ACK1 tyrosine kinase interacts with histone demethylase KDM3A to regulate the mammary tumor oncogene HOXA1

Abstract

Hormone therapy with the selective estrogen-receptor modulator tamoxifen provides a temporary relief for patients with estrogen receptor α (ER)-positive breast cancers. However, a subset of patients exhibiting overexpression of the HER2 receptor tyrosine kinase displays intrinsic resistance to tamoxifen therapy. Therefore, elucidating the mechanisms promoting the estrogen (E2)-independent ER-regulated gene transcription in tamoxifen-resistant breast tumors is essential to identify new therapeutic avenues to overcome drug resistance and ameliorate poor prognosis. The non-receptor tyrosine kinase, ACK1 (also known as TNK2), has emerged as a major integrator of signaling from various receptor tyrosine kinases including HER2. We have uncovered that heregulin-mediated ACK1 activation promoted ER activity in the presence of tamoxifen, which was significantly down-regulated upon ACK1 knockdown or inhibition of ACK1 by small molecule inhibitors, AIM-100 or Dasatinib. We report that ACK1 phosphorylates the ER co-activator, KDM3A, a H3K9 demethylase, at an evolutionary conserved tyrosine 1114 site in a heregulin-dependent manner, even in the presence of tamoxifen. Consistent with this finding, ACK1 activation resulted in a significant decrease in the deposition of dimethyl H3K9 epigenetic marks. Conversely, inhibition of ACK1 by AIM-100 or Dasatinib restored dimethyl H3K9 methylation marks and caused transcriptional suppression of the ER-regulated gene HOXA1. Thus, by its ability to regulate the epigenetic activity of an ER co-activator KDM3A, ACK1 modulates HOXA1 expression in the absence of E2, conferring tamoxifen resistance. These data reveal a novel therapeutic option, suppression of ACK1 signaling by AIM-100 or Dasatinib, to mitigate HOXA1 up-regulation in breast cancer patients displaying tamoxifen resistance.

Keywords: Breast Cancer; Cancer Therapy; Oncogene; Signal Transduction; Transcription Regulation; Tyrosine-Protein Kinase (Protein-tyrosine Kinase).

FIGURE 1.

Heregulin-mediated HER2 activation leads to ACK1 activation. A, MCF-7 cells were treated with heregulin (10 ng/ml) for various time points, and equal amounts of protein lysates were subjected to immunoprecipitation with ACK1 (top panel) and HER-2 (second panel) antibodies followed by immunoblotting with pTyr antibodies. The lysates were also subjected to immunoblotting with KU antibodies to confirm equal loading of protein samples. p indicates phospho. B, activated ACK1 interacts with and Tyr-phosphorylates KDM3A. HEK293 cells were transfected with FLAG-tagged KDM3A and ACK1 constructs as described. 48 h after transfection, equal amounts of protein lysates were subjected to immunoprecipitation with FLAG beads followed by immunoblotting with anti-ACK1 monoclonal Abs (top panel) or with anti-pTyr Abs to detect phosphorylated KDM3A (second panel). The remaining panels represent cell lysates subjected to immunoblotting with the indicated antibodies. vec, empty vector; mock, mock-transfected. C, ACK1 activation regulates ER·ACK1 complex formation in the presence of 4HT. 293T cells were transfected with Myc-tagged ACK1 and FLAG-tagged ER and KDM3A constructs as shown. 36 h after transfection, the cells were treated with 4HT for 16 h. Cells were harvested, and equal amounts of protein lysates were immunoprecipitated with Myc beads followed by immunoblotting with anti-FLAG antibodies (top panel). The remaining panels represent cell lysates subjected to immunoblotting with the indicated antibodies. D, schematic representation of ACK1, KDM3A and their deletion constructs. SAM, sterile α motif; C, Cdc42/Rac interactive binding domain; CL, clathrin-interacting domain; MHR, MIG6 homology region; UBA, ubiquitin association (UBA) domain. ZF, zinc finger; L, LXXLL motif; JmjC, JmjC domain. E, HEK293 cells were transfected with FLAG-tagged KDM3A and Myc-tagged ACK1 deletion construct. 48 h after transfection, equal amounts of protein lysates were subjected to immunoprecipitation with FLAG beads followed by immunoblotting with anti-Myc Abs to detect interaction (top panel) or anti-ACK1 polyclonal Abs to detect expression of deletion constructs (second panel). F, HEK293 cells were transfected with FLAG-tagged aKDM3A or cKDM3A and ACK1 constructs. 48 h after transfection, equal amounts of protein lysates were subjected to immunoprecipitation with FLAG beads followed by immunoblotting with anti-pTyr Abs to detect Tyr phosphorylation (top panel) or anti-FLAG monoclonal Abs to detect expression of deletion constructs (second panel). G, activated ACK1 interacts with endogenous KDM3A. MCF-7 ells were treated with AIM-100 (5 μm, 16 h) followed by heregulin (10 nm, 40 min). Cell lysates were immunoprecipitated with ACK1 antibodies followed by blotting with anti-KDM3A Abs (top panel). The remaining panels represent cell lysates subjected to immunoblotting with the indicated antibodies (bottom panels).

