Inhibition of the NF-kB Pathway

In This Section:

• NF-kB in Normal Cells
• NF-kB in Cancer Cells
• Inhibiting NF-kB
• Self Test

NF-kB in Normal Cells

The balance between the synthesis and degradation of key regulatory proteins determines the activities of several cellular signaling pathways. The ubiquitin-proteasome system is one mechanism cells use to degrade damaged or unneeded proteins. This system targets proteins involved in such key cellular processes as cell proliferation, growth, and survival.

One example of a protein that is regulated by the ubiquitin-proteasome system is NF-kB. NF-kB is normally sequestered in the cell's cytoplasm by a protein called Inhibitor of NF-kB, or IkB. In this state, the NF-kB pathway is inactive.

A number of signaling pathways activate NF-kB. They do so by phosphorylating IkB, which makes it a target for ubiquitinylation. The added ubiquitin molecules mark IkB for degradation by proteasomes.

Once IkB has been degraded, NF-kB is free to move to the cell's nucleus, where it helps induce expression of a number of genes that promote cell survival and proliferation.

NF-kB in Cancer Cells

The NF-kB signaling pathway is highly active in multiple myeloma as well as in many other cancers.

Inhibition of this pathway has been shown to undermine the survival of myeloma cells, making NF-kB an attractive therapeutic target.

Inhibiting NF-kB

Interfering with the activity of the ubiquitin-proteasome system is one strategy for inhibiting NF-kB activity in cancer cells. For reasons not fully understood, cancer cells seem to be more sensitive to proteasome inhibition than normal cells.

Velcade® (bortezomib) is an example of a proteasome inhibitor. Velcade® binds strongly to the proteasome, preventing it from degrading IkB and other target proteins. This allows IkB to accumulate in the cytoplasm and keep NF-kB in its inactive state.

The reduction in NF-kB activity and the modification of other signaling pathways upon proteasome inhibition collectively reduce cell proliferation and increase the apoptosis of multiple myeloma cells.

Velcade® has been approved by the FDA for the treatment of multiple myeloma based on evidence from clinical trials that the drug can delay disease progression and increase overall survival in myeloma patients.

Velcade® continues to be studied in clinical trials for multiple myeloma and other cancers as both a single agent and in combination with standard therapies, including immune system modulators such as Thalidomid® and Synovir®, and other targeted therapies.

Self Test

Questions

1. Velcade® directly inhibits NF-kB.
   1. True
   2. False

Answers

1. Correct Answer: b
2. 1. True - Incorrect.
      Velcade® inhibits NF-kB indirectly by interfering with the activity of the cell's proteasome. Inhibition of the proteasome will alter the activity of several signaling pathways, not only the NF-kB signaling pathway.
   2. False - Correct.
      Velcade® inhibits NF-kB indirectly by interfering with the activity of the cell's proteasome. Inhibition of the proteasome will alter the activity of several signaling pathways, not only the NF-kB signaling pathway.

Inhibition of Heat Shock Protein 90 (HSP90)

In This Section:

• HSP90 in Normal Cells
• HSP90 in Cancer Cells
• Inhibiting HSP90
• Self Test

HSP90 in Normal Cells

Heat shock proteins, or HSPs, are called molecular chaperones because they help maintain the stability and activity of cellular proteins by modulating their three-dimensional shapes.

HSP90, like most heat shock proteins, acts as part of a multiprotein complex that includes other molecular chaperones. HSP90 stabilizes its so-called client proteins so they can participate more effectively in their signaling pathways. Thus, although not a signaling molecule itself, HSP90 can enhance the activity of certain signaling pathways.

HSP90 and other heat shock proteins also help stabilize cellular proteins in the presence of environmental stresses, such as increased temperatures or glucose deprivation, that can threaten cell survival.

HSP90 in Cancer Cells

Multiple myeloma cells and many other types of cancer cells have more HSP90 than normal cells. This is most likely because cancer cells must cope with numerous external and internal stressors that are not experienced by normal cells, such as low levels of oxygen or mutation of important regulatory proteins.

There is evidence that HSP90 is an integral part of the machinery that allows cancer cells to grow uncontrollably. Its client proteins are associated with processes that contribute to all of the hallmarks of cancer: growth factor independence, resistance to antigrowth signals, unlimited replicative potential, tissue invasion and metastasis, avoidance of apoptosis, and angiogenesis. Because HSP90 supports many of the biochemical mechanisms used by cancer cells to survive and grow, drugs that interfere with HSP90 may provide better cancer control than drugs that target only a single pathway.

