Feb 15, 2024 19 min read Pathology

Cancer

1 About Tumour

Tumour is the uncontrolled & unplanned growth or proliferation of cells to form a mass. This growth is both quantitative & qualitative (quantitative as the cells grow in number & qualitative as the cells are morphologically & functionally differently from the original tissue). Tumours can be both benign or metastatic. Benign tumour is the tumour that has not spread. Metastatic or malignant tumour is the one that has spread to other locations. Metastatic or malignant tumour is also called Cancer. Cancer is caused due to mutation in genes that control cell proliferation, cell differentiation & survival. E.g.,

• Gain of function (GOF) mutation in the oncogenes e.g., Ras family
• Loss of function (LOF) mutation in the tumour suppressor genes e.g., P53

Tumour can form in any tissue of the body. Common tumour in females is breast cancer, common tumour in male is prostate gland. The deadliest cancer is lung cancer. Carcinoma, cancer of the epithelial tissue is the most aggressive form of cancer. It fast growing with powerful invasion of other tissues. More aggressive the tumour; more aggressive the treatment needed for e.g., surgery + adjuvant therapy (chemotherapy, immunotherapy).

1.1 Cancer Hallmarks

Cancer hallmarks can be defined as the main phenotypic characteristics of cancer that are commonly observed in cancer cells. They are:

• Cellular autonomy: Cancer can sustain proliferative signalling & grow without the mitogens or GFs. (note, healthy cells always need mitogens to grow)
• Avoid anti-proliferative signals: Cancer can avoid anti-proliferative signals such as DNA damage or contact inhibition. These antiproliferative signals would normally stop cell growth and proliferation, but cancer can avoid them & keep growing.
• Reprogram cellular metabolism: Cancerous cells are able to reprogram cellular metabolism when they run out of energy. They perform aerobic glycolysis even in presence of oxygen to gain metabolic intermediates that can be used in proliferation pathways (like ribose for nucleotide synthesis). This is called "Warburg effect". This hallmark is used for PET scans of tumours that need more glucose compared to normal cells. But it has been found that tumoral cells may redirect metabolic pathways & can use other alternate energy sources like fatty acids, lipids, etc.
• Resistance to apoptosis: Cancer cells are resistant to apoptosis. In healthy cells, dangerous mutations usually lead to apoptosis. But in cancer cells, the cells become resistant to apoptosis (by inhibiting proapoptotic factors & promoting anti apoptotic factors).
• Dysregulated autophagy: Autophagy also called self-eating is the process by which cells break down damaged organelles, protein aggregates, etc. in order to recycle cell components. Cancer cells exhibit dysregulated autophagy.
• Unlimited replicative potential & avoid senescence: Cancer cells are able to extend their telomeres by the help of telomerase enzymes, this helps them to keep replicating & avoid senescence. Thus, they have unlimited replicative potential & avoid senescence.
• Invasion and metastasis: Invasion refers to the ability of cancer cells to penetrate surrounding tissues and invade adjacent structures, while metastasis is the process by which cancer cells spread from the primary tumor to distant sites in the body, where they can establish secondary tumors.
• Angiogenesis: Angiogenesis is an important hallmark of cancer. Typically, cancers can only grow to a size of about 1 to 2 millimetres in diameter without angiogenesis. Angiogenesis is stimulated by Hypoxia. Hypoxia occurs when the tumour cells use all the oxygen available.
• Escape from immune destruction: Some cancer cells may be destroyed by the immune system but some of them develop ways to block immune cells, thus, they are tolerated by our body.
• Tumour-promoting inflammation: Chronic inflammation within the tumour microenvironment can contribute to various aspects of cancer development and progression, including tumour growth, invasion, angiogenesis, and metastasis. So, tumour promoting inflammation is recognized as a cancer hallmark.

Understanding of cancer hallmarks is necessary because cancer hallmarks can be targeted to produce new anti-cancer therapies.
For e.g.,

VEGF signalling is necessary for angiogenesis, a cancer hallmark. Inhibitors of VEGF signalling > inhibits angiogenesis > inhibit cancer.
EGFR promotes sustained proliferation. EGFR Inhibitors > inhibit sustained proliferation> inhibit cancer.
Telomerase enzymes promote sustained proliferation. Inhibitors of Telomerase > limit the replication potential > inhibit cancer.

