The altered activity of P53 signaling pathway by STK11 gene mutations and its cancer phenotype in Peutz-Jeghers syndrome

Materials in this study were collected retrospectively. A total of 154 PJS patients were ascertained in Airforce General Hospital of PLA between May 2013 and April 2016. Blood samples of the probands and all available family members were collected after obtaining informed consent. This study was approved by the Medical Ethics Committee, Airforce General Hospital of PLA. Only PJS patients with both complete information and mutation detected and successfully constructed were included in final analysis.

Clinical diagnosis for PJS was based on the presence of any one of the following clinical findings which was recommended by WHO [22]: (1) three or more histologically confirmed PJ polyps (PJP) in the gastrointestinal tract, (2) any number of PJPs detected with a positive family history of PJS, (3) characteristic mucocutaneous pigmentation with a positive family history of PJS, and (4) any number of PJPs together with characteristic mucocutaneous pigmentation (MP).

Genomic DNA of peripheral blood leucocytes was extracted routinely by animal genomic DNA kit (TSP201, TsingKe Biotech, Beijing, China) according to the manufacturer’s instructions. All 9 coding exons and their flanking sequences of the STK11 gene were amplified by the use of primers listed in Additional file 1: Table. S1. Polymerase chain reactions (PCR) of STK11 exons were performed in a 50-ul reaction which contained 0.4 uM of each primer, 50 ng genomic DNA, and 25ul 2 × modified DNA polymerase mix (TSE004, TsingKe Biotech, Beijing, China). The PCRs were performed under the following conditions: denaturation at 95 °C for 4 min, followed by 35 thermal cycles, each composed of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 45 s.

All available family members underwent STK11 germline mutation test to confirm cosegregation of the mutation with the disease. For frameshift mutation, T vector assay (pClone007 Vector Kit, TSV-007, TsingKe Biotech, Beijing, China) was used to identify each haplotype by constructing monoclonal cells [23]. In order to rule out polymorphisms and to confirm the pathogenic effects of the variations, 100 chromosomes from 50 unrelated ethnicity-matched healthy individuals were also screened for the presence of the mutations. Allele frequency of each mutation in ExAC was checked [24].

The PCR products were gel- and column-purified and directly sequenced. The purified PCR fragments were then sequenced using BigDye Terminator on an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA) by Majorbio Co. Ltd. (Shanghai, China). The results were used to performance sequence alignment according to STK11 gene sequence (NP_000446.1 and NM_000455.4 in GRCh38.p7). All experiment details above had been reported previously [23, 25].

To predict the effects of mutations in STK11, PolyPhen-2 (http://​genetics.​bwh.​harvard.​edu/​pph2) and SIFT (http://​sift.​jcvi.​org/​) were used for specific mutations.

To investigate the impact on P53 activity, we employed dual-luciferase reporter gene technique. Human STK11 cDNA was amplified from the HEK293T cells, and cloned into pcDNA3.1 (+) (V790–20, Carlsbad, CA) vector to create expression vectors of wild type (wt) and mutant (mu) of STK11 gene. The mutant site was generated by site-directed substitution (D0206, Beyotime, Nantong, Jiangsu, China).

The luciferase reporter plasmid pp53-TA-luc which contained a P53-responsive element (D2223, Beyotime, Nantong, Jiangsu, China) was transfected into the cells, together with pRL-TK vector (E2241, Promega, Madison, WI) and pcDNA3.1(+)-STK11 expression vector, using the Lipo6000 transfection reagent (C0526, Beyotime, Nantong, Jiangsu, China). pRL-TK vector expresses wildtype Renilla luciferase reporter and is used as internal control by cotransfected with pp53-TA-luc. Twenty-four hours after transfection, cells were lysed in a passive lysis buffer, which was a component of Dual-Luciferase® Reporter Assay Systems (E1910, Promega, Madison, WI) [26]. According to the manufacturer’s protocol, luciferase activity was measured with the Synergy 2 modular multi-mode reader (BioTek Instruments, Winooski, VT). The ratio of pp53 to pRL-TK was calculated in each assay. Each experiment was repeated triple times from cell seeding to luciferase activity measurement. Data were showed as mean ± standard deviation. Student’s t test was used to test the significance, and p < 0.05 was considered as significant.