FIGURE 2.

ACK1 Tyr-phosphorylates KDM3A. A, activated ACK1 Tyr-phosphorylates endogenous KDM3A. Cells were treated with heregulin (10 nm, 60 min). Cell lysates were immunoprecipitated with KDM3A antibodies followed by blotting with anti-pTyr Abs (top panel). The remaining panels represent cell lysates subjected to immunoblotting with the indicated antibodies (bottom panel). B, cells were electroporated (Amaxa) with ACK1 or control siRNA, and 48 h after transfection, cells were treated with heregulin (10 nm, 40 min). Cells were harvested, and lysates were immunoprecipitated with KDM3A antibodies followed by blotting with anti-pTyr Abs (top panel). Bottom panels represent cell lysates subjected to immunoblotting with the indicated antibodies. C, activated ACK1 (p-ACK1) Tyr-phosphorylates endogenous KDM3A in the presence of 4HT. MCF-7 cells were treated with heregulin (10 nm, 40 min), 4HT (100 nm, 40 min), and Dasatinib (0.4 μm, 16 h). Cells were harvested, and equal amounts of protein lysates were subjected to immunoprecipitation with KDM3A antibodies followed by immunoblotting with anti-pTyr Abs (top panel). The protein lysates were also subjected to immunoprecipitation with ACK1 (third panel) or HER2 antibodies (bottom panel) followed by immunoblotting with anti-pTyr antibodies. The remaining panels represent cell lysates subjected to immunoblotting with the indicated antibodies. D, Tyr phosphorylation of endogenous KDM3A is sensitive to ACK1 inhibitor. T47D cells were treated with heregulin (10 nm, 40 min), 4HT (100 nm, 16 h), and AIM-100 (5 μm, 16 h). Cells were harvested, and equal amounts of protein lysates were subjected to immunoprecipitation with KDM3A antibodies followed by immunoblotting with anti-pTyr Abs (top panel). The protein lysates were also subjected to immunoprecipitation with ACK1 (third panel) or HER2 antibodies (bottom panel) followed by immunoblotting with anti-pTyr antibodies. The remaining panels represent cell lysates subjected to immunoblotting with the indicated antibodies.

FIGURE 3.

ACK1 phosphorylates KDM3A at an evolutionarily conserved tyrosine 1114 site. A, alignment of KDM3A protein sequences from various organisms indicate that tyrosine residue at 1114 position is invariant from Xenopus to humans. B, the sequence homology between the JmjC domains of KMD3A (residues 1058–1281) and KMD3B (residues 1498–1721) is shown. C, crystal structure of JmjC domain of human histone 3 lysine-specific demethylase 3B (KDM3B) shown in ribbon form. The sequence DRRVGTTN is shown in green with valine-1554 highlighted in yellow. The manganese atom is shown in magenta, and co-factor mimic N-oxalylglycine is shown in green. In KDM3A, the critical tyrosine 1114 residue is located in a similar position to that occupied by valine-1554 in KDM3B. PyMOL was used to generate the picture of the protein. D, schematic representation of KDM3A and Y1114F point mutant proteins. ZF, Zink finger domain (662–687 amino acids); L, LXXLL motif (885–889); JmjC, JmjC domain (1058–1281 amino acids). E, 293T cells were transfected with FLAG-tagged KDM3A or KDM3A point mutant (Y114F) and caAck1 and harvested after 48 h. Equal amounts of protein lysates were immunoprecipitated with FLAG Abs. Immunoprecipitates were resolved by SDS-PAGE followed by blotting with anti-pTyr Abs (top panel). The lower panel represents cell lysates blotted with total KDM3A antibodies.

FIGURE 4.

