B-Myb is expressed at high levels in several human malignant tumor tissues (Table I). For example, there are five molecular subtypes of breast cancer, basal-like, human epidermal growth factor receptor 2 positive (HER2+)/estrogen receptor negative (ER-), luminal A, luminal B and normal-like (7). Microarray analyses of B-Myb in breast cancer tissues have demonstrated that its expression levels significantly differ among the five subtypes, with the highest level in basal-like tumors (7). Furthermore, overexpression of B-Myb has been observed in 83% of primary tumors and all cell lines of non-small cell lung cancer (NSCLC) (8), and B-Myb has also been demonstrated to modulate cell cycle and proliferation (15). Frau et al (9) assessed B-Myb mRNA and protein expression levels across different stages of hepatocarcinogenesis and demonstrated that B-Myb expression levels were substantially higher in precancerous lesions, early proliferative nodules, advanced proliferative nodules and hepatocellular carcinoma compared with normal liver tissues. In addition, the highest levels of B-Myb were observed in hepatocellular carcinoma, among the four different stages. Nakajima et al (16) reported amplification of the B-Myb gene copy number in 36/66 cases of primary hepatocellular carcinoma.

In the absence of gene amplification, malignant tumors of the prostate exhibit elevated B-Myb expression levels, and B-Myb expression is markedly higher in metastatic prostate cancer compared with non-metastatic prostate cancer (11,17). Based on a study involving 180 patients with colorectal cancer, B-Myb mRNA and protein expression levels were notably higher in cancer tissues compared with adjacent normal tissues, and B-Myb expression was positively associated with tumor size and clinical stage (18).

Qin et al (19) demonstrated that B-Myb is amplified in esophageal cancer, based on whole-genome sequencing (10 pairs) and whole-exome sequencing (57 pairs) of esophageal cancer tissues and matched adjacent normal tissues selected from high-incidence areas of esophageal cancer in China. Additionally, Qin et al (20) reported that B-Myb protein was expressed at higher levels in esophageal squamous cell carcinoma (ESCC) tissues compared with adjacent normal tissues in a Chinese population of 107 patients with ESCC, based on immunohistochemistry.

Among patients with neuroblastoma, individuals with malignant metastasis and poor prognosis express B-Myb at significantly elevated levels; this phenomenon suggests that B-Myb expression is associated with the risk of developing neuroblastoma (21). Notable, inhibition of B-Myb expression prevents the proliferation of normal human cells and cancer cells (21–24). In addition, B-Myb expression levels are significantly higher in glioma tissues compared with adjacent normal tissues and are positively associated with the grade of glioma, based on the results of reverse transcription-PCR (RT-PCR) and western blot analyses from 79 patients with glioma (25).

In renal cell carcinoma, metastatic tumor tissues highly express B-Myb when metastasis occurs in primary tumors negative for B-Myb expression (26). B-Myb is also amplified in high-grade bladder cancer (27). In addition, high B-Myb expression has been implicated in leukemias (28), gallbladder cancer (29), fibrosarcoma (30), ovarian cancer (12) and aggressive T-cell lymphoma (31).

Increasing evidence suggests that B-Myb is overexpressed in different types of human cancer, including breast cancer (32), cervical cancer (33), colorectal cancer (18), liver cancer (34), leukemia cells (28) and lung cancer (35). In these types of cancer, B-Myb promotes cell proliferation and/or cell cycle progression (18,34). Thomas et al (36) observed a positive correlation between B-Myb mRNA expression and Ki-67 proliferation index in breast cancer. In addition, the breast cancer cell line, MDA-MB-231, exhibits remarkably decreased abilities to form colonies, migrate and invade following knockdown of B-Myb with short-hairpin RNA (37). Flow cytometric analysis demonstrated that the cell cycle is arrested at S and G₂/M phases, while in vivo experiments indicated that both the rate of tumor formation and the weight of tumor mass are significantly lower in breast cancer compared with the control group (32).

Jin et al (13) demonstrated that overexpression of B-Myb promotes the proliferation of NSCLC cells; both extracellular regulated MAP kinase (ERK) and phosphorylated-protein kinase B (Akt) signaling pathways participate in the modulation of NSCLC by B-Myb. Liang et al (29) reported that B-Myb expression is upregulated in gallbladder cancer tissues, which in turn facilitates the proliferation of gallbladder cancer cells by facilitating cell cycle progression through the S and G₂/M phases (30).

