The main reason for the high mortality rate of malignancy is the lack of effective treatments to overcome cancer radio- and chemo-resistance (1–3). Studies on the cell cycle suggest that resistance to therapy can be categorized into two types: primary resistance and acquired resistance (4, 5). Some sub-populations of tumor cells exhibit deficiencies in sensitivity to therapy depending on their specific cell cycle phase. Other sub-populations may adopt various DNA repair mechanisms or rely on faulty checkpoints to reverse or escape DNA damage by cancer therapies. Cancer stem cells (CSCs), a minority of cancer cells that can self-renew, are considered the main cause of tumor occurrence, growth, metastasis, and recurrence (6–8). CSCs inherently resist therapy due to their slow cycling, anti-apoptotic mechanism, effective DNA repair systems, and persistent stemness features (9–11). CD133 is one of the many molecules marking cancer stem cell resistance (12, 13).

CD133, or prominin-1, is a 120 kDa pentaspanning transmembrane glycoprotein, composed of five transmembrane domains, two large N-glycosylated extracellular loops, an extracellular N-terminal domain, and a cytoplasmic C-terminal domain (14). It is predominantly localized to the cell membrane, particularly within cholesterol-enriched microdomains associated with membrane protrusions (15). The extracellular N-terminal region and the two large loops are exposed to the cell exterior, whereas the C-terminal domain and two smaller loops are cytoplasmic, where they interact with the cytoskeleton to regulate cell morphology and migration (16, 17). Following glycosylation in the endoplasmic reticulum and Golgi apparatus, CD133 is integrated into the cell membrane, where it plays a crucial role in cellular signaling (18–20). Multiple transcript variants encoding distinct isoforms of this gene have been identified (21–23). Mutations in CD133 have been implicated in retinitis pigmentosa (24–26) and Stargardt disease (24, 26). It is well-known as a cell surface biomarker that allows for the recognition and sorting of stem cells from tissues or organs (27). CD133 is also a distinguishing CSC biomarkers in malignant tumors. Most CSCs markers, such as CD24, CD44, and CD133, are located in lipid rafts. CD133 is positive for CD29, CD44, CD73, and CD90. The two most important biomarkers associated with CSC are CD44 and CD133, which play important roles in diagnosis, treatment, and prognosis. However, CD133 and CD44 are differentially expressed in various solid tumors. While CD133 is often expressed in specific histological tumor types and is associated with higher tumor levels, the expression of CD44 is not related to tumor histology or tumor grading, making CD133 more clinically relevant than CD44 (28, 29).

Most research now has primarily focused on using CD133 to identify CSCs rather than understanding how CD133 operates and which pathways it may influence for optimal treatment approaches. This has sparked a debate about its role in cancer biology. Beyond its role as a reliable marker for identifying CSC populations, accumulating evidence suggests that CD133⁺ cancer cells are responsible for tumor initiation and radio- and chemo-resistance (30–32). Some studies have found higher resistance in cancer cells expressing CD133, with a growing consensus regarding the involvement of CD133 in tumor resistance. However, the exact mechanism by which above situation occurs remains unclear, with various possibilities (33, 34). It is widely accepted that cell cycle arrest can contribute to radio- and chemo-resistance. The cell cycle is the process of cell division. It is regulated by a family of proteins known as cyclin-dependent kinases (CDKs), which include CDK1, CDK2 and Cyclin dependent kinase 4/6 (CDK4/6), and cyclins (CCNs) A, B, D and E. These kinases are positively regulated by CCNs and negatively controlled by cyclin-dependent kinase inhibitors (CDKIs), such as P16, P15, p18, P19, P21, P27Kip1, and p57 (35). CDKs, their activated CCNs, and CDKIs together regulate the progression and completion of the cell cycle. Most notably, CDKs and their upstream regulatory pathways, such as PI3K/AKT, mitogen activated protein kinase (MAPK), WNT/β-catenin, Notch, and others (36–41), play key roles in the mechanism of treatment resistance (42). These transduction pathways are complex and interfere with each other (43–45). Even if the same signal transduction pathway is regulated, different tumor cells exhibit different cell cycle arrests, and the impact on CD133 or CD133 affecting them also varies.

