Fig 2: DKC1 is under transcriptional regulation of canonical WNT signaling.a Heatmap depicting DKC1 expression in CRC patients across CMS categories and its association with WNT target genes; data derived and reanalyzed from TCGA-COADREAD. The color bar indicates z-score values. b Relative expression of DKC1 in CRC patient samples (N = 76) categorized based on CMS classification; data retrieved from the Clinical Proteomic Tumor Analysis Consortium (CPTAC). c Box and whiskers plot showing Dkc1 expression in murine colon tissue (n = 3) based on Apc alteration status (GSE65461) across different time points of Apc floxing. d Bar plot showing DKC1 transcript upon siRNA-mediated silencing of CTNNB1 (β-catenin) in SW620, WiDr, and Colo320HSR cell lines. e Same as in (d), except protein expression. CD44 was used as a positive control. f Immunoblots showing subcellular DKC1 expression upon WNT signaling stimulation across three independent CRC patient-derived organoids (PDOs). Lamin A was immunoblotted separately as a nuclear loading control, and α-tubulin was used as a cytosolic loading control, respectively. p-β-Catenin (Ser675) was used as a positive control for assessing active WNT signaling. g Schema depicting the chromosomal location of β-Catenin Binding Motif (CBM 1 to 4) at the DKC1 promoter (top). ChIP-qPCR data showing enrichment of β-catenin on DKC1 promoter using SW620 cells (P < 0.0001); LGR5 and ANKARD5 were used as positive controls, and CD70 was used as a negative control (bottom). h Schema representing the proposed canonical WNT signaling-driven DKC1 oncogenicity and the positive feedback loop. For panels, (b) One-way ANOVA, Dunnett’s multiple comparisons test was used; c One-way ANOVA, Sidak’s multiple comparisons test was used; (d, g) Unpaired Student’s two-tailed t test was applied. For panels, (b, c), data are presented as box-and-whisker plots indicating median (middle line), 25th and 75th percentile (box) and minimum and maximum values (whiskers). Data shown in the panels (3d–g) are from one representative experiment out of three independent replicates with similar results, each experiment includes 3 biological replicates per group. Data expressed as mean ± SEM. Figure 3h was created in BioRender. KUNDU, S. (2026) https://BioRender.com/h9o2vi8. Source data are provided as a Source Data file.

Fig 3: DKC1 confers oncogenicity by positively regulating SGPP2 in colorectal cancer.a Heatmap depicting DKC1 and SGPP2 levels in Indian CRC specimens (N = 49). b Immunohistochemical staining of DKC1 and SGPP2 in CRC specimens (N = 75); scale bar is 100 μm (whole section), and inset 40 μm (inset). c Contingency analysis for the same as in (b). d Bar plot showing quantification of DKC1 and SGPP2, as in (b). e Immunofluorescence staining for SGPP2 in tumor (N = 6) and adjacent normal (N = 5) tissues of Indian CRC patients. Scale bar represents 5 μm. f Same as (e), except bar plot depicting quantification. g Transcript (bottom) and protein (top) levels of SGPP2 upon DKC1 knockdown in SW620 cells. h Immunoprecipitation for DKC1 showing interaction with SOX2 using SW620 cells. i ChIP-PCR data for SOX2 binding on SGPP2 promoter using shSCRM and shDKC1 SW620 cells. j Relative cell proliferation using SGPP2 knockdown SW620 cells at the indicated time points. k Same as (j), except representative images for foci formation assay (top) and data quantification (bottom). Scale bar represents 0.5 mm. l Same as (j), except using stable isogenic SW620 cells; control shSCRM and shDKC1 stably transfected with vector backbone (EV), and shDKC1 with ectopic expression of SGPP2. m Same as (k), except using cells as in (l). n Bar plot showing S1P levels using same cells as in (i). o Transcript expression of CD44 using the same cells as in (j). p Same as (o), except CTNNB1 expression in the same cells as in (l). q Same as (o), except using DKC1-silenced; SGPP2-rescued SW620 cells versus control. r Same as (p), except immunoblot for protein levels in nuclear and cytoplasmic fractionations. Lamin A was immunoblotted separately as a nuclear loading control. For panels, (c) Two-tailed Fisher’s exact test for contingency analysis was used; (d) Two-sided Chi square test for comparison of frequencies was used for both panels; (f) Paired two-tailed t test was used; (g, i, m, n, and q) Unpaired Student’s two-tailed t test was used; (j, l) Two-way ANOVA, Dunnett’s multiple comparisons test was used; (k, o, and p) One-way ANOVA, Dunnett’s multiple comparisons test was used. Data shown in the panels (5a, f–r) are from one representative experiment out of three independent replicates with similar results; each experiment includes 3 biological replicates per group. Data expressed as mean ± SEM. Source data are provided as a Source Data file.

