Abstract
Introduction
Colorectal cancer (CRC) is a complex disease that ranks second in mortality and third in morbidity rates among common malignancies. It is estimated that over 1.9 million new cases occurred in 2020.1,2 A range of risk factors, including physical activity, dietary habits, genetics, and others such as smoking, alcohol consumption, and caffeine intake, may contribute to the development of CRC. The oncogenic factors prompt the epithelial cells of colorectal mucosa to undergo a sequence of transformations, culminating in the development of CRC. This progression involves hyperplasia, dysplasia, adenomas, and ultimately carcinoma.3,4 While surgery and chemotherapy remain the primary treatment modalities for CRC, the prognosis for advanced stages remains unsatisfactory, highlighting the need for more effective therapeutic strategies. By contrast, early-stage CRC often has a more favorable prognosis. Despite the promising initial therapeutic effect of neoadjuvant therapy in advanced CRC patients, the relapse rate remains high, primarily due to drug resistance developed by tumor cells during treatment. 5 Therefore, there is an urgent need to delve into the mechanisms underlying drug resistance in CRC to enhance treatment efficacy and improve patient outcomes.
Cancer drug resistance ensues from cancer cells' continuous adaptation within the tumor microenvironment (TME), giving rise to diverse inhibitory networks and mechanisms that undermine the effectiveness of antitumor therapy. 6 Cell resistance in cancer encompasses tumor heterogeneity, oncogenic mutations, epigenetic modifications, plasticity of cancer stem cells, and tumor-host interactions. 7 After antitumor therapy, residual cancer cells may remain dormant and then reactivate due to adaptive responses, leading to cancer recurrence. 8 Drug resistance in CRC is a complex phenomenon involving altered metabolism, target modifications, aberrant activation of resistance-related signaling pathways, and changes in TME.9-11 Abnormal expression of drug resistance-linked microRNAs (miRNAs) disrupts hormonal signaling, leading to therapeutic resistance. These miRNAs can be targeted to develop anticancer drugs that overcome resistance in cancer progression. 12
Stem cells, characterized by their long-term self-renewal and differentiation capabilities triggered by soluble factors, play a pivotal role in organ and tissue proliferation and regeneration.13-15 Cancer stem cells (CSCs), a subpopulation within tumors, possess these same stem cell traits, but with the addition of resistance to radiotherapy and chemotherapy. 16 Furthermore, a subset of CSCs with metastatic potential develops resistance to anticancer drugs, posing a significant challenge in cancer treatment. 17 Chemotherapy often leaves residual CSCs in tumors, 18 causing rapid tumor regrowth once treatment stops. CSCs rapidly reconstitute the original tumor mass in the absence of drug-induced damage particularly in colon cancer. By conducting serial transplantation in mice, residual CRCs were enriched for colorectal cancer stem cells (CCSCs) with increased ALDH1 activity, potentially conferring resistance to cyclophosphamide. Colon cancer cells produce IL-4, upregulating anti-apoptotic genes and promoting cancer cell survival. These findings suggest a role for ALDH1 and IL-4 in CRC stem cell phenotype and resistance development. IL-4 functions to safeguard colon CSCs and promote their resistance to cell death. The subset of CSCs that manage to survive chemotherapy plays a pivotal role in the recurrence of tumors.19-21 Moreover, the administration of cisplatin (CDDP) contributes to the enrichment of CSCs, potentially through upregulation of the TRIB oncogene and histone deacetylase class I (HDAC) activity, or activation of the NF-κB/TNFα/PIK3CA signaling pathway. 22 The process of chemotherapy and subsequent acquisition of drug resistance are implicated in the proliferation of CSCs. The potential of targeted therapy lies in its ability to effectively eliminate both progenitor cells and CSCs, thereby enhancing clinical outcomes for cancer patients. 23 In CRC, understanding the mechanisms of drug resistance, especially as it pertains to CSCs, is crucial in identifying promising therapeutic targets and ultimately enhancing treatment efficacy.
Mechanisms of Drug Resistance in Colorectal Cancer
Cancer chemotherapy often fails due to drug resistance, a significant obstacle in treatment. Multidrug resistance (MDR) particularly poses a challenge, arising from tumors’ capacity to develop resistance to multiple drugs with diverse origins, structures, and mechanisms. MDR can be either intrinsic or acquired, both contributing to the failure of cancer treatment. The natural resistance of cancer cells to treatment is known as intrinsic drug resistance, whereas acquired drug resistance arises when treated cells gradually become insensitive to drugs.24,25 Resistance in CRC can be attributed to changes in drug efflux, targeting, disrupted apoptotic pathways, TME, CSCs, and signaling pathways.9,26 The main mechanisms of CRC drug resistance are described below (Figure 1). Mechanisms of drug resistance in colorectal cancer. (A) Increased drug efflux. (B) Drug target changes. (C) Apoptosis. A: Antiapoptotic protein. p: proapoptotic protein. (D) LncRNAs can promote CRC drug resistance by regulating certain miRNAs. (E) Extracellular vesicles. (F) Tumor stem cell. sp: signaling pathway.
