Immunohistochemical analysis of the mechanistic target of rapamycin and hypoxia signalling pathways in basal cell carcinoma and trichoepithelioma

Background: Basal cell carcinoma (BCC) is the most common cancer in Caucasians. Trichoepithelioma (TE) is a benign neoplasm that strongly resembles BCC. Both are hair follicle (HF) tumours. HFs are hypoxic microenvironments, therefore we hypothesized that hypoxia-induced signalling pathways could be involved in BCC and TE as they are in other human malignancies. Hypoxia-inducible factor 1 (HIF1) and mechanistic/mammalian target of rapamycin (mTOR) are key players in these pathways. Objectives: To determine whether HIF1/mTOR signalling is involved in BCC and TE. Methods: We used immunohistochemical staining of formalin-fixed paraffin-embedded BCC (n = 45) and TE (n = 35) samples to assess activity of HIF1, mTORC1 and their most important target genes. The percentage positive tumour cells was assessed manually in a semi-quantitative manner and categorized (0%, 80%). Results: Among 45 BCC and 35 TE examined, expression levels were respectively 81% and 57% (BNIP3), 73% and 75% (CAIX), 79% and 86% (GLUT1), 50% and 19% (HIF1 alpha), 89% and 88% (pAKT), 55% and 61% (pS6), 15% and 25% (pMTOR), 44% and 63% (PHD2) and 44% and 49% (VEGF-A). CAIX, Glut1 and PHD2 expression levels were significantly higher in TE when only samples with at least 80% expression were included. Conclusions: HIF and mTORC1 signalling seems active in both BCC and TE. There are no appreciable differences between the two with respect to pathway activity. At this moment immunohistochemical analyses of HIF, mTORC1 and their target genes does not provide a reliable diagnostic tool for the discrimination of BCC and TE

Immunohistochemical stains for hypoxia inducible factor 1α (HIF1α), phosphorylated mechanistic/mammalian target of rapamycin (pmTOR) and their target genes in basal cell carcinoma (BCC).

<p>BCL-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) (<b>a</b>); carbonic anhydrase IX (CAIX) (<b>b</b>); glucose transporter 1 (GLUT1) (<b>c</b>); HIF1α (<b>d</b>); phosphorylated protein kinase B (pAKT) (<b>e</b>);phosphorylated ribosomal protein S6 (pS6) (<b>f</b>); pmTOR (<b>g</b>); prolyl hydroxylase domain protein 2 (PHD2) (<b>h</b>); vascular endothelial growth factor (VEGF-A) (<b>i</b>). Original magnification: (a–i) x 200.</p

Simplified depiction of pathways affected by hypoxia.

<p>Under hypoxic conditions the expression of prolyl hydroxylase domain proteins (PHDs) is reduced leading to an induction of hypoxia inducible factor 1α (HIF1α) expression, which becomes stable and active as a transcription factor, together with hypoxia inducible factor 1β (HIF1β). HIF1 activation regulates the expression of several target genes whose products address the needs of oxygen starved cells, such as vascular endothelial growth factor (VEGF-A), BCL-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3), glucose transporter 1 (GLUT1) and glycolytic enzymes such as carbonic anhydrase IX (CAIX). Hypoxia also influences mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signalling mainly mediated through hypoxic activation of the TSC1-TSC2 complex by REDD1. First, phosphatidylinositol 3 kinase (PI(3)K) and protein kinase B (AKT) have been implicated in the activation of the mTOR protein kinase. One critical target of AKT that regulates mTOR is the tumour suppressor protein, tuberin (TSC2). Tuberin negatively regulates mTOR signalling, and AKT activation circumvents this inhibition. Constitutive mTOR signalling positively stimulates S6 kinase (S6K), a downstream effector of mTOR pathway, which mainly drives cell growth and proliferation. Also, mTOR enhances the protein levels of HIF and consequently enhances the expression of HIF target genes. Second, under conditions of hypoxia, intracellular ATP levels drop and AMP levels rise. AMP directly binds to a subunit of AMP activated protein kinase (AMPK), which is then phosphorylated by serine/threonine protein kinase 11 (STK11/LKB1). Elevated concentrations of AMPK can cause a complete inhibition of mTOR (mTORC1) activity without affecting PI(3)K-AKT signalling.</p

Tumour characteristics.

