Fig 1: RCCD1 modulates autophagy activity and WNT5A secretion in CCD‐18Co fibroblasts. CCD‐18Co fibroblasts were transfected with sh‐RCCD1 (shRNA targeting RCCD1), sh‐NC (control shRNA), OE‐RCCD1 (RCCD1 expression vector), or OE‐NC (empty vector) constructs. Cells harvested 48 h post‐transfection. (A) RT‐qPCR analysis of autophagy genes (ATG5, BECN1, MAP1LC3B, ATG7, and p62). Expression normalized to GAPDH using 2^‐ΔΔCt method. (B) Western blot analysis of RCCD1, Beclin‐1, and p62 with β‐actin loading control. (C) LC3B immunofluorescence in isolated fibroblasts. Nuclear stain: DAPI (blue). Quantification from ≥100 cells per condition. Scale bar: 15 µm. (D) Western blot analysis of phosphorylated and total AMPK (Thr172), mTOR (Ser2448), and ULK1 (Ser555). Quantification shows phospho‐to‐total protein ratios. (E) ELISA quantification of WNT5A in culture supernatants using Human WNT5A ELISA Kit. Statistical analysis: one‐way ANOVA with Tukey's post‐hoc test; data presented as mean ± SD from n = 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 2: Schematic diagram of the RCCD1‐autophagy‐WNT5A signaling axis mediating CAF‐tumor cell crosstalk in colon cancer progression. In cancer‐associated fibroblasts (CAFs), RCCD1 overexpression activates the AMPK/mTOR/ULK1 signaling cascade, leading to enhanced autophagy characterized by increased Beclin‐1 and LC3B‐II expression and decreased p62 levels. This RCCD1‐mediated autophagy promotes WNT5A secretion from CAFs into the tumor microenvironment. Secreted WNT5A acts as a paracrine factor that binds to receptors on colon cancer cells, activating the noncanonical Wnt/CaMKII/ERK signaling pathway. This activation induces epithelial‐mesenchymal transition (EMT), ultimately enhancing malignant behaviors including proliferation, invasion, and migration. The RCCD1‐autophagy‐WNT5A axis can be therapeutically targeted using 3‐methyladenine (3‐MA) to block autophagy in CAFs or anti‐WNT5A neutralizing antibodies to disrupt paracrine signaling, both of which effectively reverse cancer progression. These data indicate the critical role of the RCCD1‐autophagy‐WNT5A axis in mediating protumorigenic crosstalk between CAFs and tumor cells in the colon cancer microenvironment.
Fig 3: Autophagy inhibition and WNT5A neutralization reverse RCCD1‐induced fibroblast‐mediated cancer cell malignant behaviors. Transwell coculture system: CCD‐18Co fibroblasts (upper chamber) transfected with OE‐NC or OE‐RCCD1, treated with vehicle, 3‐MA (5 mM), or anti‐WNT5A antibody (10 μg/mL); HCT116 cells (lower chamber). Five groups: HCT116 alone, coculture with OE‐NC, OE‐RCCD1, OE‐RCCD1 + 3‐MA, or OE‐RCCD1 + anti‐WNT5A fibroblasts. Coculture duration: 48 h. All assays performed on HCT116 cells. (A) RT‐qPCR analysis of EMT markers (E‐cadherin, Vimentin, and N‐cadherin). Expression normalized to GAPDH using 2^‐ΔΔCt method. (B) Western blot analysis of phosphorylated and total CaMKII (Thr286) and ERK1/2 (Thr202/Tyr204) with β‐actin loading control. Quantification shows phospho‐to‐total ratios. (C) Transwell invasion assay in Matrigel‐coated chambers using different groups of HCT116 cells for 24 h. Invaded cells were stained with crystal violet and counted in five random fields. (D) Wound healing assay in scratched monolayers cultured in serum‐free medium; images at 0 and 48 h. Quantification shows relative wound areas. (E) CCK‐8 proliferation assay in different groups of HCT116 cells at 0, 24, 48, and 72 h. Absorbance was measured at 450 nm. Statistical analysis: one‐way ANOVA with Tukey's post‐hoc test for panels A–D; two‐way ANOVA with Tukey's post‐hoc test for panel E; data presented as mean ± SD from n = 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 4: Clinical validation confirms RCCD1 upregulation and enhanced autophagy in tumor‐associated fibroblasts. Paired tumor and adjacent normal tissues from colon cancer patients were analyzed, with fibroblasts isolated using magnetic bead separation. (A) RT‐qPCR analysis of RCCD1 mRNA expression in tissue lysates. Expression normalized to GAPDH using 2^‐ΔΔCt method. (B) Western blot analysis of RCCD1 protein in tissue lysates with β‐actin loading control. Quantification by densitometry normalized to β‐actin. (C) ELISA quantification of WNT5A protein in tissue lysates using Human WNT5A ELISA Kit. (D) Flow cytometry validation of fibroblast purity using FAP staining on BD FACSCanto II system. Gates show FAP + cell percentages. (E) RT‐qPCR analysis of autophagy genes (ATG5, BECN1, MAP1LC3B, ATG7, and p62), RCCD1, and WNT5A in isolated fibroblasts. Expression normalized to GAPDH using 2^‐ΔΔCt method. (F) Western blot analysis of RCCD1, WNT5A, Beclin‐1, and p62 in isolated fibroblasts. Quantification by densitometry normalized to β‐actin. (G) LC3B immunofluorescence in isolated fibroblasts. Nuclear stain: DAPI (blue). Quantification from ≥100 cells per condition. Scale bar: 15 µm. Statistical analysis: t‐test for all comparisons; data presented as mean ± SD from n = 10 patient samples in A–C; data presented as mean ± SD from 3 experiments using 3 batchs of isolsated fibroblasts in D‐G. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 5: Autophagy inhibition attenuates RCCD1‐induced autophagy in CCD‐18Co fibroblasts. CCD‐18Co fibroblasts transfected with OE‐NC or OE‐RCCD1 were treated with vehicle, 3‐MA (autophagy inhibitor, 5 mM), or anti‐WNT5A antibody (10 μg/mL) for 48 h. (A) RT‐qPCR analysis of autophagy genes (ATG5, BECN1, MAP1LC3B, ATG7, and p62). Expression normalized to GAPDH using 2^‐ΔΔCt method. (B) Western blot analysis of RCCD1, Beclin‐1, and p62 with β‐actin loading control. Quantification by densitometry. (C) LC3B immunofluorescence in isolated fibroblasts. Nuclear stain: DAPI (blue). Quantification from ≥100 cells per condition. Scale bar: 15 µm. (D) Western blot analysis of phosphorylated and total AMPK (Thr172), mTOR (Ser2448), and ULK1 (Ser555). Quantification shows phospho‐to‐total ratios. (E) ELISA quantification of WNT5A in culture supernatants. Statistical analysis: one‐way ANOVA with Tukey's post‐hoc test; data presented as mean ± SD from n = 3 independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001.
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