The renin angiotensin system (RAS) mediates bifunctional growth regulation in melanoma and is a novel target for therapeutic intervention.

Despite emergence of new systemic therapies, metastatic melanoma remains a challenging and often fatal form of skin cancer. The renin-angiotensin system (RAS) is a major physiological regulatory pathway controlling salt-water equilibrium, intravascular volume and blood pressure. Biological effects of the RAS are mediated by the vasoactive hormone angiotensin II (AngII) via two receptor subtypes, AT1R (encoded by AGTR1) and AT2R (encoded by AGTR2). We report decreasing expression and increasing CpG island methylation of AGTR1 in metastatic versus primary melanoma and detection in serum of methylated genomic DNA from the AGTR1 CpG island in metastatic melanoma implying that AGTR1 encodes a tumour suppressor function in melanoma. Consistent with this hypothesis, antagonism of AT1R using losartan or shRNA-mediated knockdown in melanoma cell lines expressing AGTR1 resulted in acquisition of the ability to proliferate in serum-free conditions. Conversely, ectopic expression of AGTR1 in cell lines lacking endogenous expression inhibits proliferation irrespective of the presence of AngII implying a ligand-independent suppressor function for AT1R. Treatment of melanoma cell lines expressing endogenous AT2R with either AngII or the AT2R-selective agonist Y6AII induces proliferation in serum-free conditions whereas the AT2R-specific antagonists PD123319 and EMA401 inhibit melanoma growth and angiogenesis and potentiate inhibitors of BRAF and MEK in cells with BRAF V600 mutations. Our results demonstrate that the RAS has both oncogenic and tumour suppressor functions in melanoma. Pharmacological inhibition of AT2R may provide therapeutic opportunities in melanomas expressing this receptor and AGTR1 CpG island methylation in serum may serve as a novel biomarker of metastatic melanoma.

Collagen Prolyl Hydroxylases Are Bifunctional Growth Regulators in Melanoma.

Appropriate post-translational processing of collagen requires prolyl hydroxylation, catalyzed by collagen prolyl 3-hydroxylase and collagen prolyl 4-hydroxylase, and is essential for normal cell function. Here we have investigated the expression, transcriptional regulation, and function of the collagen prolyl 3-hydroxylase and collagen prolyl 4-hydroxylase families in melanoma. We show that the collagen prolyl 3-hydroxylase family exemplified by Leprel1 and Leprel2 is subject to methylation-dependent transcriptional silencing in primary and metastatic melanoma consistent with a tumor suppressor function. In contrast, although there is transcriptional silencing of P4HA3 in a subset of melanomas, the collagen prolyl 4-hydroxylase family members P4HA1, P4HA2, and P4HA3 are often overexpressed in melanoma, expression being prognostic of worse clinical outcomes. Consistent with tumor suppressor function, ectopic expression of Leprel1 and Leprel2 inhibits melanoma proliferation, whereas P4HA2 and P4HA3 increase proliferation, and particularly invasiveness, of melanoma cells. Pharmacological inhibition with multiple selective collagen prolyl 4-hydroxylase inhibitors reduces proliferation and inhibits invasiveness of melanoma cells. Together, our data identify the collagen prolyl 3-hydroxylase and collagen prolyl 4-hydroxylase families as potentially important regulators of melanoma growth and invasiveness and suggest that selective inhibition of collagen prolyl 4-hydroxylase is an attractive strategy to reduce the invasive properties of melanoma cells.

ARNT controls the expression of epidermal differentiation genes through HDAC- and EGFR-dependent pathways.

Previously we showed that spatial and developmental modulation of ARNT (HIF1ß) expression in mouse epidermis is essential for maintenance of keratinocyte differentiation, proper formation of the barrier and normal desquamation. Here, using lentiviral suppression or induction of ARNT in TERT-immortalized (N-TERT) and HaCaT cells we assessed the nature and mechanisms of ARNT involvement in control of differentiation in human epidermal keratinocytes. ARNT depletion did not affect the levels of basal keratins K5 and K14, but significantly induced expression of several key differentiation markers (an effect abolished by EGF supplementation). Furthermore, ARNT deficiency resulted in the downregulation of amphiregulin (AREG) - the most highly expressed EGFR ligand in human keratinocytes - whereas upregulation of ARNT showed the opposite. In ARNT-deficient monolayer cultures and 3D epidermal equivalents, the downregulation of AREG was concurrent with a decline of EGFR and ERK1/2 phosphorylation. TSA, a potent suppressor of HDAC activity, abolished the effects of ARNT deficiency, implying a role for HDACs in ARNT-dependent modulation of the AREG-EGFR pathway and downstream epidermal genes. Total HDAC activity was significantly increased in ARNT-depleted cells and decreased with ARNT overexpression. ARNT-dependent shifts in HDAC activity were specifically attributed to significant changes in the levels of HDAC1, HDAC2 and HDAC3 proteins (but not mRNA) in both monolayer and 3D cultures. Collectively, our results suggest that ARNT controls AREG expression and the downstream EGFR-ERK pathway in keratinocytes, at least in part, by modulating HDAC activity. This novel regulatory pathway targeting advanced stages of epidermal differentiation might have important implications for skin pathology such as psoriasis, atopic dermatitis and cancer.

