PRKCSH enhances colorectal cancer radioresistance via IRE1α/XBP1s-mediated DNA repair

Abstract

Neoadjuvant radiotherapy is the standard treatment for locally advanced rectal cancer, but resistance to this therapy remains a significant clinical challenge. Understanding the molecular mechanisms of radioresistance and developing strategies to enhance radiosensitivity are crucial for improving treatment outcomes. This study investigated the role of PRKCSH in colorectal cancer radioresistance and its underlying mechanisms. Our results demonstrate that PRKCSH is upregulated in colorectal cancer cells following ionizing radiation. Inhibiting PRKCSH sensitized these cells to radiation by reducing clonogenic survival, promoting apoptosis, and impairing DNA damage repair. Mechanistically, PRKCSH inhibition reduced p53 ubiquitination and degradation by activating the ER stress IRE1α/XBP1s pathway after radiation exposure, which enhanced DNA repair and contributed to radioresistance. In preclinical CRC models, PRKCSH depletion suppressed tumor growth and increased radiosensitivity. Similarly, in patient-derived organoid models, PRKCSH knockdown reduced organoid growth post-radiotherapy. In rectal cancer patients receiving neoadjuvant radiotherapy, higher PRKCSH expression in post-treatment samples correlated with reduced tumor regression. These findings suggest that targeting PRKCSH diminishes radioresistance by impairing DNA repair through the modulation of ER stress. Furthermore, PRKCSH expression may serve as a biomarker for evaluating radiotherapy efficacy and clinical outcomes in rectal cancer patients undergoing neoadjuvant therapy.

Fig. 1. PRKCSH is upregulated in CRC samples and correlates with radioresistance.

A PRKCSH mRNA levels in 24 human colorectal cancer (CRC) samples and matched normal tissues were analyzed using NCBI GEO microarray data. Results are expressed as mean ± SD and analyzed using a two-tailed Student’s t-test (***P < 0.001). B PRKCSH mRNA expression in radiation-resistant (n = 6) and radiation-sensitive (n = 6) CRC tissues was quantified. Data are presented as mean ± SD, with statistical analysis performed using a two-tailed Student’s t-test (*P < 0.05). C Western blot analysis confirmed PRKCSH expression levels at different time points post-8 Gy irradiation in HCT116 cells, with gray value analysis shown below. D Western blot analysis confirmed PRKCSH expression levels at different time points post-8 Gy irradiation in RKO cells, with gray value analysis shown below. E Western blot analysis confirmed PRKCSH knockdown efficiency in HCT116 and RKO CRC cell lines. F, G Cell proliferation in various CRC groups post-8 Gy irradiation was evaluated using the CCK-8 assay. H, I The CCK-8 assay was performed to evaluate cell proliferation across different radiation doses, including NC and PRKCSH-KD groups. Cell viability was measured at 72 h post-irradiation. J, K The scratch wound healing assay was used to assess the migration ability of HCT116 cells, including NC and PRKCSH-KD groups, at 24 h post-irradiation. L, M The Transwell invasion assay was conducted to evaluate the invasive capacity of HCT116 cells, including NC and PRKCSH-KD groups, at 24 h post-irradiation. Data represent mean ± SD from three independent experiments. Error bars indicate SD. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. Knockdown of PRKCSH improves radiosensitivity of CRC cells.

A–C Representative images and quantitative analysis of colony formation survival assays were performed on PRKCSH knockdown (PRKCSH-KD) and negative control (NC) cells following radiation at 0, 2, 4, and 6 Gy. D–F Apoptosis in colorectal cancer cells from different groups was assessed by flow cytometry at 24 and 48 h post-8 Gy radiation. In (E, F) apoptotic cells include both early and late apoptotic cells, as determined by Annexin V-APC positivity. G Western blot analysis detected the expression of apoptosis-related proteins in NC and PRKCSH-KD cells at various time points post-8 Gy radiation. H The relative Bcl2/Bax ratio was evaluated. Data are presented as mean ± standard deviation from three independent experiments. Error bars indicate standard deviation. Statistical significance is indicated as *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. Knockdown of PRKCSH inhibits radiation-induced ER stress response.

A Representative images of Western Blot of the protein expression in IRE1α/XBP1s pathway in HCT116 cells after 8 Gy IR at different time points. B, D Quantitative analysis of GRP78, p-IRE1α, IRE1α, and XBP1s protein levels. E Relative expression of DGAT2 mRNA in HCT116 cells at different time points post-irradiation. F Representative images of Western Blot of the protein expression in UPR pathway in NC and PRKCSH-KD cells after 8 Gy IR at different time points. G Relative OD values at 450 nm for HCT116 cells after 8 Gy irradiation, treated with NC, PRKCSH-KD, PRKCSH-KD + Vector, PRKCSH-KD + XBP1s, and the IRE1 inhibitor ATF-083010 (25 μM and 50 μM). Data are representative of three independent experiments with similar results. Error bars, SD. **P < 0.01, ***P < 0.001.

Fig. 4. Knockdown of PRKCSH attenuates the radiation-induced DNA damage repair response.

