Cell Cycle- and Apoptosis-regulatory Protein-1 Is Involved in Apoptosis Signaling by Epidermal Growth Factor Receptor

Abstract

CARP-1, a novel apoptosis inducer, regulates apoptosis signaling by diverse agents, including adriamycin and growth factors. Epidermal growth factor receptor (EGFR)-related protein (ERRP), a panErbB inhibitor, inhibits EGFR and stimulates apoptosis. Treatments of cells with ERRP or Iressa (an EGFR tyrosine kinase inhibitor) results inProgrammed cell death (apoptosis) is essential for the development and maintenance of cellular homeostasis. The pathways regulating apoptosis serve as important targets for many anti-cancer agents currently utilized for treatment of diverse malignancies. Recently, we reported identification of a novel perinuclear protein CARP-1, also known as CCAR1 (1). CARP-1 regulates apoptosis signaling by chemotherapeutic agent adriamycin and by a novel retinoid [3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437). [3-(1-adamantyl)-4-Hydroxyphenyl]-2-naphthalene carboxylic acid belongs to a novel class of apoptogenic adamantyl retinoids that induce growth arrest and stimulate apoptosis in a wide variety of malignant cell types, including breast and prostate cancer and leukemia, by an retinoic acid receptor/retinoid X receptor-independent mechanism (2-4). Expression of CARP-1, on the other hand, induces apoptosis while causing elevated levels of CDKI p21 WAF1CIP1 and inhibiting the expression of a number of cell cycle-regulatory proteins such as c-Myc, cyclin B, and topoisomerase II␣ (1). Whereas it is evident that CARP-1 is an important apoptosis signal transducer, it is unclear how CARP-1-dependent apoptosis is accomplished. Understanding the apoptosis-inducing pathways utilized by CARP-1 will define mechanism(s) of action of agents such as adriamycin. Members of the EGFR 2 family of receptor tyrosine kinases, which includes EGFR, ErbB-2/HER-2, ErbB-3/HER-3, and ErbB-4/HER-4, collectively referred to as EGFRs, serve as critical mediators of the cellular communication network regulating complex biological processes such as growth, differentiation, motility, or death (5, 6). Since deregulated signaling by EGFRs is frequently noted in a variety of human cancers (7-9), interference with growth factor receptor activation and/or with intracellular growth factor-activated signal transduction pathways represents a promising strategy for the development of novel and selective anti-cancer therapies (10 -12). Small molecule inhibitors of EGFR, such as gefitinib (Iressa) and erlotinib (Tarceva; OSI-774), monoclonal antibodies to EGFR (cetuximab/IMC-C225/Erbitux), and HER-2 (trastuzumab/Herceptin) are being utilized as anti-cancer therapeutics.ERRP, a recently isolated pan-EGFR inhibitor, is a 53-55-kDa protein that possesses substantial homology to the extracellular ligand-binding domain of EGFR and its family members (13). ERRP is a secretory protein that forms an inactive heterodimer with EGFR, causing inhibition of EGFR-dependent signaling events (14). ERRP inhibits proliferation and induces apoptosis as well as attenuates ligand-induced activation of EGFR and HER-2 in cancer cells th...

Transactivator of transcription–tagged cell cycle and apoptosis regulatory protein-1 peptides suppress the growth of human breast cancer cells in vitro and in vivo

“…That CARP-1 is a key and specific regulator of apoptosis induced by these agents was underscored by the observations in which antisensedependent loss of CARP-1 resulted in the abrogation of apoptosis by adriamycin or EGFR-related protein, but not cisplatin (13,15). Increased expression of CARP-1 results in the repression of various cell cycle and proliferation-related genes such as p21Rac1, c-Myc, cyclin B1, and topoisomerase IIa, although causing elevated levels of CDKI p21 WAF1/CIP1 , and activation of caspase-3 and caspase-9 (13,15). Based on our observations thus far, CARP-1 seems to be a transducer of diverse pathways of cell growth regulation.…”

“…CARP-1 amino acids 1 -198 harbor one such apoptosis-inducing subdomain, and regulate apoptosis signaling by EGFRs in vitro (15). Because EGFR signaling pathways are often deregulated in breast cancer, we hypothesized that apoptosis-promoting peptide(s) of CARP-1 will suppress human breast cancer growth, and could be potential anticancer agents alone or in conjunction with currently used therapies.…”

“…The cell growth inhibition was assessed by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, whereas apoptosis levels were determined by using either DNA fragmentation -based ELISA kit (Roche Diagnostics) or Live/Dead assay kit (Molecular Probes) essentially following manufacturer-suggested guidelines and our published protocols (13,15,17). Apoptosis levels in tumor explants were determined by either immunostaining of the sections with anti -phospho-p38 stress-activated protein kinase (SAPK) antibody following the methodology detailed below or using the terminal deoxynucleotidyl transferase -mediated nick end labeling assay kit (Roche Diagnostics).…”

“…CARP-1 regulates apoptosis by diverse agents including chemotherapeutics, adriamycin and etoposide. Moreover, inhibition of epidermal growth factor receptors (EGFR) by EGFR-related protein, a recently reported pan-ErbB inhibitor (14), causes the induction of apoptosis that involves increased CARP-1 expression and tyrosine phosphorylation (15). That CARP-1 is a key and specific regulator of apoptosis induced by these agents was underscored by the observations in which antisensedependent loss of CARP-1 resulted in the abrogation of apoptosis by adriamycin or EGFR-related protein, but not cisplatin (13,15).…”

“…Moreover, inhibition of epidermal growth factor receptors (EGFR) by EGFR-related protein, a recently reported pan-ErbB inhibitor (14), causes the induction of apoptosis that involves increased CARP-1 expression and tyrosine phosphorylation (15). That CARP-1 is a key and specific regulator of apoptosis induced by these agents was underscored by the observations in which antisensedependent loss of CARP-1 resulted in the abrogation of apoptosis by adriamycin or EGFR-related protein, but not cisplatin (13,15). Increased expression of CARP-1 results in the repression of various cell cycle and proliferation-related genes such as p21Rac1, c-Myc, cyclin B1, and topoisomerase IIa, although causing elevated levels of CDKI p21 WAF1/CIP1 , and activation of caspase-3 and caspase-9 (13,15).…”