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Negative Breast Cancers with the Protein Kinase D Inhibitor CRT0066101 Sahra Borges1, Edith A

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Negative Breast Cancers with the Protein Kinase D Inhibitor CRT0066101 Sahra Borges1, Edith A Published OnlineFirst April 7, 2015; DOI: 10.1158/1535-7163.MCT-14-0945 Small Molecule Therapeutics Molecular Cancer Therapeutics Effective Targeting of Estrogen Receptor– Negative Breast Cancers with the Protein Kinase D Inhibitor CRT0066101 Sahra Borges1, Edith A. Perez1,2, E. Aubrey Thompson1, Derek C. Radisky1, Xochiquetzal J. Geiger3, and Peter Storz1 Abstract Invasive ductal carcinomas (IDC) of the breast are associated mechanism of inhibition of PKD3 expression. Loss of ER with altered expression of hormone receptors (HR), amplifi- results in upregulation of PKD3, leading to all hallmarks of cation or overexpression of HER2, or a triple-negative pheno- aggressive IDC, including increased cell proliferation, migra- À type. The most aggressive cases of IDC are characterized by a tion, and invasion. This identifies ER breast cancers as ideal high proliferation rate, a great propensity to metastasize, and for treatment with the PKD inhibitor CRT0066101. We show their ability to resist to standard chemotherapy, hormone that similar to a knockdown of PKD3, treatment with this therapy, or HER2-targeted therapy. Using progression tissue inhibitor targets all tumorigenic processes in vitro and microarrays, we here demonstrate that the serine/threonine decreases growth of primary tumors and metastasis in vivo. kinase protein kinase D3 (PKD3) is highly upregulated in Our data strongly support the development of PKD inhibitors À À estrogen receptor (ER)–negative (ER ) tumors. We identify for clinical use for ER breast cancers, including the triple- direct binding of the ER to the PRKD3 gene promoter as a negative phenotype. Mol Cancer Ther; 14(6); 1306–16. Ó2015 AACR. Introduction mesenchymal and stem cell features, for which no specific targeted therapies are available (7–9). Thus, identification of Invasive ductal carcinomas (IDC) of the breast are among the tissue biomarkers that may lead to novel therapeutic strategies most aggressive types of cancer affecting women. IDCs with the is of utmost importance. worst outcome are associated either with loss of expression of The protein kinase D (PKD) family members PKD1, PKD2, and hormone receptors, estrogen receptor (ER) and progesterone PKD3 have been implicated in the progression of breast cancer. receptor (PR), overexpression, or amplification of the human PKD1 contributes to breast cancer cell proliferation (10), but epidermal growth factor receptor-2 (HER2/ErbB2), or a triple- inhibits the invasive phenotype (11, 12). This is mediated by negative phenotype [lack of expression of HER2 and both inhibition of epithelial-to-mesenchymal transition (EMT) hormone receptors (HR]. The most aggressive cases of IDC are through phosphorylation of Snail (13–16), downregulation of characterized by a high proliferation rate, a great propensity to the expression of several matrix metalloproteinases (MMP), and metastasize, and their ability to resist to standard chemother- negative-regulation of F-actin reorganization at the leading edge apy, hormone therapy, or HER2-directed agents such as tras- at multiple levels (12, 13, 17, 18). Consequently, PRKD1 (encod- tuzumab (1, 2). Particularly, triple-negative breast cancers ing PKD1) is silenced by hypermethylation in the most aggressive (TNBC), which represent approximately 20% of all cases, are breast cancers, including the TNBC subtype (11, 19). In contrast to difficult to treat, because molecular targets are lacking (3, 4). PKD1, the two other isoforms PKD2 and PKD3, in breast cancer Cytotoxic radio- and chemotherapy currently are the only cell lines seem to drive all aspects of oncogenic transformation, options for patients with TNBC (5). However, the rate of including cell proliferation, migration, invasion, and chemore- recurrence is high after such treatment (6). The high resistance sistance (20–22). Similar opposing functions in breast cancer to therapy has been partially attributed to tumors that show have been described for other kinases such as members of the Akt/ PKB kinase family (23, 24). However, how subtypes of the same 1Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida. kinase family, which recognize the same substrate phosphoryla- 2Department of Hematology/Oncology, Mayo Clinic, Jacksonville, tion motif, can have opposite cellular functions remains unclear. 