Mechanisms in Cancer

The past few decades have witnessed remarkable progress in understanding the molecular and cellular framework of cancer forming the basis for the novel innovative and efficient oncological therapies, during later years with a particular emphasis on harnessing anti-tumor immunity. As a result, several tumor markers are currently being used for a wide range of cellular processes in cancer.

Cancer is one of the world’s most significant health problems, contributing to more than 9 million deaths yearly. The tissue of origin generally defines the tumor class. Carcinomas, which are epithelial malignancies, are the most frequent solid tumors. Squamous carcinomas arise from benign, precancerous lesions of the epidermis and non-secretory epithelia. Corresponding malignancies derived from hyperplastic glandular epithelia (adenomas) are called adenocarcinomas

Tumors originating from the non-hematopoietic mesodermal lineage, e.g., connective tissue, skeleton, muscles, are collectively called sarcomas. Leukemias and lymphomas arise from the blood-forming (hematopoietic) cells in the bone marrow and from cells of the immune system, respectively. Finally, gliomas represent the most frequent types of malignant tumors of the central nervous system.

The tumor cells of different cancer types share several common properties and are characterized by their dedifferentiated state. These properties typically include sustained proliferation (immortality), escape from programmed cell death, genomic instability, as well as enhanced ability to move away from the sites of origin and the ability to invade other tissues in the body. The carcinogenesis can often be triggered by genetic mutations within the regulatory regions or reading frames of genes resulting in altered translation products or non-coding RNAs. In addition to this, the epigenetic regulation of gene activity may also induce cancer.

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Figure 1: A. IHC staining of human colorectal cancer using the Anti-p53 monoclonal antibody (AMAb90956) shows strong nuclear positivity in tumor cells but not in normal mucosa. B. WB analysis in U-251MG cells transfected with control siRNA, target-specific siRNA probe #1 and #2, using the Anti-p53 monoclonal antibody (AMAb90956). Remaining relative intensity is presented. Loading control: Anti-PPIB. C. ICC-IF staining in A431 cell line with the Anti-p53 monoclonal antibody (AMAb90956), showing cell cycle-dependent nuclear (without nucleoli) staining in green. Microtubules are visualized in red.

Cell Cycle

Cancer lesions commonly originate from the aberrant cellular proliferation due to e.g., mutations or amplifications in the open-reading frames or regulatory domains of oncogenes and tumor suppressors that regulate cell growth. In addition, gross chromosomal alterations may generate fusion proteins with de novo transforming potential.

The cell cycle is a progressive set of molecular events that culminate into altered cell growth and mitotic rate, i.e., by which frequency one cell divides into two daughter cells. Cell cycle progression is positively regulated and enforced by a family of serine/threonine protein kinases collectively referred to as cyclin-dependent kinases (CDKs), which are activated by binding to respective cyclin partners and by phosphorylation.

These checkpoint pathways recognize and sense whether or not to initiate and/ or promote cellular proliferation under a particular set of external and internal conditions, including timing.

Crucial components of these pathways are mainly proteins encoded by some of the checkpoint genes, including the tumor suppressor protein TP53.

Figure 2: A. IHC staining of human lung cancer with the Anti-PARP1 monoclonal antibody (AMAb90959) shows strong nuclear immunoreactivity, in brown. B. WB analysis in RT-4 cells transfected with control siRNA, target-specific siRNA probe #1 and #2, using the Anti-PARP1 monoclonal antibody (AMAb90959). Remaining relative intensity is presented. Loading control: Anti- GAPDH. C. ICC-IF staining in HeLa cell line with the Anti-PARP1 monoclonal antibody (AMAb90959), showing cell cycle-dependent nuclear staining in green. Microtubules are visualized in red.

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Readings

Barnum K.J. and O’Connell M.J. (2014) Cell Cycle Regulation by Checkpoints. Methods Mol Biol. 1170: 29–40.

Feitelson M.A. et al. (2015) Sustained proliferation in cancer: mechanisms and novel therapeutic targets. Semin Cancer Biol. 35(Suppl): S25–S54

López-Sáez J.F. et al. (1998) Cell proliferation and cancer. Histol Histopathol.13(4):1197-214

Tobias O. and Sicinski P. (2017) Cell cycle proteins as promising targets in cancer therapy. Nature Rev Cancer 17:93-115

Cell Death

On the morphological basis, cell death can be achieved by apoptosis, necrosis, and autophagy. Apoptosis and autophagy are regarded as “programmed cell death” while necrosis is considered as “unprogrammed cell death” due to deregulated activity. Another type of cell death termed necroptosis, exhibits morphological features of both apoptosis and necrosis. An inappropriate level of cell death (either too little or too much) is a decisive factor in many human disease conditions, including cancer.

Apoptosis

Apoptosis (from the Greek meaning “falling off”) remains the most well-studied mechanism of programmed cell death. Apoptosis is a regulated, ATP-dependent cell death mechanism crucial for the removal of surplus and/or aberrant cells. This process is ultimately dependent on the actions of the activated form of the protease caspase-3.