Induction of Drug Transporter Protein Expression
The absorption, distribution, and efflux of various drugs are modulated by drug transporter proteins, significantly influencing drug efficacy. Notably, drug efflux, enabled by these proteins, promotes the expulsion of substrates and therapeutic drugs from cells, leading to diminished concentrations of antitumor agents within cancer cells and the subsequent emergence of drug resistance.27,28 Transporter proteins, categorized into solute carrier protein (SLC) and ABC types, are key players in drug efflux, with the latter group playing a dominant role. 29 These ABC transporter proteins, including P-glycoprotein (P-gp), multidrug resistance protein (MRP1), and breast cancer resistance protein (BRCP), 30 contribute significantly to drug resistance in colon cancer cells. This resistance is enhanced by the expression of these proteins, which is induced by the IRE1α-XBP1 axis. The upregulation of these transporter proteins by anti-cancer drugs can lead to reduced drug efficacy and increased cancer cell survival. 31
Changes of Drug Targets
Targeted drugs aim to minimize toxicity and side effects in the treatment of CRC. They typically target DNA, cell surface receptors, and other intracellular structures. Modifications to these sites can disrupt the drug’s normal pathway, contributing to drug resistance in CRC.
High expression of thymidylate synthase
DNA replication relies on thymidylate synthase (TS), which catalyzes the conversion of dUMP to dTMP, essential for thymine biosynthesis. 32 As thymine is a fundamental component of nucleic acids and DNA, TS inhibition represents a promising approach for anticancer drug design, particularly given the elevated DNA synthesis activity observed in cancerous cells. The primary mode of action of 5-fluorouracil (5-FU) as an anticancer agent involves the inhibition of TS. A distinctive form of drug resistance associated with 5-FU is characterized by elevated levels of TS expression. 33 TS binds to its mRNA, maintaining a negative feedback loop that suppresses protein translation. 5-FU metabolism generates fluoro deoxyuridine monophosphate (5-FdU), which stably binds to TS, preventing its mRNA interaction. This disrupts the feedback loop, elevating TS protein levels. In vitro studies reveal that a rapid TS surge enhances resistance to 5-FU. 34
Low expression of DNA topoisomerase 1
DNA topoisomerases (DNA TOPOs) alleviate the torsional strain by temporarily cleaving and resealing DNA strands, enabling the resolution of topological challenges associated with the extended nature of human double-helical DNA polymers.35,36 The antitumor activity of irinotecan is enabled by its conversion into SN-38 by carboxylesterase (CES), which inhibits Topoisomerase 1 (TOPO-1), a type I topoisomerase that cleaves only one DNA strand. Furthermore, the resistance of irinotecan against CRC may be associated with the low expression of TOPO-1.37,38
Mutations of epidermal growth factor receptor structure
Epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase (RTK) protein family, sharing a conserved architecture comprising an extracellular structural domain (ECD), a transmembrane structural domain (TMD), a juxtamembrane domain (JMD), a tyrosine kinase domain, and a C-terminal tail. Signal transduction is mediated by diverse ligands, including high-affinity ligands such as EGF, transforming growth factor alpha (TGF-α), and heparin-binding EGF-like growth factor (HB-EGF). 39 Therapeutically, monoclonal antibodies like panitumumab and cetuximab target the EGFR by binding to its ECD, thereby preventing ligand binding and exerting a therapeutic effect. 40 However, ECD mutations can impact cetuximab’s binding, leading to resistance against this EGFR monoclonal antibody.41,42
Alteration of Microtubule Structure
Microtubules, dynamic cytoskeletal fibers made up of tubulin subunits, play a key role in mitotic spindle microtubule formation, making them a prime target for anticancer therapy. Current microtubule-targeting agents, such as paclitaxel and Vinca alkaloids, function by inhibiting microtubule dynamics. Mutations in microtubule structure, altered tubulin expression, and regulation of microtubule-associated proteins can all influence microtubule dynamics. Furthermore, structural changes can affect drug binding, dynamics, and structure, potentially explaining drug resistance mechanisms.43,44
Imbalance of Apoptosis-Regulating Proteins
Apoptosis, the programmed death of cells that eliminate harmful or damaged ones, 45 is the desired endpoint of cytotoxic chemotherapy. Central to apoptosis regulation is the BCL-2 family, whose proteins maintain a delicate balance. Disturbances in this equilibrium, often caused by overexpression of BCL-2 and its homologs, can lead to drug resistance. To improve patient prognosis, new therapies aim to normalize cellular pathways while mitigating mitochondrial activation, thereby restoring apoptotic function. 46
Dysregulation or Mutation of the Signaling Pathways
Cellular signaling, a crucial process that triggers responses between cells, involves various signaling pathways such as Wnt/β-catenin, PI3K/AKT/mTORC1, and JAK/STAT. These pathways play a regulatory role in CRC development.47-49 However, dysregulation or mutations in these pathways can lead to further alterations in other cellular processes, ultimately resulting in drug resistance. CRC progression is typically associated with mutations in the Wnt/β-catenin pathway, regulating tumor stemness and metabolism, thus affecting chemoresistance. 50 Aberrant Wnt signaling plays a significant role in cancer chemoresistance. This resistance arises from various mechanisms such as ligand upregulation of the Wnt pathway, miRNA level changes, transporter gene expression, and β-catenin gene mutations.51,52 Progress in drug resistance often involves Wnt7b modulating the upregulation of multidrug resistance genes like ABCB1 and ABCG2, facilitating drug efflux. Targeting these mechanisms offers potential therapeutic avenues to overcome cancer chemoresistance. 53
