Peripheral blood samples of PJS patients (n=21) from 6 PJS families and 5 sporadic cases were collected between January 2012 and September 2015 at Nanfang Hospital (Guangzhou, China). The patients were diagnosed according to previously published criteria (6). Endoscopic or surgical polypectomy and histopathological examination (by ≥2 pathologists) were performed in all patients. Endoscopic or surgical polypectomy were performed in all patients. The tissues were fixed in 10% formalin solution for 24 h at 4°C and embedded in paraffin for 0.5 h at 65°C. Serial sections (4 µm) were cut and prepared for hematoxylin-eosin (HE) staining and immunohistochemistry, as previously described (6). All HE-stained sections were reviewed by two experienced pathologists independently, who defined the hamartomatous polyps. Surgical resection was performed on other organs, including the breasts and cervix, in 3 patients due to the presence of benign or malignant tumors. The family cancer history was collected from the patients' medical records. All patients provided informed consent for this study, and the principles outlined in the Declaration of Helsinki were followed. Ethical approval was obtained from the Medical Ethics Committee at Nanfang Hospital.

Genomic DNA was extracted from peripheral blood using the commercially available QiAamp DNA Blood Midi kit (Qiagen GmbH, Hilden, Germany). PCR primers were designed using the software program Primer (version 3.0; http://frodo.wi.mit.edu/primer3/) to amplify the STK11 exons (RefSeq accession number NM_000455.4) and intron-exon boundaries. Sanger sequencing was performed for each index case in the 6 familial and 5 sporadic cases, as previously described (6).

All STK11 exons (exon1-9) were amplified (for primers see Table I) using regular Taq polymerase (Takara Ex Taq; Takara Bio, Inc., Otsu, Japan). PCR amplification comprised an initial denaturation cycle at 95°C for 15 min, followed by 38 amplification cycles consisting of 95°C for 30 sec, annealing at 60°C for 45 sec, and extension at 72°C for 7 min. Sanger sequencing was performed for each index case in the 6 familial and 5 sporadic cases, as previously described (6). In familial cases, identified mutations were further tested in all available family members to confirm segregation of the mutation with the disease. All variants identified were screened against the single nucleotide polymorphism database (www.ncbi.nlm.nih.gov/SNP) to exclude the possibility of these variants being polymorphisms.

An MLPA assay was performed for large intragenic deletions using the MLPA test kit (SALSA P101-B1 STK11; MRC-Holland, Amsterdam, Netherlands) as previously described (6). Deletion screening was performed according to the manufacturer's instructions, and the results were analyzed using the GeneMarker^®HID STR Human Identity software (version 3.0; SoftGenetics, LLC., State College, PA, USA). Values of 0.85 to 1.15 indicate normal results (presence of two copies), and values of 0.35 to 0.65 or 1.35 to 1.65 indicate a deletion or duplication, respectively. All identified deletions were confirmed in a second independent reaction and confirmed to segregate within the relevant family members.

The novel missense mutations were all predicted using the PolyPhen-2 web tool (PolyPhen2: Http://genetics.bwh.harvard.edu/pph2/). The PolyPhen-2 score represents the probability that a substitution is damaging. The novel missense mutation (p.Leu290Pro) is predicted to be highly likely to be damaging with a score of 1.0, and the other (p.Asp30Asn) is predicted to be possibly damaging with a score of 0.775. Variants with scores of 0.0 are predicted to be benign. Values closer to 1.0 are more confidently predicted to be deleterious.

Immunohistochemistry (IHC) was performed as previously described (6). Tissue sections were deparaffinized and incubated with a phosphorylated (p)-S6 (Ser240/244) primary antibody (1:400; catalog no. 2903; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. After three washes with PBST (2,000 ml PBS+4 ml Tween 20), the sections were incubated with biotin-conjugated goat anti-rabbit immunoglobulin G (1:300; catalog no. SPN-9001; Invitrogen; Thermo Fisher Scientific, Inc.) for 10 min at room temperature, and then incubated with streptavidin-peroxidase for 10 min at room temperature. A known p-S6 positive colorectal cancer tissue, from one patient who had undergone endoscopic at Nanfang Hospital. HE stained specimens were reviewed by a senior pathologist, who determined the polyp histology and tumor type, was used as a positive control for p-S6 staining. p-S6 staining was scored as positive when >10% of the cells exhibited nuclear expression.

Total RNA was extracted using Trizol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and cDNA was synthesized by reverse transcription from 1 µg total RNA using oligo(dT) primers (Promega Corporation, Madison, WI, USA). RT-qPCR was performed using the All-in-One^™ qPCR mix (GeneCopoeia Inc., Rockville, MD, USA) on a LightCycler 480 system (Roche Diagnostics, Basel, Switzerland). Primer sequences for STK11 were as follows: Forward, 5′-AGGGCCGTCAAGATCCTCAA-3′ and reverse, 5′-GCATGCCACACACGCAGTA-3′. Primer sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: Forward, 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse, 5′-GAAGATGGTGATGGGATTTC-3′. The housekeeping gene GAPDH was used as an internal control for normalization. The relative expression of STK11 was calculated using the 2^−ΔΔCq method (23). Experiments were repeated three times and the mean of the data was determined.