Characterization of the anti-pTyr-1114 KDM3A antibodies. A, HEK293 cells were treated with EGF ligand (1 nm) for various time intervals. Cells were harvested, and equal amounts of protein lysates were subjected to immunoblotting with pTyr-1114 KDM3A antibodies (pKDM3A) at 1:1500 dilution (top panel). The bottom panel represents cell lysates subjected to immunoblotting with the antibodies as indicated. pEGFR, phospho-EGFR. B, MCF-7 cells were treated with heregulin (Hrg) ligand (10 nm) for 45 min. Cells were harvested, and equal amounts of protein lysates were subjected to immunoblotting with pTyr-1114 KDM3A antibodies as described above. C, MCF-7 cells were electroporated (Amaxa) with ACK1 or control siRNA, and 48 h after transfection, cells were treated with heregulin, and lysates were subjected to immunoblotting with pTyr-1114 KDM3A antibodies (pKDM3A(Tyr-1114)) as described above. D, MCF-7 cells were electroporated (Amaxa) with KDM3A or control siRNA, and 48 h after transfection, cells were treated with heregulin, and lysates were subjected to immunoblotting with pTyr-1114 KDM3A antibodies as described above. E, MCF-7 cells were treated with ACK1 inhibitor AIM-100 or Dasatinib followed by heregulin ligand treatment. Cell lysates were subjected to immunoblotting with pTyr-1114 KDM3A antibodies as described above.

FIGURE 5.

Activated ACK1 promotes ER transcriptional activation. A, MCF-7 cells were transfected with the 3×ERE-vitellogenin-LUC reporter construct. The cells were serum-starved and treated with Hrg (10 nm, 1 h), 4HT (100 nm, 2 h before and during), kinase inhibitor Dasatinib (100 nm, 16 h), or E₂ (10 nm, 2 h). The cells were lysed, and luciferase activity was determined. The experiment was performed three times, and representative data are shown. *, p < 0.05. B, ACK1 modulates ER transcriptional activation in tamoxifen-treated breast cells. MCF-7 cells were transfected with 0.2 μm of control or ACK1 siRNA. On the next day, cells were transfected with 25 ng of 3×ERE-vitellogenin-LUC reporter in serum-free media. Cells were either untreated or treated with heregulin (3 h), 100 nm Dasatinib (16 h), or 100 nm 4HT (3 h before and during heregulin treatment), and luciferase activity was measured. The experiment was performed three times, and a representative data are shown. *, p < 0.05. C, ACK1 inhibitors suppress breast cancer cell proliferation. MCF-7 cells were treated with various agents (AIM-100, 10 μm; Dasatinib, 10 μm; heregulin, 10 nm; 4HT, 200 nm; E₂, 10 nm) for 72 h, and cells were counted by trypan blue viability assay. Data shown are a percentage of the cell number as compared with DMSO-treated sample (control). *, p < 0.05. D, ACK1 inhibitors suppress T47D breast cancer cell proliferation. T47D cells were treated with various agents as described above for 48 h, and cells were counted by trypan blue viability assay. Data shown are a percentage of the cell number as compared with DMSO-treated sample (control). *, p < 0.05.

FIGURE 6.

ACK1 recruits KDM3A and ER for transcriptional activation of HOXA1 in a tamoxifen-rich environment. A, quantitative RT-PCR analysis of HOXA1 expression in serum-depleted MCF-7 cells treated with heregulin (Hrg; 10 nm, 2 h), heregulin plus AIM-100 inhibitor (10 μm, 16 h), or heregulin plus Dasatinib (1 μm, 16 h). *, p < 0.05. B, MCF-7 cells were serum-starved and treated with heregulin (10 nm, 1 h) and/or AIM-100 inhibitor (10 μm, 16 h). The cells were cross-linked, and ChIP was performed using anti-ER antibodies. The ChIP DNA was subjected to real-time PCR using HOXA1 primers. Three independent experiments were, performed and representative data are shown. *, p < 0.05. C, MCF-7 cells were serum-starved and treated with heregulin (10 nm, 1 h), AIM-100 (10 μm, 16 h), or Dasatinib (10 μm, 16 h). The cells were cross-linked, and ChIP was performed using anti-dimethyl H3K9 antibodies. The ChIP DNA was subjected to real-time PCR using HOXA1 primers. Three independent experiments were performed, and representative data are shown. *, p < 0.05.

FIGURE 7.

A model showing that ACK1 regulates HOXA1 transcription in an estrogen-deficient environment. ACK1 activation by HER2 bypasses the requirement of estrogen for ER transcriptional activation. Activated ACK1·ER complex bound to HOXA1 intron 1 region and recruited and phosphorylated histone demethylase KDM3A, resulting in HOXA1 transcription even in the absence of estrogen. pY indicates pTyr.