Other Resistance Mechanisms
While miRNAs, non-coding RNAs (ncRNAs), regulate crucial cellular processes such as angiogenesis, signaling pathways, and epithelial-mesenchymal transition (EMT) linked to CRC, several miRNAs are emerging as promising therapeutic targets for combating colon cancer, influencing cell proliferation, metastasis, and chemoresistance. 54 LINC00473, a cancer-related Long-strand non-coding RNAs (LncRNAs), enhances CRC drug resistance by suppressing miR-15a, a tumor suppressor miRNA. This downregulation of miR-15a leads to activation of the BCL-2-associated anti-apoptotic pathway, enhancing paclitaxel resistance and inhibiting tumor regression. 55
Extracellular vesicles (EVs) are diverse membranous structures that are secreted by cells. 56 EVs play a significant role in promoting cancer invasion and progression in CRC. These vesicles remodel TME by secreting cytokines, chemokines, and ncRNAs, which enhance tumor growth and drug resistance. EVs also induce apoptosis in cytotoxic T cells (CD8+), further facilitating tumor evasion from immune surveillance. Understanding the role of EVs in CRC progression holds promise for developing novel therapeutic strategies.57,58
CSCs play a crucial role in CRC drug resistance due to their self-renewing abilities. Multiple mechanisms linked to CSC phenotypes could underlie CRC drug resistance.59,60 Understanding CSCs in CRC holds promise for discovering novel therapeutic approaches to treating tumors and their metastases.
Mechanisms of Tumor Stem Cell Drug Resistance
Concept and Origin of Tumor Stem Cells
CSCs exhibit a high level of proliferation and lack of differentiation, originating from genetic mutations or disrupted genetic processes in regular stem or progenitor cells. These cells possess distinct characteristics including markers of stemness, proteins associated with metastasis, drug transporters, and heightened expression of DNA repair mechanisms, which contribute to their involvement in tumor initiation, advancement, invasion, relapse, and resistance to treatment.61-63 Furthermore, the invasive and metastatic capabilities of CSCs may be acquired through the activation of the EMT process. 64 The dynamic transition between stem and non-stem-cell states in certain cancer cells plays a crucial role in influencing the effectiveness of treatments. These cells, often inaccurately labeled as CSCs, possess the ability to switch between states reversibly. This plasticity in stemness is a fundamental factor that impacts therapeutic outcomes. 65 Additionally, the process of chemotherapy-induced cellular senescence, along with the influence of nitric oxide and other soluble signaling factors in TME, further enhances the acquisition of stemness in cancer cells.66,67 CSC was initially introduced by Makino 68 and colleagues in 1956, suggesting that all cancer cells are derived from self-renewing CSCs. This hypothesis offers a plausible explanation for the resistance to treatment and quiescent nature observed in various tumors.69,70 John E. Dick’s research team was the first to identify and characterize CSCs in myeloid leukemia,71,72 demonstrating their ability for self-renewal and differentiation. After years of similar experiments, researchers confirmed the existence of CSCs in CRC and other solid tumors.73,74 Their pivotal role in chemoresistance underscores the urgency of exploring alternative treatment options. Recently, the advancement of CSC models has garnered increasing attention. During the transition from normal colonic epithelial cells to cancer cells, stem cell characterization is crucial. By exploring the similarities and differences between normal stem cells and CSCs, reliable stem cell models can be developed. These models offer a platform for Mechanism-based research and innovative drug discovery, to overcome drug resistance challenges. The development of drug-resistant stem cell models for colon cancer is a crucial area of research, with sulindac (SUL) and 5-FU being commonly used inducers. These models assess the acquisition of stem cell properties by measuring the expression levels of key molecules and markers like CD44, CD133, and ALDH1.75,76
Mechanisms of Tumor Stem Cell Resistance
CSCs possess the ability to trigger cancer development during tumor cell resistance and replenish cancer cells under specific conditions, making them resistant to treatment and contributing to tumor recurrence.77,78 This resistance is linked to quiescence, exaggerated DNA repair mechanisms, suppression of apoptosis, the acquisition of an EMT phenotype, the elimination of reactive oxygen species (ROS), and the upregulation of aldehyde dehydrogenase (ALDH) activity79-83 (Figure 2). Mechanisms of drug resistance in tumor stem cells. (A) Three ecological niches of CSCs in a quiescent state. (B) DNA repair mechanisms: H: homologous reorganization N: non-homologous end joining B: base excision repair R: Reduced programmed cell death activity. (C) EMT phenotype. (D) Scavenging of ROS. (E) Enhancement of ALDH activity. sp: signaling pathway.