In ESCC, an EdU-retention assay demonstrated that downregulation of B-Myb expression decreases the DNA synthesis ability of EC9706 cells, while a Cell Counting Kit-8 assay demonstrated that overexpression of B-Myb also promotes the proliferation of KYSE510 cells (20). These findings suggest that B-Myb can promote the proliferation and DNA synthesis of ESCC cells. Based on a colony formation assay, overexpression of B-Myb in the low-grade glioma cell line, Hs683, considerably increased the number of colonies, whereas knockout of B-Myb in the high-grade glioma cell line, U251, decreased the number of colonies (25). Subsequently, a MTT assay demonstrated that the proliferative ability of glioma U251 cells is enhanced following transfection with small interfering (si)RNAs (25).

B-Myb has been demonstrated to play a role in EMT, a process whereby epithelial cells lose their polarity, migrate and increase polarity (38). In breast cancer cells, downregulation of B-Myb expression can recover the expression of the epithelial marker, E-cadherin and promote the formation of intercellular adhesion, in addition to inhibiting cell invasion, anchorage-dependent growth and tumor formation (32). Conversely, overexpression of B-Myb can decrease E-cadherin expression and increase the expression of mesenchymal markers (14). In addition, it has been demonstrated that B-Myb can upregulate the expression of Snail, a key regulator of EMT, thereby mediating the promotion of EMT and cancer cell invasion (14).

The role of B-Myb in colorectal cancer invasion and metastasis has also been proven and is associated with EMT (18). For example, EMT inhibits B-Myb activity in colorectal cancer cells, thereby upregulating the expression of the epithelial marker, E-cadherin and downregulating the expression of the mesenchymal marker, Vimentin and matrix metalloproteinase 9 (MMP9) (18). Based on a western blot assay on the expression of EMT markers in glioma cells, interference with B-Myb expression inhibits the protein expression levels of N-cadherin, Vimentin, MMP2 and MMP9, while upregulating the protein levels of E-cadherin and zinc finger E-box binding homeobox 1 (25). Taken together, these findings suggest that B-Myb plays an important role in the promotion of EMT in several malignant tumors, thereby facilitating cancer cell infiltration and metastasis.

B-Myb inhibits cancer cell apoptosis potentially through multiple pathways. First, B-Myb may perform its anti-apoptotic function by positively regulating the expression of the anti-apoptotic gene, Clusterin (30), also known as apolipoprotein J (39). A study demonstrated that inhibition of Clusterin gene expression promotes the apoptosis of fibrosarcoma cells (30). Secondly, B-Myb may inhibit apoptosis by positively regulating the expression of the anti-apoptotic gene, Bcl-2 (40), a critical regulator of apoptosis (41). In support of these pathways, the anti-apoptotic function of B-Myb has been observed in different cancer cell lines, including colorectal cancer (18) and liver cancer (34).

Ren et al (18) reported that Bcl-2 protein expression is downregulated in SW480 colorectal cancer cells via interference with B-Myb expression. Calvisi et al (34) demonstrated that B-Myb exerts an anti-apoptotic function, and interference with B-Myb expression via transfection with siRNA induces apoptosis in four different hepatocellular carcinoma cell lines. In glioma U251 cells transfected with B-Myb siRNA, Zhang et al (25) detected an increase in the percentage of apoptotic cells by Annexin V-FITC/PI and hochest 3342 staining. Furthermore, Zhang et al (25) assessed the effects of B-Myb silencing on apoptosis-related proteins, including caspase-3/9, Bcl/Bax, PTEN and P53. Western blot analysis demonstrated that B-Myb silencing decreases Bcl-2 expression, while increasing the expression levels of Bax, PTEN and P53, and activates caspase-3/9 activity. Taken together, these results suggest that downregulation of B-Myb can induce apoptosis in glioma cells.

The role of B-Myb in tumor resistance to therapy is associated with its pro-apoptotic function (42,43). Overexpression of B-Myb promotes the expression of the anti-apoptotic gene, Bcl-2 in IL2-dependent murine CTLL-2 cells, thereby increasing the therapeutic resistance to drugs, including doxorubicin, ceramide and dexamethasone (42). Similarly, Levenson et al (43) demonstrated that B-Myb expression is notably upregulated in fibrosarcoma cells with therapeutic resistance induced by chemotherapeutic drugs that inhibit DNA synthesis, including hydroxyurea, cysteine, etoposide and adriamycin. Furthermore, B-Myb regulates the expression of the anti-apoptotic gene, Apolipoprotein J/Clusterin in neuroblastoma cells to resist apoptosis caused by doxycycline (30).

