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Novel anticancer mechanisms in animals

Author: Himanshu Sadulwad


Cancer is the second leading cause of deaths in the world next to cardiovascular diseases. Even more agonizing than the mortality rate is the physical and mental suffering associated with it. The question commonly asked is, 'Will there ever be a cure for cancer?' The answer to this simple question is difficult, because cancer is not one disease but many disorders that share a profound growth dysregulation. The only hope for containing this disease is to study its development and pathogenicity. Many model organisms have been incorporated to study these properties. While studying these organisms it was observed that certain organisms such as the naked mole rat, blind mole rat, certain bats, elephants and whales are resistant to cancer. Studying these organisms can help us understand the onset of the disease and the natural defense mechanisms of the body against these.


Cancer and it's onset


Cancer is characterized by loss of control of cellular growth and development leading to excessive proliferation and spread of cells.

Characteristics of cancer cells

  1. Loss of contact inhibition: Normal cells are characterized by contact inhibition that is they form monolayers and cannot move away from each other. Cancer cells can form multiple layers.

  2. Metastasis: It refers to the spread of cancer cells from the primary site of origin to other tissues of the body where they produce secondaries.

  3. Loss of anchorage dependence

  4. Increased rate of replication and transcription

  5. Increased glycolysis

Molecular basis

It is caused by genetic changes in a single cell resulting in its uncontrolled multiplication.

Oncogenes

The genes capable of causing cancer are called oncogenes. Their sequences in a normal cell are termed as protooncogenes.

Activation of a protooncogene to an oncogene

Mechanisms include:

  1. Viral insertion into chromosome

  2. Chromosomal translocation

  3. Gene amplification

  4. Point mutation

Factors causing oncogene activation

  1. Environmental factors

  2. Mutations

  3. Oncogenic viruses

  4. Inactivation of antioncogenes

Defense mechanisms against cancer

Different species require different number of mutations 'hits' that is inactivation of a specific gene for malignant transformation. Two hits are required for transformation of mouse fibroblasts, namely inactivation of either Trp53 or Rb1 and activation of Hras.

In contrast, 5 hits are required to transform human fibroblasts: Inactivation of:

  • TP53 (Tumor protein 53 or p53 is a tumor suppressor protein)

  • RB1 (Retinoblastoma associated protein)

  • PP2A (Protein phosphatase 2A)

Constitutive activation of:

  • Telomerase

  • HRAS (Harvey rat sarcoma)

The need for anticancer mechanisms

This data suggests that humans have evolved more robust anticancer defense mechanisms than these mice.

Evolutionary pressure to evolve anticancer mechanisms is very strong because an animal developing cancer prior to its reproductive age would leave no progeny. Thus, animals developed efficient anticancer mechanisms to delay the onset of tumors until post-reproductive age. Hence, cancer becomes more frequent in aged animals once they are no longer subject to natural selection. This implies that animals with a longer lifespan will develop more robust anticancer defenses which keep them cancer free until after their reproductive ages.

Another factor influencing the risk of cancer is body size. Larger animals have more somatic cells and can accumulate more mutations, thus statistically increasing the risk of cancer development. To counteract this risk large-bodied species have evolved more efficient tumor suppressor mechanisms. Therefore, novel and more sophisticated anti-cancer strategies are found in long-lived and large-bodied mammals.

The molecular mechanisms of cancer resistance are an area of interest for cancer research. These mechanisms have been evolutionarily selected over millions of years. Understanding these may hold the key to enhance cancer resistance in humans.


General study of anticancer mechanisms in species


Telomerase is a ribonucleoprotein that replicates the repetitive sequences at the ends of chromosomes, known as telomeres. It must be de-repressed to transform human cells. But it is constitutively active in the mouse. DNA polymerases cannot fully replicate chromosome ends, as they require an RNA primer to start. This is referred to as the ‘end replication problem'. Rebuilding chromosome ends is accomplished by telomerase, which carries its own RNA template. In most human somatic cells, expression of the protein component of telomerase TERT is silenced during embryonic differentiation. Due to this when cells divide, their telomeres shorten which eventually leads to replicative senescence. This is an important tumor suppressor mechanism which limits cell proliferation. Thus, mice are already a step closer to malignant transformation as they constitutively express telomerase. There is a defined mass threshold of 5,000 to 10,000 g after which telomerase activity is repressed in the majority of somatic cells. This shows that to counteract the statistical probability of developing tumors due to a larger body mass, these organisms evolved the mechanism of replicative senescence.

