Colorectal cancer stemness and ERK
Larry H. Bernstein, MD, FCAP, Curator
LPBI
KCTD12 Regulates Colorectal Cancer Cell Stemness through the ERK Pathway
Liping Li, Tingmei Duan, Xin Wang, Ru-Hua Zhang, Meifang Zhang, Suihai Wang,Fen Wang, Yuanzhong Wu, Haojie Huang & Tiebang Kang
Scientific Reports 2016; 6(20460) http://dx.doi.org:/10.1038/srep20460
Targeting cancer stem cells (CSCs) in colorectal cancer (CRC) remains a difficult problem, as the regulation of CSCs in CRC is poorly understood. Here we demonstrated that KCTD12, potassium channel tetramerization domain containing 12, is down-regulated in the CSC-like cells of CRC. The silencing of endogenous KCTD12 and the overexpression of ectopic KCTD12 dramatically enhances and represses CRC cell stemness, respectively, as assessed in vitro and in vivo using a colony formation assay, a spheroid formation assay and a xenograft tumor model. Mechanistically, KCTD12 suppresses CRC cell stemness markers, such as CD44, CD133 and CD29, by inhibiting the ERK pathway, as the ERK1/2 inhibitor U0126 abolishes the increase in expression of CRC cell stemness markers induced by the down-regulation of KCTD12. Indeed, a decreased level of KCTD12 is detected in CRC tissues compared with their adjacent normal tissues and is an independent prognostic factor for poor overall and disease free survival in patients with CRC (p = 0.007). Taken together, this report reveals that KCTD12 is a novel regulator of CRC cell stemness and may serve as a novel prognostic marker and therapeutic target for patients with CRC.
Colorectal carcinoma (CRC) remains one of the most aggressive cancers in the world. Every year, more than 1.2 million patients with CRC are diagnosed, and almost 50% die from the disease. Surgery, radiotherapy and chemotherapy are still the predominant therapeutic strategies1. Although surgery combined with chemoradiotherapy represents a viable treatment option for early stage tumors, the majority of patients are not diagnosed until the late stage, for which the 5 year survival rate post-surgery decreased from 69.2% to 11.7%2.
Cancer stem cells (CSCs) are considered to be responsible for recurrence and metastasis during CRC tumorigenesis3. CSCs, possessing self-renewal characteristics, initiate tumor growth and promote chemotherapy and radiation resistance, which are considered to be responsible for CRC progression and recurrence4,5. Targeting the determinants of CRC cell stemness has been proposed as a therapeutic strategy6.
KCTD12 (potassium channel tetramerization domain containing 12, pfetin), which contains a voltage-gated potassium (K+) channel tetramerization T1 domain and a BTB/POZ (Bric-a-brac, Tram-track, Broad complex poxvirus and zinc finger) domain, belongs to the KCTD family and was initially identified in cochlea. In addition to being a K+ channel protein that responds to membrane potential7, KCTD12 also acts as an auxiliary subunit of GABAB (γ-aminobutyric acid type B) receptors, which regulate emotionality and neuronal excitability8,9. Interestingly, high KCTD12 expression indicates a favorable prognosis and could act as an independent prognostic factor for GIST (gastrointestinal stromal tumors)10, most likely due to the control of tumor and tumor stem cell proliferation by GABA signaling11. In addition, other KCTD family members, such as KCTD21, 11, and 6, have been shown to regulate the growth of MB (medulloblastoma) stem cells through the histone deacetylase HDAC1 and Hh/Gli12,13,14. However, there is no information about whether KCTD family members play crucial roles in CRC cell stemness. As described here, using HT29 cells and their spheroids, we observed that KCTD12 was the most altered member of the KCTD family in the spheroids of HT29 cells, leading to our speculation that KCTD12 plays a crucial role in CRC cell stemness. In verification of this hypothesis, our data support that KCTD12 is a potential regulator of CRC cell stemness at a cellular level, in an animal model and in clinical samples.
