Apoptosis and Cancer

Healthy cells have a finely tuned early warning system that monitors for cell defects, halts the replication process while trying to activate repair, and removes the dangerous cells if repair fails. Apoptosis is the self-destruction program for safely removing the defective or damaged cells before they can spread the abnormality by dividing and replicating. The cell can be safely discarded without incurring any collateral damage (inflammation) to healthy neighboring cells.

Disruption of Apoptosis

This system is a very effective defense mechanism against cancer, since genetic defects occur early in the cancerous process. Apoptosis is the natural or healthy end of a malignant or pre-malignant cancer cell. For cancer to become dangerous, it must develop ways to override the program that leads to apoptosis. Interfering with the apoptotic pathway itself is the most direct approach cancer uses to override this protective process.

Pivotal Role of Bcl-2 Family of Proteins

In recent years, scientists have identified many of the proteins involved in the apoptotic pathways. Two protein families, one pro- and the other anti-apoptotic, compete to control the apoptotic process, and a shift in this balance of power can push cells toward cancer. The Bcl-2 proteins (Bcl-2 stands for B-cell lymphoma) comprise the best known anti-apoptotic group and they are known to be associated with cancer. Most cancer cells have found a way to elevate these proteins and slow down or stop apoptosis, making the cells resistant to natural and therapeutic mechanisms. These mutated cancer cells can replicate, multiply, invade and kill normal tissues.

There are at least five well characterized members of the Bcl-2 family, the anti-apoptotic proteins. Over-expression of these proteins, and/or increases in the ratio of these proteins to the pro-apoptotic proteins, has been observed in many cancers. Three family members are most implicated in cancer progression and resistance to conventional therapies: Bcl-2, Bcl-XL and Mcl-1.

1. Inhibitors of the Bcl-2 Family Proteins

The Bcl-2 family of proteins is pivotal to regulating apoptosis. Defective regulation of apoptosis is central to cancer pathogenesis and progression, and has been associated with resistance to standard therapy. The Bcl-2 family includes pro-apoptotic (e.g., Bax, Bak, Bad, Bid, Bim) and anti-apoptotic (e.g., Bcl-2, Bcl-xL, Bcl-W, Mcl-1) proteins. The activation of downstream signals caused by the interactions of these anti- and pro-apoptotic proteins is critical to the ultimate sensitivity of cells to various apoptotic stimuli. The anti-apoptotic (e.g., Bcl-2, Bcl-xL, Mcl-1, Bcl-W) and some pro-apoptotic (e.g., Bax, Bak and Bok) proteins are homologous within conserved Bcl-2 homology (BH) regions (BH1, BH2, BH3, BH4), which control the ability of the Bcl-2 proteins to bind to each other to form homodimers and heterodimers.

Many data indicate that Bcl-2 and Bcl-xL bind to the BH3 domains of pro-apoptotic family members, sequestering them and thereby inhibiting their ability to promote cell death. Other pro-apoptotic Bcl-2 family proteins (e.g., Bad, Bid, Bim), the “BH3-only” proteins, derive their activity from their ability to activate Bax and Bak and/or to sequester Bcl-2. Numerous peptide and non-peptide BH3 mimetics have been shown in laboratory studies to inhibit the heterodimerization of Bcl-2 or Bcl-xL to pro-apoptotic BH3 family members, with the attendant expected effects of increasing mitochondrial permeability and cytochrome c release, activation of effector caspases, and apoptosis.

Anti-apoptotic Bcl-2 family proteins are overexpressed in many cancers, and have been proposed as therapeutic targets in non-Hodgkin’s lymphoma (Bcl-2), myeloma (Mcl-1), chronic lymphocytic leukemia (Bcl-2 and Mcl-1), prostate cancer (Bcl-xL, Bcl-2), melanoma (Bcl-2), and other cancers. New drugs that inhibit the function of anti-apoptotic Bcl-2 family proteins may be promising cancer medicines, as single agents or in combination with other agents or modalities. However, no such drugs have yet been approved in the United States.

2. Small Molecules Inhibiting the p53-MDM2 Interaction

Another potential drug target in the apoptotic pathway is the pro-apoptotic p53 tumor suppressor protein, which plays a central role in controlling cell cycle progression and apoptosis. Mutations or deletions of the TP53 gene which lead to inactivation of p53 function are found in approximately 50% of human tumors, with the other 50% of human tumors retaining a wild-type p53 genotype. In these cancer cells, p53 activity is held in check by an endogenous cellular inhibitor, the MDM2 protein, which binds directly to p53. Inhibition of this interaction can stimulate p53 activity in these wild-type cancer cells driving them towards apoptosis. A new and promising approach for the development of novel anticancer agents is the inhibition of the MDM2-p53 interaction using non-peptide small-molecule inhibitors.

X-ray crystallography of the p53-MDM2 complex demonstrates this protein-protein interaction is primarily mediated by a few key amino acids of p53 and a small but deep hydrophobic cleft in MDM2. This interface is an ideal target for small-molecule inhibitors that block the p53-MDM2 interaction such as the ones currently in development at Ascenta.

These inhibitors are potent, non-peptide, small-molecule inhibitors of the MDM2-p53 interaction that bind to MDM2 with extremely high affinity and induce apoptosis in cancer cells with wild-type p53. This effect is highly selective for cancer cells with wild-type p53 protein relative to cancer cells with mutated p53 or normal cells. Our preliminary results suggest that these potent, non-peptide, small-molecule inhibitors of the MDM2-p53 interaction may have great therapeutic potential for the treatment of human cancer.

3. XIAP Inhibitors or Smac Mimetics

The X-linked inhibitors of apoptosis proteins (XIAPs) are a class of central apoptosis regulators and potent endogenous apoptosis inhibitors. XIAPs suppress apoptosis in cancer cells against a large variety of therapeutic agents and make cancer cells resistant to most current therapeutic agents. XIAP proteins represent new and highly promising molecular targets for anti-cancer drug design. Smac/DIABLO, a recently identified protein, directly interacts with XIAP proteins, antagonising their apoptosis-inhibiting effects and causing cells to undergo apoptosis. In pre-cancerous and cancerous cells, it is believed that overexpression of XIAPs makes it difficult for cancer cells to eliminate themselves, instead allowing them to proliferate, metastasize, and accumulate additional oncogenic mutations. Inhibition of XIAP activity using anti-sense oligonucleotides has demonstrated anti-tumor activity in human tumor xenograft animal models.

One way XIAPs suppress apoptosis is by binding to and inhibiting the apoptosis initiator, caspase 9. In order for apoptosis to proceed, XIAP needs to be removed from the caspase. This is accomplished by a member of another class of apoptosis-regulating protein, Smac (second mitochondria-derived activator of caspases), which binds to and inhibits XIAP, preventing it from suppressing caspase-9. High-resolution, experimental three-dimensional structures of XIAP in complex with Smac protein/peptide show that the Smac-XIAP interaction is mediated by only four amino acid residues on the Smac protein and a well-defined surface groove on a key domain of XIAP. This well-defined interaction site is ideal for the design of non-peptide, small molecule Smac mimetics that target XIAP proteins and disrupt this interaction. Several recent studies have demonstrated that Smac mimetics (XIAP inhibitors) may have great therapeutic potential for the treatment of human cancer.