Apoptosis is important during development, organogenesis, and aging, and serves as a mechanism to maintain cellular homeostasis within tissues.

Cancer cells can surpass apoptosis by various mechanisms such as: upregulation of the anti-apoptotic (Bcl2) pathway or inhibition of the pro-apoptotic proteins through genetic or epigenetic mechanisms.

From a therapeutic point of view, apoptosis resistance is a major issue when it comes to treatment failure during cancer chemotherapy.

Autophagy

Autophagy (from the Greek “self-eating”) plays a housekeeping role in removing misfolded/aggregated proteins and damaged organelles and depends upon their lysosomal proteolysis.

In healthy tissues, autophagy contributes to the homeostasis of the cells. The dysregulation of autophagy in cancer contributes to the protection of cells undergone malignant transformation. As a result, sustained survival and proliferation of tumor cells occur, supporting tumor growth, invasion, and metastasis.

Necroptosis

Necroptosis is the caspase-independent “cellular suicide” or “regulated” necrosis. It is an alternative mode of regulated cell deathmimickingfeaturesofbothapoptosis and necrosis. Pieces of evidence based on a mouse model reveal that the de- regulation of necroptosis is associated with pathological conditions like cancer. Necroptosis is primarily regulated by proteins RIPK1 and RIPK3, and it may trigger and amplify antitumor immunity in cancer therapy.

Figure 3: A. IHC staining of the human tonsil with the Anti-BCL2 monoclonal antibody (AMAb91492) shows strong positivity in non-germinal center cells. B. IHC staining of the human testis with the Anti-BCL2 monoclonal antibody (AMAb91492) shows no positivity in cells in seminiferous tubules, as expected (negative control).

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Readings

Glick D. et al. (2010) Autophagy: cellular and molecular mechanisms. J Pathol. 221(1): 3–12

Green DR. et al. (2002) A matter of life and death. Cancer Cell. 1(1):19-30

Guo JY., et al. (2013) Autophagy-mediated tumor promotion. Cell 155(6):1216-9

Kroemer G. et al. (2010) Autophagy and the integrated stress response. Mol Cell. 40(2):280-93

Lopez J. et al. (2015) Mitochondrial apoptosis: killing cancer using the enemy within. Br J Cancer. 112(6):957-62

McIlwain D.R. et al. (2013) Caspase Functions in Cell Death and Disease. Cold Spring Harb Perspect Biol. 5(4): a008656

Choi M.E. et al. (2019) Necroptosis: a crucial pathogenic mediator of human disease. JCI Insight. 4(15): e128834.

Metastasis

A unique and clinically decisive feature of cancer cells is their capability to disseminate from their point of origin invading secondary and distal tissue sites, a set of events known as metastasis.

The metastasis cascade refers to the sequential occurrences enabling cancer cells to detach from each other in situ and escape from the primary tumor site. The final destination of metastatic cells is non-random and depends on both tissue vascularization and the expression of particular homing proteins in the respective cell surfaces.

The metastatic tumor cells lose their adhesive properties and contacts with the basement membrane. This process depends on e.g., expression of matrix metalloproteases (MMPs) which degrade

the basement membrane, allowing cells to disseminate, reach their distal destinations via vascular routes (blood and lymph vessels) and, finally, to converge the formation of clinically detectable micro- or macroscopic secondary tumors in distal organs (metastases).

Upon exit from the primary site and during dissemination, metastatic cells are reprogrammed into a mesenchymal phenotype through the so-called Epithelial- to-Mesenchymal-Transition (EMT). Upon EMT, cancers of epithelial origin acquire molecular signatures usually associated with a “stem cell-like” phenotype, which includes, for instance, induction of TGF- beta, transcription factors Snail1- and -2, Twist, as well as the intermediate filament protein vimentin.

The histopathological analysis may enable the distinction of the invading tumor cells from the healthy local tissue. Sometimes, the presence of invading metastases is revealed by the mere differences in size and nuclear morphology (degree of pleomorphism) of the tumor cells versus the healthy surrounding tissue. To that end, conventional histochemical staining like H&E is employed.

However, to define the origin of the metastatic tumor cells using only conventional histopathology can be challenging. Therefore, specific phenotypic IHC-markers like e.g., cytokeratins KRT7 and KRT20 can be used, often as a panel of different markers.

Figure 4: Multiplexed IHC-IF staining of a human breast cancer section Figure 5. IHC analysis in the human bone marrow and endometrium tissues using the Anti-AR monoclonal antibody (AMAb91547, nuclear staining in using the Anti-MMP9 monoclonal antibody (AMAb90804). Corresponding red) and the Anti-KRT7 polyclonal antibody (HPA007272, cytoplasmatic MMP9 RNA-seq data are presented for the same tissues.
staining in green). DAPI is used as a counterstain (in blue).

