Overview of Cancer Progression Pathways - US (2024)

Progression of a normal cell to a cancerous state is a multi-step process initiated by single and/or multiple somatic mutations, as well as microenvironmental cues. These changes lead to a cell’s physiological deregulation pertaining to several key regulatory pathways involved in cell proliferation, differentiation, and death.

Although physiological deregulation in cancer cells present a random and highly complex front, there have been efforts to classify them into several key processes or cancer progression pathways. Hanahan & Weinberg proposed the existence of several hallmarks that define cancer progression. These hallmarks include self-sufficiency in growth signaling, insensitivity to anti-growth signals, evading apoptosis, limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis, deregulating cellular energetics, and avoiding immune destruction.

Antibodies are a cornerstone for research in cancer biology, playing a vital role in cancer diagnosis, prognosis, and therapeutic strategies. Conventional histological grading of tumor biopsies has been coupled with antibodies against tumor-specific antigens, oncogenes, tumor suppressor genes, and cell proliferation markers to obtain a detailed molecular profiling of tumors, and to effectively predict therapeutic response. They are also extensively used in academic research to decipher cancer-specific regulatory pathways, and the function of individual components of these pathways. Each of the sections below will be accompanied by data using Invitrogen antibodies to show what is possible when using antibodies to study cancer pathways and targets.

Aberrant growth receptor expression is another strategy adopted by cancerous cells towards growth signaling autonomy. This can be achieved by:

• receptor gene amplification leading to overexpression.
• mutations leading to constitutive overexpression.
• irregular signaling caused by chromosomal translocations.
• impairment in receptor recycling and degradation machinery.

The epidermal growth factor receptor (EGFR) family, also known as the Erb family, have been extensively studied in cancer progression (Tiash et al., 2015). Human epidermal growth factor receptor 2 (HER2) has been found to be overexpressed in about 25% of breast cancers and is one of the markers used in the molecular sub-typing of breast cancer. Below is data for an antibody against HER2 (ErbB2) used for western blotting.

Cellular quiescence and growth arrest are important in the maintenance of tissue homeostasis. In order to attain unchecked proliferative capability, cancer cells rely on the inactivation of several key players of cell cycle progression, as well as extracellular matrix (ECM) components that act as antagonists to growth signaling pathways. To achieve uncontrolled division capability, the regulatory mechanism for cell division must be overridden. Those regulatory mechanisms include the cyclin-dependent kinases (CDKs), important in the cycle progression, and the cycle checkpoint regulators. For example, the retinoblastoma (Rb) pathway for regulation of G1S cell cycle progression involves the activation of cyclin D by mitogenic signals and leads to the downstream activation of CDK4/6. This activation promotes the phosphorylation of the Rb protein, which leads to the dissociation of the transcription factor E2F1 from the Rb-E2F1 complex. The dissociation allows E2F1 to transcribe and express the genes required for S-phase. p16INK4a opposes the activation of the cyclin D–CDK4/6 complex and acts as a major regulator in this process.

The amplification of the cyclin D1 gene and overexpression of the cyclin D1 protein have been implicated in a wide spectrum of human cancers. The upregulation of CDK4 and cyclin D1 have been reported in the vast majority of breast carcinomas. Several malignancies also display a loss of function, mutation, and/or silencing of Rb proteins and p16INK4a (Deshpande et al., 2005; Collins et al., 1997).

The TP53 gene is another tumor suppressor gene that plays a role in cell growth arrest and apoptosis induction. p53 transcriptionally activates p21, which in turn binds to the cyclinD1/CDK4 complex. This binding inhibits the phosphorylation of the Rb protein and downstream S phase progression. About 50% of all cancers display mutations leading to a loss of function in the TP53 gene, which is indicative of the importance it has in cell cycle regulation (Chen et al., 2016). Data below shows western blot analysis of cyclin D1 and CDK4 in multiple cells lines, tissues, and whole cell extracts.

