Keratin 23 promotes telomerase reverse transcriptase expression and human colorectal cancer growth

Abstract

The overexpression of human telomerase reverse transcriptase (hTERT) has been associated with the proliferation and migration of colorectal cancer (CRC) cells. We investigated the roles of KRT23 and hTERT in promoting CRC cell proliferation and migration. We verified the relationship between KRT23 and hTERT in CRC using streptavidin-agarose pulldown and chromatin immunoprecipitation (ChIP) assays. One hundred and fifty-four human CRC specimens were analyzed using immunohistochemistry. The roles of KRT23 and hTERT in cell growth and migration were studied using siRNA and lentiviruses in vivo and in vitro. Western blot and wound scratch analyses were used to determine the signaling pathway for KRT23-mediated activation of CRC growth and migration. Telomerase activity was measured by using the TeloTAGGG Telomerase PCR ELISA PLUS Kit. We identified KRT23 as a new hTERT promoter-binding protein. Patients with high KRT23 and hTERT expression had markedly shorter overall survival. Overexpression of KRT23 upregulated the expression of hTERT protein, hTERT promoter-driven luciferase and telomerase activity in CRC. Conversely, inhibition of KRT23 by a KRT23-specific siRNA repressed the endogenous hTERT protein, the expression of hTERT promoter-driven luciferase and telomerase activity. Overexpression of KRT23 also promoted CRC proliferation and migration. By contrast, KRT23 inhibition significantly inhibited tumor cell growth in vitro and in vivo. KRT23 promoted cancer stem cell properties and increased the expression of CD133 and CD44. These results demonstrate that KRT23 is an important cellular factor that promotes CRC growth by activating hTERT expression and that KRT23 is a potential novel therapeutic target for CRC.

Figure 1

KRT23 was identified and validated as a transcription factor of hTERT in CRC cells. (a) A streptavidin–biotin pulldown assay was performed to identify the specific proteins that bind to the hTERT promoter. Nuclear extracts prepared from human CRC cells (SW620, RKO, DLD1 and LoVo) and hepatoma carcinoma cells (SNU, HEPG2 and BEL) were incubated with a biotin-labeled hTERT promoter probe and streptavidin–agarose beads. The DNA–protein complexes were separated by SDS-PAGE, and the protein bands were visualized by silver staining. The protein bands (at ~35 kDa) were excised from the gel and identified by the mass spectrum analysis. We predicted that the candidate CRC-specific hTERT promoter-binding protein was KRT23. (b) Immunoblot assay for detecting KRT23 binding to the hTERT promoter probe. KRT23 protein in the DNA–protein complexes was detected by western blot assay using an anti-KRT23 antibody. (c) Chromatin immunoprecipitation (CHIP) assays were performed using the hTERT promoter from human CRC cells (SW620, RKO, DLD1 and LoVo). PCR products were separated on 1% agarose gels. Normal immunoglobulin G (IgG) was the negative control for the KRT23 antibody

Figure 2

KRT23 and hTERT were highly expressed in CRC tissues and CRC cells. (a) Protein samples extracted from human colorectal tissues and adjacent tissues were used to evaluate the expression of KRT23 and hTERT. (b) The correlation between the KRT23 and hTERT expression in CRC tissues from 154 patients. (c) The expression and distribution of KRT23 and hTERT in CRC tissues and their adjacent non-cancer tissues through immunofluorescence analysis (× 200). (d) Kaplan–Meier analysis of the overall survival of CRC patients with different KRT23 expression levels (P<0.05, log-rank test). (e) Kaplan–Meier analysis of the overall survival of CRC patients with different hTERT expression levels (P<0.05, log-rank test). (f) Kaplan–Meier analysis of the overall survival of CRC patients with different KRT23 and hTERT expression levels (P<0.05, log-rank test). (g) KRT23 and hTERT expression levels in CRC cells were measured by western blot using anti-KRT23 and hTERT antibodies. (h) The expression levels of KRT23 and hTERT in CRC cells (LoVo and SW620) were detected by immunofluorescence assay. The localization of KRT23 and hTERT is shown. Green, KRT23; red, hTERT; blue, nuclei (× 1200)