1.2 Monoclonal origin of Cancer

In theory, Cancer can have two origins: monoclonal (cancer originates from one cell) or polyclonal (cancer originates from multiple cells/ different cells. According to evidences, cancer is monoclonal.

• The Barr body is an inactive X chromosome in females. Females are mosaic in terms of the expression X chromosome. In some cells, the X chromosome inherited from father is inactivated, in other cells, the one from mother is inactivated. According to research, cancer cells have the same inactivated X chromosome, which suggests that the cancer is originated from a single cell i.e., monoclonal theory of oncogenesis.
• Another evidence comes from the analysis of cancer cells with cytogenetics techniques ( which includes SKY (spectral karyotyping), M-Fish (Multicolor-Fish), etc). Many cancers have chromosomal aberrations (aneuploidies, translocations, inversions, etc. ). In cytogenetic analysis of cancer cells, these aberrations are found to be the same in all the cells which implies monoclonal origin of cancer.

1.2 Molecular Basis of Cancer

Cancer is a complex and multifaceted disease involving both genetics & environmental factors. Cancer can be called as a genetic disease. It arises due to genetic mutations in a cell. So, genetic mutations are the basis for all Cancers. These genetic mutations affect the genes that regulate cell growth, proliferation, and DNA repair. So, these mutations lead to uncontrolled cell proliferation, resulting in the formation of tumours. DNA mutations can be acquired as somatic mutations (in somatic cells) or can be inherited in germline as predisposing mutations. The predisposing mutations are also called as hereditary mutations as they are passed from parents to offspring. They increase cancer risk & susceptibility. The genetic mutations that cause cancer arise due to DNA damage. DNA damage can be caused by endogenous or exogenous factors:

• Endogenous factors
  Genomic instability: It is an endogenous source of DNA mutation..
  Cellular metabolism: produces mutagens like ROS. (mutagens=cause mutations)
• Exogenous factors
  Physical agents = e.g., UV rays (from sun)
  Chemical agents = e.g., tobacco from cigarette
  Microbiological agents = e.g., virus & bacteria
  
  Test for mutagens-AMES Test

AMES Test is a Mutagen test. It is a technique to determine mutagenic potential i.e., whether an agent is mutagenic or not & the grade of mutagenesis it induces. The test uses a particular strain of Salmonella (whose operon for the synthesis of histidine has been mutated) & is defined as His -. The compound whose mutagenic potential is to be determined is called test compound.
His- bacteria has mutation in histidine so, it doesn’t have its own supply of histidine so, it cannot replicate (grow) without external supply of histidine. These bacteria are then exposed to a test compound.

Principle: if the test compound is mutagenic, it is able to induce mutation in at least some of the His- bacteria, some of them will grow & form colonies. Thus, if we see some colonies, we can say that some of the His- bacteria has gained DNA mutations (as they produce histidine now) , which means that the test compound has induced DNA mutations in the bacteria. Thus, we can conclude that the test compound is mutagenic.
This happens due to a process called complementation. The induced DNA mutation is able to revert the phenotype of an original mutation by acting in cis (reversing the first mutation back to normal), or in trans (creating a different gene product that will work with the mutated gene or compensate for it). These mutagenic compounds are usually inactive in the environment, so they wouldn’t cause DNA mutations in bacteria. They are mutagenic only in animals because the liver enzymes processes them and activate (like some drugs which are activated in our body). So for this experiment, rat liver enzymes are added to the test compound in order to activate them.

(Pic)

1.3 Cancer progression

Cancer is a multistep disease that progresses in several steps:
First the normal cells undergo hyperplasia (excessive proliferation) , then they undergo mild dysplasia (formation of cells with abnormal or changed morphology of cells), then they undergo severe dysplasia (also called carcinoma in situ). At this stage, tumour is theoretically treatable. Carcinoma in situ progresses to invasion & metastasis of other cells & tissues. This is called Cancer or malignant state. (At this stage, tumour has metastasised & is difficult to treat. Treatment includes surgery, investigation of nearby lymph nodes and adjuvant treatment). So, a summary of cancer progression: Normal cells > hyperplasia (excessive proliferation) > mild dysplasia (formation of abnormal morphology/change of morphology of cells) > severe dysplasia (carcinoma in situ) > cancer (invasive tumour/metastasis/malignant state).

