The sun, cholesterol and Orai1 conspire in melanoma

How does skin cancer become metastatic? A recent study by Gross et al (2022) reports a novel pathway by which UV radiation acts on cholesterol biosynthesis to control Ca²⁺ influx by Orai1, causing protein O‐GlcNAcylation that promotes transformation to invasive melanoma.

Recent work reveals unexpected links between UV radiation, lipid biosynthesis, and Orai1‐mediated Ca²⁺ influx promoting skin cancerogenesis and metastasis.

Ca²⁺ signaling regulates virtually all cellular activities, and aberrant Ca²⁺ signaling is associated with numerous diseases. The receptor‐evoked Ca²⁺ signal is initiated by activation of PLC and generation of IP₃ that releases Ca²⁺ from the endoplasmic reticulum (ER), which causes clustering of the ER Ca²⁺ sensor STIM1 at the ER/ plasma membrane (PM) junctions. STIM1, in turn, activates the PM Ca²⁺ influx channel Orai1 (Berridge, 2016) and facilitates the activation of Ca²⁺ permeable TRPC channels (Liu et al, 2022). Increased [Ca²⁺] _i activates the plasma membrane (PMCA) and ER (SERCA) Ca²⁺ pumps to clear part of cytoplasmic Ca²⁺, thereby preventing Ca²⁺ toxicity (Berridge, 2016). The ER Ca²⁺ store is limited, and therefore, the Ca²⁺ signal must be sustained by Ca²⁺ influx through the PM Ca²⁺ influx channels. Ca²⁺ influx mediated by Orai1 feeds all cellular organelles, including the ER and mitochondria, and is essential for cellular metabolism, immune responses, regulated exocytosis, proliferation and differentiation, transcription, and other vital cellular functions (Prakriya & Lewis, 2015).

In recent years, it has become clear that aberrant Ca²⁺ signaling is associated with cancer. Ca²⁺ influx by the STIM1‐activated Orai1 generally promotes cancer via its role in cell transformation and tumor progression (Hammad & Machaca, 2021). In melanoma, a malignancy of pigment‐producing skin cells, the Orai and STIM proteins regulate cells within the tumor environment and control the integrity of the extracellular matrix to facilitate metastasis (Zhang et al, 2022). Melanoma is characterized by progressive phases in which melanocytes first become hyperplastic, then transform into melanoma in situ, and finally progress to highly proliferative and invasive cells, culminating in fatal metastasis. Several mutations have been associated with malignant transformation and metastatic progression, and the primary trigger is exposure to UV radiation (Zhang et al, 2022). Various components of the Ca²⁺ signaling pathway are also implicated in malignant transformation, including Ca²⁺ permeable channels, Ca²⁺ pumps, Orai1, STIM2, and mitochondrial Ca²⁺ uptake (Zhang et al, 2019, 2022). However, the role of Ca²⁺ influx in UV radiation‐triggered malignant transformation is not well‐defined. A study published in this issue of the EMBO Journal by the group of Jonathan Soboloff (Gross et al, 2022) reports a novel and intricate pathway that confers metastatic potential to transformed melanocytes (Fig 1).

Figure 1. Orai1‐STIM1 complexes at the ER/PM junction of melanocytes mediate essential component of the receptor‐stimulated Ca²⁺ influx.

UV radiation activates the mevalonate pathway in naïve or transformed melanocytes to increase cholesterol at the ER/PM junctions that are assembled by tether proteins that include lipid transfer proteins and where STIM1‐Orai1 complexes are formed. Among other effects, cholesterol binds to Orai1, partially inhibiting its activity and reducing Ca²⁺ influx at the ER/PM junction. Reduced Ca²⁺ influx leads to increased glucose uptake, which is shunted to the hexosamine pathway to increase UDP‐GlcNAc synthesis and O‐GlcNAcylation of proteins to promote malignant transformation of melanocytes and increase invasiveness.

Gross et al (2022) used several melanoma cell lines and compared the effect of brief exposure to UV radiation on store‐operated Ca²⁺ entry (SOCE) 2 and 5 weeks later. All cell lines that gained metastatic activity had reduced SOCE activity that developed over time. Interestingly, pharmacological reduction in SOCE to the level caused by UV radiation was sufficient to confer invasive potential, while restoration of SOCE and complete SOCE repression both prevented metastasis. This indicated that partial SOCE inhibition was sufficient for the invasive transformation, and it was necessary for melanoma. SOCE inhibition was not due to repressed Orai or STIM expression but was rather due to a change in SOCE activity. Comparing the transcriptomes of noninvasive and metastatic melanoma cells revealed upregulation of the mevalonate pathway in invasive cells. This finding correlates with the association of melanoma with mevalonate pathway dysregulation indicated by the Cancer Genome Atlas. The cholesterol (CHL) biosynthesis pathway is a key part of the mevalonate pathway; accordingly, UV radiation markedly increased CHL content in metastatic cells independent of SOCE, and inhibition of CHL biosynthesis increased SOCE and inhibited invasiveness. Moreover, metastasis of melanoma cells was increased in Ldlr ^−/− mice that have high CHL and was further enhanced by administration of high‐fat diet. CHL is thought to bind to the N terminus of Orai1 and inhibit channel function (Derler et al, 2016). Thus, CHL‐mediated inhibition of Orai1 could underlie the suppression of SOCE in melanoma seen by (Gross et al, 2022).