It is also observed that larger and longer lived species require more hits for transformation as compared to smaller and shorter lived species. Small and shorter lived species require inactivation of Trp53 or Rb1 along with an activating mutation in Hras to form tumors. However, small species which have longer lifespans required both Trp53 and Rb1 to be inactivated. In contrast larger species require constitutive activation of telomerase along with the aforementioned changes to develop a tumor. Larger and longer lived species further required the inactivation of PP2A. This indicates that body mass and lifespan play a vital role in shaping the various tumor suppressor mechanisms.

It can be argued that replicative senescence should have been evolutionarily selected in small animals to prevent tumor growth. This problem can be solved with the simple hypothesis that in small organisms, a benign tumor arising prior to short-telomere mediated growth arrest would be hazardous for a small organism. A 3g tumor greatly impedes the movements of a 30g mouse but would be inconsequential to a 50kg organism. Hence, small bodied, long lived organisms developed a mechanism which restricts cell proliferation early that is at the hyperplasia stage.

There exist some animals who possess such robust anticancer mechanisms that they are almost cancer resistant. Let us study the defense mechanisms in some of them.


Naked mole rat

The naked mole rat (Heterocephalus glaber) is a mouse sized rodent that inhabits subterranean tunnels in East Africa. Due to a constant underground temperature it has no need for insulation and has lost its fur. It is the longest living rodent with a lifespan of 32 years in captivity. Out of thousands of these animals observed only 6 cases of neoplasms were reported which occurred due to exposure to greater light and temperature ranges.

The naked mole rat is a small, long-lived mammal and hence does not rely on replicative senescence. Rather it relies on early acting, anti hyperplastic tumor suppressor mechanisms. Its arsenal of anticancer mechanisms include:

Early contact inhibition

It has a modified form of contact inhibition which is early acting and arrests cell proliferation at stages earlier to the formation of a dense monolayer. It is triggered by activation of p16INK4A rather than p27 which is the activator in humans. If the gene encoding p16INK4A which is Cdkn2aINK4A is silenced, normal contact inhibition occurs via p27. To completely nullify contact inhibition loss of both the genes Cdkn2aINK4A and Cdkn1b (which codes for p27) is required. Thus these rats have an increased level of protection.

pALT

The Cdkn2a-Cdkn2b is a locus that contains key tumor suppressor genes. In humans it encodes cyclin dependent kinase (CDK) inhibitors p15INK4B, p16INK4A and a p53 activator protein ARF. However, in the naked mole rat due to alternative splicing, pALT is produced which acts as a potent CDK inhibitor.

High molecular mass hyaluronan

Hyaluronan is a linear glucosaminoglycan the major non-protein component of the extracellular matrix. Longer molecules of hyaluronan have anti-proliferative, anti-inflammatory and anti-metastatic properties. Naked mole rats have hyaluronan molecules 6 to 30 times longer than those in humans. This occurs due to two factors. The hyaluronan synthase 2 gene (Has2) has a unique sequence leading to higher production. The second is that hyaluronidases have reduced activity in their tissues.

Inactivation of Tp53 and Rb1

The inactivation of these tumor suppressors causes apoptosis in naked mole rat cells as opposed to rapid proliferation which occurs in human cells. Similarly, inactivation of Cdkn2aARF, which reduces activity of p53 also triggers senescence in them.


Additional mechanisms of cancer resistance in naked mole rat cells include high fidelity protein synthesis, more active antioxidant pathways and more active proteolysis.


Blind mole rat

The blind mole rat (Spalax ehrenbergi) has a lifespan of 21 years and is resistant to cancer. The modifications in this organism include

Reduced p53 activity

The strictly subterranean life of the blind mole rat resulted in its increased tolerance to hypoxia. To avoid hypoxia induced apoptosis, it has a modified Tp53 sequence which weakens p53.

Concerted cell death

It was observed that after 12-15 population doubling, the entire culture of blind mole rat cells dies within 3-4 days via a combination of necrotic and apoptotic processes. It is mediated by a massive release of INFß into the medium. This suggests that blind mole rat cells are acutely sensitive to hyperplasia.

