Fundamentals of Immuno-oncology

Investigation of the underlying causes of cancer

Although cancer is a group of highly complex diseases, cancer cells show a number of different features that allow them to grow in tumor types and metastasize to distant organs. ¹

When considered together, these distinctive features provide a comprehensive framework of mechanisms that contain information about the transformation of a premalignant cell into lethal metastatic analogue in a multi-step process.¹

Activation of invasion and metastasis

Tissue invasion and metastasis are integral components of the method of tumor cells leaving the primary site and spreading to distant organs. Although the tissue invasion and metastasis process is not fully understood, it generally involves changes in how cells attach to other cells and the extracellular matrix. ¹

This process has several steps including¹

• Local tissue invasion
• the intravasation
• Transition through the blood and lymphatic system
• Foreign tissue colonization

The molecular communication bridge between the tumor cells and the neoplastic stroma indicates that metastases are not caused by an autonomous model for the cell, but require input from the surrounding tissue. ¹

• An example of this is the involvement of tumor-associated macrophages that provide epidermal growth factor (EGF) and colony stimulating factor 1 (CSF-1) to tumor cells and assist with intravasation.

The results of molecular cancer research on the complexity of metastatic growth also show that different features appear in different malignancies:^1,2

• Different invasion models are seen in metastatic diseases and non-metastatic diseases. Although the difficulty in explaining the reasons for this continues, it is possible that it may be caused by different cell biology programs.
• Genetic pathways such as tumor necrosis factor α (TNF-α), which plays a role in bone dissemination, can facilitate metastasis of the tumor to preferred organ targets.

Tumor cell migration is partially promoted through the paracrine cycle, which includes CSF-1, EGF and their respective receptors that are expressed differently in carcinoma cells, and macrophages found in the tumor microenvironment.³

Gaining replicative immortality

Normal cells have a limited replicative ability. The intrinsic cellular mechanism allows a limited number of normal cells to divide and blocks cell division that exceeds a certain limit.¹

Cancer cells overcome this limitation by over-expression of the telomerase enzyme, which allows the telomere (protects the chromosome ends and allows the cell to continue proliferation). Tumor suppressor genes such as p53 also help this process.¹

In recent years, molecular cancer studies have revealed additional functions of telomerase, which are independent of telomere continuity and can help tumor growth:¹

• Increased cell proliferation and / or resistance to apoptosis
• DNA damage repair
• RNA dependent RNA polymerase function
• Connection with chromatin

Figure 2. Replicative immortality gain^1,4

The shortening of telomere length activates replicative aging in normal cells; however, tumor cells eliminate the limitation of replicative ability through over-expression of the telomerase enzyme, which maintains telomere length.^1,4

Escape from growth suppressors

Cell proliferation in normal cells is a tightly controlled process in which pro- and anti-proliferation signals are coordinated at the cell cycle level. In particular, the G₁ phase of the cell cycle is a very important control point where anti-growth signals exert their effects on blocking cell proliferation. 5⁵

Anti-growth signals in normal cells can block proliferation in several ways:^5,6

• Triggering the G₀ phase
• Triggering a post-mitotic condition that mostly involves terminal cell differentiation

However, most cancer cells escape normal growth suppressors to maintain proliferation.¹

The two most commonly regulated tumor suppressors in cancer cells are retinoblastoma protein (Rb) and p53. In normal tissue, these proteins are part of a large network that controls the cell cycle.^1,5

• Rb inhibits cell passage through the restriction point in the G₁ phase of the cell cycle
• In cancer cells containing mutated Rb, the transition controller is removed and cell proliferation continues.
• p53 functions as an important apoptosis regulator because it stops the cell cycle in cells with DNA damage
• The disappearance of p53 allows the cell cycle to progress despite the presence of DNA damage and cellular stresses

Figure 3. Escape from growth suppressors⁷

Rb and p53 are 2 common tumor suppressors that are inactivated in tumor cells, and their inactivation leads to uncontrolled growth and proliferation.⁷

Escape from immune destruction

Immune surveillance is the basic cellular process that proactively prevents tumor formation in the human body. In preclinical studies, it has been shown that the active immune system continuously recognizes and eliminates the vast majority of cancer cells before they take a place and form a tumor mass.^1,8

However, cancer immuno-shaping, a newly identified reference feature, includes three key phases: elimination, balance and escape.⁹

• In the process defined as the elimination phase, the immune system successfully recognizes and eliminates cancer cells.
• Tumor cells that are not eliminated by the immune system progress to the equilibrium phase, in this phase the immune system can keep cancer cell growth under control, but cannot completely eliminate the transformed cells.
• Tumor cells that are not susceptible to immune destruction enter the escape phase. In this phase the tumor clones that "escape" continue to divide and grow - not effectively detected and eliminated by the immune system.