Inhibiting HSP90

HSP90 inhibitors have been examined in preclinical models of multiple myeloma and other types of cancer. These studies have shown that HSP90 inhibitors reduce the viability of myeloma cells, even those that are resistant to standard therapies for this cancer.

As expected, numerous proteins and pathways are affected by HSP90 inhibitors, including many suspected to play a role in multiple myeloma. One protein affected by HSP90 inhibition is Akt. The HSP90 complex binds to and stabilizes Akt in cancer cells, allowing the protein to promote cell proliferation and survival.

However, in the presence of an HSP90 inhibitor, the interaction between HSP90 and Akt is altered. As a result, Akt becomes a target for ubiquitinylation and degradation by proteasomes, a fate shared by many other HSP90 client proteins when the activity of this molecular chaperone is inhibited.

Although no HSP90 inhibitors have been approved by the FDA, several are being tested in clinical trials of multiple myeloma.

Self Test

Questions

1. HSP90 supports cell survival by activating Akt.
   1. True
   2. False

Answers

1. Correct Answer: b
2. 1. True - Incorrect.
      HSP90 does not activate Akt. Rather, the HSP90 complex stabilizes Akt. Although this increased stability makes it more likely that Akt will be activated by other signaling molecules, HSP90 cannot directly activate Akt.
   2. False - Correct.
      HSP90 does not activate Akt. Rather, the HSP90 complex stabilizes Akt. Although this increased stability makes it more likely that Akt will be activated by other signaling molecules, HSP90 cannot directly activate Akt.

Inhibition of Histone Deacetylases (HDACs)

In This Section:

• HDACs in Normal Cells
• HDACs in Cancer Cells
• Inhibiting HDACs
• Self Test

HDACs in Normal Cells

The activity of proteins can be altered in several ways, including by chemical modification. Phosphorylation is one common type of modification. Another common modification is acetylation, in which acetyl chemical groups are added to proteins.

Acetylation--and deacetylation, the removal of acetyl groups--can influence the stability or function of proteins or alter their capacity to interact with other molecules.

One group of proteins that is frequently modified by acetylation is the histone family. Histones are proteins that interact closely with DNA and help package it inside the nucleus.

Genes located within regions of the tightly wound DNA that is associated with unacetylated histones are usually not expressed because the DNA is so tightly packaged that they are inaccessible to the cellular machinery that drives gene expression. Acetylation of histones loosens the close association between these proteins and DNA, thereby allowing the DNA structure to relax. Consequently, other proteins are able to reach the DNA and activate gene expression.

On the other hand, gene expression can be shut down if cellular enzymes called histone deacetylases, or HDACs, remove the acetyl groups from the histones.

Although named for their interaction with histones, HDACs participate in the regulation of acetylation of a wide variety of proteins that are involved in virtually all cellular processes.

HDACs in Cancer Cells

The activities and expression of many proteins implicated in cancer are regulated by acetylation. The importance of acetylation in cancer is illustrated by the finding that cancer cells cultured in the laboratory undergo cell cycle arrest and ultimately die when treated with HDAC inhibitors, whereas normal cells are relatively unaffected.

Inhibiting HDACs

The apoptotic death of myeloma cells in response to treatment with HDAC inhibitors is likely due to changes in the activities and expression of numerous proteins. For example, through their effects on histones, HDAC inhibitors are thought to promote expression of p21, a cell cycle inhibitor, and Bax, a protein that promotes apoptosis.

In addition, HDAC inhibitors affect the activity of HSP90, one of a number of cytoplasmic proteins regulated by acetylation. Acetylated HSP90 is unable to form stable complexes with its client proteins, leading to their degradation by proteasomes.

HDAC inhibitors in combination with standard chemotherapy or other targeted therapies are being tested in clinical trials of multiple myeloma.

Self Test

Questions

1. Acetylation of proteins can:
   1. Reduce protein stability
   2. Modify protein-protein interactions
   3. Modify gene expression
   4. All of the above

Answers

1. Correct Answer: d
2. 1. Reduce protein stability - Partially correct.
      There is a better answer.
   2. Modify protein-protein interactions - Partially correct.
      There is a better answer.
   3. Modify gene expression - Partially correct.
      There is a better answer.
   4. All of the Above - Correct.
      Protein acetylation can influence protein stability and protein-protein interactions. Acetylation of histones can also have an effect on gene expression.