Cancer stem cells (CSCs) exist within the large majority of tumor cells. The CSC theory proposes the idea that CSCs are responsible for driving and promoting tumor growth. Driving the CSC population are embryonic signaling pathways (ESPs) that includes the following mechanisms responsible for regulating normal stem cells and organ development: Hedgehog (Hh), Notch, and Wnt.
Hh signalling is active during embryonic development, a stage in which it controls the growth and patterning of various tissues and development organs by sending signals that act as both mitogens and morphogens. Similarly, the pathway also plays a critical role in the development and activity of endocrine glands. The Hh signalling pathway becomes activated by interaction between Hh ligands (either Sonic Hh (SHh), Indian, Hh, and Desert Hh) and receptors, such as the Patched (Ptc) complex. The consequence of this interaction at the cell membrane results in the discontinuation of repression activity by Ptc on Smoothened (Smo). As a consequence, downstream signalling by Smo is permitted. Facilitating the signalling cascade in the cytoplasm, that ensues upon Smo activation, is the interaction between Smo and the Suppressor fused (Sufu) that is bound to the glioma-associated oncogene homolog (Gli) transcription factors (Gli 1-3). This interaction liberates Sufu from Gli that is followed by the translocation of Gli to the nucleus to permit gene transcription.
Aberrant Hh signalling is observed in a wide range of cancer types, although the aberrant activity associated with tumors varies by cancer type. In general, abnormal Hh signalling can be categorized as ligand-dependent or ligand-independent. Ligand-dependent drivers of Hh signalling activity includes the overexpression of Hh ligands by the tumor or stromal cells (using autocrine or paracrine mechanisms). Examples of ligand-dependent drivers include stromal cells, small cell lung cancer (SCLC), and gastrointestinal tract tumors. In contrast, ligand-independent mechanisms are noted where mutations can occur at various points along the activation pathway. For instance, in basal cell carcinoma and medulloblastomas it is observed that SMO mutations and Ptc1 loss of function are implicated in disease progression. A mutation of SUFU, while less common, is another possibility in addition to downregulation of micro RNAs that inhibit SUFU or Gli1.
While Hh signalling is important to cell growth and clonogenicity during embryogenicity under normal circumstances and its abberant activity is responsible for some cancers, an additional signalling pathway that plays a role in the same arena as Hh signalling is the Notch signalling pathway. Indeed, during embryonic development and the adult tissue lifecycle functions such as cell-to-cell communication, cell fate determination, cellular proliferation, and cell death are all outcomes influenced by Notch signalling. In particular, neural progenitor maintenance is a feature of Notch signalling that results in the inhibition of neural differentiation, however, the induction of the differentiation of glial cells and the development of a range of organs is activated by Notch signalling.
The Notch signalling pathway is activated by receptor-ligand interactions. Interestingly, the relevant receptors (Notch receptors, Notch1-4) and transmembrane ligands (Notch ligands Delta-like (DI) 1, 3, and 4; Jagged 1 and 2) are located on separate cells. Structurally, Notch receptors have been found to contain an extracellular subunit, called NEC, that has multiple EGF-like repeats. In addition, NEC has three specialized Lin-Notch repeats that interact with a transmembrane subunit known as NTM via hydrophobic mechanisms. Separately, a heterodimerization domain (HD) contains a cleavage site for ‘A disintegrin and metalloproteinase’ (ADAM).
The consequence of ligand-receptor interactions is the breakup of NEC and NTM linkage. Following this NTM becomes susceptible to ADAM cleavage by ADAM 17 or ADAM 10, as it is transendocytosed by the ligand-bearing cell to yield NEXT – the truncated intermediate of NTM. Following this, gamma secretase is able to cleave NEXT to produce notch intracellular domain (NCID). Localization of NCID in the nucleus is the next step where it comes into contact with the core binding factor-1 (CBF-1) and becomes associated with CBF-1 Suppressor of Hairless and lymphocyte activation gene 1 (CSL) and transcriptional co-activators such as Mastermind-like 1, 2, and 3 (MAML-1, -2, -3). At this junction in the Notch signalling pathway a number of transcription events related to CSL-target genes takes places, including the transcriptional regulators hairy and enhancer of split (HES) and hairy/enhancer-of-split related with YRPW motif (HEY) family members, oncogenes c-Myc and p21, and Ephrin B2.
