Inactivation of Tumour Suppressor Genes: Rb

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INACTIVATION OF TUMOR SUPPRESSOR GENES: RB

Introduction

Many genes control cell division; either by promoting it or inhibiting it through different cellular pathways. Since cell division is tightly controlled by cell-cycle-regulating proteins, mutations that occur can cause abnormal overexpression or under expression of genes, thereby altering their ability to control cell division. In this section, the function of tumor suppressor genes, the role of tumor suppressor genes in cell cycle and an example of a mutation that cause tumor onset is addressed.

A tumor suppressor gene (TSG) is classified as a gene that affect cell growth by limiting cell cycle and inducing apoptosis or repairing damaged DNA. A TSG is recessive at cellular level, meaning mutations in both copies are required for a complete loss of function mutation at the protein level. The elimination of wild type copies can be achieved by spontaneous mutations, mitotic recombination, gene conversion, promoter methylation and other changes in the genomic integrity of the cell. Loss of heterozygosity (LOH) is required for abnormal phenotype, such as tumor development, where cell cycle control is lost in tumor cells, resulting in uncontrolled growth.

There are different ways to detect LOH mutation, including genotyping, microsatellite polymorphism and SNP array. In order to determine whether a tumor is caused by mutation in a tumor suppressor gene or its counterpart, an oncogene, a cell fusion assay can be performed by fusing tumor cells with normal tissue. Mutations in oncogenes are dominant whereas mutations in tumor suppressor genes are recessive. By performing a cell fusion assay, those that remain tumorigenic imply a mutation in the oncogene since it acts in a dominant fashion and those that lost the tumorigenic activity are the ones with mutation in TSG.

Since TSGs act in a recessive manner, the two-hit hypothesis model of carcinogenesis by Knudsons proposed an explanation for the difference in age of onset for familial and sporadic cases of retinoblastoma. He suggested that familial cases of retinoblastoma have one copy of the gene mutation as a germline mutation, whereas sporadic cases do not have such mutation. The time required for a familial case to obtain one mutation in any single cell in the eye is much less than what it would take for a sporadic case of retinoblastoma to accumulate two mutations within the same cell to lose both copies of retinoblastoma. As a result, children born with familial retinoblastoma will often develop tumors in at least one eye (unilateral), and sometimes both (bilateral), within the first few years of life (need source).

Retinoblastoma gene (Rb)

Retinoblastoma gene Rb was the first tumor suppressor gene to be identified and was later found to be absent or mutated in more than 30% of human tumors. Knudsons suggested that in familial cases of retinoblastoma, affected individuals are predisposed to this disease with one mutation in Rb that is passed on from the parents, in other words, a germ line mutation. Between birth and the onset of the disease, another mutation is acquired in the same gene that leads to the onset of the disease. Although TSG is recessive at the cellular level, it is dominant in the organismic level since there are enough cells within the predisposed individual’s eye that as long as one of them contains a mutation with in the Rb gene, retinoblastoma is resulted. Familial retinoblastoma hence has a much earlier onset than the sporadic cases, where non predisposed individuals have to accumulate at least two mutations within the Rb gene for the development of the disease to inactivate both copies of Rb gene.

Rb gene is a repressor for E2F, a transcription factor that controls transcription of cellular genes essential for cell division. Rb gene is normally regulated by phosphorylation, with hyperphosphorylation of Rb releasing E2F, hence promoting cell division. RB can also bind to various effector proteins, leading to a coordinated control of downstream related pathways.

Besides RB, other genes that act as TSG includes p53, brca1 and brca2, where brca1 and brca2 are frequently mutated in breast cancer. Rb is an example of a gatekeeper since it directly inhibit tumor growth, as demonstrated in tissue culture1. Tumor suppressor genes can be classified by their function into two main categories: Gatekeeper and Caretaker. Gatekeeper TSGs promote tumor death directly and inactivation of gatekeepers directly leads to tumor formation and progression1. A caretaker on the other hand, does not directly enhance tumor development, but inactivation of caretakers increases the chance of genomic mutations, and hence may inactivate TSG or oncogenes1. Some tumor suppressor genes such as brca1 brca2 above can be classified as either and therefore the two categories are not mutually exclusive from one another.

To summarize, tumor suppressor gene is a recessive mutation in which both copies have to be lost, causing loss of heterozygosity within the cell either by a germline mutation and a somatic mutation or two somatic mutations, with the former example of familial retinoblastoma and the latter sporadic case of retinoblastoma. The two main categories of TSG are caretaker and gatekeeper, acting directly or indirectly to enhance tumor growth when inactivated and an example of TSG is Rb, responsible for causing retinoblastoma, a tumor growth in the eye.

