PRR14 overexpression promotes cell growth, epithelial to mesenchymal transition and metastasis of colon cancer via the AKT pathway

Background PRR14 (Proline rich protein 14) was firstly identified for its ability to specify and localize heterochromatin during cell cycle progression. Aberrant expression of PRR14 is associated with the tumorigenesis and progression of lung cancer. However, its involvement in colon cancer remains unknown. Herein, we report the role of PRR14 in colon cancer. Methods Colon cancer tissue microarray was used to analyze and compare the expression of PRR14 among some clinicopathological characteristics of colon cancer. HCT116 and RKO cells were transfected with siRNA to downregulate PRR14 expression. The roles of PRR14 in proliferation, migration and invasion of the cell lines were determined using cell counting kit-8, colony formation assay, wound healing assay and transwell assays respectively. The expression of PRR14 was measured using immunofluorescence, qRT- PCR and western blot. Epithelial-mesenchymal transition (EMT) markers were determined by western blot. Results PRR14 was highly expressed in colon cancer tissues, and the expression level was correlated with tumor size, distant metastasis and Tumor Node Metastasis stages. Functional study revealed that downregulation of PRR14 inhibited colon cancer cells growth, migration and invasion. Furthermore, knockdown of PRR14 inhibited epithelial-mesenchymal transition (EMT) process, cell cycle-associated proteins expression and p-AKT level. Conclusion PRR14 may promote the progression and metastasis of colon cancer, and may be a novel prognostic and therapeutic marker for the disease.

detected it and added a set of images into the manuscript ( Figure 2C).Please kindly find the revision in: Section: Results -Knockdown of PRR14 inhibited cell growth in vitro and in vivo. Pages: 12 Lines:238 Figure:2 The results showed that PRR14 staining was located in the nucleus, nuclear membrane and a small amount in the cytoplasm. Immunofluorescence staining of PRR14 protein has more power in localizing the PRR14 protein cellular level. This result is consistent well with the Proteinatlas database, https://www.proteinatlas.org/ENSG00000156858-PRR14/tissue/colon#img We also inserted magnified figures of PRR14 staining in Figure 1E. It showed that PRR14's localization is mainly in the nucleus. However, we thought the intensive IHC staining of PRR14 in nuclear membrane might cover up the hematoxylin staining in nucleus. Hence, IHC could semi-quantify the PRR14 protein level in the tissue but not cellular level in this study.
2. What is effect of PRR14 knockdown on other signaling pathways such as ERK or JNK. Does PRR14 have a global role in intracellular signaling?
Response: As per your kind suggestions, We examined the effect of PRR14 knockdown on ERK and JNK pathways. Western blot results and semi-quantitative analysis of the histogram showed that no significant differences in phosphorylated and total protein levels of ERK and JNK were found in knockdown and controls cells. The figures and histogram were attached below. Our results indicate that PRR14 might not has close relation with ERK and JNK signaling pathway in HCT116 and RKO cell lines. Therefore, we did not put these results in revised manuscript. 3. What is the cell cycle profile with PRR14 (G1/S block?).

Response:
As per the reviewer's comments and suggestions, we additionally examined the effect of PRR14 expression on cell cycle progression, and the results were put in Figure 3. The results showed that PRR14 knockdown led to G1 phase arrest. Brdu labeling assay showed significant decline in percentage of S phase. Please kindly find the revision in Section: Methods -Cell cycle and apoptosis analysis, immunofluorescent assay Pages:8 and 9 Paragraph: 1 and 5 Lines: 145 and 183 Section: Results -Knockdown of PRR14 resulted in cell cycle arrest at G1 phase Pages: 13 Paragraph: 4 Lines:257 Figure:3 4. Further comment on differences in PRR14 kd in 2 cell lines. E.g., p21/p27 mRNA; despite no change p27 mRNA, protein increased in RKO.

Response:
We appreciate your critical comments on inconsistent expression of P21 and P27 in PRR14 kd in 2 cell lines. We think that the inconsistent mRNA expression of P21 and P27 after PRR14 knockdown indicates that the mechanism of action may be inconsistent between the two cells in mRNA level. But we do not know why and feel sorry for not studying it further since its protein level changes may make sense. The western blot results showed that the expression of P21 and P27 increased after PRR14 knockdown in both cell lines. We speculate that PRR14 plays a more important role in the post-translation level. We reinterpreted this in the paper. Please kindly find the revision in Section: Results -Knockdown of PRR14 affected the expression of cell cycle-related genes and AKT pathway genes. Pages: 16 Paragraph: 3 Lines:311 Section: Discussion Pages: 17 Paragraph: 3 Lines:339 5. Table 2 distant mets n=4 all with high PRR14 (100%), whereas low n=0 yet number in parenthesis is 48.8%; should be 0. How explain P=0.029 for TNM but 0.079 ln mets?
Response: we appreciate the reviewer's meticulous evaluation. We apologize for this mistake.The original Table 2 has been changed to Table 1, The correct number in Table 1 parenthesis is n=0. We have corrected it accordingly. Please kindly find the revision in Section: Results -PRR14 is highly expressed in colon cancer, and this correlates with poor prognosis of the disease. Pages: 11 Paragraph:3 Lines:228 Table:1 Group: Distant metastasis-PRR14 low As for the P value, in TNM staging system, T,N,M represents three indicators respectively: primary tumor, regional lymph node involvement and distant metastasis. TNM staging is a combination of the three indicators. Therefore, the two values may be inconsistent.
6. Tumor xenografts---presumably kd is transient if using siRNA. Level of PRR14 in tumors? How long after siRNA transfection does PRR14 kd last in vitro?

Response:
We greatly appreciate your critical comment in strengthening this study. We tested the interference effect of HCT116 cells on 2 to 10 days after transfection with siRNA, and the results were shown in below ( Figure A). The Western blot results and semi-quantitative analysis showed that PRR14 expression was gradually increased during 10 days following knock down by siRNA, but still significantly lower than the control cells. We think the PRR14 expression would be comparable in siRNA kd cells and control cells if time went on.
Since siRNA could transiently knock down gene expression as mentioned by reviewer and others, and siRNA might not be such effective like shRNA. We were curious about our results, firstly, as per reviewer's kind suggestions, we then detected the expression of PRR14 in subcutaneous tumors by Western blot. We pooled tumors of two groups (No.9 and 10) since their small size. The results showed PRR14 expression was significantly lower in siRNA tumors than controls ( Figure B). We postulated that although some cells restored their PRR14 expression in siRNA kd xenograft cells, their proliferation has been inhibited in the first 10 days following grafting. Therefore, the size in siRNA kd xenograft was smaller than controls. SiRNA has been also used in tumor xenografts in some of qualified studies which further convinced our results were correct. Here we listed several of them. https://www.cell.com/molecular-therapy-family/nucleic-acids/pdf/S2162-2531(16)30300-6.pdf https://www.ncbi.nlm.nih.gov/pubmed/23095742 https://www.nature.com/articles/s41598-017-07973-4 Figure: (A) Western blot was used to test the interference effect of HCT116 cells on 2 to 10 days after transfection with siRNA, and the results were statistically analyzed. (B) Western blot was used to detect the PRR14 expression in subcutaneous tumors, and the results were statistically analyzed. (*: p < 0.05; **: p < 0.01).