This study examines the molecular mechanisms through which CD133 regulates the cell cycle and its contribution to radio- and chemo-resistance in cancer stem cells. A comprehensive literature search was performed using PubMed and Web of Science, employing keywords such as PIK3/AKT, MAPK, WNT/β-catenin, Notch, and miRNAs, among others. The search was restricted to publications from the past 20 years and utilized both electronic and manual retrieval methods. A total of 414 publications were identified, of which 124 were included in this systematic review. We summarize the earlier research on CD133 as they are crucial for understanding its novel roles, and describe the latest evidence proving that this unique molecule is involved in signal pathway, which affect various aspects of cellular homeostasis and cancer development.

Phosphatidylinositol-3-kinase (PI3K) is a family of proteins that catalyze transfer of the γ-phosphate of ATP to the D3 position of phosphoinositides. AKT is the primary mediator of PI3K-initiated signaling. Regarding cell cycle progression and cell growth, several targets of AKT are involved in protein synthesis, glycogen metabolism, and cell cycle regulation. As shown in Figure 1, these targets include the mammalian target of rapamycin (mTOR), glycogen synthase kinase-3 (GSK3), and the P21 and P27Kip1. AKT regulates the G₁/S cell cycle transition through the inactivation of GSK3-β, which up-regulates Cyclin D1 (CCND1). Akt phosphorylated and inhibited tumor suppressor tuberin (TSC2), ultimately leading to a decrease in P27Kip1 (46–49). After years of research, the signaling pathways regulating the cell cycle via PI3K/AKT and mTOR are well understood.

CD133 can regulate the PI3K/Akt and MAPK pathways. Silencing CD133 suppresses PI3K/Akt and MAPK signaling pathways, leading to the downregulation of downstream targets, including RAF1, MAP2K1, MAPK3, PIK3CA, AKT3, mTOR and c-MYC, and induces cell cycle arrest at the sub-G₁ phase (50–52). Based on this relationship, new inhibitors, arylidene derivatives, and Chinese herbal extracts have been designed to interfere with the cell cycle through these pathways, reducing the resistance of cancer stem cells. For instance, siRNA targeting midkine (a tumor promoting factor, MDK) (iMDK) (53) induces cell cycle arrest at the S and G₂/M phases, decreases p-Akt levels, and significantly up-regulates PTEN expression in CD133-positive population of prostate cancer cells (53). Additionally, a novel arylidene derivative, IOX-101, has been shown to deactivate Akt/mTOR/NF-κB signaling and increase the sub-G₀ phase in the cell cycle of CD133⁺ A549 cells (54). Novel derivative of matrine (MASM), suppresses the PI3K/AKT/mTOR pathway, induces cell cycle arrest at G₀/G₁ phase, and reduces the population of CD133⁺ cancer cells (54). Bufalin inhibits PI3K/AKT pathway, reduces the expression of multiple stemness-associated proteins, including OCT4, Sox2 and the stem cell-surface marker proteins CD133, and induces cell cycle arrest at the G₂/M phase in gallbladder cancer cells (55).

In recent years, new pathways involving CD133 and PI3K/AKT have been discovered, as illustrated in Figure 1. For example, glucose-regulated protein 78 (GRP78), belonging to the HSP70 family, translocates and anchors at the lung cancer cell surface membrane by binding to the ER-cochaperone MTJ1. The GRP78/MTJ1 complex and CD133 are concomitantly increased in senescent H460 cells after treatment with cisplatin (DDP). The total GRP78 protein level is downregulated, while MTJ1 and downstream regulator p-AKT/AKT ratio are up-regulated (56). Additionally, CD133 physically interacts with the epidermal growth factor receptor (EGFR) in pancreatic cancer cells. CD133 appears to function as an activator of EGFR, with CD133-EGFR interaction specifically inducing the activation of PI3K/AKT downstream signaling pathways (57). Studies also show that PI3K/AKT signaling acts as an upstream signal of Glioma-associated oncogene homolog 1 (GLI1), and the upregulation of GLI1 can increase CD133 expression (58).

