cell irradiation, MKP1 translocates into mitochondria, where it prevents apoptotic induction by limiting accumulation of phosphorylated active forms of the stress kinase JNK. Increased levels of mitochondrial MKP1 after irradiation occurred in the mitochondrial inner membrane space. Notably, cell survival regulated by mitochondrial MKP1 was responsible for conferring radioresistance in HER2-overexpressing breast cancer cells, due to the fact that MKP1 serves as a major downstream effector in the HER2-activated RAF-MEK-ERK pathway. Clinically, we documented MKP1 expression exclusively in HER2-positive breast tumors, relative to normal adjacent tissue from the same patients. MKP1 overexpression was also detected in irradiated HER2-positive breast cancer stem-like cells (HER2(+)/CD44(+)/CD24(-/low)) isolated from a radioresistant breast cancer cell population after long-term radiation treatment. MKP1 silencing reduced clonogenic survival and enhanced radiosensitivity in these stem-like cells. Combined inhibition of MKP1 and HER2 enhanced cell killing in breast cancer. Together, our findings identify a new mechanism of resistance in breast tumors and reveal MKP1 as a novel therapeutic target for radiosensitization.

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Figures

Figure 1
Figure 1
Mitochondrial MKP1 is enhanced by genotoxic stress in MEFs to inhibit mitochondria-initiated apoptosis via reduction of pJNK. A, Mitochondrial translocation of MKP1 in sham or 10 Gy irradiated MEFs. COXIV was used as the marker for mitochondria and IκB was used as the cytoplasmic marker. B, The purity of mitochondrial preparations in these experiments were further analyzed by immunoblots of MKP1, COXIV, α-tubulin (cytoplasmic marker), Histone H3 (nuclear marker), and Giantin (Golgi marker). C, Sub-mitochondrial localization of MKP1 detected by alkaline extraction (33). Total input (T), soluble matrix proteins (S), and membrane pellets (P) were immunoblotted for MKP1, TOM40 (an outer membrane protein), TIMM13 (an inter-space protein), and HSP60 (a matrix protein). D, Sub-mitochondrial localization of MKP1 detected via mitoplasting and protease protection assay (49). The total (T), pellet (P), and supernatant (S) fractions were subjected to western blotting with indicated antibodies. m (E, measured by fluorescent probe JC-1; n=3, **p<0.01), Caspase 3 cleavage (F), and cytochrome c release (G) in sham or 10 Gy irradiated MEFs. H, Increased MKP1 and decreased pJNK levels in mitochondrial fractions 4h post 10 Gy IR. I, JNK phosphorylation in the mitochondria of MKP1 knock-out and wt MEFs 4h after 10 Gy of radiation. pJNK levels were normalized to that of JNK levels and represented under the blots. J, Clonogenic survival analysis of wt versus MKP1−/− MEFs (n=3, *p<0.05, **p<0.01).
Figure 2
Figure 2
ERK1/2-dependent mitochondrial localization and phosphorylation of MKP1 under radiation insult. Immunoblots of mitochondrial MKP1 in IR-treated breast cancer MCF7 (A), MDA-MB-231 and SKBR3 (B) cells and colon cancer HCT116 cell (C). D, Co-IP of mitochondrial MKP1 and JNK in sham or IR-treated MCF7 cells. E, Enhanced mitochondrial MKP1 and decreased pJNK levels in radioresistant MCF7/C6 and MCF7/HER2 cells compared to wt MCF7s. F, pERK and ERK levels in sham or 10 Gy irradiated wt and radioresistant MCF7s. G, MKP1 mitochondrial translocation was inhibited by the MEK inhibitor, U0126. MCF7 cells were pre-treated with 10μM of U0126 for 1 h before IR and harvested 2 h post-IR. H, ERK-mediated phosphorylation of MKP1 determined via co-IP using anti-MKP1 for IP and anti-pSer antibody for IB; and I, anti-JNK for IP and anti-pMKP for IB. MKP1 phosphorylation in irradiated MCF7 cells (J) and their mitochondria (K).
Figure 3
Figure 3
Co-activation of HER2 and MKP1 in HER2+ and HER2− breast cancer tissues and radioresistant HER+ breast cancer stem cells (HER2+ BCSCs). A, The co-expression of HER2 and MKP1 in a panel of paired human normal and tumor breast tissues (N = tumor surrounding normal breast tissue). B, The expression of MKP1, pMKP1 (S359) and HER2 in clinically diagnosed breast cancer patients with differential HER2 status. C, MKP1 expression levels in HER2+/CD44+/CD24− and HER2−/CD44+/CD24− breast cancer stem cells (HER2+ BCSCs and HER2− BCSCs) that were live-sorted from MCF7/C6 cells according to Duru et al. (22). D, Clonogenic survival of HER2-positive breast CSCs with or without MKP1 siRNA transfection (n=3, **p<0.01).