As shown in Table 1, the 13 probands from unrelated families were 7 familial and 6 sporadic patients, and their native places were various in China (Additional file 1: Table S2). The median age of diagnosis, first polypectomy and at present was 25, 28 and 33 years. Seven of these families (PJS03, 05, 06, 07, 09, 10 and 12) showed autosomal dominant pattern and the remaining 6 cases (PJS01, 02, 04, 08, 11 and 13) are sporadic (Fig. 1a). MP on the lips, buccal mucosa, and digits were observed at all patients (Fig. 1b). Twelve probands in these families underwent laparotomy or polypectomy due to intussusception or intestinal obstruction (Fig. 1c and d). Three probands of these families (PJS03, 06 and 11) in this study had developed colon cancer before 40 years old (Fig. 1e), and cancer history existed in 3 families (PJS06, 09 and 12). Detail information is showed in Table 2.

Sequencing analysis identified 13 mutations of STK11 gene in the 13 probands which were included in the function analysis (Table 2, Fig. 2a). Most of the mutations have been previously reported [25, 27‐29] except for c.924G > A (Fig. 3). The 13 mutations included 8 truncating mutations (4 frameshift and 4 nonsense) and 5 missense mutations. Regarding frameshift mutations, those of PJS05, 06 and 07 (p.P281Rfs*6, p.P281Pfs*4 and p.L285Lfs*2) led to a partial loss of the kinase domain and a completed loss of the C-terminal, and the one in PJS11 (p.P321Hfs*38) led to a partial loss of the α-helix of the C-terminus. PJS02, 03, 04 and 10 possessed nonsense mutations of STK11 that resulted in the production of truncated proteins (p.E65*, p.K84*, p.Y118* and p.W308*) (Additional file 2: Figure S2A). Among the PJS cases owning the 13 mutations, 7 were familial and 6 were sporadic, and 5 of them were associated with cancer history. Tested in 100 chromosomes from control individuals, 16 of the detected mutations were not discovered and only c.1062C > G was detected in 5/100 controls. c.1062C > G and c.1225C > T were recorded in ExAC database with allele frequencies of 0.005757 and 0.000147, and all other mutations were not recorded (Table 2).

Analyzed using PolyPhen-2, missense mutation c.911 was predicted as probably damaging, mutations c.56, c.862 and c.1225 were predicted as possible damaging, while c.1062 was benign (Table 2).

Analyzed using SIFT, missense mutation c.56 and c.1225 were predicted as intolerated, and mutations c.862, c.911 and c.1062 were tolerated (Table 2).

P53 activity was measured using dual-luciferase reporter gene technique after cotransfection of pp53-TA-luc, pRL-TK vector, and pcDNA3.1 (+)-STK11-wt or -mu. Luciferase activity was significantly elevated when STK11-wt was transfected into HEK293T cells. Using the elevation caused by STK11-wt as control, elevations by mutants c.193, c.250, c.354, c.842, c.843 and c.855 were significantly reduced, while those by mutants c.56, c.862, c.911, c.924, c.962_963, c.1062 and c.1225 were not changed significantly (Fig. 2b).

There were total 5 families with cancer history (PJS03, 06, 09, 11 and 12). Within the 5 cancer families, P53 activity was reduced and cancer occurred before 40 in 2 families (PJS03 and 06), while in the other 3 families, P53 activity was not significantly changed and cancers happened after 45 years old (PJS09, 11 and 12) (Table 3).

Through Fisher exact test, none of the factors (sex, family history and P53 activity change) was significantly associated with cancer in PJS (Additional file 1: Table S3).