The mitogen-activated protein kinase (MAPK) family in vertebrates includes p38, extracellular signal-regulated kinase (59), and c-Jun NH2-terminal kinase (JNK). Each MAPK signaling pathway consists of a MAPK kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK. These signaling pathways orchestrate numerous cellular processes, including cell growth, survival, differentiation, and apoptosis (52, 60–62). In the context of the relationship between MAPK and the cell cycle, some anticancer drugs are used to inhibit the progression of resistant cancer cells. For instance, Cedrol, a sesquiterpene alcohol, can reduce drug resistance proteins and CD133 expression in glioblastoma cells, causing cell cycle arrest at the G₀/G₁ phase, whereas temozolomide (TMZ)-treated cells are arrested at the G₂/M phase. Combination treatment induces cell cycle arrest at the G₀/G₁ phase at 6–24 h and at the G₂/M phase at 48 h. The expression of TP53 and P21 is increased, whereas that of CDK4, CCND1, CKD2, Cyclin A (CCNA), and Cyclin B1 (CCNB1) are decreased via regulation of the AKT and MAPK signaling pathways (63). Berberine can attenuate CD133 and potentiate G₀/G₁ cell cycle arrest by inhibiting PI3K/AKT and MAPK/ERK signaling and subsequently upregulating p38-MAPK in neuroblastoma cells (64) as showed in Figure 1.

The relationship between CD133 and p38-MAPK involves cell cycle regulation, as illustrated in Figure 1. Cyclophilin A (CypA), belonging to the immunophilin family, catalyzes the isomerization of peptidyl–prolyl bonds. Secreted CypA can bind to CD147, and forced down-regulation of CypA expression by natural inhibitors can inhibit CD133 expression and induce cell cycle arrest at the G₀/G₁ phase. This is associated with the regulation of CypA/CD147-mediated AKT and MAPK signaling pathways (65, 66). Platelet-derived growth factors (PDGFs) include five dimeric forms derived from pairs of A, B, C, and D peptide chains. PDGF dimers activate two specific receptors: PDGFRA, which binds the A, B, and C chains, and PDGFRB, which binds the B and D chains. PDGF receptors (PDGFRs) A and B are transmembrane glycoproteins that belong to the type III receptor tyrosine kinase family. This family also includes KIT, FLT3, and c-Fms1 (67). Stem cell factor is a dimeric molecule that exerts its biological functions by binding to and activating the receptor tyrosine kinase c-Kit. Data indicate that knockdown of PDGFRB and c-Kit expression in glioblastoma multiforme cells leads to inhibited CD133 expression and a normal-like cell cycle distribution driven by the MAPK/ERK signaling pathway rather than the PI3K/AKT pathway (68). MAPK can inhibit CD133 expression, whereas CD133 overexpression significantly activates Erk. The mechanism involves exogenous CXCL3, which induces Erk1/2 phosphorylation and further promotes CD133 expression. Sustained ERK signaling facilitates G₂ cell cycle exit and primes cells for whole-genome duplication.