3 Florida. Laboratory Medicine and Pathology, Mayo Clinic, Jackson- On the basis of the recent studies for PKD enzymes, it seems that a ville, Florida. number of different parameters, such as their relative level of Note: Supplementary data for this article are available at Molecular Cancer expression or activity, their cellular localization, and/or their Therapeutics Online (http://mct.aacrjournals.org/). ability to form complexes, can differentially influence cellular Corresponding Author: Peter Storz, Mayo Clinic, Griffin Room 306, 4500 San phenotypes (25). Pablo Road, Jacksonville, FL 32224. Phone: 904-953-6909; Fax: 904-953-0277; Using progression tissue microarrays (TMA), here we dem- E-mail: [email protected] onstrate that a switch toward the isoform PKD3 is associated doi: 10.1158/1535-7163.MCT-14-0945 with the aggressiveness of breast cancer. Although PKD1 is Ó2015 American Association for Cancer Research. downregulated and PKD2 is expressed homogeneously at low 1306 Mol Cancer Ther; 14(6) June 2015 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst April 7, 2015; DOI: 10.1158/1535-7163.MCT-14-0945 Targeting of ERÀ Breast Cancer with CRT0066101 levels in different breast cancer subtypes as well as in normal was produced in HEK293FT cells using the ViraPower Lentiviral À tissue, PKD3 is highly upregulated in ER-negative (ER )tumors. Expression System (Life Technologies). MDA-MB-231 cells We identify estrogen-dependent signaling as the mechanism were infected with PKD3-shRNA lentivirus to generate stable cell of inhibition of PKD3 expression in ER-expressing ductal cancer lines. After infection, cell pools were selected using puromycin cells. Loss of ER results in upregulation of PKD3, leading to (1 mg/mL) for 15 days. increased cell proliferation, migration, and invasion. These data À identify ER breast cancers as ideal cancers for treatment with Plasmids and transfections the PKD inhibitor CRT0066101, because they express little or To generate a PKD3 promoter-luciferase reporter, the human no PKD1 and high levels of PKD3. We show that, similar to a PRKD3 promoter region (À1000 to þ3) was cloned in pGL3 knockdown of PKD3, treatment with this inhibitor targets plasmid from Promega via BglII and XhoI restriction sites, using most tumorigenic processes in vitro, and also decreases growth 50-TTTTTTGTCCCTTCTGTTTTTGAT-30 and 50-GACGGAAAGAAA- of primary tumors and prevents metastasis in vivo.Thus,because TTAGAAAATTTT-30 as primers. The pRL-CMV-Renilla luciferase it can be given orally, it may be developed for treatment plasmid was from Promega. The ERa (pEGFP-C1-ERa; #28230) of PKD1-negative breast cancers, including the triple-negative expression plasmid was from Addgene. The pSuper-PKD2-shRNA phenotype. plasmid was obtained by cloning the oligonucleotides 50-GAT- CCCCGTTCCCTGAGTGTGGCTTCTTCAAGAGAGAAGCCACAC- Materials and Methods TCAGGGAACTTTTTGGAAA-30 and 50-AGCTTTTCCAAAAAGTT- CCCTGAGTGTGGCTTCTCTCTTGAAGAAGCCACACTCAGGGA- Cell lines, antibodies, and reagents ACGGG-30 into pSuper. GenJet from SignaGen was used for The MDA-MB-231-luc2 cell line was obtained from PerkinEl- transfection of breast cancer cells. mer in May 2009. All other cells lines were obtained from the American Type Culture Collection in August 2008. All cell lines Cell lysates and Western blot analysis were not further authenticated. MCF-7, MDA-MB-231, and Cells were washed twice with ice-cold phosphate-buffered T47D cells were maintained in Dulbecco's Modified Eagle saline (PBS; 140 mmol/L NaCl, 2.7 mmol/L KCl, 8 mmol/L Medium (DMEM) with 10% fetal bovine serum (FBS). MDA- Na HPO , 1.5 mmol/L KH PO , pH 7.2) and lysed with Buffer MB-468 and HCC1954 were maintained in RPMI-1640 with 2 4 2 4 A (50 mmol/L Tris–HCl, pH 7.4, 1% Triton X-100, 150 mmol/L 10% FBS. BT20 cells were maintained in Eagle's minimal essen- NaCl, 5 mmol/L EDTA, pH 7.4) plus Protease Inhibitor Cocktail tial medium with 10% FBS, 2 mmol/L L-glutamine, 1.5 g/L (Sigma-Aldrich). Lysates were used for Western blot analysis as sodium bicarbonate, 0.1 mmol/L nonessential amino acids described previously (12). (NEAA), and 1 mmol/L sodium pyruvate. MCF-10A cells were maintained in DMEM/Ham's F-10 medium (50:50 v/v) with 5% Immunofluorescence horse serum, 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100 Cells were seeded in 8-well ibiTreat m-Slides (ibidi) and treated ng/mL cholera toxin, 10 mg/mL insulin, and 1% penicillin/ as indicated. Before fixation with 4% paraformaldehyde (20 min- streptomycin. NEAAs were obtained from Mediatech, EGF from utes, 4C), cells were washed twice with PBS. Fixed cells were PeproTech, and insulin and hydrocortisone from Sigma-Aldrich. washed three times in PBS, permeabilized with 0.1% Triton X-100 Anti-b-actin antibody was obtained from Sigma-Aldrich; anti- in PBS (2 minutes, room temperature), and then blocked with Ki67 from Dako; anti-cleaved PARP, anti-cleaved caspase-3, and blocking solution [3% bovine serum albumin (BSA) and 0.05% anti-MMP9 from Cell Signaling Technology; anti-COX-2 from Tween 20 in PBS] for 30 minutes at room temperature. F-actin was Cayman Chemical; anti-smooth muscle actin (SMA), anti-GFP, stained with Alexa Fluor 633–Phalloidin (Life Technologies), and anti-cyclin D1 from Abcam; anti-Snail from Abgent; anti- nuclei with 40,6-diamidino-2-phenylindole dihydrochloride vimentin (for Western blotting) from Santa Cruz Biotechnology; (DAPI; Sigma-Aldrich) in blocking solution. After extensive and anti-N-cadherin and anti-vimentin [for immunohistochem- washes with PBS, cells were mounted in ibidi mounting medium istry (IHC)] from Epitomics. The rabbit polyclonal antibody (ibidi). Samples were examined using an IX81 DSU Spinning specific for PKD2 was from Upstate Biotechnology and the Disc Confocal from Olympus with a 40Â objective.
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