Fig 2: GALNT7 is upregulated in the urine and blood from men with prostate cancer.Detection of GALNT7 in patient urine and blood samples from 3 patient cohorts using pre-validated sandwich ELISA assays. A, B GALNT7 protein levels detected by sandwich ELISA assays in urine and matched plasma samples from men with suspected prostate cancer. Compared to men with benign disease, GALNT7 urine levels are higher in men with prostate cancer (n = 27, unpaired t test, p = 0.082) (AUC 0.78, sensitivity 87%, specificity 58%). Additionally, GALNT7 plasma levels are significantly higher in men with prostate cancer (n = 27, unpaired t test, p = 0.0172)(AUC 0.79, sensitivity 93%, specificity 42%). C GALNT7 levels are significantly higher in plasma samples from men with prostate cancer compared to men with benign disease (n = 305, unpaired t test, p < 0.0001)(AUC 0.83, sensitivity 85%, specificity 65%). D GALNT7 urine levels were monitored via sandwich ELISA assays in 180 men with suspected prostate cancer taking part in the INNOVATE clinical trial [31]. GALNT7 urine levels are significantly higher in men who were diagnosed with prostate cancer, compared to men who received a ‘no cancer’ diagnosis (n = 180, unpaired t test, p < 0.0001). GALNT7 urine level was more accurate than serum PSA at identifying men with prostate cancer (urine GALNT7 AUC: 0.73, sensitivity 85%, specificity 41%)(serum PSA AUC: sensitivity 85%, specificity 38%). E For the 180 men with suspected prostate cancer taking part in the INNOVATE clinical trial, the combination of serum PSA and urine GALNT7 had the greatest diagnostic power to identify those with prostate cancer (AUC 0.76, sensitivity 85%, specificity 58%). F For men taking part in the INNOVATE study that received mpMRI scored Likert 3, GALNT7 levels were significantly higher in men with prostate cancer (n = 59, unpaired t test, p = 0.0001)(AUC 0.78).

Fig 3: GALNT7 promotes prostate tumour growth and correlates with cell cycle and immune signalling pathways in prostate cancer cells.A Knockdown of GALNT7 using shRNA significantly reduces the growth of CWR22RV1 tumours xenografts in a subcutaneous xenograft model. B Upregulation of GALNT7 in PC3 cells significantly increases the growth of subcutaneous xenograft tumours. C Knockdown of GALNT7 decreases prostate cancer cell invasion and upregulation of GALNT7 promotes prostate cancer cell invasion. D RNAseq analysis of CWR22RV1 cells with knockdown of GALNT7 and DU145 cells with upregulated GALNT7 identified 457 genes that dynamically change in response to GALNT7. Gene Ontology analysis of the 457 genes regulated by GALNT7 shows an enrichment of genes with roles in the ‘cell cycle’ and a de-enrichment of genes with roles in ‘immune signalling’. E Proteomics analysis of DU145 cells with upregulation of GALNT7 identified 249 proteins with a significant change in expression levels. Revigo analysis of these 249 proteins identified ‘cell cycle’ and ‘immune signalling’ as significantly enriched pathways. F Analysis of the SU2C mCRPC cohort [33] identified immune related pathways (including interferon gamma response, interferon alpha response, complement, allograft rejection, IL6 signalling, TNFA Signaling via NFKB and inflammatory response) with attenuated enrichment with GALNT7 expression.

Fig 4: GALNT7 can modify O-glycosylation in prostate cancer cells.A Lectin flow cytometry shows PC3 cells with upregulated GALNT7 have increased binding to SBA lectin (which recognises terminal GalNAc or Tn antigen). B Detection of the cancer-associated Tn antigen by immunofluorescence in DU145 cells show upregulation of GALNT7 promotes increased expression of the Tn antigen. C Analysis of extracellular vesicles (EVs) in conditioned media from LNCaP and DU145 cells with knockdown or overexpression of GALNT7 suggests a correlation between GALNT7 and the Tn antigen in the prostate cancer secretome. D Detection of glycosylation of secreted proteins in samples from PC3 control cells or GALNT7-KO cells using GALNT7 bump-and-hole engineering. The active site of GALNT7 was engineered by mutation (IIe and Leu to 2xAla). The mutant (termed BH) uses a chemically modified analogue of the native substrate UDP-GalNAc. Following glycosylation, the chemical modification can be traced by methods of click chemistry.

Fig 5: GALNT7 is upregulated in prostate cancer tissue.Analysis of GALNT7 gene and protein levels in clinical prostate tissue. A GALNT7 mRNA levels in the TCGA PRAD cohort [24] are significantly higher in prostate cancer tissue relative to normal prostate tissue (n = 549, unpaired t test, p = <0.001). B Real-time PCR analysis of GALNT7 in RNA samples extracted from FFPE prostate tissue. GALNT7 is significantly upregulated in cancer relative to benign tissue (n = 12, unpaired t test, p = 0.0174). C Real-time PCR analysis of GALNT7 mRNA in matched normal and prostate tumour samples. GALNT7 is upregulated in tumour tissue relative to matched normal tissue from the same patient (n = 20, paired t test, p = 0.0357). D Immunohistochemistry analysis of GALNT7 protein in a previously published prostate cancer tissue microarray (TMA) [25]. Each section was scored using the Histoscore method [77, 78]. GALNT7 is upregulated in prostate cancer tissue relative to benign prostate hyperplasia tissue (BPH) (n = 147, unpaired t test, p < 0.001). Scale bar is 100 µm. E Immunohistochemistry analysis of GALNT7 protein in a previously published TMA containing matched tumour and normal tissue from the same patient [79]. GALNT7 protein is significantly increased in prostate tumour tissue relative to matched normal tissue from the same patient (n = 200, paired t test, p < 0.001). Scale bar is 100 µm.