LGR5 expression is controled by IKKα in basal cell carcinoma through activating STAT3 signaling pathway

Abstract

Basal cell carcinomas (BCC) of the skin are the most common of human cancers. The noncanonical NF-κB pathway is dependent on IKKα. However, the role of IKKα in BCC has not been elucidated. We show here that IKKα is expressed in the nucleus in BCC and non-malignant diseases. Nuclear IKKα could directly bind to the promoters of inflammation factors and LGR5, a stem cell marker, in turn, upregulating LGR5 expression through activation of STAT3 signaling pathway during cancer progression. Activation of STAT3 signaling pathway contributes LGR5 expression in dependent of IKKα after the interplay between STAT3 and IKKα. Meanwhile knockdown of IKKα inhibits tumor growth and transition of epithelial stage to mescheme stage. Taken together, we demonstrate that IKKα functions as a bone fide chromatin regulator in BCC, whose promoted expression contributes to oncogenic transformation via promoting expression stemness- and inflammatory- related genes. Our finding reveals a novel viewpoint for how IKKα may involve in BCCs tumor progression in the inflammatory microenvironment.

Keywords: BCC; EGF; IKKα; LGR5; STAT3.

Figure 1. IKKα was dysregulated and delocalized in skin-related tumors

A.. Anti- IKKα staining intensity was quantified in three microscopic fields for each tissues section and expression level of IKKα in skin-related diseases as indicated. n, number of analyzed samples. B.. A series of tissues samples was subjected to IHC with IKKα-specific antibody. The panels shown here were representative of IKKα staining in skin-related diseases as indicated. (Magnification: 40×.) C.. Distribution of IKKα in the cytoplasm and nucleus was quantified in BCC, SCC and metastasis. D. H & E staining was used to show different tissue type of BCC and keratin SCC. E. Immunohistochemical analysis was used to examine the level of IKKα in BCC and keratin SCCs. F. Expression level of IKKα in normal skin, keratin SCC and BCC as indicated. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2. Inhibition of IKKα reduces LGR5 expression

A.. A431cells with the treatment of IKKi-II were analyzed for their ability to migrate in a wound healing assay at indicated time points. B.. FACS analysis was used to detect cell cycle progression in A431 cells after the treatment of IKKi-II. C.. Statics of FACS analysis in A431 cells after the treatment of IKKi-II. * p < 0.05, ** p < 0.01. D.. A431 (Left) and HaCaT (Right) with treatment of IKKi-II were examined for the expression of LGR6, LGR5, IKKα, p-STAT3, STAT3 and β-actin by Western analysis.

Figure 3. Knockdown of IKKα decreased LGR5 expression

A431 A. and HaCaT B. cells were examined for the expression of proteins as indicated by Western analysis after an inducible knockdown Tet-on system of IKKα the cells were stably transfected. C.. Immunohistochemical analysis was used to examine the level of LGR5 protein in skin-related diseases. LGR5 was expressed at high levels whereas IKKα was also expressed at higher levels in skin BCC tissues (Tumor, Metastasis). (Magnification: 40×.) D.. Summary of LGR5 and IKKα protein levels in 35 tissue samples. Fisher's test, two-tailed p < 0.01.

Figure 4. Inflammatory factors activated STAT3 signaling pathway that was controlled by IKKα

The MTT assay was performed to assess cell viability in A431 cells that were stably transfected with an inducible Tet-on IKKα shRNAs and the cells were treated with EGF A., IL-6 C., and Cxcl1 E.. The MTT assay was performed to assess cell viability in HaCaT cells that were stably transfected with an inducible Tet-on IKKα shRNAs and the cells were treated with EGF B., IL-6 D., and Cxcl1 F.. G.. A431 cells with an inducible knockdown Tet-on system of IKKα were examined for the expression of proteins as indicated by Western analysis after the treatment of inflammatory factors for 24 h. * p < 0.05, ** p < 0.01.

Figure 5. Activation of STAT3 signaling pathway was involved in the regulation of LGR5 expression

The MTT assay was performed to assess cell viability in A431 cells A. and HaCaT cells B. that were stably that were stably transfected with an inducible Tet-on IKKα shRNAs and the cells were treated with Stattic. C.. A luciferase reporter assay was carried out to evaluate LGR5 promoter activity in 293 cells after the treatment of IKKi-II and/or Stattic. All promoter luciferase intensity was normalized to the pRL renilla luciferase control reporter. * p < 0.05, ** p < 0.01. D.. ChIP analysis in HaCaT cells and chemicals treatment indicated was performed to detect p-STAT3 binding to LGR5 gene. E.. ChIP analysis in A431 cells after Tet-on shIKKα transfected and chemicals treatment indicated was performed to detect p-STAT3 binding to LGR5 promoter gene. F.. A431 (Left) and HaCaT (Right) with treatment of Stattic were examined for the expression of selected proteins by Western analysis. ** p < 0.01

Figure 6. IKKα targeted to the inflammatory factors and LGR5

RT-PCR analysis was conducted to detect selected inflammatory factors using total RNA obtained from A431 cells A. and HaCaT cells B. after Tet-on shIKKα transfected. C. RT-PCR analysis was conducted to detect selected inflammatory factors using total RNA obtained from both HaCaT and A431 cells after the treatment of IKK-I II. D.. A luciferase reporter assay was carried out to evaluate LGR5 promoter activity in 293 cells. All promoter luciferase intensity was normalized to the pRL renilla luciferase control reporter. E.. ChIP analysis of A431 cells and chemicals treatment indicated was performed to detect IKKα binding to selected genes. F. Co-IP detected the intact complex of IKKα and STAT3 G. Cell proliferation in A431 was performed after the knockdown of LGR5. LGR5 protein levels, as detected by Western analysis, were shown in the inlet figure. H. A xenograft model of tumor weight was established in nude mice to evaluate the ability of knockdown of LGR5 in A431 cells and shCTRL cells to form cells. * p < 0.05, ** p < 0.01.

Figure 7. Inhibition of IKKα reduced tumor growth in vivo

A.. A xenograft model of tumor growth was established in nude mice to evaluate the ability of knockdown of IKKα in A431 cells and shCTRL cells to form cells. IKKα protein levels, as detected by Western analysis, were shown in the inlet figure. * p < 0.05, * p < 0.01, *** p < 0.001. B.. Tumor status was analyzed by H & E staining. C.. Immunohistochemical analysis was used to examine the level of IKKα, Ki67, gp130, p-STAT3 and LGR5 in tumor samples from nude mice. (Magnification: 40×.) D.. Immunohistochemical analysis was used to examine the level of EMT markers as indicated in tumor samples from nude mice. (Magnification: 40×.).