KCTD12 is down-regulated in the spheroids of HT29 cells
To investigate whether KCTD family members are involved in the stemness of CRC cells, we first enriched for stemness characteristics of HT29 cells by culturing them as spheroids for 8 days15. As shown in Fig. 1A,B, the percentages of cells expressing CD133 and CD44, two well-known stemness markers, were dramatically increased as shown by flow cytometry assay and western blotting. Subsequently, the mRNA levels of KCTD family members were compared between the spheroids and the parental HT29 cells. As the results in Fig. 1C show, the mRNA levels of KCTD1, 5 and 12 were decreased, while the levels of KCTD21 were increased in spheroids of HT29 cells. Notably, KCTD12 was the most significantly decreased family member in spheroids of HT29 cells (Fig. 1C), which was further confirmed by the observation that the KCTD12 protein was also significantly decreased in the spheroids of HT29 cells (Fig. 1D). However, KCTD8 and KCTD19 could not be detected in HT29 cells. These findings indicate that KCTD12 may play a crucial role in the stemness of CRC cells.
Figure 1: KCTD12 is down-regulated in CSC-like HT29 cells.
(A) Representative flow cytometry plots and quantitative analysis showing the percentages of CD44+ and CD133+ cells in normal adherent and spheroid cultures of HT29 cells. (B) CD44 and CD133 expressions were analyzed by Western blotting in adherent and spheroid cultures of HT29 cells. (C) Quantitative real time PCR analysis of the relative mRNA levels of the KCTD family members in adherent and spheroid cultures of HT29 cells. (D) KCTD12 expression was analyzed by Western blotting in adherent and spheroid cultures of HT29 cells. The results are presented as the means ± SD, and all data are representative of three independent experiments. *P < 0.05, **P < 0.01.
KCTD12 regulates CRC cell stemness in cell lines
Next, we asked whether KCTD12 influences stemness using CRC cell lines with varying levels of KCTD12 (Fig. 2A). HT29 cells and DLD1 cells were chosen to knockdown and overexpress KCTD12, respectively (Fig. 2B). As shown in Fig. 2C, the silencing and the overexpression of KCTD12 were capable of increasing and decreasing, respectively, the well-known CRC cell stemness markers CD44, CD133 and CD29 at the protein and mRNA levels. Consistently, the percentages of cells positive for CD44 or CD133 were dramatically increased when KCTD12 was knocked down in HT29 cells (Fig. 2D). In addition, the silencing and the overexpression of KCTD12 in HT29 or DLD1 cells increased and decreased the sizes of spheres, respectively, while having no effect on the numbers of spheres (Fig. 2E,F). Taken together, these results indicate that KCTD12 is critical to the stemness of CRC cells.
Figure 2: KCTD12 suppresses the stemness of CRC cells.
(A) KCTD12 protein was analyzed by Western blotting in the indicated CRC cell lines. (B) The indicated stable cell lines with silencing or overexpression of KCTD12 were analyzed by Western blotting. α-tubulin or HSP70 was used as the loading control. (C–E) CD44, CD133 and CD29 levels were analyzed by Western blotting, qRT-PCR and flow cytometry, in the indicated stable cell lines. Red lines indicating the mean intensity of fluorescence of CD44+ or CD133+ were quantified by Flow-J software in the flow cytometry analysis. The mean intensity of fluorescence of CD44+ or CD133+ was calculated in triplicates. (F,G) Images and quantification of the number and size of spheres formed from the indicated stable cell lines in the absence of serum for 7 days. Original magnification in F, 40×(upper), 400×(lower). Original magnification in G, 40×. Scale bars, 100 μm. The results are presented as the means ± SD, and all data are representative of three independent experiments. *P < 0.05, **P < 0.01.
KCTD12 is involved in the self-renewal ability of CRC cells in vitro and in the tumorigenesis of CRC cells in vivo
We further explored the functions of KCTD12 in the self-renewal and tumorigenesis of CRC cells. First, as shown in Fig. 3A by the colony formation assay, the knockdown of KCTD12 in HT29 cells significantly enhanced the cells’ colony formation capacity, whereas the overexpression of KCTD12 in both DLD1 and HCT116 cells reduced this capacity (Fig. 3B). However, the alteration of KCTD12 in these cells did not affect their proliferation (Fig. 3C). These results indicate that KCTD12 is involved in the self-renewal ability of CRC cells in vitro. Second, as shown inFig. 4, the knockdown of KCTD12 in HT29 cells promoted, whereas the overexpression of KCTD12 in DLD1 cells inhibited, tumor growth in nude mice, as measured by tumor volumes and weights. These results suggest that KCTD12 plays a crucial role in CRC tumorigenesis in vivo.
