CARP-1 Functional Mimetics: A Novel Class of Small Molecule Inhibitors of Medulloblastoma Cell Growth

<div><p>Medulloblastomas (MBs) constitute an aggressive class of intracranial pediatric tumors. Current multimodality treatments for MBs include surgery, ionizing radiation, and chemotherapy. Toxic side effects of therapies coupled with high incidence of recurrence and the metastatic spread warrant development of more effective, less toxic therapies for this disease. CARP-1/CCAR1 is a peri-nuclear phospho-protein that is a co-activator of the cell cycle regulatory anaphase promoting complex/cyclosome (APC/C) E3 ligase. CARP-1 functional mimetics (CFMs) are a novel class of small molecule compounds that interfere with CARP-1 binding with APC/C subunit APC-2, and suppress growth of a variety of cancer cells in part by promoting apoptosis. Here we investigated MB growth inhibitory potential of the CFMs and found that CFM-4 inhibits growth of MB cells in part by inducing CARP-1 expression, promoting PARP cleavage, activating pro-apoptotic stress-activated protein kinases (SAPK) p38 and JNK, and apoptosis. Gene-array-based analysis of the CFM-4-treated Daoy MB cells indicated down-regulation of a number of key cell growth and metastasis-promoting genes including cell motility regulating small GTP binding protein p21Rac1, and extracellular matrix metallopeptidase (MMP)-10. Moreover, CFM-4 treatment stimulated expression of a number of molecules such as neurotrophin (NTF)3, and NF-κB signaling inhibitors ABIN1 and 2 proteins. Overexpression of NTF3 resulted in reduced MB cell viability while knock-down of NTF3 interfered with CFM-4-dependent loss of viability. CFMs also attenuated biological properties of the MB cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Together our data support anti-MB properties of CFM-4, and provide a proof-of-concept basis for further development of CFMs as potential anti-cancer agents for MBs.</p></div

CFMs suppress MB survival and metastasis promoting genes while enhance expression of pro-apoptotic CARP-1.

<p>(A, B) Indicated MB cells were either untreated (Control) or treated with CFM-4 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066733#pone-0066733-g002" target="_blank">figure 2</a>. Staining of the cells was performed using anti-CARP-1 (α2) antibody as detailed in Methods. Presence of CARP-1 is indicated by intense brown staining in the nuclei and cytosol of the treated cells. MB cells were either untreated (Control) or treated with different agents for indicated dose and time, and cell lysates were analyzed by western blotting for levels of CARP-1 (C), c-Myc (D), Cyclin B1 (E), and p21Rac1 (F) as in Methods.</p

NTF3 expression regulates MB growth inhibitory signaling by CFMs.

<p>(A-C) CFM treatments result in elevated NTF3 levels in MB cells. In panel A, cells were either untreated (Control) or treated with noted doses of respective CFMs for 6 h. Staining of the cells was performed using anti-NTF3 antibody as detailed in Methods. Presence of NTF3 is indicated by intense brown staining in the nuclei and cytosol of the treated cells. In panels B and C, cells were either untreated (Control) or treated with CFMs for indicated dose and time, and cell lysates were analyzed by western blotting for levels of pro-NTF3, NTF3, and actin proteins as in Methods. (D, E) Knock-down of NTF3 interferes with MB cell growth inhibitory effects of CFM-4. MB cells were transfected with scrambled or NTF3 siRNAs as in Methods. In panel D, the cell lysates were analyzed for levels of pro-NTF3 and actin proteins by western blotting. In panel E, MB cells were transfected with siRNAs as in D, and were either untreated (Control) or treated with indicated doses of CFM-4 for 3 h. Determination of viable/live cells was carried out by MTT assay as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066733#pone-0066733-g001" target="_blank">figure 1</a>. The data in the histogram represents means of three independent experiments; bars, S.E. (∗, p = <0.03 relative to CFM-4-treated, scrambled siRNA-transfected cells). (F) NTF3 expression suppresses MB cell growth and enhances inhibitory effects of CFMs. MB cells were transiently transfected with vector or NTF3 expression plasmid as in Methods. Cells were either untreated (denoted as Vector-Control or NTF3 Plasmid) or treated with respective CFM for the noted dose and time. Determination of viable/live cells was carried out by MTT assay as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066733#pone-0066733-g001" target="_blank">figure 1</a>. The data in the histogram represent means of three independent experiments; bars, S.E.</p

CFMs stimulate apoptosis in MB cells.