Clinical examples, in which the prognosis has been shown to be better in colorectal cancer and ovarian cancer patients with enhanced immune response, compared to patients with reduced immune response, also support this finding.¹

Figure 4. Escape from immune destruction^10,11

Cancer immune shaping, a newly determined reference feature, consists of 3 key phases: elimination, balance and escape. Cancer cells that successfully pass these phases gain the ability to escape immune destruction.¹⁰

Genome instability and mutation

Numerous changes in the genomes of cancer cells serve as the basis for many oncogenic processes. Increasing mutation rates provides an advantage for cancer cells in terms of accumulation of several mutations needed to promote tumorigenesis. This happens through the following^1,12

• Increased sensitivity to mutagenic agents
• Disruption of DNA repair mechanisms mediated by genes such as p53 or breast cancer type 1 sensitivity protein (BRCA1) of one or more cells
• Combination of these factors

The accumulation of these mutations is accelerated through the modification of the DNA maintenance mechanism or "caring" genes. These genes are responsible for the following¹

• Detection of DNA damage and activation of the repair mechanism
• Repair of directly damaged DNA
• Inactivation or inhibition of mutagenic molecules

By inactivating or suppressing caring genes, tumor cells can increase the rate of mutations followed by tumorigenesis.¹

Analyzes of cancer cell genomes also reveal function-altering mutations and show that genome instability increases during tumor progression.¹

Figure 5. Genome instability and mutation¹²

Mutations in DNA repair pathways provide an advantage for cancer cells in terms of increased genomic instability. The drawing above shows such a mechanism resulting from the defective BRCA signal transmission path.¹²

Triggering angiogenesis

The angiogenesis process or formation of new blood vessels in tumor cells is critical for continued tumor growth and metastasis. Tumor angiogenesis is a multi-step process and includes signal input from several pro-angiogenic growth factors. ^13,14 The moment when a tumor begins to over-express pro-angiogenic factors such as vascular endothelial growth factor (VEGF) is often described as an "angiogenic key." 1¹

Continuing without interruption, angiogenesis provides enlargement of the tumor and local invasion by:¹³

• Delivery of oxygen and nutrients
• Production of growth factors that benefit tumor cells

The findings of molecular cancer studies also show that metastases can eventually enter the systemic circulation through the new tumor vasculature.¹³

Two additional components play a role in tumor neovasculature:¹

• Pericytes are supporting cells that are associated with normal tissue vasculature; However, the findings of recent studies show that the pericyte coverage is also important for tumor angiogenesis.
• Data from molecular cancer studies also show that bone marrow-derived cells, such as macrophages and neutrophils, are aggregated into lesions and can help initiate the angiogenic key

Figure 6. Triggering angiogenesis^13,14

Tumor angiogenesis is a function consisting of the sum of a large number of signals from various cell types found in the tumor microenvironment.^13,14

Reprogramming of energy metabolism

Reprogramming of energy metabolism has been described as a distinctive feature that occurs in cancer cells.¹

Cancer cells make adjustments in energy production using the following ways to maintain uncontrolled proliferation:¹

• Reprogramming of glucose metabolism
• Increased glucose transporters such as glucose transporter 1 (GLUT1)
• Binding to alternative metabolic pathways

Limiting energy production to the glycolysis phase also reduces the amount of adenosine triphosphate (ATP) produced, but it also allows cancer cells to direct glycolic intermediates to various pathways, including those needed to assemble new cells.¹

The findings of molecular cancer studies also show that various activating mutations are detected in glioblastoma conditions, enzymes, which provides an advantage for changing tumor cell energy.¹⁵

In addition, reprogramming of energy metabolism is now widely practiced in clinical conditions through the use of [ ¹⁸ F] fluorodeoxyglucose positron emission tomography (FDG-PET) technology, which helps to obtain tumor images with increased glucose uptake . 16¹⁶

