Beta catenin and cytokine pathway dysregulation in patients with manifestations of the "PTEN hamartoma tumor syndrome"

Abstract

Background: The "PTEN hamartoma tumor syndrome" (PHTS) includes a group of syndromes caused by germline mutations within the tumor suppressor gene "phosphatase and tensin homolog deleted on chromosome ten" (PTEN), characterized by multiple polyps in the gastrointestinal tract and by a highly increased risk of developing malignant tumours in many tissues. The current work clarifies the molecular basis of PHTS in three unrelated Italian patients, and sheds light on molecular pathway disregulation constitutively associated to PTEN alteration.

Methods: We performed a combination of RT-PCR, PCR, sequencing of the amplified fragments, Real Time PCR and western blot techniques.

Results: Our data provide the first evidence of β-catenin accumulation in blood cells of patients with hereditary cancer syndrome caused by germ-line PTEN alteration. In addition, for the first time we show, in all PHTS patients analysed, alterations in the expression of TNFα, its receptors and IL-10. Importantly, the isoform of TNFRI that lacks the DEATH domain (TNFRSF1β) was found to be overexpressed.

Conclusion: In light of our findings, we suggest that the PTEN pathway disregulation could determine, in non-neoplastic cells of PHTS patients, cell survival and pro-inflammatory stimulation, mediated by the expression of molecules such as β-catenin, TNFα and TNFα receptors, which could predispose these patients to the development of multiple cancers.

Figure 1

Alterations of PTEN gene in PHTS patients. a) Real Time PCR quantification analysis of PTEN mRNA. Real Time RT-PCR quantification analysis was performed for the PTEN messenger. H_1-20: mean value between all healthy samples used as calibrator to measure the relative expression; H₁, H₂, ... H₂₀: healty subject supposedly not deleted for the target genes. The graph shows quantification of the PTEN mRNA of the PHTS patients and twenty healty subjects. b) Copy number quantification of PTEN gene and PTENP1 pseudogene. Real Time PCR quantification analysis was performed for PTEN and PTENP1 genomic sequences. IVS9: amplified fragment inside intron 9 of the PTEN gene; IVS5: amplified fragment at the boundaries of exon 5 and IVS5 of the PTEN gene; PHTS1, PHTS2 and PHTS3: patients affected by PHTS syndrome, as reported in Table 1. H1, H2 and H3: three healthy subjects supposedly not deleted for the target genes.

Figure 2

Molecular alterations of PI3K/Akt and WNT pathways associated with PHTS syndrome. a) Western blot assay of β-catenin protein. H_1-5, H_6-10, H_11-15, H_15-20: mixes of healthy subjects. PHTS1, PHTS2 and PHTS3: patients affected by PHTS syndrome, as reported in Table 1. b) Real Time PCR quantification analysis of COX2, CCND1, cMYC, and APC messengers. H_1-5, H_6-10, H_11-15, H_15-20: mixes of healthy subjects; H_m: mean value between all healthy samples used as calibrator to measure the relative expression; PHTS1, PHTS2 and PHTS3: patients affected by PHTS syndrome, as reported in Table 1.

Figure 3

Cytokine disregulation on peripheral blood cells of PHTS patientsa) Real Time PCR quantification analysis of IL10 and TNFα mRNA. b) Western blot assay of TNFR1 and TNFR2. H_1-5, H_6-10, H_11-15, H_15-20: mixes of healthy subjects; H_m: mean value between all healthy samples used as calibrator to measure the relative expression; PHTS1, PHTS2 and PHTS3: patients affected by PHTS syndrome, as reported in Table 1. c) RT-PCR analysis of the TNFRSF1Aβ isoform. S.M.: genomic molecular weight marker; B: amplified sample without cDNA template. d) sequence analysis of the amplified fragments showed in Figure 3d. The genomic sequence GenBank accession number is: EU927389. The underlined nucleotide are those showed in the electropherogram.