Figure 4
Figure 4
MKP1 is a potential therapeutic target for HER2-positive breast cancer cells. A, Immunoblots of MKP1 in MCF7 cells incubated with indicated concentrations of siRNA for 48h. Clonogenic survival of MCF7 (B), MCF7/C6 (C), HER2-negative breast cancer MDA-MB-231 cells (D); and HER2-positive breast cancer SKBR3 cells (E) treated with MKP1 siRNA for 48h followed by sham or 10 Gy IR. Cells were seeded 2 h post-IR and clonogenic survival was determined on 14th day (n=3, *p<0.05, **p<0.01). The bright field images of cells 48h post siRNA treatment were shown in left panels of D and E.
Figure 5
Figure 5
MKP1 inhibition via Sanguinarine treatment enhances cell death and radiosensitivity in breast cancer cells. A, MDA-MB-231 (HER2 negative), B, MCF7 (HER2 low), C, SKBR3 (HER2 overexpressing), and D, MCF7/C6 (HER2 overexpressing) cells were treated with 2μM and 5μM of Sanguinarine and the cell viability was determined by trypan blue assay (n=3, **p<0.01). E, Clonogenic survival of the MCF7, SKBR3, and MCF7/C6 cells after 24h of 2μM Sanguinarine treatment with or without 10 Gy IR (n=3, *p<0.05, **p<0.01). F, MCF7 (HER2 low) and G, SKBR3 (HER2 overexpressing) cells were transfected with GFP-tagged MKP1 construct and the clonogenic survival was determined with or without 10 Gy of IR (n=3, *p<0.05, **p<0.01). The transfection efficiency was shown in left panels.
Figure 6
Figure 6
Simultaneous inhibition of HER2 and MKP1 in HER2-positive breast cancer cells. A, MCF7/C6, B, SKBR3 and C, MCF7 wt cells were treated with MKP1 siRNA (10nM for 48h) alone or in combination with Lapatinib (RTK inhibitor, indicated concentrations for 72 h) and cell viability was determined by trypan blue assay. D, MCF7/C6, E, SKBR3 and F, MCF7 wt cells were treated with HER2 siRNA alone or in combination with Sanguinarine (MKP1 inhibitor) and cell viability was determined by trypan blue assay. G, MCF7/C6, H, SKBR3 and I, HER2+/CD44+/CD24− BCSCs were treated with Sanguinarine, Lapatinib, or their combination and cell viability was determined by trypan blue assay before and 24h after 10 Gy of IR (n=3, *p<0.05, **p<0.01).
Figure 7
Figure 7
Activation of HER2/ERK/MKP1 pathway in therapy-resistant breast cancer cells. We have previously reported that radiation therapy induces the expression of HER2 and MKP1 via NF-κB-mediated gene promoter activation in breast cancer cells (4, 5, 50). Here we identified that MKP1 mitochondrial relocation is enhanced by radiation to target mitochondrial pJNK. The dephosphorylation and inactivation of JNK leads to the attenuation of the pro-apoptotic signals from JNK, resulting in the inhibition of mitochondria-mediated apoptosis and radioresistant phenotype of breast cancer cells with HER2 status, including the HER2-positive breast cancer stem cells. The mitochondrial MKP1 is thus a potential target for therapy-resistant breast cancer cells, especially for recurrent/metastatic lesions with adaptive resistance to anti-HER2 therapy.
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References

    1. Recht A, Come SE, Henderson IC, Gelman RS, Silver B, Hayes DF, et al. The sequencing of chemotherapy and radiation therapy after conservative surgery for early-stage breast cancer. N Engl J Med. 1996;334:1356–61. - PubMed
    1. Liang K, Lu Y, Jin W, Ang KK, Milas L, Fan Z. Sensitization of breast cancer cells to radiation by trastuzumab. Mol Cancer Ther. 2003;2:1113–20. - PubMed
    1. Debeb BG, Xu W, Woodward WA. Radiation resistance of breast cancer stem cells: understanding the clinical framework. J Mammary Gland Biol Neoplasia. 2009;14:11–7. - PubMed
    1. Cao N, Li S, Wang Z, Ahmed KM, Degnan ME, Fan M, et al. NF-kappaB-mediated HER2 overexpression in radiation-adaptive resistance. Radiat Res. 2009;171:9–21. - PMC - PubMed
    1. Wang Z, Cao N, Nantajit D, Fan M, Liu Y, Li JJ. Mitogen-activated protein kinase phosphatase-1 represses c-Jun NH2-terminal kinase-mediated apoptosis via NF-kappaB regulation. J Biol Chem. 2008;283:21011–23. - PMC - PubMed

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