<p>(A, B) Indicated MB cells were either untreated (Control) or treated with different CFMs for indicated dose and time. Staining of the cells was performed using TUNEL assay as detailed in Methods. Dark brown staining represents fragmented cell nuclei. (C) MB cells were either untreated (Control) or treated with indicated agents for noted time and dose, and levels of PARP, cleaved PARP, and actin proteins were determined by Western blotting. (D) CFMs induce caspase activation in MB cells. Activities of the noted caspases were profiled as in Methods. Columns in histogram represent fold activities of caspases relative to the corresponding controls and are derived from means of two independent experiments.</p

CFMs reduce viabilities of the human MB cells.

<p>Cells were treated with vehicle (Control) or indicated doses of CFMs for 12 h (A) or 24 h (B). In panel C and D, the cells were treated with respective CFM for noted dose and time. Determination of viable/live cells was carried out by MTT assay. The data in the histograms represent means of three independent experiments; bars, S.E. ∗, p = <0.05 relative to Control (A, B) or 0.00 (C, D).</p

List of select CFM-4-regulated genes in MB cells.

<p>List of select CFM-4-regulated genes in MB cells.</p

CFMs activate pro-apoptotic MAPK p38 in MB cells.

<p>(A, B) Indicated MB cells were either untreated (Control) or treated with noted agents as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066733#pone-0066733-g002" target="_blank">figure 2</a>. Staining of the cells was performed using anti-phospho-p38 antibody as detailed in Methods. Presence of p38 is indicated by intense brown staining in the nuclei and cytosol of the treated cells. (C, D) MB cells were either untreated (Control) or treated with indicated agents for noted time and dose, and levels of phosphorylated p38 (noted as p-p38), and total p38 proteins were determined by Western blotting.</p

CFMs inhibit MB Cell Growth in Soft Agar, invasion and MMP activities.

<p>(A) MB cells were either untreated (Control) or treated with indicated dose of respective CFMs, and were subjected to the subjected to the scratch assays (indicated as wound healing assay; upper panel) or soft-agar assay (lower panel). The cells in the scratch assay or the colonies of cells in soft-agar were photographed as described in Methods. Representative photomicrographs of untreated and CFM-treated UW-228-1 cells are shown. (B) The indicated MB cells were either untreated [Control (DMSO)] or treated with CFM-4 or CFM-5 for noted dose and time. Cell lysates were analyzed for activities of various MMPs as detailed in Methods. The data in the histogram represents means of the activities of the noted MMPs from three independent experiments; bars, S.E. (C) The MB cells were separately seeded in chambers with matrigel-coated membranes, and treated with buffer (Control) or with 10 µM dose of respective CFMs as noted in Methods. Live cells migrating across the matrigel-coated membranes were dissociated, and quantitated by an MTT-based assay. The columns in histogram represent MTT OD of the CFM-treated MB cells relative to untreated controls. (#, p =  <0.05 relative to Control (DMSO)-treated cells).</p

CFM-4 activates pro-apoptotic JNK while causes a biphasic regulation of canonical NF-κB signaling.

<p>Cells were either untreated (Control) or treated with CFM-4 for indicated dose and time, and cell lysates were analyzed by western blotting for levels of pro-apoptotic, phospho-JNK, phosphorylated p38, total JNK1/3, total p38 (A, C), ABIN1, ABIN2, APC-2, IκBα, IκBβ and actin (B, C) proteins as indicated in Methods.</p