Plk2 Loss Commonly Occurs in Colorectal Carcinomas but not Adenomas: Relationship to mTOR Signaling

Abstract

Plk2 is a target of p53. Our previous studies demonstrated that with wild-type p53, Plk2 impacts mTOR signaling in the same manner as TSC1, and Plk2-deficient tumors grew larger than control. Other investigators have demonstrated that Plk2 phosphorylates mutant p53 in a positive feedback loop. We investigated Plk2's tumor suppressor functions in relationship to mTOR signaling. Archival specimens from 12 colorectal adenocarcinomas were stained for markers including Plk2, phosphorylated mTOR (serine 2448) and ribosomal S6 (Serine 235/236). We show that Plk2 is expressed in normal colon, with a punctate staining pattern in supranuclear cytoplasm. In colorectal adenocarcinoma, Plk2 demonstrates complete or partial loss of expression. Strong expression of phosphorylated mTOR is observed in the invasive front. Phosphorylated S6 expression partially correlates with phosphorylated mTOR expression but appears more diffuse in some cases. p53 and Ki67 expression is diffuse, in the subset of cases examined. In order to determine whether Plk2 is lost prior to the development of invasive cancer, 8 colon polyps from 6 patients were evaluated for Plk2 expression. All polyps are positive for Plk2. A Cancer Genome Atlas search identified Plk2 mutations to be infrequent in colorectal adenocarcinomas. Neither Plk2 methylation (in the gene body) nor copy number variations correlated with changes in mRNA expression levels. Loss of Plk2 expression along with accentuated expression of phosphorylated mTOR and phosphorylated S6 at the invasive front in some colorectal carcinomas is consistent with previous findings that an interaction between Plk2 and TSC1 / mTOR signaling molecules plays a role in tumor suppression. Plk2 protein expression is lost at the same stage in colorectal carcinogenesis as p53. The p53 dependence of Plk2 loss and tumor suppressor function in relationship to mTOR signaling may have therapeutic implications.

Figure 1

Plk2 loss in colorectal adenocarcinomas. Plk2 expression is punctuated in distinct areas / intracellular structures adjacent to the nucleus (arrows). A) Normal adjacent colon (patient 1), 400×), B) Colorectal adenocarcinoma: center of the tumor, the area with the greatest Plk2 loss (stage IV, patient 1), 400×. Plk2 loss was more prominent in C) some areas of the tumor than D) other areas (stage IIIB, patient 11). Panels A-B are from one patient with stage IV colorectal adenocarcinoma shown in Figure 2, A–D. Proteins detected by antibody: brown color; Hematoxylin nuclear counterstain: blue.

Figure 2

The mTOR pathway is activated in colorectal adenocarcinomas. A) P~mTOR ^{Ser 2448} in the colorectal adenocarcinoma invasive front (stage IIIB, patient 11), 400×. B) Lower power version of A, 100×. C) P~mTOR ^{Ser 2448} in the colorectal adenocarcinoma invasive front (stage IV, patient 1), 200×. D) P~S6 ^{Ser 235/236} in the colorectal adenocarcinoma invasive front (stage IIIB, patient 11), 400×. E) Lower power version of D, 100×. F) P~S6 ^{Ser 235/236} in the colorectal adenocarcinoma invasive front (stage IV, patient 1), 200×. Phospho-protein detected by antibody: reddish-brown color; Hematoxylin counterstain of nuclei: blue. Arrows point to invasive front.

Figure 3

Ki67 and p53 expression are diffuse in colorectal adenocarcinomas. A) p53 protein expression, 200×, B) Ki67 protein expression (Ki67 proliferation index= 85%), 200×. Tissue sections are from the same representative stage IV colorectal adenocarcinoma tumor (patient 1).

Figure 4

Plk2 is present in tubular adenomas. Plk2 expression in A) Adjacent normal and B) Tubular adenoma (patient E), 400×, C) Adjacent normal and D) Tubular Adenoma of a different patient than panels A & B (patient F), 400×. Center top panel: Adjacent normal (patient F), 200×; Center bottom panel: zoom section of Panel B.

Figure 5

Plk2 presence in tubular adenomas coincides with the absence of activated mTOR. A) Plk2 expression in tubular adenoma (patient C), 200×, B) Phosphorylated mTOR ^ser2448 is negative in the same tumor area as panel A, 200×.

Figure 6

Plk2 gene body methylation and mRNA expression remain unaltered in colorectal cancer as compared to normal: TCGA. Plk2 Gene methylation normal versus colorectal tumors: A) 5′UTR to the First Exon (P=0.2), B) Transcription Start Site (TSS)(P=0.9), C) Gene Body (P<0.001, and D) Plk2 mRNA expression (RSEM values) in normal versus colorectal tumors (P=0.003). Panels A-C: RNA seq by expectation-maximization (RSEM). Beta-values above 0.4 were considered methylated. Cancer Genome Atlas (TCGA) search yielded 312 colorectal cancers with methylation data of which 305 cases also had mRNA data.

Figure 7

Plk2 mRNA expression does not correlate with Plk2 gene methylation or copy number variation: TCGA. A) Plk2 mRNA expression (RSEM values) versus 5′UTR methylation (Beta values), B) Plk2 mRNA expression (RSEM values) versus transcription start site (TSS) methylation and C) Plk2 mRNA expression (RSEM values) versus gene body methylation. D) Plk2 mRNA expression versus copy number variation (CNV). Top panels: normal; Bottom panels: colorectal cancer. Cancer Genome Atlas (TCGA) search identified 312 colorectal cancers with methylation data of which 305 cases also had mRNA expression data. Of the 309 cases with copy number variation (CNV) data and 305 cases with mRNA data, 302 cases over-lapped. A-C: 309 patients. D: 302 patients. RNA seq by expectation-maximization (RSEM); Gene methylation expressed as Beta-values.

Figure 8

Structural analysis of human Polo-like kinase 2. A). Domain schematic of PLK2. Experimental structures are denoted covering the kinase domain (residues 71-335) and its ordered C-terminal tail (dark blue, residues 336-355) and the Polo-box domains and adjacent ordered N-terminal tail and linkers (residues 469-682). The graph shows the prediction of disordered regions with the program IUPred. Values over 0.5 are predicted to be disordered and are marked in red in the domain schematic. Mutations in TCGA colorectal tumors are marked. B). Modeled structure of full length PLK2 based on structural superposition of the kinase domain (PDB: 4I6H) and the Polo-box domains (PDB: 4R6S) onto the experimental structure of zebrafish PLK1 (PDB: 4J7B). The zebrafish structure provides the relative orientation of the kinase and Polo-box domains of Polo-like kinases. In addition, the structure of the phosphopeptide bound Polo-box domains of human PLK1 (PDB: 1Q4K) was superimposed in order to locate the likely position of phosphopeptides bound to the Polo-box domains of human PLK2. The phosphopeptide is shown in green spheres. The N and C-terminal helices of the Polo-box region are indicated in yellow and dark-red respectively, adjacent to the second Polo-box domain.