Inflammation‐dependent overexpression of c‐Myc enhances CRL4^DCAF4 E3 ligase activity and promotes ubiquitination of ST7 in colitis‐associated cancer

Abstract

Inflammation is well known as an important driver of the initiation of colitis-associated cancer (CAC). Some cytokines, such as IL-6 and TNF- can activate expression of the oncogene c-Myc (MYC) and regulate its downstream effects. Cullin-RING E3 Ligases (CRLs) are emerging as master regulators controlling tumorigenesis.Here, we demonstrate that two cullin genes, CUL4A and CUL4B, but not other members, are specifically overexpressed in CAC tumour samples and positively correlate with levels of the proinflammatory cytokines IL-1 and IL-6. In vitro experiments revealed that the transcription factor c-Myc can specifically activate the expression of CUL4A and CUL4B by binding to a conserved site (CACGTG) located in their promoters. Additionally, we found that both CUL4A and CUL4B can form an E3 complex with DNA damage-binding protein 1 (DDB1) and DDB1-CUL4-associated factor 4 (DCAF4). In vitro and in vivo ubiquitination analyses indicate that CRL4 DCAF4 E3 ligase specifically directs degradation of ST7 (suppression of tumorigenicity 7). Overexpression of c-Myc in human colon epithelial cells resulted in the accumulation of CUL4A, CUL4B and DCAF4, but degradation of ST7. In contrast, knockdown of c-Myc, CUL4A or CUL4B in the colon adenocarcinoma cell line HT29 caused accumulation of ST7 and inhibition of cell proliferation, colony formation ability and in vivo tumour growth. Collectively, our results provide in vitro and in vivo evidence that c-Myc regulates CRL4 DCAF4 E3 ligase activity to mediate ubiquitination of ST7, whose presence is physiologically essential for CAC tumorigenesis.No conflicts of interest were declared. ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) [16,17]. The human genome encodes two E1s, ∼40 E2s and more than 600 E3s [18,19]. Of these E3 ligases, Cullin-RING

The CARM1-p300-c-Myc-Max (CPCM) transcriptional complex regulates the expression of CUL4A/4B and affects the stability of CRL4 E3 ligases in colorectal cancer

“…Both CUL4A and CUL4B can assemble an E3 ligase with DDB1, RBX1, and DCAF4. These two complexes are termed as CRL4 DCAF4 E3 ligases, which can specifically ubiquitinate a tumor suppressor ST7 [27]. However, we did not reveal how c-Myc activated the expression of CUL4A/4B in this process.…”

“…The mammalian CUL4A and CUL4B share over 80% protein sequence identity, however, current findings indicate that they do not show obvious functional redundancy [27]. In the same type of cancer cells, only either CUL4A or CUL4B is overexpressed [27]. One exception is our recent finding in which CUL4A/4B are both overexpressed in colorectal cancer [27].…”

“…2)] [20][21][22][23], cell cycle progression (e.g., p21 and p27) [24,25], and tumor suppression (e.g., PTEN and ST7] [26,27]. The mammalian CUL4A and CUL4B share over 80% protein sequence identity, however, current findings indicate that they do not show obvious functional redundancy [27].…”

“…In the same type of cancer cells, only either CUL4A or CUL4B is overexpressed [27]. One exception is our recent finding in which CUL4A/4B are both overexpressed in colorectal cancer [27]. The mechanical investigation demonstrates that intracellular inflammatory environment induces the expression of a transcription factor c-Myc, which specifically binds to the promoters of CUL4A/4B and activates their expression.…”

“…Cells were grown in DMEM containing 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA, #F2442) and 50 U/mL penicillin-streptomycin (PS) (Sigma-Aldrich, #P4333). The source and growth condition of the human colon epithelial cell line (HCEC-1CT) were the same as described previously [27]. Cells were cultured in a 37°C humidified atmosphere supplemented with 5% CO 2 and cells were split twice one week.…”