Figure 3: KCTD12 inhibits the colony formation of CRC cells in vitro.
(A,B) The colony formation assays were performed in the indicated stable cell lines. (C) The cell proliferation was measured by MTT in the indicated stable cell lines. The results are presented as the means ± S.E. of three independent experiments. *P < 0.05, **P < 0.01.
Figure 4: KCTD12 represses the tumorigenicity of CRC cells in vivo.
(A,B) A xenograft model consisting of nude mice with HT29 cells harboring KCTD12 silencing injected into the armpits of 4 week old mice (n = 7/group). The images of mice harboring tumors (left) and tumors from the mice (right). Tumor volumes were measured every two days (left). Mean tumor weights were calculated. (D,E) A xenograft model consisting of nude mice with DLD1 cells overexpressing KCTD12 were injected into the armpits of 4 week old mice (n = 5/group). The images of mice harboring tumors (left) and tumors from the mice (right). Tumor volumes were measured every two days (left). Mean tumor weights were calculated. The results are presented as the means ± SD. *P < 0.05, **P < 0.01. (C,F) H&E staining of tumors and IHC staining for KCTD12 protein in these cells. Original magnification, 200×. Scale bars, 100 μm.
Silencing of KCTD12 enhances the drug resistance of CRC cells
Figure 5: Silencing of KCTD12 enhances the drug resistance to both imatinib and 5-FU in HT29 cells.
KCTD12 regulates CRC cell stemness via the ERK pathway
Given that KCTD12 acts as a component of the GABABcomplex, downstream of which is the ERK pathway, we sought to determine whether the ERK pathway is involved in the KCTD12-mediated regulation of CRC cell stemness. As shown in Fig. 6A,B, phosph-ERK1/2 levels were dramatically increased in HT29 cells with silenced KCTD12 and decreased in DLD1 cells with overexpressed KCTD12. Moreover, U0126, an ERK 1/2 inhibitor, abrogated the increases in CD44, CD133 and CD29 levels in HT29 cells induced by the knockdown of KCTD12 (Fig. 6C). Likewise, the inhibition of the ERK pathway by U0126 reduced the sizes of spheres in KCTD12 knockdown HT29 cells (Fig. 6D). These results indicate that KCTD12 regulates CRC cell stemness via the ERK pathway.
Figure 6: KCTD12 regulates stemness of CRC cells via the ERK signaling pathway
(A,B) Phosphorylation of ERK1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/2) were detected using western blotting in the indicated stable cell lines. (C) HT29 cells with silenced KCTD12 were treated with U0126 (30 μM) for 24 h. Western blotting was performed to detect t-ERK1/2, p-ERK1/2, CD44, CD133 and CD29. Hsp70 was used as a loading control. (D) The sphere formation assays were performed in HT29 cells with silenced KCTD12 and treated with U0126 or DMSO for 7 days. Images and quantification of the numbers and sizes of spheres formed were calculated. The experiments were repeated three times. *P < 0.05, **P < 0.01. Scale bars, 200 μm (left) and 100 μm (right).
Low KCTD12 expression indicates a poor prognosis of patients with CRC
Finally, we analyzed the clinical relevance of KCTD12 in CRC samples. As shown in Fig. 7A, the protein level of KCTD12 was significantly higher in normal tissues than in CRC tumor tissues. …
Figure 7: Low expression of KCTD12 was detected in human colorectal cancer tissues.
In this report, the down-regulation of KCTD12 is detected in colorectal CSC-like cells, and a low level of KCTD12 is associated with a poor prognosis of patients with CRC. Functionally, KCTD12 regulates CRC cell stemness characteristics, such as self-renewal, tumorigenesis and drug resistance, through the ERK pathway. This is the first report to reveal that KCTD12 regulates CRC cell stemness through the ERK pathway.
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