Total protein was extracted using radioimmunoprecipitation assay lysis buffer with protease and phosphatase inhibitors (both, 1:100; Roche, Nutley, NJ, USA). Total protein was quantified using bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). Total protein (25 µg) was resolved by 10% SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% BSA (Bio-Rad Laboratories, Inc.) in PBS with 0.1% Tween-20 for 2 h at room temperature. Membranes were incubated with primary antibodies against STK11 (1:1,000; catalog no. 3047) S6K1 (1:1,000; catalog no. 2903) S6 (1:1,000; catalog no. 1155), p-S6K1 (Thr389; 1:1,000; catalog no. 9234) and p-S6 (Ser240/244; 1:1,000; catalog no. 2903) (all Cell Signaling Technology, Inc.) overnight at 4°C. Mouse polyclonal anti-GAPDH antibody (1:3,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used as the loading control. The membranes were subsequently incubated for 2 h at room temperature with either horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2,000; catalog no. 2903; Cell Signaling Technology, Inc.) or HRP-conjugated goat anti-mouse IgG (1:5,000; catalog no. sc-2005; Santa Cruz Biotechnology, Inc.). Protein expression was detected using an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.).

Human colon cancer (SW1116, HT29, SW620, SW480, LoVo, Caco2, and HCT116), human cervical carcinoma (HeLa), normal human gastric epithelial (GES-1), and human kidney (293T) cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and maintained in a humidified atmosphere of 5% CO₂ at 37°C. All cell lines used in the present study were obtained from the American Type Culture Collection, Rockville, MD, USA.

SW117 and HeLa cells were used for transfection and subsequent experiments. The sequences of all STK11 gene mutant plasmids were confirmed by genetic sequencing. The constructs pGCMV-green fluorescent protein (GFP) (empty vector), pGCMV-GFP-STK11 WT (wild-type), pGCMV-GFP-STK11 p.Leu290Pro and pGCMV-GFP-STK11 p.Asp30Asn were synthesized by Shanghai GenePharma Co., Ltd., (Shanghai, China). Cells were seeded (4×10⁵) in tissue culture plates and grown to 70 to 80% confluence. Subsequently, the cells were transfected with the vectors using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Gene overexpression was examined by RT-qPCR, and protein expression was analyzed by western blotting at 24 to 48 h post-transfection. Stable transfections were performed at 48 h post-transfection, and the transfected cells were grown with G418 (400 mg/ml; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) for 4 to 8 weeks to allow for positive selection.

To determine the effect of novel STK11 mutants on cell proliferation, transfected cells were measured using CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocols. Proliferation was assessed by colony formation assay. Transfected cells were counted and seeded into 6-well plates (1,000 cells/well). The number of colonies containing >50 cells were stained with 0.5% crystal violet in isopropanol and counted 2 weeks after seeding. The relative cell colony numbers were evaluated as the ratio of the last visible colonies to those at initialization.

Cell apoptosis was quantitated using the Annexin V-APC and 7-aminoactinomycin D (7-AAD) double dye cell apoptosis kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Cells (1×10⁶ cells/well) were collected at 48 h post-transfection with the aforementioned STK11 plasmids and washed twice with ice-cold PBS. The cells were re-suspended in 500 µl 1X binding buffer, stained with 5 µl Annexin V-APC and 5 µl 7-AAD and incubated on ice for 15 min in the dark. Apoptotic cells (10,000 cells/test) were quantified by flow cytometer (BD Biosciences).

All statistical analyses were performed using SPSS (version 17.0; SPSS Inc., Chicago, IL, USA). The data are expressed as the mean ± standard deviation unless otherwise noted. Comparisons among multiple groups were determined using one-way analysis of variance, followed by least significance difference or Dunnett's T3 test. P<0. 05 was considered to indicate a statistically significant difference.

The present study identified 6 different germline point mutations in 6 out of 11 index cases (52.4%; Table II). A number of mutations (3/6, 50%) were associated with colon or esophagus cancer in either index patients or in relatives with PJS. A total of 4 mutations were identified in familial cases (4/6, 66.7%) and 2 in sporadic cases (2/5, 40%) of PJS. Of the 6 mutations identified, 4 were truncation mutations and 2 were missense mutations. To the best of our knowledge, 3 of the mutations [c.143_144insA (p.Try49Valfs*114), c.869T>C (p.Leu290Pro) and c.88G>A (p.Asp30Asn)] have previously been unreported and are therefore novel (Fig. 1; Table II).

MLPA was used to determine the presence or absence of exonic rearrangements in the 5 PJS probands, where no mutations were identified by Sanger sequencing. This resulted in the identification of two large deletions, with one being associated with the presence of breast cancer in the relative of the proband (PJS7; Table II). The two deletions were predicted to affect the kinase domain of the protein. The total mutation detection rate in the index cases was 72.7% (8/11), which indicated that genetic alterations in STK11 in individuals with PJS may increase the risk of developing PJS-associated cancer.