Sottile et al (44) reported that individuals with B-Myb overexpression or MYCN amplification are more sensitive to therapy with camptothecins (irinotecan and topotecan) among patients with neuroblastoma. B-Myb is a downstream target of MYCN; MYCN amplification promotes B-Myb overexpression, while B-Myb overexpression in turn promotes an upregulation of MYCN expression, thus these two factors regulate each other (21). Camptothecins selectively downregulates B-Myb and MYCN expression, while upregulation of B-Myb decreases the killing effect of camptothecins, suggesting that B-Myb is an important target of camptothecins (44). In addition, B-Myb is overexpressed in cetuximab-resistant NSCLC, suggesting that B-Myb overexpression is associated with cetuximab resistance (45).

High B-Myb expression is associated with tumor growth and poor prognosis of patients, making it a potential clinical marker for poor prognosis (20,46). Based on microRNA (miRNA/miR) prediction and RT-PCR analyses, B-Myb may have a negative regulatory association with miR-30 family members, and biochemical relapse-free survival time is shortened in patients with acute myeloid leukemia highly overexpressing B-Myb; thus, B-Myb can be a predictive marker for the prognosis of patients with acute myeloid leukemia (46). In addition, B-Myb expression is elevated in ESCC tissues and negatively associated with postoperative overall survival in patients with ESCC, as revealed by a Kaplan-Meier analysis (20).

A prognostic analysis of breast cancer and its different subtypes revealed that the B-Myb high expression group has a worse prognosis compared with the low expression group (7). High B-Myb expression also increases the risk of poor prognosis, decreases the differentiation ability of cells, and promotes tumor development in neuroblastoma (47). In HepG2 and HuH7 hepatocellular carcinoma cell lines, overexpression of B-Myb increases the cell proliferative ability and facilitates G₁-S and G₂-M transitions, whereas interference of B-Myb with siRNA results in cell cycle arrest at G₀-G₁ and G₂-M phases (9).

Among patients with colorectal cancer, the 5-year survival rate is notably lower in individuals with high B-Myb expression than those with low B-Myb expression; B-Myb expression and clinical stage of the tumor can be used as independent prognostic factors of colorectal cancer (18). With regards to the prognosis and survival of 79 patients with glioma, Kaplan-Meier analysis and log-rank test results indicated that high B-Myb expression is negatively associated with survival, and is a poor prognostic factor in patients with glioma (25).

B-Myb plays a role in the clinical diagnosis and treatment of malignant tumors. B-Myb can be used as a biological marker of cervical cancer to make up for the limitations of conventional cytology for cervical intraepithelial neoplasia and cervical cancer diagnosis in routine cervical cytology testing (43,44). Astbury et al (48) measured MYBL2 expression levels in cervical cancer cell lines, cervical intraepithelial neoplasia and cervical glandular epithelium using genomics and proteomics technology, and assessed the potential of B-Myb as a biomarker. Previous studies (47) have demonstrated that patients with neuroblastoma, with MYBL2 overexpression, are more sensitive to camptothecin therapy (44). Camptothecin drugs can selectively downregulate the expression of B-Myb. Conversely, upregulating B-Myb can decrease the killing effect of camptothecin drugs (44). Collectively, these results suggest that B-Myb is an important target of camptothecin drugs (44).

miRNAs are a group of small non-coding single stranded RNAs that have been extensively observed in animals and plants, typically 18–22 nucleotides in length (49). Both misregulation and mutation of miRNAs can cause tumorigenesis, and they contribute to the function of oncogenes through targeted downregulation of tumor suppressor genes or activation of oncogene transcription factors (50,51). B-Myb is mainly regulated by the miR-29 and miR-30 families (52). In breast cancer cell lines, miRNA binds to the 3′-untranslated region of B-Myb and thereby inhibits B-Myb expression (53). Additionally, miR-29a expression is negatively correlated with B-Myb expression, and cyclin A2 and cyclin D1 expression is positively correlated with B-Myb expression (54). These results indicate that miR-29a suppresses tumor growth by downregulating B-Myb expression (54).