 

Reference

1.           Oliveira, A. M., Fletcher, J. a. & Ross, J. S. Tumor Suppressor Genes in Breast Cancer : The Gatekeepers and the Caretakers. Pathology Patterns Reviews 124, S16–S28 (2005).

 


Targeted Cancer Therapy

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TARGETED CANCER THERAPY

In previous section we described hallmarks of cancer which are acquired during the process of tumorigenesis. Since cells need to acquire all these characteristics in order to become cancerous, we can use this knowledge to our advantage by developing drugs which target each of the hallmark capabilities. Their role is to inhibit tumour growth and progression. Many target specific cancer drugs are already in in use and new ones are being developed.1
 

What are targeted cancer therapy drugs and how they work

Most of these drugs are either small molecules or monoclonal antibodies specific for different molecular targets. Most small-molecule drugs act on intracellular targets by diffusion into cells. On the other hand, monoclonal antibodies can only act on targets outside of the cell or on the cell surface, as they are not able to enter a cell. But in order to develop targeted therapy drugs, researchers need to first identify a potential target. A good target molecule is one that has an important role in cancer cell growth and survival. Many of the targeted therapy drugs interfere with proteins involved in cell signaling pathways in order to block cancer cell division and spread.2 Individual drugs have different modes of action and affect different molecular targets. Examples of possible targeted therapies are given in the figure below.1

Advantages and disadvantages of targeted cancer therapies

Targeted cancer therapy interferes with specific molecular targets that are involved in enabling particular hallmark capability of cancer cell.1 Not all molecular targets are present in all cancers, not even in all tumours of particular type. This allows doctors to tailor treatment based on the unique set of molecular targets that are present in the patient’s tumor. It results in more effective treatment and reduced nonspecific toxicity because targeted cancer therapy is more selective against cancer cells then normal cells. Less side effects result in improved quality of life of cancer patient.2

However, it is important to note that there are also some limitations to targeted therapies. Main one is that cancer cells may develop resistance to a drug directed against specific molecular targetresulting in tumour regrowth and clinical relapse. This adaptation seems to be a result of selective pressure in combination with mutations, epigenetic reprogramming and changes in microenvironment. It may happen because there are multiple parallel pathways which regulate each of the core hallmark capabilities, and if one becomes blocked, cancer cell may start to use another pathway that is not inhibited.  Therefore, therapies which target all of these supporting pathways should be used to prevent resistance development. There is also a possibility that the cancer cell will develop reduced dependence on a particular hallmark capability and start to rely more on another. An example would be a case where after treating tumour with angiogenesis inhibitors, tumour stops to rely on angiogenesis and instead increases its invasive and metastatic abilities. To prevent this, combination of targeted therapies which interfere with different hallmark capabilities of cancer cells should be used. These strategies could help reduce the probability of cancer cells developing adaptive resistance and result in more effective therapy for human cancer.1
 

Examples of targeted therapies already approved by FDA

Drugs which inhibit uncontrolled cell growth by e.g. targeting tyrosine kinase enzymes which participate in signal transduction (Gleevec®, Sprycel®).

Drugs which induce cell to undergo apoptosis, eg by proteasome inhibition, since proteasome controls the degradation of many proteins involved in cell proliferation regulation (Velcade®).

Drugs which block angiogenesis by e.g. binding VEGF so that VEGF cannot interact with endothelial cells’ receptors. This blocks development of new blood vessels (Avastin®).2

 

 

References

1. Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011;144(5):646-674.

2. Targeted cancer therapies. National Cancer Institute at the National institutes of Health Web site. http://www.cancer.gov/cancertopics/factsheet/Therapy/targeted. Updated 2012. Accessed February 26, 2013.


 

TUMORIGENESIS IS A MULTI-STEP PROCESS

Malignant transformation or tumorigenesis is a process by which normal cells become cancerous. It is characterized by changes on a genetic and cellular level resulting in uncontrolled cell division. Tumor formation is a multistep process involving alterations of cell's control mechanisms (inactivation of TSGs and activation of oncogenes). Because of its complexity, it usually takes many decades for most human cancers to develop.

