ST6GAL1 is associated with poor response to chemoradiation in rectal cancer

Abstract

Introduction: Colorectal cancer is the third most common cause of cancer death. Rectal cancer makes up a third of all colorectal cases. Treatment for locally advanced rectal cancer includes chemoradiation followed by surgery. We have previously identified ST6GAL1 as a cause of resistance to chemoradiation in vitro and hypothesized that it would be correlated with poor response in human derived models and human tissues.

Methods: Five organoid models were created from primary human rectal cancers and ST6GAL1 was knocked down via lentivirus transduction in one model. ST6GAL1 and Cleaved Caspase-3 (CC3) were assessed after chemoradiation via immunostaining. A tissue microarray (TMA) was created from twenty-six patients who underwent chemoradiation and had pre- and post-treatment specimens of rectal adenocarcinoma available at our institution. Immunohistochemistry was performed for ST6GAL1 and percent positive cancer cell staining was assessed and correlation with pathological grade of response was measured.

Results: Organoid models were treated with chemoradiation and both ST6GAL1 mRNA and protein significantly increased after treatment. The organoid model targeted with ST6GAL1 knockdown was found to have increased CC3 after treatment. In the tissue microarray, 42 percent of patient samples had an increase in percent tumor cell staining for ST6GAL1 after treatment. Post-treatment percent staining was associated with a worse grade of treatment response (p = 0.01) and increased staining post-treatment compared to pre-treatment was also associated with a worse response (p = 0.01).

Conclusion: ST6GAL1 is associated with resistance to treatment in human rectal cancer and knockdown in an organoid model abrogated resistance to apoptosis caused by chemoradiation.

Keywords: Radiation resistance; Rectal cancer; ST6GAL1; Sialylation.

Fig. 1

ST6GAL1 mRNA levels and protein expression increase after chemoradiation in human organoid models. A. Brightfield image of a primary rectal cancer organoid model B. ST6GAL1 mRNA increases after chemoradiation. Representative graph of organoid model. Standard deviation error bars C. ST6GAL1 protein increases after chemoradiation. Representative graph of organoid model. Standard deviation error bars. Representative western blot image of organoid model. 0 Gy no chemoradiation, 5 Gy chemoradiation (5FU+5 Gy).

Fig. 2

Organoids transduced with shRNA for ST6GAL1 have decreased ST6GAL1 protein expression and are less resistant to apoptosis after chemoradiation. A. ST6GAL1 mRNA expression is decreased with targeted knockdown. B. ST6GAL1 protein is decreased with knock down and this persists even after chemoradiation. Representative western blot shown of organoid model. C. Representative images of one organoid model with Cleaved Caspase-3 staining for apoptosis. Images are 4x, scale bar 1mm. C. Representative analysis of 3 experiments of staining an organoid model for percent Cleaved Caspase-3. Minimum of 5 images were taken at high power (60X) for analysis. Standard deviation error bars. One-way ANOVA p < 0.05. CV control vector, KD ST6GAL1 knockdown organoid model.

Fig. 3

ST6GAL-1 is increased in higher grade rectal cancer tumors on TMA. A. Example of cancer cells marked by red dots that are positive for ST6GAL-1 and black dots that are negative. Scale bar is 100 μm. B. Post-treatment specimen Grade 1 response. Scale bar is 20 μm. C. & D. Post-treatment specimens Grade 3 response. Scale bar is 20 μm. Magnification 40X. Figure photos are from tissue microarrays.

Fig. 4

ST6GAL1 is increased in higher grade rectal cancer tumors on TMA. A. Percent ST6GAL1 stained pre-treatment patient samples compared to grade of response. B. Percent ST6GAL1 staining in chemoradiation post-treatment patients compared to grade of response. C. Difference between pre- and post-treatment patient grade of response.