The existence of Stationary States
The specialized microenvironments within tumors known as CSC ecotopes support and sustain CSCs. 84 The quiescent nature of these CSCs is closely linked to the hypoxic and perivascular niches within the TME, which foster resistance to treatment. CSCs can thrive in both niches, enabling them to persist and contribute to tumor recurrence and metastasis. The ecological niche at the invasion edge supports CSCs, although the exact role remains speculative.85,86 Under hypoxic conditions, hypoxia-inducible factor (HIF) signaling is activated in the hypoxic zone, leading to increased CSC proliferation by upregulating embryonic stem cell transcription factors such as SOX2, Oct3/4, and Nanog. 87
Overexpression of DNA-Repair Mechanisms
The genetic stability of CSCs is crucial, as DNA damage leads to mutations that affect their survival. 88 Despite this, cancer cells often show resistance to DNA damage therapy, potentially due to their enhanced DNA repair mechanisms, 89 including homologous recombination, non-homologous end joining (NHJ), base excision repair (BER), and decreased activity of programmed cell death. To overcome drug resistance in chemo or radiotherapy, inhibiting at least two DNA repair mechanisms concurrently is crucial. 79
The Acquisition of EMT Phenotypes
EMT is a process that involves the loss of apical-basal polarity and enhanced motility of epithelial cells, leading to increased cell migration and invasion capabilities. 90 EMT plays a crucial role in cancer invasion, metastatic spread, and acquisition of therapeutic resistance. Transforming growth factor-β (TGF-β) induces EMT in cancer pathogenesis by activating signaling pathways in CSCs, such as the Wnt signaling pathway. 91 EMT facilitates the emergence of drug-resistant cancer cells resembling CSCs by inducing stem cell-like properties and overexpressing ABC transporter proteins. Conversely, triggering EMT in a targeted manner can abolish CSC stemness and drug resistance. 80
Removal of ROS
ROS, a chemically reactive byproduct of oxygen metabolism, is produced primarily in mitochondria and peroxisomes. Low ROS levels sustain normal signaling functions, whereas elevated concentrations cause cellular harm.92-94 The stemness marker NANOG plays a crucial role in inhibiting oxidative phosphorylation in CSCs, leading to decreased mitochondrial ROS generation and enhanced fatty acid oxidation. This helps maintain CSC self-renewal and drug resistance. 95 Furthermore, genes involved in ROS scavenging, such as glutathione peroxidase (GPX), are upregulated in breast cancer CSCs, enhancing their resistance to radiotherapy. 96 Hence, maintaining low ROS levels is a significant mechanism underlying CSC drug resistance.
Enhancement of ALDH Activity
Aldehyde dehydrogenase (ALDHs), a type of NADP-dependent detoxifying enzymes, oxidize toxic aldehydes into carboxylic acids, vital for retinoic acid (RA) synthesis in cellular processes. 97 These enzymes play a significant role in CSCs, promoting DNA repair and reducing ROS, thereby contributing to the CSC phenotype. 98
Promotion of Tumor Stem Cells in CRC Drug Resistance
Currently, there’s evidence indicating that cancers in humans, including CRC, can be viewed as stem cell disorders. 99 CCSCs, derived from ISC and transporter-expanded cells, play a significant role in CRC heterogeneity. CCSCs are characterized by unique surface markers like CD133, which plays a vital role in primitive cell differentiation and EMT. 59 CSCs, linked to tumor angiogenesis, are key drivers of CRC growth, metastasis, drug resistance, and recurrence. 100 Hence, personalized CRC therapy should prioritize targeting these cells.
Colorectal cancer stem cells (CCSCs), similar to other tumor stem cells, exhibit aberrant proliferative signaling like Wnt, Notch, and Hedgehog, along with heightened tumorigenicity. Like intestinal stem cells, they maintain self-renewal and differentiation abilities.101,102 Prolonged drug therapy can increase CSCs by 30%, leading to treatment resistance. The dysregulation of Wnt, Notch, and TGF-β signaling pathways in intestinal epithelial cells, along with the upregulation of specific miRNAs in CCSCs, together promote stemness in these cells.
