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.

Explore Cancer Research Antibodies  Explore Cancer Pathways 


Self-sufficiency in growth signaling

Normal cells require mitogenic growth signals from their microenvironment to obtain proliferative abilities. Cancerous cells exhibit decreased dependency on microenvironmental cues for proliferation, which is achieved by the following processes:

Autonomy in growth factor production and autocrine growth signaling

Very often, both tumor cells and the stromal cells that form an integral part of the tumor microenvironment acquire the ability to produce aberrant levels of growth factors. These abnormal growth factors stimulate the tumor cells via autocrine/paracrine signaling into a state of uncontrolled proliferation. Platelet derived growth factor (PDGF), transforming growth factor alpha (TGF-α), and insulin-like growth factor (IGF) are a few examples of common growth factors that exhibit anomalous expression, both in terms of levels, as well as tissue specificity. The secretion of anti-proliferative ligands stimulates de-differentiation and tumor microenvironment remodeling. An example is transforming growth factor beta (TGF-β), a conventional anti-growth ligand that has roles in epithelial to mesenchymal transition (EMT) and tumor invasiveness (Fouad et al., 2017). Antibodies recognizing growth factors are most used to neutralize their effects. Below is data showing a neutralizing antibody against TGF-β.

Expression of mitogenic signal receptors

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.

Alteration in downstream effectors of growth signaling

Alteration in downstream effectors of growth signaling is one of the most common alterations that results in growth signaling autonomy. The Ras proteins, a family of small GTPases that participate in SOS-Ras-Raf-MAPK cascade, serves as an illustrative example. Three prominent members, H-Ras, N-Ras, and K-Ras, have proven essential for proliferation, differentiation, and survival of eukaryotic cells. An altered, constitutively active form of K-Ras proteins is known to be expressed in most pancreatic carcinomas (Fernández-Medarde et al., 2011). Below is data that depicts immunoblot analysis using a K-Ras antibody with siRNA-based specificity verification.

K-Ras antibody in western blot. Knockdown of K-Ras was achieved by transfecting HCT 116 cells with K-Ras specific validated siRNA. Western blot analysis (Fig a) was performed using whole cell extracts from K-Ras knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2), and untransfected cells (Lane 1). The blot was probed with K-Ras Recombinant Rabbit Monoclonal Antibody (11H35L14) (Cat. No. 703345, 1:5,000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A27036, 0.25 µg/mL, 1:4,000 dilution). Densitometric analysis of this western blot is shown in the histogram (Fig b). Loss of signal upon siRNA mediated knockdown confirms that antibody is specific to K-Ras.

Insensitivity to anti-growth signals

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.

Western blot data for cyclin D1 and CDK4. (A) Western blot was performed using Cyclin D1 Recombinant Rabbit Monoclonal Antibody (SP4) (Cat. No. MA5-14512) and a 33 kDa band corresponding to cyclin D1 was observed across all the cell lines and tissues tested. The blot was probed with the primary antibody (1:1,000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A27036, 1:4,000 dilution) using the iBright FL 1000 (Cat. No. A32752). (B) Western blot analysis was performed on whole cell extracts. The blots were probed with CDK4 Antibody Cocktail (Cat. No. MA5-13720, 1–3 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Cat. No. 62-6520, 1:4,000 dilution). A 33 kDa band corresponding to CDK4 was observed across cell lines tested.


Evading apoptosis

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.

In the intrinsic pathway of apoptosis, caspase activation is carried out by the distinct members of the Bcl family. Apoptosis via the intrinsic pathway leads to the permeabilization of the outer membrane of the mitochondria, which is brought about by several cytotoxic stimuli and pro-apoptotic signal transduction. The Bcl family plays an important role in regulating this process. These proteins can be pro-survival (e.g., Bcl-xl, Mcl-1) or pro-apoptotic (e.g., Bim, Bax), and a balanced level of both is required for normal tissue homeostasis. Cancer cell survival and drug resistance have been associated with a higher expression of pro-survival factors and loss of expression and function mutations in the pro-apoptotic factors (Fulda et al., 2006).


Replication stress and genome instability in cancer

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 homologous 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 Chk1 and PARP1. Western blot analysis of (A) Chk1 (55kDa) using Chk1 Monoclonal Antibody (2G1D5) (Cat. No. MA5-15239, 1:1,000 dilution) and (B) PARP1 (116 kDa, 86 kDa) using PARP1 Monoclonal Antibody (C-2-10) (Cat. No. MA3-950, 1:500 dilution). The blot was probed with the primary antibodies and the bands of interest were detected using Goat anti-Mouse IgG (H+L) Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A28177, 1:4,000 dilution). A band of interest was obtained across the panel at the reported molecular weight, and cell treatment in PARP1 yielded a cleaved fragment of PARP1 at 86 kDa.