Production of HMM-HA

The high molecular mass hyaluronan slows proliferation of tumor cells.

Reduced activity of heparanase

Heparanase is an endoglycosylase that degrades heparin sulphate on the cell surface and in the ECM. The blind mole rat expresses a splice variant of heparanase that acts as a dominant negative which inhibits matrix degradation. This along with abundant expression of HMM-HA results in a more structured ECM that restricts tumor growth and metastasis.


Elephants and whales


Peto's paradox

In 1977, Peto noted that while humans have 1000 times more cells than a mouse and are much longer-lived, human cancer risk is not higher than that in the mouse. This observation was seemingly inconsistent with the multistage carcinogenesis model according to which individual cells become cancerous after accumulating a specific number of mutational hits. This contradiction became known as Peto’s paradox. An answer to Peto’s paradox is that different species do not need the same number of mutational hits. In other words, large-bodied and long-lived animal species have evolved additional tumor suppressor mechanisms to compensate for the increased numbers of cells. Furthermore, many large animals are also long-lived, hence they need additional protection from cancer over their lifespan.


Anticancer mechanisms in elephants

Elephants possess 19 extra copies of the TP53 gene. All the additional copies appear to be pseudogenes and contain deletions. Some of these are transcribed from neighboring transposable element derived promoters. Transcripts from two of the 19 TP53 pseudogenes are translated in elephant fibroblasts. However, all the additional copies of TP53 are missing DNA binding domains and the nuclear localization signal and, therefore, cannot function as transcription factors.

Elephant cells have an enhanced p53-dependent DNA damage response leading to an increased induction of apoptosis, compared to smaller members of the same family, such as armadillo and aardvark. Although the precise mechanism of action of the novel forms of TP53 is not known, it was proposed that their protein products may act to stabilize the wild type p53 protein by binding to either the wild type p53 molecule itself or to its endogenous inhibitors, the MDM2 proteins.


Anticancer mechanisms in whales

Comparative genomic and transcriptomic studies in the bowhead whale identified genes under positive selection linked to cancer and aging, as well as bowhead whale-specific changes in gene expression, including genes involved in insulin signaling pathways. Notable examples of positively selected genes are excision repair cross complementation group 1 (ERCC1), which encodes a DNA repair protein and uncoupling protein 1 (UCP1), which encodes a mitochondrial protein of brown adipose tissue. In addition, these studies identified copy number gains and losses involving genes associated with cancer and aging, notably a duplication of proliferating cell nuclear antigen (PCNA). Since both ERCC1 and PCNA are involved in DNA repair, these proteins may protect from cancer by lowering mutation rates; thus whales may not need extra copies of TP53 because their cells do not accumulate cancer causing mutations and do not reach a pre-neoplastic stage.

Slower metabolism of the largest mammals may lead to lower levels of cellular damage and mutations, and thus contribute to lower cancer incidence.


Conclusion

The reason for diversity in tumor suppressive mechanisms is that the need for more efficient anticancer defenses has arisen independently in different phylogenetic groups. As species evolved larger body mass and longer lifespan, depending on their ecology, the tumor suppressor mechanisms had to adjust to become more efficient. In each case, the ecology and unique requirements of individual species would determine the outcome.

While the ultimate goal of cancer research is to develop safe and efficient anticancer therapies as well as preventative strategies, what can be learnt from tumor-prone models has its limitations. Mice simply do not possess anticancer mechanisms that humans do not already have. With regard to inherently cancer resistant species, the potential for improving the development of anticancer therapies is much greater. Anticancer adaptations that evolved in these species may be missing in humans and if introduced into human cells could result in increased cancer resistance. For example, humans did not evolve HMM-HA, as they do not lead a subterranean lifestyle; hence, activating similar mechanisms in humans may be beneficial. HA is a natural component of human bodies and is well tolerated. Therefore, identifying strategies to systemically upregulate HMM-HA in human bodies may serve in cancer prevention for predisposed individuals or as a cancer treatment.

Nature is a treasure trove of resources and while we seek an ideal anticancer mechanism, the answer may already be out there. Understanding the molecular mechanisms of multiple anticancer adaptations that evolved in different species and then developing medicines reconstituting these mechanisms in humans could lead to new breakthroughs in cancer treatment and prevention.


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