Apoptosis is a vital pathway in developmental biology and tissue homeostasis. It is regulated by the expression of several cell survival factors and pro-apoptotic genes. Apoptosis can occur by either one of two pathways: the intrinsic (or mitochondrial) and extrinsic (or death receptor) pathways. Although both culminate in caspase activation, the mechanisms by which this occurs are distinct. Apoptosis is an important hallmark of cancer progression, determining treatment outcome, and drug resistance in cancer cells.

Mutations in key players of the apoptotic pathway are linked to poor prognosis and reduced treatment sensitivity. CD95 receptor/CD95L is a key player in the extrinsic pathway of cell death. CD95 transmembrane receptor is expressed on activated lymphocytes, on tissues of lymphoid/non-lymphoid origin, and on tumor cells. CD95 ligand is produced by activated T cells and triggers autocrine suicide or paracrine death in lymphocytes or other target cells. CD95L expression has been suggested to be a method of immune system evasion displayed by tumor cells. Down regulation of CD95 expression in drug-resistant leukemia or neuroblastoma cells suggest that it may have an impact on drug sensitivity. This downregulation can be achieved by genetic mechanisms, such as mutations in the gene loci, or by epigenetic methods including gene promoter hypermethylation and histone modifications, leading to chromatin condensation (Fulda et al., 2006). Below, a western blot analysis of CD95 (FAS receptor) in multiple cells lines is shown.

The eukaryotic replication process is a heavily regulated process. When replication is interfered with, as in the case of human carcinomas, it can lead to a significant amount of DNA damage, replicative stress, and accelerated mutation rates. Several checkpoints exist to ensure the accuracy of the replication process and to invoke a repair mechanism if errors are detected. Cancer cells can override these checkpoints and repair mechanisms, leading to an accumulation of mutations and genome instability.

The ataxia telangiectasia and Rad3-related (ATR) protein complex, upon activation, invokes downstream kinases like checkpoint kinase 1 (Chk1), and is important for stalling DNA replication, ssDNA repair, and stabilization of the replicative fork. However, in cases where cells fail to recruit the respective repair machinery, this can lead to DNA mutations and chromosomal rearrangements, as often seen in human cancers. In such cases, the damaged site is repaired by an error-prone translesion DNA polymerase that inaccurately synthesizes DNA to complete bulk genome replication. This error-prone DNA repair system leads to an accumulation of replication errors, which enhances genomic instability. Mutations in the ATR-Chk1 pathway leads to a reduction in the activity of the ATR pathway, favoring the accumulation of mutations in the replicated cells (Gaillard et al., 2015).

PARPs, important in BER/SSBR for DNA repair, are targeted by therapeutics to prevent cancer cells from repairing DNA damage sites. Major players in the hom*ologous repair mechanism (which enables repair of erroneous DNA replication), BRCA1 and BRCA2, are found mutated in several cancers including breast and ovarian. Apart from this, cancer cells also have enhanced telomerase activity, which extends the telomeric sequences, delaying cell senescence (Gaillard et al., 2015). Below, there is data for antibodies against Chk1 and PARP1. Cell treatment has been used to show specificity of the antibody to PARP1, where the cleaved form of PARP1 can be detected in Staurosporine-treated Hela and Jurkat cells.

Western blot analysis of SNAIL, E-cadherin, and Vimentin.The bands of interest were obtained in accordance to the reported molecular weight and expression levels were concordant of the target biology. The blots were probed with (A)SNAIL Monoclonal Antibody (F.31.8) (Cat. No. MA5-14801, 1:1,000 dilution), (B)E-cadherin Monoclonal Antibody (HECD-1) (Cat. No. 13-1700, 5 µg/mL), and (C)Vimentin Polyclonal Antibody (Cat. No. PA5-27231, 1:2,000 dilution). The bands were detected by host specific secondary antibodies Goat anti-Mouse IgG (H+L) Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A28177, 1:4,000 dilution) or Goat anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A27036, 1:4,000 dilution).