Fig 4: Comprehensive overview of DKC1-mediated CRC pathogenesis, underlying mechanistic circuitries, and its potential in diagnostic and therapeutic implications.Schematic showing the oncogenic traits associated with elevated DKC1 in CRC. Mechanistically, WNT-regulated DKC1 perturbs sphingolipid metabolism via SGPP2, activates canonical WNT signaling, forms a positive feedback loop, and confers chemoresistance in CRC patients. Presence of very-long-chain fatty acid ceramides in DKC1 high patients may serve as a potential diagnostic biomarker. Finally, dual targeting of DKC1 and WNT signaling offers a therapeutic strategy to overcome drug resistance. Figure 7 was created in BioRender. KUNDU, S. (2026) https://BioRender.com/99ih9wu.

Fig 5: High DKC1 expression in colorectal cancer patients associate with aggressive disease, and poor survival.a Representative image for the Immunohistochemical (IHC) analysis of DKC1 expression using a tissue microarray (TMA) comprising CRC patient specimens (N = 75); scale bar is 100 μm (whole section), and 40 μm (inset). b Bar plot showing distribution of CRC patients based on IHC staining intensity using the same Tissue microarray as in (a). c H-score distribution (0–300) across patient samples used for evaluating DKC1 staining intensity classified into high (N = 23), medium (N = 34), and low (N = 18) based on quartile-cutoff using the same specimens as in (a). d Dot plot showing expression of DKC1 in Ki-colon cohort (GSE6988) in normal (N = 28) and primary tumor (N = 52) tissues. e Dot plot indicating densitometric quantification for DKC1 immunoblot normalized to the β-actin levels using CRC specimens and adjacent normal tissues obtained from Indian patients (N = 13). f Representative images showing immunofluorescence staining for DKC1 in the tumor (N = 6) and adjacent normal (N = 5) tissues of Indian CRC patient specimens. Scale bar represents 5 μm. g Bar plot depicting quantification for DKC1 mean fluorescence intensity relative to DAPI for the same specimens as in (f). h Kaplan-Meier plot showing recurrence-free survival probability of DKC1-high (red) vs DKC1-low (blue) patients (n = 243) using data from the TCGA COAD cohort; the number of patients at risk at a respective time point is given at the bottom of the plot. i Upregulated biological processes of 265 upregulated genes associated with high DKC1 expression analyzed by DAVID functional annotation clustering; bars (blue) represent −log10(P-value) and frequency polygon (red line) represents the number of genes. For panels, (b) Two-sided Chi-square test for comparison of frequencies was used; (c) One-way ANOVA, Tukey’s multiple comparisons test was used; (d) Unpaired Student’s two-tailed t test was applied. e, g Paired two-tailed t test was applied. h Cox proportional hazard analysis was performed between DKC1-high and DKC1-low groups. i Fisher exact test, as calculated by the DAVID database was used. Data are expressed as mean ± SEM, unless specified otherwise. Source data are provided as a Source Data file.