In total, 3 of the truncation mutations identified in the present study have previously been described (7,24,25), and 1 is novel [c.143_144insA (p.Try49Valfs*114)] (PJS2; Table II). The novel mutation involves an insertion of nucleotide A between nucleotides 143 and 144 in exon 1. This results in a 433-amino acid sequence with a frameshift after nucleotide 49 and premature termination at nucleotide 114. Therefore, the alteration leads to a complete loss of the kinase domain of STK11 (26).

The novel missense mutation [c.869T>C (p.Leu290Pro)] leads to an amino acid change at codon 290 in exon 7, which is located in the catalytic kinase region of STK11 (PJS4 II-3; Fig. 1C; Table II). The other novel missense mutation [c.88G>A (p.Asp30Asn)] leads to an alteration at nucleotide 88 (G<A) and an amino acid change at codon 30 in exon 1, which is located at the N-terminal of STK11 (PJS5 III-2; Fig. 1D; Table II). This results in the substitution of aspartate by asparagine. The two mutations were predicted to be pathogenic by PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/).

To investigate whether the novel missense mutations affect the phosphorylation of mTOR pathway key downstream target gene S6, IHC was performed to detect p-S6 (Ser240/244) expression in colon hamartomatous polyp and colorectal mucosal epithelium tissues obtained from individuals with or without STK11 mutations. p-S6 staining was observed in the nuclei of epithelial cells in colonic hamartoma obtained from patient PJS4 II-3 with the mutation c.869T>C (p.Leu290Pro) (Fig. 2B) and in the breast intraductal papillary tumor from the patient PJS5 III-2 with the mutation c.88G>A (p.Asp30Asn) (Fig. 2E). By contrast, p-S6 staining was not observed in colorectal mucosa derived from PJS4 III-3, who is a relative of PJS4 II-3 (Fig. 2C) and in breast hyperplasia glandular epithelial obtained from PJS5 II-2, who is a relative of PJS5 III-2 (Fig. 2F). PJS4 III-3 and PJS5 II-2 did not carry STK11 mutations. These findings suggest that the two novel missense mutations may disrupt the protein function of STK11 serine/threonine kinase and induce S6 phosphorylation.

RT-qPCR and western blotting were used to detect endogenous expression levels of STK11 in human cell lines. As shown in Fig. 3A, STK11 expression levels were nearly undetectable in HeLa and SW1116 cells when compared with all other tested cell lines. Subsequently, HeLa and SW1116 cells were used for further studies, and the effects of the novel STK11 mutations were investigated.

STK11 plasmids (empty vector, wild-type vector, STK11 mutant p.Leu290Pro vector and STK11 mutant p.Asp30Asn vector) were transfected into HeLa and SW1116 cells, and STK11 expression was examined by RT-qPCR and western blot analysis. As shown in Fig. 3B and C, the expression of STK11 in HeLa and SW1116 cells transfected with the STK11 mutants and the wild-type vector was significantly increased when compared with cells transfected with the empty vector. However, there were no significant differences in STK11 expression in cells transfected with the wild-type vector and the STK11 mutants. Taken together, these results suggest that the novel STK11 mutants have negligible effects on STK11 transcription and translation.

The effects of the two novel STK11 missense mutations on gene function and whether the mutations result in dysregulation of the downstream mTOR-mediated pathway was investigated. S6K1 and S6 are substrates of mTORC1. Phosphorylation of S6K1 at Thr389 and S6 at Ser240/244 was analyzed using western blotting. As shown in Fig. 4, there was increased expression of p-S6K1 (Thr389) and p-S6 (Ser240/244) in HeLa and SW1116 cells transfected with the two novel missense STK11 mutants compared with cells transfected with the empty vector. By contrast, cells transfected with the wild-type vector exhibited a marked decrease in the levels of p-S6K1 and p-S6. These results demonstrated that the novel STK11 missense mutations disrupted the endogenous protein kinase activity of STK11, which led to the activation of the mTORC1 signaling pathway. Consequently, this results in the dysregulation of the STK11-S6K1 axis. We hypothesized that this disruption leads to abnormal de-repression of protein synthesis and promotion of cell growth.

To further determine whether the novel missense STK11 mutations are able to effectively suppress cell growth and proliferation, CCK-8 and colony formation assays were performed. As shown in Fig. 5, HeLa and SW1116 cells transfected with wild-type vectors exhibited a marked reduction in cell growth compared with cells transfected with the STK11 mutant vectors. The growth of HeLa and SW1116 cells transfected with the STK11 mutants (p.Leu290Pro and p.Asp30Asn) was significantly increased (P<0. 01) when compared with the empty vector control (Fig. 5A and C). These results suggest that STK11 gene is a tumor suppressor gene in the two cell lines. The two novel STK11 mutations abrogated cellular growth suppression function of the STK11 gene, which contributes to tumorigenesis. Furthermore, the effect of the novel STK11 missense mutations on cell apoptosis was investigated. Compared with the empty vector control, the wild-type vector promoted apoptosis. However, there was no statistically significant difference between the two novel missense STK11 mutants and the control (data not shown). These findings indicate that although the STK11 gene exerts a role in promoting apoptosis in HeLa and SW1116 cells, the two novel STK11 missense mutations have no significant effect.