Normal cell energetics rely on oxidative phosphorylation coupled with the citric acid (TCA) cycle for the generation of ATP and beta-oxidation for ATP generation from fatty acids, both being mitochondria-dependent processes. However, in tumor cells, hypoxia and mitochondrial dysfunction require a shift to anaerobic respiration for fulfilling the cellular energy requirements, even in the presence of adequate oxygen (also known as the Warburg effect).

Mitochondrial dysfunction may be brought about by point mutations, changes in copy number in the mtDNA, or by mutations in the genes encoding elements for components of the aerobic respiration cycle. As a result, the cell switches to the glycolytic pathway for sustaining the cell’s energy requirements. Alterations in oncogenes and tumor suppressors, like HIF-1 and TP53, can regulate this process. The activation of HIF1α in a tumor can upregulate the expression of several glycolytic pathway enzymes including pyruvate dehydrogenase kinase 1 (PDK1), lactate dehydrogenase A (LDHA), glucose transporters, and glycolytic enzymes. TP53, on the other hand, represses the expression of the glycolytic pathway enzymes and is known to induce the expression of Cytochrome c and AIF; these both favor mitochondrial respiration. Therefore, mutations in TP53 lead to a dependence on glycolysis for the cell’s energy requirements (Hsu et al., 2016). Western blot data for antibodies against PDK1, LDHA, and p53 can be seen below.

Western blot analysis of PDK1, LDHA, and p53.The blots were probed with (A)PDK1 Recombinant Rabbit Monoclonal Antibody (JA67-30) (Cat. No. MA5-32702, 1:1,000 dilution), (B)LDHA Polyclonal Antibody (Cat. No. PA5-27406, 1 µg/mL), and (C)p53 Antibody co*cktail (Cat. No. MA5-14067, 1–2 µg/mL). The band of interest was detected using host specific secondary antibodies Goat anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A27036, 1:4,000 dilution) or Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Cat. No. 62-6520, 1:4,000 dilution). The bands of interest were picked up at the expected molecular weights for(A) PDK1: 49.4 kDa, (B) LDHA: 35 kDa, and (C) p53: 53kDa. The protein levels were in accordance to reports of the target expression.

The immune system interacts with the tumor microenvironment through all stages of tumor progression and invasion. In order to overcome destruction by the immune system, cancer cells adopt several mechanisms to evade immunosurveillance.

Immunoediting—this mechanism of immune evasion involves the selection of tumor cells that can escape recognition by the immune cells. The immune system detects and destroys a clone of the cancer cells that expresses recognized tumor antigens. This leads to the elimination of the clone and the tumor mass is replenished by the proliferation of a small fraction of cells that do not express the tumor antigen. These then form the bulk of the tumor and maintain an immune-suppressed tumor microenvironment.

Regulatory T cells—tumor cells import the host’s T-reg cells via tumor cell-mediated cytokine production to provide immune-suppressive function to the tumor microenvironment. Studies have reported that tumor derived T-reg cells have a higher suppressive activity than naturally occurring T-reg cells. TGF-β, produced by the various cells within the tumor microenvironment, can convert CD4⁺T cells into suppressive T-reg cells in situ.

Inflammation and immunosuppression—recruitment of inflammatory elements of the immune system, like myeloid-derived suppressor cells (MDSCs), modulated dendritic cells (DCs), and activated M1 and M2 macrophages create an inflammatory microenvironment within the tumor. This microenvironment can mediate tumor initiation, angiogenesis, metastasis, and a resistance to apoptosis in MDSCs, which suppresses CD8⁺ T cell-mediated antitumor immunity.

Cytokines—tumors can evade immune surveillance by inhibiting cytotoxic T lymphocyte (CTL) functionality via several immune-suppressive cytokines like tumor necrosis factor (TNF)-a, IL-1, IL-6, IL-8, IL-10, and type I IFNs. These cytokines are secreted either by the tumor cells or the stromal cells within the tumor microenvironment.

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