F. Javier Piedrafita, Ph.D, Assistant Professor

Dr. Piedrafita's Active Grants
Dr. Piedrafita's Publications
jpiedrafita@skcc.org

Retinoid Program

MECHANISM OF RETINOID-INDUCED APOPTOSIS IN CANCER CELLS

Laboratory Staff: Maria Ortiz, Ph.D., Yolanda Bayon, Ph.D., and Francisco Lopez, Ph.D.

Vitamin A (retinol) and its derivatives, the retinoids, are essential regulators of many biological events including cell growth and differentiation, development, homeostasis and carcinogenesis. Because of their antiproliferative activity, retinoids have been proposed as cancer preventive and chemotherapeutic agents. Retinoid signals are mediated by the retinoid receptors, which belong to the superfamily of nuclear hormone receptors. Retinoids show high potential for the treatment of diseases, including cancer. However, most natural and synthetic retinoid derivatives exhibit side effects that have limited their therapeutic use. The discovery of RAR subtypes (RARa, ß, and ?) stimulated a tremendous chemical effort to develop retinoids that selectively activate or inhibit one of the receptors, with the presumption that they would activate only one subset of biological responses and minimize RAR-dependent secondary effects. During the last decade, several agonists and antagonists of RARa, mixed RARß/?, and selective RAR?, as well as RXR-selective retinoids have been described. Selective retinoids have thus helped in deciphering the biological role of retinoid receptors. Moreover, some of these selective molecules show enhanced antiproliferative activity against cancer cells, which correlates with their ability to induce growth arrest and apoptosis.

Three types of synthetic retinoids and one natural metabolite (anhydroretinol) have been reported to induce apoptosis in cancer cells and are particularly interesting for future clinical evaluation. 4-HPR induces apoptosis in HL-60 cells independently of the retinoid receptors, probably via the generation of reactive oxygen species (ROS) . A second class of selective retinoids with strong pro-apoptotic activity is represented by the RRM CD437, which is a RARg-selective agonist. CD437 and several analogs such as MX2870-1, MX3350-1, and CD2325, have been found to induce apoptosis in numerous cancer cell lines. CD437 shows toxicity in animal models and has been found to inhibit growth in non-tumorigenic cells. In contrast, MX3350-1 was effective against solid tumors derived from non-small cell lung cancer cells in animal models with no major side effects, although the effects on normal cells remains to be examined. A third type of RRM, unrelated to CD437, is MX781. This is an RAR antagonist that induces apoptosis and showed exceptional anticancer activity against estrogen-independent breast cancer cells, although high concentrations of the compound are required in vitro to induce apoptosis.

We are interested in deciphering how these two types of selective RRMs, agonists (CD437 analogs) and antagonist (MX781) induce apoptosis in cancer cells. The fact that CD437 induced apoptosis in RA-resistant cells and this effect was not inhibited by retinoid antagonists indicated an RAR-independent mechanism of action. Moreover, CD437 induced apoptosis does not involve gene transcription or protein synthesis in human leukemia cells MCB 17, 6348. CD437 and the analogs CD2325, MX3350-1, and MX2870-1 induce a strong and sustained activation of JNK and p38 stress kinases that precedes the release of cytochrome c and subsequent induction of apoptosis Cancer Res 61, 8504. Although JNK activation induces AP-1 activity and this is required for CD437-induced apoptosis in lung cancer cells, activation of AP-1 is not necessary in Jurkat cells. This agrees with a transcription-independent mechanism proposed for JNK-mediated apoptosis . Another cell signaling pathway that is affected by the apoptotic RRMs is IKK/NFkB, which evokes survival signals. The antagonist MX781 and the agonist CD2325 are strong inhibitors of IKK in vitro, while only the antagonist shows efficient inhibition of IKK/NFkB signals in several cancer cells lines investigated MCB 23, 1061. Targeting IKK/NFkB pathways by means of kinase dead IKK mutants or non phosphorylable form of IkBa induced cell death, further supporting an important role for IKK and NFkB activity in cell survival by inhibiting apoptosis.

A striking difference between both agonist and antagonist RRMs is the apical caspase utilized to initiate the process of cell death. Using a caspase 9 mutant, we have recently shown that agonist RRMs (CD2325) induce apoptosis via the intrinsic pathway and require caspase 9 activity for the effective induction of apoptosis. In contrast, the antagonist MX781 induces apoptosis via caspase 2, which in turn causes mitochondrial damage and subsequent cytochrome c release and caspase 9 activation CDD 2003. We are further examining the mechanism of caspase 2 activation by MX781.

A major focus in the laboratory is to isolate and identify the primary targets of RRM action. We are currently investigating how RRMs induce JNK/p38 kinase activation and how the RRMs inhibit IKK/NFkB signaling in vivo. We continue to analyze the role of oxidative stress in RRM-induced apoptosis. Understanding the mechanism of RRM-induced cell death and analyzing the anticancer activity of novel CD2325 and MX781 analogs will provide us with important clues to develop more potent and selective retinoid-like anticancer drugs with improved therapeutic potential.