KLHL5 Contributes to Colorectal Cancer Cell Survival by Promoting Cell Cycle Progression and Suppressing Apoptotic Cell Death

Abstract

Kelch-like protein 5 (KLHL5) is highly expressed in colorectal cancer (CRC) compared to that in adjacent normal mucosa, and its expression level increases with CRC stage, showing a correlation with poor prognostic factors. However, its functional role in the malignant progression still remains unknown. To elucidate the role of KLHL5 in CRC, we characterized human CRC cell lines, including HCT116 and SW480, under KLHL5-depleted conditions. KLHL5-depleted HCT116 and SW480 cells suppressed their growth and migration in culture. Further duration induced cell death characterized by apoptotic cell death with down-regulation of antiapoptotic factor Bcl-2 and up-regulation of proapoptotic factors Bac, Boc, Puma, Bid, Noxa, and Bik. Proteomic analyses indicated KLHL5 depletion suppressed cell cycle progression by affecting multiple pathways, including the activation of the G2/M DNA damage pathway and inhibition of the G1/S transition. Further biochemical and cell biological analyses revealed the downregulation of CDT1 and CDC6 proteins, which are essential factors for the initiation of DNA replication, and the downregulation of cyclins A and B, which are essential factors for the progression of S and G2/M phases. Arrested cells undergo apoptotic cell death. Taken together, these data strongly indicate that KLHL5 expression in CRC serves as a survival factor to strengthen the cell cycle and protect against apoptotic cell death under harsh tumor microenvironments.

Keywords: KLHL5; anti‐cell death; biomarker; cell cycle; colorectal cancer.

FIGURE 1

Effect of KLHL5 knockdown on mRNA expression, cell viability, migration, and anoikis in CRC cell lines. Relative expression of KLHL5 mRNA in different colorectal cancer cell lines (HCT116, HT‐29, Caco2, Widr, and SW480) after KLHL5 knockdown using siRNA. KLHL5 mRNA knockdown using specific siRNA in HCT116 and SW480 cells resulted in reduced KLHL5 mRNA expression in both cell lines. Effect of KLHL5 knockdown on the viability of HCT116 and SW480 cells using the MMT assay. Effect of KLHL5 knockdown on HCT116 and SW480 cell numbers using the anoikis assay. The impact of KLHL5 knockdown on cell migration in HCT116 and SW480 cells. Statistical significance: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

FIGURE 2

Impact of KLHL5 knockdown on cell proliferation and LDH leakage in CRC cell lines. (A) Micrograph of the effect of KLHL5 knockdown on proliferation in HCT116 and SW480 cells. Scale bar: 50 μM. (B) Effect of KLHL5 knockdown on protein expression profiles of EMT markers in HCT116 cells at day 4. (C) Changes in cell number of KLHL5 knockdown on proliferation of HCT116 and SW480 cells. (D) LDH leakage as a marker of cell death in HCT116 and SW480 cells after KLHL5 knockdown. Statistical significance: **p < 0.01, *p < 0.05. LDH, lactate dehydrogenase.

FIGURE 3

Role of KLHL5 in apoptotic cell death with and without caspase inhibition in HCT1116. KLHL5 knockdown led to an increase in cleaved‐caspase 3, suggesting enhanced apoptosis. Schematic representation of the apoptotic signaling pathway. Apoptotic stimuli activate BH3‐only proteins, which are categorized as sensitizers or activators. Sensitizer proteins inhibit anti‐apoptotic proteins, such as Bcl‐2, whereas activator proteins directly promote the activity of pro‐apoptotic proteins, leading to apoptosis. Western blotting analysis of proapoptotic BH3‐only and antiapoptotic Bcl‐2 proteins. Representative images of HCT116 cells after KLHL5 knockdown, treated with or without the caspase inhibitor zVAD (40 μM). Scale bar: 50 μM. Quantification of LDH leakage in HCT116 cells after KLHL5 knockdown, with and without zVAD treatment. Statistical significance: **p < 0.01.

FIGURE 4

Rescue experiment showing effects of KLHL5 wild type and mutant constructs on cell viability. Western blot analysis showing the expression of KLHL5 and GAPDH in HCT116 cells transfected with an empty vector, KLHL5 wild‐type (KLHL5‐wt), or KLHL5 mutant (KLHL5‐mut) constructs. The cells were then treated with control or KLHL5 siRNA. Representative microscopic images of differently transfected HCT116 cells showing the effect of KLHL5 knockdown. Scale bar: 50 μM. Quantification of cell viability (WST‐1 assay) was performed by absorbance (A440‐A660) under the same conditions. Statistical significance: ****p < 0.0001, ***p < 0.001, ns = not significant.

FIGURE 5

Pathway analysis of KLHL5 knockdown cell using proteomic data and IPA software. Upstream analysis of proteome data with KLHL5 knockdown cells using IPA software. Enrichment pathway analysis of proteome data from KLHL5 knockdown cells using IPA software. The enrichment scores were shown as −log₁₀ (p‐value). The network represented the correlations among signature terms in IPA.

FIGURE 6

Effects of KLHL5 knockdown on the cell cycle. Flow cytometry analysis of HCT116 cells transfected with control siRNA or KLHL5 siRNA at 24, 48, and 72 h post‐transfection. Western blot analysis of cell cycles regulatory proteins in HCT116 cells transfected with control or KLHL5‐specific siRNA.