Identification and Testing of Novel CARP-1 Functional Mimetic Compounds as Inhibitors of Non-Small Cell Lung and Triple Negative Breast Cancers.

The triple negative breast cancer (TNBCs) and non-small cell lung cancers (NSCLCs) often acquire mutations that contribute to failure of drugs in clinic and poor prognosis, thus presenting an urgent need to develop new and improved therapeutic modalities. Here we report that CARP-1 functional mimetic (CFMs) compounds 4 and 5, and 4.6, a structurally related analog of CFM-4, are potent inhibitors of TNBC and NSCLC cells in vitro. Cell growth suppression by CFM-4 and -4.6 involved interaction and elevated expression of CARP-1/CCAR1 and Death Effector Domain (DED) containing DNA binding (DEDD)2 proteins. Apoptosis by these compounds also involved activation of pro-apoptotic stress-activated kinases p38 and JNK1/2, cleavage of PARP and loss of mitotic cyclin B1. Both the CFMs inhibited abilities of NSCLC and TNBC cells to migrate, invade, and form colonies in suspension, while disrupting tubule formation by the human umbilical vein endothelial cells (HUVECs). Nano-lipid formulation of CFM-4 (CFM-4 NLF) enhanced its serum bioavailability when compared with the free CFM-4. Oral administration of CFM-4 NLF reduced weights and volume of the xenografted tumors derived from A549 NSCLC and MDA-MB-231 TNBC cells. Although no gross tissue or histological toxicities were noticed, the immuno-histochemical analysis revealed increased CARP-1 and DNA fragmentation in tumors of the CFM-4 NLF-treated animals. In conclusion, while stimulation of pro-apoptotic CARP-1 and DEDD2 expression and their binding underscore a novel mechanism of apoptosis transduction by CFM compounds, our proof-of-concept xenograft studies demonstrate therapeutic potential of CFM-4 for TNBC and NSCLC.

Mechanisms of neuroblastoma cell growth inhibition by CARP-1 functional mimetics.

Neuroblastomas (NBs) are a clinically heterogeneous group of extra cranial pediatric tumors. Patients with high-risk, metastatic NBs have a long-term survival rate of below 40%, and are often resistant to current therapeutic modalities. Due to toxic side effects associated with radiation and chemotherapies, development of new agents is warranted to overcome resistance and effectively treat this disease in clinic. CARP-1 functional mimetics (CFMs) are an emerging class of small molecule compounds that inhibit growth of diverse cancer cell types. Here we investigated NB inhibitory potential of CFMs and the molecular mechanisms involved. CFM-1, -4, and -5 inhibited NB cell growth, in vitro, independent of their p53 and MYCN status. CFM-4 and -5 induced apoptosis in NB cells in part by activating pro-apoptotic stress-activated kinases (SAPKs) p38 and JNK, stimulating CARP-1 expression and cleavage of PARP1, while promoting loss of the oncogenes C and N-myc as well as mitotic cyclin B1. Treatments of NB cells with CFM-4 or -5 also resulted in loss of Inhibitory κB (IκB) α and ß proteins. Micro-RNA profiling revealed upregulation of XIAP-targeting miR513a-3p in CFM-4-treated NB, mesothelioma, and breast cancer cells. Moreover, exposure of NB and breast cancer cells to CFM-4 or -5 resulted in diminished expression of anti-apoptotic XIAP1, cIAP1, and Survivin proteins. Expression of anti-miR513a-5p or miR513a-5p mimic, however, interfered with or enhanced, respectively, the breast cancer cell growth inhibition by CFM-4. CFMs also impacted biological properties of the NB cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Our studies indicate anti-NB properties of CFM-4 and 5, and suggest that these CFMs and/or their future analogs have potential as anti-NB agents.

CARP-1 functional mimetics are a novel class of small molecule inhibitors of malignant pleural mesothelioma cells.

Malignant pleural mesothelioma (MPM) is an asbestos-related thoracic malignancy that is characterized by late metastases, and resistance to therapeutic modalities. The toxic side-effects of MPM therapies often limit their clinical effectiveness, thus necessitating development of new agents to effectively treat and manage this disease in clinic. CARP-1 functional mimetics (CFMs) are a novel class of compounds that inhibit growth of diverse cancer cell types. Here we investigated MPM cell growth suppression by the CFMs and the molecular mechanisms involved. CFM-1, -4, and -5 inhibited MPM cell growth, in vitro, in part by stimulating apoptosis. Apoptosis by CFM-4 involved activation of pro-apoptotic stress-activated protein kinases (SAPKs) p38 and JNK, elevated CARP-1 expression, cleavage of PARP1, and loss of the oncogene c-myc as well as mitotic cyclin B1. Treatments of MPM cells with CFM-4 resulted in depletion of NF-κB signaling inhibitor ABIN1 and Inhibitory κB (IκB)α and ß, while increasing expression of pro-apoptotic death receptor (DR) 4 protein. CFM-4 enhanced expression of serine-phosphorylated podoplanin and cleavage of vimetin. CFMs also attenuated biological properties of the MPM cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Both podoplanin and vimentin regulate processes of cell motility and invasion, and their expression often correlates with metastatic disease, and poor prognosis. The fact that phosphorylation of serines in the cytoplasmic domain of podoplanin interferes with processes of cellular motility, CFM-4-dependent elevated phosphorylated podoplanin and cleavage of vimentin underscore a metastasis inhibitory property of these compounds, and suggest that CFMs and/or their future analogs have potential as anti-MPM agents.