Figure 7. Reprogramming of energy metabolism¹⁷

Cancer cells, regardless of the presence of oxygen, convert existing glucose to lactate (Warburg effect), thereby directing glucose metabolites to beneficial anabolic processes that accelerate cell proliferation.¹⁷

Escape from cell death

Normal cells can initiate apoptosis in response to DNA damage, as well as other cellular stresses. In contrast, cancer cells generally have a lower sensitivity to similar stress factors and tend to escape apoptosis.¹⁸

Apoptosis occurs through 2 pathways: intrinsic that connects with intracellular stresses or extrinsic which begins by binding of mitochondrial pathway and cell surface receptors to specific ligands, death receptor path.¹⁸

Intrinsic pathway can be important in cancer, as many cellular stress factors faced by cancer cells are intrinsic pathway activators. These factors include DNA damage and growth factor deprivation, as well as treatment with chemo- and immunotherapeutic agents. The intrinsic pathway is tightly regulated by a related protein group called the BCL-2 family.

Consistent with its role in the regulation of apoptosis, many cancers can resist the apoptotic pathway by disrupting the regulation of BCL-2 family members. Cancer cells are thought to do this through 2 basic mechanisms: decreased pro-apoptotic proteins or increased BCL-2 expression.¹⁹

However, cancer cells can also avoid higher levels of apoptosis by decreasing stress signals leading to the onset of the BCL-2 pathway or, conversely, by decreasing effector molecules downstream. For example, mutation of the p53, which can link cellular stress to increased expression of pro-apoptotic proteins in normal state, leads to decreased cell susceptibility to DNA damage; Decrease of ²⁰ caspase-3 has been associated with apoptotic resistance in some tumor types^21,22

There are pathways similar to other cell death or cell death-like -such as autophagy and necrosis- and their regulation may be impaired through cancer cell survival; however, their specific roles are not fully understood yet.¹⁸

Figure 8. Escape from cell death

Ongoing proliferative signal transmission

Growth signal transduction in normal cells is an extremely well regulated process in which proliferative signals are activated whenever necessary and deactivated when it is no longer needed; this strict regulation provides cell homeostasis. However, this regulation weakens in cancer cells.¹

One of the main characteristics of cancer cells is that they can proliferate without the need for controlled signal input. The cells in question do this in several ways:¹

• Growth of growth factor production
• Stimulation of normal cells in the microenvironment to provide growth factors to cancer cells
• Increased number of receptors on the cell surface
• Structural change of receptors to facilitate signal transmission of the cancer cell
• Activation of proteins in the downstream signal transduction pathway

The findings of recent research also highlight that cancer cells can disrupt negative feedback loops, which in every case of hyperactivation in the mitogenic signal, constitute a safety mechanism to neutralize a signal transmission pathway. An important example of this is Ras oncoprotein.¹

• The oncogenic activity of Ras protein is not the result of overactive Ras signal transmission but rather a defect in normal negative feedback mechanisms that work through oncogenic GTPase.
• Other examples of this process include dysfunction mutations in phosphatase and tensin homolog (PTEN) which enhances phosphatidylinositol 3-kinase (PI3K) signal transmission.

Figure 9. Ongoing proliferative signal transduction²³

Tumor cells disrupt negative feedback loops in the oncogenic Ras signaling pathway, leading to ongoing proliferative signaling in tumor cells.²³

Tumor supporting inflammation

Natural and adaptive immune system cells often infiltrate the tumor microenvironment which allows tumors to resemble inflammatory conditions seen in normal tissues. The findings of molecular cancer research suggest that this tumor-associated inflammation can help tumor growth.¹

Data from studies show that tumor-associated inflammation can help tumor growth by providing the tumor microenvironment by:¹

• Growth factors
• Survival factors
• Pro-angiogenic factors
• Extracellular matrix (ECM) modifying enzymes that promote angiogenesis, invasion and metastasis
• Inductive signals that activate epithelial-mesenchymal transition (EMT) and other mechanisms that facilitate the distinctive feature

In addition, inflammation often occurs in the early stages of neoplastic disease. In early inflammation chemicals can be released into the tumor microenvironment and this inflammation can lead to genetic mutations that promote and accelerate tumor formation.¹

Figure 10. Tumor-promoting inflammation²⁴

Tumor-related inflammation may support tumor growth by providing tumor factors to growth factors, survival factors and angiogenesis.²⁴