 

 

 

Cell needs to acquire six biological capabilities (hallmarks) to become malignant: sustained proliferative signaling, growth suppressor evasion, invasion activation and metastasis, replicative immortality, angiogenesis and resistance to apoptosis. These hallmarks allow cancer cells to survive, proliferate and spread. Their acquisition is enabled by genomic instability, resulting in random mutations, and chronic inflammation in premalignant and malignant lesions.1

The order of hallmarks acquired as well as the ways of achieving this are likely quite variable in different cancer types and subtypes (Figure 1).2 Moreover, two additional cell characteristics which seem to be involved in cancer pathogenesis are emerging: energy metabolism reprogramming and evasion of immune destruction.1

Below is a brief characterization of the six core hallmarks as well as two emerging ones. In the following chapters each of the hallmarks will be discussed in more detail. 

 

Proliferation of normal cells is regulated by growth promoting signals which allow the cells to leave quiescence.3 Cells are in a quiescent (resting) state when they enter G0 phase of cell cycle.  Quiescent cells are not dividing and can undergo apoptosis or re-enter cell cycle depending on the signals they receive. The production and release of the mitogenic growth signals that allow cells to enter cell cycle is tightly controlled in order to maintain a homeostasis of cell number.1 Tumor cells are capable of gaining growth signal autonomy in order to sustain chronic proliferation. An example of this would be an activation of Ras oncogene which causes constitutive stimulation of cell proliferation.3




Growth suppressor evasion
Growth suppressing signals are important for maintaining normal cells in a non-dividing state.3 Negative regulation of cell proliferation is achieved by the actions of tumour suppressor genes. Cancer cells are characterized by their insensitivity to anti-growth signals where they achieve this by knock-out or missense mutations of tumour suppressor genes such as the gene encoding for RB and TP53 proteins. RB and TP53 are part of two complementary pathways which regulate whether a cell enters senescence, undergoes proliferation or apoptosis.1

Invasion activation and metastasis


Most human cancers will eventually invade adjacent tissues or spread to distant sites to form metastases. Metastases are responsible for 90% of deaths in cancer patients.2 Cancer cells can acquire invasive and metastatic abilities through a developmental regulatory program called epithelial-mesenchymal transition (EMT). EMT is a process by which cancer cells loose loss of cell-cell adherence and become motile.6 EMT upregulation can be triggered by factors such as inflammation or hypoxia. Mutation in molecules important for cell-to-cell adherence (e.g. E cadherin) is one example how can cancer cell acquire invasive ability.1

Replicative immortality

Due to the telomere shortening, cells are able to replicate only a limited number of times before, as seen in cell culture, they enter senescence. Cancer cells need to acquire infinite proliferative potential in order to form macroscopic tumours.1 In human cancers 80% show increased telomerase expression. Telomerase is an enzyme which lengthens telomeres which results in a cell with unlimited replicative potential.3

Angiogenesis 



Most types of cancers need to induce angiogenesis to sustain growth (an example of cancer which does not engage blood vessels would be pancreatic ductal adenocarcinoma which is resistant to hypoxic conditions).5 Angiogenetic switch is regulated by a balance between factors which induce and oppose angiogenesis. A well-known inducer of angiogenesis is vascular endothelial growth factor-A (VEGF-A) which induces new blood vessel growth via binding to VEGF receptors. Through oncogenic signaling or during hypoxia VEGF gene expression can be upregulated resulting in angiogenesis stimulation in tumours.1

Resistance to apoptosis
In normal cells programmed cell death is triggered in response to signals such as DNA damage, hypoxia or overexpression of oncogenes.3 There are two types of apoptosis regulation: extrinsic (through receptors on a cell surface) and intrinsic (signals which arise within the cell). Mutations in genes involved in regulation of apoptosis (mutation in TP53 tumor suppressor gene) can result in a cell death resistance.1

Energy metabolism reprogramming


This is one of two attributes of cancer cells (immune system evasion being the other one) which might become part of the core hallmarks. It involves adjustment of cellular energy metabolism to fuel continuous cell growth and proliferation. An example of this would be switching to glycolysis in cancer cells under hypoxic conditions.1

Evasion of immune destruction
It is believed that immune system constantly monitors cells and tissues and is able to attack and eliminate majority of cancer cells. Therefore it seems that those cancer cells which form tumours must somehow be able to evade the immune system. One of possible mechanisms to do so might be suppressing the action of NK cells or CTLs (natural killer cells and cytotoxic T lymphocytes respectively).1

 

 

References

 

1. Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011;144(5):646-674.

2. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100(1):57-70.

3. Spencer SL, Gerety RA, Pienta KJ, Forrest S. Modeling somatic evolution in tumorigenesis. PLoS Comput Biol. 2006;2(8):e108.