Geng et al (55) reported that miR-30a expression is significantly downregulated in NSCLC tissues compared with adjacent normal tissues, and B-Myb is a target gene of miR-30a based on a double-luciferase reporter gene assay; miR-30a can suppress proliferation and growth of NSCLC through targeted inhibition of B-Myb expression. Li et al (56) demonstrated a close association between B-Myb overexpression and low levels of miR-30a, miR-30b and miR-30c in 291 patients with acute myeloid, based on RT-qPCR analysis of B-Myb, miR-29 family and miR-30 family genes. Li et al (56) reported that the long non-coding RNA, LINC01139, upregulates B-Myb by competitively binding to the miR-30 family, thereby promoting the progression of hepatocellular carcinoma.

Zauli et al (28) demonstrated that the cell cycle is arrested at G₁ following downregulation of B-Myb expression in primary leukemia cells and P53 wild-type myeloid and lymphoblastic cells; miR-34a plays a pivotal regulatory role in this process. Lee et al (57) reported that inhibition of miR-34a recovers B-Myb expression, while miR-34a mimics downregulates B-Myb expression in HCT116 colorectal cancer cells. In addition, Li et al (56) and Yu et al (58) have concluded that the expression of G₁/S-related genes, Ezh2 and B-Myb, are suppressed by miR-34c overexpression in the cancer cell line cultured from a mouse model of tubal high-grade serous ovarian carcinoma, leading to cell cycle arrest at G₁ phase and induction of apoptosis.

Wang et al (59) demonstrated that post-transcriptional regulation of MALAT1 is regulated by miR-101 and miR-217 in ESCC. Specifically, post-transcriptional silencing of MALAT1 significantly inhibits ESCC cell proliferation by arresting the G₂/M phase, while the migratory and invasive abilities of ESCC cells decrease following overexpression of miR-101 and miR-217. This may be due to MALAT1-mediated upregulation of P21 and P27 expression and inhibition of B-Myb expression (60). In addition, Chen et al (61) indicated that miR-143-3p negatively regulates B-Myb in breast cancer cells, and modulates cancer cell proliferation and apoptosis (62).

The E2F transcription factor family plays a crucial role in the regulation of cell cycle progression, DNA replication and apoptosis (63). The E2F family can be divided into two groups, E2F1-3a are transcription factors that activate the cell cycle and mainly encode genes that promote the progression of G₁ to S phase, while E2F3b and E2F4-8 mainly promote cells to exit the cell replication cycle and facilitate cell differentiation (64,65). B-Myb is a typical cell cycle regulator that is rarely expressed in the G₀ phase (66). When external growth factor-mediated signal pathways, such as ERK1/2, are activated, they promotes the release and activation of E2F1-3 from pRB through CCND1; these factors drive their target genes to encode G₁/S-related cytokines, and B-Myb expression is also induced by E2F1-3 (4,67,68). The induction of B-Myb expression at the end of the G₁ phase is due to a substantial increase in gene transcription, suggesting that B-Myb may be a gene regulated by the E2F transcription factors (62).

The promoters of both human and murine B-Myb genes contain completely conserved E2F-binding sites, which is key for B-Myb to participate in the transcription regulation of the cell cycle (69,70). A chromatin immunoprecipitation assay of NIH3T3 in fibroblasts revealed that both E2F4/P107 and E2F4/P130 are associated with the B-Myb promoter in the G₀ phase, while E2F4/P107 is associated with the B-Myb promoter in the early G₁ phase (69). In human malignant glioma T98G cells, E2F1 and E2F3 are associated with the B-Myb promoter in the late G₁ phase (71). B-Myb transcriptionally activates its own promoter through the SP1-binding site adjacent to the transcription initiation site (72). SP1 is coupled with E2F1 to promote transcriptional activation of the B-Myb promoter (73,74). It is suggested that E2F1 and E2F3 play a role in promoting B-Myb transcription in at least some cells (75).

Nakajima et al (16) demonstrated that knockout of E2F1 in JHH-5 hepatocellular carcinoma cells decreases the expression levels of B-MYB, CCNE1, MYC, TK1 and RRM1. The kinetics of B-Myb interaction with G₂-regulated promoters coincides with the activation of the genes; a decrease in RNAi-regulated B-Myb expression can inhibit cyclin B1 and cell division cycle 2 (cdc2) expression, arrest cell cycle at G₂/M, and increase apoptosis (76). The interaction of B-Myb with the cdc2 promoter is dependent on the complete E2F-binding site, and the B-Myb gene is regulated by E2F at G₁/S transition, thereby modulating the target genes associated with G₁/S and G₂/M transitions, including cdc2, cyclin A2 and cyclin B1 (77).