Figure 3

KRT23 promoted hTERT expression. (a and b) Downregulation of hTERT mRNA and protein expression by siKRT23 were analyzed by RT-PCR and western blot in LoVo cells. Luciferase activity (c) and telomerase activity (d) were measured after transfection with siKRT23-1/3 in LoVo cells. Upregulation of hTERT mRNA and protein expression by LV-KRT23 were analyzed by RT-PCR (e) and western blot (f) in RKO cells. Luciferase activity (g) and telomerase activity (h) were detected after infection of RKO cells with LV-KRT23

Figure 4

Knockdown of KRT23 inhibited the proliferative capacity and migration of LoVo CRC cells. (a) Cell viability was analyzed by MTT in LoVo cells that were transfected with siKRT23 or NSP for 48 h. (b) Colony formation assay of LoVo cells transfected with siKRT23 or NSP two times a week for 2 weeks. (c) Cell migration was analyzed by a wound-healing assay. LoVo cells were seeded in 6-well plates and grown to full confluence (50 ×). (d) The phosphorylated and total ERK and p38 levels in LoVo cells transfected with siKRT23 were detected by western blot assay using antibodies against p-p38, p38, p-ERK and ERK (*P<0.05, **P<0.01 and ***P<0.001)

Figure 5

Overexpression of KRT23 promoted proliferation and migration in RKO CRC cells. (a) Cell viability was analyzed by MTT in RKO cells that were infected with or the NSP LV-NC. (b) Colony formation assay of RKO cells infected with LV-KRT23 or the NSP LV-NC. (c) Cell migration was analyzed by a wound-healing assay. RKO cells were seeded in 6-well plates and grown to full confluence (50 ×). (d) The phosphorylated and total ERK and p38 levels in RKO cells infected with LV-KRT23 were measured by a western blot assay using antibodies against p-p38, p38, p-ERK and ERK. (e) Cell viability was analyzed by CCK-8 in RKO cells that were treated with U0126 and/or LV-KRT23. (f) Cell viability was analyzed by CCK-8 in RKO cells that were treated with SB203580 and/or LV-KRT23. (g) Cell migration was analyzed by a wound-healing assay. RKO cells were treated with SB203580 and/or LV-KRT23. (h) Cell migration was analyzed by a wound-healing assay. RKO cells were treated with U0126 and/or LV-KRT23. (i) The phosphorylated and total p38 levels in RKO cells treated with SB203580 and/or LV-KRT23 were measured by a western blot assay using antibodies against p-p38 and p38. (j) The phosphorylated and total ERK 1/2 levels in RKO cells treated with U0126 and/or LV-KRT23 were measured by a western blot assay using antibodies against p-ERK 1/2 and ERK 1/2 (*P<0.05, **P<0.01 and ***P<0.001)

Figure 6

KRT23 promoted CSC properties and increased CD133 and CD44 expression levels. (a) Representative images of tumor cell spheres formed by LoVo cells after transfection with siKRT23-1, 2, and 3 for 2 weeks, respectively (scale bars, 100 μm). (b and c) Diagrams show the diameter and number of tumor cell spheres (>50 μm). (d) The CD44 and CD133 protein levels in the cell lysates were measured by western blot analysis after transfection with siKRT23-1, 2 and 3. (e) Representative images of tumor cell spheres formed by RKO cells after infection with LV-KRT23 for 2 weeks (scale bars, 100 μm). (f and g) Diagrams showing the diameter and number of tumor cell spheres (>50 μm). (h) The CD44 and CD133 protein levels in the cell lysates were measured by western blot analysis after infection with LV-KRT23 (**P<0.01 and ***P<0.001)

Figure 7

Inhibition of tumor growth by KRT23 knockdown in mice. (a) The tumor volume of each group of nude mice was measured and calculated as V=(width² × length)/2, with n=6 and **P<0.01, ***P<0.001. (b) The weight of tumor grafts 17 days after the intratumoral injection of PBS, NSP or siKRT23-3, ***P<0.001. (c) The proteins were extracted from tumor xenografts. The KRT23, hTERT, CD44, CD133, PCNA, GAPDH expression levels were measured by western blot analysis. (d) The morphology of the tumor xenografts in each nude mouse after 17 days of treatment. (e) An immunohistochemistry assay of KRT23 and hTERT expression in tumor xenografts from each group of nude mice (× 200)