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

Abstract

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.

CARP-1 / CCAR1: A biphasic regulator of cancer cell growth and apoptosis

“…Consistent with our previous observations with HBC cells, CFM-4 and CFM-5 compounds inhibited the growth of MPM, MB, NB, and NSCLC cells [68–71] in part by activating apoptosis signaling and diminishing the levels of key cell cycle regulatory proteins including cyclin-B1, c- and N-myc. Both CFM-4 and CFM-5 compounds enhanced expression of CARP-1/CCAR1 along with activation of pro-apoptotic SAPKs (p38α/β and JNK1/2) [68–71].…”

PTHrP attenuates osteoblast cell death and apoptosis induced by a novel class of anti-cancer agents

“…The DEDD and DEDD2 proteins activate apoptosis by (a) binding and activating caspases-8, -10, and promoting activated caspase-8 translocation to the nucleus [61], (b) binding with cytosolic caspase-3 and enhancing its cleavage [62], and (c) trans-locating to nucleus and activating caspase-6 [63]. Our group has recently found that CFMs induce DEDD2 in MDA-MB-468 triple negative breast cancer, Daoy medulloblastoma, HepG2 liver, and HeLa cervical cancer cells [32]. Since DEDD and DEDD2 may be important mediators for death receptors and that these proteins often translocate activated caspases either in the nucleus or in the cytoplasm, understanding PTHrP's role in DEDD/DEDD2-dependent regulation of caspase-3 and nuclear-cytoplasmic translocation of CARP-1 as well as PTHrP antagonism of CFM's action in osteoblast warrants extensive investigation.…”

“…Similar to proliferating cells CFM-1 did not reduce the viability of differentiated MC-4 osteoblasts (data not shown). The ineffectiveness of CFM-1 in osteoblast viability may be explained by the fact that the chemical structure, identity of CFM-1, and its interaction with CARP-1 and anaphase promoting complex/Cyclosome (APC/C) E3 ligase are different and distinct from CFM-4 and CFM-5 and also dependent on cell types [30,32].…”

“…5). Considering the current understanding on the requirement of various caspase activities in Death Effector Domain (DED) and DED containing DNA-binding protein (DEDD-and DEDD2)-dependent cytoplasmic and nuclear apoptotic effects [44,45], and that CFMs induce DED family of proteins [32], it is likely that PTHrP regulation and CARP-1 localization in osteoblast play an important role in possible DED-mediated osteoblast survival and death.…”

“…CARP-1 functional mimetics (CFMs) suppress growth of a variety of cancer cells in part by stimulating apoptosis [28,30,31]. The studies thus far suggest CFMs potential as a novel class of anti-cancer agents both in vitro and in vivo [30,32].…”