Loss of Arnt (Hif1ß) in mouse epidermis triggers dermal angiogenesis, blood vessel dilation and clotting defects.

Targeted ablation of Aryl hydrocarbon receptor nuclear translocator (Arnt) in the mouse epidermis results in severe abnormalities in dermal vasculature reminiscent of petechia induced in human skin by anticoagulants or certain genetic disorders. Lack of Arnt leads to downregulation of Egln3/Phd3 hydroxylase and concomitant hypoxia-independent stabilization of hypoxia-induced factor 1α (Hif1α) along with compensatory induction of Arnt2. Ectopic induction of Arnt2 results in its heterodimerization with stabilized Hif1α and is associated with activation of genes coding for secreted proteins implicated in control of angiogenesis, coagulation, vasodilation and blood vessel permeability such as S100a8/S100a9, S100a10, Serpine1, Defb3, Socs3, Cxcl1 and Thbd. Since ARNT and ARNT2 heterodimers with HIF1α are known to have different (yet overlapping) downstream targets our findings suggest that loss of Arnt in the epidermis activates an aberrant paracrine regulatory pathway responsible for dermal vascular phenotype in K14-Arnt KO mice. This assumption is supported by a significant decline of von Willebrand factor in dermal vasculature of these mice where Arnt level remains normal. Given the essential role of ARNT in the adaptive response to environmental stress and striking similarity between skin vascular phenotype in K14-Arnt KO mice and specific vascular features of tumour stroma and psoriatic skin, we believe that further characterization of Arnt-dependent epidermal-dermal signalling may provide insight into the role of macro- and micro-environmental factors in control of skin vasculature and in pathogenesis of environmentally modulated skin disorders.

The reduction of water-soluble tetrazolium salt reagent on the plasma membrane of epidermal keratinocytes is oxygen dependent.

The water-soluble tetrazolium salt (WST-1) assay is frequently used to assess cell proliferation. However, our study showed that in normal and cancerous keratinocytes, this assay is more responsive to changes in oxygenation than to rates of cell growth. Stimulation of keratinocyte proliferation by low Ca(2+) and suppression of proliferation by nocodazole resulted in modest changes in WST-1 readings, whereas gradually reducing the level of oxygen in the cellular environment from ambient (21%) to near anoxic (0.1%) revealed a very strong negative correlation between cell oxygenation and WST-1 reagent reduction. In contrast, the very similar MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay, which uses a different tetrazolium salt, showed no sensitivity to the level of oxygen. Unlike MTT, WST-1 reagent is reduced extracellularly through trans-plasma membrane transport (tPMET), thereby suggesting that tPMET is oxygen dependent. We propose that the WST-1 assay can be developed into a sensitive quantitative method to evaluate cell oxygenation in vitro and used to study the role of hypoxia and tPMET in homeostasis and disease (e.g., cancer). At the same time, WST-1 assay should be used cautiously to assess cell viability or proliferation because readings can be affected by certain extrinsic (low atmospheric oxygen or high density culture) or intrinsic (defects in oxygen-sensing pathways) factors.

Hypoxia-mediated control of HIF/ARNT machinery in epidermal keratinocytes.

Transcriptional activity of hypoxia-induced factor 1 (HIF1) - a heterodimer of HIF1α and ARNT (HIF1ß) - is essential for cellular adaptation to environmental stress and plays an important role in skin development, wound healing, tumorigenesis and barrier function. Using primary mouse and human epidermal keratinocytes at ambient or hypoxic (1% O(2)) conditions we studied effects of hypoxia upon HIF protein expression. Significant nuclear levels of ARNT and HIF1α along with high HIF1 activity in normoxic keratinocytes suggest an as yet uncharacterised oxygen-independent role for HIF pathway in the epidermis. Acute hypoxia results in an instant but transient increase of HIF1α protein accompanied by a gradual decrease in its mRNA, while ARNT expression remains unchanged. In prolonged (chronic) hypoxia both HIF1α and Arnt are downregulated along with decline of HIF1 activity. However, expression of classical HIF1 targets such as Selenbp1 and Vegfa remains high. Thus, keratinocytes respond to acute hypoxia with immediate block of HIF1α protein degradation and concomitant increase of HIF activity, while under chronic hypoxia pro-angiogenic signalling is maintained through HIF1-independent pathway(s). Decline of HIF1α during chronic exposure is controlled at both mRNA and protein levels, while Arnt is downregulated post-translationally. Distinct transcription levels of Hif1α and Hif3α splice variants under normoxia and their differential response to hypoxia suggest functional diversity of Hif-α isoforms and highlight the complexity of HIF machinery control in epidermal keratinocytes.