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Readings

Wittekind C. and Neid M. (2005) Cancer Invasion and Metastasis. Oncology 69:14–16

Seyfried T.N. and Huysentruyt L.C. (2013) On the Origin of Cancer Metastasis. Crit Rev Oncolog.18(1-2): 43–73

Wang R.A. et al. (2015) Reasons for cancer metastasis: A holistic perspective. Mol Clin Oncol. 3(6): 1199–1202

Lee S.L.C. et al. (2009) Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, invasion, and metastasis in a zebrafish tumor model. PNAS 106(46):19485- 19490

Tumor Angiogenesis and Lymphoangiogenesis

Cancer cells differ from healthy cells and benign tumor cells in their metabolic characteristics. Paradoxically, despite sufficient oxygen (O2) levels, aggressive cancers preferentially produce lactate.

This distinguishing feature is known as the “Warburg effect.” The metabolic state of these tumors arises from changes in the tumor microenvironment (TME), coupled among other things, to O2 availability. Ultimately, the increased growth and metabolic demand of malignant tumors lead to intra-tumoral hypoxia (lowered partial O2 pressure).

Hypoxia is associated with metabolic reprogramming and enhanced glycolysis. A chief mechanism regulating aerobic glycolysis in cancer cells involves the Hypoxia-Inducing Factor (HIF-1alpha), which is responsible for the altered state of metabolism in cancer by triggering the transcription of a multitude of factors.

Angiogenesis is the life-long formation of blood vessels from the existing vasculature. It occurs throughout life in both health and disease. Neo-angiogenesis, including lymphoangiogenesis (formation of lymph vessels), plays a significant role in both tumor growth and metastasis.

In fact, like all living cells, cancer cells strongly depend on an adequate supply of O2 and nutrients and the removal of waste products for their survival. Endogenous regulators of angiogenesis include, among others, growth factors, cytokines, proteases, protease inhibitors, and oncogenes.

Recognition that control of angiogenesis (decreasing or inhibiting) could have therapeutic value has stimulated considerable interest during the past 40 years.

However, the clinical outcome of this therapeutic approach has been limited, not least due to resistance development. Also, anti-angiogenic therapy may be of a disadvantage because many drugs reach the tumor through a vascular route.

Figure 6: A. IHC analysis in human breast cancer using the Anti-VWF monoclonal antibody (AMAb90931).
B. IHC analysis in human fallopian tube and liver using the Anti-VWF monoclonal antibody (AMAb90931). Corresponding VWF RNA-seq data are presented for the same tissues.

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Readings

Lu J. et al. (2015) The Warburg effect in tumor progression: mitochondrial oxidative metabolism as an anti-metastasis mechanism. Cancer Lett. 356 (2A):156-64

Rajabi M. and Mousa S.A. (2017) The Role of Angiogenesis in Cancer Treatment. Biomedicines 5(2):34

Romero-Garcia S. et al. (2011) Tumor cell metabolism - an integral view. Cancer Biol Ther.12(11):939–948

Tonini T. et al. (2003) Review: Molecular basis of angiogenesis and cancer. Oncogene 22:6549–6556

Yin Z. et al. (2019) Targeting T cell metabolism in the tumor microenvironment: an anti-cancer therapeutic strategy.
J Exp&Clin Cancer Res. 38:403

Tumor Proliferation

Abnormal proliferation is one of the cardinal features of cancer and is indispensable for tumor development and progression. The processes that restrain healthy regulated cell growth are often compromised or lost in cancer.

Many aggressive tumors, particularly carcinomas, have a very rapid proliferation rate while others, like some neuroendocrine tumors, grow very slowly. Hyperproliferation is often triggered by constitutively activated signal transduction pathways that promote uncontrolled proliferation.

These include growth factor signaling pathways mediated by, for example, tyrosine kinase receptors (EGFR, PDGFR, IGF-1R, Wnt, Bcr-Abl, PI3K/Akt, etc.).

The fact that cancer cells show increased proliferation rate, makes them particularly sensitive to cytostatic agents. One of the most common therapy in cancer is the use of cytotoxic (cytostatic) drugs.

Other therapeutic options may include small molecules, e.g., tyrosine kinase inhibitors or therapeutic antibodies like trastuzumab (Herceptin) against HER2.

Figure 7: A. IHC staining of human breast cancer using the Anti-ESR1 monoclonal antibody (AMAb90867) shows strong nuclear immunoreactivity in a subset of tumor cells. B. WB analysis in MCF-7 cells transfected with control siRNA, target-specific siRNA probe #1 and #2, using the Anti-ESR1 antibody (AMAb90867). Remaining relative intensity is presented. Loading control: Anti-GAPDH. C. ICC-IF staining of MCF-7 cells using the Anti-ESR1 antibody (AMAb90867), showing specific staining in the nucleoplasm in green. Microtubules are visualized in red.

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