The passage from one stage to another is acquired by the DNA mutations (activation of an oncogene or inactivation of a tumour suppressor genes). These are called driver mutations. The driver mutations between carcinomas (tumours of epithelial tissue) and sarcomas (tumours of mesenchymal tissue). In carcinoma (tumours of epithelial tissue), we need about 7-8 driver mutations to change a cell from normal to malignant one, for sarcomas (tumours of mesenchymal tissue) we need less. This is because mesenchymal tissues are vascularized and motile, while epithelia are non-vascularized and non-motile. So, carcinomas usually occur later in life compared to sarcomas. The process of driver mutations is found to be closely related to Darwinian evolution: pressure from the environment (presence of O2, presence of vessels, cancer therapy) > determines selection & survival of the cells with the “correct” mutation > so, only few cells survive but they will be very aggressive (due to positive selection from the others).

1.4 Gene mutations in cancer

The gene mutations that can lead to cancer can be divided into four main types. They are:

1. Oncogenes: Oncogenes are the mutated form of cellular protooncogenes. Protooncogenes encode for proteins that positively regulate cellular growth, survival, motility & ability to induce angiogenesis. When protooncogenes are mutated, they may promote uncontrolled cell growth & proliferation which may lead to tumour formation. Those mutations are dominant and have a Gain of function (GOF) effect i.e., mutation of only one allele is sufficient to induce cancer. E.g., of oncogenes are Ras, PI3K, etc.
2. Tumour suppressors genes (=gatekeepers): Mutations in tumour suppressor genes can cause tumours. Tumour suppressor genes encode for proteins that negatively regulate cellular growth, survival, motility and ability to induce angiogenesis. When tumour suppressor genes are mutated, the negative regulators of cell growth are dysfunctional leading to uncontrolled cell growth & proliferation. E.g., of tumour suppressor genes are Rb, TP53, etc. Those mutations are usually recessive and have a loss of function (LOF) effect i.e., mutation of both alleles is necessary to induce cancer like for Rb gene. Exceptions to this recessive LOF effects are genes that cause LOF with haploinsufficiency or negative dominance like the TP53 gene.
3. DNA damage response (DDR) genes (=caretakers or housekeepers): DDR genes are a class of tumour suppressor genes that control genomic instability & accumulation of gene mutations. They are also called caretakers or housekeepers. DDR gene mutations also have a LOF effect. Loss of function of DDR genes causes an increased rate of mutations and this promotes development of cancer. E.g., of DDR genes are BRCA 1 & BRCA 2
4. miRNA genes: A class of genes recently identified, codes for microRNA that modulate the expression of other three classes of genes, either positively or negatively. So we can have both oncogenic miRNA & tumour suppressors miRNA.

How cells gain cellular autonomy

CELL CYCLE

To understand how cells acquire autonomous growth & genomic instability we have to go back to the cell cycle and its control. The decision of cells to divide or not (= or entry into cell cycle) depends on several internal and external signals. External signals such as Growth factors(mitogens), nutrition, etc. work as positive regulators of cell cycle. External signals such as TGF-β, contact inhibition, etc work as negative regulators of cell cycle. Internal signals such as cyclin-dependent kinases (CDKs) & cyclin proteins work as positive regulators of cell cycle. Internal signals such as DNA damage work as negative regulators even with mitogens present.

Autonomous proliferation of cancer

In normal cells, the cell cycle is activated in the presence of mitogens & the cell cycle checkpoints control them. But, In cancer cells, the mitogenic signalling is activated even in the absence of mitogens, leading to uncontrolled proliferation. So, Cancer cells exploit the physiological proliferation pathways but in the wrong ways. They are able to undergo autonomous proliferation by the mutation of oncogenes & tumour suppressors. An example is a mutation in a Rb gene, which is a tumour suppressor gene that negatively controls the cell cycle > thus, inhibits proliferation. Mutation of Rb > causes uncontrolled cell cycle progression > hyper proliferation > tumour.

Cell cycle checkpoints

Cell cycle is precisely controlled to prevent uncontrolled proliferation that may cause cell death or malignancies. The cell has 4 cell cycle checkpoints that control the cell cycle progression. Alteration in the cell cycle checkpoints may lead to Cancer. The checkpoints are:

between G1 and S (R point or Restriction point)

Cell checks for DNA damage, nutrients, GFs.

If there is a problem > it will not enter the cycle.

in S

Cell checks for DNA damage. If DNA is damaged > replication will stop.

