Functional mechanisms for human tumor suppressors.

Tumor suppressors refer to a large group of molecules that are capable of controlling cell division, promoting apoptosis, and suppressing metastasis. The loss of function for a tumor suppressor may lead to cancer due to uncontrolled cell division. Because of their importance, extensive studies have been undertaken to understand the different functional mechanisms of tumor suppressors. Here, we briefly review the four major mechanisms, inhibition of cell division, induction of apoptosis, DNA damage repair, and inhibition of metastasis. It is noteworthy that some tumor suppressors, such as p53, may adopt more than one mechanism for their functions.

Introduction

Cancer cells are basically transformed from normal cells 1. The transformation usually requires genetic mutations in proto-oncogenes and/or tumor suppressors 1. These mutations can be directly induced by specific cancer-causing agents such as radiations, chemicals, hormones, viruses and genetic factors 2. After transformation, the cancer cells lose their ability to control cell division, but still maintain certain characteristics of the cells from which they are derived. Tumor suppressors prevent cancer by reliably controlling cell division, promoting apoptosis, and suppressing metastasis 3. Once a tumor suppressor becomes inactive, cell division may proceed out of control and this may lead to cancer. Tumor suppressors affect both the initiation and the development of cancer, and generally follow the classic “two-hit” model 3-5. They are encoded by two alleles because chromosomes are paired in humans 3. If one allele becomes mutated (heterozygosis) or underexpressed, the other can still express the appropriate tumor suppressor to inhibit cell division 5. When both alleles are mutated or underexpressed, the inhibition of cell division is reduced and even lost 5. However, there are also exceptions, such as tumor suppressor p27. The production of p27 from the unmutated allele is not sufficient enough to bring the cell to its original condition in the heterozygote, and the cell mitosis can only be arrested when both alleles are unmutated 6.

Mechanisms of Tumor Suppression

Many tumor suppressors have activity in both normal and tumor cells; whereas the others, such as p53, are inactive in normal cells and only activated by potential cancer risks. A tumor suppressor may possess multiple mechanisms to suppress cancer cell growth 3. For example, the most important tumor suppressor p53, which is associated with about 50% of human cancer cases 7, can trigger DNA repair processes, induce the transcription of other tumor suppressors, such as p21 and p16, and initiate cell apoptosis 3, 8, 9. Despite the tremendous growth in cancer research and identification of numerous tumor suppressors 10, the exact underlying mechanisms through which the tumor suppressors function are not always clearly revealed. To date, four major mechanisms have been revealed for tumor suppressors: suppression of cell division, induction of apoptosis, DNA damage repair and inhibition of metastasis.

Suppression of Cell Division

Suppression of cell division is the main mechanism for most tumor suppressors. The tumor suppressors that adopt this mechanism include retinoblastoma protein (Rb), adenomatosis polyposis coli (APC), alternate reading frame (ARF), RIZ1, p15, p16, p18, p19, p21, p27, and p53 8, 9, 11-21. Rb, which is the first discovered tumor suppressor, inhibits the transcription of specific genes required for mitosis through binding to transcription factors such as E2Fs, which are key cell proliferation regulators 12, 13. Tumor suppressor p53, which can also bind to DNA, stimulates the expression of other genes, such as WAF1/CIP1 encoding p21 8, 22. APC stabilizes microtubules to inhibit mitosis 15 and interferes with cell adhesion to its growing matrix 23. The interference with cell adhesion results in indirect suppression of cell division due to contact inhibition. It has been observed that a few tumor suppressors may act in cooperation to inhibit cell mitosis 3. Tumor suppressors p15, p16, p18, p19, p21 and p27 inhibit cyclin-dependent kinases (CDKs), which, in turn, inhibit Rb 11, 24. When CDKs are inhibited, Rb is kept active to suppress cell division 24. ARF is capable of relocating the murine double minute (MDM2), a critical negative regulator of p53 inhibitor, into nuclei to activate cellular p53 25. In addition, epigenetic regulation may play a role in tumor suppressing actions. Histone methyltransferases are recently deemed as a special group of tumor suppressors, which can directly inhibit mitosis via changing the conformation of histone to block double-strand DNA unwinding 26.