WNT/β-catenin signaling is known to control many aspects of cell behavior, including a direct link with the cell cycle machinery. An aberrant WNT signaling pathway is associated with a wide array of tumor types and plays an important role in the drug resistance of CSCs (69–71). The relationship between CD133, WNT, and the cell cycle is shown in Figure 2. Enhancer of zeste homolog 2 (EZH2) is highly expressed in colorectal cancer stem-like cells. CD133 regulates the cell cycle in colorectal cancer stem-like cells by activating the WNT/β-catenin pathway via EZH2, which upregulates P21cip1 and promotes G₁/S phase arrest. EZH2 knockdown significantly reduces the CD133⁺/CD44⁺ subpopulation (72). The upregulation of glycosyltransferase 8 domain-containing 1 (GLT8D1) in cancerous cells, induced by hypoxia or HIF-1α, is associated with more aggressive disease in gliomas. GLT8D1 knockdown promotes cell cycle arrest at the G₂/M phase and cellular apoptosis in glioma stem cell. GLT8D1 impedes CD133 degradation through the endosomal-lysosomal pathway by N-linked glycosylation and protein-protein interactions. By directly blocking the GLT8D1/CD133 complex formation or inhibiting GLT8D1 expression, WNT/β-catenin signaling-dependent tumorigenesis is suppressed (73). The WNT signaling pathway inhibitor XAV939 significantly decreases the percentage of cells in the G₀/G₁ phase and increases apoptosis induced by 5-fluorouracil (5-FU)/DDP, accompanied by altered protein expression levels of β-catenin and Axin (74). Another small molecule inhibitor of WNT/β-catenin pathway, FH535, represses the expression of CD133, significantly decreasing the proportion of colon cancer cells in the S phase while increasing those in the G₀/G₁ phase, and effectively down-regulating target genes including CCND1 and survivin (75).

The Notch pathway is implicated in cell fate allocation and cell proliferation. Depending on the context, Notch signaling can either inhibit (76–78) or promote (79, 80) cell cycle progression. Notch target genes include key cell cycle regulators such as CCND1, P21, and MYC (81–85).

The role of Notch in CD133⁺ cell cycle is illustrated in Figure 2. Notch blockade suppresses the expression of HES1 and CD133 expression (86). Notch signaling is expressed in both CD133⁺ and CD133⁻ lung cancer cells. However, Notch1 and Notch2 are less expressed in CD133⁺ cells, causing cell cycles arrest at the G₀/G₁ phase spontaneously. This is because HES1 is expressed in CD133⁻ cells but not in CD133⁺ cells, indicating that the effect of Notch on CD133 is mediated by HES1 (45). Additionally, reducing the expression of transient receptor potential melastatin-related 7 (TRPM7), a non-specific divalent cation channel with a functional serine/threonine-protein kinase domain at its C-terminus, slows cells progression into certain phases of cell cycle (S and G₂/M) and encourages apoptosis. TRPM7 expression is positively linked to Notch1 signaling activity and CD133 expression. Kinase-inactive mutants of TRPM7 result in reduced activation of Notch1 signaling and decreased CD133 expression compared to wild-type TRPM7 (87). In pancreatic cancer, leptin (LEP) increases CD133 expression, the number of cells in the S phase, and overall progression and proliferation through increased expression of Notch receptors, ligands, and target molecules (NOTCH1-4, DLL4, JAG1, BIRC5 [survivin], and HEY2). LEP regulation of Notch may be related to Ras mutations (88). In addition, elevated ALDH1A1 expression prolongs the G₁ stage of the cell cycle and inhibits the cell cycle progression by suppressing the Notch/CDK2/CCNE1 in lung adenoma stem cells (89).