103
TME in CRC offers a protected niche for CSCs, within which matrix components secrete active factors that impact CRC cell and CSC properties via multiple pathways of action.104,105 In recent years, numerous studies have explored these signaling pathways, miRNA aberrations, and the role of the TME in CRC. This review summarizes and synthesizes these findings, aiming to provide valuable insights for the development of effective CRC treatments (Figure 3). Hope that they can help in the treatment of CRC. Mechanisms of cellular resistance in colorectal cancer tumor stem cells. (A) Regulation of signaling pathways represented by the Wnt/β-catenin pathway. (B) miRNA regulation. (C) Tumor microenvironment regulation. ie: expression enhancement. fe: functional enhancement. CCRC-A-M:CCRC-associated-marker.
Promoting Drug Resistance in CCSCs by Modulating Relevant Signaling Pathways
The resistance to treatment in CRC is partly attributed to aberrant signaling pathway activation in CSCs. The key pathways include Notch, Wnt/β-catenin, and Hedgehog (Hh), regulating CSC stemness. Resistance in CRC cells is linked to these signaling pathways and their cross-talk.106,107 Therefore, they also contribute to drug resistance in CCSCs.
Activation of the WNT/β-Catenin Signaling Pathway
The Wnt pathway’s aberrant activation is a pivotal driver in human cancer development, especially CRC. 108 This pathway, which regulates stem cell survival, proliferation, and self-renewal, suggests that disrupting Wnt signaling could halt CSC maintenance.109,110 Real-time visualization of stem cells reveals that more differentiated cells can replace damaged stem cells, reverting to the stem cell niche and regaining stemness, possibly mediated by activated Wnt signaling. 111 The Wnt signaling pathway plays a crucial role in stem cell formation and carcinogenesis, acting to initiate CSCs. CD44 and CD24, surface markers of cancer stem cells, are Wnt target genes. 52 CD44-positive cells exhibit translocation of β-linker proteins to the nucleus, expressing β-linker target genes, whereas CD44-negative cells lack nuclear β-linker proteins. This observation suggests that Wnt signaling may be a critical component in CSC initiation. 112 Although compelling evidence exists, further exploration of the Wnt signaling pathway’s role in CSCs’ development is essential. This pathway, both typical and atypical, plays a pivotal role in cancer’s progression. The typical Wnt pathway regulates β-catenin levels during gene expression. Anomalous activation or genetic alteration of the conventional Wnt signaling pathway results in increased β-catenin activity, promoting tumorigenesis and the sustenance of CSCs.113,114 Nevertheless, the accumulation of β-catenin in the cytoplasm in the absence of Wnt signaling is impeded by the degradation of a complex protein structure comprising adenomatous polyposis coli (APC) and other constituents of this pathway. 115 Notably, mutations in the APC gene are detected in approximately 80%-85% of sporadic CRCs, and the inactivation of this gene serves as a trigger for the onset of colorectal neoplasia. A study has discovered that CRCs exhibiting intact APC genes harbor activating mutations in β-catenin. APC plays a crucial role in maintaining low nuclear β-catenin levels through promoting its export, which disrupts the binding of β-catenin with TCF/LEF and subsequently curbs the transcription of genes implicated in CRC progression.116-118
The Wnt signaling target Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), a CCSC marker, can be stimulated with MEK1/2 inhibitors to elevate LGR5 levels. This enhances CSC quiescence and drug toxicity evasion.119,120 Blocking the Wnt/β-catenin pathway diminishes ABC transporter mRNA expression in CRC cells, thereby reducing protein levels and heightening sensitivity to chemotherapy. 121 Increased glycolysis is observed in cancer cells that are resistant to chemotherapy. The activation of the Wnt/β-catenin signaling pathway is linked to drug resistance as it enhances glycolysis. 122 Stem cells residing in a hypoxic environment stimulate glycolysis through the activation of the Wnt/β-catenin signaling pathway, resulting in the increased expression of solute carrier family 2. 121 The Wnt signaling pathway is crucial in regulating drug resistance in CSCs. By stimulating the expression of Lgr5, ABC transporter proteins, and glycolysis, the pathway enhances CSC drug resistance. Various regulatory factors influence CCSCs via the Wnt pathway, further modulating resistance. Among these, β-catenin’s highly homologous coactivators, CBP and p300, play a significant role. While p300/β-catenin binding fosters CSC differentiation, CBP/β-catenin association preserves CSC potency.123,124 Suppressing miR-148a upregulates CCSC markers via the Wnt/β-catenin pathway, minimizing cisplatin resistance. 