Induction of Apoptosis

Apoptosis, or programmed cell death, is another functional mechanism of tumor suppression. Examples of this group of tumor suppressors are p53, APC, cluster of differentiation 95 (CD95), bridging integrator 1 (Bin1) and phosphatase and tensin homolog (PTEN) 27-31. Unlike premature cell death, apoptosis maintains normal homeostasis and suppresses cancer 32, 33. It is regulated by many different pathways integrating both positive and negative regulations 34. p53 mediates apoptosis through two major pathways, the extrinsic pathway, which activates a caspase cascade including caspase-9, -3, -6 and -7, and the intrinsic pathway, which promotes the apoptosome formation via the Bcl-2 family 28, 35. APC, which has been observed to be frequently mutated in colorectal cancer, promotes transcription-independent apoptosis via caspase 8 27. The APC-mediated apoptosis can be abolished by caspase-8 inhibitor Z-IETD-FMK 27. CD95 is overexpressed in cancer cells and acts as an important receptor for cell death 29, 36. Upon being recognized immunogenically by a specific ligand expressed in the cytotoxic T killer cells, CD95 initiates apoptosis in the cancer cells 37. Bin1, which is a cell death agent, mediates apoptosis to suppress cancer by c-Myc 30. In contrary to the above discussed tumor suppressors that directly induce apoptosis, PTEN utilizes an alternative mechanism to promote apoptosis. It inactivates phosphatidylinositol 3,4,5-triphosphate (PIP3), which is important for anti-apoptosis and aids in cancer cell survival 31. Although some tumor suppressors can both inhibit mitosis and induce apoptosis, apoptosis is not necessarily induced by the inhibition of mitosis 38.

DNA Damage Repair

The tumor suppressors that can help in DNA damage repair include mutS homolog 2 (MSH2), mutL homolog 1 (MLH1), Ataxia-telangiectasia-mutated gene product (ATM), breast cancer protein (BRCA), Nijmegen breakage syndrome 1 (NBS1), Fanconi-Anemia−related tumor suppressor (FA), and p53 3, 39-43. They are able to fix DNA damages, including mismatch and vast damage to one of the DNA double strands. Generally, p53 can induce nucleotide excision repair to remove damaged DNA portions and mediate synthesis from the other strand; whereas MSH2 and MLH1 can repair DNA mismatch 3, 39, 40. ATM is a general sensor to DNA damage and phosphorylates p53, BRCP, NBS1 and FA to activate the DNA repair process 3, 41. BRCA and p53 work together in nucleotide excision repair of DNA adducts 42. NBS1 and FA make DNA resistant to crosslinking, and may amplify the phosphorylation signal from ATM 43. In addition, thymine-DNA glycosylase (TDG) is proposed as a tumor suppressor candidate 44-46. Previous studies have shown that 5-methylation of cytosine could lead to spontaneous hydrolytic deamination to produce the C→T transition mutation and T-G mismatches 44-46. TDG recognizes the T-G mismatch, removes the mismatched T through hydrolysis of its N-glycosidic bond, and initiates a nucleotide excision repair 44-46. The C→T transition mutation happens frequently in human tumors and counts for about 24% of p53 mutations 44, 47-50. Recently, TDG was shown to be a co-activator of p53 51.

Inhibition of Metastasis

The majority of cancer death is caused by metastasis 52. During metastasis, tumor cells have signal interactions with endothelial cells to initiate angiogenesis and break down vascular walls. These actions promote their spread. Tumor suppressors that can inhibit metastasis consist of metastin, breast cancer metastasis suppressor 1 (BRMS1), tissue inhibitor of metalloproteinase (TIMP), cofactor required for specificity protein 1 activation (CRSP), and KAL1/CD82 53-64. The binding of metastin to the metastin receptor (orphan G protein-coupled receptor GPR54) increases the expression and activity of focal adhesion kinase (FAK) and inhibits the metastasis of melanoma cells 53. It has also been shown that metastin triggers phospholipase C activation, arachidonic acid release and extracellular signal-regulated kinase (ERK) phosphorylation in Chinese hamster ovary (CHO) cells overexpressing metastin receptor 54, 55. BRMS1 regulates gene transcription through interaction with the mSin3 histone deacetylase (HDAC) complex 57. TIMPs can bind not only all matrix metalloproteinases (MMPs) in their active forms but also MMP-2 and MMP-9 in their latent forms. TIMPs reduce cancer metastasis by inhibiting the cancer cell-released MMPs, maintaining the integrity of extracellular matrices, and preventing the penetration of cancer cells through the base membrane of blood vessels 58-60. CRSP can up-regulate the expression of metastin 61. CD82 is a cell surface glycoprotein activated by p53 62, 63. The expression levels of CD82 and p53 are strongly correlated 63. The exact mechanism for CD82 to inhibit cancer metastasis is still unclear; however, it may inhibit cancer cell migration/invasion through the FAK-Lyn-p130CAS-CrkII pathway 64.

Summary

In summary, we briefly reviewed the four major functional mechanisms for human tumor suppressors. Some tumor suppressors, such as p53, may utilize more than one mechanism for their suppressing functions. Recent progress in structural and functional studies of tumor suppressors and their interactions with other molecules has benefited the design and discovery of novel anticancer agents. For example, quite a few CDK inhibitors have been developed and some are already in clinical trials.