In between G2 and M

Cell checks for DNA damage. If DNA hasn’t correctly duplicated during the previous phase > replication will be halted

The first 3 checkpoints are activated when there is DNA damage.

SAC (spindle assembly checkpoint)

Cell checks if all the sister chromatids are correctly aligned in the metaphasic plate & each chromatid is attached to the spindle of the opposite direction. (to make sure that each of the sister chromatids will migrate in opposite directions and the genome is divided exactly in the two daughter cells). SAC is activated by the tension. Alteration of proteins in SAC will result in aneuploidies.

• Oncogenic lesions(tumour) target 1st checkpoint( G1-S/R) for autonomous proliferation.
• They target DNA damage checkpoints (checkpoints 1,2, & 3) for genomic instability. If the first 3 checkpoints are dysregulated > DNA mutations will remain.
• If the 4th checkpoint is dysregulated > number of chromosomes is altered.

CDC2 (is also CDK1)

DECISION TO ENTER THE CYCLE

G1 is divided in two different phases: early G1 & late G1. At early G1, cells are sensitive to external stimuli (pro & anti mitogenic signals). At the late G1 stage, G1-S checkpoint (R point) occurs. At R point, commitment of entering/not in the cell cycle occurs. R point is temporarily mapped at 2–3 hours prior to the onset of DNA synthesis in S phage. Crossing the R point, means the cell has decided to divide & will synthesize DNA. Cancer cells can undergo autonomous proliferation when there is no control on R checkpoint.

Cyclins & CDKsCyclins (the regulators) and CDKs (cyclin-dependent kinases are enzymes) are important molecular apparatus that allow cells to proceed along the cell cycle phase. In the cyclin-CDK complex: CDK has enzymatic activity & Cyclin activates the enzymatic activity of CDK. In each cell cycle phase, a different cyclin-CDK complex is activated. (They are regulated by activatory & inhibitory phosphorylation/dephosphorylation). Cyclins are called so because they cycle during cell division. The production of cyclins E, A & B are activated by the internal signals in the cell whereas the production of cyclin D is dependent on the external factors (growth signals). When Cyclin D is present, CDK4/6 is activated, and the Cyclin D-CDK4/6 complex allows the passage to the R point. Cyclin D is dependent on external factors & this dependence must be eliminated in cancer for autonomous proliferation.

CAK= Cdk-activating kinase= is an activating kinase

Wee1 = inactivating kinase

Cdc25 = inhibit effect of Wee1

Cyclin inhibitors

To maintain the cell in G1 phase, cyclin-CDK complexes are inhibited by inhibitor proteins. We have two families of inhibitor proteins, INK4 & Cip/Kip. Inhibitors are also called tumor suppressors.

Summary table

Inhibitor family

members

action

INK4 family

includes p15, p16, p18 & p19

inhibits cyclin D-CDK4/6

Cip/Kip family

includes p21, p27 and p57

inhibit the cyclin E, A, B -CDK complexes

activate cyclin D-CDK4/6

How does the cell overcome the R point?

To overcome the R point & enter the S-phage, we need activation of the cyclin E-CDK2 complex.

Entry into S phage

During the G1 phase, we have a basal production of cyclin E-CDK2 complexes (which could overcome R point). But, it is inhibited by Cip/Kip inhibitors.

How mitogens function:

1. Mitogens induce synthesis/transcription of cyclin D > the Cyclin D-CDK4/6 forms > it will shut inhibitors > stops the inhibition of the cyclin E-CDK2 by inhibitors > cyclin E-CDK2 is free > by positive feedback, transcription of more cyclins E > entry into the S phase.
2. Mitogens activate the kinase PKB (protein kinase B = Akt) > PKB will phosphorylate & inhibit the Cip/Kip inhibitors > cyclin E-CDK2 is free/ activated.

Glossary

• Genomic instability (=instability of genome)
  Genomic instability refers to mutations occurring within the genome. These mutations occur due to occasional errors during DNA replication, recombination & repair. So, our genome is unstable & this is beneficial because it contributes to genetic diversity, immune diversity, environmental adaptation & evolution. But the normal instability of the genome is insufficient to acquire all the mutations that leads to cancer. To gain high number of mutations seen in cancer, we need to acquire an important hallmark called genome instability. Genomic instability is achieved through the mutation of tumour suppressors that we call care takers. The care taker genes are the genes involved in DNA damage repair.