114 Through examination of gene expression patterns in CRC patients, it has been observed that the cluster of differentiation 45 (CD45), which is encoded by the protein tyrosine phosphatase receptor type C (PTPRC) gene, is preferentially expressed in CCSCs. The phosphatase activity of CD45 is implicated in enhancing Wnt transcriptional activity by stabilizing β-catenin, thereby promoting stemness and drug resistance characteristics. This mechanism sheds light on how cancer stem cells sustain abnormal activation of the Wnt signaling pathway and suggests a potential therapeutic approach for treating CRC. 125 Additionally, Sec62, a newly identified target associated with chemoresistance in CRC, is a protein-coding gene that also contributes to the stabilization of β-catenin and the promotion of cancer stemness through β-catenin signaling. Targeting Sec62 has the potential to enhance the effectiveness of chemotherapy in CRC patients by inhibiting β-catenin signaling. 126 Furthermore, GANT61, a selective inhibitor of Gli (a transcription factor in the Hedgehog signaling pathway closely linked to CRC pathology), has been shown to simultaneously block the Wnt/β-catenin and Notch signaling pathways in CRC, leading to the elimination of CCSCs. 127 In addition to in vivo gene expression studies, recent research has also explored the effects of bioactive compounds from exotic plants. For instance, Procyanidin has been found to down-regulate surface markers and transcriptional factors associated with stemness in cancer stem cells, as well as inhibit their proliferation through the Wnt/β-catenin pathway. 128 Without mutations, the activation of the Wnt/β-catenin pathway is precluded, affecting APC genes and other Wnt-regulating proteins. Surface markers and β-catenin, however, serve to preserve stemness in CSCs.
Bidirectional regulation of CSCs via the notch signaling pathway
When the Notch signaling pathway is disrupted, it can also foster tumor growth, alongside the Wnt pathway. Notch is involved in diverse cellular processes, promoting survival, proliferation, and suppressing apoptosis. Whether it behaves as an oncogene or tumor suppressor gene depends on the cellular microenvironment.129,130 The discovery of Notch signaling genes in the wing notch of Drosophila melanogaster in 1917 has linked them to leukemia and various cancers, such as CRC. 131 The Notch pathway is bifurcated into canonical and non-canonical subtypes. The canonical Notch pathway stimulates transcription of target genes, retards differentiation, and preserves stem cell properties. Alternatively, the non-canonical Notch pathway emerges from atypical ligands, creating the CSL-NICD-Deltex complex that triggers MAG transcription and enhances differentiation. 132
Notch signaling maintains the properties of CCSCs and inhibits apoptosis by repressing the cell cycle inhibitor p27. 133 JAG2, a significant ligand of the Notch pathway, is utilized to sustain a stem cell-like state. Depletion of JAG2 leads to the inhibition of the stem cell-like marker CD133. 134 Disruption of the PRC2 complex formation in CSCs can have a significant regulatory impact on Notch signaling. 135 Additionally, certain agents have been identified to modulate drug resistance in CSCs through the Notch pathway. Nanotechnology has been employed to transport anticancer medications, enhancing their efficacy and stability. α-Mangostin (αM) exhibits anti-proliferative and apoptotic properties in cancer cells. At low concentrations, αM hinders CCSCs and counteracts the 5-FU-induced promotion of CSCs via the Notch signaling pathway. 136 α-Mangostin nanoparticles (αM-PLGA nanoparticles) impede the self-renewal ability of CCSCs and CRC carcinogenesis through the Notch pathway. 137 Jin 135 and colleague’s study discovered that serine-threonine kinase receptor-associated protein (STRAP) epigenetically controls the Notch pathway, maintaining stem cell-like features of CRC cells by boosting HES1 and transcriptional-level stemness markers. PER3, a circadian clock gene, plays a key role in this process. When overexpressed, it downregulates stemness markers, Notch1, and β-catenin, thus promoting CCSCs’ self-renewal and chemoresistance. 138 Significantly, prolactin (PRL) has the ability to stimulate Notch signaling via the JAK2-STAT3 or JAK2-ERK1/2 pathways, thereby augmenting stem cell activity in 3 colon cancer cell lines. 139 The oncogenic histone cluster 2 H2B family member F (HIST2H2BF) plays a role in upregulating the expression of NICD, an activated form of Notch1, which in turn facilitates downstream gene transcription and the phenotype of CCSCs. 140 There is promise in the utilization of Traditional Chinese Medicine within cancer treatment settings. For instance, Evodiamine, a compound with dual-targeting capabilities against both colon cancer cells and CSC, exhibits inhibitory effects on Wnt and Notch signaling pathways in CSC. 141 These investigations underscore potential novel therapeutic targets for CRC.
