Sample characteristics.

*<p>FFPE =  Formalin-fixed, paraffin-embedded, FF =  Fresh- frozen.</p

Low levels of nuclear ß-catenin coincide with high levels of E-cadherin in BCC.

<p><b>A.</b> Microphotographs of selected sample of ß-catenin, showing nuclear staining only at the periphery or the tumor. Bar = 200 µm. <b>B.</b> Microphotographs of selected sample of E-cadherin showing lowered expression of the tumor compared with the normal epidermis.</p

Expression of SHH, APC, SFRP5 and RASSF1A is reduced in BCC versus healthy skin control tissue.

<p><b>A.</b> Relative expression levels of <i>APC</i> and <i>SFRP5 i</i>n BCC tissues as compared to the expression level in normal skin (n = 6) (2−^ΔΔCt). All reactions were done in triplicates, standard error of the mean (SEM) is shown as error bars. Expression levels were normalized to Cyclophilin A. U, unmethylated sample; M, methylated sample. *p≤0.05, **p≤0.001. <b>B.</b> Relative mRNA expression in unmethylated versus methylated BCC samples for either <i>APC</i> or <i>SFRP5. </i><b>C.</b> Microphotographs of selected samples of SHH, APC and RASSF1A. Bar = 200 µm.</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

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

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

Tumour characteristics.

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