Keratin23 (KRT23) Knockdown Decreases Proliferation and Affects the DNA Damage Response of Colon Cancer Cells

<div><p>Keratin 23 (KRT23) is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression <i>in vitro.</i> Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed that KRT23 depletion affected molecules of the cell cycle and DNA replication, recombination and repair. <i>In vitro</i> analyses confirmed that KRT23 depletion significantly decreased the cellular proliferation of SW948 and LS1034 cells and markedly decreased the expression of genes involved in DNA damage response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced proliferation of the KRT23 depleted cells compared to irradiated control cells.</p></div

Irradiation of colon cancer cells.

<p>A) SW948-ctrl or SW948-sh1506 with stable KRT23 knockdown were irradiated with 0 GY or 5 GY of γ-rays, respectively and seeded on RTCA16-well plates with 16.000 cells/well (n = 4). Non-irradiated SW948-sh1506 cells showed a reduced proliferation rate compared to non-irradiated SW948-ctrl cells. Irradiated SW948-ctrl cells continued proliferation after a short lag period, while the proliferation of the irradiated KRT23-depleted SW948-sh1506 cells decreased after 72 h post-irradiation. The Cell Index CI of the irradiated SW948-sh1506 cells markedly dropped down at about 96 h post-irradiation suggesting a detaching of the cells, possibly induced by cell death upon irradiation of the KRT23 depleted cells. B) A MTT viability assay co-performed at 120 h post-irradiation together with RTCA showed that the viability of KRT23 depleted SW948-sh1506 cells was reduced by 60% upon irradiation with 5GY (p = 8.1E-08) compared to 30% in the SW948-ctrl cells (p = 6.4E-05). C) Visual inspection at 7 days post-irradiation showed a markedly reduced number of cells in KRT23 depleted SW948-sh1506 cells irradiated with 5GY compared to non-irradiated cells.</p

Knockdown of KRT23.

<p><b>A</b>) Western Blot of freshly made SW948-ctrl and SW948-sh1506 cells extracts with 20 µg extract per lane using a monospecific anti-K23 antibody in a 1∶150 dilution, Marker Biorad All Blue. Stable knockdown of K23 in the SW948-sh1506 cells resulted in >80% reduced K23 protein expression compared to the SW948-ctrl cells. Beta-actin was used as loading control. B) Immunofluorescence analysis confirmed a decreased K23 expression in SW948-sh1506 cells; anti-K23 antibody 1∶500, detection with Alexa 488, nuclear stain Hoechst, magnification 630x C) Visual inspection indicated a lower cell density for SW948-sh1506 cells 48 h post seeding. D) SW948-sh1506 cells showed less nuclear expression of the proliferation marker KI67 (green); anti-KI67 1:100 E) SW948-ctrl and SW948-sh1506 cells were seeded on 96-well plates with 4000 cells per well (n = 12) and proliferation was analyzed post-seeding at five time-points. Proliferation of SW948-sh1506 cells was significantly (Fisher’s exact t-test, p<0.0001) decreased at 96 hours and 120 hours post-seeding. F) The MTT assay was repeated by seeding 16000 cells per well (n = 11) on a 96 well plate and proliferation/viability was analyzed at 48 h post-seeding and percentage viability was measured. The proliferation of KRT23 depleted cells was significantly (p = 2.6E-06) decreased by about 30%. G) SW948-ctrl and SW948-sh1506 cells were seeded on 96-well RTCA-plates with 8000 cells/well (n = 3). Values are shown as medians and standard deviations for each group at selected time points for a representative experiment.</p

Ingenuity pathway analyses and protein expression.

<p>A) KRT23 knockdown affected main regulatory pathways, and decreased the expression of key molecules. Key molecules decreased upon KRT23 knockdown essential for the assembly of replication and repair complexes comprise E2F1, BRCA1, BRCA2, RAD51, MRE11A and RPA. blue = decreased, yellow = increased transcript expression. B) Western blotting. MRE11A, E2F1, BRCA1 and RAD51 proteins were markedly decreased in KRT23 depleted SW948-sh1506 cells compared to control cells. 20µg nuclear (nuc) or cytoplasmic extract (cyt) was loaded. Anti-MRE11A antibody 1∶1000 dilution (nuclear extracts), 1∶500 dilution (nuclear and cytoplasmic extracts). Marker All Blue BioRad; C) Immunofluorescence staining of SW948-ctrl and SW948-sh1506 cells also showed that KRT23 knockdown resulted in a decreased expression of E2F1, MRE11A, RAD51 and BRCA1.</p

KRT23 knockdown affects canonical pathways involved in DNA damage control.

<p>Data were obtained by microarray expression profiling followed by RMA normalization, comparison of SW948 control cells versus SW948-sh1506 with KRT23 knockdown. All molecules are located in the nucleus.</p

Colon cancer cells depleted from KRT23 compared to control cells.

*<p>number of molecules differentially expressed to number of molecules in given pathway.</p

Methylation versus expression profiling of KRT23.

<p>Comparison of KRT23 transcription data from Exon 1.0 ST arrays (–•–) to methylation data from 2 probes, cg22392708 and cg06378617 from the Illumina Bead arrays (□) showed a negative correlation between methylation and transcription in the 40 tissue samples analyzed (Spearman rank correlation coefficient of −0.64 and −0.74, respectively).</p

Assessment of in vivo biocompatibility/safety.

<p>(<b>A</b>) Body weights were recorded for all mice throughout the first 100 days of the experiment. (<b>B</b>) White blood cell counts of all mice were assessed at the end of the administration period of the triple combination.</p

Comparison of the <i>in vitro</i> and <i>in vivo</i> effects of Pegylated Gemcitabine.

<p>(<b>A</b>) <i>In vitro</i> effects of Gem and PEG-Gem on apoptosis and cell death as well as CD133 expression (cumulative results of cells obtained from different xenografts). (<b>B</b>) Kaplan-Meier Curve depicting cumulative survival time of all mice pooled by treatment group. For illustrative purposes, selected survival curves of Fig. 2D are depicted again. (<b>C</b>) Tumor growth curves for primary whole-tissue xenografts implanted subcutaneously and orthotopically, respectively. Continuous line depicts Gem+vehicle, dashed line depicts Gem+ SIBI, dotted line depicts Gem+SIBI+Rapa. (<b>D</b>) Kaplan-Meier Curve depicting cumulative survival time of all mice pooled by treatment group. For illustrative purposes, selected survival curves of Fig. 2D are depicted again.</p

Effect of combination therapy on tumor composition.

<p>(<b>A</b>) Representative histological pictures showing stroma content in the respective treatment groups in gemcitabine resistant orthotopic tumors (<b>PDAC-185, upper panel</b>), (<b>Pax22, lower panel</b>). (<b>B</b>) Quantification of stroma content throughout the different treated xenografts.</p