Maintenance of CSCs in a Proliferative State by Hh Signaling Pathway
Cancer, particularly colon cancer, is linked to dysregulation of the Hh signaling pathway. This dysregulation arises from mutations in genes related to the pathway or increased expression of Hh molecules, with the difference requiring a ligand for signal transduction in the latter. 142 Despite the rare activation of Hedgehog signaling in CRC due to negative regulation of the Wnt pathway, Hh signaling maintains the undifferentiated, clonally proliferative state of CSCs, serving as a potential foundation for other tumor studies, including CRC.143,144 Moreover, non-classical Hedgehog signaling enhances Wnt gene expression and hinders CSC differentiation in vivo. 145 The extent or rate at which the Hh signaling pathway promotes self-renewal of CCSCs plays a key role in tumor maintenance and expansion. 146 This pathway involves 3 genes: Sonic Hedgehog (SHh), Indian Hedgehog (IHh), and Desert Hedgehog (DHh). 142 A compound known as Physciosporin has been identified as an inhibitor of BCC stemness and ALDH1 expression, which is mediated through the SHH and Notch signaling cascades. ALDH1, a distinct marker for CCSCs, promotes metastasis and proliferation of these cells via various well-established biological pathways.147,148 Gli proteins serve as downstream effectors of the classical Hh signaling pathway, facilitating the transcriptional activation of target genes within this pathway. 142 The distinctive spherical morphology of HCT-116 cells is associated with CSCs, and the administration of cyclobenzaprine, a Sonic hedgehog (Shh) inhibitor, induces or elevates the mRNA levels of CHD1 (a gene expressing E-calmodulin) within the colon cancer spheres of the HCT-116 cell line by suppressing Gli1. 149 Garcinone C has been found to impede CCSCs by interacting with Gli1 and promoting its degradation, resulting in a notable reduction of stemness markers such as CD44, CD133, ALDH1, and Nanog. 150 Conversely, the transcription factor RUNX3 diminishes the expression of Gli1-mediated stemness markers in stem-like CRC cells, thereby suppressing their stemness characteristics. 151
Other Signaling Pathways with Different Mechanisms
In CRC resistance, there exist alternative signaling paths regulating CCSCs. APC and K-Ras mutations enhance CCSC activation, promoting tumorigenesis. High RAS protein levels correlate with β-catenin expression. CDP0857 mitigates CCSC activation by modulating Wnt/β-catenin and Ras/signal-regulated kinase (ERK) signaling. 152 The Radiation-induced PI3K/AKT pathway plays a key role in SOX2-induced CCSC activation, highlighting its therapeutic potential against radiation-activated CCSCs. 153 The JAK/STAT signaling pathway plays a pivotal role in the growth of CCSCs post-radiotherapy (RT). JAK2 overexpression in CCSCs influences CRC cell radio-resistance and stemness via targeted regulation of CCND2, a crucial cell cycle protein.154,155
The signaling pathways connecting the preceding and following elements serve as bridging mediators in CCSCs' drug resistance mechanism. Targeting these pathways to affect CSC properties could provide a viable approach for treating CRC patients.
The Role of miRNAs Regulation in the Drug Resistance Process of CCSCs
MicroRNAs, short noncoding RNAs, play a crucial role in cancer pathogenesis. 156 Their expression in CSCs contributes to colorectal cancer heterogeneity and is linked to uncontrolled proliferation and metastasis. 157 CCSC-related miRNAs primarily target signaling pathways and molecules key for CCSC maintenance and oncogenic functions, often inversely correlating with cellular expression. 158 CCSCs demonstrate bidirectional miRNA expression regulation. Comprehending this phenomenon which is the underlying factors that influence tumor heterogeneity is critical for guiding targeted miRNA drug delivery, eliminating cancer stem cells, and enhancing cancer treatment efficacy, thereby supporting better cancer management. 159
Functions of miRNAs in the CRC and CSC.
Interaction Between Tumor Microenvironment and CCSCs to Regulate Drug Resistance
Tumor microenvironment (TME) serves as a dynamic interaction hub for tumor cells, including vascular components, immune cells, and other elements. 174 As previously documented, CSCs reside in specific niches where a cocktail of cytokines and soluble factors govern their stem-like properties, fueling their self-renewal capabilities.175,176 The maintenance of CSC stemness within the TME shields these cells from immune surveillance while inducing EMT, which promotes their migratory potential and facilitates the establishment of secondary tumors. 177 Activated cancer-associated fibroblasts (CAFs) release IL-11 and TGF-β signals to modify the TME and stimulate CRC progression, consequently inducing resistance to therapeutic drugs.178,179 CAFs facilitate the transmission of exosomes to CRC cells, thereby enhancing the expression of cell stemness markers such as CD133 and CD44. This process results in an elevation in the population of CD133- and CD44-positive CSCs and the initiation of an EMT phenotype in CRC cells. Consequently, this leads to an increased propensity for metastasis and chemoresistance in CRC cells. 180 The study of the relationship between TME and CSCs, which share a similar mechanism of drug resistance, holds significant implications for guiding clinical drug use in CRC resistance.