To search for potential consequences of reduced SOCE activity that might promote invasive melanoma, Gross et al (2022) further analyzed the transcriptomes of UV‐transformed invasive and noninvasive melanoma cells to identify genes associated with both SOCE suppression and invasiveness. Both were associated with marked upregulation of genes involved in metabolic functions, particularly glucose uptake. However, invasive cells had minimal changes in glycolytic activity and oxygen consumption. Rather, increased glucose uptake appeared to fuel the hexosamine pathway culminating in the synthesis of UDP‐GlcNAc to increase post‐transcriptional O‐GlcNAcylation mediated by an increase in UDP‐GlcNAc transferase (OGT) activity. Post‐transcriptional O‐GlcNAcylation promotes epithelial‐mesenchymal transformation, increases cell proliferation and cell survival, and thus likely promotes malignant transformation. Accordingly, knockdown of OGT reduced UV‐mediated invasiveness. Finally, the clinical relevance of these findings was supported by the observation that cells obtained from patient tumors showed reduced SOCE. Reducing intracellular CHL and inhibiting OGT increased SOCE and reduced melanoma invasiveness in in vitro invasion assays (Gross et al, 2022).

The findings of Gross et al (2022) suggest the model depicted in Fig 1. UV radiation that penetrates the skin acts on naïve or transformed melanocytes to induce CHL biosynthesis via the mevalonate pathway, thereby increasing CHL levels at the ER/PM junctions where STIM1‐Orai1 complexes are formed. CHL binds to Orai1, partially inhibiting its activity and reducing Ca²⁺ influx at the ER/PM junction. Reduced Ca²⁺ influx through Orai1 increases uptake of glucose, which is shunted to the hexosamine biosynthetic pathway to increase UDP‐GlcNAc synthesis. O‐GlcNAcylation of proteins at their serine and threonine residues promotes malignant transformation of melanocytes as well as increases their invasiveness.

The interesting findings of Gross et al (2022) pose several questions and open several areas for future investigation. These include the mechanisms by which UV radiation induces the mevalonate pathway, and the potential role of other physiologic Orai1‐inhibitors in malignant transformation. It is unclear whether inhibition of Orai1 is exclusively by CHL due to the paucity of reports showing inhibition of Orai1 by CHL and reports that CHL at the ER/PM junctions is required for Orai1‐STIM1 interaction. It was also quite surprising that enhanced glucose uptake had no effect on glycolysis and cellular respiration, but was instead routed to the hexosamine pathway. It is possible that the increased glucose uptake and the hexosamine pathway are co‐compartmentalized to explain the specific use of this glucose uptake. More generally, the roles of lipids in modulating Orai1 and STIM1 will require future investigation. Orai1‐STIM1 interaction is restricted to the ER/PM junction domain where lipids, including CHL, are delivered by lipid transfer proteins (LTPs) (Muallem et al, 2017; Arora et al, 2022). For CHL to have a specific effect on Orai1, it should be enriched at the ER/PM junction. It will, therefore, be important to determine how LTPs deliver high local concentrations of CHL to modulate Orai1 function. The association of reduced SOCE with invasiveness is also at odds with several observations that tumorigenesis is associated with increased Ca²⁺ influx (Hammad & Machaca, 2021). This raises the possibility that Orai1 has divergent roles in different phases of tumor initiation and metastasis. Increased Orai1‐mediated Ca²⁺ influx may promote tumor induction, while decreased Orai1 activity may promote metastasis. Because CHL concentration is elevated in many solid organ tumors, further investigating these mechanisms in other malignancies could therefore expand the relevance of Gross et al (2022) findings.

Tumorigenesis and metastasis are complicated biological processes that are likely affected by multiple pathways. A tight communication exists between Orai1‐mediated Ca²⁺ influx and mitochondrial Ca²⁺ uptake by the mitochondrial Ca²⁺ channel MCU complex that, in turn, regulates Ca²⁺ influx by Orai1 (Prakriya & Lewis, 2015). Ca²⁺ uptake by MCU regulates mitochondrial redox homeostasis, which has been implicated in melanoma (Zhang et al, 2019). The connection between Ca²⁺ influx by Orai1 and Ca²⁺ uptake by MCU suggests a role for the latter in melanoma tumorigenesis and metastasis. Indeed, a parallel study by the group of Ivan Bogeski (Stejerean‐Todoran et al, 2022) reports correlation between reduced MCU expression and patient survival and that reduced MCU increases melanoma cells migration and invasion, leading to enhanced lung metastases. Reduced MCU activity was associated with enhanced antioxidants that is necessary and sufficient for the enhanced melanoma invasiveness. Reduced MCU activity increases Ca²⁺ at the mouth of Orai1 to decrease its activity (Prakriya & Lewis, 2015), which may contribute to the reduced Orai1 activity reported by Gross et al (2022). Thus, the findings of Gross et al (2022) and Stejerean‐Todoran et al (2022) provide two cytoplasmic Ca²⁺ related and likely linked targets to be considered for developing a new therapy for invasive melanoma.

Disclosure and competing interests statement

The authors declare that they have no conflict of interest.

The EMBO Journal (2022) 41: e112250.