4. Weinberg RA. Chapter 11 multi-step tumorigenesis. In: The biology of cancer. New York, USA: Garland Science, Taylor & Francis Group, LLC; 2007:399.

5. Bernatchez PN. Angiogenesis and tumors. [Lecture notes]. https://www.vista.ubc.ca/webct/urw/tp0.lc5116011/cobaltMainFrame.dowebct. 2013.

 

 


Applications of Transformation

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APPLICATIONS OF CELL TRANSFORMATION

Cell transformation assay (CTA)

Several in vitro cell transformation assays (CTAs) have been developed as quicker and more cost effective alternative methods for detection of carcinogenic potential.  They are currently used by chemical, agrochemical, cosmetic and pharmaceutical industries for screening purposes on top of rodent bioassay. Researchers also use CTAs to investigate basic mechanisms of carcinogenicity. Cell transformation is an essential step in tumourgenesis, and CTAs mimic some key stages of in vivo multistep carcinogenesis and have been shown to have a good concordance with rodent bioassay results, detecting both genotoxic and non-genotoxic carcinogens.  However, several limitations of the assay still remain, such as reproducibility of results, the subjective nature of the assessment, and the fact that the assay could not reveal the mechanisms underlying transformation.

Morphological difference between transformed cells and normal cells is often used by scientists to detect cell transformation caused by potential carcinogen. Cytotoxicity of the chemical can be first evaluated to determine the dose required in morphological transformation assay. Cells treated with potential carcinogens are fixed and stained by a Giemsa solution, which specifically bonds for phosphate groups of DNA in high A-T region, and the colonies were counted under a stereo microscope. Or cells can be fixed with formalin and stained with crystal violet to read under a spectrophotometer. Cells treated with selected dose of the chemical are then seeded, fixed and stained. Foci of certain morphological characteristics are counted and recorded, such as dense multilayers of cells and invasive growth at the edge of foci (3).

Transformed cells are usually not inhibited by cell-cell contact, so the detection of anchorage-independent growth is another accurate and stringent assays in vitro for detecting malignant transformation.  Cells treated with potential carcinogens are seeded and allowed to grow in soft agar media. After a period of time, sizable colonies formed are manually counted, which is a time-consuming and highly subjective to give meaningful results. Recently, more commercialized cell transformation assay kits are developed using fluorescent or colourimetric dyes to detect cells in soft agar after being solubilized and lysed, which on some extent improve the reproducibility and lessen the subjective nature of the assay (2).

Fig. 1. Transformation assay for mouse fibroblasts. Normal and non-transformed cells shown in (a) and (b) do not progress past a four cell stage, while transformed cells will form a focus or colony. The transformation of the mouse fibroblasts occurs in the absence of Krupple-like factor 4 (KLF4) which has tumour suppressor activity. (4)

Ames’ tumorgenecity assay and its application in screening of carcinogens

Ames’ assay was developed in 1970s particularly to test tumorigenecity of chemical compounds. It used one characteristic of transformed cells: the ability to grow and replicate even in absence of growth factors, to screen for carcinogenic chemicals.  Using this assay, Bruce Ames has discovered many naturally occurring carcinogenic compounds in diet. This assay is still adopted today as one of the transformation assay to predict chemical carcinogenicity by the International Conference of Pharmaceuticals for Human Use (5).

 

 

Fig. 2. Ames’ tumorigenecity assay (6).

 

  1. Creton S, Aardema MJ, Carmichael PL and et al. Cell transformation assays for prediction of carcinogenic potential: state of the science and future research needs. Mutagenesis27(1): 93–101. (2012)
  2. Cell Transformation Assay. Biocat. Retrieved from http://www.biocat.com/cgibin/page/sub2.plsub1=cell_transformation&sub2=cell_transformation_assay&main_group=cell_biology on Mar. 10, 2013.
  3. Sasaki, K. et al. Recommended protocol for the BALB/c 3T3 cell transformation assay. Mutat Res. 744(1): 30-5. (2012)
  4. Chow, A. Y. Cell cycle control by oncogenes and tumor Suppressors: driving the transformation of normal cells into cancerous cells. Nature Education 3(9):7. (2010)
  5. Ohmori, K. In vitro assays for the prediction of tumorigenic potential of nongenotoxic carcinogens. Journal of Health Science55(1) 20-30. (2009)
  6. Weinberg, R. A. The Biology of Cancer. Garland Science. (2007)