Phenethyl isothiocyanate (PEITC) has the potential to regulate inflammatory chemokines, thereby inhibiting CSCs and hindering the progression of CRC. 181 Kim 182 and colleagues suggest that the augmentation of CSCs in myofibroblasts is facilitated by the activation of HES1 induced by IL-6 and IL-8 within the TME. CRC is constantly exposed to mechanical stimuli, primarily emanating from compression, matrix stiffness, and hydrodynamic forces. The stiffness of the matrix enhances the potential for distant metastasis in CRCs, altering CCSC characteristics. 183 Elevated YAP activation in CCSCs under stiffness promotes stemness marker expression, such as CD133, ALDH1, and Lgr5, and contributes to chemoresistance and cancer recurrence. 184 Cellular stiffness also affects CRC chemoresistance. Down-regulating CX43 in CRC cells reduces cellular stiffness, enhancing stemness and drug resistance. 185 Resveratrol counters TME-induced CCSC markers, promoting apoptosis and sensitizing CRC cells to 5-FU. 186 SMC1A plays a key role in CRC proliferation, drug resistance, immune microenvironment regulation, and bidirectional targeting switches in CSCs. 187 Innate immune signals from CSCs activate Fusobacterium nucleatum (Fn), leading to a proinflammatory secretory response that promotes a tumorigenic microenvironment while also exerting tumor suppressor effects. 188
The tumorigenesis and progression of cancers within the TME involve complex interactions between tumor cells and mesenchymal cells, leading to the emergence of CSCs, further sustaining the tumor as well as resistance to cytotoxic drugs. 189 These interactions play a significant role in the establishment of drug resistance in CRC, as they are mediated by various components of the TME that together form mechanisms that enhance drug resistance.
Conclusion
CSCs play a crucial role in tumor formation and metastasis, often leading to cancer recurrence and drug resistance. Traditional tumor therapies often lack precision, making it challenging to target specific sites within the drug-resistant process of CSCs. By identifying and targeting these sites, we can improve the efficiency of treatment and overcome resistance to chemotherapy, thus simplifying CRC treatment and enhancing the therapeutic process. CCSCs are key players in drug resistance of CRC, indicating their potential as targets for effective therapy. Elucidating their role in this process could aid in discovering tailored treatment strategies.
This paper highlights the significance of signaling pathways, TME, and miRNA in regulating drug resistance in CCSCs. These signaling pathways primarily act by relaying upstream signals to downstream effectors in the drug resistance mechanism. Regulatory factors play a pivotal role in governing these pathways, indirectly modulating CCSC drug resistance. The central concept of the paragraph is that miRNAs play crucial regulatory roles in CCSCs surface markers and signaling pathways, distinct from signaling pathways in terms of their influence on CRC drug resistance via distinct proteins and processes like gene transcription. The regulation of CCSCs often involves regulatory factors or protein complexes interacting with signaling pathways or alternative routes. These interactions indirectly impact cell stemness through miRNAs, modulating the expression of critical factors. Therefore, a comprehensive understanding of the intricate relationship between miRNAs and regulatory factors is necessary to further explore the underlying mechanism of CCSC drug resistance. The TME is more macroscopic than the aforementioned ones, regulating drug resistance not solely through its internal components, but also by leveraging its innate attributes, including matrix stiffness and the establishment of a tailored microenvironment. Hence, comprehension of the TME’s macroscopic properties and modulation of its component interactions hold key clinical relevance for mitigating drug resistance and enhancing therapeutic outcomes.
In summary, clinical CRC studies have identified relevant targets for therapeutic action. Targeted therapies have significantly boosted CRC patient survival rates, and reducing drug resistance in CSCs holds promise for expanding treatment options. The studies above outline potential targets for clinical CRC interventions. Targeted therapies have already boosted CRC patient survival rates, and reducing drug resistance in CSCs represents a promising avenue for further expanding treatment options. Nevertheless, it is important to acknowledge the heterogeneity of tumors and the ongoing reliance on animal studies in some research. These constraints should be considered when designing new clinical trials, emphasizing the necessity for additional investigation into viable and efficient treatment strategies.
Footnotes
Acknowledgments
We acknowledge the Department of Oncology of Jiangxi University of Chinese Medicine.
Author Contributions
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by National Natural Science Foundation of China (Grant number 82205221, 82160925), Jiangxi Provincial Natural Science Foundation (Grant number 20224BAB206097), Jiangxi University of Traditional Chinese Medicine School-level Science and Technology Innovation Team Development Program Project (Grant number No.CXTD22011) and Jiangxi Provincial Traditional Chinese Medicine Youth Key Talents (the Fourth Tranche) Training Program (Gan Traditional Chinese Medicine Science and Education 2022 No.7).