In cancer biology, abnormalities in Notch signalling function results in pro-tumorigenic and pro-angiogenic effect as first observed in T-cell acute lymphoblastic leukemia (T-ALL) where activating mutations of Notch 1 are implicated. As well, Notching signalling overexpression is observed in melanoma, NSCLC, pancreatic, breast, cervical, and prostate cancers, gliomas and B cell neoplasias. For instance, amplification of Notch 1 receptor and Jagged 1 ligand act in combination to down regulate Numb, a negative regulator of Notch signalling that contributes to breast carcinomas. In melanoma, tumor progression and migration is associated with Notch signalling. Specifically, Notch 1 can transform normal melanocytes into tumors while Notch 4 is noted to contribute to be involved in metastatic melanoma. Notch 4 contributes to aggressive tumor behavior in melanoma by positively regulating Nodal expression – a TGF-β superfamily member that is instrumental to tumor cell plasticity and aggression.
Similar to Hh and Notch signalling pathways, the Wnt pathway is critical to embryonic development of a variety of organs and the tissue self-repair process. Similar to the effect of Notch signalling, Wnt signalling plays a role in cell differentiation. Activation of Wnt signalling occurs via the canonical Wnt/β-catenin pathway, as well as non-canonical planar cell polarity pathway, and the Wnt/Ca2+ pathway. However, as it relates to oncology research the Wnt/β-catenin pathway is the most relevant.
Normally, regulation of the Wnt/β-catenin signalling pathway occurs by a large protein assembly called the β-catenin destruction complex that maintains low concentrations of β-catenin in the cytoplasm, and consequently in the nucleus. The β-catenin destruction complex is comprised of glycogen synthase kinase 3 (GSK3α/ GSK3β), casein kinase Iα (CKIα), Axin1/Axin2 scaffolding, and adenomatous polyposis coli (APC) – a tumor suppressor protein. Recruitment of β-catenin to the destruction complex leads to the phosphorylation of β-catenin N-terminus residues by CKIα and GSK3, following which ubiquitination and degradation events occur. Sequestering β-catenin at several N-terminal serine and threonine residues to the β-catenin destruction complex are Axin and APC. Similarly, it is believed these constituents of the β-catenin destruction complex regulate β-catenin efflux from the nucleus. The low concentration of β-catenin in the cell restricts β-catenin to the essential role of cadherin-mediated cell adhesion. An additional mechanism controlling the expression of Wnt signalling in the cell involves the inhibition of Wnt specific gene transcription by the T-cell factor (TCF) family of proteins.
Activation of the Wnt/β-catenin signalling pathway is signified by the formation of a “Wnt signalosome” – a large protein complex that develops following the recognition of Wnt protein on the cell surface by a set of proteins called the seven-pass transmembrane Frizzled (Fz) family and its co-receptor, low density lipoprotein receptor related protein (LRP5/LRP6). The Wnt signalosome inhibits the activity of the β-catenin destruction complex by recruiting several components of the destruction complex to the membrane. As a consequence, the buildup of β-catenin’s unphosphorylated form results in the cytoplasm and subsequently in the nucleus. It is in the nucleus where rising concentrations of β-catenin combine with TCF proteins to transform this protein from an inhibitory protein to an activator of Wnt-signalling pathway by encouraging the transcription of the Wnt-responsive gene.
In colorectal cancer, the Wnt/β-catenin signalling pathway is observed to play a significant contribution to tumorigenesis. Mutations in APC in the familial adenomatous polyposis (FAP) leads to the production of colorectal polyps and raises the risk of tumor development. Similarly, mutations in APC, Axin 1, or β-catenin is known to contribute to colorectal cancer as well, in addition to hepatocellular carcinoma, pancreatic cancer, prostate cancer and medulloblastoma. Other causal factors related to abnormal Wnt/β-catenin signalling pathway functioning includes overexpression of Wnt-ligands, β-catenin, LRP5 or FZD; any of these factors can deregulate the Wnt/β-catenin signalling pathway. As well, downregulation of negative regulators is another opportunity for tumorigenesis to occur.
The role of CSCs has been determined in a select number of hematologic and solid tumors. However, current research has been unable to address the origins of CSCs, define the CSC niche, and explain the regulation of the CSC network. Related to the absence of fully understanding CSCs in tumor biology is the need to better qualify biomarkers of CSCs to develop relevant diagnostic tests, and knowledge of the complexity of cross-talk events with tumor-related non-CSC signalling pathways. Nevertheless, a number of compounds are currently in development to target the Hh, Notch, and Wnt signalling pathways for therapeutic application in cancer treatment.