miRNAs have been implicated in maintaining the CSCs phenotype through their ability to influence the expression of genes and proteins that regulate cell proliferation and/or cell death (90–92). Crucial miRNAs impacting the cell cycle and CD133 are shown in Figure 3. miR-142-3p, miR-374a, and miR-134-3p are correlated with CD133 expression. A study has found that miR-150 expression is significantly up-regulated in CD133⁺ cancer cell subgroups (93). miR-150 interacts with the 3'UTR of c-MYB and P27^Kip1 mRNA (93), leading to the down-regulation of c-MYB and P27^Kip1 protein levels and inducing G₀/G₁ phase arrest. There is an interaction between CD133 mRNA and miR-142-3p (94). Transfection of miR-142-3p and miR-150 down-regulated CCND1 expression and induced G₁ phase cell cycle arrest (94, 95). High expression of miR-374a down-regulated CCND1 and CDK4 protein expression, and increased the number of cells in the G₂/M phase in CD133⁺ human glioblastoma stem cells. Additionally, Expression of miR-134-3p in human ovarian cancer stem cells not only inhibited the expression of RAB27A but also induced G₀/G₁ phase arrest (96, 97). The study demonstrated that miR-95 expression was significantly higher in the ALDH1⁺CD133⁺ subpopulation (98). Both miR-363-3p and miR-95 can up-regulate the level of CD133 expression, leading to induction of S phase cell cycle arrest. However, the study suggested that miR-363-3p was significantly down-regulated in CD133⁺ larynx cancer stem cells (99). Overexpression of HIF-2α significantly increased levels of miR-363-3p, and the expression of CD133 and HIF-2α is positively correlated (100). The target gene of miR-363-3p is P21, a key inhibitor of S-phase DNA synthesis and cell cycle progression (101). miR-95 negatively regulates DUSP5, inhibits MAPK pathway activation, and increases proportion of gastric cancer cells in the S phase. Curcumin treatment induces high miR-145 expression and inhibits the expression of lncRNA-ROR. Both lncRNA-ROR and Oct4 mRNA contain miR-145 binding sites and directly compete for miR-145, leading to curcumin-induced inhibition of CCND1, CDK4 and CD133 expressions, and G₂/M phase arrest (102). The transcription factor Oct4 significantly increases wild-type CCND1 promoter activity. There are also other RNAs involved. CD133⁺ tumor stem cells exhibit up-regulated expression of N6-methyladenosine (m6A) mRNA and its writer METTL3. METTL3 enhances the stability of PARP1 by mediating m6A modification of PARP1 mRNA and recruiting YTHDF1 to its 3'-untranslated region (3'-UTR), exerting the DNA damage repair ability of PARP1. Knocking down METTL3 arrests the cell cycle in the G₂/S phase (103, 104). The up-regulation of circRNA-ABCC1 (circ-ABCC1) contributes to the colorectal cancer cell stemness, spheroid formation, and metastasis of CD133⁻ colorectal cancer cells. The interaction between β-catenin and circ-ABCC1 can activate WNT/β-catenin and promote the progression of colorectal cancer (105).

Other pathways involved in CD133 and cell cycle regulation are discussed in Figure 4.

Therapeutic resistance remains a critical challenge in cancer treatment, largely driven by the persistence of CSCs, which exhibit intrinsic resistance to conventional therapies. Among CSC markers, CD133 has emerged as a key player, closely associated with mechanisms of therapy resistance. However, its molecular and biological functions remain poorly defined, fueling ongoing debate. Current evidence links CD133 to the regulation of cell cycle dynamics and therapy resistance, particularly in radio- and chemo-resistant tumors. Recent studies have illuminated the connection between CD133 and key signaling pathways, including PI3K/AKT, WNT/β-catenin, and Notch, which govern cell cycle checkpoints and tumor progression. CD133⁺ cells exhibit distinct mechanisms of cell cycle arrest, influenced by the genetic context of the tumor, underscoring the complexity of its role in therapy resistance. Targeting CD133 has been shown to disrupt oncogenic signaling and enhance the sensitivity of CSCs to therapeutic interventions, identifying CD133 as a compelling target for overcoming resistance. Emerging research highlights the potential of CD133-targeted therapies combined with radio- or chemotherapy to enhance treatment efficacy. By modulating the cell cycle and promoting CSC proliferation, these strategies may increase CSC susceptibility to therapeutic agents. Furthermore, novel drugs targeting CD133 and its signaling networks show promise in addressing radio-resistance, a major barrier in cancer treatment. Despite these advances, current findings remain fragmented, with unresolved questions about the interactions between CD133 and key cell cycle regulators, such as cyclins, CDKs, and checkpoint proteins. Future studies should focus on delineating the precise mechanisms by which CD133 regulates the cell cycle and contributes to therapy resistance. Such insights are vital for developing combinatorial approaches integrating CD133-targeted agents with standard treatments to overcome CSC-mediated resistance and improve therapeutic outcomes. A deeper understanding of these mechanisms will provide critical guidance for optimizing cancer treatment strategies.