<p>*Date are presented as n (%).</p><p>Tumour characteristics.</p

The expression levels and staining intensity of BNIP3, CAIX, GLUT1, Hif1α, pAKT, PHD2, pmTOR, pS6 and VEGF-A in hair follicle tumours.

<p>BNIP3, BCL-2/adenovirus E1B 19 kDa-interacting protein 3; CAIX, carbonic anhydrase IX; GLUT1, glucose transporter 1; Hif1α, hypoxia inducible factor 1α; pAKT, phosphorylated protein kinase B; pS6, phosphorylated ribosomal protein S6, pMTOR, phosphorylated mechanistic target of rapamycin; PHD2, prolyl hydroxylase domain protein 2; VEGF-A, vascular endothelial growth factor; BCC, basal cell carcinoma; TE, trichoepithelioma. Expression levels were graded semiquantitatively as 0%, 1–30%, 30–80% or >80% positive tumour cells.</p><p>The expression levels and staining intensity of BNIP3, CAIX, GLUT1, Hif1α, pAKT, PHD2, pmTOR, pS6 and VEGF-A in hair follicle tumours.</p

Percentage of positive specimens between basal cell carcinoma and trichoepithelioma.

<p>Panel a represents all tissue samples being either positive or negative. In panel b the same results are shown, however here a cut off value of 80% of the tumour cells being positive was used. * (P<0·05), basal cell carcinoma (BCC); trichoepithelioma (TE).</p

Antibodies used for immunohistochemical analysis.

<p>BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), carbonic anhydrase IX (CAIX), glucose transporter member 1 (GLUT1), hypoxia-inducible factor 1-alpha (HIF1α), phosphorylated -protein kinase B (pAKT), phosphorylated-S6 (pS6), phosphorylated-mechanistic target of Rapamycin (pMTOR), prolyl hydroxylase domain protein 2 (PHD2), vascular endothelial growth factor (VEGF-A).</p><p>Antibodies used for immunohistochemical analysis.</p

Novel loci for adiponectin levels and their influence on type 2 diabetes and metabolic traits: A multi-ethnic meta-analysis of 45,891 individuals

Circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are highly heritable and are inversely associated with type 2 diabetes mellitus (T2D) and other metabolic traits. We conducted a meta-analysis of genome-wide association studies in 39,883 individuals of European ancestry to identify genes associated with metabolic disease. We identified 8 novel loci associated with adiponectin levels and confirmed 2 previously reported loci (P = 4.5×10−8- 1.2 ×10−43). Using a novel method to combine data across ethnicities (N = 4,232 African Americans, N = 1,776 Asians, and N = 29,347 Europeans), we identified two additional novel loci. Expression analyses of 436 human adipocyte samples revealed that mRNA levels of 18 genes at candidate regions were associated with adiponectin concentrations after accounting for multiple testing (p<3×10−4). We next developed a multi-SNP genotypic risk score to test the association of adiponectin decreasing risk alleles on metabolic traits and diseases using consortia-level meta-analytic data. This risk score was associated with increased risk of T2D (p = 4.3×10−3, n = 22,044), increased triglycerides (p = 2.6×10−14, n = 93,440), increased waist-to-hip ratio (p = 1.8×10−5, n = 77,167), increased glucose two hours post oral glucose tolerance testing (p = 4.4×10−3, n = 15,234), increased fasting insulin (p = 0.015, n = 48,238), but with lower in HDL- cholesterol concentrations (p = 4.5×10−13, n = 96,748) and decreased BMI (p = 1.4×10−4, n = 121,335). These findings identify novel genetic determinants of adiponectin levels, which, taken together, influence risk of T2D and markers of insulin resistance