A Comet assay images depict the conditions of NC and KD cells 8 h after exposure to 8 Gy IR. B, C Tail moments in NC and KD cells were quantified using CASP 1.2.3b2 software. D Immunofluorescence staining was employed to detect γ-H2AX foci in HCT116 and RKO cells following 8 Gy IR. E, F The number of γ-H2AX foci per cell was quantified in HCT116 (E) and RKO (F) cells, with 30 cells per group at each time point. G Western blot images show the expression of DNA damage response and cell cycle proteins in HCT116-NC and HCT116-KD cells at various time points post-8 Gy IR. H, I The cell cycle distribution in HCT116 cells, treated with NC and PRKCSH-KD, was assessed at 0 and 24 h post-irradiation. Data are from three independent experiments and are presented with SD error bars. **P < 0.01, ***P < 0.001.

Fig. 5. PRKCSH promotes DNA damage repair by activating the IRE1α/XBP1s pathway.

A ssGSEA analysis reveals a significant positive correlation between the IRE1α/XBP1s signaling pathway and DNA damage repair. B Western blot analysis was performed to detect changes in key protein expression within the IRE1α/XBP1s and DDR pathways in Vector, PRKCSH-OE, PRKCSH-OE+si-NC, and PRKCSH-OE+si-IRE1α groups following ionizing radiation. C Comet assay was conducted to assess DNA damage in HCT116 cells transfected with Vector, PRKCSH-OE, PRKCSH-OE+si-NC, and PRKCSH-OE+si-IRE1α after ionizing radiation. D Tail moments in the different cell groups were quantified using CASP 1.2.3b2 software. Error bars represent SD. ns indicates no significant difference. **P < 0.01, ***P < 0.001.

Fig. 6. PRKCSH inhibits ubiquitinated degradation of p53 via XBP1s to promote DNA damage repair.

A Immunofluorescence was employed to observe the expression and localization of XBP1s and p53 in HCT116 cells immediately and 24 h after 8 Gy irradiation. B PRKCSH, XBP1s, and p53 protein levels in PRKCSH-overexpressing HCT116 cells were measured by Western blot. C Immunoprecipitation of irradiated HCT116-NC and HCT116-PRKCSH-KD cell lysates revealed the co-precipitation of p53 with PRKCSH and XBP1s. D Similarly, XBP1s co-precipitated with PRKCSH and p53 post-irradiation. E Immunofluorescence staining showed the expression and localization of XBP1s (green) and p53 (red) in NC and PRKCSH-KD cells 24 h post-irradiation. F The impact of PRKCSH on p53 ubiquitination was assessed by Western blot. G DNA damage, indicated by γ-H2AX foci per cell, was measured in HCT116 cells transfected with Vector, PRKCSH-OE, PRKCSH-OE+si-NC, PRKCSH-OE+si-XBP1s, and PRKCSH-OE+si-p53 following IR exposure. H Quantitative analysis of γ-H2AX foci per cell was conducted across groups, with 30 cells per group at each time point. Error bars represent SD. “ns” indicates no significant difference; **P < 0.01, ***P < 0.001.

Fig. 7. Inhibition of PRKCSH enhances the sensitivity of colorectal cancer xenograft tumor to radiation in vivo.

A Overview of the animal experiment design. B Photographs of xenograft tumors taken 28 days after 15 Gy radiotherapy in the pelvic cavity. C Tumor volume growth in PRKCSH-NC and PRKCSH-KD groups, with and without ionizing radiation (IR). D Tumor weight measured at the time of sacrifice. E Western blot analysis of the IRE1α pathway and DNA damage repair signaling, showing data from a representative animal in each group. F–J Immunohistochemical images showing γH2AX, TUNEL, and Ki67 staining in tumor sections, based on representative tumors from each group. G–K Quantitative analysis of mean density for γH2AX, TUNEL, and Ki67 in tumors. Data are from three independent experiments. Error bars represent SD. ns indicates no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 8. PRKCSH promotes radioresistance and poor prognosis in rectal cancer.

A PRKCSH expression levels in colon adenocarcinoma (COAD) were analyzed using TCGA datasets, comparing normal tissue (n = 349) with colorectal adenocarcinoma tissue (n = 275). B A survival analysis of colorectal cancer patients was conducted based on PRKCSH expression, comparing high (n = 116) and low expression (n = 322) groups. C Representative patient-derived organoid (PDO) images showing tumor growth in NC and PRKCSH-KD groups at Day 1 and Day 3. PRKCSH knockdown validation was performed via Western blot (top). D Quantitative analysis of relative surface area of PDOs on Day 1 and Day 3. E Immunohistochemical (IHC) staining of PRKCSH in rectal cancer tissues, comparing radiosensitive (TRG = 0–1) and radioresistant (TRG = 2–3) tumors, as well as adjacent normal tissues. F Statistical analysis of PRKCSH-positive cell rates in tumors and adjacent normal tissues. G Comparison of PRKCSH-positive cell rates between radiotherapy-sensitive (TRG: 0–1) and resistant (TRG: 2–3) rectal cancer tissues. H PRKCSH was identified as a regulator of colorectal cancer radiosensitivity, promoting DNA damage repair via the IRE1α/XBP1s signaling pathway. Statistical significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001.