Sustained angiogenesis

Tissue vascularization is an important process in maintenance of organ homeostasis. The tightly regulated process is instrumental in coordinating the functions of the endothelial cells to repair damaged blood vessels and expansion of new blood vessels from the endothelial cell sprouting. Angiogenesis in tumor cells is essential to cope with the increased nutrient stress and hypoxia observed within expanding solid tumors and to facilitate the dissemination of cells for distant site colonization and secondary growth formation. Increased levels of hypoxia within a tumor activates Hypoxia Inducible Factor 1a (HIF1a) expression, which has been linked to the expression of Vascular Endothelial Growth Factor (VEGF), a potent angiogenic cytokine that leads to vascular endothelial proliferation within the tumor microenvironment (Tonini et al., 2003; Krock et al., 2011). Below is representative data for antibodies against HIF1a and VEGF, respectively.

Immunofluorescent analysis of HIF1A in HeLa cells

Immunofluorescence analysis of HIF1A in HeLa cells. Cells were treated with 150 µM of CoCl2 for 48 hours. Then, the cells were labeled with HIF1A Polyclonal Antibody (Cat. No. PA1-16627) at 5 µg/mL and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. A27034) at a dilution of 1:2,000. Treatment with CoCl2 results in an expected translocation of HIF1A from the cytoplasm to the nucleus.


Tissue invasion and metastasis

Tumor cells undergo phenotypic changes enabling them to break from the tumor mass, intravasate into blood and lymph vessels, and migrate to distant organs where they re-establish cell-cell connections and proliferate to form a secondary tumor. In order to invade neighboring and distant tissues, tumor cells undergo a loss of inherent epithelial characteristics and gain a mesenchymal phenotype, a process known as Epithelial to Mesenchymal Transition (EMT). This transition is characterized as a loss of cell-cell connections, cell polarity, and cytoskeletal remodeling. EMT is transcriptionally regulated by transcription factors such as SNAIL, SLUG, Zeb1, and Twist1. The alteration in phenotype is characterized by a loss of epithelial markers like E-cadherin and ZO-1, and a gain of mesenchymal markers like N-cadherin and Vimentin (Nieto et al., 2016). The data below shows western blot data for SNAIL, E-cadherin, and Vimentin.

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).


Deregulating cellular energetics

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 Cocktail (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.


Mechanisms for immune evasion

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.
 


Recommended reading

  1. Hanahan, Douglas et al. Hallmarks of Cancer: The Next Generation. Cell, Volume 144, Issue 5, 646 – 674 (2011).
  2. Yousef Ahmed Fouad, Carmen Aanei. Revisiting the hallmarks of cancer. Am J Cancer Res 2017;7(5):1016-1036.
  3. Tiash S, Chowdhury EH. Growth factor receptors: promising drug targets in cancer. J Cancer Metastasis Treat 2015;1:190-200.
  4. Fernández-Medarde A, Santos E. Ras in cancer and developmental diseases. Genes Cancer. 2011;2(3):344-358.
  5. Kathleen Collins, Tyler Jacks, Nikola P. Pavletich. The cell cycle and cancer. Proceedings of the National Academy of Sciences 1997, 94 (7) 2776-2778.
  6. Deshpande, A., Sicinski, P. & Hinds, P. Cyclins and cdks in development and cancer: a perspective. Oncogene 24, 2909–2915 (2005).
  7. Chen J. The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and Progression. Cold Spring Harb Perspect Med. 2016;6(3): a026104. Published 2016 Mar 1.
  8. Fulda, S., Debatin, K. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25, 4798–4811 (2006).
  9. Gaillard, H., García-Muse, T. & Aguilera, A. Replication stress and cancer. Nat Rev Cancer 15, 276–289 (2015).
  10. Tonini, T., Rossi, F. & Claudio, P. Molecular basis of angiogenesis and cancer. Oncogene 22, 6549–6556 (2003). 
  11. Krock BL, Skuli N, Simon MC. Hypoxia-induced angiogenesis: good and evil. Genes Cancer. 2011;2(12):1117-1133.
  12. M. Angela Nieto, Ruby Yun-Ju Huang, Rebecca A. Jackson, Jean Paul Thiery. EMT: 2016. Cell, Volume 166, Issue 1, 2016, Pages 21-45.
  13. Hsu CC, Tseng LM, Lee HC. Role of mitochondrial dysfunction in cancer progression. Exp Biol Med (Maywood). 2016;241(12):1281-1295.
  14. Dass S. Vinay, Elizabeth P. Ryan, Graham Pawelec, et al. Immune evasion in cancer: Mechanistic basis and therapeutic strategies. Seminars in Cancer Biology, 2015, Volume 35, Pages S185-S198.